During my PhD research at Ghent University I studied the interaction between bacteriophages and the human immune response. It is known that the oral administration of phages to animals results in the translocation of phages to systemic tissues, whereby phages are able to cross cell monolayers. This suggests that mammals have mechanisms for the uptake and delivery of phages that may allow intestinal phages to elicit innate and adaptive immune responses.
If phages are present everywhere, the question can be asked whether they interact with our immune system. More specifically do they have anti-inflammatory properties. Otherwise how can they work systemically? These are questions I was trying to answer during my PhD, under the supervision of Prof. Mario Vaneechoutte and Prof. Rob Lavigne. We found that our studied phage have indications that they induce an immune response. Not only do the phages that we studied induce pro-inflammatory properties, we have indications that they might induce anti-inflammatory responses as well.
During my research I developed a profound interest in phage biology and phage therapy, more specifically in the interaction between phages and the mammalian host. I’m amazed by the possibilities of science, and fascinated by what is still to be discovered.
I'm looking forward to further elucidate the interaction of phages with the human immune response, and the potential impacts this could have on phage therapy or human health.
Doctor of Science, Universiteit Gent (2017)
Current Research and Scholarly Interests
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
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
Pro- and anti-inflammatory responses of peripheral blood mononuclear cells induced by Staphylococcus aureus and Pseudomonas aeruginosa phages.
2017; 7 (1): 8004
The ability of bacteriophages to kill bacteria is well known, as is their potential use as alternatives to antibiotics. As such, bacteriophages reach high doses locally through infection of their bacterial host in the human body. In this study we assessed the gene expression profile of peripheral blood monocytes from six donors for twelve immunity-related genes (i.e. CD14, CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, IL10, LYZ, SOCS3, TGFBI and TNFA) induced by Staphylococcus aureus phage ISP and four Pseudomonas aeruginosa phages (i.e. PNM, LUZ19, 14-1 and GE-vB_Pae-Kakheti25). The phages were able to induce clear and reproducible immune responses. Moreover, the overall immune response was very comparable for all five phages: down-regulation of LYZ and TGFBI, and up-regulation of CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, SOCS3 and TNFA. The observed immune response was shown to be endotoxin-independent and predominantly anti-inflammatory. Addition of endotoxins to the highly purified phages did not cause an immune response comparable to the one induced by the (endotoxin containing) phage lysate. In addition, the use of an intermediate level of endotoxins tipped the immune response to a more anti-inflammatory response, i.e. up-regulation of IL1RN and a strongly reduced expression of CXCL1 and CXCL5.
View details for PubMedID 28808331
View details for PubMedCentralID PMC5556114
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
Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells.
2020; 15 (2): e0229423
RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (NucliSENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit-blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs.
View details for DOI 10.1371/journal.pone.0229423
View details for PubMedID 32084228
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
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
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
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
Development of a qPCR platform for quantification of the five bacteriophages within bacteriophage cocktail 2 (BFC2).
2019; 9 (1): 13893
To determine phage titers accurately, reproducibly and in a non-laborious and cost-effective manner, we describe the development of a qPCR platform for molecular quantification of five phages present in bacteriophage cocktail 2 (BFC2). We compared the performance of this molecular approach, with regard to quantification and reproducibility, with the standard culture-based double agar overlay method (DAO). We demonstrated that quantification of each of the five phages in BFC2 was possible by means of qPCR, without prior DNA extraction, but yields were significantly higher in comparison to DAO. Although DAO is assumed to provide an indication of the number of infective phage particles, whereas qPCR only provides information on the number of phage genomes, the difference in yield (qPCR/DAO ratio) was observed to be phage-dependent and appeared rather constant for all phages when analyzing different (freshly prepared) stocks of these phages. While DAO is necessary to determine sensitivity of clinical strains against phages in clinical applications, qPCR might be a valid alternative for rapid and reproducible quantification of freshly prepared stocks, after initial establishment of a correction factor towards DAO.
View details for DOI 10.1038/s41598-019-50461-0
View details for PubMedID 31554892
- Phage Interaction with the Mammalian Immune System PHAGE THERAPY: A PRACTICAL APPROACH 2019: 91–122
- Interactions between Bacteriophage, Bacteria, and the Mammalian Immune System VIRUSES-BASEL 2019; 11 (1)
- Interactions between Bacteriophage, Bacteria, and the Mammalian Immune System Phage Therapy: A Practical Approach 2019
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
A comparative study of different strategies for removal of endotoxins from bacteriophage preparations
JOURNAL OF MICROBIOLOGICAL METHODS
2017; 132: 153-159
Bacterial endotoxins have high immunogenicity. Phage biology studies as well as therapeutic phage applications necessitate highly purified phage particles. In this study, we compared combinations of seven different endotoxin removal strategies and validated their endotoxin removal efficacy for five different phages (i.e. four Pseudomonas aeruginosa phages and one Staphylococcus aureus phage). These purification strategies included Endotrap HD column purification and/or CsCl density centrifugation in combination with Endotrap purification, followed by organic solvent (1-octanol), detergent (Triton X-100), enzymatic inactivation of the endotoxin using alkaline phosphatase and CIM monolytic anion exchange chromatography. We show that CsCl density purification of the P. aeruginosa phages, at an initial concentration of 1012-1013pfu/ml, led to the strongest reduction of endotoxins, with an endotoxin removal efficacy of up to 99%, whereas additional purification methods did not result in a complete removal of endotoxins from the phage preparations and only yielded an additional endotoxin removal efficacy of 23 to 99%, sometimes accompanied with strong losses in phage titer.
View details for DOI 10.1016/j.mimet.2016.11.020
View details for Web of Science ID 000393017100027
View details for PubMedID 27913133
Stability of bacteriophages in burn wound care products.
2017; 12 (7): e0182121
Bacteriophages could be used along with burn wound care products to enhance antimicrobial pressure during treatment. However, some of the components of the topical antimicrobials that are traditionally used for the prevention and treatment of burn wound infection might affect the activity of phages. Therefore, it is imperative to determine the counteraction of therapeutic phage preparations by burn wound care products before application in patients. Five phages, representatives of two morphological families (Myoviridae and Podoviridae) and active against 3 common bacterial burn wound pathogens (Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus) were tested against 13 different products commonly used in the treatment of burn wounds. The inactivation of the phages was quite variable for different phages and different products. Majority of the anti-infective products affected phage activity negatively either immediately or in the course of time, although impact was not always significant. Products with high acidity had the most adverse effect on phages. Our findings demonstrate that during combined treatment the choice of phages and wound care products must be carefully defined in advance.
View details for PubMedID 28750102
View details for PubMedCentralID PMC5531522
Haemophilus influenzae biofilm formation in chronic otitis media with effusion
EUROPEAN ARCHIVES OF OTO-RHINO-LARYNGOLOGY
2016; 273 (11): 3553-3560
Otitis media with effusion (OME) is a highly prevalent disease in children, but the exact pathogenesis and role of bacteria are still not well understood. This study aimed to investigate the presence of otopathogenic bacteria in the middle ear effusion (MEE) and adenoid of children with chronic OME (COME), and to investigate in vivo whether these bacteria, especially Haemophilus influenzae, are organized as a biofilm in the middle ear fluid. MEE and adenoid samples were collected from 21 patients with COME. Extensive bacterial culturing and genotyping was performed on all middle ear and adenoid samples. Fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) was used to visualize possible biofilm structures for a selection of middle ear effusion samples. 34 MEE samples were collected from 21 patients of which 64.7 % were culture positive for bacteria and 47.0 % were culture positive for Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and/or Streptococcus pneumoniae. All 21 adenoid samples were culture positive for one or more of these four otopathogens. H. influenzae (35.3 %) and S. pneumoniae (76.2 %) were the most frequently cultured bacteria in the MEE and adenoid samples, respectively. The same bacterial species was found in MEE and adenoid for 84.6 % of the patients and in 81.2 % of the cases where the same species was found in more than one site it involved the same bacterial genotype. FISH and CLSM demonstrated the presence of H. influenzae specific biofilm structures in five of the eight culture positive MEEs that were tested, but in none of the two culture negative MEEs. The findings in this study indicate that the adenoid acts as a reservoir for bacteria in MEE and confirms that biofilms, in at least half of the cases consisting of H. influenzae, are indeed present in the MEE of children with COME. Biofilms may thus play a crucial role in the pathogenesis of COME, which is important in the understanding of this disease and the development of potential future treatment options.
View details for DOI 10.1007/s00405-016-3958-9
View details for Web of Science ID 000385202600010
View details for PubMedID 26946303