Investigating the role of Fas (CD95) signalling in the modification of innate immune induced inflammation

Background/Aim: It has been demonstrated that a number of pathologies occur as a result of dysregulation of the immune system. Whilst classically associated with apoptosis, the Fas (CD95) signalling pathway plays a role in inflammation. Studies have demonstrated that Fas activation augments TLR4-mediated MyD88-dependent cytokine production. Studies have also shown that the Fas adapter protein FADD is required for RIG-I-induced IFNβ production. As a similar signalling pathway exists between RIG-I, TLR3 and the MyD88- independent of TLR4, we hypothesised that Fas activation may modulate both TLR3- and TLR4-induced cytokine production. Results: Fas activation reduced poly I:C-induced IFNβ, IL-8, IL-10 and TNFα production whilst augmenting poly I:C-, poly A:U- and Sendai virus-induced IP-10 production. TLR3-, RIG-I- and MDA5-induced IP-10 luciferase activation were inhibited by the Fas adapter protein FADD using overexpression studies. Poly I:C-induced phosphorylation of p-38 and JNK MAPK were reduced by Fas activation. Overexpression of FADD induced AP-1 luciferase activation. Point mutations in the AP-1 binding site enhanced poly I:C-induced IP- 10 production. LPS-induced IL-10, IL-12, IL-8 and TNFα production were enhanced by Fas activation, whilst reducing LPS-induced IFNβ production. Absence of FADD using FADD-/- MEFs resulted in impaired IFNβ production. Overexpression studies using FADD augmented TLR4-, MyD88- and TRIF-induced IFNβ luciferase activation. Overexpression studies also suggested that enhanced TLR4-induced IFNβ production was independent of NFκB activation. Conclusion: Viral-induced IP-10 production is augmented by Fas activation by reducing the phosphorylation of p-38 and JNK MAPKs, modulating AP-1 activation. The Fas adapterprotein FADD is required for TLR4-induced IFNβ production. Studies presented here demonstrate that the Fas signalling pathway can therefore modulate the immune response. Our data demonstrates that this modulatory effect is mediated by its adapter protein FADD, tailoring the immune response by acting as a molecular switch. This ensures the appropriate immune response is mounted, thus preventing an exacerbated immune response.

The Innate Immune System in Acute and Chronic Wounds.

Significance: This review article provides an overview of the critical roles of the innate immune system to wound healing. It explores aspects of dysregulation of individual innate immune elements known to compromise wound repair and promote nonhealing wounds. Understanding the key mechanisms whereby wound healing fails will provide seed concepts for the development of new therapeutic approaches. Recent Advances: Our understanding of the complex interactions of the innate immune system in wound healing has significantly improved, particularly in our understanding of the role of antimicrobials and peptides and the nature of the switch from inflammatory to reparative processes. This takes place against an emerging understanding of the relationship between human cells and commensal bacteria in the skin. Critical Issues: It is well established and accepted that early local inflammatory mediators in the wound bed function as an immunological vehicle to facilitate immune cell infiltration and microbial clearance upon injury to the skin barrier. Both impaired and excessive innate immune responses can promote nonhealing wounds. It appears that the switch from the inflammatory to the proliferative phase is tightly regulated and mediated, at least in part, by a change in macrophages. Defining the factors that initiate the switch in such macrophage phenotypes and functions is the subject of multiple investigations. Future Directions: The review highlights processes that may be useful targets for further investigation, particularly the switch from M1 to M2 macrophages that appears to be critical as dysregulation of this switch occurs during defective wound healing.

Contribution of bacterial outer membrane vesicles to innate bacterial defense.

BACKGROUND: Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria throughout growth and have proposed roles in virulence, inflammation, and the response to envelope stress. Here we investigate outer membrane vesiculation as a bacterial mechanism for immediate short-term protection against outer membrane acting stressors. Antimicrobial peptides as well as bacteriophage were used to examine the effectiveness of OMV protection. RESULTS: We found that a hyper-vesiculating mutant of Escherichia coli survived treatment by antimicrobial peptides (AMPs) polymyxin B and colistin better than the wild-type. Supplementation of E. coli cultures with purified outer membrane vesicles provided substantial protection against AMPs, and AMPs significantly induced vesiculation. Vesicle-mediated protection and induction of vesiculation were also observed for a human pathogen, enterotoxigenic E. coli (ETEC), challenged with polymyxin B. When ETEC with was incubated with low concentrations of vesicles concomitant with polymyxin B treatment, bacterial survival increased immediately, and the culture gained resistance to polymyxin B. By contrast, high levels of vesicles also provided immediate protection but prevented acquisition of resistance. Co-incubation of T4 bacteriophage and OMVs showed fast, irreversible binding. The efficiency of T4 infection was significantly reduced by the formation of complexes with the OMVs. CONCLUSIONS: These data reveal a role for OMVs in contributing to innate bacterial defense by adsorption of antimicrobial peptides and bacteriophage. Given the increase in vesiculation in response to the antimicrobial peptides, and loss in efficiency of infection with the T4-OMV complex, we conclude that OMV production may be an important factor in neutralizing environmental agents that target the outer membrane of Gram-negative bacteria.

Kassorins: Novel innate immune system peptides from skin secretions of the African hyperoliid frogs, Kassina maculata and Kassina senegalensis

From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.8 Da), respectively. The primary structure and organisation of the biosynthetic precursors of kassorins M and S were deduced from cloned skin secretion-derived cDNA. Both open-reading frames encoded a single copy of kassorin M and S, respectively, located at the C-terminus. Kassorins display limited structural similarities to vespid chemotactic peptides (7/13 residues), temporin A (5/13 residues), the N-terminus of Lv-ranaspumin, a foam nest surfactant protein of the frog, Leptodactylus vastus, and an N-terminal domain of the equine sweat surfactant protein, latherin. Both peptides elicit histamine release from rat peritoneal mast cells. However, while kassorin S was found to possess antibacterial activity against Staphylococcus aureus, kassorin M was devoid of such activity. In contrast, kassorin M was found to contract the smooth muscle of guinea pig urinary bladder (EC50 = 4.66 nM) and kassorin S was devoid of this activity. Kassorins thus represent the prototypes of a novel family of peptides from the amphibian innate immune system as occurring in defensive skin secretions.

Expression of Toll-Like Receptors 2 and 4 is Upregulated During Hospital Admission in Traumatic Patients:Lack of Correlation with Blunted Innate Immune Responses

Background: There are reports with conflicting results on the expression of toll-like receptors (TLRs) in trauma patients. In addition, these studies analyzed TLR expression only at patients hospital admission but not later when complications usually arise. Objectives: To analyze the surface expression of TLR2 and TLR4 on circulating monocytes from trauma patients during the hospitalization period and to correlate this with cytokine production after stimulation with TLR2 and TLR4 agonists. The phagocytic capacity of monocytes was analyzed at the same time points of TLR expression analysis; to correlate these molecular findings with the presence or absence of infections. Methods: Prospective and observational study from June 2005 to June 2007. In all analysis, a control group composed of healthy subjects was included. Results: We studied 70 trauma patients admitted to the intensive care unit (ICU) of a tertiary hospital, and 30 healthy volunteers. Blood samples were collected at hospital admission, on day 7 and 14. Forty-four patients (63%) developed at least one episode of infection. Monocytes from trauma patients expressed higher levels of TLR2 and TLR4 than monocytes from control subjects at all time points. Expression of TLR2 and TLR4 in monocytes from those patients who developed any infection was significantly lower than in those patients without infection but still significantly higher than in control subjects. Cellular responses to TLR4 agonist were impaired. Monocytes from traumatic patients phagocytosized less efficiently than monocytes from control subjects. Conclusions: These results indicate that trauma patients present a dysregulation of the innate immune system that persists during the first 14 days after hospital admission. Copyright © 2010 by Lippincott Williams & Wilkins.

Lack of effect of glutamine administration to boost the innate immune system response in trauma patients in the intensive care unit

The use of glutamine as a dietary supplement is associated with a reduced risk of infection. We hypothesized that the underlying mechanism could be an increase in the expression and/or functionality of Toll-like receptors (TLR), key receptors sensing infections. The objective of this study was to evaluate whether glutamine supplementation alters the expression and functionality of TLR2 and TLR4 in circulating monocytes of trauma patients admitted to the intensive care unit (ICU).

Identification of the Flagellin Glycosylation System in Burkholderia cenocepacia and the Contribution of Glycosylated Flagellin to Evasion of Human Innate Immune Responses

Burkholderia cenocepacia is an opportunistic pathogen threatening patients with cystic fibrosis. Flagella are required for biofilm formation, as well as adhesion to and invasion of epithelial cells. Recognition of flagellin via the Toll-like receptor 5 (TLR5) contributes to exacerbate B. cenocepacia-induced lung epithelial inflammatory responses. In this study, we report that B. cenocepacia flagellin is glycosylated on at least 10 different sites with a single sugar, 4,6-dideoxy-4-(3-hydroxybutanoylamino)-d-glucose. We have identified key genes that are required for flagellin glycosylation, including a predicted glycosyltransferase gene that is linked to the flagellin biosynthesis cluster and a putative acetyltransferase gene located within the O-antigen lipopolysaccharide cluster. Another O-antigen cluster gene, rmlB, which is required for flagellin glycan and O-antigen biosynthesis, was essential for bacterial viability, uncovering a novel target against Burkholderia infections. Using glycosylated and nonglycosylated purified flagellin and a cell reporter system to assess TLR5-mediated responses, we also show that the presence of glycan in flagellin significantly impairs the inflammatory response of epithelial cells. We therefore suggest that flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria.

Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

Chapter Three – Host–Parasite Interactions and the Evolution of Immune Defense

Parasites and pathogens are ubiquitous and act as an important selection pressure on animals. Here, drawing primarily on our own research, mostly on insects, we illustrate how host-parasite interactions have played a role in the evolution of a range of phenomena, including animal coloration, social behavior, foraging ecology, sexual selection, and life-history tradeoffs, as well as how variation in host behavior and ecology can drive variation in parasitism risk and host allocation of resources to immunity and other antiparasite defenses. We conclude by identifying key areas for future study.

Innate immune activation in neovascular Age-related Macular Degeneration

Purpose: To compare white blood cell populations from persons with neovascular age-related macular degeneration (nAMD) with that of age-matched controls.

Methods: Immunophenotyping for white blood cell populations (including CD14++CD16-, CD14++CD16+ and CD14+CD16++ monocytes, CD4 and CD8 T-lymphocytes, CD56 natural killer cells, CD19 B-lymphocytes and CD16+HLA-DR- neutrophils), chemokine receptor expression analysis (CX3CR1 and CCR2) as well as cell activation analysis (MHC-II, HLA-DR, CD62L, STAT3) was performed using samples of peripheral blood from nAMD patients and age- and gender-matched controls.

Results: The percentage of CD4+ T cells was significantly reduced while the percentage of CD11b+ cells and CD16+HLA-DR- neutrophils was significantly increased in nAMD patients compared to controls. The percentage of classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocytes was similar between nAMD patients and controls, however there was a significant increase of CX3CR1 on the intermediate monocyte subset and on CD16+HLA-DR- neutrophils in nAMD compared to controls. HLA-DR was significantly increased in all monocyte subsets in nAMD compared to controls. Activation of Signal Transducer and Activator of Transcription 3 (STAT3) was significantly increased in nAMD patients compared to controls following stimulation with IL6.

Conclusions: Our results suggest an increased activation of the innate immune system in patients with nAMD. A better understanding of the role of the innate immune system in the pathogenesis of nAMD may help identify novel biomarkers and thus development of improved therapeutic strategies.

The effect of a selective CXCR2 antagonist (AZD5069) on human blood neutrophil count and innate immune functions.

Abstract AIMS: The aim of the present study was to investigate whether selective antagonism of the cysteine-X-cysteine chemokine receptor-2 (CXCR2) receptor has any adverse effects on the key innate effector functions of human neutrophils for defence against microbial pathogens. METHODS: In a double-blind, crossover study, 30 healthy volunteers were randomized to treatment with the CXCR2 antagonist AZD5069 (100 mg) or placebo, twice daily orally for 6 days. The peripheral blood neutrophil count was assessed at baseline, daily during treatment and in response to exercise challenge and subcutaneous injection of granulocyte-colony stimulating factor (G-CSF). Neutrophil function was evaluated by phagocytosis of Escherichia coli and by the oxidative burst response to E. coli. RESULTS: AZD5069 treatment reversibly reduced circulating neutrophil count from baseline by a mean [standard deviation (SD)] of -1.67 (0.67) ×10(9) l(-1) vs. 0.19 (0.78) ×10(9) l(-1) for placebo on day 2, returning to baseline by day 7 after the last dose. Despite low counts on day 4, a 10-min exercise challenge increased absolute blood neutrophil count, but the effect with AZD5069 was smaller and not sustained, compared with placebo treatment. Subcutaneous G-CSF on day 5 caused a substantial increase in blood neutrophil count in both placebo- and AZD5069-treated subjects. Superoxide anion production in E. coli-stimulated neutrophils and phagocytosis of E. coli were unaffected by AZD5069 (P = 0.375, P = 0.721, respectively vs. baseline, Day 4). AZD5069 was well tolerated. CONCLUSIONS: CXCR2 antagonism did not appear adversely to affect the mobilization of neutrophils from bone marrow into the peripheral circulation, phagocytosis or the oxidative burst response to bacterial pathogens. This supports the potential of CXCR2 antagonists as a treatment option for diseases in which neutrophils play a pathological role.

Bacterial peptidoglycan biosynthesis and recognition by the innate immune system

Dissertation presented to obtain the Ph.D degree in Biology

Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica

The inhalation of airborne pollutants, such as asbestos or silica, is linked to inflammation of the lung, fibrosis, and lung cancer. How the presence of pathogenic dust is recognized and how chronic inflammatory diseases are triggered are poorly understood. Here, we show that asbestos and silica are sensed by the Nalp3 inflammasome, whose subsequent activation leads to interleukin-1beta secretion. Inflammasome activation is triggered by reactive oxygen species, which are generated by a NADPH oxidase upon particle phagocytosis. (NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate.) In a model of asbestos inhalation, Nalp3-/- mice showed diminished recruitment of inflammatory cells to the lungs, paralleled by lower cytokine production. Our findings implicate the Nalp3 inflammasome in particulate matter-related pulmonary diseases and support its role as a major proinflammatory "danger" receptor