All Publications

  • Bacteriophage populations mirror those of bacterial pathogens at sites of infection. mSystems Haddock, N. L., Barkal, L. J., Bollyky, P. L. 2023: e0049723


    Bacteriophages, viruses that parasitize bacteria, are known to be abundant at sites of bacterial colonization, but the relationship between phages and bacteria at sites of infection is unclear. Bacteriophages are highly specific to their bacterial host species, and so we hypothesize that phage populations would mirror those of bacterial pathogens within infected tissues. To test this, here we study publicly available cell-free DNA (cfDNA) generated using next-generation sequencing of infected bodily fluids, including urine, joint fluid, peritoneal fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, and abscess fluid, as well as uninfected control samples. These were analyzed using a computational pipeline for identifying bacteriophage sequences in cfDNA. We find that bacteriophage sequences are present in both infected and uninfected bodily fluids and represent a variety of bacteriophage morphologies and bacterial hosts. Additionally, phages from Escherichia coli, Streptococcus, and Staphylococcus aureus are overrepresented both in terms of proportion and diversity in fluids infected with these same pathogens. These data indicate that phages reflect the relative abundance of their bacterial hosts at sites of infection. Bacteriophage sequences may help inform future investigative and diagnostic approaches that utilize cell-free DNA to study the microbiome within infected tissues. IMPORTANCE Bacteriophages are an active area of investigation in microbiome research, but most studies have focused on phage populations at sites of bacterial colonization. Little is known about bacteriophage ecology at sites of active infection. To address this gap in knowledge, we utilized a publicly available data set to study bacteriophage populations in cell-free DNA collected from sites of infection. We find that phages reflect the relative abundance of their bacterial hosts at sites of infection. These studies may lead to future investigative and diagnostic approaches that incorporate phages as well as bacterial cell-free DNA.

    View details for DOI 10.1128/msystems.00497-23

    View details for PubMedID 37526425

  • Bacteriophage DNA in blood provides species-level insight into bacterial infections Nature Microbiology Haddock, N. L., Bollyky, P. L. 2023

    View details for DOI 10.1038/s41564-023-01422-x

    View details for PubMedCentralID 7196304

  • Phage diversity in cell-free DNA identifies bacterial pathogens in human sepsis cases. Nature microbiology Haddock, N. L., Barkal, L. J., Ram-Mohan, N., Kaber, G., Chiu, C. Y., Bhatt, A. S., Yang, S., Bollyky, P. L. 2023


    Bacteriophages, viruses that infect bacteria, have great specificity for their bacterial hosts at the strain and species level. However, the relationship between the phageome and associated bacterial population dynamics is unclear. Here we generated a computational pipeline to identify sequences associated with bacteriophages and their bacterial hosts in cell-free DNA from plasma samples. Analysis of two independent cohorts, including a Stanford Cohort of 61 septic patients and 10 controls and the SeqStudy cohort of 224 septic patients and 167 controls, reveals a circulating phageome in the plasma of all sampled individuals. Moreover, infection is associated with overrepresentation of pathogen-specific phages, allowing for identification of bacterial pathogens. We find that information on phage diversity enables identification of the bacteria that produced these phages, including pathovariant strains of Escherichia coli. Phage sequences can likewise be used to distinguish between closely related bacterial species such as Staphylococcus aureus, a frequent pathogen, and coagulase-negative Staphylococcus, a frequent contaminant. Phage cell-free DNA may have utility in studying bacterial infections.

    View details for DOI 10.1038/s41564-023-01406-x

    View details for PubMedID 37308590

    View details for PubMedCentralID 5594678

  • Reprogramming Cancer into Antigen Presenting Cells as a Novel Immunotherapy. Cancer discovery Linde, M. H., Fan, A. C., Kohnke, T., Trotman-Grant, A. C., Gurev, S. F., Phan, P., Zhao, F., Haddock, N. L., Nuno, K. A., Gars, E. J., Stafford, M., Marshall, P. L., Dove, C. G., Linde, I. L., Landberg, N., Miller, L. P., Majzner, R. G., Zhang, T. Y., Majeti, R. 2023


    Therapeutic cancer vaccination seeks to elicit activation of tumor-reactive T cells capable of recognizing tumor-associated antigens (TAAs) and eradicating malignant cells. Here, we present a cancer vaccination approach utilizing myeloid lineage reprogramming to directly convert cancer cells into tumor reprogrammed-antigen presenting cells (TR-APCs). Using syngeneic murine leukemia models, we demonstrate that TR-APCs acquire both myeloid phenotype and function, process and present endogenous TAAs, and potently stimulate TAA-specific CD4+ and CD8+ T cells. In vivo TR-APC induction elicits clonal expansion of cancer-specific T cells, establishes cancer-specific immune memory, and ultimately promotes leukemia eradication. We further show that both hematologic cancers and solid tumors, including sarcomas and carcinomas, are amenable to myeloid-lineage reprogramming into TR-APCs. Finally, we demonstrate the clinical applicability of this approach by generating TR-APCs from primary clinical specimens and stimulating autologous patient-derived T cells. Thus, TR-APCs represent a cancer vaccination therapeutic strategy with broad implications for clinical immuno-oncology.

    View details for DOI 10.1158/2159-8290.CD-21-0502

    View details for PubMedID 36856575

  • Hyaluronan in the Pathogenesis of Acute and Post-Acute COVID-19 Infection. Matrix biology : journal of the International Society for Matrix Biology Barnes, H. W., Demirdjian, S., Haddock, N. L., Kaber, G., Martinez, H. A., Nagy, N., Karmouty-Quintana, H., Bollyky, P. L. 2023


    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently emerged as the cause of a global pandemic. Infection with SARS-CoV-2 can result in COVID-19 with both acute and chronic disease manifestations that continue to impact many patients long after the resolution of viral replication. There is therefore great interest in understanding the host factors that contribute to COVID-19 pathogenesis. In this review, we address the role of hyaluronan (HA), an extracellular matrix polymer with roles in inflammation and cellular metabolism, in COVID-19 and critically evaluate the hypothesis that HA promotes COVID-19 pathogenesis. We first provide a brief overview of COVID-19 infection. Then we briefly summarize the known roles of HA in airway inflammation and immunity. We then address what is known about HA and the pathogenesis of COVID-19 acute respiratory distress syndrome (COVID-19 ARDS). Next, we examine potential roles for HA in post-acute SARS-CoV-2 infection (PASC), also known as "long COVID" as well as in COVID-associated fibrosis. Finally, we discuss the potential therapeutics that target HA as a means to treat COVID-19, including the repurposed drug hymecromone (4-methylumbelliferone). We conclude that HA is a promising potential therapeutic target for the treatment of COVID-19.

    View details for DOI 10.1016/j.matbio.2023.02.001

    View details for PubMedID 36750167

  • Bacteriophage and Bacterial Susceptibility, Resistance, and Tolerance to Antibiotics. Pharmaceutics Chen, Q., Dharmaraj, T., Cai, P. C., Burgener, E. B., Haddock, N. L., Spakowitz, A. J., Bollyky, P. L. 2022; 14 (7)


    Bacteriophages, viruses that infect and replicate within bacteria, impact bacterial responses to antibiotics in complex ways. Recent studies using lytic bacteriophages to treat bacterial infections (phage therapy) demonstrate that phages can promote susceptibility to chemical antibiotics and that phage/antibiotic synergy is possible. However, both lytic and lysogenic bacteriophages can contribute to antimicrobial resistance. In particular, some phages mediate the horizontal transfer of antibiotic resistance genes between bacteria via transduction and other mechanisms. In addition, chronic infection filamentous phages can promote antimicrobial tolerance, the ability of bacteria to persist in the face of antibiotics. In particular, filamentous phages serve as structural elements in bacterial biofilms and prevent the penetration of antibiotics. Over time, these contributions to antibiotic tolerance favor the selection of resistance clones. Here, we review recent insights into bacteriophage contributions to antibiotic susceptibility, resistance, and tolerance. We discuss the mechanisms involved in these effects and address their impact on bacterial fitness.

    View details for DOI 10.3390/pharmaceutics14071425

    View details for PubMedID 35890320

  • Leishmania donovani infection suppresses Allograft Inflammatory Factor-1 in monocytes and macrophages to inhibit inflammatory responses. Scientific reports da Silva, R. L., Elizondo, D. M., Brandy, N. Z., Haddock, N. L., Boddie, T. A., de Oliveira, L. L., de Jesus, A. R., de Almeida, R. P., de Moura, T. R., Lipscomb, M. W. 2021; 11 (1): 946


    Macrophages and monocytes are important for clearance of Leishmania infections. However, immune evasion tactics employed by the parasite results in suppressed inflammatory responses, marked by deficient macrophage functions and increased accumulation of monocytes. This results in an ineffective ability to clear parasite loads. Allograft Inflammatory Factor-1 (AIF1) is expressed in myeloid cells and serves to promote immune responses. However, AIF1 involvement in monocyte and macrophage functions during parasitic infections has not been explored. This study now shows that Leishmania donovani inhibits AIF1 expression in macrophages to block pro-inflammatory responses. Mice challenged with the parasite had markedly reduced AIF1 expression in splenic macrophages. Follow-up studies using in vitro approaches confirmed that L. donovani infection in macrophages suppresses AIF1 expression, which correlated with reduction in pro-inflammatory cytokine production and increased parasite load. Ectopic overexpression of AIF1 in macrophages provided protection from infection, marked by robust pro-inflammatory cytokine production and efficient pathogen clearance. Further investigations found that inhibiting AIF1 expression in bone marrow cells or monocytes impaired differentiation into functional macrophages. Collectively, results show that AIF1 is a critical regulatory component governing monocyte and macrophage immune functions and that L. donovani infection can suppress the gene as an immune evasion tactic.

    View details for DOI 10.1038/s41598-020-79068-6

    View details for PubMedID 33441583

  • Filamentous Bacteriophages and the Competitive Interaction between Pseudomonas aeruginosa Strains under Antibiotic Treatment: a Modeling Study. mSystems Pourtois, J. D., Kratochvil, M. J., Chen, Q., Haddock, N. L., Burgener, E. B., De Leo, G. A., Bollyky, P. L. 2021: e0019321


    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

  • Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection. Matrix biology : journal of the International Society for Matrix Biology Marshall, P. L., Nagy, N. n., Kaber, G. n., Barlow, G. L., Ramesh, A. n., Xie, B. J., Linde, M. H., Haddock, N. L., Lester, C. A., Tran, Q. L., de Vries, C. n., Hargil, A. n., Malkovskiy, A. n., Gurevich, I. n., Martinez, H. A., Kuipers, H. F., Yadava, K. n., Zhang, X. n., Evanko, S. P., Gebe, J. A., Wang, X. n., Vernon, R. B., de la Motte, C. n., Wight, T. N., Engleman, E. G., Krams, S. M., Meyer, E. n., Bollyky, P. L. 2020


    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

  • Pf Bacteriophage and Their Impact on Pseudomonas Virulence, Mammalian Immunity, and Chronic Infections. Frontiers in immunology Secor, P. R., Burgener, E. B., Kinnersley, M. n., Jennings, L. K., Roman-Cruz, V. n., Popescu, M. n., Van Belleghem, J. D., Haddock, N. n., Copeland, C. n., Michaels, L. A., de Vries, C. R., Chen, Q. n., Pourtois, J. n., Wheeler, T. J., Milla, C. E., Bollyky, P. L. 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

  • Allograft Inflammatory Factor-1 Governs Hematopoietic Stem Cell Differentiation Into cDC1 and Monocyte-Derived Dendritic Cells Through IRF8 and RelB in vitro. Frontiers in immunology Elizondo, D. M., Brandy, N. Z., da Silva, R. L., Haddock, N. L., Kacsinta, A. D., de Moura, T. R., Lipscomb, M. W. 2019; 10: 173


    The multistep differentiation process from hematopoietic stem cells through common myeloid progenitors into committed dendritic cell (DC) subsets remains to be fully addressed. These studies now show that Allograft Inflammatory Factor-1 (AIF1) is required for differentiation of classical DC type 1 (cDC1) subsets and monocyte-derived DC (Mo-DC). Phenotypic studies found that AIF1 expression increased in committed subsets differentiating from common myeloid progenitors (CMP). However, silencing AIF1 expression in hematopoietic stem progenitors restrained the capacity to differentiate into Mo-DC and cDC1 cell subsets under GM-CSF or Flt3-L stimuli conditions, respectively. This was further marked by restrained expression of IRF8, which is critical for development of Mo-DC and cDC1 subsets. As a result, absence of AIF1 restrained the cells at the Lin-CD117+FcγR-CD34+ CMP stage. Further biochemical studies revealed that abrogating AIF1 resulted in inhibition of the NFκB family member RelB expression and p38 MAPK phosphorylation during differentiation of Mo-DC. Lastly, protein binding studies identified that AIF1 interacts with protein kinase C (PKC) to influence downstream signaling pathways. Taken together, this is the first report showing a novel role of AIF1 as a calcium-responsive scaffold protein that supports IRF8 expression and interacts with PKC to drive NFκB-related RelB for successfully differentiating hematopoietic progenitor cells into cDC and Mo-DC subsets under Flt3-L and GM-CSF stimuli, respectively.

    View details for DOI 10.3389/fimmu.2019.00173

    View details for PubMedID 30800127

    View details for PubMedCentralID PMC6375893

  • Drebrin 1 in dendritic cells regulates phagocytosis and cell surface receptor expression through recycling for efficient antigen presentation IMMUNOLOGY Elizondo, D. M., Andargie, T. E., Haddock, N. L., Boddie, T. A., Lipscomb, M. W. 2019; 156 (2): 136–46


    Phagocytosis, macropinocytosis and antigen presentation by dendritic cells (DC) requires reorganization of the actin cytoskeleton. Drebrin (Dbn1) is an actin binding and stabilizing protein with roles in endocytosis, formation of dendrite spines in neurons and coordinating cell-cell synapses in immune cells. However, its role in DC phagocytosis and antigen presentation is unknown. These studies now report that silencing of Dbn1 in DC resulted in restrained cell surface display of receptors, most notably MHC class I and II and co-stimulatory molecules. This, as expected, resulted in impaired antigen-specific T-cell activation and proliferation. Studies additionally revealed that knockdown of Dbn1 in DC impaired macropinocytosis and phagocytosis. However, there was a concomitant increase in fluid-phase uptake, suggesting that Dbn1 is responsible for the differential control of macropinocytosis versus micropinocytosis activities. Taken together, these findings now reveal that Dbn1 plays a major role in coordinating the actin cytoskeletal activities responsible for antigen presentation in DC.

    View details for DOI 10.1111/imm.13010

    View details for Web of Science ID 000455609300005

    View details for PubMedID 30317558

    View details for PubMedCentralID PMC6328995

  • Filamentous bacteriophages are associated with chronic Pseudomonas lung infections and antibiotic resistance in cystic fibrosis. Science translational medicine Burgener, E. B., Sweere, J. M., Bach, M. S., Secor, P. R., Haddock, N. n., Jennings, L. K., Marvig, R. L., Johansen, H. K., Rossi, E. n., Cao, X. n., Tian, L. n., Nedelec, L. n., Molin, S. n., Bollyky, P. L., Milla, C. E. 2019; 11 (488)


    Filamentous bacteriophage (Pf phage) contribute to the virulence of Pseudomonas aeruginosa infections in animal models, but their relevance to human disease is unclear. We sought to interrogate the prevalence and clinical relevance of Pf phage in patients with cystic fibrosis (CF) using sputum samples from two well-characterized patient cohorts. Bacterial genomic analysis in a Danish longitudinal cohort of 34 patients with CF revealed that 26.5% (n = 9) were consistently Pf phage positive. In the second cohort, a prospective cross-sectional cohort of 58 patients with CF at Stanford, sputum qPCR analysis showed that 36.2% (n = 21) of patients were Pf phage positive. In both cohorts, patients positive for Pf phage were older, and in the Stanford CF cohort, patients positive for Pf phage were more likely to have chronic P. aeruginosa infection and had greater declines in pulmonary function during exacerbations than patients negative for Pf phage presence in the sputum. Last, P. aeruginosa strains carrying Pf phage exhibited increased resistance to antipseudomonal antibiotics. Mechanistically, in vitro analysis showed that Pf phage sequesters these same antibiotics, suggesting that this mechanism may thereby contribute to the selection of antibiotic resistance over time. These data provide evidence that Pf phage may contribute to clinical outcomes in P. aeruginosa infection in CF.

    View details for PubMedID 30996083

  • IL-10 producing CD8(+) CD122(+) PD-1(+) regulatory T cells are expanded by dendritic cells silenced for Allograft Inflammatory Factor-1 JOURNAL OF LEUKOCYTE BIOLOGY Elizondo, D. M., Andargie, T. E., Haddock, N. L., Louzada da Silva, R. L., de Moura, T., Lipscomb, M. W. 2019; 105 (1): 123–30


    Allograft Inflammatory Factor-1 (AIF1) is a cytoplasmic scaffold protein that contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes in immune cells. The protein plays a dominant role in both macrophage- and dendritic cell (DC)-mediated inflammatory responses. This study now reports that AIF1 expression in DC is important in directing CD8+ T cell effector responses. Silencing AIF1 expression in murine CD11c+ DC suppressed antigen-specific CD8+ T cell activation, marked by reduced CXCR3, IFNγ and Granzyme B expression, and restrained proliferation. These primed CD8+ T cells had impaired cytotoxic killing of target cells in vitro. In turn, studies identified that AIF1 silencing in DC robustly expanded IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells that suppressed neighboring immune effector responses through both IL-10 and PD-1-dependent mechanisms. In vivo studies recapitulated bystander suppression of antigen-responsive CD4+ T cells by the CD8+ Tregs expanded from the AIF1 silenced DC. These studies further demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and present a novel target for engineering tolerogenic DC-based immunotherapies.

    View details for DOI 10.1002/JLB.1A0118-010RR

    View details for Web of Science ID 000454409400012

    View details for PubMedID 30512224

    View details for PubMedCentralID PMC6310075