Dr. Bollyky is an Immunologist and Infectious Disease specialist at Stanford Medical Center.
- Infectious Disease
- Wound infections
- Microbial biofilms
- Diabetic wounds
Honors & Awards
Fellow, Infectious Disease Society of America (2019)
Elected to membership in the American Society of Clinical Investigators, (ASCI) (2018)
Harrington Scholar Innovator, Harrington Discovery Institute (2017)
Transformative Research Award, Dr. Ralph and Marian Falk Medical Research Trust (2017)
Grand Challenges Award, Bill & Melinda Gates Foundation (2016)
Catalyst Award, Dr. Ralph and Marian Falk Medical Research Trust (2015)
Outstanding Faculty Mentor Award, Stanford Immunology (2015)
Career Development Award, Juvenile Diabetes Research Foundation (2012)
Elected to membership, Western Society of Clinical Investigators (2012)
Young Investigator Award, University of Washington Diabetes Research Council (2012)
Excellence in Teaching Award, Harvard Medical School (2004)
Soma Weiss Prize for Student Research, Harvard Medical School (2001)
North Senior Scholar, St. John's College, Oxford (1997)
Marshall Scholar, British Marshall Scholarship Fund (1994)
Board Certification: American Board of Internal Medicine, Infectious Disease (2019)
Fellowship: University of Washington Infectious Disease Program (2007) WA
Residency: Brigham and Women's Hospital Internal Medicine Residency (2004) MA
Medical Education: Harvard Medical School (2001) MA
PhD, Oxford University (1998)
B.A., Columbia University, Biology (1994)
Current Research and Scholarly Interests
The Bollyky lab studies the innate immune response to injury and infection. Our goals are to gain insight into fundamental disease mechanisms and to generate novel therapies to improve human health.
One area of active research involves bacteriophages – viruses that infect bacteria. The human body contains more bacteriophages than human cells but their impact on human biology is largely unknown. Current efforts are focused on understanding how bacteriophage interact with mammalian cells, how this infection influences local immunity, and how phage shape the immune response to commensal and pathogenic bacteria. We also have active efforts involved in characterizing the "phageome" - the collection of phages present in the body.
A second area of active research involves biofilms - the communities of polymers and bacteria that colonize infected tissues. The extracellular matrix present in biofilms exerts important but poorly understood effects on chronic infection. We are interested in how biofilms modulate human immunity, prevent diffusion of antibiotics, and contribute to chronic infection.
A third area of investigation is the tissue extracellular matrix at sites of injury and infection. In particular, we are interested in the roles that hyaluronan and other extracellular matrix polymers play in immune regulation. We use models of autoimmunity and infection to learn how the extracellular matrix and inflammation influences innate immunity and local immune regulation.
Looking for a lab?
We are actively recruiting structural biologists, biochemists, immunologists, and microbiologists for post-doctoral positions in the lab. If interested, please contact Dr. Bollyky at firstname.lastname@example.org
For Stanford Undergraduates interested in joining the lab, we require a minimum commitment of a full year (including full-time work in the lab for at least one summer).
We take high school and undergraduate students through the SSRP program: https://biosciences.stanford.edu/prospective/diversity/ssrp/
A Study of Oral Hymecromone and Hyaluronan Synthesis
The purpose of this study is to add further understanding to the doses of hymecromone that effectively and safely lead to the inhibition of hyaluronan synthesis. In this study we will investigate both circulating hyaluronan in the serum, as well as tissue hyaluronan, using sputum samples as a non-invasive surrogate. This is a parallel, open-label, single-center, dose-response study of hymecromone in healthy adults 18 years of age or older. Up to 18 participants will be enrolled. Participants will be treated for 4 days with study drug. Safety as well as biomarkers of pharmacokinetic and pharmacodynamic response will be monitored during therapy.
Stanford is currently not accepting patients for this trial.
Independent Studies (12)
- Directed Reading in Immunology
IMMUNOL 299 (Win, Spr)
- Directed Reading in Medicine
MED 299 (Win, Spr)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Medicine
MED 280 (Win, Spr)
- Graduate Research
IMMUNOL 399 (Aut, Win, Spr, Sum)
- Graduate Research
MED 399 (Win, Spr)
- Graduate Research
MI 399 (Aut, Win, Spr)
- Immunology Research Seminars for Medical Students
IMMUNOL 210 (Aut, Win)
- Medical Scholars Research
MED 370 (Win, Spr, Sum)
- Teaching in Immunology
IMMUNOL 290 (Win, Spr)
- Undergraduate Research
IMMUNOL 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
MED 199 (Win, Spr)
- Directed Reading in Immunology
- Prior Year Courses
Med Scholar Project Advisor
Doctoral Dissertation Reader (AC)
Justin Arredondo-Guerrero, Jason Nideffer, Sarah Ruddle
Postdoctoral Faculty Sponsor
Qingquan Chen, Arya Khosravi, Kevin Kim, Nina Pennetzdorfer
Doctoral Dissertation Advisor (AC)
Tejas Dharmaraj, Naomi Haddock, Hunter Martinez
Postdoctoral Research Mentor
Layla Barkal, Qingquan Chen, Arya Khosravi, Kevin Kim, Nina Pennetzdorfer
Bacteriophages and the Immune System.
Annual review of virology
Bacteriophages-viruses that infect bacteria-are abundant within our bodies, but their significance to human health is only beginning to be explored. Here, we synthesize what is currently known about our phageome and its interactions with the immune system. We first review how phages indirectly affect immunity via bacterial expression of phage-encoded proteins. We next review how phages directly influence innate immunity and bacterial clearance. Finally, we discuss adaptive immunity against phages and its implications for phage/bacterial interactions. In light of these data, we propose that our microbiome can be understood as an interconnected network of bacteria, bacteriophages, and human cells and that the stability of these tri-kingdom interactions may be important for maintaining our immunologic and metabolic health. Conversely, the disruption of this balance, through exposure to exogenous phages, microbial dysbiosis, or immune dysregulation, may contribute to disease. Expected final online publication date for the Annual Review of Virology, Volume 8 is September 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
View details for DOI 10.1146/annurev-virology-091919-074551
View details for PubMedID 34014761
- Filamentous bacteriophages are associated with chronic Pseudomonas lung infections and antibiotic resistance in cystic fibrosis SCIENCE TRANSLATIONAL MEDICINE 2019; 11 (488)
Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection.
Science (New York, N.Y.)
2019; 363 (6434)
Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-β (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.
View details for PubMedID 30923196
Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization.
Proceedings of the National Academy of Sciences of the United States of America
2016; 113 (5): 1339-1344
The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3(+) regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.
View details for DOI 10.1073/pnas.1525086113
View details for PubMedID 26787861
Filamentous Bacteriophage Promote Biofilm Assembly and Function
CELL HOST & MICROBE
2015; 18 (5): 549-559
Biofilms-communities of bacteria encased in a polymer-rich matrix-confer bacteria with the ability to persist in pathologic host contexts, such as the cystic fibrosis (CF) airways. How bacteria assemble polymers into biofilms is largely unknown. We find that the extracellular matrix produced by Pseudomonas aeruginosa self-assembles into a liquid crystal through entropic interactions between polymers and filamentous Pf bacteriophages, which are long, negatively charged filaments. This liquid crystalline structure enhances biofilm function by increasing adhesion and tolerance to desiccation and antibiotics. Pf bacteriophages are prevalent among P. aeruginosa clinical isolates and were detected in CF sputum. The addition of Pf bacteriophage to sputum polymers or serum was sufficient to drive their rapid assembly into viscous liquid crystals. Fd, a related bacteriophage of Escherichia coli, has similar biofilm-building capabilities. Targeting filamentous bacteriophage or the liquid crystalline organization of the biofilm matrix may represent antibacterial strategies.
View details for DOI 10.1016/j.chom.2015.10.013
View details for Web of Science ID 000365113100008
View details for PubMedID 26567508
View details for PubMedCentralID PMC4653043
Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis.
The Journal of clinical investigation
We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.
View details for DOI 10.1172/JCI79271
View details for PubMedID 26368307
IL-10 promotes endothelial progenitor cell infiltration and wound healing via STAT3.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
2022; 36 (7): e22298
Endothelial progenitor cells (EPCs) contribute tode novoangiogenesis, tissue regeneration, and remodeling. Interleukin 10 (IL-10), an anti-inflammatory cytokine that primarily signals via STAT3, has been shown to drive EPC recruitment to injured tissues. Our previous work demonstrated that overexpression of IL-10 in dermal wounds promotes regenerative tissue repair via STAT3-dependent regulation of fibroblast-specific hyaluronan synthesis. However, IL-10's role and specific mode of action on EPC recruitment, particularly in dermal wound healing and neovascularization in both normal and diabetic wounds, remain to be defined.Therefore, inducible skin-specific STAT3knockdown mice were studied to determine IL-10's impact on EPCs, dermal wound neovascularization and healing, and whether it is STAT3-dependent. We show that IL-10 overexpression significantly elevated EPC counts in the granulating wound bed, which was associated with robust capillary lumen density and enhanced re-epithelialization of both control and diabetic (db/db) wounds at day 7. We noted increased VEGF and high C-X-C motif chemokine 12 (CXCL12) levels in wounds and a favorable CXCL12gradient at day 3 that may support EPC mobilization and infiltration from bone marrow to wounds, an effect that was abrogated in STAT3knockdown wounds. These findings were supportedin vitro. IL-10 promoted VEGF and CXCL12synthesis in primary murine dermal fibroblasts, with blunted VEGF expression upon blocking CXCL12 in the media by antibody binding. IL-10-conditioned fibroblast media also significantly promoted endothelial sprouting and network formation. In conclusion, these studies demonstrate that overexpression of IL-10 in dermal wounds recruits EPCs and leads to increased vascular structures and faster re-epithelialization.
View details for DOI 10.1096/fj.201901024RR
View details for PubMedID 35670763
Biochemical, biophysical, and immunological characterization of respiratory secretions in severe SARS-CoV-2 infections.
2022; 7 (12)
Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e., resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We found the percentages of solids and protein content were greatly elevated in COVID-19 compared with heathy control samples and closely resembled levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) were major components of respiratory secretions in COVID-19 and were likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors, with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observed increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factor-stimulated gene-6 staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicated that increases in HA and DNA in COVID-19 respiratory secretion samples correlated with enhanced inflammatory burden and suggested that DNA and HA may be viable therapeutic targets in COVID-19 infection.
View details for DOI 10.1172/jci.insight.152629
View details for PubMedID 35730564
Filamentous bacteriophage delays healing of Pseudomonas-infected wounds.
Cell reports. Medicine
2022; 3 (6): 100656
Chronic wounds infected by Pseudomonas aeruginosa (Pa) are characterized by disease progression and increased mortality. We reveal Pf, a bacteriophage produced by Pa that delays healing of chronically infected wounds in human subjects and animal models of disease. Interestingly, impairment of wound closure by Pf is independent of its effects on Pa pathogenesis. Rather, Pf impedes keratinocyte migration, which is essential for wound healing, through direct inhibition of CXCL1 signaling. In support of these findings, a prospective cohort study of 36 human patients with chronic Pa wound infections reveals that wounds infected with Pf-positive strains of Pa are more likely to progress in size compared with wounds infected with Pf-negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian cells. These findings suggest Pf may have potential as a biomarker and therapeutic target in chronic wounds.
View details for DOI 10.1016/j.xcrm.2022.100656
View details for PubMedID 35732145
Oral hymecromone decreases hyaluronan in human study participants.
The Journal of clinical investigation
2022; 132 (9)
BACKGROUNDHyaluronan (HA), an extracellular matrix glycosaminoglycan, has been implicated in the pathophysiology of COVID-19 infection, pulmonary hypertension, pulmonary fibrosis, and other diseases, but is not targeted by any approved drugs. We asked whether hymecromone (4-methylumbelliferone [4-MU]), an oral drug approved in Europe for biliary spasm treatment that also inhibits HA in vitro and in animal models, could be repurposed as an inhibitor of HA synthesis in humans.METHODSWe conducted an open-label, single-center, dose-response study of hymecromone in healthy adults. Subjects received hymecromone at 1200 (n = 8), 2400 (n = 9), or 3600 (n = 9) mg/d divided into 3 doses daily, administered orally for 4 days. We assessed safety and tolerability of hymecromone and analyzed HA, 4-MU, and 4-methylumbelliferyl glucuronide (4-MUG; the main metabolite of 4-MU) concentrations in sputum and serum.RESULTSHymecromone was well tolerated up to doses of 3600 mg/d. Both sputum and serum drug concentrations increased in a dose-dependent manner, indicating that higher doses lead to greater exposures. Across all dose arms combined, we observed a significant decrease in sputum HA from baseline after 4 days of treatment. We also observed a decrease in serum HA. Additionally, higher baseline sputum HA levels were associated with a greater decrease in sputum HA.CONCLUSIONAfter 4 days of exposure to oral hymecromone, healthy human subjects experienced a significant reduction in sputum HA levels, indicating this oral therapy may have potential in pulmonary diseases where HA is implicated in pathogenesis.TRIAL REGISTRATIONClinicalTrials.gov NCT02780752.FUNDINGStanford Medicine Catalyst, Stanford SPARK, Stanford Innovative Medicines Accelerator program, NIH training grants 5T32AI052073-14 and T32HL129970.
View details for DOI 10.1172/JCI157983
View details for PubMedID 35499083
A Filamentous Bacteriophage Protein Inhibits Type IV Pili To Prevent Superinfection of Pseudomonas aeruginosa.
Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in a variety of settings. Many P. aeruginosa isolates are infected by filamentous Pf bacteriophage integrated into the bacterial chromosome as a prophage. Pf virions can be produced without lysing P. aeruginosa. However, cell lysis can occur during superinfection, which occurs when Pf virions successfully infect a host lysogenized by a Pf prophage. Temperate phages typically encode superinfection exclusion mechanisms to prevent host lysis by virions of the same or similar species. In this study, we sought to elucidate the superinfection exclusion mechanism of Pf phage. Initially, we observed that P. aeruginosa that survive Pf superinfection are transiently resistant to Pf-induced plaquing and are deficient in twitching motility, which is mediated by type IV pili (T4P). Pf utilize T4P as a cell surface receptor, suggesting that T4P are suppressed in bacteria that survive superinfection. We tested the hypothesis that a Pf-encoded protein suppresses T4P to mediate superinfection exclusion by expressing Pf proteins in P. aeruginosa and measuring plaquing and twitching motility. We found that the Pf protein PA0721, which we termed Pf superinfection exclusion (PfsE), promoted resistance to Pf infection and suppressed twitching motility by binding the T4P protein PilC. Because T4P play key roles in biofilm formation and virulence, the ability of Pf phage to modulate T4P via PfsE has implications in the ability of P. aeruginosa to persist at sites of infection. IMPORTANCE Pf bacteriophage (phage) are filamentous viruses that infect Pseudomonas aeruginosa and enhance its virulence potential. Pf virions can lyse and kill P. aeruginosa through superinfection, which occurs when an already infected cell is infected by the same or similar phage. Here, we show that a small, highly conserved Pf phage protein (PA0721, PfsE) provides resistance to superinfection by phages that use the type IV pilus as a cell surface receptor. PfsE does this by inhibiting assembly of the type IV pilus via an interaction with PilC. As the type IV pilus plays important roles in virulence, the ability of Pf phage to modulate its assembly has implications for P. aeruginosa pathogenesis.
View details for DOI 10.1128/mbio.02441-21
View details for PubMedID 35038902
Interleukin-10 Producing T Lymphocytes Attenuate Dermal Scarring.
Annals of surgery
2021; 274 (4): 627-636
OBJECTIVE: Demonstrate the impact of IL-10 producing T lymphocytes on mediating dermal scarring.SUMMARY BACKGROUND DATA: We demonstrated that CD4+ cells are essential to improving postinjury wound healing and preventing fibrosis. CD4+ subsets secrete differential cytokine and growth factor profiles, though their role in fibrosis is not known. IL-10, a key anti-inflammatory cytokine shown to promote regenerative wound healing, is secreted by some CD4+ subsets. We, therefore, hypothesize that IL-10 producing CD4+ T lymphocyte subsets selectively attenuate dermal wound fibrosis.METHODS: IL-10-/- and wild-type murine splenocytes were enriched for CD4+ lymphocytes and adoptively transferred into severe combined immunodeficient (SCID) mice that received full-thickness wounds which were analyzed at days 7 and 28 for inflammation and collagen content. We then sorted CD4+CD44int/lowFoxP3-CD62L+ T cells (Tnaive) or CD4+CD44HiFoxP3- type 1 regulatory (Tr1) T cell subsets from 10BiT murine splenocytes, activated them, and transferred them into wounds. In vitro, dermal fibroblasts were cocultured with Tnaive or Tr1 and the effect on extracellular matrix (ECM) regulation was analyzed.RESULTS: The anti-inflammatory and antifibrotic effects of CD4+ cells on SCID wounds were lost with cells from IL-10-/- mice. Adoptive transfer of Tr1 into SCID mice resulted in accelerated wound closure at d7 with reduced fibrosis at d28, with Tr1 favoring hyaluronan production by fibroblasts, an ECM molecule implicated in IL-10-induced regenerative healing.CONCLUSIONS: IL-10 producing T-lymphocytes, specifically Tr1, regulate inflammatory cell cytokine expression to promote HA-rich ECM deposition and attenuate fibrosis. Promoting IL-10 producing lymphocytes in wounds may be a therapeutic target to promote regenerative wound healing.
View details for DOI 10.1097/SLA.0000000000004984
View details for PubMedID 34506318
Congenital Heart Disease: An Immunological Perspective.
Frontiers in cardiovascular medicine
2021; 8: 701375
Congenital heart disease (CHD) poses a significant global health and economic burden-despite advances in treating CHD reducing the mortality risk, globally CHD accounts for approximately 300,000 deaths yearly. Children with CHD experience both acute and chronic cardiac complications, and though treatment options have improved, some remain extremely invasive. A challenge in addressing these morbidity and mortality risks is that little is known regarding the cause of many CHDs and current evidence suggests a multifactorial etiology. Some studies implicate an immune contribution to CHD development; however, the role of the immune system is not well-understood. Defining the role of the immune and inflammatory responses in CHD therefore holds promise in elucidating mechanisms underlying these disorders and improving upon current diagnostic and treatment options. In this review, we address the current knowledge coinciding CHDs with immune and inflammatory associations, emphasizing conditions where this understanding would provide clinical benefit, and challenges in studying these mechanisms.
View details for DOI 10.3389/fcvm.2021.701375
View details for PubMedID 34434978
View details for PubMedCentralID PMC8380780
The Immune and Inflammatory Basis of Acquired Pediatric Cardiac Disease
FRONTIERS IN CARDIOVASCULAR MEDICINE
2021; 8: 701224
Children with acquired heart disease face significant health challenges, including a lifetime of strict medical management, multiple cardiac surgeries, and a high mortality risk. Though the presentation of these conditions is diverse, a unifying factor is the role of immune and inflammatory responses in their development and/or progression. For example, infectious agents have been linked to pediatric cardiovascular disease, leading to a large health burden that disproportionately affects low-income areas. Other implicated mechanisms include antibody targeting of cardiac proteins, infection of cardiac cells, and inflammation-mediated damage to cardiac structures. These changes can alter blood flow patterns, change extracellular matrix composition, and induce cardiac remodeling. Therefore, understanding the relationship between the immune system and cardiovascular disease can inform targeted diagnostic and treatment approaches. In this review, we discuss the current understanding of pediatric immune-associated cardiac diseases, challenges in the field, and areas of research with potential for clinical benefit.
View details for DOI 10.3389/fcvm.2021.701224
View details for Web of Science ID 000683132500001
View details for PubMedID 34386532
View details for PubMedCentralID PMC8353076
Treatment with a neutrophil elastase inhibitor and ofloxacin reduces P. aeruginosa burden in a mouse model of chronic suppurative otitis media.
NPJ biofilms and microbiomes
2021; 7 (1): 31
Chronic suppurative otitis media (CSOM) is a widespread, debilitating problem with poorly understood immunology. Here, we assess the host response to middle ear infection over the course of a month post-infection in a mouse model of CSOM and in human subjects with the disease. Using multiparameter flow cytometry and a binomial generalized linear machine learning model, we identified Ly6G, a surface marker of mature neutrophils, as the most informative factor of host response driving disease in the CSOM mouse model. Consistent with this, neutrophils were the most abundant cell type in infected mice and Ly6G expression tracked with the course of infection. Moreover, neutrophil-specific immunomodulatory treatment using the neutrophil elastase inhibitor GW 311616A significantly reduces bacterial burden relative to ofloxacin-only treated animals in this model. The levels of dsDNA in middle ear effusion samples are elevated in both humans and mice with CSOM and decreased during treatment, suggesting that dsDNA may serve as a molecular biomarker of treatment response. Together these data strongly implicate neutrophils in the ineffective immune response to P. aeruginosa infection in CSOM and suggest that immunomodulatory strategies may benefit drug-tolerant infections for chronic biofilm-mediated disease.
View details for DOI 10.1038/s41522-021-00200-z
View details for PubMedID 33824337
Dynamic light scattering microrheology for soft and living materials.
We present a method for using dynamic light scattering in the single-scattering limit to measure the viscoelastic moduli of soft materials. This microrheology technique only requires a small sample volume of 12 muL to measure up to six decades in time of rheological behavior. We demonstrate the use of dynamic light scattering microrheology (DLSmuR) on a variety of soft materials, including dilute polymer solutions, covalently-crosslinked polymer gels, and active, biological fluids. In this work, we detail the procedure for applying the technique to new materials and discuss the critical considerations for implementing the technique, including a custom analysis script for analyzing data output. We focus on the advantages of applying DLSmuR to biologically relevant materials: breast cancer cells encapsulated in a collagen gel and cystic fibrosis sputum. DLSmuR is an easy, efficient, and economical rheological technique that can guide the design of new polymeric materials and facilitate the understanding of the underlying physics governing behavior of naturally derived materials.
View details for DOI 10.1039/d0sm01597k
View details for PubMedID 33427280
Skin-specific knockdown of hyaluronan in mice by an optimized topical 4-methylumbelliferone formulation.
2021; 28 (1): 422–32
Hyaluronan (HA) is abundant in the skin; while HA can be synthesized by the synthases (HAS1-3), HAS2 is the leading contributor. Dysregulation and accumulation of HA is implicated in the pathogenesis of diseases such as keloid scarring, lymphedema and metastatic melanoma. To understand how HA synthesis contributes to skin physiology, and pathologic and fibrotic disorders, we propose the development of skin-specific HA inhibition model, which tests an optimal delivery system of topical 4-methylumbelliferone (4-MU). A design-of-experiments (DOE) approach was employed to develop an optimal 4-MU skin-delivery formulation comprising propylene glycol, ethanol, and water, topically applied to dorsal skin in male and female C57BL/6J wildtype mice to determine the effect on HAS gene expression and HA inhibition. Serum and skin samples were analyzed for HA content along with analysis of expression of HAS1-3, hyaluronidases (HYAL 1-2), and KIAA1199. Using results from DOE and response surface methodology with genetic algorithm optimization, we developed an optimal topical 4-MU formulation to result in ∼70% reduction of HA in dorsal skin, with validation demonstrating ∼50% reduction in HA in dorsal skin. 4-MU topical application resulted in significant decrease in skin HAS2 expression in female mice only. Histology showed thicker dermis in male mice, whereas female mice had thinner dermal layer with more adiposity; and staining for HA-binding protein showed that topical 4-MU resulted in breakdown in HA. Our data suggest a topical 4-MU formulation-based dermal HA inhibition model that would enable elucidating the skin-specific effects of HA in normal and pathologic states.
View details for DOI 10.1080/10717544.2021.1886376
View details for PubMedID 33605181
Filamentous Bacteriophages and the Competitive Interaction between Pseudomonas aeruginosa Strains under Antibiotic Treatment: a Modeling Study.
Pseudomonas aeruginosa (Pa) is a major bacterial pathogen responsible for chronic lung infections in cystic fibrosis patients. Recent work has implicated Pf bacteriophages, nonlytic filamentous viruses produced by Pa, in the chronicity and severity of Pa infections. Pf phages act as structural elements in Pa biofilms and sequester aerosolized antibiotics, thereby contributing to antibiotic tolerance. Consistent with a selective advantage in this setting, the prevalence of Pf-positive (Pf+) bacteria increases over time in these patients. However, the production of Pf phages comes at a metabolic cost to bacteria, such that Pf+ strains grow more slowly than Pf-negative (Pf-) strains in vitro. Here, we use a mathematical model to investigate how these competing pressures might influence the relative abundance of Pf+ versus Pf- strains in different settings. Our model suggests that Pf+ strains of Pa cannot outcompete Pf- strains if the benefits of phage production falls onto both Pf+ and Pf- strains for a majority of parameter combinations. Further, phage production leads to a net positive gain in fitness only at antibiotic concentrations slightly above the MIC (i.e., concentrations for which the benefits of antibiotic sequestration outweigh the metabolic cost of phage production) but which are not lethal for Pf+ strains. As a result, our model suggests that frequent administration of intermediate doses of antibiotics with low decay rates and high killing rates favors Pf+ over Pf- strains. These models inform our understanding of the ecology of Pf phages and suggest potential treatment strategies for Pf+ Pa infections. IMPORTANCE Filamentous phages are a frontier in bacterial pathogenesis, but the impact of these phages on bacterial fitness is unclear. In particular, Pf phages produced by Pa promote antibiotic tolerance but are metabolically expensive to produce, suggesting that competing pressures may influence the prevalence of Pf+ versus Pf- strains of Pa in different settings. Our results identify conditions likely to favor Pf+ strains and thus antibiotic tolerance. This study contributes to a better understanding of the unique ecology of filamentous phages in both environmental and clinical settings and may facilitate improved treatment strategies for combating antibiotic tolerance.
View details for DOI 10.1128/mSystems.00193-21
View details for PubMedID 34156288
Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist.
Interleukin-2 is a pleiotropic cytokine that mediates both pro- and anti-inflammatory functions. Immune cells naturally differ in their sensitivity to IL-2 due to cell type and activation state-dependent expression of receptors and signaling pathway components. To probe differences in IL-2 signaling across cell types, we used structure-based design to create and profile a series of IL-2 variants with the capacity to titrate maximum signal strength in fine increments. One of these partial agonists, IL-2-REH, specifically expanded Foxp3+ regulatory T cells with reduced activity on CD8+ T cells due to cell type-intrinsic differences in IL-2 signaling. IL-2-REH elicited cell type-dependent differences in gene expression and provided mixed therapeutic results: showing benefit in the in vivo mouse dextran sulfate sodium (DSS) model of colitis, but no therapeutic efficacy in a transfer colitis model. Our findings show that cytokine partial agonists can be used to calibrate intrinsic differences in response thresholds across responding cell types to narrow pleiotropic actions, which may be generalizable to other cytokine and growth factor systems.
View details for DOI 10.7554/eLife.65777
View details for PubMedID 34003116
Plasma Microbial Cell-free DNA Next Generation Sequencing in the Diagnosis and Management of Febrile Neutropenia.
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
Standard testing fails to identify a pathogen in most patients with febrile neutropenia (FN). We evaluated the ability of the Karius microbial cell-free DNA (mcfDNA) sequencing Test (KT) to identify infectious etiologies of FN and its impact on antimicrobial management.This prospective study (ClinicalTrials.gov; NCT02912117) enrolled and analyzed 55 patients with FN. Up to 5 blood samples were collected per subject within 24h of fever onset (T1) and every 2-3 days. KT results were compared to blood culture (BC) and standard microbiological testing (SMT) results.Positive agreement was defined as KT identification of ≥1 isolate also detected by BC. At T1, positive and negative agreement were 90% (9/10) and 31% (14/45) respectively; 61% of KT detections were polymicrobial. Clinical adjudication by 3 independent infectious diseases specialists categorized Karius results as: unlikely to cause FN (N=0); Definite (N=12): KT identified ≥1 organism also found by SMT within 7 days; Probable (N=19): KT result was compatible with a clinical diagnosis; Possible (N=10): KT result was consistent with infection but not considered a common cause of FN. Definite, probable and possible cases were deemed true positives. Following adjudication, KT sensitivity and specificity were 85% (41/48) and 100% (14/14) respectively. Calculated time to diagnosis was generally shorter with KT (87%). Adjudicators determined real-time KT results could have allowed early optimization of antimicrobials in 47% of patients, by addition of antibacterials (20%) (mostly against anaerobes [12.7%]), antivirals (14.5%) and/or antifungals (3.6%); and antimicrobial narrowing in 27.3% of cases.KT shows promise in the diagnosis and treatment optimization of FN.
View details for DOI 10.1093/cid/ciab324
View details for PubMedID 33870413
Tolerant Small-colony Variants Form Prior to Resistance Within a Staphylococcus aureus Biofilm Based on Antibiotic Selective Pressure.
Clinical orthopaedics and related research
The treatment of periprosthetic joint infection (PJI) is focused on the surgical or chemical removal of biofilm. Antibiotics in isolation are typically ineffective against PJI. Bacteria survive after antibiotic administration because of antibiotic tolerance, resistance, and persistence that arise in the resident bacteria of a biofilm. Small-colony variants are typically slow-growing bacterial subpopulations that arise after antibiotic exposure and are associated with persistent and chronic infections such as PJI. The role of biofilm-mediated antibiotic tolerance in the emergence of antibiotic resistance remains poorly defined experimentally.We asked: (1) Does prior antibiotic exposure affect how Staphylococcus aureus survives within a developing biofilm when exposed to an antibiotic that penetrates biofilm, like rifampicin? (2) Does exposure to an antibiotic with poor biofilm penetration, such as vancomycin, affect how S. aureus survives within a developing biofilm? (3) Do small-colony variants emerge from antibiotic-tolerant or-resistant bacteria in a S. aureus biofilm?We used a porous membrane as an in vitro implant model to grow luminescent S. aureus biofilms and simultaneously track microcolony expansion. We evaluated the impact of tolerance on the development of resistance by comparing rifampicin (an antibiotic that penetrates S. aureus biofilm) with vancomycin (an antibiotic that penetrates biofilm poorly). We performed viability counting after membrane dissociation to discriminate among tolerant, resistant, and persistent bacteria. Biofilm quantification and small-colony morphologies were confirmed using scanning electron microscopy. Because of experimental variability induced by the starting bacterial inoculum, relative changes were compared since absolute values may not have been statistically comparable.Antibiotic-naïve S. aureus placed under the selective pressure of rifampicin initially survived within an emerging biofilm by using tolerance given that biofilm resident cell viability revealed 1.0 x 108 CFU , of which 7.5 x 106 CFU were attributed to the emergence of resistance and 9.3 x 107 CFU of which were attributed to the development of tolerance. Previous exposure of S. aureus to rifampicin obviated tolerance-mediate survival when rifampicin resistance was present, since the number of viable biofilm resident cells (9.5 x 109 CFU) nearly equaled the number of rifampicin-resistant bacteria (1.1 x 1010 CFU). Bacteria exposed to an antibiotic with poor biofilm penetration, like vancomycin, survive within an emerging biofilm by using tolerance as well because the biofilm resident cell viability for vancomycin-naïve (1.6 x 1010 CFU) and vancomycin-resistant (1.0 x 1010 CFU) S. aureus could not be accounted for by emergence of resistance. Adding rifampicin to vancomycin resulted in a nearly 500-fold reduction in vancomycin-tolerant bacteria from 1.5 x 1010 CFU to 3.3 x 107 CFU. Small-colony variant S. aureus emerged within the tolerant bacterial population within 24 hours of biofilm-penetrating antibiotic administration. Scanning electron microscopy before membrane dissociation confirmed the presence of small, uniform cells with biofilm-related microstructures when unexposed to rifampicin as well as large, misshapen, lysed cells with a small-colony variant morphology [29, 41, 42, 63] and a lack of biofilm-related microstructures when exposed to rifampicin. This visually confirmed the rapid emergence of small-colony variants within the sessile niche of a developing biofilm when exposed to an antibiotic that exerted selective pressure.Tolerance explains why surgical and nonsurgical modalities that rely on antibiotics to "treat" residual microscopic biofilm may fail over time. The differential emergence of resistance based on biofilm penetration may explain why some suppressive antibiotic therapies that do not penetrate biofilm well may rely on bacterial control while limiting the emergence of resistance. However, this strategy fails to address the tolerant bacterial niche that harbors persistent bacteria with a small-colony variant morphology.Our work establishes biofilm-mediated antibiotic tolerance as a neglected feature of bacterial communities that prevents the effective treatment of PJI.
View details for DOI 10.1097/CORR.0000000000001740
View details for PubMedID 33835090
The Role of the Anti-Inflammatory Cytokine Interleukin-10 in Tissue Fibrosis.
Advances in wound care
2020; 9 (4): 184-198
Significance: Fibrosis is the endpoint of chronic disease in multiple organs, including the skin, heart, lungs, intestine, liver, and kidneys. Pathologic accumulation of fibrotic tissue results in a loss of structural integrity and function, with resultant increases in morbidity and mortality. Understanding the pathways governing fibrosis and identifying therapeutic targets within those pathways is necessary to develop novel antifibrotic therapies for fibrotic disease. Recent Advances: Given the connection between inflammation and fibrogenesis, Interleukin-10 (IL-10) has been a focus of potential antifibrotic therapies because of its well-known role as an anti-inflammatory mediator. Despite the apparent dissimilarity of diseases associated with fibrotic progression, pathways involving IL-10 appear to be a conserved molecular theme. More recently, many groups have worked to develop novel delivery tools for recombinant IL-10, such as hydrogels, and cell-based therapies, such as ex vivo activated macrophages, to directly or indirectly modulate IL-10 signaling. Critical Issues: Some efforts in this area, however, have been stymied by IL-10's pleiotropic and sometimes conflicting effects. A deeper, contextual understanding of IL-10 signaling and its interaction with effector cells, particularly immune cells, will be critical to future studies in the field. Future Directions: IL-10 is clearly a gatekeeper of fibrotic/antifibrotic signaling. The development of novel therapeutics and cell-based therapies that capitalize on targets within the IL-10 signaling pathway could have far-reaching implications for patients suffering from the consequences of organ fibrosis.
View details for DOI 10.1089/wound.2019.1032
View details for PubMedID 32117582
View details for PubMedCentralID PMC7047112
The Immune Response to Chronic Pseudomonas aeruginosa Wound Infection in Immunocompetent Mice.
Advances in wound care
2020; 9 (2): 35-47
Objective: Our goal was to develop a chronic wound model in mice that avoids implantation of foreign material or impaired immunity and to use this to characterize the local and systemic immune response associated with Pseudomonas aeruginosa infection. Approach: We generated bilateral full-thickness dermal wounds in healthy 10-12-week-old C57Bl6 mice. We waited 24 h to inoculate the developing wound eschar at these sites. We performed careful titration experiments with luminescent strains of P. aeruginosa to identify bacterial inoculation concentrations that consistently established stable infections in these animals. We performed flow cytometry-based immunophenotyping of immune cell infiltrates at the wound site, spleen, and draining lymph nodes over time. Finally, we compared inflammatory responses seen in wound inoculation with planktonic bacteria, preformed biofilm, and heat-killed (HK) P. aeruginosa. Results: Using this delayed inoculation model and 7.5 ± 2.5 × 102 CFU/mL of PAO1 we consistently established stable infections that lasted at 10 days in duration. During early infection, we detected a strong upregulation of inflammatory cytokines and neutrophil infiltration at the wound site, while natural killer (NK) cells and dendritic cells (DCs) were reduced. At the systemic level, only plasmacytoid DCs were increased early in infection. During later stages, there was systemic upregulation of B cells, T cells, and macrophages, whereas NK cells and interferon killer DCs were reduced. Infections with P. aeruginosa biofilms were not more virulent than infections with planktonic P. aeruginosa, whereas treatment with HK P. aeruginosa only induces a short-term inflammatory state. Innovation: We describe a versatile wound model of chronic P. aeruginosa infection that lasts 10 days without causing sepsis or other excessive morbidity. Conclusion: This model may facilitate the study of chronic wound infections in immunocompetent mice. Our findings also highlight the induction of early innate immune cell populations during P. aeruginosa infection.
View details for DOI 10.1089/wound.2019.1039
View details for PubMedID 31903297
View details for PubMedCentralID PMC6940591
Phage Therapy for Limb-threatening Prosthetic Knee Klebsiella pneumoniae Infection: Case Report and In Vitro Characterization of Anti-biofilm Activity.
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
Prosthetic joint infection (PJI) is a potentially limb-threatening complication of total knee arthroplasty. Phage therapy is a promising strategy to manage such infections including those involving antibiotic-resistant microbes, and to target microbial biofilms. Experience with phage therapy for infections associated with retained hardware is limited. A 62-year-old diabetic man with a history of right total knee arthroplasty 11 years prior who had suffered multiple episodes of prosthetic knee infection despite numerous surgeries and prolonged courses of antibiotics, with progressive clinical worsening and development of severe allergies to antibiotics, had been offered limb amputation for persistent right prosthetic knee infection due to Klebsiella pneumoniae complex. Intravenous phage therapy was initiated as a limb-salvaging intervention.The patient received 40 intravenous doses of a single phage (KpJH46Φ2) targeting his bacterial isolate, alongside continued minocycline (which he had been receiving when he developed increasing pain, swelling, and erythema prior to initiation of phage therapy). Serial cytokine and biomarker measurements were performed before, during, and after treatment. The in vitro anti-biofilm activity of KpJH46Φ2, minocycline and the combination thereof was evaluated against a preformed biofilm of the patient's isolate and determined by safranin staining.Phage therapy resulted in resolution of local symptoms and signs of infection and recovery of function. The patient did not experience treatment-related adverse effects and remained asymptomatic 34 weeks after completing treatment while still receiving minocycline. A trend in biofilm biomass reduction was noted 22 hours after exposure to KpJH46Φ2 (P = .063). The addition of phage was associated with a satisfactory outcome in this case of intractable biofilm-associated prosthetic knee infection. Pending further studies to assess its efficacy and safety, phage therapy holds promise for treatment of device-associated infections.
View details for DOI 10.1093/cid/ciaa705
View details for PubMedID 32699879
A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection.
Journal of visualized experiments : JoVE
Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7-10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.
View details for DOI 10.3791/60599
View details for PubMedID 32150161
Pf Bacteriophage and Their Impact on Pseudomonas Virulence, Mammalian Immunity, and Chronic Infections.
Frontiers in immunology
2020; 11: 244
Pf bacteriophage are temperate phages that infect the bacterium Pseudomonas aeruginosa, a major cause of chronic lung infections in cystic fibrosis (CF) and other settings. Pf and other temperate phages have evolved complex, mutualistic relationships with their bacterial hosts that impact both bacterial phenotypes and chronic infection. We and others have reported that Pf phages are a virulence factor that promote the pathogenesis of P. aeruginosa infections in animal models and are associated with worse skin and lung infections in humans. Here we review the biology of Pf phage and what is known about its contributions to pathogenesis and clinical disease. First, we review the structure, genetics, and epidemiology of Pf phage. Next, we address the diverse and surprising ways that Pf phages contribute to P. aeruginosa phenotypes including effects on biofilm formation, antibiotic resistance, and motility. Then, we cover data indicating that Pf phages suppress mammalian immunity at sites of bacterial infection. Finally, we discuss recent literature implicating Pf in chronic P. aeruginosa infections in CF and other settings. Together, these reports suggest that Pf bacteriophage have direct effects on P. aeruginosa infections and that temperate phages are an exciting frontier in microbiology, immunology, and human health.
View details for DOI 10.3389/fimmu.2020.00244
View details for PubMedID 32153575
View details for PubMedCentralID PMC7047154
High molecular weight hyaluronan attenuates tubulointerstitial scarring in kidney injury.
Renal fibrosis features exaggerated inflammation, extracellular matrix (ECM) deposition, and peritubular capillary loss. We previously showed that IL-10 stimulates high molecular weight hyaluronan (HMW-HA) expression by fibroblasts, and we hypothesize that HMW-HA attenuates renal fibrosis by reducing inflammation and ECM remodeling. We studied the effects of IL-10 overexpression on HA production and scarring in mouse models of unilateral ureteral obstruction (UUO) and ischemia/reperfusion (I/R) to investigate whether IL-10 anti-fibrotic effects are HA-dependent. C57BL/6J mice were fed with the HA synthesis inhibitor, 4-methylumbelliferone (4-MU), prior to UUO. We observed that in vivo injury increased intratubular spaces, ECM deposition, and HA expression at day 7 and onwards. IL-10 overexpression reduced renal fibrosis in both models, promoted HMW-HA synthesis and stability in UUO, and regulated cell proliferation in I/R. 4-MU inhibited IL-10-driven anti-fibrotic effects, indicating that HMW-HA is necessary for cytokine-mediated reduction of fibrosis. We also found that IL-10 induces in vitro HMW-HA production by renal fibroblasts via STAT3-dependent upregulation of HA synthase 2. We propose that IL-10-induced HMW-HA synthesis plays cytoprotective and anti-fibrotic roles in kidney injury, thereby revealing an effective strategy to attenuate renal fibrosis in obstructive and ischemic pathologies.
View details for DOI 10.1172/jci.insight.136345
View details for PubMedID 32396531
Methods for Extraction and Detection of Pf Bacteriophage DNA from the Sputum of Patients with Cystic Fibrosis.
PHAGE (New Rochelle, N.Y.)
2020; 1 (2): 100–108
Background: There is increasing interest in the pulmonary microbiome's bacterial and viral communities, particularly in the context of chronic airway infections in cystic fibrosis (CF). However, the isolation of microbial DNA from the sputum from patients with CF is technically challenging and the optimal protocols for the analysis of viral species, including bacteriophage, from clinical samples remains difficult. Materials and Methods: In this study, we evaluate a set of methods developed for processing and analyzing sputum from patients with CF with the goal of detecting Pf bacteriophage virion-derived nucleic acid. We evaluate the impact of bead beating, deoxyribonuclease digestion, and heating steps in these protocols focusing on the quantitative assessment of Pseudomonas aeruginosa and Pf bacteriophage in sputum. Results: Based on these comparative data, we describe an optimized protocol for processing sputum from patients with CF and isolating DNA for polymerase chain reaction or sequencing-based studies. Conclusion: These studies demonstrate the assessment of a specific bacteriophage and bacteria in sputum from patients with CF.
View details for DOI 10.1089/phage.2020.0003
View details for PubMedID 32626852
View details for PubMedCentralID PMC7327540
Weekly injection of IL-2 using an injectable hydrogel reduces autoimmune diabetes incidence in NOD mice.
IL-2 injections are a promising therapy for autoimmune type 1 diabetes but the short half-life of this cytokine in vivo limits effective tissue exposure and necessitates frequent injections. Here we have investigated whether an injectable hydrogel could be used to promote prolonged IL-2 release in vivo.Capitalising on the IL-2-binding capabilities of heparin, an injectable hydrogel incorporating clinical-grade heparin, collagen and hyaluronan polymers was used to deliver IL-2. The IL-2-release kinetics and in vivo stability of this material were examined. The ability of soluble IL-2 vs hydrogel-mediated IL-2 injections to prevent autoimmune diabetes in the NOD mouse model of type 1 diabetes were compared.We observed in vitro that the hydrogel released IL-2 over a 12-day time frame and that injected hydrogel likewise persisted 12 days in vivo. Notably, heparin binding potentiates the activity of IL-2 and enhances IL-2- and TGFβ-mediated expansion of forkhead box P3-positive regulatory T cells (FOXP3+ Tregs). Finally, weekly administration of IL-2-containing hydrogel partially prevented autoimmune diabetes while injections of soluble IL-2 did not.Hydrogel delivery may reduce the number of injections required in IL-2 treatment protocols for autoimmune diabetes. Graphical abstract.
View details for DOI 10.1007/s00125-020-05314-1
View details for PubMedID 33125521
Phages in vaccine design and immunity; mechanisms and mysteries.
Current opinion in biotechnology
2020; 68: 160–65
Bacteriophages have attracted extensive interest in vaccine design. This includes the use of phage display technology to select antigens, the use of engineered phages displaying target antigens in vaccine formulations, and phage DNA vaccines. However, the development of these approaches is limited in part by uncertainty regarding the underlying mechanisms by which phages elicit immunity. This has stymied the clinical development of this technology. Here we review the immunology of phage vaccines and highlight the gaps in our knowledge regarding the underlying mechanisms. First, we review the basic biology of phages and their use in vaccines. Next we discuss what is known about the mechanisms of immunity against engineered phages and phage DNA. Finally, we highlight the gaps in our understanding regarding the immunogenicity of these preparations. We argue that mechanistic insight into the immunology of phage vaccines is essential for the further development and clinical utility of these technologies.
View details for DOI 10.1016/j.copbio.2020.11.002
View details for PubMedID 33316575
Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection.
Matrix biology : journal of the International Society for Matrix Biology
A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.
View details for DOI 10.1016/j.matbio.2020.12.001
View details for PubMedID 33290836
The Role of an IL-10/Hyaluronan Axis in Dermal Wound Healing.
Frontiers in cell and developmental biology
2020; 8: 636
Scar formation is the typical endpoint of postnatal dermal wound healing, which affects more than 100 million individuals annually. Not only do scars cause a functional burden by reducing the biomechanical strength of skin at the site of injury, but they also significantly increase healthcare costs and impose psychosocial challenges. Though the mechanisms that dictate how dermal wounds heal are still not completely understood, they are regulated by extracellular matrix (ECM) remodeling, neovascularization, and inflammatory responses. The cytokine interleukin (IL)-10 has emerged as a key mediator of the pro- to anti-inflammatory transition that counters collagen deposition in scarring. In parallel, the high molecular weight (HMW) glycosaminoglycan hyaluronan (HA) is present in the ECM and acts in concert with IL-10 to block pro-inflammatory signals and attenuate fibrotic responses. Notably, high concentrations of both IL-10 and HMW HA are produced in early gestational fetal skin, which heals scarlessly. Since fibroblasts are responsible for collagen deposition, it is critical to determine how the concerted actions of IL-10 and HA drive their function to potentially control fibrogenesis. Beyond their independent actions, an auto-regulatory IL-10/HA axis may exist to modulate the magnitude of CD4+ effector T lymphocyte activation and enhance T regulatory cell function in order to reduce scarring. This review underscores the pathophysiological impact of the IL-10/HA axis as a multifaceted molecular mechanism to direct primary cell responders and regulators toward either regenerative dermal tissue repair or scarring.
View details for DOI 10.3389/fcell.2020.00636
View details for PubMedID 32850791
View details for PubMedCentralID PMC7396613
A novel mouse model of chronic suppurative otitis media and its use in preclinical antibiotic evaluation.
2020; 6 (33): eabc1828
Chronic suppurative otitis media (CSOM) is a neglected pediatric disease affecting 330 million worldwide for which no new drugs have been introduced for over a decade. We developed a mouse model with utility in preclinical drug evaluation and antimicrobial discovery. Our model used immune-competent mice, tympanic membrane perforation and inoculation with luminescent Pseudomonas aeruginosa that enabled bacterial abundance tracking in real-time for 100 days. The resulting chronic infection exhibited hallmark features of clinical CSOM, including inhibition of tympanic membrane healing and purulent ear discharge. We evaluated the standard care fluoroquinolone ofloxacin and demonstrated that this therapy resulted in a temporary reduction of bacterial burden. These data are consistent with the clinical problem of persistent infection in CSOM and the need for therapeutic outcome measures that assess eradication post-therapeutic endpoint. We conclude that this novel mouse model of CSOM has value in investigating new potential therapies.
View details for DOI 10.1126/sciadv.abc1828
View details for PubMedID 32851190
View details for PubMedCentralID PMC7428333
The Rationale for Using Bacteriophage to Treat and Prevent Periprosthetic Joint Infections.
Frontiers in microbiology
2020; 11: 591021
Prosthetic joint infection (PJI) is a devastating complication after a joint replacement. PJI and its treatment have a high monetary cost, morbidity, and mortality. The lack of success treating PJI with conventional antibiotics alone is related to the presence of bacterial biofilm on medical implants. Consequently, surgical removal of the implant and prolonged intravenous antibiotics to eradicate the infection are necessary prior to re-implanting a new prosthetic joint. Growing clinical data shows that bacterial predators, called bacteriophages (phages), could be an alternative treatment strategy or prophylactic approach for PJI. Phages could further be exploited to degrade biofilms, making bacteria more susceptible to antibiotics and enabling potential combinatorial therapies. Emerging research suggests that phages may also directly interact with the innate immune response. Phage therapy may play an important, and currently understudied, role in the clearance of PJI, and has the potential to treat thousands of patients who would either have to undergo revision surgery to attempt to clear an infections, take antibiotics for a prolonged period to try and suppress the re-emerging infection, or potentially risk losing a limb.
View details for DOI 10.3389/fmicb.2020.591021
View details for PubMedID 33408703
The Heparan Sulfate Mimetic PG545 Modulates T Cell Responses and Prevents Delayed-Type Hypersensitivity.
Frontiers in immunology
2020; 11: 132
The heparan sulfate mimetic PG545 (pixatimod) is under evaluation as an inhibitor of angiogenesis and metastasis including in human clinical trials. We have examined the effects of PG545 on lymphocyte phenotypes and function. We report that PG545 treatment suppresses effector T cell activation and polarizes T cells away from Th17 and Th1 and toward Foxp3+ regulatory T cell subsets in vitro and in vivo. Mechanistically, PG545 inhibits Erk1/2 signaling, a pathway known to affect both T cell activation and subset polarization. Interestingly, these effects are also observed in heparanase-deficient T cells, indicating that PG545 has effects that are independent of its role in heparanase inhibition. Consistent with these findings, administration of PG545 in a Th1/Th17-dependent mouse model of a delayed-type hypersensitivity led to reduced footpad inflammation, reduced Th17 memory cells, and an increase in FoxP3+ Treg proliferation. PG545 also promoted Foxp3+ Treg induction by human T cells. Finally, we examined the effects of other heparan sulfate mimetics PI-88 and PG562 on lymphocyte polarization and found that these likewise induced Foxp3+ Treg in vitro but did not reduce Th17 numbers or improve delayed-type hypersensitivity in this model. Together, these data indicate that PG545 is a potent inhibitor of Th1/Th17 effector functions and inducer of FoxP3+ Treg. These findings may inform the adaptation of PG545 for clinical applications including in inflammatory pathologies associated with type IV hypersensitivity responses.
View details for DOI 10.3389/fimmu.2020.00132
View details for PubMedID 32117279
View details for PubMedCentralID PMC7015948
T Lymphocytes Attenuate Dermal Scarring by Regulating Inflammation, Neovascularization, and Extracellular Matrix Remodeling.
Advances in wound care
2019; 8 (11): 527-537
Objective: While tissue injury and repair are known to involve adaptive immunity, the profile of lymphocytes involved and their contribution to dermal scarring remain unclear. We hypothesized that restoration of T cell deficiency attenuates dermal scarring. Approach: We assessed the temporal-spatial distribution of T lymphocytes and their subtypes during the physiological dermal wound repair process in mice. Also, we compared the scarring outcomes between wild-type (WT) and severe combined immunodeficient (SCID) mice, which are lymphocyte deficient. Complementary gain-of-function experiments were performed by adoptively transferring lymphocyte subsets to validate their contribution to tissue repair in wounded SCID mice. Results: CD4+ T lymphocytes were present within dermal wounds of WT mice beginning on day 1 and remained through day 30. Wounds of SCID mice exhibited accelerated closure, increased inflammation, limited neovascularization, and exacerbated scarring compared with WT mice. Conversely, transfer of either mixed B and T lymphocytes or CD4+ lymphocytes alone into SCID mice resulted in moderated healing with less inflammation, collagen deposition, and scarring than control SCID wounds. In contrast, transfer of other lymphocyte subsets, including helper T lymphocytes (CD3+CD4+CD25-), CD8+ T cells and B cells, or regulatory T lymphocytes (CD4+CD25+CD127low), did not reduce scar. Innovation: The finding that lymphocytes delay wound healing but reduce scar is novel and provides new insights into how dermal scarring is regulated. Conclusion: Our data support a suppressive role for CD4+ T cells against inflammation and collagen deposition, with protective effects in early-stage dermal wound healing. These data implicate adaptive immunity in the regulation of scarring phenotypes.
View details for DOI 10.1089/wound.2019.0981
View details for PubMedID 31637099
View details for PubMedCentralID PMC6798809
Quantitative assessment of bacterial growth phase utilizing flow cytometry.
Journal of microbiological methods
Flow cytometry is currently underutilized for bacterial phenotyping and standard microbiological techniques do not provide phenotypic information about the state of the bacterial disease. Pseudomonas aeruginosa is a human pathogen of increased importance in public health due to both the ability to cause chronic diseases and the prevalence of functionally different subsets that can be difficult to treat and diagnose. In the present study, we used flow cytometry to analyze the growth phase of P. aeruginosa. A simple method for single cell quantitative detection of bacterial biofilm and planktonic cells was established with a combination of membrane permeable (SYTO 60) and impermeable (TOTO-1) dyes plus the addition of polystyrene counting beads. The specificity of the dye combination for biofilm detection was determined by comparison with impaired biofilm forming strains of P. aeruginosa LasI/RhlI-/- and ∆PfPhage. Results suggest that flow cytometric bacterial phenotyping serves as an expandable platform that may be useful for enumeration of population level variation in P. aeruginosa studies.
View details for DOI 10.1016/j.mimet.2019.105760
View details for PubMedID 31678132
- Hydrogel-based delivery of Il-10 improves treatment of bleomycin-induced lung fibrosis in mice BIOMATERIALS 2019; 203: 52–62
4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition.
The Journal of biological chemistry
4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibiting HA synthesis. In particular, 4-MU in mice has a short half-life causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3+ regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent 4-MU:4-MUG ratios in serum, liver and pancreas, indicating that 4-MU and 4-MUG reach an equilibrium in these tissues. LC-tandem MS experiments revealed that 4-MUG is hydrolyzed to 4-MU in serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intra-vital 2-photon microscopy, we found that 4-MUG (a non-fluorescent molecule) undergoes conversion into 4-MU (a fluorescent molecule) and that 4-MU is extensively tissue bound in the liver, fat, muscle, and pancreas of treated mice. 4-MUG also suppressed HA synthesis independently of its conversion into 4-MU and without depletion of the HA precursor UDP-glucuronic acid (GlcUA). Together, these results indicate that 4-MUG both directly and indirectly inhibits HA synthesis and that the effective bioavailability of 4-MU is higher than previously thought. These findings greatly alter the experimental and therapeutic possibilities for HA synthesis inhibition.
View details for PubMedID 30914479
- Perspectives of Phage Therapy in Non-bacterial Infections FRONTIERS IN MICROBIOLOGY 2019; 9
4-Methylumbelliferone improves the thermogenic capacity of brown adipose tissue.
2019; 1 (5): 546–59
Therapeutic increase of brown adipose tissue (BAT) thermogenesis is of great interest as BAT activation counteracts obesity and insulin resistance. Hyaluronan (HA) is a glycosaminoglycan, found in the extracellular matrix, which is synthesized by HA synthases (Has1/Has2/Has3) from sugar precursors and accumulates in diabetic conditions. Its synthesis can be inhibited by the small molecule 4-methylumbelliferone (4-MU). Here, we show that the inhibition of HA-synthesis by 4-MU or genetic deletion of Has2/Has3 improves BAT`s thermogenic capacity, reduces body weight gain, and improves glucose homeostasis independently from adrenergic stimulation in mice on diabetogenic diet, as shown by a magnetic resonance T2 mapping approach. Inhibition of HA synthesis increases glycolysis, BAT respiration and uncoupling protein 1 expression. In addition, we show that 4-MU increases BAT capacity without inducing chronic stimulation and propose that 4-MU, a clinically approved prescription-free drug, could be repurposed to treat obesity and diabetes.
View details for DOI 10.1038/s42255-019-0055-6
View details for PubMedID 31602424
View details for PubMedCentralID PMC6786893
The Innate Sense of Bacteriophages.
Cell host & microbe
2019; 25 (2): 177–79
Perturbation of the gut microbiota is associated with inflammatory bowel disease, obesity, and even cancer. In this issue of Cell Host & Microbe, Gogokhia et al. 2019 sought to kill cancer-causing bacteria with bacteriophages. However, the bacteriophages themselves directly stimulated an immune response, which worsened inflammatory bowel disease pathogenesis.
View details for PubMedID 30763530
Adenosine and hyaluronan modulate lung fibrosis and pulmonary hypertension in combined pulmonary fibrosis and emphysema (CPFE).
Disease models & mechanisms
Combined pulmonary fibrosis and emphysema (CPFE) is a syndrome that predominantly affects male smokers or ex-smokers and it has a mortality rate of 55% and a median survival of 5 years. Pulmonary hypertension (PH) is a highly fatal complication of CPFE. Despite this dismal prognosis, no curative therapies exist for patients with CPFE outside of lung transplantation and no therapies are recommended to treat PH. This highlights the need to develop novel treatment approaches for CPFE. Studies from our group have demonstrated that adenosine and its receptor, ADORA2B, are elevated in chronic lung diseases. Activation of ADORA2B leads to elevated levels of hyaluronan synthases and increased hyaluronan, a glycosaminoglycan which contributes to chronic lung injury. We hypothesize that ADORA2B and hyaluronan contribute to CPFE. Using isolated CPFE lung tissue, we characterized expression levels of ADORA2B and hyaluronan synthases (HAS). Next using a unique model of experimental lung injury that replicates features of CPFE, namely airspace enlargement, PH and fibrotic deposition, we investigated whether 4MU, a hyaluronan synthase inhibitor, was able to inhibit features of CPFE. Increased protein levels of ADORA2B and HAS3 were detected in CPFE and in our experimental model of CPFE. Treatment with 4MU was able to attenuate PH and fibrosis but not airspace enlargement. This was accompanied by a reduction of HAS3-positive macrophages. We have generated pre-clinical data demonstrating the capacity of 4MU, a FDA-approved drug, to attenuate features of CPFE in an experimental model of chronic lung injury.
View details for PubMedID 31036697
Salivary thiocyanate as a biomarker of Cystic Fibrosis Transmembrane Regulator function.
Improved methods are needed to reliably assess CFTR function in vivo in light of recent therapeutic developments targeting the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. Saliva from patients with cystic fibrosis (CF) and healthy controls (HC) was studied using colorimetry and non-resonant Raman spectroscopy. Colorimetry experiments showed only a 36% decrease in thiocyanate (SCN-) concentration, but a sharp Raman peak at 2068 cm-1, attributable to (SCN-) vibrations, normalized to C-H peak, was on average 18 times higher for HC samples. Samples from patients under-going treatment with CFTR modulators including ivacaftor, lumacaftor and tezacaftor, showed a high normalized peak in response to therapy. The peak intensity was consistent in longitudinal samples from single donors and in stored samples. The Raman peak ratio is a more sensitive, convenient, non-invasive biomarker for assessments of the therapeutic efficacy of drugs targeting CFTR and provides a value that is in much better agreement with theoretical expectations of saliva SCN- concentrations, compared to colorimetry. Moreover, samples from patients can be collected remotely, as they are stable and can be shipped in dry state. This insight may greatly facilitate assessments of CFTR modulator efficacy in individual pa-tients as well as development of new ones.
View details for DOI 10.1021/acs.analchem.9b01800
View details for PubMedID 31117414
Natural Tr1-like cells do not confer long-term tolerogenic memory.
IL-10-producing Tr1 cells promote tolerance but their contributions to tolerogenic memory are unclear. Using 10BiT mice that carry a Foxp3-eGFP reporter and stably express CD90.1 following IL-10 production, we characterized the spatiotemporal dynamics of Tr1 cells in a house dust mite model of allergic airway inflammation. CD90.1+Foxp3-IL-10+ Tr1 cells arise from memory cells and rejoin the tissue-resident memory T-cell pool after cessation of IL-10 production. Persistent antigenic stimulation is necessary to sustain IL-10 production and Irf1 and Batf expression distinguishes CD90.1+Foxp3-IL-10+ Tr1 cells from CD90.1+Foxp3-IL-10- 'former' Tr1. Depletion of Tr1-like cells after primary sensitization exacerbates allergic airway inflammation. However, neither transfer nor depletion of former Tr1 cells influences either Tr1 numbers or the inflammatory response during subsequent allergen memory re-challenge weeks later. Together these data suggest that naturally-arising Tr1 cells do not necessarily give rise to more Tr1 upon allergen re-challenge or contribute to tolerogenic memory. This phenotypic instability may limit efforts to re-establish tolerance by expanding Tr1 in vivo.
View details for DOI 10.7554/eLife.44821
View details for PubMedID 31603425
Extracellular matrix and the maintenance and loss of peripheral immune tolerance in autoimmune insulitis.
Current opinion in immunology
2018; 55: 22–30
There is a growing appreciation that the extracellular matrix (ECM) contributes to both the maintenance of immune tolerance in healthy tissues and to its loss at sites of autoimmunity. Here, we review recent literature on the role of ECM and particularly the glycosaminoglycans hyaluronan and heparan sulfate in the development of autoimmune, type 1 diabetes (T1D). Data from transplant models suggest that healthy islets are embedded within an intact ECM that supports beta-cell homeostasis and provides physical and immunoregulatory barriers against immune infiltration. However, studies of human insulitis as well as the non-obese diabetic (NOD) and DORmO mouse models of T1D indicate that autoimmune insulitis is associated with the degradation of basement membrane structures, the catabolism of the islet interstitium, and the accumulation of a hyaluronan-rich, pro-inflammatory ECM. Moreover, in these models of autoimmune diabetes, either the pharmacologic inhibition of heparan sulfate catabolism, the reduction of hyaluronan synthesis, or the targeting of the pathways that sense these ECM changes can all prevent beta-cell destruction. Together these data support an emerging paradigm that in healthy islets the local ECM contributes to both immune tolerance and beta-cell homeostasis while in chronic inflammation the islet ECM is permissive to immune infiltration and beta-cell destruction. Therapies that support ECM-mediated 'barrier tolerance' may have potential as adjunctive agents in combination regimens designed to prevent or treat autoimmunity.
View details for PubMedID 30248522
Hyaluronan levels are increased systemically in human type 2 but not type 1 diabetes independently of glycemic control.
Matrix biology : journal of the International Society for Matrix Biology
Hyaluronan (HA), an extracellular matrix glycosaminoglycan, is implicated in the pathogenesis of both type 1 diabetes (T1D) as well as type 2 diabetes (T2D) and has been postulated to be increased in these diseases due to hyperglycemia. We have examined the serum and tissue distribution of HA in human subjects with T1D and T2D and in mouse models of these diseases and evaluated the relationship between HA levels and glycemic control. We found that serum HA levels are increased in T2D but not T1D independently of hemoglobin-A1c, C-peptide, body mass index, or time since diabetes diagnosis. HA is likewise increased in skeletal muscle in T2D subjects relative to non-diabetic controls. Analogous increases in serum and muscle HA are seen in diabetic db/db mice (T2D), but not in diabetic DORmO mice (T1D). Diabetes induced by the beta-cell toxin streptozotozin (STZ) lead to an increase in blood glucose but not to an increase in serum HA. These data indicate that HA levels are increased in multiple tissue compartments in T2D but not T1D independently of glycemic control. Given that T2D but not T1D is associated with systemic inflammation, these patterns are consistent with inflammatory factors and not hyperglycemia driving increased HA. Serum HA may have value as a biomarker of systemic inflammation in T2D.
View details for PubMedID 30196101
DIFFERENTIAL REGULATION OF EXOSOME PRODUCTION BY MECHANICAL TENSION INFLUENCES FIBROGENIC PHENOTYPES
WILEY. 2018: A11
View details for Web of Science ID 000451464000050
Chitin, chitinases, and chitin lectins: Emerging roles in human pathophysiology.
Pathophysiology : the official journal of the International Society for Pathophysiology
Chitin is a simple beta-linked repeating sugar polymer prominent in the building block structures of a wide variety of organisms, from the yeast cell wall to the exoskeleton and shells of arthropods and other forms of invertebrate life. It had previously been assumed that vertebrates did not contain chitins. However, chitin and chitinases are now documented to occur in vertebrate tissues. Chitin, chitinases and particularly chitinase-like proteins are involved in important human pathologies, though the mechanisms by which these function is unknown. These chitinase-like proteins bind to chitin and function as chitin lectins in that they bind to chitin but have lost the ability to degrade it. Emphasis is placed on one of the chitinase-like proteins, CHI3L1, that has acquired wide clinical importance. The purpose of this review is to place an array of bewildering observations associated with various human disorders into a framework, particularly the pathologies of the human gastro-intestinal tract. A reasonably cohesive story may eventually emerge.
View details for PubMedID 30266339
Hyaluronan: A Master Switch Between Vascular Homeostasis and Inflammation.
2018; 122 (10): 1341–43
View details for PubMedID 29748364
Interactions between Bacteriophage, Bacteria, and the Mammalian Immune System.
2018; 11 (1)
The human body is host to large numbers of bacteriophages (phages)⁻a diverse group of bacterial viruses that infect bacteria. Phage were previously regarded as bystanders that only impacted immunity indirectly via effects on the mammalian microbiome. However, it has become clear that phages also impact immunity directly, in ways that are typically anti-inflammatory. Phages can modulate innate immunity via phagocytosis and cytokine responses, but also impact adaptive immunity via effects on antibody production and effector polarization. Phages may thereby have profound effects on the outcome of bacterial infections by modulating the immune response. In this review we highlight the diverse ways in which phages interact with human cells. We present a computational model for predicting these complex and dynamic interactions. These models predict that the phageome may play important roles in shaping mammalian-bacterial interactions.
View details for PubMedID 30585199
Macrophages and innate immune memory against Staphylococcus skin infections.
Proceedings of the National Academy of Sciences of the United States of America
View details for PubMedID 30389708
Hyaluronan in immune dysregulation and autoimmune diseases.
Matrix biology : journal of the International Society for Matrix Biology
The tissue microenvironment contributes to local immunity and to the pathogenesis of autoimmune diseases - a diverse set of conditions characterized by sterile inflammation, immunity against self-antigens, and destruction of tissues. However, the specific factors within the tissue microenvironment that contribute to local immune dysregulation in autoimmunity are poorly understood. One particular tissue component implicated in multiple autoimmune diseases is hyaluronan (HA), an extracellular matrix (ECM) polymer. HA is abundant in settings of chronic inflammation and contributes to lymphocyte activation, polarization, and migration. Here, we first describe what is known about the size, amount, and distribution of HA at sites of autoimmunity and in associated lymphoid structures in type 1 diabetes, multiple sclerosis, and rheumatoid arthritis. Next, we examine the recent literature on HA and its impact on adaptive immunity, particularly in regards to the biology of lymphocytes and Foxp3+ regulatory T-cells (Treg), a T-cell subset that maintains immune tolerance in healthy individuals. We propose that HA accumulation at sites of chronic inflammation creates a permissive environment for autoimmunity, characterized by CD44-mediated inhibition of Treg expansion. Finally, we address potential tools and strategies for targeting HA and its receptor CD44 in chronic inflammation and autoimmunity.
View details for PubMedID 29625181
Implant-Associated Bacterial Biofilm and Quorum Sensing in Periprosthetic Joint Infections.
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Periprosthetic joint infection (PJI) continues to be a common complication after total knee arthroplasty and total hip arthroplasty leading to severe morbidity and mortality. With an aging population and increasing prevalence of total joint replacement procedures, the burden of PJI will be felt not only by individual patients, but in increased healthcare costs. Current treatment of PJI is inadequate resulting in incredibly high failure rates. This is believed to be largely mediated by the presence of bacterial biofilms. These polymicrobial bacterial colonies form within secreted extracellular matrices, adhering to the implant surface and local tissue. The biofilm architecture is believed to play a complex and critical role in a variety of bacterial processes including nutrient supplementation, metabolism, waste management, and antibiotic and immune resistance. The establishment of these biofilms relies heavily on the quorum sensing communication systems utilized by bacteria. Early stage research into disrupting bacterial communication by targeting quorum sensing show promise for future clinical applications. However, prevention of the biofilm formation via early forced induction of the biofilm forming process remains yet unexplored. This article is protected by copyright. All rights reserved.
View details for PubMedID 29663554
Inhibition of Hyaluronan Synthesis Attenuates Pulmonary Hypertension Associated with Lung Fibrosis.
British journal of pharmacology
Group III Pulmonary hypertension (PH) is a highly lethal and widespread lung disorder that is a common complication in idiopathic pulmonary fibrosis (IPF) where it is considered to be the single most significant predictor of mortality. While increased levels of hyaluronan have been observed in IPF patients, hyaluronan-mediated vascular remodelling and the hyaluronan-mediated mechanisms promoting PH associated with IPF are not fully understood.Explanted lung tissue from patients with IPF with and without a diagnosis of PH was used to identify increased levels of hyaluronan. In addition, an experimental model of lung fibrosis and PH was used to test the capacity of 4-methylumbeliferone (4MU), a hyaluronan synthase inhibitor to attenuate PH. Human pulmonary artery smooth muscle cells (PASMC) were used to identify the hyaluronan-specific mechanisms that lead to the development of PH associated with lung fibrosis.In patients with IPF and PH, increased levels of hyaluronan and expression of hyaluronan synthase genes are present. Interestingly, we also report increased levels of hyaluronidases in patients with IPF and IPF with PH. Remarkably, our data also show that 4MU is able to inhibit PH in our model either prophylactically or therapeutically, without affecting fibrosis. Studies to determine the hyaluronan-specific mechanisms revealed that hyaluronan fragments result in increased PASMC stiffness and proliferation but reduced cell motility in a RhoA dependent manner.Taken together, our results show evidence of a unique mechanism contributing to PH in the context of lung fibrosis.
View details for DOI 10.1111/bph.13947
View details for PubMedID 28688167
A Consensus Definitive Classification of Scavenger Receptors and Their Roles in Health and Disease
JOURNAL OF IMMUNOLOGY
2017; 198 (10): 3775-3789
Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the United States National Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.
View details for DOI 10.4049/jimmunol.1700373
View details for Web of Science ID 000401137200002
View details for PubMedID 28483986
Role of dendritic cell maturation factors produced by human invariant NKT cells in immune tolerance
JOURNAL OF LEUKOCYTE BIOLOGY
2017; 101 (4): 989-1003
In this study, we used the culture supernatant of iNKT cells to identify human myeloid DC maturation factors produced by human CD4(+) iNKT cells. S100A8 had a strong maturation effect. Notably, the recombinant S100A8 protein displayed properties of DC maturation functioning, and the induction of DC differentiation by both the purified and the recombinant protein were blocked by anti-S100A8 and anti-TLR-4 mAbs. DC differentiation induced by anti-major histocompatibility complex class II/CD1d Ab, S100A8, or both was qualitatively indistinguishable from that induced by the coculture of DCs and iNKT cells or via culture supplementation with supernatants from activated CD4(+) iNKT cells. S100A8 also induced CD4(+)/CD25(+)/Foxp3(+) Treg cells from naïve T cells. S100A8 may contribute to DC differentiation by elevating transcription factors or activating transcription factor-2, heat shock factor-1, or both, in mature DCs. S100A8 is a novel candidate iNKT cell-dependent DC maturation factor.
View details for DOI 10.1189/jlb.1A0416-164RRR
View details for Web of Science ID 000398503100018
Interleukin-10-mediated regenerative postnatal tissue repair is dependent on regulation of hyaluronan metabolism via fibroblast-specific STAT3 signaling
2017; 31 (3): 868-881
The cytokine IL-10 has potent antifibrotic effects in models of adult fibrosis, but the mechanisms of action are unclear. Here, we report a novel finding that IL-10 triggers a signal transducer and activator of transcription 3 (STAT3)-dependent signaling pathway that regulates hyaluronan (HA) metabolism and drives adult fibroblasts to synthesize an HA-rich pericellular matrix, which mimics the fetal regenerative wound healing phenotype with reduced fibrosis. By using cre-lox-mediated novel, inducible, fibroblast-, keratinocyte-, and wound-specific STAT3 knockdown postnatal mice-plus syngeneic fibroblast cell-transplant models-we demonstrate that the regenerative effects of IL-10 in postnatal wounds are dependent on HA synthesis and fibroblast-specific STAT3-dependent signaling. The importance of IL-10-induced HA synthesis for regenerative wound healing is demonstrated by inhibition of HA synthesis in a murine wound model by administering 4-methylumbelliferone. Although IL-10 and STAT3 signaling were intact, the antifibrotic repair phenotype that is induced by IL-10 overexpression was abrogated in this model. Our data show a novel role for IL-10 beyond its accepted immune-regulatory mechanism. The opportunity for IL-10 to regulate a fibroblast-specific formation of a regenerative, HA-rich wound extracellular matrix may lead to the development of innovative therapies to attenuate postnatal fibrosis in organ systems or diseases in which dysregulated inflammation and HA intersect.-Balaji, S., Wang, X., King, A., Le, L. D., Bhattacharya, S. S., Moles, C. M., Butte, M. J., de Jesus Perez, V. A., Liechty, K. W., Wight, T. N., Crombleholme, T. M., Bollyky, P. L., Keswani, S. G. Interleukin-10-mediated regenerative postnatal tissue repair is dependent on regulation of hyaluronan metabolism via fibroblast-specific STAT3 signaling.
View details for DOI 10.1096/fj.201600856R
View details for Web of Science ID 000395671200004
Phosphorylation of aB-crystallin supports reactive astrogliosis in demyelination.
Proceedings of the National Academy of Sciences of the United States of America
2017; 114 (9): E1745-E1754
The small heat shock protein αB-crystallin (CRYAB) has been implicated in multiple sclerosis (MS) pathogenesis. Earlier studies have indicated that CRYAB inhibits inflammation and attenuates clinical disease when administered in the experimental autoimmune encephalomyelitis model of MS. In this study, we evaluated the role of CRYAB in primary demyelinating events. Using the cuprizone model of demyelination, a noninflammatory model that allows the analysis of glial responses in MS, we show that endogenous CRYAB expression is associated with increased severity of demyelination. Moreover, we demonstrate a strong correlation between the expression of CRYAB and the extent of reactive astrogliosis in demyelinating areas and in in vitro assays. In addition, we reveal that CRYAB is differentially phosphorylated in astrocytes in active demyelinating MS lesions, as well as in cuprizone-induced lesions, and that this phosphorylation is required for the reactive astrocyte response associated with demyelination. Furthermore, taking a proteomics approach to identify proteins that are bound by the phosphorylated forms of CRYAB in primary cultured astrocytes, we show that there is clear differential binding of protein targets due to the specific phosphorylation of CRYAB. Subsequent Ingenuity Pathway Analysis of these targets reveals implications for intracellular pathways and biological processes that could be affected by these modifications. Together, these findings demonstrate that astrocytes play a pivotal role in demyelination, making them a potential target for therapeutic intervention, and that phosphorylation of CRYAB is a key factor supporting the pathogenic response of astrocytes to oligodendrocyte injury.
View details for DOI 10.1073/pnas.1621314114
View details for PubMedID 28196893
View details for PubMedCentralID PMC5338510
Modified High-Molecular-Weight Hyaluronan Promotes Allergen-Specific Immune Tolerance
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY
2017; 56 (1): 109-120
The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, high-molecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-κB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.
View details for DOI 10.1165/rcmb.2016-0111OC
View details for Web of Science ID 000392133000012
View details for PubMedCentralID PMC5248962
Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo
INFECTION AND IMMUNITY
2017; 85 (1)
Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as occurs in the lungs of patients with cystic fibrosis and non-healing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage, similar to those produced by biofilms under in vitro conditions, would affect pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage trap P. aeruginosa within the lung. In vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa were less prone to phagocytosis by macrophages compared to bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alter the progression of the inflammatory response and promote phenotypes typically associated with chronic infection.
View details for DOI 10.1128/IAI.00648-16
View details for Web of Science ID 000394607700021
Pseudomonas phage inhibition of Candida albicans.
Microbiology (Reading, England)
Pseudomonas aeruginosa (Pa) and Candida albicans (Ca) are major bacterial and fungal pathogens in immunocompromised hosts, and notably in the airways of cystic fibrosis patients. The bacteriophages of Pa physically alter biofilms, and were recently shown to inhibit the biofilms of Aspergillus fumigatus. To understand the range of this viral-fungal interaction, we studied Pa phages Pf4 and Pf1, and their interactions with Ca biofilm formation and preformed Ca biofilm. Both forms of Ca biofilm development, as well as planktonic Ca growth, were inhibited by either phage. The inhibition of biofilm was reversed by the addition of iron, suggesting that the mechanism of phage action on Ca involves denial of iron. Birefringence studies on added phage showed an ordered structure of binding to Ca. Electron microscopic observations indicated phage aggregation in the biofilm extracellular matrix. Bacteriophage-fungal interactions may be a general feature with several pathogens in the fungal kingdom.
View details for PubMedID 28982395
Hyaluronan synthase 3 promotes plaque inflammation and atheroprogression.
Matrix biology : journal of the International Society for Matrix Biology
Hyaluronan (HA) is a prominent component of the provisional extracellular matrix (ECM) present in the neointima of atherosclerotic plaques. Here the role of HA synthase 3 (HAS3) in atheroprogression was studied.It is demonstrated here that HAS isoenzymes 1, -2 and -3 are expressed in human atherosclerotic plaques of the carotid artery. In Apolipoprotein E (Apoe)-deficient mice Has3 expression is increased early during lesion formation when macrophages enter atherosclerotic plaques. Importantly, HAS3 expression in vascular smooth muscle cells (VSMC) was found to be regulated by interleukin 1 β (IL-1β) in an NFkB dependent manner and blocking antibodies to IL-1β abrogate Has3 expression in VSMC by activated macrophages. Has3/Apoe double deficient mice developed less atherosclerosis characterized by decreased Th1-cell responses, decreased IL-12 release, and decreased macrophage-driven inflammation.Inhibition of HAS3-dependent synthesis of HA dampens systemic Th1 cell polarization and reduces plaque inflammation. These data suggest that HAS3 might be a promising therapeutic target in atherosclerosis. Moreover, because HAS3 is regulated by IL-1β, our results suggest that therapeutic anti-IL-1β antibodies, currently tested in human clinical trials, may exert their beneficial effects on inflammation in post-myocardial infarction patients via effects on HAS3. in post-myocardial infarction patients who remain at high cardiovascular risk due to persistent elevated inflammatory biomarkers.
View details for DOI 10.1016/j.matbio.2017.09.005
View details for PubMedID 28987865
Human Chitotriosidase Does Not Catabolize Hyaluronan.
International journal of biological macromolecules
Humans express an enzyme that degrades chitin, called chitotriosidase, despite the fact that we do not produce chitin. One possible explanation for this is that chitinase also degrades hyaluronan, a polysaccharide that is abundant in human tissues and shares structural attributes in common with chitinase. The objective of this study was to determine whether human chitotriosidase is capable of hydrolyzing hyaluronan. Hyaluronan of various sizes under a range of pH conditions displayed no degradation when incubated with various chitinases over a period of 5 days, while commercial hyaluronidase readily digested the hyaluronan. Under the same conditions, recombinant chitinase but not our negative control chitinase, was able to digest chitosan. We conclude that human chitinase does not digest hyaluronan. Because chitin is a prominent component of certain fungi and insects, it seems likely that human chitinase evolved for roles in host defense rather than serving to catabolize the endogenous polymer hyaluronan.
View details for PubMedID 29247734
Hyaluronan content governs tissue stiffness in pancreatic islet inflammation.
The Journal of biological chemistry
We have identified a novel role for hyaluronan (HA), an extracellular matrix (ECM) polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes (T1D) of mice and humans is preceded by intra-islet accumulation of HA, a highly hygroscopic polymer. Using the DORmO double transgenic (DO11.10 x RIPmOVA) mouse model of T1D, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared to controls. Conversely, treatment with 4-methylumbelliferone (4-MU), a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses.
View details for PubMedID 29183997
The formation and function of tertiary lymphoid follicles in chronic pulmonary inflammation.
2016; 149 (3): 262-269
Tertiary lymphoid follicles (TLFs) can develop in the respiratory tract in response to infections or chronic inflammation. However, their functional relevance remains unclear as they are implicated in both protective and pathologic responses. In contrast to homeostatic conditions, external antigens and damage to the lung tissue may drive TLF formation in inflamed lungs, and once established, the presence of pulmonary TLFs may signal the progression of chronic lung disease. This novel concept will be discussed in light of recent work in chronic obstructive pulmonary disease (COPD) and how changes in the pulmonary microbiota may be drive and direct TLF formation and function. We will also discuss the cellularity of TLFs at the pulmonary mucosa, with emphasis on the potential roles of LTi's, B and T cell aggregates and examine the function of key chemokines and cytokines including CXCL13 and IL-17, in the formation and maintenance of pulmonary TLFs. This article is protected by copyright. All rights reserved.
View details for DOI 10.1111/imm.12649
View details for PubMedID 27441396
The pharmacokinetics and dosing of oral 4-methylumbelliferone for inhibition of hyaluronan synthesis in mice.
Clinical and experimental immunology
2016; 185 (3): 372-381
Recently, there has been considerable interest in using 4-methylumbelliferone (4-MU) to inhibit hyaluronan synthesis in mouse models of cancer, autoimmunity, and a variety of other inflammatory disorders where hyaluronan (HA) has been implicated in disease pathogenesis. In order to facilitate future studies in this area, we have examined the dosing, treatment route, treatment duration, and metabolism of 4-MU in both C57BL/6 and BALB/c mice. Mice fed chow containing 5% 4-MU, a dose calculated to deliver 250 mg/mouse/day, initially lose substantial weight but typically resume normal weight gain after one week. It also takes up to a week to see a reduction in serum HA in these animals, indicating that at least a one-week loading period on the drug is required for most protocols. At steady state, over 90% of the drug is present in plasma as the glucuronidated metabolite 4-methylumbelliferyl glucuronide (4-MUG), with the sulfated metabolite, 4-methylumbelliferyl sulfate (4-MUS) comprising most of the remainder. Chow containing 5% but not 0.65% 4-MU was effective at preventing disease in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis as well as in the DORmO mouse model of autoimmune diabetes. While oral 4-MU was effective at preventing EAE, daily intraperitoneal injections of 4-MU were not. Factors potentially affecting 4-MU uptake and plasma concentrations in mice include its taste, short half-life and low bioavailability. These studies provide a practical resource for implementing oral 4-MU treatment protocols in mice. This article is protected by copyright. All rights reserved.
View details for DOI 10.1111/cei.12815
View details for PubMedID 27218304
Pf4 bacteriophage produced by Pseudomonas aeruginosa inhibits Aspergillus fumigatus metabolism via iron sequestration
2016; 162 (9): 1583-1594
Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are major human pathogens known to interact in a variety of disease settings, including airway infections in cystic fibrosis. We recently reported that clinical CF isolates of Pa inhibit the formation and growth of Af biofilms. Here, we report that the bacteriophage Pf4, produced by Pa, can inhibit the metabolic activity of Af biofilms. This phage-mediated inhibition was dose dependent, ablated by phage denaturation, and was more pronounced against preformed Af biofilm rather than biofilm formation. In contrast, planktonic conidial growth was unaffected. Two other phages, Pf1 and fd, did not inhibit Af, nor did supernatant from a Pa strain incapable of producing Pf4. Pf4, but not Pf1, attaches to Af hyphae in an avid and prolonged manner, suggesting that Pf4-mediated inhibition of Af may occur at the biofilm surface. We show that Pf4 binds iron, thus denying Af a crucial resource. Consistent with this, the inhibition of Af metabolism by Pf4 could be overcome with supplemental ferric iron, with preformed biofilm more resistant to reversal. To our knowledge, this is the first report of a bacterium producing a phage that inhibits the growth of a fungus and the first description of a phage behaving as an iron chelator in a biological system.
View details for DOI 10.1099/mic.0.000344
View details for Web of Science ID 000385273100008
View details for PubMedID 27473221
Filamentous bacteriophage organize the Pseudomonas aeruginosa biofilm matrix into a liquid crystal.
2016; 3 (1; 49-52)
View details for DOI 10.15698/mic2016.01.475
Chemokine Involvement in Fetal and Adult Wound Healing.
Advances in wound care
2015; 4 (11): 660-672
Significance: Fetal wounds heal with a regenerative phenotype that is indistinguishable from surrounding skin with restored skin integrity. Compared to this benchmark, all postnatal wound healing is impaired and characterized by scar formation. The biologic basis of the fetal regenerative phenotype can serve as a roadmap to recapitulating regenerative repair in adult wounds. Reduced leukocyte infiltration, likely mediated, in part, through changes in the chemokine milieu, is a fundamental feature of fetal wound healing. Recent Advances: The contributions of chemokines to wound healing are a topic of active investigation. Recent discoveries have opened the possibility of targeting chemokines therapeutically to treat disease processes and improve healing capability, including the possibility of achieving a scarless phenotype in postnatal wounds. Critical Issues: Successful wound healing is a complex process, in which there is a significant interplay between multiple cell types, signaling molecules, growth factors, and extracellular matrix. Chemokines play a crucial role in this interplay and have been shown to have different effects in various stages of the healing process. Understanding how these chemokines are locally produced and regulated during wound healing and how the chemokine milieu differs in fetal versus postnatal wounds may help us identify ways in which we can target chemokine pathways. Future Directions: Further studies on the role of chemokines and their role in the healing process will greatly advance the potential for using these molecules as therapeutic targets.
View details for PubMedID 26543680
Angiopoietin-1 improves endothelial progenitor cell-dependent neovascularization in diabetic wounds
2015; 158 (3): 846-856
The diabetic phenotype of wound healing is in part characterized by impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Angiopoietin-1 (Ang-1) is a potent mobilizer of EPCs from the bone marrow (BM). A suggested mechanism for EPC mobilization from the BM is mediated by matrix metalloproteinase 9 (MMP-9) and stem cell factor (SCF). Taken together, we hypothesized that overexpression of Ang-1 in diabetic wounds will recruit EPCs and improve neovascularization and wound healing.An endothelial lineage BM-labeled murine model of diabetes was developed to track BM-derived EPCs. FVBN mice were lethally irradiated and then reconstituted with BM from syngeneic Tie2/LacZ donor mice. Diabetes was induced with streptozotocin. Dorsal wounds in BM-transplanted mice were treated with Ad-Ang-1, Ad-GFP, or phosphate-buffered saline. At day 7 after injury, wounds were harvested and analyzed. A similar experiment was conducted in EPC mobilization deficient MMP-9 -/- mice to determine whether the effects of Ang-1 were EPC-dependent.Overexpression of Ang-1 resulted in greatly improved re-epithelialization, neovascularization, and EPC recruitment in diabetic BM-transplanted wounds at day 7. Ang-1 treatment resulted in increased serum levels of proMMP-9 and SCF but had no effect on vascular endothelial growth factor levels. According to our FACS results, peripheral blood EPC (CD34(+)/Cd133(+)/Flk1(+)) counts at day 3 after wounding showed impaired EPC mobilization in MMP-9 -/- mice compared with those of wild-type controls. EPC mobilization was rescued by SCF administration, validating this model for EPC-mobilization-deficient mechanistic studies. In MMP-9 -/- mice, Ad-Ang-1 accelerated re-epithelialization in a similar manner, but had no effect on neovascularization.Our results show that Ang-1 administration results in improved neovascularization which is dependent on EPC recruitment and has direct effects on wound re-epithelialization. These data may represent a novel strategy to correct the phenotype of impaired diabetic neovascularization and may improve diabetic wound healing.
View details for DOI 10.1016/j.surg.2015.06.034
View details for Web of Science ID 000359755000033
View details for PubMedID 26266763
View details for PubMedCentralID PMC4549153
- The Role of Interleukin-10 and Hyaluronan in Murine Fetal Fibroblast Function In Vitro: Implications for Recapitulating Fetal Regenerative Wound Healing PLOS ONE 2015; 10 (5)
The role of interleukin-10 and hyaluronan in murine fetal fibroblast function in vitro: implications for recapitulating fetal regenerative wound healing.
2015; 10 (5)
Mid-gestation fetal cutaneous wounds heal scarlessly and this has been attributed in part to abundant hyaluronan (HA) in the extracellular matrix (ECM) and a unique fibroblast phenotype. We recently reported a novel role for interleukin 10 (IL-10) as a regulator of HA synthesis in the fetal ECM, as well as the ability of the fetal fibroblast to produce an HA-rich pericellular matrix (PCM). We hypothesized that IL-10-mediated HA synthesis was essential to the fetal fibroblast functional phenotype and, moreover, that this phenotype could be recapitulated in adult fibroblasts via supplementation with IL-10 via an HA dependent process.To evaluate the differences in functional profile, we compared metabolism (MTS assay), apoptosis (caspase-3 staining), migration (scratch wound assay) and invasion (transwell assay) between C57Bl/6J murine fetal (E14.5) and adult (8 weeks) fibroblasts. We found that fetal fibroblasts have lower rates of metabolism and apoptosis, and an increased ability to migrate and invade compared to adult fibroblasts, and that these effects were dependent on IL-10 and HA synthase activity. Further, addition of IL-10 to adult fibroblasts resulted in increased fibroblast migration and invasion and recapitulated the fetal phenotype in an HA-dependent manner.Our data demonstrates the functional differences between fetal and adult fibroblasts, and that IL-10 mediated HA synthesis is essential for the fetal fibroblasts' enhanced invasion and migration properties. Moreover, IL-10 via an HA-dependent mechanism can recapitulate this aspect of the fetal phenotype in adult fibroblasts, suggesting a novel mechanism of IL-10 in regenerative wound healing.
View details for DOI 10.1371/journal.pone.0124302
View details for PubMedID 25951109
View details for PubMedCentralID PMC4423847
Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways.
International journal of cell biology
2015; 2015: 614297-?
Cyclosporine A (CSA) is an immunosuppressive agent that specifically targets T cells and also increases the percentage of pro-tolerogenic CD4+Foxp3+ regulatory T cells (Treg) through unknown mechanisms. We previously reported that CD44, a receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA), promotes Treg stability in IL-2-low environments. Here, we asked whether CD44 signaling also promotes Treg resistance to CSA. We found that CD44 cross-linking promoted Foxp3 expression and Treg viability in the setting of CSA treatment. This effect was IL-2 independent but could be suppressed using sc-355979, an inhibitor of Stat5-phosphorylation. Moreover, we found that inhibition of HA synthesis impairs Treg homeostasis but that this effect could be overcome with exogenous IL-2 or CD44-cross-linking. Together, these data support a model whereby CD44 cross-linking by HA promotes IL-2-independent Foxp3 expression and Treg survival in the face of CSA.
View details for DOI 10.1155/2015/614297
View details for PubMedID 26448755
View details for PubMedCentralID PMC4581548
- 4-methylumbelliferone treatment and hyaluronan inhibition as a therapeutic strategy in inflammation, autoimmunity, and cancer. Frontiers in immunology 2015; 6: 123-?
Hyaluronan and hyaluronan-binding proteins accumulate in both human type 1 diabetic islets and lymphoid tissues and associate with inflammatory cells in insulitis.
2014; 63 (8): 2727-2743
Hyaluronan is an extracellular matrix glycosaminoglycan that is present in pancreatic islets but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and non-diabetic organ donors differ in the amount and distribution of hyaluronan and hyaluronan binding proteins (hyaladherins) such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor-stimulated gene-6 (TSG-6). Hyaluronan was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, hyaluronan was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in hyaluronan-rich areas of islets and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of less than 10 years. Furthermore, hyaluronan and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D compared to controls. Our observations highlight potential roles for hyaluronan and hyaladherins in the pathogenesis of diabetes.
View details for DOI 10.2337/db13-1658
View details for PubMedID 24677718
Adenoviral-mediated gene transfer of insulin-like growth factor 1 enhances wound healing and induces angiogenesis
JOURNAL OF SURGICAL RESEARCH
2014; 190 (1): 367-377
Chronic wounds are characterized by a wound healing and neovascularization deficit. Strategies to increase neovascularization can significantly improve chronic wound healing. Insulin-like growth factor (IGF)-1 is reported to be a keratinocyte mitogen and is believed to induce angiogenesis via a vascular endothelial growth factor (VEGF)-dependent pathway. Using a novel ex vivo human dermal wound model and a diabetic-impaired wound healing murine model, we hypothesized that adenoviral overexpression of IGF-1 (Ad-IGF-1) will enhance wound healing and induce angiogenesis through a VEGF-dependent pathway.Ex vivo: 6-mm full-thickness punch biopsies were obtained from normal human skin, and 3-mm full-thickness wounds were created at the center. Skin explants were maintained at air liquid interface. Db/db murine model: 8-mm full-thickness dorsal wounds in diabetic (db/db) mice were created. Treatment groups in both human ex vivo and in vivo db/db wound models include 1 × 10(8) particle forming units of Ad-IGF-1 or Ad-LacZ, and phosphate buffered saline (n = 4-5/group). Cytotoxicity (lactate dehydrogenase) was quantified at days 3, 5, and 7 for the human ex vivo wound model. Epithelial gap closure (hematoxylin and eosin; Trichrome), VEGF expression (enzyme-linked immunosorbent assay), and capillary density (CD 31 + CAPS/HPF) were analyzed at day 7.In the human ex vivo organ culture, the adenoviral vectors did not demonstrate any significant difference in cytotoxicity compared with phosphate buffered saline. Ad-IGF-1 overexpression significantly increases basal keratinocyte migration, with no significant effect on epithelial gap closure. There was a significant increase in capillary density in the Ad-IGF-1 wounds. However, there was no effect on VEGF levels in Ad-IGF-1 samples compared with controls. In db/db wounds, Ad-IGF-1 overexpression significantly improves epithelial gap closure and granulation tissue with a dense cellular infiltrate compared with controls. Ad-IGF-1 also increases capillary density, again with no significant difference in VEGF levels in the wounds compared with control treatments.In two different models, our data demonstrate that adenoviral-mediated gene transfer of IGF-1 results in enhanced wound healing and induces angiogenesis via a VEGF-independent pathway. Understanding the underlying mechanisms of IGF-1 effects on angiogenesis may help produce novel therapeutics for chronic wounds or diseases characterized by a deficit in neovascularization.
View details for DOI 10.1016/j.jss.2014.02.051
View details for Web of Science ID 000338444700053
Comparison of interleukin 10 homologs on dermal wound healing using a novel human skin ex vivo organ culture model
JOURNAL OF SURGICAL RESEARCH
2014; 190 (1): 358-366
Anti-inflammatory cytokine interleukin (IL)-10 has been shown to induce regenerative healing in postnatal wounds. A viral homolog of IL-10 produced by human cytomegalovirus (CMV IL-10) similarly generates potent immunoregulatory effects, but its effects on wound healing have not been investigated. Currently, there are limited cost-effective methods of screening vulnerary therapeutics. Taken together, we aim to develop and validate a novel human ex vivo dermal wound model and hypothesize that CMV IL-10 will enhance dermal wound healing.Full-thickness circular (6-mm) explants were taken from surgical skin samples and 3-mm full-thickness wounds were created. Explants were embedded in collagen I matrix and maintained in specially formulated media with the epidermis at air-liquid interface, and treated with human IL-10 or CMV IL-10 (200 ng/mL). The viability of cultured explants was validated by histology and lactate dehydrogenase (LDH) activity. Epithelial gap, epithelial height, basal keratinocyte migration, vascular endothelial growth factor levels, and neovascularization were measured at days 3 and 7 to determine IL-10 effects on wound healing.Culture explants at day 7 appeared similar to fresh skin in morphology, cell, and vessel density. By day 14, the epidermis separated from the dermis and the cell density diminished. Day 7 wounds appeared viable with advancing epithelial and basal keratinocyte migration with no evidence of necrosis. Cytotoxicity analysis via the quantification of LDH revealed no differences between controls and treated groups. There was a slight increase in the quantity of LDH in media at day 3; however, this decreased at day 5 and continued to decline up to day 21. CMV IL-10 treatment resulted in a significant decrease in the epithelial gap and an increase in epithelial height. There were no differences in the rates of basal keratinocyte migration at day 7 between treated and control groups. Interestingly, human IL-10 increased vascular endothelial growth factor expression and neovascularization compared with controls.The human ex vivo wound model provides a simple and viable design to study dermal wound healing. Both IL-10 homologs demonstrate vulnerary effects. The viral homolog demonstrates enhanced effects on wound closure compared with human IL-10. These data represent a novel tool that can be used to screen therapeutics, such as CMV IL-10, before preclinical studies.
View details for DOI 10.1016/j.jss.2014.02.027
View details for Web of Science ID 000338444700052
Tissue integrity signals communicated by high-molecular weight hyaluronan and the resolution of inflammation.
2014; 58 (2-3): 186-192
The extracellular matrix polysaccharide hyaluronan (HA) exerts size-dependent effects on leukocyte behavior. Low-molecular weight HA is abundant at sites of active tissue catabolism and promotes inflammation via effects on Toll-like receptor signaling. Conversely, high-molecular weight HA is prevalent in uninjured tissues and is anti-inflammatory. We propose that the ability of high-molecular weight but not low-molecular weight HA to cross-link CD44 functions as a novel form of pattern recognition that recognizes intact tissues and communicates "tissue integrity signals" that promote resolution of local immune responses.
View details for DOI 10.1007/s12026-014-8495-2
View details for PubMedID 24614953
Evaluation of in vivo T cell kinetics: use of heavy isotope labelling in type 1 diabetes.
Clinical and experimental immunology
2013; 172 (3): 363-374
CD4(+) memory cell development is dependent upon T cell receptor (TCR) signal strength, antigen dose and the cytokine milieu, all of which are altered in type 1 diabetes (T1D). We hypothesized that CD4(+) T cell turnover would be greater in type 1 diabetes subjects compared to controls. In vitro studies of T cell function are unable to evaluate dynamic aspects of immune cell homoeostasis. Therefore, we used deuterium oxide ((2) H(2)O) to assess in vivo turnover of CD4(+) T cell subsets in T1D (n = 10) and control subjects (n = 10). Serial samples of naive, memory and regulatory (T(reg)) CD4(+) T cell subsets were collected and enrichment of deoxyribose was determined by gas chromatography-mass spectrometry (GC-MS). Quantification of T cell turnover was performed using mathematical models to estimate fractional enrichment (f, n = 20), turnover rate (k, n = 20), proliferation (p, n = 10) and disappearance (d*, n = 10). Although turnover of T(regs) was greater than memory and naive cells in both controls and T1D subjects, no differences were seen between T1D and controls in T(reg) or naive kinetics. However, turnover of CD4(+) memory T cells was faster in those with T1D compared to control subjects. Measurement and modelling of incorporated deuterium is useful for evaluating the in vivo kinetics of immune cells in T1D and could be incorporated into studies of the natural history of disease or clinical trials designed to alter the disease course. The enhanced CD4(+) memory T cell turnover in T1D may be important in understanding the pathophysiology and potential treatments of autoimmune diabetes.
View details for DOI 10.1111/cei.12064
View details for PubMedID 23600824
Reactivation of latent viruses in individuals receiving rituximab for new onset type 1 diabetes
JOURNAL OF CLINICAL VIROLOGY
2013; 57 (2): 115-119
Rituximab has been successfully used as an experimental therapy in different autoimmune diseases. Recently, a double-blind placebo-controlled phase-2 study in early onset type 1 diabetes showed that rituximab delayed progression of the disease. However, like with any immunosuppressive therapy, there is a concern of opportunistic viral reactivations with the use of rituximab, including herpes and polyomaviruses.To study the incidence of new infections and reactivations with BK, JC, Epstein-Barr and cytomegalovirus (BKV, JCV, EBV and CMV) in T1D participants in the phase-2 rituximab study.Subjects received 4 weekly doses of rituximab (N = 57) or placebo (N = 30) during the first month of study. Blood samples obtained at weeks 0, 12, 26, 56 and 78 were assayed for CMV, EBV, BKV and JCV by real-time DNA PCR and serology.EBV reactivations were diagnosed by PCR in 25% of placebo, but none of rituximab recipients (p < 0.01). There were no episodes of CMV viremia in either treatment group. BKV viremias were significantly more common in the rituximab recipients (9%) compared with placebo controls (0, p < 0.01). No JCV reactivations were detected in this study, but among 6 rituximab and 2 placebo recipients who seroconverted for JCV during the study, only one rituximab recipient had detectable viremia. All infections were asymptomatic.Four doses of rituximab administered to individuals with early onset T1D decreased the incidence of asymptomatic EBV reactivations, as predicted by the rituximab-mediated elimination of memory B-cells, but increased the frequency of asymptomatic viremias caused by polyomaviruses.
View details for DOI 10.1016/j.jcv.2013.01.016
View details for Web of Science ID 000318563900005
View details for PubMedID 23422292
IL-10 induction from implants delivering pancreatic islets and hyaluronan.
Journal of diabetes research
2013; 2013: 342479-?
Local induction of pro-tolerogenic cytokines, such as IL-10, is an appealing strategy to help facilitate transplantation of islets and other tissues. Here, we describe a pair of implantable devices that capitalize on our recent finding that hyaluronan (HA) promotes IL-10 production by activated T cells. The first device is an injectable hydrogel made of crosslinked HA and heparan sulfate loaded with anti-CD3/anti-CD28 antibodies and IL-2. T cells embedded within this hydrogel prior to polymerization go on to produce IL-10 in vivo. The second device is a bioengineered implant consisting of a polyvinyl alcohol sponge scaffold, supportive collagen hydrogel, and alginate spheres mediating sustained release of HA in fluid form. Pancreatic islets that expressed ovalbumin (OVA) antigen were implanted within this device for 14 days into immunodeficient mice that received OVA-specific DO.11.10 T cells and a subsequent immunization with OVA peptide. Splenocytes harvested from these mice produced IL-10 upon re-challenge with OVA or anti-CD3 antibodies. Both of these devices represent model systems that will be used, in future studies, to further evaluate IL-10 induction by HA, with the objective of improving the survival and function of transplanted islets in the setting of autoimmune (type 1) diabetes.
View details for DOI 10.1155/2013/342479
View details for PubMedID 23971054
View details for PubMedCentralID PMC3736405
- Science and government. Obama and the promotion of international science. Science 2012; 338 (6107): 610-612
The Role of Hyaluronan and the Extracellular Matrix in Islet Inflammation and Immune Regulation
CURRENT DIABETES REPORTS
2012; 12 (5): 471-480
Type 1 diabetes (T1D) is a disease that in most individuals results from autoimmune attack of a single tissue type, the pancreatic islet. A fundamental, unanswered question in T1D pathogenesis is how the islet tissue environment influences immune regulation. This crosstalk is likely to be communicated through the extracellular matrix (ECM). Here, we review what is known about the ECM in insulitis and examine how the tissue environment is synchronized with immune regulation. In particular, we focus on the role of hyaluronan (HA) and its interactions with Foxp3+ regulatory T-cells (Treg). We propose that HA is a "keystone molecule" in the inflammatory milieu and that HA, together with its associated binding proteins and receptors, is an appropriate point of entry for investigations into how ECM influences immune regulation in the islet.
View details for DOI 10.1007/s11892-012-0297-0
View details for Web of Science ID 000308286400004
View details for PubMedID 22810951
Hyaluronan and versican in the control of human T-lymphocyte adhesion and migration
2012; 31 (2): 90-100
The ability of lymphocytes to migrate freely through connective tissues is vital to efficient immune function. How the extracellular matrix (ECM) may affect T-cell adhesion and migration is not well understood. We have examined the adhesion and migration of activated human T-lymphocytes on ECM made by fibroblast-like synoviocytes and lung fibroblasts. These cells were minimally interactive until treated with a viral mimetic, Poly I:C. This treatment promoted myofibroblast formation and engendered a higher-order structured ECM, rich in versican and hyaluronan, to which T-cells avidly adhered in a hyaluronidase-sensitive manner. This Poly I:C-induced matrix impeded T-cell spreading and migration on and through synoviocyte monolayers, while hyaluronidase treatment or adding versican antibody during matrix formation reversed the effect on T-cell migration. Hyaluronidase also reversed the spread myofibroblast morphology. These data suggest that the viscous hyaluronan- and versican-rich matrix binds and constrains T-lymphocytes. Using purified matrix components and solid state matrices of defined composition, we uncovered a role for versican in modulating hyaluronan-T-cell interactions. Versican prevented T-cell binding to soluble hyaluronan, as well as the amoeboid shape change on hyaluronan-coated dishes and T-cell penetration of collagen gels. Together, these data suggest that hyaluronan and versican play a role in T-cell trafficking and function in inflamed tissues.
View details for DOI 10.1016/j.matbio.2011.10.004
View details for Web of Science ID 000301632900003
View details for PubMedID 22155153
Reversal of Diabetes in Mice With a Bioengineered Islet Implant Incorporating a Type I Collagen Hydrogel and Sustained Release of Vascular Endothelial Growth Factor
2012; 21 (10): 2099-2110
We have developed a bioengineered implant (BI) to evaluate strategies to promote graft survival and function in models of islet transplantation in mice. The BI, sized for implantation within a fold of intestinal mesentery, consists of a disk-shaped, polyvinyl alcohol sponge infused with a type I collagen hydrogel that contains dispersed donor islets. To promote islet vascularization, the BI incorporates a spherical alginate hydrogel for sustained release of vascular endothelial growth factor (VEGF). BIs that contained 450-500 islets from syngeneic (C57Bl/6) donors and 20 ng of VEGF reversed streptozotocin (STZ)-induced diabetes in 100% of mice (8/8), whereas BIs that contained an equivalent number of islets, but which lacked VEGF, reversed STZ-induced diabetes in only 62.5% of mice (5/8). Between these "+VEGF" and "-VEGF" groups, the time to achieve normoglycemia (8-18 days after implantation) did not differ statistically; however, transitory, postoperative hypoglycemia was markedly reduced in the +VEGF group relative to the -VEGF group. Notably, none of the mice that achieved normoglycemia in these two groups required exogenous insulin therapy once the BIs began to fully regulate levels of blood glucose. Moreover, the transplanted mice responded to glucose challenge in a near-normal manner, as compared to the responses of healthy, nondiabetic (control) mice that had not received STZ. In future studies, the BIs described here will serve as platforms to evaluate the capability of immunomodulatory compounds, delivered locally within the BI, to prevent or reverse diabetes in the setting of autoimmune (type 1) diabetes.
View details for DOI 10.3727/096368912X636786
View details for Web of Science ID 000313227300001
View details for PubMedID 23231959
Effect of rituximab on human in vivo antibody immune responses.
The Journal of allergy and clinical immunology
2011; 128 (6): 1295-1302.e5
B-lymphocyte depletion with rituximab has been shown to benefit patients with various autoimmune diseases. We have previously demonstrated that this benefit is also apparent in patients with newly diagnosed type 1 diabetes.The effect of rituximab on in vivo antibody responses, particularly during the period of B-lymphocyte depletion, is incompletely determined. This study was designed to assess this knowledge void.In patients with recent-onset type 1 diabetes treated with rituximab (n = 46) or placebo (n = 29), antibody responses to neoantigen phiX174 during B-lymphocyte depletion and with hepatitis A (as a second neoantigen) and tetanus/diphtheria (as recall antigens) after B-lymphocyte recovery were studied. Anti- tetanus, diphtheria, mumps, measles, and rubella titers were measured before and after treatment by means of ELISA. Antibody titers and percentage IgM versus percentage IgG to phiX174 were measured by means of phage neutralization. B-lymphocyte subsets were determined by means of flow cytometry.No change occurred in preexisting antibody titers. Tetanus/diphtheria and hepatitis A immunization responses were protective in the rituximab-treated subjects, although significantly blunted compared with those seen in the controls subjects, when immunized at the time of B-lymphocyte recovery. Anti-phiX174 responses were severely reduced during the period of B-lymphocyte depletion, but with B-lymphocyte recovery, anti-phiX174 responses were within the normal range.During the time of B-lymphocyte depletion, rituximab recipients had a decreased antibody response to neoantigens and significantly lower titers after recall immunization with diphtheria and tetanus toxoid. With recovery, immune responses return toward normal. Immunization during the time of B-lymphocyte depletion, although ineffective, does not preclude a subsequent response to the antigen.
View details for DOI 10.1016/j.jaci.2011.08.008
View details for PubMedID 21908031
View details for PubMedCentralID PMC3659395
Low-Dose Antigen Promotes Induction of FOXP3 in Human CD4(+) T Cells
JOURNAL OF IMMUNOLOGY
2011; 187 (7): 3511-3520
Low Ag dose promotes induction and persistence of regulatory T cells (Tregs) in mice, yet few studies have addressed the role of Ag dose in the induction of adaptive CD4(+)FOXP3(+) Tregs in humans. To this end, we examined the level of FOXP3 expression in human CD4(+)CD25(-) T cells upon activation with autologous APCs and varying doses of peptide. Ag-specific T cells expressing FOXP3 were identified by flow cytometry using MHC class II tetramer (Tmr). We found an inverse relationship between Ag dose and the frequency of FOXP3(+) cells for both foreign Ag-specific and self Ag-specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr(+)FOXP3(+) T cells was not due to expansion of natural Tregs, but instead, we found that induction, proliferation, and persistence of FOXP3(+) cells was similar in high- and low-dose cultures, whereas proliferation of FOXP3(-) T cells was favored in high Ag dose cultures. The frequency of FOXP3(+) cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3(+) cells was maintained with IL-2, but not upon restimulation with Ag. Together, these data suggest that low Ag dose favors the transient generation of human Ag-specific adaptive Tregs over the proliferation of Ag-specific FOXP3(-) effector T cells. These adaptive Tregs could function to reduce ongoing inflammatory responses and promote low-dose tolerance in humans, especially when Ag exposure and tolerance is transient.
View details for DOI 10.4049/jimmunol.1003880
View details for Web of Science ID 000295036400009
View details for PubMedID 21865550
Antigen-based therapy with glutamic acid decarboxylase (GAD) vaccine in patients with recent-onset type 1 diabetes: a randomised double-blind trial
2011; 378 (9788): 319-327
Glutamic acid decarboxylase (GAD) is a major target of the autoimmune response that occurs in type 1 diabetes mellitus. In animal models of autoimmunity, treatment with a target antigen can modulate aggressive autoimmunity. We aimed to assess whether immunisation with GAD formulated with aluminum hydroxide (GAD-alum) would preserve insulin production in recent-onset type 1 diabetes.Patients aged 3-45 years who had been diagnosed with type 1 diabetes for less than 100 days were enrolled from 15 sites in the USA and Canada, and randomly assigned to receive one of three treatments: three injections of 20 μg GAD-alum, two injections of 20 μg GAD-alum and one of alum, or 3 injections of alum. Injections were given subcutaneously at baseline, 4 weeks later, and 8 weeks after the second injection. The randomisation sequence was computer generated at the TrialNet coordinating centre. Patients and study personnel were masked to treatment assignment. The primary outcome was the baseline-adjusted geometric mean area under the curve (AUC) of serum C-peptide during the first 2 h of a 4-h mixed meal tolerance test at 1 year. Secondary outcomes included changes in glycated haemoglobin A(1c) (HbA(1c)) and insulin dose, and safety. Analysis included all randomised patients with known measurements. This trial is registered with ClinicalTrials.gov, number NCT00529399.145 patients were enrolled and treated with GAD-alum (n=48), GAD-alum plus alum (n=49), or alum (n=48). At 1 year, the 2-h AUC of C-peptide, adjusted for age, sex, and baseline C-peptide value, was 0·412 nmol/L (95% CI 0·349-0·478) in the GAD-alum group, 0·382 nmol/L (0·322-0·446) in the GAD-alum plus alum group, and 0·413 nmol/L (0·351-0·477) in the alum group. The ratio of the population mean of the adjusted geometric mean 2-h AUC of C-peptide was 0·998 (95% CI 0·779-1·22; p=0·98) for GAD-alum versus alum, and 0·926 (0·720-1·13; p=0·50) for GAD-alum plus alum versus alum. HbA(1c), insulin use, and the occurrence and severity of adverse events did not differ between groups.Antigen-based immunotherapy therapy with two or three doses of subcutaneous GAD-alum across 4-12 weeks does not alter the course of loss of insulin secretion during 1 year in patients with recently diagnosed type 1 diabetes. Although antigen-based therapy is a highly desirable treatment and is effective in animal models, translation to human autoimmune disease remains a challenge.US National Institutes of Health.
View details for DOI 10.1016/S0140-6736(11)60895-7
View details for Web of Science ID 000293615800029
View details for PubMedID 21714999
Co-stimulation modulation with abatacept in patients with recent-onset type 1 diabetes: a randomised, double-blind, placebo-controlled trial
2011; 378 (9789): 412-419
The immunopathogenesis of type 1 diabetes mellitus is associated with T-cell autoimmunity. To be fully active, immune T cells need a co-stimulatory signal in addition to the main antigen-driven signal. Abatacept modulates co-stimulation and prevents full T-cell activation. We evaluated the effect of abatacept in recent-onset type 1 diabetes.In this multicentre, double-blind, randomised controlled trial, patients aged 6-45 years recently diagnosed with type 1 diabetes were randomly assigned (2:1) to receive abatacept (10 mg/kg, maximum 1000 mg per dose) or placebo infusions intravenously on days 1, 14, 28, and monthly for a total of 27 infusions over 2 years. Computer-generated permuted block randomisation was used, with a block size of 3 and stratified by participating site. Neither patients nor research personnel were aware of treatment assignments. The primary outcome was baseline-adjusted geometric mean 2-h area-under-the-curve (AUC) serum C-peptide concentration after a mixed-meal tolerance test at 2 years' follow-up. Analysis was by intention to treat for all patients for whom data were available. This trial is registered at ClinicalTrials.gov, NCT00505375.112 patients were assigned to treatment groups (77 abatacept, 35 placebo). Adjusted C-peptide AUC was 59% (95% CI 6·1-112) higher at 2 years with abatacept (n=73, 0·378 nmol/L) than with placebo (n=30, 0·238 nmol/L; p=0·0029). The difference between groups was present throughout the trial, with an estimated 9·6 months' delay (95% CI 3·47-15·6) in C-peptide reduction with abatacept. There were few infusion-related adverse events (36 reactions occurred in 17 [22%] patients on abatacept and 11 reactions in six [17%] on placebo). There was no increase in infections (32 [42%] patients on abatacept vs 15 [43%] on placebo) or neutropenia (seven [9%] vs five [14%]).Co-stimulation modulation with abatacept slowed reduction in β-cell function over 2 years. The beneficial effect suggests that T-cell activation still occurs around the time of clinical diagnosis of type 1 diabetes. Yet, despite continued administration of abatacept over 24 months, the decrease in β-cell function with abatacept was parallel to that with placebo after 6 months of treatment, causing us to speculate that T-cell activation lessens with time. Further observation will establish whether the beneficial effect continues after cessation of abatacept infusions.US National Institutes of Health.
View details for DOI 10.1016/S0140-6736(11)60886-6
View details for Web of Science ID 000293615900032
View details for PubMedID 21719096
ECM components guide IL-10 producing regulatory T-cell (TR1) induction from effector memory T-cell precursors
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (19): 7938-7943
We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3- IL-10-producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10-dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4(+)CD62L(-)FoxP3(-), suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation.
View details for DOI 10.1073/pnas.1017360108
View details for Web of Science ID 000290439500058
View details for PubMedID 21518860
View details for PubMedCentralID PMC3093524
Isolated Infection of a Decommissioned Penile Prosthesis Reservoir with Actinomyces neuii
JOURNAL OF SEXUAL MEDICINE
2011; 8 (3): 923-926
Inflatable penile prostheses (IPPs) are a well-established and reliable treatment for medication refractory erectile dysfunction. The most serious complication with IPPs is infection, with the reported incidence after primary placement 1% to 3% and after revision surgery 8% to 18%.The aim of this report is to describe an infected decommissioned IPP reservoir with Actinomyces neuii with successful preservation of a functioning implant.After 9 years of successful use with an IPP (AMS 700 CX) for Peyronie's disease and organic erectile dysfunction, a 79-year-old man underwent replacement with an AMS 700 LGX. The decommissioned reservoir was kept in the right prevesical space, and the new reservoir was placed in the left prevesical space. Three months later, he presented with right inguinal pain and swelling.He was found to have an infected right reservoir with A. neuii, sparing his new IPP. After removal of the right reservoir, he had an uneventful recovery and has shown no evidence of infection in the new device.Revision surgery for IPPs carries a higher risk for implant infection. This is the first report of a genitourinary implant infection with A. neuii. Aggressive surgical and medical treatment may allow preservation of the functioning implant, despite gross infection of the decommissioned reservoir.
View details for DOI 10.1111/j.1743-6109.2010.02144.x
View details for Web of Science ID 000287703100031
View details for PubMedID 21143418
Peptide-MHC Cellular Microarray with Innovative Data Analysis System for Simultaneously Detecting Multiple CD4 T-Cell Responses
2010; 5 (6)
Peptide:MHC cellular microarrays have been proposed to simultaneously characterize multiple Ag-specific populations of T cells. The practice of studying immune responses to complicated pathogens with this tool demands extensive knowledge of T cell epitopes and the availability of peptide:MHC complexes for array fabrication as well as a specialized data analysis approach for result interpretation.We co-immobilized peptide:DR0401 complexes, anti-CD28, anti-CD11a and cytokine capture antibodies on the surface of chamber slides to generate a functional array that was able to detect rare Ag-specific T cell populations from previously primed in vitro T cell cultures. A novel statistical methodology was also developed to facilitate batch processing of raw array-like data into standardized endpoint scores, which linearly correlated with total Ag-specific T cell inputs. Applying these methods to analyze Influenza A viral antigen-specific T cell responses, we not only revealed the most prominent viral epitopes, but also demonstrated the heterogeneity of anti-viral cellular responses in healthy individuals. Applying these methods to examine the insulin producing beta-cell autoantigen specific T cell responses, we observed little difference between autoimmune diabetic patients and healthy individuals, suggesting a more subtle association between diabetes status and peripheral autoreactive T cells.The data analysis system is reliable for T cell specificity and functional testing. Peptide:MHC cellular microarrays can be used to obtain multi-parametric results using limited blood samples in a variety of translational settings.
View details for DOI 10.1371/journal.pone.0011355
View details for Web of Science ID 000279259900023
View details for PubMedID 20634998
Th1 cytokines promote T-cell binding to antigen-presenting cells via enhanced hyaluronan production and accumulation at the immune synapse
CELLULAR & MOLECULAR IMMUNOLOGY
2010; 7 (3): 211-220
Hyaluronan (HA) production by dendritic cells (DCs) is known to promote antigen presentation and to augment T-cell activation and proliferation. We hypothesized that pericellular HA can function as intercellular 'glue' directly mediating T cell-DC binding. Using primary human cells, we observed HA-dependent binding between T cells and DCs, which was abrogated upon pre-treatment of the DCs with 4-methylumbelliferone (4-MU), an agent which blocks HA synthesis. Furthermore, T cells regulate HA production by DCs via T cell-derived cytokines in a T helper (Th) subset-specific manner, as demonstrated by the observation that cell-culture supernatants from Th1 but not Th2 clones promote HA production. Similar effects were seen upon the addition of exogenous Th1 cytokines, IL-2, interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). The critical factors which determined the extent of DC-T cell binding in this system were the nature of the pre-treatment the DCs received and their capacity to synthesize HA, as T-cell clones which were pre-treated with monensin, added to block cytokine secretion, bound equivalently irrespective of their Th subset. These data support the existence of a feedforward loop wherein T-cell cytokines influence DC production of HA, which in turn affects the extent of DC-T cell binding. We also document the presence of focal deposits of HA at the immune synapse between T-cells and APC and on dendritic processes thought to be important in antigen presentation. These data point to a pivotal role for HA in DC-T cell interactions at the IS.
View details for DOI 10.1038/cmi.2010.9
View details for Web of Science ID 000277282200008
View details for PubMedID 20228832
Assessment of Seasonal Influenza A Virus-Specific CD4 T-Cell Responses to 2009 Pandemic H1N1 Swine-Origin Influenza A Virus
JOURNAL OF VIROLOGY
2010; 84 (7): 3312-3319
Very limited evidence has been reported to show human adaptive immune responses to the 2009 pandemic H1N1 swine-origin influenza A virus (S-OIV). We studied 17 S-OIV peptides homologous to immunodominant CD4 T epitopes from hemagglutinin (HA), neuraminidase (NA), nuclear protein (NP), M1 matrix protein (MP), and PB1 of a seasonal H1N1 strain. We concluded that 15 of these 17 S-OIV peptides would induce responses of seasonal influenza virus-specific T cells. Of these, seven S-OIV sequences were identical to seasonal influenza virus sequences, while eight had at least one amino acid that was not conserved. T cells recognizing epitopes derived from these S-OIV antigens could be detected ex vivo. Most of these T cells expressed memory markers, although none of the donors had been exposed to S-OIV. Functional analysis revealed that specific amino acid differences in the sequences of these S-OIV peptides would not affect or partially affect memory T-cell responses. These findings suggest that without protective antibody responses, individuals vaccinated against seasonal influenza A may still benefit from preexisting cross-reactive memory CD4 T cells reducing their susceptibility to S-OIV infection.
View details for DOI 10.1128/JVI.02226-09
View details for Web of Science ID 000275307400017
View details for PubMedID 20071564
Defects in IL-2R Signaling Contribute to Diminished Maintenance of FOXP3 Expression in CD4(+) CD25(+) Regulatory T-Cells of Type 1 Diabetic Subjects
2010; 59 (2): 407-415
In humans, multiple genes in the interleukin (IL)-2/IL-2 receptor (IL-2R) pathway are associated with type 1 diabetes. However, no link between IL-2 responsiveness and CD4(+)CD25(+)FOXP3(+) regulatory T-cells (Tregs) has been demonstrated in type 1 diabetic subjects despite the role of these IL-2-dependent cells in controlling autoimmunity. Here, we address whether altered IL-2 responsiveness impacts persistence of FOXP3 expression in Tregs of type 1 diabetic subjects.Persistence of Tregs was assessed by culturing sorted CD4(+)CD25(hi) natural Tregs with IL-2 and measuring FOXP3 expression over time by flow cytometry for control and type 1 diabetic populations. The effects of IL-2 on FOXP3 induction were assessed 48 h after activation of CD4(+)CD25(-) T-cells with anti-CD3 antibody. Cytokine receptor expression and signaling upon exposure to IL-2, IL-7, and IL-15 were determined by flow cytometry and Western blot analysis.Maintenance of FOXP3 expression in CD4(+)CD25(+) Tregs of type 1 diabetic subjects was diminished in the presence of IL-2, but not IL-7. Impaired responsiveness was not linked to altered expression of the IL-2R complex. Instead, IL-2R signaling was reduced in Tregs and total CD4(+) T-cells of type 1 diabetic subjects. In some individuals, decreased signal transducer and activator of transcription 5 phosphorylation correlated with significantly higher expression of protein tyrosine phosphatase N2, a negative regulator of IL-2R signaling.Aberrant IL-2R signaling in CD4(+) T-cells of type 1 diabetic subjects contributes to decreased persistence of FOXP3 expression that may impact establishment of tolerance. These findings suggest novel targets for treatment of type 1 diabetes within the IL-2R pathway and suggest that an altered IL-2R signaling signature may be a biomarker for type 1 diabetes.
View details for DOI 10.2337/db09-0694
View details for Web of Science ID 000274435900011
View details for PubMedID 19875613
Intact extracellular matrix and the maintenance of immune tolerance: high molecular weight hyaluronan promotes persistence of induced CD4+CD25+regulatory T cells
JOURNAL OF LEUKOCYTE BIOLOGY
2009; 86 (3): 567-572
The composition of the ECM provides contextual cues to leukocytes in inflamed and healing tissues. One example of this is HA, where LMW-HA, generated during active inflammation, is a TLR ligand and an endogenous "danger signal," and HMW-HA, predominant in healing or intact tissues, functions in an inverse manner. Our data suggest that HMW-HA actively promotes immune tolerance by augmenting CD4+CD25+ T(Reg) function, and LMW-HA does not. Using a human iT(Reg) model, we demonstrate that HMW-HA but not LMW-HA provides a costimulatory signal through cross-linking CD44 which promotes Foxp3 expression, a critical signaling molecule associated with T(Reg). This effect, in part, may be mediated by a role for intact HMW-HA in IL-2 production, as T(Reg) are highly IL-2-dependent for their survival and function. We propose that HMW-HA contributes to the maintenance of immune homeostasis in uninjured tissue and effectively communicates an "all-clear" signal to down-regulate the adaptive immune system through T(Reg) after tissue matrix integrity has been restored.
View details for DOI 10.1189/jlb.0109001
View details for Web of Science ID 000269377200014
View details for PubMedID 19401397
CD44 Costimulation Promotes FoxP3(+) Regulatory T Cell Persistence and Function via Production of IL-2, IL-10, and TGF-beta
JOURNAL OF IMMUNOLOGY
2009; 183 (4): 2232-2241
Work by our group and others has demonstrated a role for the extracellular matrix receptor CD44 and its ligand hyaluronan in CD4(+)CD25(+) regulatory T cell (Treg) function. Herein, we explore the mechanistic basis for this observation. Using mouse FoxP3/GFP(+) Treg, we find that CD44 costimulation promotes expression of FoxP3, in part through production of IL-2. This promotion of IL-2 production was resistant to cyclosporin A treatment, suggesting that CD44 costimulation may promote IL-2 production through bypassing FoxP3-mediated suppression of NFAT. CD44 costimulation increased production of IL-10 in a partially IL-2-dependent manner and also promoted cell surface TGF-beta expression. Consistent with these findings, Treg from CD44 knockout mice demonstrated impaired regulatory function ex vivo and depressed production of IL-10 and cell surface TGF-beta. These data reveal a novel role for CD44 cross-linking in the production of regulatory cytokines. Similar salutary effects on FoxP3 expression were observed upon costimulation with hyaluronan, the primary natural ligand for CD44. This effect is dependent upon CD44 cross-linking; while both high-molecular-weight hyaluronan (HA) and plate-bound anti-CD44 Ab promoted FoxP3 expression, neither low-molecular weight HA nor soluble anti-CD44 Ab did so. The implication is that intact high-molecular weight HA can cross-link CD44 only in those settings where it predominates over fragmentary LMW-HA, namely, in uninflamed tissue. We propose that intact but not fragmented extracellular is capable of cross-linking CD44 and thereby maintains immunologic tolerance in uninjured or healing tissue.
View details for DOI 10.4049/jimmunol.0900191
View details for Web of Science ID 000268906500007
View details for PubMedID 19635906
The Toll-Like Receptor Signaling Molecule Myd88 Contributes to Pancreatic Beta-Cell Homeostasis in Response to Injury
2009; 4 (4)
Commensal flora and pathogenic microbes influence the incidence of diabetes in animal models yet little is known about the mechanistic basis of these interactions. We hypothesized that Myd88, an adaptor molecule in the Toll-like-receptor (TLR) pathway, regulates pancreatic beta-cell function and homeostasis. We first examined beta-cells histologically and found that Myd88-/- mice have smaller islets in comparison to C57Bl/6 controls. Myd88-/- mice were nonetheless normoglycemic both at rest and after an intra-peritoneal glucose tolerance test (IPGTT). In contrast, after low-dose streptozotocin (STZ) challenge, Myd88-/-mice had an abnormal IPGTT relative to WT controls. Furthermore, Myd88-/- mice suffer enhanced beta-cell apoptosis and have enhanced hepatic damage with delayed recovery upon low-dose STZ treatment. Finally, we treated WT mice with broad-spectrum oral antibiotics to deplete their commensal flora. In WT mice, low dose oral lipopolysaccharide, but not lipotichoic acid or antibiotics alone, strongly promoted enhanced glycemic control. These data suggest that Myd88 signaling and certain TLR ligands mediate a homeostatic effect on beta-cells primarily in the setting of injury.
View details for DOI 10.1371/journal.pone.0005063
View details for Web of Science ID 000265500600019
View details for PubMedID 19357791
Cutting edge: High molecular weight hyaluronan promotes the suppressive effects of CD4(+)CD25(+) regulatory T cells
JOURNAL OF IMMUNOLOGY
2007; 179 (2): 744-747
Hyaluronan is a glycosaminoglycan present in the extracellular matrix. When hyaluronan is degraded during infection and injury, low m.w. forms are generated whose interactions influence inflammation and angiogenesis. Intact high m.w. hyaluronan, conversely, conveys anti-inflammatory signals. We demonstrate that high m.w. hyaluronan enhances human CD4(+)CD25(+) regulatory T cell functional suppression of responder cell proliferation, whereas low m.w. hyaluronan does not. High m.w. hyaluronan also up-regulates the transcription factor FOXP3 on CD4(+)CD25(+) regulatory T cells. These effects are only seen with activated CD4(+)CD25(+) regulatory T cells and are associated with the expression of CD44 isomers that more highly bind high m.w. hyaluronan. At higher concentrations, high m.w. hyaluronan also has direct suppressive effects on T cells. We propose that the state of HA in the matrix environment provides contextual cues to CD4(+)CD25(+) regulatory T cells and T cells, thereby providing a link between the innate inflammatory network and the regulation of adaptive immune responses.
View details for Web of Science ID 000247752100007
View details for PubMedID 17617562
- Histoplasmosis presenting as an isolated spinal cord lesion ARCHIVES OF NEUROLOGY 2006; 63 (12): 1802-1803
CD1d-restricted T-cell subsets and dendritic cell function in autoimmunity
IMMUNOLOGY AND CELL BIOLOGY
2004; 82 (3): 307-314
CD1-restricted T cells have been shown to play a critical role in host defence, tumour surveillance, and maintenance of tolerance. However, immunologic outcomes resulting from activation of CD1d-restricted T cells can be either beneficial or deleterious. A major mechanism by which CD1d-restricted T cells are thought to exert immunoregulatory control is via effects on dendritic cell (DC) differentiation and migration. Important functional subsets of CD1d-restricted T cells are also known to exist and the potential implications for preferential subset activations are discussed.
View details for DOI 10.1111/j.1440-1711.2004.01253.x
View details for Web of Science ID 000221866600011
View details for PubMedID 15186262
Multiple differences in gene expression in regulatory V alpha 24J alpha Q T cells from identical twins discordant for type 1 diabetes
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2000; 97 (13): 7411-7416
Quantitative and qualitative defects in CD1d-restricted T cells have been demonstrated in human and murine autoimmune diseases. To investigate the transcriptional consequences of T cell receptor activation in human Valpha24JalphaQ T cell clones, DNA microarrays were used to quantitate changes in mRNA levels after anti-CD3 stimulation of clones derived from identical twins discordant for type 1 diabetes and IL-4 secretion. Activation resulted in significant modulation of 226 transcripts in the IL-4 secreting clone and 86 in the IL-4-null clone. Only 28 of these genes were in common. The differences observed suggest both ineffective differentiation of diabetic Valpha24JalphaQ T cells and a role for invariant T cells in the recruitment and activation of cells from the myeloid lineage.
View details for Web of Science ID 000087811600087
View details for PubMedID 10840051
Reconstructing the complex evolutionary history of hepatitis B virus
JOURNAL OF MOLECULAR EVOLUTION
1999; 49 (1): 130-141
A detailed analysis of the evolutionary history of hepatitis B virus (HBV) was undertaken using 39 mammalian hepadnaviruses for which complete genome sequences were available, including representatives of all six human genotypes, as well as a large sample of small S gene sequences. Phylogenetic trees of these data were ambiguous, supporting no single place of origin for HBV, and depended heavily on the underlying model of DNA substitution. In some instances genotype F, predominant in the Americas, was the first to diverge, suggesting that the virus arose in the New World. In other trees, however, sequences from genotype B, prevalent in East Asia, were the most divergent. An attempt was also made to determine the rate of nucleotide substitution in the C open reading frame and then to date the origin of HBV. However, no relationship between time and number of substitutions was found in two independent data sets, indicating that a reliable molecular clock does not exist for these data. Both the pattern and the rate of nucleotide substitution are therefore complex phenomena in HBV and hinder any attempt to reconstruct the past spread of this virus.
View details for Web of Science ID 000080974500014
View details for PubMedID 10368441
Recombination between sequences of hepatitis B virus from different genotypes
JOURNAL OF MOLECULAR EVOLUTION
1996; 42 (2): 97-102
A comparison of 25 hepatitis B virus (HBV) isolates for which complete genome sequences are available revealed two that occupied different positions in phylogenetic trees reconstructed from different open reading frames. Further analysis indicated that this incongruence was the result of recombination between viruses of different genomic and antigenic types. Both putative recombinants originated from geographic regions where multiple genotypes are known to cocirculate. A search of the sequence databases showed evidence of similar intergenotypic recombinants. These observations indicate that recombination between divergent strains may represent an important source of genetic variation in HBV.
View details for Web of Science ID A1996UC69800005
View details for PubMedID 8919861