Distinct inactivated bacterial-based immune modulators vary in their therapeutic efficacies for treating disease based on the organ site of pathology.

Recent developments in understanding how the functional phenotype of the innate immune system is programmed has led to paradigm-shifting views on immunomodulation. These advances have overturned two long-held dogmas: (1) only adaptive immunity confers immunological memory; and, (2) innate immunity lacks specificity. This work describes the observation that innate immune effector cells appear to be differentially recruited to specific pathological sites when mobilized by distinct inactivated bacterial-based stimuli administered subcutaneously. The studies presented suggest that the immune system, upon detecting the first signs of a potential infection by a specific pathogen, tends to direct its resources to the compartment from which that pathogen is most likely originating. The findings from this work puts forth the novel hypothesis that the immunotherapeutic efficacy of a microbial-based stimulus for innate immune mobilization depends on the correct selection of the microbial species used as the stimulant and its relationship to the organ in which the pathology is present.

Immune Stimulation Using a Gut Microbe-Based Immunotherapy Reduces Disease Pathology and Improves Barrier Function in Ulcerative Colitis.

Background: Current ulcerative colitis (UC) treatments are focused on symptom management primarily via immune suppression. Despite the current arsenal of immunosuppressant treatments, the majority of patients with UC still experience disease progression. Importantly, aggressive long-term inhibition of immune function comes with consequent risk, such as serious infections and malignancy. There is thus a recognized need for new, safe and effective treatment strategies for people living with UC that work upstream of managing the symptoms of the disease. The objective of this study was to evaluate a microbial-based treatment, QBECO, that functions to productively activate rather than suppress mucosal immune function as a novel approach to treat UC. Methods: Two established models of experimental colitis, namely chemically-induced DSS colitis and the spontaneous colitis that develops in Muc2 deficient mice, were used to assess whether QBECO treatment could ameliorate gastrointestinal disease. A small exploratory 16-week QBECO open-label trial was subsequently conducted to test the safety and tolerability of this approach and also to determine whether similar improvements in clinical disease and histopathology could be demonstrated in patients with moderate-to-severe UC. Results: QBECO treatment successfully reduced inflammation and promoted mucosal and histological healing in both experimental models and in UC patients. The preclinical models of colitis showed that QBECO ameliorated mucosal pathology, in part by reducing inflammatory cell infiltration, primarily that induced by neutrophils and inflammatory T cells. The most rapid and noticeable change observed in QBECO treated UC patients was a marked reduction in rectal bleeding. Conclusion: Collectively, this work demonstrates for the first time that strategically activating immune function rather than suppressing it, not only does not worsen colitis induced-damage, but may lead to an objective reduction in UC disease pathology.

15-epi-Lipoxin A4, Resolvin D2, and Resolvin D3 Induce NF-κB Regulators in Bacterial Pneumonia.

Specialized proresolving mediators (SPMs) decrease NF-κB activity to prevent excessive tissue damage and promote the resolution of acute inflammation. Mechanisms for NF-κB regulation by SPMs remain to be determined. In this study, after LPS challenge, the SPMs 15-epi-lipoxin A4 (15-epi-LXA4), resolvin D1, resolvin D2, resolvin D3, and 17-epi-resolvin D1 were produced in vivo in murine lungs. In LPS-activated human bronchial epithelial cells, select SPMs increased expression of the NF-κB regulators A20 and single Ig IL-1R-related molecule (SIGIRR). Of interest, 15-epi-LXA4 induced A20 and SIGIRR in an lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2) receptor-dependent manner in epithelial cells and in murine pneumonia. This SPM regulated NF-κB-induced cytokines to decrease pathogen-mediated inflammation. In addition to dampening lung inflammation, surprisingly, 15-epi-LXA4 also enhanced pathogen clearance with increased antimicrobial peptide expression. Taken together, to our knowledge these results are the first to identify endogenous agonists for A20 and SIGIRR expression to regulate NF-κB activity and to establish mechanisms for NF-κB regulation by SPMs for pneumonia resolution.

Resolvin D3 and Aspirin-Triggered Resolvin D3 Are Protective for Injured Epithelia.

Acute lung injury is a life-threatening condition caused by disruption of the alveolar-capillary barrier leading to edema, influx of inflammatory leukocytes, and impaired gas exchange. Specialized proresolving mediators biosynthesized from essential fatty acids, such as docosahexaenoic acid, have tissue protective effects in acute inflammation. Herein, we found that the docosahexaenoic acid-derived mediator resolvin D3 (RvD3): 4S,11R,17S-trihydroxydocosa-5Z,7E,9E,13Z,15E,19Z-hexaenoic acid was present in uninjured lungs, and increased significantly 24 to 72 hours after hydrochloric acid-initiated injury. Because of its delayed enzymatic degradation, we used aspirin-triggered (AT)-RvD3: 4S,11R,17R-trihydroxydocosa-5Z,7E,9E,13Z,15E,19Z-hexaenoic acid, a 17R-epimer of RvD3, for in vivo experiments. Histopathological correlates of acid injury (alveolar wall thickening, edema, and leukocyte infiltration) were reduced in mice receiving AT-RvD3 1 hour after injury. AT-RvD3-treated mice had significantly reduced edema, as demonstrated by lower wet/dry weight ratios, increased epithelial sodium channel γ expression, and more lymphatic vessel endothelial hyaluronan receptor 1-positive vascular endothelial growth factor receptor 3-positive lymphatic vessels. Evidence for counterregulation of NF-κB by RvD3 and AT-RvD3 was seen in vitro and by AT-RvD3 in vivo. Increases in lung epithelial cell proliferation and bronchoalveolar lavage fluid levels of keratinocyte growth factor were observed with AT-RvD3, which also promoted cutaneous re-epithelialization. Together, these data demonstrate protective actions of RvD3 and AT-RvD3 for injured mucosa that accelerated restoration of epithelial barrier and function.

Goblet Cell Derived RELM-ß Recruits CD4+ T Cells during Infectious Colitis to Promote Protective Intestinal Epithelial Cell Proliferation.

Enterohemorrhagic Escherichia coli and related food and waterborne pathogens pose significant threats to human health. These attaching/effacing microbes infect the apical surface of intestinal epithelial cells (IEC), causing severe diarrheal disease. Colonizing the intestinal luminal surface helps segregate these microbes from most host inflammatory responses. Based on studies using Citrobacter rodentium, a related mouse pathogen, we speculate that hosts rely on immune-mediated changes in IEC, including goblet cells to defend against these pathogens. These changes include a CD4+ T cell-dependent increase in IEC proliferation to replace infected IEC, as well as altered production of the goblet cell-derived mucin Muc2. Another goblet cell mediator, REsistin-Like Molecule (RELM)-ß is strongly induced within goblet cells during C. rodentium infection, and was detected in the stool as well as serum. Despite its dramatic induction, RELM-ß's role in host defense is unclear. Thus, wildtype and RELM-ß gene deficient mice (Retnlb-/-) were orally infected with C. rodentium. While their C. rodentium burdens were only modestly elevated, infected Retnlb-/- mice suffered increased mortality and mucosal ulceration due to deep pathogen penetration of colonic crypts. Immunostaining for Ki67 and BrDU revealed Retnlb-/- mice were significantly impaired in infection-induced IEC hyper-proliferation. Interestingly, exposure to RELM-ß did not directly increase IEC proliferation, rather RELM-ß acted as a CD4+ T cell chemoattractant. Correspondingly, Retnlb-/- mice showed impaired CD4+ T cell recruitment to their infected colons, along with reduced production of interleukin (IL)-22, a multifunctional cytokine that directly increased IEC proliferation. Enema delivery of RELM-ß to Retnlb-/- mice restored CD4+ T cell recruitment, concurrently increasing IL-22 levels and IEC proliferation, while reducing mucosal pathology. These findings demonstrate that RELM-ß and goblet cells play an unexpected, yet critical role in recruiting CD4+ T cells to the colon to protect against an enteric pathogen, in part via the induction of increased IEC proliferation.

Metalloprotease NleC suppresses host NF-κB/inflammatory responses by cleaving p65 and interfering with the p65/RPS3 interaction.

Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p65¹â»³8 fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p65¹â»³8 fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a 'specifier' subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.

Noncanonical inflammasome activation of caspase-4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens.

Inflammasome-mediated host defenses have been extensively studied in innate immune cells. Whether inflammasomes function for innate defense in intestinal epithelial cells, which represent the first line of defense against enteric pathogens, remains unknown. We observed enhanced Salmonella enterica serovar Typhimurium colonization in the intestinal epithelium of caspase-11-deficient mice, but not at systemic sites. In polarized epithelial monolayers, siRNA-mediated depletion of caspase-4, a human ortholog of caspase-11, also led to increased bacterial colonization. Decreased rates of pyroptotic cell death, a host defense mechanism that extrudes S. Typhimurium-infected cells from the polarized epithelium, accounted for increased pathogen burdens. The caspase-4 inflammasome also governs activation of the proinflammatory cytokine, interleukin (IL)-18, in response to intracellular (S. Typhimurium) and extracellular (enteropathogenic Escherichia coli) enteric pathogens, via intracellular LPS sensing. Therefore, an epithelial cell-intrinsic noncanonical inflammasome plays a critical role in antimicrobial defense at the intestinal mucosal surface.

A novel mouse model of Campylobacter jejuni gastroenteritis reveals key pro-inflammatory and tissue protective roles for Toll-like receptor signaling during infection.

Campylobacter jejuni is a major source of foodborne illness in the developed world, and a common cause of clinical gastroenteritis. Exactly how C. jejuni colonizes its host's intestines and causes disease is poorly understood. Although it causes severe diarrhea and gastroenteritis in humans, C. jejuni typically dwells as a commensal microbe within the intestines of most animals, including birds, where its colonization is asymptomatic. Pretreatment of C57BL/6 mice with the antibiotic vancomycin facilitated intestinal C. jejuni colonization, albeit with minimal pathology. In contrast, vancomycin pretreatment of mice deficient in SIGIRR (Sigirr(-/-)), a negative regulator of MyD88-dependent signaling led to heavy and widespread C. jejuni colonization, accompanied by severe gastroenteritis involving strongly elevated transcription of Th1/Th17 cytokines. C. jejuni heavily colonized the cecal and colonic crypts of Sigirr(-/-) mice, adhering to, as well as invading intestinal epithelial cells. This infectivity was dependent on established C. jejuni pathogenicity factors, capsular polysaccharides (kpsM) and motility/flagella (flaA). We also explored the basis for the inflammatory response elicited by C. jejuni in Sigirr(-/-) mice, focusing on the roles played by Toll-like receptors (TLR) 2 and 4, as these innate receptors were strongly stimulated by C. jejuni. Despite heavy colonization, Tlr4(-/-)/Sigirr(-/-) mice were largely unresponsive to infection by C. jejuni, whereas Tlr2(-/-)/Sigirr(-/-) mice developed exaggerated inflammation and pathology. This indicates that TLR4 signaling underlies the majority of the enteritis seen in this model, whereas TLR2 signaling had a protective role, acting to promote mucosal integrity. Furthermore, we found that loss of the C. jejuni capsule led to increased TLR4 activation and exaggerated inflammation and gastroenteritis. Together, these results validate the use of Sigirr(-/-) mice as an exciting and relevant animal model for studying the pathogenesis and innate immune responses to C. jejuni.

Intestinal epithelium-specific MyD88 signaling impacts host susceptibility to infectious colitis by promoting protective goblet cell and antimicrobial responses.

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 RELMß). 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.

DNBS/TNBS colitis models: providing insights into inflammatory bowel disease and effects of dietary fat.

Inflammatory Bowel Diseases (IBD), including Crohn's Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48 hr and rats fed SO by 72 hr. Weight gain resumed after 72 hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5 days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

CD4+ T cells drive goblet cell depletion during Citrobacter rodentium infection.

Both idiopathic and infectious forms of colitis disrupt normal intestinal epithelial cell (IEC) proliferation and differentiation, although the mechanisms involved remain unclear. Recently, we demonstrated that infection by the attaching and effacing murine pathogen Citrobacter rodentium leads to a significant reduction in colonic goblet cell numbers (goblet cell depletion). This pathology depends on T and/or B cells, as Rag1(-/-) mice do not suffer this depletion during infection, instead suffering high mortality rates. To address the immune mechanisms involved, we reconstituted Rag(-/-) mice with either CD4(+) or CD8(+) T cells. Both T cell subsets increased Rag1(-/-) mouse survival during infection, with mice that received CD8(+) T cells developing colonic ulcers but not goblet cell depletion. In contrast, mice that received CD4(+) T cells showed goblet cell depletion in concert with exaggerated IEC proliferation. To define the possible involvement of T cell-derived cytokines, we infected gamma interferon receptor gene knockout (IFN-γR(-/-)) mice and wild-type mice given interleukin 17A (IL-17A) neutralizing antibodies and found that IFN-γ signaling was required for both goblet cell depletion and increased IEC proliferation. Immunostaining revealed that C. rodentium cells preferentially localized to nonhyperplastic crypts containing numerous goblet cells, whereas hyperplastic, goblet cell-depleted crypts appeared protected from infection. To address whether goblet cell depletion benefits the C. rodentium-infected host, we increased goblet cell numbers using the γ-secretase inhibitor dibenzazepine (DBZ), which resulted in greatly increased pathogen burdens and mortality rates. These results demonstrate that goblet cell depletion reflects host immunomodulation of IEC homeostasis and reflects a novel host defense mechanism against mucosal-adherent pathogens.

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.

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.

Regulated virulence controls the ability of a pathogen to compete with the gut microbiota.

The virulence mechanisms that allow pathogens to colonize the intestine remain unclear. Here, we show that germ-free animals are unable to eradicate Citrobacter rodentium, a model for human infections with attaching and effacing bacteria. Early in infection, virulence genes were expressed and required for pathogen growth in conventionally raised mice but not germ-free mice. Virulence gene expression was down-regulated during the late phase of infection, which led to relocation of the pathogen to the intestinal lumen where it was outcompeted by commensals. The ability of commensals to outcompete C. rodentium was determined, at least in part, by the capacity of the pathogen and commensals to grow on structurally similar carbohydrates. Thus, pathogen colonization is controlled by bacterial virulence and through competition with metabolically related commensals.

Innate host responses to enteric bacterial pathogens: a balancing act between resistance and tolerance.

Infection by enteric bacterial pathogens activates pathogen recognition receptors, leading to innate responses that promote host defence. While responses that promote host 'resistance' to infection, through the release of antimicrobial mediators, or the recruitment of inflammatory cells aimed at clearing the infection are best known, recent studies have begun to identify additional innate driven responses that instead promote intestinal tissue repair and host survival. Described as infection 'tolerance' responses, we and others have primarily studied these responses in the Citrobacter rodentium infection model. In this review we discuss the impact of innate resistance mechanisms on host defence, and describe how 'tolerance' responses act primarily on the intestinal epithelium, triggering epithelial cell proliferation, repair or promoting barrier function. Resistance and tolerance responses appear to work together, with tolerance repairing the tissue injury caused by resistance driven inflammation. Tolerance responses fit a pattern where innate immunity and inflammation are tightly regulated in the gastrointestinal tract. Moreover, tolerance may have developed due to the successful subversion and avoidance of host resistance by enteric bacterial pathogens. Further studies are needed to clarify the contribution of different pathogen recognition receptors to tolerance and resistance responses against bacterial pathogens, in the gut or in other host tissues.

Attaching and effacing bacterial effector NleC suppresses epithelial inflammatory responses by inhibiting NF-κB and p38 mitogen-activated protein kinase activation.

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.

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.

Muc2 protects against lethal infectious colitis by disassociating pathogenic and commensal bacteria from the colonic mucosa.

Despite recent advances in our understanding of the pathogenesis of attaching and effacing (A/E) Escherichia coli infections, the mechanisms by which the host defends against these microbes are unclear. The goal of this study was to determine the role of goblet cell-derived Muc2, the major intestinal secretory mucin and primary component of the mucus layer, in host protection against A/E pathogens. To assess the role of Muc2 during A/E bacterial infections, we inoculated Muc2 deficient (Muc2(-/-)) mice with Citrobacter rodentium, a murine A/E pathogen related to diarrheagenic A/E E. coli. Unlike wildtype (WT) mice, infected Muc2(-/-) mice exhibited rapid weight loss and suffered up to 90% mortality. Stool plating demonstrated 10-100 fold greater C. rodentium burdens in Muc2(-/-) vs. WT mice, most of which were found to be loosely adherent to the colonic mucosa. Histology of Muc2(-/-) mice revealed ulceration in the colon amid focal bacterial microcolonies. Metabolic labeling of secreted mucins in the large intestine demonstrated that mucin secretion was markedly increased in WT mice during infection compared to uninfected controls, suggesting that the host uses increased mucin release to flush pathogens from the mucosal surface. Muc2 also impacted host-commensal interactions during infection, as FISH analysis revealed C. rodentium microcolonies contained numerous commensal microbes, which was not observed in WT mice. Orally administered FITC-Dextran and FISH staining showed significantly worsened intestinal barrier disruption in Muc2(-/-) vs. WT mice, with overt pathogen and commensal translocation into the Muc2(-/-) colonic mucosa. Interestingly, commensal depletion enhanced C. rodentium colonization of Muc2(-/-) mice, although colonic pathology was not significantly altered. In conclusion, Muc2 production is critical for host protection during A/E bacterial infections, by limiting overall pathogen and commensal numbers associated with the colonic mucosal surface. Such actions limit tissue damage and translocation of pathogenic and commensal bacteria across the epithelium.

Loss of single immunoglobulin interlukin-1 receptor-related molecule leads to enhanced colonic polyposis in Apc(min) mice.

BACKGROUND & AIMS: Commensal bacteria can activate signaling by the Toll-like and interleukin-1 receptors (TLR and IL-1R) to mediate pathogenesis of inflammatory bowel diseases and colitis-associated cancer. We investigated the role of the single immunoglobulin IL-1 receptor-related (SIGIRR) molecule, a negative regulator of TLR and IL-1R signaling, as a tumor suppressor to determine whether SIGIRR controls cell-cycle progression, genetic instability, and colon tumor initiation by modulating commensal TLR signaling in the gastrointestinal tract. METHODS: We analyzed adenomatous polyposis coli (Apc)min/+/Sigirr-/- mice for polyps, microadenomas, and anaphase bridge index. Commensal bacteria were depleted from mice with antibiotics. Akt, mammalian target of rapamycin (mTOR), and beta-catenin pathways were examined by immunoblotting and immunohistochemistry. Loss of heterozygosity of Apc and expression of cytokines and proinflammatory mediators were measured by nonquantitative or quantitative polymerase chain reaction. RESULTS: Apcmin/+/Sigirr-/- mice had increased loss of heterozygosity of Apc and microadenoma formation, resulting in spontaneous colonic polyposis, compared with Apcmin/+/Sigirr+/+ mice. The increased colonic tumorigenesis that occurred in the Apcmin/+/Sigirr-/- mice depended on the presence of commensal bacteria in the gastrointestinal tract. Cell proliferation and chromosomal instability increased in colon crypt cells of the Apcmin/+/Sigirr-/- mice. Akt, mTOR, and their substrates were hyperactivated in colon epithelium of Apcmin/+/Sigirr-/- mice in response to TLR or IL-1R ligands. Inhibition of the mTOR pathway by rapamycin reduced formation of microadenomas and polyps in the Apcmin/+/Sigirr-/- mice. CONCLUSIONS: SIGIRR acts as a tumor suppressor in the colon by inhibiting TLR-induced, mTOR-mediated cell-cycle progression and genetic instability.