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Current therapies for chronic inflammatory diseases often abrogate the immune functions required to fight infections. Human cathelicidin host defence peptide (HDP) LL-37 selectively suppresses pathogen-induced inflammation, without compromising resistance to infections. These unique dual abilities of LL-37 make it a promising candidate as an alternative therapeutic for treating chronic inflammatory diseases. The objective of this study was to investigate the effects of LL-37 and its derivative peptide IG-19 in cytokine-mediated inflammation. I demonstrated that LL-37 and IG-19 selectively suppressed cytokine IL-32γ-induced pro-inflammatory cytokines, without compromising the production of anti-inflammatory cytokines, and chemokines in human PBMC and macrophages. However, significant quantitative differences between LL-37 and IG-19-mediated chemokine productions suggested that the mechanisms underlying the activity of these two peptides were different. I showed that both peptides suppressed IL-32γ-mediated phosphorylation of the Src-kinase FYN(Y420), known to enhance inflammation. Contrastingly, phosphorylation of the dual phosphatase MKP-1(S359), a negative regulator of inflammation, was enhanced in response to both peptides. Similarly, both peptides increased the activity of p44/42MAPK, which phosphorylates and stabilizes MKP-1. These results suggested that MKP-1 may be a critical mediator of the immunomodulatory activity of these peptides. Bioinformatic interrogation revealed that direct interacting protein partners of MKP-1 were overrepresented in MAPK and NF-κB signalling pathways. Both peptides enhanced the phosphorylation of p38MAPK. However, contrasting to LL-37, IG-19 did not mediate the phosphorylation of JNK MAPK and IKK-α signaling intermediates involved in inflammation. This was consistent with observations that chemokine production was significantly lower in response to IG-19 compared to LL-37. These results suggested that IG-19 may be a better immunomodulatory therapeutic candidate compared to LL-37. As cytokine-mediated inflammation plays critical roles in the disease pathogenesis of inflammatory arthritis, I examined the effects of exogenous administration of IG-19 in a murine model of collagen-induced arthritis. Administration of IG-19 decreased disease severity, suppressed pro-inflammatory cytokines and anti-collagen antibodies, and mitigated cartilage destruction in the CIA mice. These results provide a rationale to further develop IG-19 as a therapeutic agent for chronic inflammatory arthritis. The advantage of HDP based therapy is the potential to control inflammation without compromising the patient’s ability to resolve infections. / May 2017

Although inflammation is a crucial mechanism in response to injury and pathogen clearance, inappropriate or excessive induction of the inflammatory response in pregnancy can cause initiation of the labour cascade and subsequent preterm delivery. Host Defence Peptides (HDPs) have important anti-microbial properties but are also implicated as multifunctional modulators of immunity and infection. They are predominantly secreted by mucosal epithelial cells and released by leukocytes. The specific HDPs that are the focus of this thesis are Human beta-defensin 3 (hBD3) and Human Cathelicidin (hCAP-18/LL-37). The immunomodulatory effect of HDPs in reproductive tissues in response to infection/inflammation has not been well studied. In a pregnant state, the hypothesis of this thesis is that HDPs have a dual role in preventing ascending infection, but also preventing an exacerbated inflammatory response that can cause preterm birth by initiation of the labour cascade. To explore this I determine whether bacterial stimuli can regulate HDPs expression in pregnancy tissues. I also explore what interactions HDPs have on the production/induction of important cytokines that are vital to the inflammatory response. With the aid of HDP knockout mice, the role of these peptides in infection/inflammation and continuation of pregnancy is investigated in a mouse-model of induced preterm-labour. To understand how ascending infection might be controlled by HDPs in pregnancy, I explore how HDPs regulate commensal and pathogenic bacteria. This is achieved by interrogating the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene and next generation sequencing. Results Placental explants respond to Lipopolysaccharide (LPS) challenge by increasing production of pro-inflammatory cytokines. LL-37 but not hBD3 peptide was able to modulate this inflammation by inhibiting the release of these pro-inflammatory cytokines. To establish whether HDPs are critical in the continuation of pregnancy I use a LPS induced mouse–model of preterm labour in animals lacking the genes for the HDPs, Defb14 (Defb14-/-), or Camp (Camp-/-). Intrauterine injection of LPS induced preterm labour in wildtype mice. However, the Defb14-/- and Camp-/- mice do not have an increased rate of preterm labour. Key inflammatory mediators are increased in response to LPS-induced PTL. Camp-/- animals have a similar inflammatory response to wildtype mice when given LPS during pregnancy. To understand how ascending infection might be controlled by HDPs, I interrogated the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene. I established a workflow for 16S rRNA gene sequencing on next-generation sequencing platforms and a bioinformatic pipeline for data analysis. Using this approach I was able to show the mucosal microbiome of Camp-/- animals were significantly different to that of wildtype controls, showing increased diversity in the microbes present. In murine pregnancy, there were very little global cumulative or progressive shifts in bacteria, with the exception of Candidatus arthromitus, which significantly increases with gestation compared to non-pregnancy This thesis has demonstrated that Host Defence Peptides are expressed in pregnancy tissues and have anti-inflammatory properties in response to bacterial stimuli. It is not clear whether the HDPs, hBD3 and LL-37 are fundamental to the immune defence in pregnancy by preventing excessive inflammation, Although, I have shown LL-37 may have a role in modulation of the maternal microbiota.

The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics.

The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics.

The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Cathelicidins are cationic host defence peptides (CHDP) with essential roles in the innate defence system. These peptides have antimicrobial potential and the capacity to modulate innate immunity and inflammatory processes. Neutrophils (PMN) are the main reservoir of cathelicidins and play key roles in first line defence against infection. The appropriate regulation of PMN function, death, and clearance is critical to innate immunity, and the efferocytosis of apoptotic PMN, in contrast to necrotic cells, is proposed to promote the resolution of inflammation. In this thesis I demonstrate that the human cathelicidin LL-37 rapidly induced secondary necrosis of apoptotic human PMN and identify the essential C-terminal region of LL-37 required for this activity. In addition to the induction of secondary necrosis, higher concentrations of LL-37 also promoted PMN granule contents release. LL-37-induced secondary necrosis did not affect PMN ingestion by human monocyte-derived macrophages and, in contrast to expectation, was not proinflammatory. Interestingly, the anti-inflammatory effects of apoptotic PMN on activated macrophages were retained and even potentiated where LL-37-mediated secondary necrosis induced anti-inflammatory granule content release. Consistent with the results of in vitro studies, in vivo murine sterile peritonitis model revealed the same phenomenon: LL-37-induced secondary necrosis diminished inflammatory responses with decreased PMN influx. I also present data on LL-37- mediated modulation of innate immune effector cell cytokines responses to inflammatory signals. I propose that during acute inflammation LL-37 can modulate innate immune responses through its activity on cytokine production, and that LL-37-mediated secondary necrosis of apoptotic PMN has anti-inflammatory effects, but may also mediate host damage by promoting the release of potentially harmful intracellular contents under chronic or dysregulated conditions.

Properties of Escherichia coli considered to be of importance in overcoming host defence mechanisms against urinary tract infection (OTI) were investigated. These were: 1) 0 and H serotype 2) K antigen content 3) Sensitivity to the bactericidal activity of human serum 4) Haemolysin production 5) Fimbriae production 6) Fermentation of sucrose, salicin and dulcitol 7) Sensitivity to serine, spermine and urea 8) Growth requirements 9) Mucinase production Ecoli strains isolated from normal subjects and patients attending the Nephrourological Clinic at St. Bartholomew's Hospital because of known or suspected UTI, were studied. Strains isolated from urines more frequently belonged to 0 serogroups 2,4,6,8,18ab and 75, had high K antigen titres, were haemolytic and fimbriate, and fermented salicin than periurethral strains from normal subjects. These findings support the concept of "special pathogenicity", that certain strains are more invasive for the urinary tract than others. Strains rich in these "pathogenic properties" were rarely isolated from normal subjects but were significantly more frequently isolated from periurethral swabs of patients. Periurethral strains from symptomatic, abacteriuric (urethral syndrome) patients were similar to those from bacteriuric patients when they were between infections. Previous work has not implicated bacteria in the aetiology of most cases of this disease and this finding remains unexplained. Strains isolated from the upper tracts of patients undergoing localisation tests more frequently exhibited pathogenic properties than those isolated from only the lower tract, and this was considered to reflect the superior ability of these strains to reach the upper tract or better combat host defence mechanisms.

Neutrophils are one of the main effector cells of innate immunity and were shown to kill bacteria by phagocytosis more than 100 years ago. Neutrophils are also capable of antimicrobial activity by producing extracellular structures named neutrophil extracellular traps (NETs). This thesis is an investigation of porcine neutrophils and their ability to produce NETs, as well as the antimicrobial ability of secretions from activated porcine neutrophils in combating a variety of common porcine pathogens. Porcine neutrophils were found to produce NET-like structures, and secretions from activated neutrophils were found to possess variable bactericidal activity against common pathogens of swine. Antimicrobial proteins dependent on elastase activity were shown to be partially responsible for the bactericidal activities of activated neutrophils. Several antimicrobial proteins and peptides were identified via proteomic techniques. This work allows for better understanding of innate immunity in swine, and identification of potential targets for addressing porcine health. / Ontario Ministry of Agriculture Food & Rural Affairs, Ontario Pork, Natural Sciences and Engineering Research Council of Canada

Yes / More than 2 million people/year suffer a bone fracture in the UK1. Reconstruction of bone defects represents a major clinical challenge and is addressed using a number of medical devices. Although medical device compositions and applications may differ widely, all attract microorganisms and represent niches for medical device associated infections. For open fractures, the risk of infection can be 55%2. These infections are often resistant to many of the currently available antibiotics and represent a huge and growing financial and healthcare burden. The aim of this study was a fundamental understanding of how the presence of host defence peptides (HDPs)3 and/or RGD can influence the outcome of cell vs. bacterial viability and proliferation. / Presented at the conference: eCM XVI - Bone and Implant Infection June 24-26, 2015, Convention Centre, Davos Platz, Switzerland.