Caixia Ma

MyD88 signalling plays a critical role in host defence by controlling pathogen burden and promoting epithelial cell homeostasis during Citrobacter rodentium‐induced colitis

Myeloid differentiation factor (MyD)88, an adaptor protein shared by the Toll‐interleukin 1 receptor superfamily, plays a critical role in host defence during many systemic bacterial infections by inducing protective inflammatory responses that limit bacterial growth. However, the role of innate responses during gastrointestinal (GI) infections is less clear, in part because the GI tract is tolerant to commensal antigens. The current study investigated the role of MyD88 following infection by the murine bacterial pathogen, Citrobacter rodentium. MyD88‐deficient mice suffered a lethal colitis coincident with colonic mucosal ulcerations and bleeding. Their susceptibility was associated with an overwhelming bacterial burden and selectively impaired immune responses in colonic tissues, which included delayed inflammatory cell recruitment, reduced iNOS and abrogated production of TNF‐α and IL‐6 from MyD88‐deficient macrophages and colons cultured ex vivo. Immunostaining for Ki67 and BrDU revealed that MyD88 signalling mediated epithelial hyper‐proliferation in response to C. rodentium infection. Thus, MyD88‐deficient mice could not promote epithelial cell turnover and repair, leading to deep bacterial invasion of colonic crypts, intestinal barrier dysfunction and, ultimately, widespread mucosal ulcerations. In conclusion, MyD88 signalling within the GI tract plays a critical role in mediating host defence against an enteric bacterial pathogen, by controlling bacterial numbers and promoting intestinal epithelial homeostasis.

Toll-like receptor 2 plays a critical role in maintaining mucosal integrity during Citrobacter rodentium-induced colitis.

Inflammatory bowel diseases and infectious gastroenteritis likely occur when the integrity of intestinal barriers is disrupted allowing luminal bacterial products to cross into the intestinal mucosa, stimulating immune cells and triggering inflammation. While specific Toll‐like receptors (TLR) are involved in the generation of inflammatory responses against enteric bacteria, their contributions to the maintenance of intestinal mucosal integrity are less clear. These studies investigated the role of TLR2 in a model of murine colitis induced by the bacterial pathogen Citrobacter rodentium. C. rodentium supernatants specifically activated TLR2 in vitro while infected TLR2–/– mice suffered a lethal colitis coincident with colonic mucosal ulcerations, bleeding and increased cell death but not increased pathogen burden. TLR2–/– mice suffered impaired epithelial barrier function mediated via zonula occludens (ZO)‐1 in naïve mice and claudin‐3 in infected mice, suggesting this could underlie their susceptibility. TLR2 deficiency was also associated with impaired production of IL‐6 by bone marrow‐derived macrophages and infected colons cultured ex vivo. As IL‐6 has antiapoptotic and epithelial repair capabilities, its reduced expression could contribute to the impaired mucosal integrity. These studies report for the first time that TLR2 plays a critical role in maintaining intestinal mucosal integrity during infection by a bacterial pathogen.

Colonic microbiota alters host susceptibility to infectious colitis by modulating inflammation, redox status and ion transporter gene expression

Individuals vary in their resistance to enteric infections. The role of the intestinal microbiota in altering susceptibility to enteric infection is relatively unknown. Previous studies have identified that C3H/HeOuJ mice suffer 100% mortality during Citrobacter rodentium-induced colitis, whereas C57BL/6 mice recover from infection. The basis for their differences in susceptibility is unclear and has been mainly attributed to differences in host genetics. This study investigated the role of the intestinal microbiota in altering susceptibility to C. rodentium-induced colitis. When the feces of C57BL/6 mice were gavaged into antibiotic treated C3H/HeOuJ mice, the C57BL/6 microflora led to a complete reversal in mortality patterns where 100% of the C3H/HeOuJ mice survived infection. This protection corresponded with reduced colonic pathology and less systemic pathogen load and was associated with increased inflammatory and redox responses with reduced epithelial cell death. C3H/HeOuJ mice are normally susceptible to infection-induced dehydration due to defective expression of colonic ion transporters such as Dra, CA IV, and CA I; expression of these genes was normalized when C3H/HeOuJ mice were colonized with the C57BL/6 microflora. Together, these data reveal that the colonic microbiota play a critical role in protecting against intestinal infection by inducing proinflammatory and prooxidant responses that control pathogen load as well as ion transporter gene expression previously shown to prevent fatal dehydration. Protection of mice from lethal colitis was associated with higher levels of bacteria from Bacteroidetes. This study reveals that the microbiota is sufficient to overcome inherent genetic susceptibility patterns in C3H/HeOuJ mice that cause mortality during C. rodentium infection.

Flagellin-Dependent and -Independent Inflammatory Responses following Infection by Enteropathogenic Escherichia coli and Citrobacter rodentium

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and the murine pathogen Citrobacter rodentium belong to the attaching and effacing (A/E) family of bacterial pathogens. These noninvasive bacteria infect intestinal enterocytes using a type 3 secretion system (T3SS), leading to diarrheal disease and intestinal inflammation. While flagellin, the secreted product of the EPEC fliC gene, causes the release of interleukin 8 (IL-8) from epithelial cells, it is unclear whether A/E bacteria also trigger epithelial inflammatory responses that are FliC independent. The aims of this study were to characterize the FliC dependence or independence of epithelial inflammatory responses to direct infection by EPEC or C. rodentium. Following infection of Caco-2 intestinal epithelial cells by wild-type and ΔfliC EPEC, a rapid activation of several proinflammatory genes, including those encoding IL-8, monocyte chemoattractant protein 1, macrophage inflammatory protein 3α (MIP3α), and β-defensin 2, occurred in a FliC-dependent manner. These responses were accompanied by mitogen-activated protein kinase activation, as well as the Toll-like receptor 5 (TLR5)-dependent activation of NF-κB. At later infection time points, a subset of these proinflammatory genes (IL-8 and MIP3α) was also induced in cells infected with ΔfliC EPEC. The nonmotile A/E pathogen C. rodentium also triggered similar innate responses through a TLR5-independent but partially NF-κB-dependent mechanism. Moreover, the EPEC FliC-independent responses were increased in the absence of the locus of enterocyte effacement-encoded T3SS, suggesting that translocated bacterial effectors suppress rather than cause the FliC-independent inflammatory response. Thus, we demonstrate that infection of intestinal epithelial cells by A/E pathogens can trigger an array of proinflammatory responses from epithelial cells through both FliC-dependent and -independent pathways, expanding our understanding of the innate epithelial response to infection by these pathogens.

Attaching and Effacing Bacterial Effector NleC Suppresses Epithelial Inflammatory Responses by Inhibiting NF-κB and p38 Mitogen-Activated Protein Kinase Activation

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli are noninvasive attaching and effacing (A/E) bacterial pathogens that cause intestinal inflammation and severe diarrheal disease. These pathogens utilize a type III secretion system to deliver effector proteins into host epithelial cells, modulating diverse cellular functions, including the release of the chemokine interleukin-8 (IL-8). While studies have implicated the effectors NleE (non-locus of enterocyte effacement [LEE]-encoded effector E) and NleH1 in suppressing IL-8 release, by preventing NF-κB nuclear translocation, the impact of these effectors only partially replicates the immunosuppressive actions of wild-type EPEC, suggesting another effector or effectors are involved. Testing an array of EPEC mutants, we identified the non-LEE-encoded effector C (NleC) as also suppressing IL-8 release. Infection by ΔnleC EPEC led to exaggerated IL-8 release from infected Caco-2 and HT-29 epithelial cells. NleC localized to EPEC-induced pedestals, with signaling studies revealing NleC inhibits both NF-κB and p38 mitogen-activated protein kinase (MAPK) activation. Using Citrobacter rodentium, a mouse-adapted A/E bacterium, we found that ΔnleC and wild-type C. rodentium-infected mice carried similar pathogen burdens, yet ΔnleC strain infection led to worsened colitis. Similarly, infection with ΔnleC C. rodentium in a cecal loop model induced significantly greater chemokine responses than infection with wild-type bacteria. These studies thus advance our understanding of how A/E pathogens subvert host inflammatory responses.

Cloning Vectors and Fluorescent Proteins Can Significantly Inhibit Salmonella enterica Virulence in Both Epithelial Cells and Macrophages: Implications for Bacterial Pathogenesis Studies

ABSTRACT Plasmid vectors and fluorescent protein reporter systems are commonly used in the study of bacterial pathogenesis. Here we show that they can impair the ability of Salmonella enterica serovar Typhimurium to productively infect either cultured mammalian cells or mice. This has significant implications for studies that rely on these systems.

Prolonged antibiotic treatment induces a diabetogenic intestinal microbiome that accelerates diabetes in NOD mice

Accumulating evidence supports that the intestinal microbiome is involved in Type 1 diabetes (T1D) pathogenesis through the gut-pancreas nexus. Our aim was to determine whether the intestinal microbiota in the non-obese diabetic (NOD) mouse model played a role in T1D through the gut. To examine the effect of the intestinal microbiota on T1D onset, we manipulated gut microbes by: (1) the fecal transplantation between non-obese diabetic (NOD) and resistant (NOR) mice and (2) the oral antibiotic and probiotic treatment of NOD mice. We monitored diabetes onset, quantified CD4+T cells in the Peyer’s patches, profiled the microbiome and measured fecal short-chain fatty acids (SCFA). The gut microbiota from NOD mice harbored more pathobionts and fewer beneficial microbes in comparison with NOR mice. Fecal transplantation of NOD microbes induced insulitis in NOR hosts suggesting that the NOD microbiome is diabetogenic. Moreover, antibiotic exposure accelerated diabetes onset in NOD mice accompanied by increased T-helper type 1 (Th1) and reduced Th17 cells in the intestinal lymphoid tissues. The diabetogenic microbiome was characterized by a metagenome altered in several metabolic gene clusters. Furthermore, diabetes susceptibility correlated with reduced fecal SCFAs. In an attempt to correct the diabetogenic microbiome, we administered VLS#3 probiotics to NOD mice but found that VSL#3 colonized the intestine poorly and did not delay diabetes. We conclude that NOD mice harbor gut microbes that induce diabetes and that their diabetogenic microbiome can be amplified early in life through antibiotic exposure. Protective microbes like VSL#3 are insufficient to overcome the effects of a diabetogenic microbiome.

Active vitamin D (1,25-dihydroxyvitamin D3) increases host susceptibility to Citrobacter rodentium by suppressing mucosal Th17 responses

Vitamin D deficiency affects more that 1 billion people worldwide and is associated with an increased risk of developing a number of inflammatory/autoimmune diseases, including inflammatory bowel disease (IBD). At present, the basis for the impact of vitamin D on IBD and mucosal immune responses is unclear; however, IBD is known to reflect exaggerated immune responses to luminal bacteria, and vitamin D has been shown to play a role in regulating bacteria-host interactions. Therefore, to test the effect of active vitamin D on host responses to enteric bacteria, we gave 1,25(OH)(2)D(3) to mice infected with the bacterial pathogen Citrobacter rodentium, an extracellular microbe that causes acute colitis characterized by a strong Th1/Th17 immune response. 1,25(OH)(2)D(3) treatment of infected mice led to increased pathogen burdens and exaggerated tissue pathology. In association with their increased susceptibility, 1,25(OH)(2)D(3)-treated mice showed substantially reduced numbers of Th17 T cells within their infected colons, whereas only modest differences were noted in Th1 and Treg numbers. In accordance with the impaired Th17 responses, 1,25(OH)(2)D(3)-treated mice showed defects in their production of the antimicrobial peptide REG3γ. Taken together, these studies show that 1,25(OH)(2)D(3) suppresses Th17 T-cell responses in vivo and impairs mucosal host defense against an enteric bacterial pathogen.

Interleukin-11 Reduces TLR4-Induced Colitis in TLR2-Deficient Mice and Restores Intestinal STAT3 Signaling

BACKGROUND & AIMS The roles of intestinal Toll-like receptors (TLR) in the pathogenesis of colitis are not known. TLR2 and TLR4 appear to protect against dextran sodium sulfate-induced colitis by promoting mucosal integrity, but it is not clear whether this method of protection occurs in other models of colitis. We investigated the roles of TLR2 and TLR4 and the cell types that express these receptors during infectious colitis. METHODS We generated chimeric mice with TLR2(-/-) or TLR4(-/-) bone marrow and infected them with the bacterial pathogen Citrobacter rodentium. We assessed their susceptibility to colitis and the mechanisms of TLR-mediated mucosal integrity. RESULTS TLR2-expressing tissue resident cells prevented lethal colitis, whereas TLR4-dependent inflammatory responses of hematopoietic cells mediated intestinal damage. TLR2 expression protected against intestinal damage by maintaining epithelial barrier function and inducing expression of interleukin (IL)-11 from tissue resident cells in the muscularis mucosae, concurrent with epithelial activation of the transcription factor STAT3. Addition of exogenous IL-11 protected against the lethal colitis in TLR2-deficient mice via STAT3 activation in intestinal epithelial cells. CONCLUSIONS TLR2-dependent cytoprotective responses from tissue resident cells maintain mucosal integrity against the ultimately lethal TLR4-dependent inflammatory responses of hematopoietic cells. Whereas TLR2 protects against various noxious agents, the role of TLR4 during colitis can be either protective or damaging, depending on the stimulus. Therefore, therapeutics that reduce innate immunity (TLR2 signaling in particular) may not be beneficial to patients with colitis; they could worsen symptoms. Therapies that stimulate cytoprotective responses, like IL-11, could have benefits for patients with colitis.

Intestinal Epithelium-Specific MyD88 Signaling Impacts Host Susceptibility to Infectious Colitis by Promoting Protective Goblet Cell and Antimicrobial Responses

ABSTRACT Intestinal epithelial cells (IECs), including secretory goblet cells, form essential physiochemical barriers that separate luminal bacteria from underlying immune cells in the intestinal mucosa. IECs are common targets for enteric bacterial pathogens, with hosts responding to these microbes through innate toll-like receptors that predominantly signal through the MyD88 adaptor protein. In fact, MyD88 signaling confers protection against several enteric bacterial pathogens, including Salmonella enterica serovar Typhimurium and Citrobacter rodentium. Since IECs are considered innately hyporesponsive, it is unclear whether MyD88 signaling within IECs contributes to this protection. We infected mice lacking MyD88 solely in their IECs (IEC-Myd88 −/−) with S. Typhimurium. Compared to wild-type (WT) mice, infected IEC-Myd88 −/− mice suffered accelerated tissue damage, exaggerated barrier disruption, and impaired goblet cell responses (Muc2 and RELMβ). Immunostaining revealed S. Typhimurium penetrated the IECs of IEC-Myd88 −/− mice, unlike in WT mice, where they were sequestered to the lumen. When isolated crypts were assayed for their antimicrobial actions, crypts from IEC-Myd88 −/− mice were severely impaired in their antimicrobial activity against S. Typhimurium. We also examined whether MyD88 signaling in IECs impacted host defense against C. rodentium, with IEC-Myd88 −/− mice again suffering exaggerated tissue damage, impaired goblet cell responses, and reduced antimicrobial activity against C. rodentium. These results demonstrate that MyD88 signaling within IECs plays an important protective role at early stages of infection, influencing host susceptibility to infection by controlling the ability of the pathogen to reach and survive at the intestinal mucosal surface.