Bacterial-epithelial contact is a key determinant of host innate immune responses to enteropathogenic and enteroaggregative escherichia coli

Background: Enteropathogenic (EPEC) and Enteroaggregative (EAEC) E. coli have similar, but distinct clinical symptoms and modes of pathogenesis. Nevertheless when they infect the gastrointestinal tract, it is thought that their flagellin causes IL-8 release leading to neutrophil recruitment and gastroenteritis. However, this may not be the whole story as the effect of bacterial adherence to IEC innate response(s) remains unclear. Therefore, we have characterized which bacterial motifs contribute to the innate epithelial response to EPEC and EAEC, using a range of EPEC and EAEC isogenic mutant strains. Methodology: Caco-2 and HEp-2 cell lines were exposed to prototypical EPEC strain E2348/69 or EAEC strain O42, in addition to a range of isogenic mutant strains. E69 [LPS, non-motile, non-adherent, type three secretion system (TTSS) negative, signalling negative] or O42 [non-motile, non-adherent]. IL-8 and CCL20 protein secretion was measured. Bacterial surface structures were assessed by negative staining Transmission Electron Microscopy. The Fluorescent-actin staining test was carried out to determine bacterial adherence. Results: Previous studies have reported a balance between the host pro-inflammatory response and microbial suppression of this response. In our system an overall balance towards the host pro-inflammatory response is seen with the E69 WT and to a greater extent O42 WT, which is in fit with clinical symptoms. On removal of the external EPEC structures flagella, LPS, BFP, EspA and EspC; and EAEC flagella and AAF, the host inflammatory response is reduced. However, removal of E69 lymphostatin increases the host inflammatory response suggesting involvement in the bacterial mediated anti-inflammatory response. Conclusion: Epithelial responses were due to combinations of bacterial agonists, with host-bacterial contact a key determinant of these innate responses. Host epithelial recognition was offset by the microbe's ability to down-regulate the inflammatory response. Understanding the complexity of this host-microbial balance will contribute to improved vaccine design for infectious gastroenteritis

Is the skin microbiota a modifiable risk factor for breast disease?: A systematic review

Purpose: High prevalence, unreliable risk discrimination and poor clinical outcomes are observed in malignant and benign breast diseases (BD). The involvement of microbial communities in the development of BD has become topical, and distal influences of microbial dysregulation in the breast have been well established. Despite advances, the role of the breast skin microbiota in BD remains unclear. Interactions between the skin microbiota and the underlying mucosal immune system are complex. In homeostasis, the skin offers a physical barrier protecting underlying breast tissue from skin commensals and noxious environmental triggers. Our review aims to illuminate the role of the skin microbiota in the development of BD. Methods: Adhering to the PRISMA protocol, a systematic review was conducted utilising the Medline and Embase search engines. Results: Through a comprehensive search of the last ten years, twenty-two studies satisfied the inclusion criteria. Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were identified as the most prevalent phyla of both breast tissue and skin in healthy controls and BD. High abundance of skin commensals, specifically some species of Staphylococcus, have been linked in breast cancer and metastases. Similarly, dysregulated microbial abundance is also seen in inflammatory and implant-associated BD. These findings raise the hypothesis that the skin microbiota plays a role in tissue homeostasis and may contribute to a range of breast pathologies. Several mechanisms of microbial transfer to underlying tissue have been proposed, including retrograde transfer through ductal systems, breakdown of the skin barrier, and migration through nipple-aspirate fluid. Conclusion: Our review provides preliminary insights into the skin microbiota as a modifiable risk factor for BD. This raises opportunities for future studies in antimicrobials/probiotics as an adjunct to, or replacement of surgery; a diagnostic and/or prognostic tool for BD; and the possibility of conditioning the microbiota to manage BD

Bacterial outer-membrane vesicles promote Vγ9Vδ2 T cell oncolytic activity

BACKGROUND: Increasing evidence suggests the immune activation elicited by bacterial outer-membrane vesicles (OMVs) can initiate a potent anti-tumor immunity, facilitating the recognition and destruction of malignant cells. At present the pathways underlying this response remain poorly understood, though a role for innate-like cells such as γδ T cells has been suggested. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with E. coli MG1655 Δpal ΔlpxM OMVs and corresponding immune activation studied by cell marker expression and cytokine production. OMV-activated γδ T cells were co-cultured with cancer cell lines to determine cytotoxicity. RESULTS: The vesicles induced a broad inflammatory response with γδ T cells observed as the predominant cell type to proliferate post-OMV challenge. Notably, the majority of γδ T cells were of the Vγ9Vδ2 type, known to respond to both bacterial metabolites and stress markers present on tumor cells. We observed robust cytolytic activity of Vγ9Vδ2 T cells against both breast and leukaemia cell lines (SkBr3 and Nalm6 respectively) after OMV-mediated expansion. CONCLUSIONS: Our findings identify for the first time, that OMV-challenge stimulates the expansion of Vγ9Vδ2 T cells which subsequently present anti-tumor capabilities. We propose that OMV-mediated immune activation leverages the anti-microbial/anti-tumor capacity of Vγ9Vδ2 T cells, an axis amenable for improved future therapeutics

Campylobacter jejuni outer membrane vesicle-associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E-cadherin and occludin.

Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram-negative bacteria. Campylobacter jejuni produces OMVs that trigger IL-8, IL-6, hBD-3 and TNF-α responses from T84 intestinal epithelial cells and are cytotoxic to Caco-2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E-cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co-incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E-cadherin and occludin. The addition of 11168H OMVs to the co-culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time-dependent and dose-dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells

A critical role for ATF2 transcription factor in the regulation of E-selectin expression in response to non-endotoxin components of Neisseria meningitidis

Vascular injury is a serious complication of sepsis due to the gram-negative bacterium Neisseria meningitidis. One of the critical early steps in initiating this injury is via the interaction of leucocytes, particularly neutrophils, with adhesion molecules expressed on inflamed endothelium. We have previously demonstrated that both lipopolysaccharide (LPS) and non-LPS components of meningococci can induce very high levels of expression of the vascular endothelial cell adhesion molecule E-selectin, which is critical for early tethering and capture of neutrophils onto endothelium under flow. Using an LPS-deficient strain of meningococcus, we showed that very high levels of expression can be induced in primary endothelial cells, even in the context of weak activation of the major host signal transduction factor [nuclear factor-κB (NF-κB)]. In this study, we show that the particular propensity for N. meningitidis to induce high levels of expression is regulated at a transcriptional level, and demonstrate a significant role for phosphorylation of the ATF2 transcription factor, likely via mitogen-activated protein (MAP) kinases, on the activity of the E-selectin promoter. Furthermore, inhibition of E-selectin expression in response to the lpxA- strain by a p38 inhibitor indicates a significant role of a p38-dependent MAPK signalling pathway in ATF2 activation. Collectively, these data highlight the role that LPS and other bacterial components have in modulating endothelial function and their involvement in the pathogenesis of meningococcal sepsis. Better understanding of these multiple mechanisms induced by complex stimuli such as bacteria, and the specific inflammatory pathways they activate, may lead to improved, focused interventions in both meningococcal and potentially bacterial sepsis more generally

Neutrophil extracellular traps formation and clearance is enhanced in fever and attenuated in hypothermia

Fever and hypothermia represent two opposite strategies for fighting systemic inflammation. Fever results in immune activation; hypothermia is associated with energy conservation. Systemic Inflammatory Response Syndrome (SIRS) remains a significant cause of mortality worldwide. SIRS can lead to a broad spectrum of clinical symptoms but importantly, patients can develop fever or hypothermia. During infection, polymorphonuclear cells (PMNs) such as neutrophils prevent pathogen dissemination through the formation of neutrophil extracellular traps (NETs) that ensnare and kill bacteria. However, when dysregulated, NETs also promote host tissue damage. Herein, we tested the hypothesis that temperature modulates NETs homeostasis in response to infection and inflammation. NETs formation was studied in response to infectious (Escherichia coli, Staphylococcus aureus) and sterile (mitochondria) agents. When compared to body temperature (37°C), NETs formation increased at 40°C; interestingly, the response was stunted at 35°C and 42°C. While CD16+ CD49d+ PMNs represent a small proportion of the neutrophil population, they formed ~45-85% of NETs irrespective of temperature. Temperature increased formyl peptide receptor 1 (FPR1) expression to a differential extent in CD16+ CD49d- vs. CD49d+ PMNSs, suggesting further complexity to neutrophil function in hypo/hyperthermic conditions. The capacity of NETs to induce Toll-like receptor 9 (TLR9)-mediated NF-κB activation was found to be temperature independent. Interestingly, NET degradation was enhanced at higher temperatures, which corresponded with greater plasma DNase activity in response to temperature increase. Collectively, our observations indicate that NETs formation and clearance are enhanced at 40°C whilst temperatures of 35°C and 42°C attenuate this response. Targeting PMN-driven immunity may represent new venues for intervention in pathological inflammation

Delineation of the Innate and Adaptive T-Cell Immune Outcome in the Human Host in Response to Campylobacter jejuni Infection

Background: Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Despite the significant health burden this infection presents, molecular understanding of C. jejuni-mediated disease pathogenesis remains poorly defined. Here, we report the characterisation of the early, innate immune response to C. jejuni using an ex-vivo human gut model of infection. Secondly, impact of bacterial-driven dendritic cell activation on T-cell mediated immunity was also sought.Methodology: Healthy, control paediatric terminal ileum or colonic biopsy tissue was infected with C. jejuni for 8-12 hours. Bacterial colonisation was followed by confocal microscopy and mucosal innate immune responses measured by ELISA. Marked induction of IFN gamma with modest increase in IL-22 and IL-17A was noted. Increased mucosal IL-12, IL-23, IL-1 beta and IL-6 were indicative of a cytokine milieu that may modulate subsequent T-cell mediated immunity. C. jejuni-driven human monocyte-derived dendritic cell activation was followed by analyses of T cell immune responses utilising flow cytometry and ELISA. Significant increase in Th-17, Th-1 and Th-17/Th-1 double-positive cells and corresponding cytokines was observed. The ability of IFN gamma, IL-22 and IL-17 cytokines to exert host defence via modulation of C. jejuni adhesion and invasion to intestinal epithelia was measured by standard gentamicin protection assay.Conclusions: Both innate and adaptive T cell-immunity to C. jejuni infection led to the release of IFN gamma, IL-22 and IL-17A; suggesting a critical role for this cytokine triad in establishing host anti-microbial immunity during the acute and effectors phase of infection. In addition, to their known anti-microbial functions; IL-17A and IL-17F reduced the number of intracellular C. jejuni in intestinal epithelia, highlighting a novel aspect of how IL-17 family members may contribute to protective immunity against C. jejuni