Andrew J. McDermott

The microbiome and regulation of mucosal immunity

The gastrointestinal tract is a mucosal surface constantly exposed to foreign antigens and microbes, and is protected by a vast array of immunologically active structures and cells. Epithelial cells directly participate in immunological surveillance and direction of host responses in the gut and can express numerous pattern recognition receptors, including Toll‐like receptor 5 (TLR5), TLR1, TLR2, TLR3, TLR9, and nucleotide oligomerization domain 2, as well as produce chemotactic factors for both myeloid and lymphoid cells following inflammatory stimulation. Within the epithelium and in the underlying lamina propria resides a population of innate lymphoid cells that, following stimulation, can become activated and produce effector cytokines and exert both protective and pathogenic roles during inflammation. Lamina propria dendritic cells play a large role in determining whether the response to a particular antigen will be inflammatory or anti‐inflammatory. It is becoming clear that the composition and metabolic activity of the intestinal microbiome, as a whole community, exerts a profound influence on mucosal immune regulation. The microbiome produces short‐chain fatty acids, polysaccharide A, α‐galactosylceramide and tryptophan metabolites, which can induce interleukin‐22, Reg3γ, IgA and interleukin‐17 responses. However, much of what is known about microbiome–host immune interactions has come from the study of single bacterial members of the gastrointestinal microbiome and their impact on intestinal mucosal immunity. Additionally, evidence continues to accumulate that alterations of the intestinal microbiome can impact not only gastrointestinal immunity but also immune regulation at distal mucosal sites.

Acute infection of mice with Clostridium difficile leads to eIF2α phosphorylation and pro-survival signalling as part of the mucosal inflammatory response

The current study sought to delineate the gene expression profile of the host response in the caecum and colon during acute infection with Clostridium difficile in a mouse model of infection, and to investigate the nature of the unfolded protein response in this process. The infected mice displayed a significant up‐regulation in the expression of chemokines (Cxcl1, Cxcl2 and Ccl2), numerous pro‐inflammatory cytokines (Ifng, Il1b, Il6, and Il17f), as well as Il22 and a number of anti‐microbial peptides (Defa1, Defa28, Defb1, Slpi and Reg3g) at the site(s) of infection. This was accompanied by a significant influx of neutrophils, dendritic cells, cells of the monocyte/macrophage lineage and all major subsets of lymphocytes to these site(s). However, CD4 T cells of the untreated and C. difficile‐infected mice expressed similar levels of CD69 and CD25. Neither tissue had up‐regulated levels of Tbx21, Gata3 or Rorc. The caeca and colons of the infected mice showed a significant increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, but neither the splicing of Xbp1 nor the up‐regulation of endoplasmic reticulum chaperones, casting doubt on the full‐fledged induction of the unfolded protein response by C. difficile. They also displayed significantly higher phosphorylation of AKT and signal transducer and activator of transcription 3 (STAT3), an indication of pro‐survival signalling. These data underscore the local, innate, pro‐inflammatory nature of the response to C. difficile and highlight eIF2α phosphorylation and the interleukin‐22–pSTAT3–RegIIIγ axis as two of the pathways that could be used to contain and counteract the damage inflicted on the intestinal epithelium.

Tryptophan Catabolism Restricts IFN-γ–Expressing Neutrophils and Clostridium difficile Immunopathology

The interplay between Clostridium difficile and the host’s metabolome is believed to influence the severity of infection. However, the mechanism for this phenomenon remains unclear. In this study, we model one of these metabolic pathways by focusing on tryptophan metabolism in the host. We found that inhibition of tryptophan catabolism in IDO1-knockout mice led to increased mucosal destruction, cecal hemorrhage, and increased production of IFN-γ in response to C. difficile infection, but no significant change in mucosal effector or regulatory T cell numbers or IL-10 mRNA expression. The increased immunopathology in infected IDO1-knockout mice was associated with a lower C. difficile burden and an increased percentage of IFN-γ–expressing neutrophils. We further demonstrated the ability of kynurenine to induce apoptosis in bone marrow–derived neutrophils, whereas the presence of tryptophan reversed this effect, providing a possible mechanism for the increased neutrophil accumulation in IDO1−/− mice. We conclude that C. difficile induces tryptophan catabolism in cecal lamina propria cells, which restricts C. difficile–associated immunopathology and the accumulation of IFN-γ–expressing neutrophils. This might represent a self-regulatory mechanism for neutrophils, via the IFN-γ–IDO1 pathway, to restrict their own accumulation during infection. These findings have important clinical implications because IDO inhibitors are used to treat cancer in clinical trials (in patients particularly susceptible to getting C. difficile infection), and treatment with IDO1 inhibitors may exacerbate the severity of C. difficile colitis.

Role of GM-CSF in the inflammatory cytokine network that regulates neutrophil influx into the colonic mucosa during Clostridium difficile infection in mice

Clostridium difficile infection in antibiotic-treated mice results in acute colitis characterized by severe intestinal histopathology, robust neutrophil influx, and increased expression of numerous inflammatory cytokines, including GM-CSF. We utilized a neutralizing monoclonal antibody (mAb) against GM-CSF in a murine model to study the role of GM-CSF during acute C. difficile colitis. Cefoperazone-treated mice were challenged with C. difficile (strain 630) spores. Expression of GM-CSF was significantly increased in animals challenged with C. difficile. Treatment with an anti-GM-CSF mAb did not alter C. difficile colonization levels, weight loss, or expression of IL-22 and RegIIIγ. However, expression of the inflammatory cytokines TNFα and IL-1β, as well as iNOS, was significantly reduced following anti-GM-CSF treatment. Expression of the neutrophil chemokines CXCL1 and CXCL2, but not the chemokines CCL2, CCL4, CXCL9, and CXCL10, was significantly reduced by anti-GM-CSF treatment. Consistent with a decrease in neutrophil-attractant chemokine expression, there were fewer neutrophils in histology sections and a reduction in the expression of secretory leukocyte protease inhibitor (SLPI), a tissue anti-protease that protects against damage by secreted neutrophil elastase. These data indicate that GM-CSF plays a role in the inflammatory signaling network that drives neutrophil recruitment in response to C. difficile infection but does not appear to play a role in clearance of the infection.

Interleukin-23 (IL-23), independent of IL-17 and IL-22, drives neutrophil recruitment and innate inflammation during Clostridium difficile colitis in mice

Our objective was to determine the role of the inflammatory cytokine interleukin‐23 (IL‐23) in promoting neutrophil recruitment, inflammatory cytokine expression and intestinal histopathology in response to Clostridium difficile infection. Wild‐type (WT) and p19−/− (IL‐23KO) mice were pre‐treated with cefoperazone in their drinking water for 5 days, and after a 2‐day recovery period were challenged with spores from C. difficile strain VPI 10463. Interleukin‐23 deficiency was associated with significant defects in both the recruitment of CD11bHigh Ly6GHigh neutrophils to the colon and the expression of neutrophil chemoattractants and stabilization factors including Cxcl1, Cxcl2, Ccl3 and Csf3 within the colonic mucosa as compared with WT animals. Furthermore, the expression of inflammatory cytokines including Il33, Tnf and Il6 was significantly reduced in IL‐23‐deficient animals. There was also a trend towards less severe colonic histopathology in the absence of IL‐23. The induction of Il17a and Il22 was also significantly abrogated in IL‐23KO mice. Inflammatory cytokine expression and neutrophilic inflammation were not reduced in IL‐17a‐deficient mice or in mice treated with anti‐IL‐22 depleting monoclonal antibody. However, induction of RegIIIg was significantly reduced in animals treated with anti‐IL‐22 antibody. Taken together, these data indicate that IL‐23, but not IL‐17a or IL‐22, promotes neutrophil recruitment and inflammatory cytokine and chemokine expression in the colon in response to C. difficile infection.

Interleukin‐22 and CD160 play additive roles in the host mucosal response to Clostridium difficile infection in mice

Our previous work has shown the significant up‐regulation of Il22 and increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the mucosal inflammatory response to Clostridium difficile infection in mice. Others have shown that phosphorylation of STAT3 at mucosal surfaces includes interleukin‐22 (IL‐22) and CD160‐mediated components. The current study sought to determine the potential role(s) of IL‐22 and/or CD160 in the mucosal response to C. difficile infection. Clostridium difficile‐infected mice treated with anti‐IL‐22, anti‐CD160 or a combination of the two showed significantly reduced STAT3 phosphorylation in comparison to C. difficile‐infected mice that had not received either antibody. In addition, C. difficile‐infected mice treated with anti‐IL‐22/CD160 induced a smaller set of genes, and at significantly lower levels than the untreated C. difficile‐infected mice. The affected genes included pro‐inflammatory chemokines and cytokines, and anti‐microbial peptides. Furthermore, histopathological and flow cytometric assessments both showed a significantly reduced influx of neutrophils in C. difficile‐infected mice treated with anti‐IL‐22/CD160. These data demonstrate that IL‐22 and CD160 are together responsible for a significant fraction of the colonic STAT3 phosphorylation in C. difficile infection. They also underscore the additive effects of IL‐22 and CD160 in mediating both the pro‐inflammatory and pro‐survival aspects of the host mucosal response in this infection.

The role of Gr-1+ cells and tumour necrosis factor-α signalling during Clostridium difficile colitis in mice

The host response to Clostridium difficile infection in antibiotic‐treated mice is characterized by robust recruitment of Gr‐1+ cells, increased expression of inflammatory cytokines including tumour necrosis factor‐α (TNF‐α), and the development of severe epithelial damage. To investigate the role of Gr‐1+ cells and TNF‐α during C. difficile colitis, we treated infected mice with monoclonal antibodies against Gr‐1 or TNF‐α. Mice were challenged with vegetative cells of C. difficile strain VPI 10463 following treatment with the third‐generation cephalosporin ceftriaxone. Ceftriaxone treatment alone was associated with significant changes in cytokine expression within the colonic mucosa but not overt inflammatory histopathological changes. In comparison, C. difficile infection following ceftriaxone treatment was associated with increased expression of inflammatory cytokines and chemokines including Cxcl1, Cxcl2, Il1b, Il17f and Tnfa, as well as robust recruitment of Ly6CMid Gr‐1High neutrophils and Ly6CHigh Gr‐1Mid monocytes and the development of severe colonic histopathology. Anti‐Gr‐1 antibody treatment resulted in effective depletion of both Ly6CMid Gr‐1High neutrophils and Ly6CHigh Gr‐1Mid monocytes: however, we observed no protection from the development of severe pathology or reduction in expression of the pro‐inflammatory cytokines Il1b, Il6, Il33 and Tnfa following anti‐Gr‐1 treatment. By contrast, anti‐TNF‐α treatment did not affect Gr‐1+ cell recruitment, but was associated with increased expression of Il6 and Il1b. Additionally, Ffar2, Ffar3, Tslp, Tff and Ang4 expression was significantly reduced in anti‐TNF‐α‐treated animals, in association with marked intestinal histopathology. These studies raise the possibility that TNF‐α may play a role in restraining inflammation and protecting the epithelium during C. difficile infection.

Role of interferon-γ and inflammatory monocytes in driving colonic inflammation during acute Clostridium difficile infection in mice

The inflammatory response to the colonic pathogen Clostridium difficile is characterized by the induction of inflammatory cytokines including Interleukin‐23 (IL‐23) and interferon‐γ (IFN‐γ) and the recruitment of myeloid cells including Ly6CHigh monocytes. IL‐23 knockout mice showed reduced expression of the monocyte chemokines Ccl4 and Ccl7, but not Ccl2, as well as reduced Ly6CHigh Ly6GMid monocyte recruitment to the colon in response to C. difficile colitis. Clostridium difficile‐infected CCR2−/− (CCR2 KO) mice showed a significant defect in Ly6CHigh Ly6GMid monocyte recruitment to the colon in response to C. difficile. Although there was no decrease in expression of the inflammatory cytokines Il1b, Il6 or Tnf or reduction in the severity of colonic histopathology associated with ablation of monocyte recruitment, Slpi and Inos expression was significantly reduced in the colons of these animals. Additionally, neutralization of IFN‐γ through the administration of anti‐IFN‐γ monoclonal antibody resulted in a significant reduction in the expression of the IFN‐γ‐inducible chemokines Cxcl9 and Cxcl10, but not a reduction in the neutrophil chemokines Cxcl1, Cxcl2 and Ccl3 or the monocyte chemokine Ccl2. Consistently, monocyte and neutrophil recruitment were unchanged following anti‐IFN‐γ treatment. Additionally, Inos and Slpi expression were unchanged following anti‐IFN‐γ treatment, suggesting that Inos and Slpi regulation is independent of IFN‐γ during C. difficile colitis. Taken together, these data strongly suggest that IL‐23 and CCR2 signalling are required for monocyte recruitment during C. difficile colitis. Additionally, these studies also suggest that monocytes, but not IFN‐γ, are necessary for full expression of Inos and Slpi in the colon.