Chronic Colitis In Mice Research Articles

Environmental Factors Modify the Severity of Acute DSS Colitis in Caspase-11-Deficient Mice.

Human and mouse studies implicate the inflammasome in the pathogenesis of inflammatory bowel diseases, though the effects in mice are variable. The noncanonical inflammasome activator caspase-11 (Casp11) reportedly attenuates acute dextran sodium sulfate (DSS) colitis in mice. However, the effects of Casp11 on chronic experimental colitis and factors that influence the impact of Casp11 on acute DSS colitis are unknown. We studied the role of Casp11 in Il10-/- mice and acute and chronic DSS colitis mouse models. We quantified colonic Casp11 mRNA using quantative polymerase chain reaction and colitis using weight loss, blinded histological scoring, IL-12/23p40 secretion by colonic explants, and fecal lipocalin-2. We determined fecal microbial composition using 16S amplicon sequencing. We detected increased colonic Casp11 mRNA in Il10-/- mice with chronic colitis, but not in mice with DSS colitis. The presence of Casp11 did not alter the severity of chronic colitis in DSS-treated or Il10-/- mice. Contrary to prior reports, we initially observed that Casp11 exacerbates acute DSS colitis. Subsequent experiments in the same animal facility revealed no effect of Casp11 on acute DSS colitis. There were pronounced stochastic changes in the fecal microbiome over this time. The majority of bacterial taxa that changed over time in wild-type vs Casp11-/- mice belong to the Clostridiales. Casp11 does not impact chronic experimental colitis, and its effects on acute DSS colitis vary with environmental factors including the microbiota, particularly Clostridiales. Stochastic drifts in intestinal microbiota composition, even in mice in the same housing facility, should be considered when interpreting studies of acute DSS colitis models.

MCC950, a specific small molecule inhibitor of NLRP3 inflammasome attenuates colonic inflammation in spontaneous colitis mice

MCC950 a potent, highly specific small molecule inhibitor of canonical and noncanonical activation of NLRP3 inflammasome has been evaluated in a multitude of NLRP3 driven inflammatory diseases. However, the effect of MCC950 on colonic inflammation has not yet been reported. In the present study we investigated the effect of MCC950 in a spontaneous chronic colitis mouse model Winnie, which mimics human ulcerative colitis. Oral administration of 40 mg/kg MCC950 commencing at Winnie week seven for three weeks significantly improved body weight gain, colon length, colon weight to body weight ratio, disease activity index and histopathological scores. MCC950 significantly suppressed release of proinflammatory cytokines IL-1β, IL-18, IL1-α, IFNγ, TNF-α, IL6, IL17, chemokine MIP1a and Nitric Oxide in colonic explants. Moreover, MCC950 resulted in a significant decrease of IL-1β release and activation of caspase-1 in colonic explants and macrophage cells isolated from Winnie. Complete inhibition with MCC950 in Winnie colonic explants shows, for the first time, the contribution of inflammatory effects resulting exclusively from canonical and noncanonical NLRP3 inflammasome activation in colitis. Taken together, our results illustrate the efficacy of MCC950 in the treatment of murine ulcerative colitis and provides avenue for a potential novel therapeutic agent for human inflammatory bowel diseases.

A characterization of pro-inflammatory cytokines in dextran sulfate sodium-induced chronic relapsing colitis mice model

Repeated cycles of dextran sulfate sodium (DSS) administration in mice, inducing chronic relapsing colitis, have been used to mimic human ulcerative colitis (UC). However, no systematic characterization of pro-inflammatory cytokines in these DSS mice has been reported. In this study, the development of colitis was examined by assessment of the disease severity and inflammation in the colon of C57BL/6 mice that received DSS. ELISA was used to analyze the levels of pro-inflammatory cytokines in serum, colon, spleen and supernatant of cultured splenocytes. mRNA levels of the above cytokines in colon and mesenteric lymph node (MLN) were measured with RT-PCR. The mice receiving three cycles of 2% DSS over a 43-day period showed a fluctuating appearance of diarrhea and bloody feces, and a significant reduction in body weight and colon length. When compared with normal control mice, an increase in TNF-α level in serum was detected in the DSS mice, along with a decrease in the amounts of TNF-α, IL-17, IL-1β and IL-6 in the colonic tissue. However, mRNA levels of these cytokines were found to be significantly increased in the colon while decreased in the MLN of the colitis mice. Further, the ELISA assay suggested a pronounced increase of TNF-α production by cultured splenocytes with PMA/ionomycin re-stimulation but no increase in its presence in spleen tissue upon DSS challenge. In conclusion, we have systematically demonstrated the dysregulation of pro-inflammatory cytokines in the DSS-induced chronic relapsing colitis model, which will provide markers to test emerging therapeutic strategies by this model.

Effects of overexpression tumor necrosis factor-related ligand-1A on T helper 9 cells in chronic experimental colitis

Objective To investigate the effects of tumor necrosis factor-related ligand-1A (TL1A) on activation of T helper 9 (Th9) cells of colonic tissues in chronic experimental colitis mice. Methods The chronic experimental colitis mice model was established with drinking dextran sulfate sodium salt (DSS). A total of 32 lymphocytes TL1A highly expressed mice and wild type (WT) mice were divided into WT control group, transgene control group, WT modeling group and transgene modeling group. The mice of control groups were administrated with distilled water. The mice of modeling groups received 3% DSS in drinking water discontinuously. The mice were sacrificed on 29 days after modeling. Body mass was measured, length of colon was recorded, scores of gross colon and the disease activity index (DAI) were calculated. The colonic morphological changes were observed by hematoxylin-eosin (H-E) staining. The lamina propria mononuclear cells (LPMC) were isolated and the number of Th9 cells was tested by flow cytometry. The levels of interleukin-9 (IL-9) in serum and LPMC were detected by enzyme-linked immunosorbent assay (ELISA). The expressions of IL-9 protein and mRNA of the colonic tissues were measured by Western blotting and real-time polymerase chain reaction (PCR), respectively. T test and single factor analysis of variance were performed for statistical analysis. Results The percentage of body mass loss of WT modeling group was lower than that of transgene modeling group (16.2%±1.0% vs 18.9%±1.2%), and the difference was statistically significant (t=4.90, P<0.05). The scores of gross colon, DAI and pathology of transgene modeling group were all higher than those of WT modeling group (2.80±0.64 vs 1.60±0.31, 2.55±0.20 vs 1.58±0.17, and 11.85±0.86 vs 9.50±0.79), and the differences were statistically significant (t=4.77, 10.45 and 5.69, all P<0.05). The number of LPMC in transgene modeling group was higher than that of WT modeling group (3.70×106±0.28×106 vs 2.65×106±0.32×106) and the difference was statistically significant (t=6.98, P<0.05). The percentage of Th9 in total CD4+ T cells of LPMC in colonic tissues of transgene modeling group was higher than that of WT modeling group (0.54%±0.04% vs 0.23%±0.03%), and the difference was statistically significant (t=17.54, P<0.05). The serum IL-9 level of transgene modeling group was higher than that of WT modeling group ((170.23±5.69) pg/mL vs (150.62±6.45) pg/mL), and the difference was statistically significant (t=6.50, P<0.05). The level of IL-9 secreted by LMPC of transgene modeling group was higher than that of WT modeling group ((265.21±8.76) pg/mL vs (237.58±10.24) pg/mL), and the difference was statistically significant (t=5.80, P<0.05). The expressions of IL-9 protein and mRNA of transgene modeling group were higher than those of WT modeling group (1.31±0.09 vs 1.18±0.03, and 8.26±1.13 vs 2.25±0.29, respectively), and the differences were statistically significant (t=3.88 and 14.57, both P<0.05). Conclusion TL1A high expression in lymphocytes can promote Th9 cells differentiation and IL-9 secretion which involved in the genesis of chronic experimental colitis. Key words: Inflammatory bowel diseases; Colitis, ulcerative; Interleukin-9; TL1A; T helper 9 cells; Mice

Intestinal Epithelial Cell Autophagy Is Required to Protect against TNF-Induced Apoptosis during Chronic Colitis in Mice

Genome-wide association studies have linked polymorphisms in the autophagy gene ATG16L1 with susceptibility to inflammatory bowel disease (IBD). However, the cell-type-specific effects of autophagy on the regulation of chronic intestinal inflammation have not been investigated. Here, we assessed theeffect of myeloid-specific or intestinal epithelial cell (IEC)-specific deletion of Atg16l1 on chronic colitis triggered by the intestinal opportunistic pathogen Helicobacter hepaticus in mice. Although Atg16l1 deficiency in myeloid cells had little effect on disease, mice selectively lacking Atg16l1 in IECs (Atg16l1VC) developed severely exacerbated pathology, accompanied by elevated pro-inflammatory cytokine secretion and increased IEC apoptosis. Using exvivo IEC organoids, we demonstrate that autophagy intrinsically controls TNF-induced apoptosis and invivo blockade of TNF attenuated the exacerbated pathology in Atg16l1VC mice. These findings suggest that the IBD susceptibility gene ATG16L1 and the process of autophagy within the epithelium control inflammation-induced apoptosis and barrier integrity to limit chronic intestinal inflammation.

P077 Aberrant Muc1 and Muc13 expression in association with Par polarity complex and Vip dysfunction in a colitis T-cell transfer model

There is emerging evidence that loss of the gastrointestinal mucosal barrier integrity is a significant contributor to the pathophysiology of inflammatory bowel disease (IBD). Mucins represent the major components of the mucus barrier and inappropriate overexpression of transmembrane mucins can affect the barrier integrity by disrupting cell polarity and cell-cell interactions resulting in tight junction dysfunction.1 Aberrant expression of Muc1, Muc4, and Muc13, in particular, is observed in colonic tissue samples of IBD patients.2, 3 Nevertheless, the role of mucins in mucosal barrier disruption in IBD remains elusive. In this study, we investigated the expression patterns of transmembrane mucins and the mediators involved in barrier homeostasis during the progression of colitis in mice. Colitis was induced in immunocompromised SCID mice by the adoptive transfer of CD4+CD25-CD62L+ T cells from the spleens of BALB/c donor mice. At 2, 4, and 6 weeks post-colitis, animals were sacrificed and colonic tissue collected for RNA and protein analysis of membrane-bound mucins (Muc1, Muc4, and Muc13), polarity proteins, and the vasointestinal polypeptide (Vip). This latter protein is implicated in the regulation of tight junction and mucin expression (Hokari et al., 2005). Apoptotic cells in tissue sections were detected using a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Our data showed that Muc1 expression was significantly increased after 4 weeks of colitis (Figure a). Interestingly, a significant decrease of Muc13 expression was observed during acute colitis (2 weeks) whereas a remarkable turnover in expression was noted during chronic colitis (4–6 weeks; Figure b). Normal intestinal MUC13 expression plays crucial roles in epithelial proliferation, differentiation and apoptosis but inappropriate overexpression of this mucin could lower the level of protection. Indeed, a higher number of apoptotic cells was seen in tissue of chronic colitis-induced mice (Figure d). Furthermore, the polarity proteins Par3 and aPKC as well as Vip (Figure c) were significantly reduced in expression from week 2 onwards suggesting a disturbance in cell polarity. Relative expression of Muc1 (a), Muc13 (b), Vip (c) in the colon of mice with acute (2w) and chronic (4–6w) colitis. (d) Number of apoptotic cells in healthy and chronic colitis mice. Asterisk indicates significance (one-way ANOVA; p < 0.05). The findings of this study highlight a potential role for Muc1 and Muc13 in mucosal barrier disruption in IBD and the underlying mechanisms involved necessitate further investigation. 1. Kufe D. Mucins in cancer: function, prognosis and therapy. Nat Rev Cancer, 2009. 2. Sheng YH, Lourie R, Lindén SK, et al.. The MUC13 cell-surface mucin protects against intestinal inflammation by inhibiting epithelial cell apoptosis. Gut, 2011. 3. Maaike V, Tim V, Ricard F, et al. Genetic and transcriptomic bases of intestinal epithelial barrier dysfunction in inflammatory bowel disease. Inflamm Bowel Dis. 2017.

P091 Inflammation in relation to mucosal barrier function in chronic colitis: a study in time

The pathogenesis of inflammatory bowel diseases (IBD) is multifactorial. Midst in the complex interaction between genetic, environmental and immunological factors is the intestinal barrier. IBD patients have been observed to have increased intestinal permeability which can partially explain the exaggerated immune response towards luminal antigens. Inflammatory mediators perpetuate this barrier defect by causing further damage to the intestinal barrier. Our aim is to investigate the interaction between permeability and inflammation in a chronic colitis mouse model. Colitis was induced in immunodeficient SCID mice by the adoptive transfer of naive T cells, isolated from BALB/c mice. Mice were sacrificed at fixed time points to study disease progression resulting in five groups: control mice (Week 0); colitis mice in week 1, 2, 4 and 6 of the experimental protocol (Week 1; Week 2; Week 4, Week 6; all n = 10). FITC-dextran was used to assess intestinal permeability, next to the messenger RNA of the tight junction proteins Claudin-1 and Occludin. After sacrifice, colonic inflammation was assessed by macro- and microscopic scoring, myeloperoxidase (MPO) activity and cytometric bead array (CBA) for TNF- α and IL-1β . Messenger RNA of transcription factors that regulate T helper (Th) cell differentiation including T-bet (Th1), GATA-3 (Th2), ROR- γt (Th17) were quantified using RT-qPCR technique. Mucosal inflammation appeared gradually in the colon of the diseased mice. Macroscopically there was a significant increase starting from Week 2 on, gradually increasing until Week 6. The same was true for the MPO activity. Microscopically, however, the first significant signs of inflammation were observed at Week 1, which then also gradually increased with the highest score at Week 6. The colonic inflammation was mainly Th1/Th2 driven, since the transcription factors T-bet and GATA-3 were significantly upregulated from respectively Week 2 and Week 4 on. TNF-α and IL-1β were significantly elevated starting from Week 1. Intestinal permeability was significantly elevated at Week 1 and remained elevated at Week 2, 4 and 6. Data are presented as mean±SEM and analysed by one-way ANOVA with multiple comparisons SNK posthoc testing. *<0.05 vs. WEEK 0 # <0.05 vs. WEEK 1. Our results show a gradual increase of intestinal inflammation over time which, for most parameters, started at Week 2. However, we observed an unexpectedly early increase in intestinal permeability at Week 1. This shows that the mucosal barrier function is of great importance in disease onset and could be an interesting therapeutic target or biomarker predicting flares.

Human filarial proteins attenuate chronic colitis in an experimental mouse model.

Encouraged by our earlier results of promising therapeutic effect of filarial recombinant proteins BmALT2, BmCys and WbL2 individually in the mouse model of acute ulcerative colitis, in this study, these proteins have been explored individually and in different combinations for their therapeutic potential in dextran sulphate sodium (DSS)-induced chronic colitis mice. These mice, treated with filarial proteins, showed reduced disease parameters including body weight loss, disease activity index, macroscopic and histopathological scores of colon and myeloperoxidase activity in colonic mucosa. Among various treatment schemes, rBmALT2+rBmCys which showed most pronounced therapeutic implication was found to downregulate the mRNA expressions of IFN-γ and TNF-α and upregulate IL-10 and TGF-β expression in the splenocytes. Also, increase in level of IgG1 and IgG2a isotypes in the sera of rBmALT2+rBmCys-treated colitis mice was noted. Activated NF-κB level was found to be reduced in the colon of treated colitis mice compared to untreated one. In conclusion, filarial proteins in combination have been shown to improve the clinicopathologic status of chronic colitis through suppression of pro-inflammatory immune response most possibly in NF-κB-dependent manner. We propose this therapeutic strategy to be tested further to be considered as an effective option in chronic colitis.

TL1A blocking ameliorates intestinal fibrosis in the T cell transfer model of chronic colitis in mice

Tumor necrosis factor like cytokine 1A (TL1A) is a member of the TNF superfamily. Accumulating evidence demonstrated the importance of TL1A in the pathogenesis of inflammatory bowel disease (IBD) and suggested a potential role of TL1A blocking in IBD therapy. Here we aimed to explore whether the anti-TL1A antibody could ameliorate intestinal inflammation and fibrosis in IBD. A T cell transfer model of chronic colitis was induced by intraperitoneal injection of CD4+CD45RBhigh naive T cells isolated from either C57BL/6 wild type (WT) mice or LCK-CD2-Tl1a-GFP transgenic (L-Tg) mice into recombinase activating gene-1-deficient (RAG−/−) mice. The colitis model mice were treated prophylactically or therapeutically with anti-Tl1a antibody or IgG isotype control. Haematoxylin and eosin staining (H&E staining), Masson's trichrome staining (MT staining) and sirius red staining were used to detect histopathological changes in colonic tissue; immunohistochemical staining was used to detect the expressions of collagen I, collagen III, TIMP1, vimentin, α-SMA and TGF-β1/Smad3. Results showed that anti-Tl1a antibody could reduce intestinal inflammation and fibrosis by inhibiting the activation of intestinal fibroblasts and reducing the collagen synthesis in the T cell transfer model of chronic colitis. The mechanism may be related to the inhibition of TGF-1/Smad3 signaling pathway.

Regulation of epithelial cell expressed C3 in the intestine – Relevance for the pathophysiology of inflammatory bowel disease?

The complement system not only plays a critical role in efficient detection and clearance of bacteria, but also in intestinal immune homeostasis as mice deficient for key complement components display enhanced intestinal inflammation upon experimental colitis. Because underlying molecular mechanisms for this observation are unclear, we investigated the crosstalk between intestinal epithelial cells (IEC), bacteria and the complement system in the course of chronic colitis.Surprisingly, mouse intestinal epithelial cell lines constitutively express high mRNA levels of complement component 3 (C3), Toll-like receptor 2 (Tlr2) and Tlr4. Stimulation of these cells with lipopolysaccharide (LPS), but not with flagellin, LD-muramyldipeptide or peptidoglycan, triggered increased C3 expression, secretion and activation. Stimulation of the C3aR on these cell lines with C3a resulted in an increase of LPS-triggered pro-inflammatory response. Tissue biopsies from C57BL/6J mice revealed higher expression of C3, Tlr1, Tlr2 and Tlr4 in colonic primary IECs (pIECs) compared to ileal pIECs, while in germ-free mice no differences in C3 protein expression was observed. In DSS-induced chronic colitis mouse models, C3 mRNA expression was upregulated in colonic biopsies and ileal pIECs with elevated C3 protein in the lamina propria, IECs and the mucus. Notably, increased C3b opsonization of mucosa-attached bacteria and decreased fecal full-length C3 protein was observed in DSS-treated compared to untreated mice. Of significant interest, non-inflamed and inflamed colonic biopsy samples from CD but not UC patients displayed exacerbated C3 expression compared to controls.These findings suggest that a novel TLR4-C3 axis could control the intestinal immune response during chronic colitis.

Transcriptional modulation of pattern recognition receptors in chronic colitis in mice is accompanied with Th1 and Th17 response

Pattern recognition receptors (PRRs) may contribute to inflammatory bowel diseases (IBD) development due to their microbial-sensing ability and the unique microenvironment in the inflamed gut. In this study, the PRR mRNA expression profile together with T cell-associated factors in the colon was examined using a chronic colitis mice model. 8–12 week old C57BL/6 mice were exposed to multiple dextran sodium sulfate (DSS) treatments interspersed with a rest period to mimic the course of chronic colitis. The clinical features and histological data were collected. The mRNA expressions of colonic PRRs, T cell-associated components were measured. Finally, the colons were scored for Foxp3+ cells. During chronic colitis, the histological data, but not the clinical manifestations demonstrated characteristic inflammatory symptoms in the distal colon. In contrast to acute colitis, the expression of all Toll-like receptors (Tlrs), except Tlr5 and Tlr9, was unaffected after repeated DSS treatments. The expression of Nod1 was decreased, while Nod2 increased. After third DSS treatment, only the expressions of Tlr3 and Tlr4 were significantly enhanced. Unlike other PRRs, decreased Tlr5 and increased Tlr9 mRNA expression persisted during the chronic colitis period. As the colitis progress, only the mRNA expression of Ifnγ and Il17 staid increased during chronic colitis, while the acute colitis-associated increase of Il23, and Il10 and Il12 was abolished. Finally, increased histological score of Foxp3+ cell in colon was found during the chronic colitis period. This study provides an expression pattern of PRRs during chronic colitis that is accompanied by a Th1- and Th17 cell-mediated immune response.

The Extracts of Morinda officinalis and Its Hairy Roots Attenuate Dextran Sodium Sulfate-Induced Chronic Ulcerative Colitis in Mice by Regulating Inflammation and Lymphocyte Apoptosis.

Morinda officinalis is beneficial for the treatment of inflammatory bowel disease (IBD). The hairy root with higher genetic and biochemical stability cultured from M. officinalis might have similar effects to treat IBD. In this study, the main chemical composition of the root extracts of M. officinalis (MORE) native plant and the hairy root extract of M. officinalis (MOHRE) was compared by quantitative HPLC. The difference of their therapeutic effects and potential mechanism was evaluated using 3% dextran sodium sulfate-induced chronic colitis in mice and T lymphocytes in vitro. The results found that MOHRE possesses many specific peaks unobserved in the chromatogram of native plant. The content of iridoids in the MORE (3.10%) and MOHRE (3.01%) is somewhat similar but quite different for their anthraquinones’s content (0.14 and 0.66%, respectively). Despite all this, treatment with both MORE and MOHRE significantly attenuated the symptoms of colitis, including diarrhea, body weight loss, colon shortening, histological damage, and decreased inflammatory cytokine levels. In addition, they dose-dependently increased the apoptosis of T lymphocyte in vivo and in vitro. And, the differences for treatment effects on ulcerative colitis (UC) between them both in this study were mostly insignificant. The results demonstrated that the effects of MORE and MOHRE for the treatment of UC are similar, although there are a few difference on their chemical composition, indicating the hairy root cultured from M. officinalis might be able to replace its native plant on treatment of UC. The successful derivation of a sustainable hairy root culture provides a model system to study the synthetic pathways for bioactive metabolites, which will make the use of bioreactors to largely produce traditional medicine become reality.

Colon tumorigenesis mediated by Tollip in neutrophils.

Abstract Innate neutrophils have ability to confer either pro- or anti-tumor immune responses. However, molecular mechanisms responsible for the cell polarization are not well understood. Here, we aim to identify and characterize the neutrophil phenotypes in chronic colitis (CC) mouse model with use of AOM and DSS. We identify that Toll-interacting-protein (Tollip) may play a key role in modulating the long-term polarization and function of neutrophils as well as pro-tumorigenesis. We observed that Tollip deficient mice had improved clinical score and reduced generation of colon polyps when subjected to prolonged challenges with AOM and DSS. At the cellular level, Tollip deficient mice challenged with AOM-DSS had distinct neutrophil activation profiles as compared to wild type mice, including the differential expression of cell surface co-stimulatory molecules, migratory behaviors, as well as cytokine expression profiles. Adoptive transfer of neutrophils from Tollip deficient mice to wild type mice recapitulated the outcome of Tollip deficient mice. Based on the in vivo and in vitro studies, we conclude that Tollip may play a key role during the polarization of innate neutrophils involved in the modulation of chronic colitic tumor environment. Further studies with regard to the molecular mechanisms may enable a better understanding of neutrophil modulation in colon tumorigenesis.

Spray-drying process preserves the protective capacity of a breast milk-derived Bifidobacterium lactis strain on acute and chronic colitis in mice

Gut microbiota dysbiosis plays a central role in the development and perpetuation of chronic inflammation in inflammatory bowel disease (IBD) and therefore is key target for interventions with high quality and functional probiotics. The local production of stable probiotic formulations at limited cost is considered an advantage as it reduces transportation cost and time, thereby increasing the effective period at the consumer side. In the present study, we compared the anti-inflammatory capacities of the Bifidobacterium animalis subsp. lactis (B. lactis) INL1, a probiotic strain isolated in Argentina from human breast milk, with the commercial strain B. animalis subsp. lactis BB12. The impact of spray-drying, a low-cost alternative of bacterial dehydration, on the functionality of both bifidobacteria was also investigated. We showed for both bacteria that the spray-drying process did not impact on bacterial survival nor on their protective capacities against acute and chronic colitis in mice, opening future perspectives for the use of strain INL1 in populations with IBD.

P044 Therapeutic effects of mesenchymal stem cells induced regulatory dendritic cells on chronic colitis model in mice

Background: Mesenchymal stem cells (MSCs), emerging cell therapeutics for inflammatory bolwel disease (IBD), have demonstrated immunomodulatory effects by interactions between T cells and dendritic cells (DCs). In this study, we investigated whether MSCs could differentiate mature DCs (mDCs) into a distinct regulatory DCs and these DCs could alleviate inflammation through a T cell modulation in DSS induced chronic colitis mouse model. Methods: DCs were derived from mouse bone marrow and different subsets of DCs were characterized by fluorescence-activated cell sorting (FACS). Quantitative polymerase chain reaction (q-PCR) for anti-inflammatory (interleukin (IL)-10 and transforming growth factor (TGF)–β) and pro-inflammatory cytokines (interferon (IFN)-γ, tumor necrosis factor (TNF)-α and IL-6) were performed in both in vitro DC subsets and in vivo. Using splenocytes co-cultured with MSC induced DCs, q-PCRs for CD4, CD8, CD25 and Foxp3, and Western blotting for Foxp3 were performed to measure the expression of regulatory T (Treg) cell markers in vitro. In vivo dextran sulfate sodium (DSS) chronic colitis model, Expression of IL-10, TGF–β, STAT3 and Foxp3 were compared among 5 groups (control, DSS-colitis, immature DCs only, MSCs, and MSC-induced DCs). Results: MSC-induced DCs expressed CD11c+ CD80lo CD86locell surface marker. These DCs secreted high levels of anti-inflammatory cytokines, whereas production of pro-inflammatory cytokines were diminished in vitro. Also, the levels of mRNA and protein of Foxp3+, CD4+ and CD25+ were increased in splenocytes co-cultured with MSC-induced DCs. In addition, intraperitoneal injection of MSC induced DCs significantly increased anti-inflammatory cytokines at both m-RNA and protein level and enhanced differentiation of FOXP3+ Treg cells, resulting in considerabe amelioration of chronic colitis and improvement of survival in DSS treated mice. Conclusions: These results showed that MSCs induced DCs alliviated chronic colitis by promotin g Treg cells differentiation and the secretion of anti-inflammatory cytokines, providing potential application of MSC-induced DCs in the treatment of IBD.

Sodium selenite ameliorates dextran sulfate sodium-induced chronic colitis in mice by decreasing Th1, Th17, and γδT and increasing CD4(+)CD25(+) regulatory T-cell responses.

AIMTo assess the effect of sodium selenite on the severity of dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice.METHODSMice were randomly divided into four groups (n = 10/group): normal group, selenium (Se) group, chronic colitis group, and Se + chronic colitis group. The mice were sacrificed on day 26. Survival rates, clinical symptoms, colon length, and histological changes were determined. The percentages and absolute numbers of immune system cells in the lamina propria lymphocytes (LPL) of the colon, the expression of mRNA in colon tissue, and the concentrations of Th1, Th17, and Treg cytokines in LPL from the large intestine, were measured.RESULTSSe significantly ameliorated the symptoms of colitis and histological injury (P < 0.05 each), increasing the proportions of neutrophils and CD4+ CD25+ T cells (P < 0.05 each) and decreasing the proportions of γδT cells, CD4+, CD4+CD44+, and CD4+ CD69+ T cells in LPL (P < 0.05 each). Moreover, Se reduced the expression of IL-6, IFN-γ, IL-17A, IL-21, T-bet, and RORγt (P < 0.05 each), but enhanced the expression of IL-10 and Foxp3 (P < 0.05 each).CONCLUSIONThese results suggest that Se protects against DSS-induced chronic colitis perhaps by increasing the number of CD4(+)CD25(+) Tregs that suppress the secretion of proinflammatory cytokines and populations of Th1, Th17, and γδT cells.

Elevated IL-23R Expression and Foxp3+Rorgt+ Cells in Intestinal Mucosa During Acute and Chronic Colitis.

BackgroundIL-23/IL-23R signaling plays a pivotal role during the course of inflammatory bowel diseases (IBD). However, the underlying mechanisms are poorly characterized. Foxp3+ regulatory T cells are critical in the maintenance of gut immune homeostasis and therefore are important in preventing the development of IBD. This study was performed to clarify the association between IL-23/IL-23R signaling and Foxp3+ regulatory T cells in colitis.Material/MethodsAcute and chronic mouse colitis models were established by administering mice DSS in drinking water. IL-23R, IL-23, IL-I7, and IFN-γ expression level, as well as regulatory T cell, Th17-, and Th1-related transcription factors Foxp3, RORγt, and T-bet were assayed by real-time PCR. The frequency of Foxp3+ RORγt+ cells in a Foxp3+ cell population in colon mucosa during acute and chronic colitis was evaluated through flow cytometry. The signaling pathway mediated by IL-23R in the colon mucosa from acute colitis mice and chronic colitis mice was monitored by Western blot analysis.ResultsWe detected elevated IL-23R, IL-23, and IFN-γ expression in colon mucosa during acute and chronic colitis and found increased IL-17 in acute colitis mice. Transcription factors Foxp3 and T-bet were elevated in colon mucosa during acute and chronic colitis. Phosphorylation of Stat3 was greatly enhanced, indicating the activation of IL-23R function in colitis mice. The percentage of Foxp3+ T cells in acute and chronic colitis mice was comparable to control mice, but there was a 2-fold increase of Foxp3+ RORγt+ cells among the Foxp3+ cell population in acute and chronic colitis mice compared to control mice.ConclusionsThese findings indicate that the induction of Foxp3+ RORγt+ T cells could be enhanced during inflammation in the intestine where IL-23R expression is greatly induced. Our study highlights the importance of IL-23R expression level and the instability of Foxp3+ regulatory T cells in the development of inflammatory bowel diseases.

Novel PPARγ Modulator GED-0507-34 Levo Ameliorates Inflammation-driven Intestinal Fibrosis

Intestinal fibrosis is mainly associated with Crohn's disease and is defined as a progressive and excessive deposition of extracellular matrix components. No specific antifibrotic therapies are available. In this study, we evaluate the antifibrotic effect of a novel 5-ASA analog able to activate the peroxisome proliferator-activated receptor γ, named GED-0507-34 Levo. Colonic fibrosis was induced in 110 C57BL/6 mice by 3 cycles of 2.5% (wt/vol) dextran sulfate sodium administration for 6 weeks. The preventive effects of oral daily GED (30 mg · kg(-1) · d(-1)) administration were evaluated using a macroscopic and histological score and also through biological endpoints. Expression of main markers of myofibroblasts activation was determined in transforming growth factor (TGF-β)-stimulated intestinal fibroblasts and epithelial cells. GED improved macroscopic and microscopic intestinal lesions in dextran sulfate sodium-treated animals and reduced the profibrotic gene expression of Acta2, COL1a1, and Fn1 by 1.48-folds (P < 0.05), 1.93-folds (P < 0.005), and 1.03-fold (P < 0.05), respectively. It reduced protein levels of main markers of fibrosis (α-SMA and Collagen I-II) and the main TGF-β/Smad pathway components. GED also decreased the interleukin-13 and connective tissue growth factor expression by 1.89-folds (P < 0.05) and 2.2-folds (P < 0.005), respectively. GED inhibited TGF-β-induced activation of both fibroblast and intestinal epithelial cell lines, by regulating mRNA expression of α-SMA and fibronectin, and restoring the TGF-β-induced loss of intestinal epithelial cell markers. GED treatment also reduced the TGF-β and ACTA1 expression in primary human intestinal fibroblasts from ulcerative colitis patients. GED ameliorates intestinal fibrosis in dextran sulfate sodium-induced chronic colitis in mice and regulates major profibrotic cellular and molecular mechanisms.

Identification of novel anti-inflammatory probiotic strains isolated from pulque.

Probiotics are live microorganisms which when administered in adequate amounts, confer health benefits on the host. Their use is more and more widespread for both prevention and treatment of diseases, including traveler’s diarrhea and inflammatory bowel diseases (IBDs). In this work, we isolated and characterized novel candidate probiotic strains from pulque (xaxtle), a traditional Mexican alcoholic fermented beverage. A total of 14 strains were obtained from xaxtle samples isolated from three different Mexican regions. Species identification was performed by biochemical methods and 16S rRNA gene targeted PCR. The isolates belonged to the Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus brevis, and Lactobacillus composti phylogenetic groups, with L. brevis being the most dominant group. Bacteria were tested for lysozyme, low pH, and bile acid resistance. Moreover, the strains were tested for adherence to human intestinal epithelial cells and screened for their immunomodulatory properties using a cellular model. Selected bacterial strains with anti-inflammatory properties were then tested in vivo in a dinitro-benzene sulfonic acid (DNBS)-induced chronic colitis mouse model, and weight loss, gut permeability, and cytokine profiles were measured as readouts of inflammation. One of the selected strains, Lactobacillus sanfranciscensis LBH1068, improved mice health as observed by a reduction of weight loss, significant decreases in gut permeability, and cytokine modulation. Altogether, our results highlighted the potential of lactobacilli isolated from pulque and in particular the strain L. sanfranciscensis LBH1068 as a novel probiotic to treat IBD.

IL-33 alleviates DSS-induced chronic colitis in C57BL/6 mice colon lamina propria by suppressing Th17 cell response as well as Th1 cell response

Interleukin (IL)-33, a member of the IL-1 cytokine family, is associated with autoimmune diseases including inflammatory bowel diseases (IBD). A few studies on animal models have shown that IL-33 can suppress Th1 cell response and improve Th2 cell response in mesenteric lymph nodes (MLN) and sera. However, there is little data published about the effect of IL-33 on Th17 cell in and Th1/Th2 cell in colon lamina propria. The aim of this study was to investigate the effect of IL-33 on Th17 cell in colon lamina propria of mice with dextran sulfate sodium (DSS) induced chronic colitis. We studied the influence of IL-33 on colonic tissue injury and clinical symptoms of colitis. The T cell subsets were measured by flow cytometry and the production of cytokines secreted by lamina propria lymphocytes (LPL) was measured by Enzyme-Linked Immunosorbent Assay (ELISA) and quantitative real-time PCR. We have found that rIL-33 treatment led to a significant alleviation of DSS induced chronic colitis as evidenced by 1) alleviation of weight loss, DAI, macroscopic changes and histological score; 2) down-regulating the rates and absolute cell numbers of Th17 and Th1 cell in LPL; 3) inducing secretion of lower levels of IFN-γ and IL-17A. It is therefore concluded that IL-33 may play a therapeutic role in DSS-induced chronic colitis in mice by suppressing Th17 response and switching Th1 to Th2 response.