Justin M. Chan

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.

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.

SIGIRR, a Negative Regulator of TLR/IL-1R Signalling Promotes Microbiota Dependent Resistance to Colonization by Enteric Bacterial Pathogens

Enteric bacterial pathogens such as enterohemorrhagic E. coli (EHEC) and Salmonella Typhimurium target the intestinal epithelial cells (IEC) lining the mammalian gastrointestinal tract. Despite expressing innate Toll-like receptors (TLRs), IEC are innately hypo-responsive to most bacterial products. This is thought to prevent maladaptive inflammatory responses against commensal bacteria, but it also limits antimicrobial responses by IEC to invading bacterial pathogens, potentially increasing host susceptibility to infection. One reason for the innate hypo-responsiveness of IEC is their expression of Single Ig IL-1 Related Receptor (SIGIRR), a negative regulator of interleukin (IL)-1 and TLR signaling. To address whether SIGIRR expression and the innate hypo-responsiveness of IEC impacts on enteric host defense, Sigirr deficient (−/−) mice were infected with the EHEC related pathogen Citrobacter rodentium. Sigirr −/− mice responded with accelerated IEC proliferation and strong pro-inflammatory and antimicrobial responses but surprisingly, Sigirr −/− mice proved dramatically more susceptible to infection than wildtype mice. Through haematopoietic transplantation studies, it was determined that SIGIRR expression by non-haematopoietic cells (putative IEC) regulated these responses. Moreover, the exaggerated responses were found to be primarily dependent on IL-1R signaling. Whilst exploring the basis for their susceptibility, Sigirr −/− mice were found to be unusually susceptible to intestinal Salmonella Typhimurium colonization, developing enterocolitis without the typical requirement for antibiotic based removal of competing commensal microbes. Strikingly, the exaggerated antimicrobial responses seen in Sigirr −/− mice were found to cause a rapid and dramatic loss of commensal microbes from the infected intestine. This depletion appears to reduce the ability of the microbiota to compete for space and nutrients (colonization resistance) with the invading pathogens, leaving the intestine highly susceptible to pathogen colonization. Thus, SIGIRR expression by IEC reflects a strategy that sacrifices maximal innate responsiveness by IEC in order to promote commensal microbe based colonization resistance against bacterial pathogens.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection. The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

Milk Fat Globule Membrane Supplementation in Formula Modulates the Neonatal Gut Microbiome and Normalizes Intestinal Development

Breast milk has many beneficial properties and unusual characteristics including a unique fat component, termed milk fat globule membrane (MFGM). While breast milk yields important developmental benefits, there are situations where it is unavailable resulting in a need for formula feeding. Most formulas do not contain MFGM, but derive their lipids from vegetable sources, which differ greatly in size and composition. Here we tested the effects of MFGM supplementation on intestinal development and the microbiome as well as its potential to protect against Clostridium difficile induced colitis. The pup-in-a-cup model was used to deliver either control or MFGM supplemented formula to rats from 5 to 15 days of age; with mother’s milk (MM) reared animals used as controls. While CTL formula yielded significant deficits in intestinal development as compared to MM littermates, addition of MFGM to formula restored intestinal growth, Paneth and goblet cell numbers, and tight junction protein patterns to that of MM pups. Moreover, the gut microbiota of MFGM and MM pups displayed greater similarities than CTL, and proved protective against C. difficile toxin induced inflammation. Our study thus demonstrates that addition of MFGM to formula promotes development of the intestinal epithelium and microbiome and protects against inflammation.

Secreted protein acidic and rich in cysteine-induced cellular senescence in colorectal cancers in response to irinotecan is mediated by P53

Cellular senescence is another mechanism that can be exploited to achieve better chemosensitivity and greater tumor regression. Unlike apoptosis, cellular senescence can be induced at much lower concentrations of chemotherapy that are better tolerated by patients. We previously revealed that secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, may function as a modulator of chemotherapy sensitivity by enhancing apoptosis. Here, we examine the effects of SPARC on cellular senescence in the presence of chemotherapy. Cellular senescence is induced only in sensitive colorectal cancer (CRC) cells with low concentrations of irinotecan (CPT-11). However, CPT-11-resistant cells exposed to endogenous or exogenous SPARC can also be triggered into cellular senescence. This induction is associated with higher levels of p16(INK4A) and phosphorylated p53. Knock down of p16(INK4A) reduces drug-induced senescence in all cells, but knock down and overexpression of p53 modulates senescence only in cells exposed to SPARC. Furthermore, treatment of mice with SPARC and CPT-11 leads to significantly increased cellular senescence and tumor regression. The chemosensitizing effects of SPARC in CRCs are, therefore, probably mediated in part by activating cellular senescence.

Investigation of Host and Pathogen Contributions to Infectious Colitis Using the Citrobacter rodentium Mouse Model of Infection.

Citrobacter rodentium is used as a model organism to study enteric bacterial infections in mice. Infection occurs via the oral-fecal route and results in the pathogen forming attaching and effacing lesions on infected epithelial cells. Moreover, infection leads to a subsequent host-mediated form of colitis. C. rodentium infection is thus an excellent model to study infectious colitis in vivo, while the ability to genetically manipulate C. rodentium virulence genes provides the opportunity to develop clear insights into the pathogenesis of this and related infectious microbes. This chapter outlines the basic techniques involved in setting up a C. rodentium infection in mice and several different methodologies to assess the severity of the infection.