Models Of Chronic Intestinal Inflammation Research Articles

Cross-species transcriptomic signatures predict response to MK2 inhibition in mouse models of chronic inflammation

SummaryInflammatory bowel diseases (IBDs) are genetically complex and exhibit significant inter-patient heterogeneity in disease presentation and therapeutic response. Here, we show that mouse models of IBD exhibit variable responses to inhibition of MK2, a pro-inflammatory serine/threonine kinase, and that MK2 inhibition suppresses inflammation by targeting inflammatory monocytes and neutrophils in murine models. Using a computational approach (TransComp-R) that allows for cross-species comparison of transcriptomic features, we identified an IBD patient subgroup that is predicted to respond to MK2 inhibition, and an independent preclinical model of chronic intestinal inflammation predicted to be non-responsive, which we validated experimentally. Thus, cross-species mouse-human translation approaches can help to identify patient subpopulations in which to deploy new therapies.

Unique Regulation of Coupled NaCl Absorption by Inducible Nitric Oxide in a Spontaneous SAMP1/YitFc Mouse Model of Chronic Intestinal Inflammation.

In the small intestine, Na:H (NHE3) and Cl:HCO3 (DRA or PAT1) exchangers present in the brush border membrane (BBM) of absorptive villus cells are primarily responsible for the coupled absorption of NaCl, the malabsorption of which causes diarrhea, a common symptom of inflammatory bowel disease (IBD). Inducible nitric oxide (iNO), a known mediator of inflammation, is increased in the mucosa of the chronically inflamed IBD intestine. An SAMP1/YitFc (SAMP1) mouse, a spontaneous model of chronic ileitis very similar to human IBD, was used to study alterations in NaCl absorption. The SAMP1 and control AKR mice were treated with I-N(6)-(1-Iminoethyl)-lysine (L-NIL) to inhibit iNO production, and DRA/PAT1 and NHE3 activities and protein expression were studied. Though Na:H exchange activity was unaffected, Cl:HCO3 activity was significantly decreased in SAMP1 mice due to a reduction in its affinity for Cl, which was reversed by L-NIL treatment. Though DRA and PAT1 expressions were unchanged in all experimental conditions, phosphorylation studies indicated that DRA, not PAT1, is affected in SAMP1. Moreover, the altered phosphorylation levels of DRA was restored by L-NIL treatment. Inducible NO mediates the inhibition of coupled NaCl absorption by decreasing Cl:HCO3 but not Na:H exchange. Specifically, Cl:HCO3 exchanger DRA but not PAT1 is regulated at the level of its phosphorylation by iNO in the chronically inflamed intestine.

T cell stemness and ST6Gal-I mediated sialylation

Abstract Populations of both CD4 and CD8 T cells with stem-like properties have been documented in the setting of autoimmunity, chronic infection, and cancer. These cells exhibit the capacity to self renew, as well as serve as progenitors to more differentiated T cell phenotypes, but the cell intrinsic factors that promote the development and maintenance of T cell stemness are not well known. In a mouse model of chronic intestinal inflammation, we observed that a subset of effector CD4 T cells displayed many of the attributes consistent with T cell stemness; these cells expressed the key stemness transcription factor TCF1, could persist long term in vivo, and further transitioned into terminal effector cells. Importantly, these stem-like effector CD4 T cells were able to both sustain and confer intestinal inflammation. To discern molecules that contribute to the stemness phenotype, we employed gene set enrichment analyses with multiple data sets and identified the glycosyltransferase ST6Gal-I, which facilitates the addition of a2,6-linked sialic acid moieties to surface N-glycans. We found that this sialyltransferase was preferentially expressed in stem-like CD4 T cells during colitis and CD8 T cells during chronic lymphocytic choriomeningitis virus infection, and that these cell populations displayed a2,6-linked sialic acids on the cell surface. Moreover, in the colitis model, we observed that genetic deletion of ST6Gal-I in T cells correlated with diminished TCF1 expression and reduced inflammation. Furthermore, constitutive expression of ST6Gal-I by T cells was associated with the ability to promote intestinal inflammation. Together, these data highlight ST6Gal-I as a potential regulator of the T cell stemness phenotype.

A44 FATTY ACID COMPOSITION OF THE MEDITERRANEAN DIET PROTECTS AGAINST SPONTANEOUS COLITIS IN THE MUCIN-2 DEFICIENT MURINE MODEL

Abstract Background A Mediterranean diet (MD) has been proven efficacious in reducing inflammation in many chronic conditions, mediated by the interaction between diet, the gut microbiota and the immune system. The role of the MD as a dietary management strategy in the management of colitis requires further elucidation. While high fat diets have been shown to result in dysbiosis, our lab has clarified that the type of fat, not total calories derived from fat, predict gut dysbiosis and immunity in murine models of colitis. The n-6 polyunsaturated fatty acids (PUFA) promote colitis, however monounsaturated fatty acids (MUFA) protect against colitis. We hypothesize that a blend of fats, similar to the MD (high MUFA, low n-6 PUFA) will promote gut health beneficial to colitis. Aims Using a murine model of chronic intestinal inflammation, the aim of this study was to investigate the effects of dietary fatty acids on colitis by studying dietary fats in isolation from each other, as well as in a fatty acid profile similar to the blend contained in the MD. Methods Mice lacking the mucin 2 gene (Muc 2-/-) were weaned on to a 9-week, high fat (41%), isocaloric, isonitrogenous diet where the fat was derived from corn oil, olive oil, milk fat or a MD fat blend (28% MUFA, 8% SFA, 4% n-6 PUFA, 1% n-3 PUFA). The MD fat blend mimicked the fat profile consumed in the human diet. Disease activity, colon histology, cytokines (serum and colonic expression), cecal short chain fatty acids, intestinal permeability, glucose tolerance and gut microbiota (16S rRNA) were analyzed. Results Muc 2-/- fed the MD were protected from developing the most severe form of colitis, showing significantly lower disease activity and the absence of rectal prolapse. Histological damage was more severe in the corn oil and milk fat groups which coincided with an increase in infiltrating inflammatory cells and increased mucosal ulcerations. MD and milk fat diets exhibited enhanced intestinal permeability, glucose tolerance, intestinal alkaline phosphatase compared to the corn oil diet. Lower colonic mRNA levels of pro-inflammatory RELM-ß and IL-6 were also seen in the MD and MF diet in comparison to corn oil diet with the milk fat eliciting unique protective immune responses as evidenced by increased expression of IL-22 and Reg3-γ. Differences in alpha-diversity were seen between the MD and milk fat diets, beta-diversity revealed differences in taxa between diet groups. Conclusions The fatty acid profile of the MD protects against the development of spontaneous colitis in the Muc2-/- mouse model. In summary, not all dietary fats aggravate colitis, and some may be beneficial during colitis. A diet low in n-6 PUFA and high in MUFA is recommended. Funding Agencies CCCCFDR

Inhibition of APE1/Ref-1 Redox Signaling Alleviates Intestinal Dysfunction and Damage to Myenteric Neurons in a Mouse Model of Spontaneous Chronic Colitis.

Inflammatory bowel disease (IBD) associates with damage to the enteric nervous system (ENS), leading to gastrointestinal (GI) dysfunction. Oxidative stress is important for the pathophysiology of inflammation-induced enteric neuropathy and GI dysfunction. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a dual functioning protein that is an essential regulator of the cellular response to oxidative stress. In this study, we aimed to determine whether an APE1/Ref-1 redox domain inhibitor, APX3330, alleviates inflammation-induced oxidative stress that leads to enteric neuropathy in the Winnie murine model of spontaneous chronic colitis. Winnie mice received APX3330 or vehicle via intraperitoneal injections over 2 weeks and were compared with C57BL/6 controls. In vivo disease activity and GI transit were evaluated. Ex vivo experiments were performed to assess functional parameters of colonic motility, immune cell infiltration, and changes to the ENS. Targeting APE1/Ref-1 redox activity with APX3330 improved disease severity, reduced immune cell infiltration, restored GI function ,and provided neuroprotective effects to the enteric nervous system. Inhibition of APE1/Ref-1 redox signaling leading to reduced mitochondrial superoxide production, oxidative DNA damage, and translocation of high mobility group box 1 protein (HMGB1) was involved in neuroprotective effects of APX3330 in enteric neurons. This study is the first to investigate inhibition of APE1/Ref-1's redox activity via APX3330 in an animal model of chronic intestinal inflammation. Inhibition of the redox function of APE1/Ref-1 is a novel strategy that might lead to a possible application of APX3330 for the treatment of IBD.

Cyclooxygenase pathway mediates the inhibition of Na-glutamine co-transporter B0AT1 in rabbit villus cells during chronic intestinal inflammation.

In the mammalian intestine, glutamine assimilation by the absorptive villus cells is mediated by Na-glutamine co-transport, specifically by B0AT1. In a rabbit model of chronic intestinal inflammation, B0AT1 is inhibited secondary to a decrease in the number of co-transporters in the brush border membrane (BBM). This inhibition can be reversed by treatment with a broad-spectrum immune modulator such as glucocorticoid suggesting that immune inflammatory mediators may regulate B0AT1 during chronic intestinal inflammation. Arachidonic acid (AA) metabolites (AAM) are increased during chronic intestinal inflammation. However, whether AAM may regulate B0AT1 during chronic intestinal inflammation is unknown. Treatment of rabbits with ATK, to prevent the release of AAM reversed the inhibition of B0AT1. AAM are products of cyclooxygenase (COX) and/or lipoxygenase (LOX) pathways. Inhibition of COX with piroxicam, therefore reduction of prostaglandin formation in the chronically inflamed intestine, reversed the inhibition of B0AT1 to its normal levels. In contrast, inhibition of LOX with MK886, thus reduction of leukotriene formation during chronic enteritis, did not affect the inhibition of B0AT1. Kinetic studies showed that the mechanism of restoration of B0AT1 by ATK or piroxicam was secondary to the restoration of BBM co-transporter numbers. Western Blot analysis also demonstrated restoration of BBM B0AT1 co-transporter numbers. In conclusion, this study demonstrates that Na-glutamine co-transport mediated by B0AT1 in villus cells is regulated by prostaglandins rather than leukotrienes in the chronically inflamed intestine.

Unique regulation of Na-glutamine cotransporter SN2/SNAT5 in rabbit intestinal crypt cells during chronic enteritis.

The only Na‐nutrient cotransporter described in mammalian small intestinal crypt cells is SN2/SNAT5, which facilitates glutamine uptake. In a rabbit model of chronic intestinal inflammation, SN2 stimulation is secondary to an increase in affinity of the cotransporter for glutamine. However, the immune regulation of SN2 in the crypt cells during chronic intestinal inflammation is unknown. We sought to determine the mechanism of regulation of Na‐nutrient cotransporter SN2 by arachidonic acid metabolites in crypt cells. The small intestines of New Zealand white male rabbits were inflamed via inoculation with Eimeria magna oocytes. After 2‐week incubation, control and inflamed rabbits were subjected to intramuscular injections of arachidonyl trifluoromethyl ketone (ATK), piroxicam and MK886 for 48 hrs. After injections, the rabbits were euthanized and crypt cells from small intestines were harvested and used. Results: Treatment of rabbits with ATK prevented the release of AA and reversed stimulation of SN2. Inhibition of cyclooxygenase (COX) with piroxicam did not affect stimulation of SN2. However, inhibition of lipoxygenase (LOX) with MK886, thus reducing leukotriene formation during chronic enteritis, reversed the stimulation of SN2. Kinetic studies showed that the mechanism of restoration of SN2 by ATK or MK886 was secondary to the restoration of the affinity of the cotransporter for glutamine. For all treatment conditions, Western blot analysis revealed no change in SN2 protein levels. COX inhibition proved ineffective at reversing the stimulation of SN2. Thus, this study provides evidence that SN2 stimulation in crypt cells is mediated by the leukotriene pathway during chronic intestinal inflammation.

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases.

Crohn's disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn's disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNFΔRE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Fecal Microbiota and Metabolome in a Mouse Model of Spontaneous Chronic Colitis: Relevance to Human Inflammatory Bowel Disease.

Dysbiosis of the gut microbiota may be involved in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms underlying the role of the intestinal microbiome and metabolome in IBD onset and its alteration during active treatment and recovery remain unknown. Animal models of chronic intestinal inflammation with similar microbial and metabolomic profiles would enable investigation of these mechanisms and development of more effective treatments. Recently, the Winnie mouse model of colitis closely representing the clinical symptoms and characteristics of human IBD has been developed. In this study, we have analyzed fecal microbial and metabolomic profiles in Winnie mice and discussed their relevance to human IBD. The 16S rRNA gene was sequenced from fecal DNA of Winnie and C57BL/6 mice to define operational taxonomic units at ≥97% similarity threshold. Metabolomic profiling of the same fecal samples was performed by gas chromatography-mass spectrometry. Composition of the dominant microbiota was disturbed, and prominent differences were evident at all levels of the intestinal microbiome in fecal samples from Winnie mice, similar to observations in patients with IBD. Metabolomic profiling revealed that chronic colitis in Winnie mice upregulated production of metabolites and altered several metabolic pathways, mostly affecting amino acid synthesis and breakdown of monosaccharides to short chain fatty acids. Significant dysbiosis in the Winnie mouse gut replicates many changes observed in patients with IBD. These results provide justification for the suitability of this model to investigate mechanisms underlying the role of intestinal microbiota and metabolome in the pathophysiology of IBD.

Molecular mechanism of regulation of villus cell Na-K-ATPase in the chronically inflamed mammalian small intestine

Na-K-ATPase located on the basolateral membrane (BLM) of intestinal epithelial cells provides a favorable intracellular Na+ gradient to promote all Na dependent co-transport processes across the brush border membrane (BBM). Down-regulation of Na-K-ATPase activity has been postulated to alter the absorption via Na-solute co-transporters in human inflammatory bowel disease (IBD). Further, the altered activity of a variety of Na-solute co-transporters in intact villus cells has been reported in animal models of chronic enteritis. But the molecular mechanism of down-regulation of Na-K-ATPase is not known. In the present study, using a rabbit model of chronic intestinal inflammation, which resembles human IBD, Na-K-ATPase in villus cells was shown to decrease. The relative mRNA abundance of α-1 and β-1 subunits was not altered in villus cells during chronic intestinal inflammation. Similarly, the protein levels of these subunits were also not altered in villus cells during chronic enteritis. However, the BLM concentration of α-1 and β-1 subunits was diminished in the chronically inflamed intestinal villus cells. An ankyrin–spectrin skeleton is necessary for the proper trafficking of Na-K-ATPase to the BLM of the cell. In the present study, ankyrin expression was markedly diminished in villus cells from the chronically inflamed intestine resulting in depolarization of ankyrin-G protein. The decrease of Na-K-ATPase activity was comparable to that seen in ankyrin knockdown IEC-18 cells. Therefore, altered localization of Na-K-ATPase as a result of transcriptional down-regulation of ankyrin-G mediates the down-regulation of Na-K-ATPase activity during chronic intestinal inflammation.

Loss of estrogen-mediated immunoprotection underlies female gender bias in experimental Crohn's-like ileitis.

The incidence and severity of Crohn’s disease (CD) are increased in female patients. Using SAMP1/YitFc (SAMP) mice, a spontaneous model of chronic intestinal inflammation that displays histologic and pathogenic similarities to human CD, we investigated the potential mechanism(s) contributing to sex differences observed in CD. Similar to gender differences observed in CD patients, SAMP female (SAMP-F) mice displayed an earlier onset and more severe ileitis compared to SAMP males (SAMP-M). Further, T regulatory cells (Tregs) from gut-associated lymphoid tissue (GALT) of SAMP-F were reduced in frequency and impaired in their in vitro and in vivo suppressive functions compared to SAMP-M Tregs. Given the interaction between sex hormones and Treg function, we investigated the possible role of estrogen (E2) in SAMP ileitis. SAMP-M responded to exogenous E2 administration by expanding Treg frequency and reducing ileal inflammation, whereas SAMP-F were resistant. Conventional T cells (Tconv) and Tregs responded differentially to estrogen signaling, leading to distinct immunoprotective effects mediated by distinct estrogen receptor (ER) isoforms. These mechanisms were impaired in T cells from SAMP-F mice. Thus, hormone signaling influences the expansion and function of GALT Tregs in an ER-dependent manner and contributes to gender-based differences in experimental CD.

Vagal nerve stimulation modulates the dendritic cell profile in posthemorrhagic shock mesenteric lymph

Previous studies have established that posthemorrhagic shock mesenteric lymph (PHSML) contains proinflammatory mediators, while the cellular basis of PHSML is less well characterized in acute models of injury. CD103 dendritic cells (DCs) have been identified in the mesenteric lymph (ML) in models of chronic intestinal inflammation, suggesting an important role in the gut response to injury. We have previously demonstrated the ability of vagal nerve stimulation (VNS) to prevent gut barrier failure after trauma/hemorrhagic shock (T/HS); however, the ability of VNS to alter ML DCs is unknown. We hypothesized that the CD103 MHC-II DC population would change in PHSML and that VNS would prevent injury-induced changes in this population in PHSML. Male Sprague-Dawley rats were randomly assigned to trauma/sham shock or T/HS. T/HS was induced by midline laparotomy and 60 minutes of HS (blood pressure, 35 mm Hg), followed by fluid resuscitation. A separate cohort of animals underwent cervical VNS after the HS phase. Gut tissue was harvested at 2 hours after injury for histologic analysis. ML was collected during the pre-HS, HS, and post-HS phase. For flow cytometric analysis, ML cells were subjected to staining with CD103 and MHC-II antibodies, and this cell population was compared in the pre-HS and post-HS phase from the same animal. The CD4Foxp3 cell (T reg) population in the ML node (MLN) was also tested to determine effects of CD103 DC modulation in the ML. VNS reduced histologic gut injury and ML flow seen after injury. The CD103 MHC-II DC population in the PHSML was significantly decreased compared with pre-HS and was associated with decreased T reg expression in the MLN. VNS prevented the injury-induced decrease in the CD103 MHC-II+ DC population in the ML and restored the T reg population in the MLN. These findings suggest that VNS mediates the inflammatory responses in ML DCs and MLN T reg cells by affecting the set point of T/HS responsiveness.

737 A Crucial Role of PU.1 Expressing Th9 Cells in Chronic Intestinal Inflammation

Introduction: Interleukin 9 is a pleiotropic pro-inflammatory cytokine mainly produced by T cells, beside B cells and mast cells. Mounting evidence suggests that there may be a specialized subset of T cells dedicated to produce IL-9 which are so called Th9 cells. In these cells the IL-9 gene is regulated by transcription factors like PU.1 and IRF4. Also many cytokines have been investigated so far in experimental models of chronic intestinal inflammation, the role of IL-9 is largely unidentified. But high concentrations of IL-9 in the colon tissue during colitis reveal the important role of this cytokine. Methods: For the analysis of IL-9 in the development of chronic intestinal inflammation IL-9 deficient mice were used in oxazolone-colitis model. Miniendoscopic analysis has been done to monitor the manifestation of the colitis. The inflamed colon was isolated and histological sections were taken for immunohistofluorescent staining and real-time PCR analysis. For therapeutically treatment wildtype mice were given 40μg of specific anti IL-9 antibody to prevent the emergence of colitis. Results: In the experimental oxazolone-colitis model the IL-9KO mice were protected. This became evident in the miniendoscopic analysis and in the HE staining. Immunofluorescence staining shows a decrease of the IL-9 regulating transcription factor PU.1 in the IL-9 deficient mice, indicating the involvement of PU.1 in the IL-9 production. This is consistent with the fact that PU.1 is higher expressed in human biopsies of colitis patients, which illustrates the pro-inflammatory role of IL-9 in patients suffering from inflammatory bowel disease. Double staining of CD4+ and PU.1 and EPCAM and PU.1 respectively suggests that there are specialized T cells expressing the transcription factor PU.1 implying a role of Th9 cells in colitis. Further analysis of the pro-inflammatory effect of IL-9 showed that the blockage of high IL-9 concentrations with a specific anti IL-9 antibody in wildtype mice lead to a protection in oxazolone-colitis model. Conclusion: Here, we have identified a central pathogenic role for Th9 cells in chronic intestinal inflammation. This is based on the fact that IL-9 is increased in inflamed colon tissue and IL-9KO mice are protected in the experimental oxazolone-colitis model. Furthermore, administration of a blocking anti IL-9 antibody before the manifestation of colitis has a protective effect. Thus IL-9 emerges as a potentially new therapeutic target for inflammatory bowel diseases.

740 The Role of SMAD Signaling in the Pathogenesis of Pain in Chronic Pancreatitis

Introduction: Interleukin 9 is a pleiotropic pro-inflammatory cytokine mainly produced by T cells, beside B cells and mast cells. Mounting evidence suggests that there may be a specialized subset of T cells dedicated to produce IL-9 which are so called Th9 cells. In these cells the IL-9 gene is regulated by transcription factors like PU.1 and IRF4. Also many cytokines have been investigated so far in experimental models of chronic intestinal inflammation, the role of IL-9 is largely unidentified. But high concentrations of IL-9 in the colon tissue during colitis reveal the important role of this cytokine. Methods: For the analysis of IL-9 in the development of chronic intestinal inflammation IL-9 deficient mice were used in oxazolone-colitis model. Miniendoscopic analysis has been done to monitor the manifestation of the colitis. The inflamed colon was isolated and histological sections were taken for immunohistofluorescent staining and real-time PCR analysis. For therapeutically treatment wildtype mice were given 40μg of specific anti IL-9 antibody to prevent the emergence of colitis. Results: In the experimental oxazolone-colitis model the IL-9KO mice were protected. This became evident in the miniendoscopic analysis and in the HE staining. Immunofluorescence staining shows a decrease of the IL-9 regulating transcription factor PU.1 in the IL-9 deficient mice, indicating the involvement of PU.1 in the IL-9 production. This is consistent with the fact that PU.1 is higher expressed in human biopsies of colitis patients, which illustrates the pro-inflammatory role of IL-9 in patients suffering from inflammatory bowel disease. Double staining of CD4+ and PU.1 and EPCAM and PU.1 respectively suggests that there are specialized T cells expressing the transcription factor PU.1 implying a role of Th9 cells in colitis. Further analysis of the pro-inflammatory effect of IL-9 showed that the blockage of high IL-9 concentrations with a specific anti IL-9 antibody in wildtype mice lead to a protection in oxazolone-colitis model. Conclusion: Here, we have identified a central pathogenic role for Th9 cells in chronic intestinal inflammation. This is based on the fact that IL-9 is increased in inflamed colon tissue and IL-9KO mice are protected in the experimental oxazolone-colitis model. Furthermore, administration of a blocking anti IL-9 antibody before the manifestation of colitis has a protective effect. Thus IL-9 emerges as a potentially new therapeutic target for inflammatory bowel diseases.

IL-1β mediates chronic intestinal inflammation by promoting the accumulation of IL-17A secreting innate lymphoid cells and CD4+ Th17 cells

Although very high levels of interleukin (IL)-1β are present in the intestines of patients suffering from inflammatory bowel diseases (IBD), little is known about the contribution of IL-1β to intestinal pathology. Here, we used two complementary models of chronic intestinal inflammation to address the role of IL-1β in driving innate and adaptive pathology in the intestine. We show that IL-1β promotes innate immune pathology in Helicobacter hepaticus-triggered intestinal inflammation by augmenting the recruitment of granulocytes and the accumulation and activation of innate lymphoid cells (ILCs). Using a T cell transfer colitis model, we demonstrate a key role for T cell-specific IL-1 receptor (IL-1R) signals in the accumulation and survival of pathogenic CD4(+) T cells in the colon. Furthermore, we show that IL-1β promotes Th17 responses from CD4(+) T cells and ILCs in the intestine, and we describe synergistic interactions between IL-1β and IL-23 signals that sustain innate and adaptive inflammatory responses in the gut. These data identify multiple mechanisms through which IL-1β promotes intestinal pathology and suggest that targeting IL-1β may represent a useful therapeutic approach in IBD.

Tu1900 Defective NOD2 Signaling and Innate Cytokine Production in Response to MDP in Experimental Crohn's Disease (CD)

PCR. IL-34 expression in the IBD mucosa was evaluated by immunohistochemical methods. In separate experiments human PBMC and human intestinal fibroblasts (HIF) were stimulated with IL-1β and TNF-α to study induction of IL-34 mRNA and protein. Protein levels were determined by immnoblotting. Finally SAMP-1/YitFc with CD-like ileitis were treated with Dexamethazone (Dex) at a dose of 5 mg/day body weight for 7 days and IL-34 and CSF-1 levels analyzed in intestinal tissue at the end of the experiment. Results: mRNA transcript levels of IL-34 were significantly increased in UC patients compared to CD patients and HC (p=0.05). In addition, IL-34 protein was detected predominantly in UC histological sections. Further characterization revealed that IL-34 mRNA is induced upon stimulation of both human PBMC as well as HIF with TNF-α and IL-1β while it is downregulated upon stimulation with TGF-β. Treatment of SAMP-1/YitFc mice with Dex was associated with downregulation of IL-34 compared to PBS-treated SAMP mice. Interestingly CSF-1 mRNA levels were elevated in Dex-treatedmice, indicating a reciprocal secretion pattern and possibly function between IL-34 and CSF-1. Conclusion: Taken together these data demonstrate that IL-34 is specifically increased in UC patients and is modulated by proand antiinflammatory factors in both human tissue and murine model of chronic intestinal inflammation.

SAMP1/YitFc mouse strain: A spontaneous model of Crohnʼs disease-like ileitis

The SAMP1/YitFc mouse strain represents a model of Crohn's disease (CD)-like ileitis that is ideal for investigating the pathogenesis of chronic intestinal inflammation. Different from the vast majority of animal models of colitis, the ileal-specific phenotype characteristic of SAMP1/YitFc mice occurs spontaneously, without genetic, chemical, or immunological manipulation. In addition, SAMP1/YitFc mice possess remarkable similarities to the human condition with regard to disease location, histologic features, incidence of extraintestinal manifestations, and response to conventional therapies. SAMP1/YitFc mice also display a well-defined time course of a predisease state and phases of acute and chronic ileitis. As such, the SAMP1/YitFc model is particularly suitable for elucidating pathways that precede the clinical phenotype that may lead to preventive, and therefore more efficacious, intervention with the natural course of disease, or alternatively, for the development of therapeutic strategies directed against chronic, established ileitis. In this review we summarize important contributions made by our group and others that uncover potential mechanisms in the pathogenesis of CD using this unique murine model of chronic intestinal inflammation.

Novel model of colitis induced by a non-inflammatory small bowel infection (166.10)

Abstract Introduction: Chronic colitis is sustained by aberrant T cells responding to normal microbiota, although how these cells develop is poorly understood. Our aim was to evaluate whether Giardia, a parasite that infects the small intestine but does not elicit inflammation, triggers colitis in a susceptible host and to elucidate mechanisms governing decisions leading to disease. Methods: IL-10-/- mice were infected with Giardia and evaluated up to 12 wks after infection. T cells and DC were isolated and characterized by FACS. Mechanistic studies were performed using neutralizing antibodies and double knockout mice. Results: Within 2 wks of Giardia infection, IL-10-/- mice developed colitis, which persisted after parasite eradication. Infection resulted in Th1 and Th17 expansion and increased expression of neutrophil-recruiting chemokines. Surprisingly, a similar response was measured in infected WT mice, which did not develop colitis. Mechanistic studies revealed that absence of T cells or Myd88 in IL-10-/- mice prevented Giardia-induced colitis. Giardia infection induced expansion of the colonic DC population and ablation of DC led to reduced severity of inflammation. Conclusions: These results suggest that Giardia functions as a microbial trigger of colitis in IL-10-/- mice. This is a powerful new model of chronic intestinal inflammation that can provide fresh insight into mechanisms controlling homeostasis and the development of colitogenic T cells.

Evaluation of the immunoregulatory activity of intraepithelial lymphocytes in a mouse model of chronic intestinal inflammation

Intraepithelial lymphocytes (IELs) represent the first line of lymphocyte defense against the intestinal bacteria. Although previous studies have demonstrated a protective role of IELs in the development of colitis, the data supporting a regulatory role for IELs are limited. The objective of this study was to examine the suppressive activity of IELs in vitro and in vivo using a mouse model of chronic small and large bowel inflammation. Adoptive transfer of CD8α(+) IELs isolated from small intestines of wild-type (WT) mice into TCR βxδ-deficient (TCR βxδ(-/-)) recipients did not prevent or delay the onset of the disease induced by WT CD4(+)CD45RB(high) T cells. On the contrary, we observed a more rapid onset of wasting and clinical signs of intestinal inflammation when compared with animals injected with CD4(+)CD45RB(high) T cells alone. Histopathological scores of small and large bowel did not differ significantly between the two groups. Transfer of IELs alone did not produce any pathological changes. Real-time PCR analysis of intestinal tissues showed up-regulation of message for T(h)1- and macrophage-derived cytokines in colon and small bowel. Using Foxp3-GFP reporter mice, we were unable to detect any Foxp3(+) cells within the CD8α(+) IELs but did find a small population of Foxp3(+)CD4(+) IELs in the small and large bowel. Using in vitro suppression assay, we found that neither TCRαβ(+)CD8αα(+), TCRαβ(+)CD8αβ(+) nor TCRγδ(+)CD8αα(+) IELs were capable of suppressing CD4(+) T-cell proliferation. Taken together, our data do not support an immunoregulatory role for CD8α(+) IELs in a mouse model of small and large bowel inflammation.

Association between blood flow and inflammatory state in a T-cell transfer model of inflammatory bowel disease in mice

Adoptive transfer of naive T-lymphocyte subsets into lymphopenic mice initiates chronic gut inflammation that mimics several aspects of inflammatory bowel disease (IBD). Patients with IBD can have profound alterations in intestinal blood flow, but whether the same is true in the T-cell transfer model has yet to be determined. In the current study, chronic intestinal inflammation was induced in recombinase-activating gene-1-deficient (RAG(-/-)) mice by adoptive transfer of CD4(+) T-lymphocytes obtained from interleukin-10 deficient (IL-10(-/-)) mice. Four weeks later, widespread colonic inflammation was observed in the reconstituted recipients, in contrast to 2 control sets of mice injected with a different subset of lymphocytes or with vehicle alone. We observed that the resulting pathology induced in the reconstituted RAG(-/-) mice was divided distinctly into 2 subsets: 1 with blood flow near normal with very high inflammation scores, and the other with severely attenuated blood flow but with much lower signs of inflammation. Colonic and ileal blood flow rates in the latter subset of CD4(+) mice averaged only approximately 30% compared to the mice with higher inflammation scores. The lower blood flow rates were associated with greatly reduced red blood cell concentrations in the tissue, suggesting a possible loss of vascular density. In this model of chronic intestinal inflammation, mild inflammation was associated with significant decreases in blood flow.