IL-18: A potential inflammation biomarker in Wiskott-Aldrich syndrome.

Analysis of serum cytokine levels in Wiskott-Aldrich syndrome patients pre- and post- treatment reveals IL-18 as a stable and reliable marker of inflammation. Definitive stem cell treatment with good myeloid correction correlates with resolution of inflammation and reduction of circulating IL-18, highlighting the importance of actin cytoskeletal regulation of myeloid cells in control of inflammation.

Salmonella exploits HLA-B27 and host unfolded protein responses to promote intracellular replication.

OBJECTIVE: Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells. METHODS: Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S.enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane. RESULTS: S.enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism. CONCLUSIONS: HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention.

The bile salt sodium taurocholate induces Campylobacter jejuni outer membrane vesicle production and increases OMV-associated proteolytic activity.

Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an arsenal of virulence-associated proteins within outer membrane vesicles (OMVs). C. jejuni OMVs contain three serine proteases (HtrA, Cj0511, and Cj1365c) that cleave the intestinal epithelial cell (IEC) tight and adherens junction proteins occludin and E-cadherin, promoting enhanced C. jejuni adhesion to and invasion of IECs. C. jejuni OMVs also induce IECs innate immune responses. The bile salt sodium taurocholate (ST) is sensed as a host signal to coordinate the activation of virulence-associated genes in the enteric pathogen Vibrio cholerae. In this study, the effect of ST on C. jejuni OMVs was investigated. Physiological concentrations of ST do not have an inhibitory effect on C. jejuni growth until the early stationary phase. Coculture of C. jejuni with 0.1% or 0.2% (w/v) ST stimulates OMV production, increasing both lipid and protein concentrations. C. jejuni ST-OMVs possess increased proteolytic activity and exhibit a different protein profile compared to OMVs isolated in the absence of ST. ST-OMVs exhibit enhanced cytotoxicity and immunogenicity to T84 IECs and enhanced killing of Galleria mellonella larvae. ST increases the level of mRNA transcripts of the OMVs-associated serine protease genes and the cdtABC operon that encodes the cytolethal distending toxin. Coculture with ST significantly enhances the OMVs-induced cleavage of E-cadherin and occludin. C. jejuni OMVs also cleave the major endoplasmic reticulum chaperone protein BiP/GRP78 and this activity is associated with the Cj1365c protease. These data suggest that C. jejuni responds to the presence of physiological concentrations of the bile salt ST that increases OMV production and the synthesis of virulence-associated factors that are secreted within the OMVs. We propose that these events contribute to pathogenesis.

Novel Campylobacter concisus lipooligosaccharide is a determinant of inflammatory potential and virulence.

The pathogenicity of Campylobacter concisus, increasingly found in the human gastrointestinal (GI) tract, is unclear. Some studies indicate that its role in GI conditions has been underestimated, whereas others suggest that the organism has a commensal-like phenotype. For the enteropathogen C. jejuni, the lipooligosaccharide (LOS) is a main driver of virulence. We investigated the LOS structure of four C. concisus clinical isolates and correlated the inflammatory potential of each isolate with bacterial virulence. Mass spectrometric analyses of lipid A revealed a novel hexa-acylated diglucosamine moiety with two or three phosphoryl substituents. Molecular and fragment ion analysis indicated that the oligosaccharide portion of the LOS had only a single phosphate and lacked phosphoethanolamine and sialic acid substitution, which are hallmarks of the C. jejuni LOS. Consistent with our structural findings, C. concisus LOS and live bacteria induced less TNF-α secretion in human monocytes than did C. jejuni Furthermore, the C. concisus bacteria were less virulent than C. jejuni in a Galleria mellonella infection model. The correlation of the novel lipid A structure, decreased phosphorylation, and lack of sialylation along with reduced inflammatory potential and virulence support the significance of the LOS as a determinant in the relative pathogenicity of C. concisus.

Role of Glycosyltransferases Modifying Type B Flagellin of Emerging Hypervirulent Clostridium difficile Lineages and Their Impact on Motility and Biofilm Formation.

Clostridium difficile is the principal cause of nosocomial infectious diarrhea worldwide. The pathogen modifies its flagellin with either a type A or type B O-linked glycosylation system, which has a contributory role in pathogenesis. We study the functional role of glycosyltransferases modifying type B flagellin in the 023 and 027 hypervirulent C. difficile lineages by mutagenesis of five putative glycosyltransferases and biosynthetic genes. We reveal their roles in the biosynthesis of the flagellin glycan chain and demonstrate that flagellar post-translational modification affects motility and adhesion-related bacterial properties of these strains. We show that the glycosyltransferases 1 and 2 (GT1 and GT2) are responsible for the sequential addition of a GlcNAc and two rhamnoses, respectively, and that GT3 is associated with the incorporation of a novel sulfonated peptidyl-amido sugar moiety whose structure is reported in our accompanying paper (Bouché, L., Panico, M., Hitchen, P., Binet, D., Sastre, F., Faulds-Pain, A., Valiente, E., Vinogradov, E., Aubry, A., Fulton, K., Twine, S., Logan, S. M., Wren, B. W., Dell, A., and Morris, H. R. (2016) J. Biol. Chem. 291, 25439-25449). GT2 is also responsible for methylation of the rhamnoses. Whereas type B modification is not required for flagellar assembly, some mutations that result in truncation or abolition of the glycan reduce bacterial motility and promote autoaggregation and biofilm formation. The complete lack of flagellin modification also significantly reduces adhesion of C. difficile to Caco-2 intestinal epithelial cells but does not affect activation of human TLR5. Our study advances our understanding of the genes involved in flagellar glycosylation and their biological roles in emerging hypervirulent C. difficile strains.

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.

Is variation in copy number of the human beta defensin gene cluster associated with preterm birth?

BACKGROUND: Increased expression of antimicrobial peptides including human beta defensins (HBD) has been reported in the amniotic fluid and vaginal secretions of women who deliver preterm. We have previously shown that these women have increased first trimester serum HBD2. The gene encoding HBD2, DEFB4A, is part of a defensin beta (DEFB) cluster on chromosome 8 that is variable in copy number. Increased serum HBD2 is associated with increased DEFB copy number. We aimed to test the hypothesis that variation in DEFB copy number is associated with preterm birth. METHODS: In a retrospective, case-control study, genomic DNA and serum were extracted from blood collected from white European women at 11-13 weeks' gestation attending King's College Hospital between March 1, 2006, and Sept 30, 2010. DEFB copy number was determined by paralogue ratio test. Serum HBD2 concentration was measured by ELISA. Data were analysed with Pearson correlation (Excel, version 2010) and binary logistic regression (SPSS, version 20). FINDINGS: Cases were 102 women who either delivered preterm in the index pregnancy or had a history of preterm delivery. Controls were 152 women who had had at least one previous term delivery and delivered at term in the index pregnancy; they had no history of preterm birth. Modal copy number was 4 (range 2-7). Serum was available from 140 women (30 cases, 54 controls, 56 not included in the genetic association study). Median HBD2 concentration was 761·5 pg/mL (IQR 449·6-1232·0). There was no association between DEFB copy number and preterm birth, nor was there a correlation between copy number and serum HBD2 concentration. INTERPRETATION: Although variation in HBD2 protein expression in the first trimester might be useful to predict risk of preterm birth, we found no association between DEFB copy number and preterm birth. Nor did we find a correlation between DEFB copy number and serum HBD2 expression in the first trimester of pregnancy; this might be due to variation in regulatory sequences-some of which are progesterone and oestrogen sensitive-between individual copies. FUNDING: Wellcome Trust, Wellbeing of Women.

Clostridium difficile-mediated effects on human intestinal epithelia: Modelling host-pathogen interactions in a vertical diffusion chamber.

Clostridium difficile infection is one of the leading causes of healthcare associated diarrhoea in the developed world. Although the contribution of C. difficile toxins to disease pathogenesis is now well understood, many facets of host-pathogen interactions between the human intestinal epithelia and the C. difficile bacterium that may contribute to asymptomatic carriage and/or clinical disease remain less clear. Herein, we tested the hypothesis that C. difficile strains mediate intestinal epithelial cell (IEC) antimicrobial immunity via toxin dependent and independent means and that the 'anaerobic' environment has a significant impact on bacterial-IEC interactions. Crosstalk between three C. difficile PCR ribotypes (RT) [RT027 (strain R20291), RT012 (strain 630) and RT017 (strains M68 and CF5)] and IEC cell-lines were investigated. All RTs showed significant engagement with human Toll-like receptors (TLR)-5, TLR2-CD14 and TLR2/6 as measured by IL-8 release from TLR-transfected HEK cells. Co-culture studies indicated minimal impact of R20291 and 630 TcdA and TcdB on bacterial adherence to Caco-2 cells. An apical anaerobic environment had a major effect on C. difficile-T84 crosstalk as significantly greater cytokine immunity and trans-epithelial electrical resistance (TEER) dysfunction was recorded when co-cultures were performed in an Ussing chamber system compared to standard 5% CO2 conditions. Overall, this study suggests that anaerobic C. difficile engagement with human IECs is a complex interplay that involves bacterial and toxin-mediated cellular events.

Pancreatic amylase is an environmental signal for regulation of biofilm formation and host interaction in Campylobacter jejuni.

Campylobacter jejuni is a commensal bacterium in the intestines of animals and birds and a major cause of food-borne gastroenteritis in humans worldwide. Here we show that exposure to pancreatic amylase leads to secretion of an α-dextran by C. jejuni and that a secreted protease, Cj0511, is required. Exposure of C. jejuni to pancreatic amylase promotes biofilm formation in vitro, increases interaction with human epithelial cell lines, increases virulence in the Galleria mellonella infection model, and promotes colonization of the chicken ileum. We also show that exposure to pancreatic amylase protects C. jejuni from stress conditions in vitro, suggesting that the induced α-dextran may be important during transmission between hosts. This is the first evidence that pancreatic amylase functions as an interkingdom signal in an enteric microorganism.

Pseudaminic acid on Campylobacter jejuni flagella modulates dendritic cell IL-10 expression via Siglec-10 receptor: a novel flagellin-host interaction.

INTRODUCTION: Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. At present the identity of host-pathogen interactions that promote successful bacterial colonisation remain ill defined. Herein, we aimed to investigate C. jejuni-mediated effects on dendritic cell (DC) immunity. RESULTS: We found C. jejuni to be a potent inducer of human and murine DC interleukin 10 (IL-10) in vitro, a cellular event that was MyD88- and p38 MAPK-signalling dependent. Utilizing a series of C. jejuni isogenic mutants we found the major flagellin protein, FlaA, modulated IL-10 expression, an intriguing observation as C. jejuni FlaA is not a TLR5 agonist. Further analysis revealed pseudaminic acid residues on the flagella contributed to DC IL-10 expression. We identified the ability of both viable C. jejuni and purified flagellum to bind to Siglec-10, an immune-modulatory receptor. In vitro infection of Siglec-10 overexpressing cells resulted in increased IL-10 expression in a p38-dependent manner. Detection of Siglec-10 on intestinal CD11c(+) CD103(+) DCs added further credence to the notion that this novel interaction may contribute to immune outcome during human infection. CONCLUSIONS: We propose that unlike the Salmonella Typhimurium flagella-TLR5 driven pro-inflammatory axis, C. jejuni flagella instead promote an anti-inflammatory axis via glycan-Siglec-10 engagement.

Campylobacter jejuni lipooligosaccharide sialylation, phosphorylation, and amide/ester linkage modifications fine-tune human Toll-like receptor 4 activation.

Campylobacter jejuni is a leading cause of acute gastroenteritis. C. jejuni lipooligosaccharide (LOS) is a potent activator of Toll-like receptor (TLR) 4-mediated innate immunity. Structural variations of the LOS have been previously reported in the oligosaccharide (OS) moiety, the disaccharide lipid A (LA) backbone, and the phosphorylation of the LA. Here, we studied LOS structural variation between C. jejuni strains associated with different ecological sources and analyzed their ability to activate TLR4 function. MALDI-TOF MS was performed to characterize structural variation in both the OS and LA among 15 different C. jejuni isolates. Cytokine induction in THP-1 cells and primary monocytes was correlated with LOS structural variation in each strain. Additionally, structural variation was correlated with the source of each strain. OS sialylation, increasing abundance of LA d-glucosamine versus 2,3-diamino-2,3-dideoxy-d-glucose, and phosphorylation status all correlated with TLR4 activation as measured in THP-1 cells and monocytes. Importantly, LOS-induced inflammatory responses were similar to those elicited by live bacteria, highlighting the prominent contribution of the LOS component in driving host immunity. OS sialylation status but not LA structure showed significant association with strains clustering with livestock sources. Our study highlights how variations in three structural components of C. jejuni LOS alter TLR4 activation and consequent monocyte activation.

Host immunity to Clostridium difficile PCR ribotype 017 strains.

Clostridium difficile is an important nosocomial pathogen and the leading cause of antibiotic-associated diarrhea. Multilocus sequence typing indicates that C. difficile strains belong to five distinct genetic clades encompassing several PCR ribotypes (RT). Since their emergence in 2003, hypervirulent RT027 strains have been a major focus of research; in contrast, our current understanding of RT017-mediated disease pathogenesis lags far behind. In this study, we aimed to characterize host immunity to CF5 and M68, two genetically well-defined RT017 strains. Both strains engaged with host Toll-like receptor 2/6 (TLR2/6), TLR2-CD14, and TLR5 to similar extents in a model cell line. Despite this, CF5 mediated significantly greater dendritic cell (DC) interleukin-12 (IL-12), IL-27, and IL-10 immunity than M68. Both strains elicited similar IL-1ß mRNA levels, and yet only M68 caused a marked increase in secretory IL-1ß. A CF5 cocultured-DC cytokine milieu drove an equipotent Th1 and Th17 response, while M68 promoted greater Th17 immunity. Human gastrointestinal ex vivo cytokine responses to both strains were characterized. Taken together, our data suggest that C. difficile strains mediate overlapping and yet distinct mucosal and DC/T cell immunity. Finally, toxin-driven IL-1ß release supports the hypothesis that this cytokine axis is a likely target for therapeutic intervention for C. difficile infection.

A Stress Reduction Intervention for Lactating Mothers Alters Maternal Gut, Breast Milk, and Infant Gut Microbiomes: Data from a Randomized Controlled Trial.

BACKGROUND: This secondary analysis of data from a randomized controlled trial (RCT) investigated how the maternal gut, breast milk, and infant gut microbiomes may contribute to the effects of a relaxation intervention, which reduced maternal stress and promoted infant weight gain. METHODS: An RCT was undertaken in healthy Chinese primiparous mother-infant pairs (340/7-376/7gestation weeks). Mothers were randomly allocated to either the intervention group (IG, listening to relaxation meditation) or the control group (CG). Outcomes were the differences in microbiome composition and the diversity in the maternal gut, breast milk, and infant gut at 1 (baseline) and 8 weeks (post-intervention) between IG and CG, assessed using 16S rRNA gene amplicon sequencing of fecal and breastmilk samples. RESULTS: In total, 38 mother-infant pairs were included in this analysis (IG = 19, CG = 19). The overall microbiome community structure in the maternal gut was significantly different between the IG and CG at 1 week, with the difference being more significant at 8 weeks (Bray-Curtis distance R2 = 0.04 vs. R2 = 0.13). Post-intervention, a significantly lower α-diversity was observed in IG breast milk (observed features: CG = 295 vs. IG = 255, p = 0.032); the Bifidobacterium genera presented a higher relative abundance. A significantly higher α-diversity was observed in IG infant gut (observed features: CG = 73 vs. IG = 113, p < 0.001). CONCLUSIONS: The findings were consistent with the hypothesis that the microbiome might mediate observed relaxation intervention effects via gut-brain axis and entero-mammary pathways; but confirmation is required.

Mannose-binding lectin genotype influences frequency and duration of infectious complications in children with malignancy.

Children with cancer receiving identical treatment differ in their experience of infection, suggesting that variations in immunity may influence susceptibility to infection. Studies of the influence of mannose-binding lectin (MBL), an important component of the innate immune system, in children with febrile neutropenia (FN) have yielded conflicting results. We examined the role of MBL in infection susceptibility in the largest cohort of children with cancer reported to date. MBL levels were measured and genotyping performed on children (≤16 y) receiving chemotherapy for cancer in London, UK. Clinical data from FN episodes were recorded prospectively. MBL status was assessed in 269 children; 513 episodes of FN were captured from 211 patients. Patients with MBL2 polymorphisms experienced more FN episodes than wildtype genotype (median 2 vs. 1, respectively; P = 0.074) and more episodes with documented infection (P = 0.045). Patients experiencing multiple FN episodes had lower MBL levels (P = 0.036). MBL genotype influenced duration of episode in some groups: high-risk MBL-deficient patients spent up to 5 nights longer/episode in hospital than equivalent wildtypes. These results indicate that MBL deficiency influences both susceptibility to and outcome of FN episodes and may be most important in those patients at higher risk of complications of FN.

Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment.

In several murine models of autoimmune arthritis, Th17 cells are the dominant initiators of inflammation. In human arthritis the majority of IL-17-secreting cells within the joint express a cytokine phenotype intermediate between Th17 and Th1. Here we show that Th17/1 cells from the joints of children with inflammatory arthritis express high levels of both Th17 and Th1 lineage-specific transcription factors, RORC2 and T-bet. Modeling the generation of Th17/1 in vitro, we show that Th17 cells "convert" to Th17/1 under conditions that mimic the disease site, namely low TGFbeta and high IL-12 levels, whereas Th1 cells cannot convert to Th17. Th17/1 cells from the inflamed joint share T-cell receptor (TCR) clonality with Th17 cells, suggesting a shared clonal origin between Th17 and Th17/1 cells in arthritis. Using CD161, a lectin-like receptor that is a marker of human Th17, we show synovial Th17 and Th17/1 cells, and unexpectedly, a large proportion of Th1 cells express CD161. We provide evidence to support a Th17 origin for Th1 cells expressing CD161. In vitro, Th17 cells that convert to a Th1 phenotype maintain CD161 expression. In the joint CD161+ Th1 cells share features with Th17 cells, with shared TCR clonality, expression of RORC2 and CCR6 and response to IL-23, although they are IL-17 negative. We propose that the Th17 phenotype may be unstable and that Th17 cells may convert to Th17/1 and Th1 cells in human arthritis. Therefore therapies targeting the induction of Th17 cells could also attenuate Th17/1 and Th1 effector populations within the inflamed joint.

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.

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.

Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.

Campylobacter jejuni infection often results in bloody, inflammatory diarrhea, indicating bacterial disruption and invasion of the intestinal epithelium. While C. jejuni infection can be reproduced in vitro using intestinal epithelial cell (IEC) lines, low numbers of bacteria invading IECs do not reflect these clinical symptoms. Performing in vitro assays under atmospheric oxygen conditions neither is optimal for microaerophilic C. jejuni nor reflects the low-oxygen environment of the intestinal lumen. A vertical diffusion chamber (VDC) model system creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured IECs, producing an in vitro system that closely mimics in vivo conditions in the human intestine. Ninefold increases in interacting and 80-fold increases in intracellular C. jejuni 11168H wild-type strain bacteria were observed after 24-h coculture with Caco-2 IECs in VDCs under microaerobic conditions at the apical surface, compared to results under aerobic conditions. Increased bacterial interaction was matched by an enhanced and directional host innate immune response, particularly an increased basolateral secretion of the proinflammatory chemokine interleukin-8 (IL-8). Analysis of the invasive ability of a nonmotile C. jejuni 11168H rpoN mutant in the VDC model system indicates that motility is an important factor in the early stages of bacterial invasion. The first report of the use of a VDC model system for studying the interactions of an invasive bacterial pathogen with IECs demonstrates the importance of performing such experiments under conditions that represent the in vivo situation and will allow novel insights into C. jejuni pathogenic mechanisms.

Campylobacter jejuni outer membrane vesicles play an important role in bacterial interactions with human intestinal epithelial cells.

Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed world; however, the molecular basis of pathogenesis is unclear. Secretion of virulence factors is a key mechanism by which enteric bacterial pathogens interact with host cells to enhance survival and/or damage the host. However, C. jejuni lacks the virulence-associated secretion systems possessed by other enteric pathogens. Many bacterial pathogens utilize outer membrane vesicles (OMVs) for delivery of virulence factors into host cells. In the absence of prototypical virulence-associated secretion systems, OMVs could be an important alternative for the coordinated delivery of C. jejuni proteins into host cells. Proteomic analysis of C. jejuni 11168H OMVs identified 151 proteins, including periplasmic and outer membrane-associated proteins, but also many determinants known to be important in survival and pathogenesis, including the cytolethal distending toxin (CDT). C. jejuni OMVs contained 16 N-linked glycoproteins, indicating a delivery mechanism by which these periplasm-located yet immunogenic glycoproteins can interact with host cells. C. jejuni OMVs possess cytotoxic activity and induce a host immune response from T84 intestinal epithelial cells (IECs), which was not reduced by OMV pretreatment with proteinase K or polymyxin B prior to coincubation with IECs. Pretreatment of IECs with methyl-beta-cyclodextrin partially blocks OMV-induced host immune responses, indicating a role for lipid rafts in host cell plasma membranes during interactions with C. jejuni OMVs. OMVs isolated from a C. jejuni 11168H cdtA mutant induced interleukin-8 (IL-8) to the same extent as did wild-type OMVs, suggesting OMV induction of IL-8 is independent of CDT.