Development Of Colitis In Mice Research Articles

P102 A small molecule selective integrin α4β7 inhibitor demonstrates efficacy in a chronic model of Inflammatory Bowel Disease

Abstract Background Integrin α4β7 regulates the recruitment of T cells to intestinal mucosa through its interaction with mucosal addressin cell adhesion molecule (MAdCAM)-1. Disruption of this interaction has been clinically validated for the treatment of inflammatory bowel diseases (IBD) by the anti-α4β7 antibody vedolizumab. The current study was aimed at elucidating the preclinical efficacy of MT-103, a potent and selective small molecule α4β7 inhibitor in the clinically relevant CD4+CD45RBhi T cell transfer (TCT) colitis mouse model. Methods Mice were administered either MT-103 via subcutaneously implanted minipumps across 3 dose cohorts (3, 10, or 30 mg/ml), or an antibody specific for IL12p40 (25 mg/kg) or α4β7 (30 mg/kg), through intraperitoneal injections for 7 weeks. Colitis development was evaluated via readouts including body weights, gross colon weight by length ratios, colonic tissue gene expression, and histopathological scores. Results MT-103 significantly inhibited the development of colitis in mice at all 3 doses equivalently, as evident from significantly improved gross colon weight/length ratios in comparison to vehicle controls which presented with moderate-severe colitis (Figure 1A). MT-103 treatment also led to a significant protection from weight loss that occurred in vehicle-treated mice. Importantly, mice treated with MT-103 exhibited remarkably improved histopathology scores, including inflammation, hyperplasia, and gland loss compared to vehicle controls (Figure 1B). Furthermore, broad gene transcriptional analysis showed that MT-103 broadly suppressed pro-inflammatory pathways and processes within colonic tissues. Specifically, MT-103-induced downregulation of Ifng, Il17a, and Il1b, besides reduced Cd3 as a surrogate metric of T cell trafficking inhibition to the colon, and conversely enabled upregulation of anti-inflammatory cytokine Il10. The colitis-protective effects of MT-103 were equivalent, if not superior, to the saturating doses of α4β7 mAb or anti-IL12p40 mAb when compared to their respective vehicle controls. Conclusion Treatment with MT-103 protects from colitis by restoring colonic tissue homeostasis as measured by body fitness and an array of immunological and histopathological assessments. These proof-of-concept data demonstrate an α4β7-specific small molecule, MT-103, can occlude pathogenic T cells from initiating disease in a chronic IBD model, equivalent to an α4β7 blocking antibody.

HSF1 promotes CD69+ Treg differentiation to inhibit colitis progression.

Regulatory T cells (Tregs) are critical for generating and maintaining peripheral tolerance. Treg-based immunotherapy is valuable for the clinical management of diseases resulting from dysregulation of immune tolerance. However, the lack of potency is a potential limitation of Treg therapy. In addition, CD69 positive-Treg (CD69+ Treg) represent a newly identified subset of Tregs with potent immune suppressive capability. Methods: Foxp3 YFP-Cre CD69 fl/fl and CD4 Cre CD69 fl/fl mice were generated to determine the relevance of CD69 to Treg. Chromatin Immunoprecipitation Assay (ChIP) and luciferase Assay were performed to detect the regulation of CD69 transcription by heat shock transcription factor 1(HSF1). Gene expression was measured by western blotting and qRT-PCR. The differentiation of naive T cells to CD69+Foxp3+ iTregs was determined by flow cytometry. The immunosuppressive ability of Tregs was analyzed by ELISA and flow cytometry. Colon inflammation in mice was reflected by changes in body weight and colon length, the disease activity index (DAI), and H&E staining of colon tissues. Results: Induced Tregs (iTregs) from CD4 Cre CD69 fl/fl mice failed to alleviate colitis. The transcription factor HSF1 interacted with the promoter of the CD69 gene to prompt its transcription during Treg differentiation. Genetic and chemical inhibition of HSF1 impaired CD69+ Treg differentiation and promoted the pathogenesis of colitis in mice. In contrast, HSF1 protein stabilized by inhibiting its proteasomal degradation promoted CD69+ Treg differentiation and alleviated colitis in mice. Moreover, adoptive transfer of iTregs with HSF1 stabilization by proteasome inhibitor (PSI) dramatically prevented the development of colitis in mice and was accompanied by decreased production of pro-inflammatory cytokines and reduced accumulation of pro-inflammatory lymphocytes in colitis tissue, whereas Tregs induced in the absence of PSI were less stable and ineffective in suppressing colitis. Conclusions: HSF1 promotes CD69+ Tregs differentiation by activating the CD69 transcription, which is critical for the immunosuppressive function of Tregs. Stabilization of HSF1 by PSIs results in the efficient generation of Tregs with high potency to treat colitis and probably other autoimmune diseases involving Tregs deficiency.

Blocking DCIR mitigates colitis and prevents colorectal tumors by enhancing the GM-CSF-STAT5 pathway.

Dendritic cell immunoreceptor (DCIR; Clec4a2), a member of the C-type lectin receptor family, plays important roles in homeostasis of the immune and bone systems. However, the intestinal role of this molecule is unclear. Here, we show that dextran sodium sulfate (DSS)-induced colitis and azoxymethane-DSS-induced intestinal tumors are reduced in Clec4a2-/- mice independently of intestinal microbiota. STAT5 phosphorylation and expression of Csf2 and tight junction genes are enhanced, while Il17a and Cxcl2 are suppressed in the Clec4a2-/- mouse colon, which exhibits reduced infiltration of neutrophils and myeloid-derived suppressor cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) administration ameliorates DSS colitis associated with reduced Il17a and enhanced tight junction gene expression, whereas anti-GM-CSF exacerbates symptoms. Furthermore, anti-NA2, a ligand for DCIR, ameliorates colitis and prevents colorectal tumors. These observations indicate that blocking DCIR signaling ameliorates colitis and suppresses colonic tumors, suggesting DCIR as a possible target for the treatment of these diseases.

MTA1 aggravates experimental colitis in mice by promoting transcription factor HIF1A and up-regulating AQP4 expression

Experimental colitis can persist as a chronic disease, accompanied with an underlying risk of development into colorectal cancer. Metastasis-associated protein 1 (MTA1), as a chromatin modifier, exerts notable association with multiple diseases, including colitis. The current study aims to investigate the mechanism of MTA1/HIF1A/AQP4 axis in experimental colitis in mice. First, experimental colitis mouse models were established using dextran sulfate sodium (DSS) and in vitro colonic epithelial cells FHC inflammation models were with lipopolysaccharide (LPS) for determination of MTA1 and HIF1A expressions. It was found that MTA1 and HIF1A were both highly-expressed in experimental colitis samples. Results of dual-luciferase reporter gene assay and ChIP assay further revealed that MTA1 activated HIF1A, and subsequently induced AQP4 transcription to up-regulate AQP4 in experimental colitis. Following loss- and gain-function, the effects of MTA1/HIF1A/AQP4 axis on apoptosis and viability of colon epithelial cells were detected by a combination of TUNEL staining and flow cytometry, and CCK-8 assay. It was observed that silencing of MAT1 in the FHC and NCM460 cells reduced IL-1β and TNF-α expressions induced by LPS. Meanwhile, AQP4 promoted LPS-induced inflammation, and exacerbated apoptosis of colon epithelial cells and augmented experimental colitis development in mice. In vivo experiments further verified that TGN-020 treatment effectively alleviated DSS-induced experimental colitis in mice and diminished apoptosis of colon epithelial cells. Altogether, MTA1 may promote AQP4 transcription by activating HIF1A, thus exacerbating DSS-induced experimental colitis in mice, which provides a novel direction for the treatment of experimental colitis.

IFN-γ+ cytotoxic CD4+ T lymphocytes are involved in the pathogenesis of colitis induced by IL-23 and the food colorant Red 40

The food colorant Red 40 is an environmental risk factor for colitis development in mice with increased expression of interleukin (IL)-23. This immune response is mediated by CD4+ T cells, but mechanistic insights into how these CD4+ T cells trigger and perpetuate colitis have remained elusive. Here, using single-cell transcriptomic analysis, we found that several CD4+ T-cell subsets are present in the intestines of colitic mice, including an interferon (IFN)-γ-producing subset. In vivo challenge of primed mice with Red 40 promoted rapid activation of CD4+ T cells and caused marked intestinal epithelial cell (IEC) apoptosis that was attenuated by depletion of CD4+ cells and blockade of IFN-γ. Ex vivo experiments showed that intestinal CD4+ T cells from colitic mice directly promoted apoptosis of IECs and intestinal enteroids. CD4+ T cell-mediated cytotoxicity was contact-dependent and required FasL, which promoted caspase-dependent cell death in target IECs. Genetic ablation of IFN-γ constrained IL-23- and Red 40-induced colitis development, and blockade of IFN-γ inhibited epithelial cell death in vivo. These results advance the understanding of the mechanisms regulating colitis development caused by IL-23 and food colorants and identify IFN-γ+ cytotoxic CD4+ T cells as a new potential therapeutic target for colitis.

CCR2 promotes monocyte recruitment and intestinal inflammation in mice lacking the interleukin-10 receptor

Macrophages are a heterogeneous population of mononuclear phagocytes abundantly distributed throughout the intestinal compartments that adapt to microenvironmental specific cues. In adult mice, the majority of intestinal macrophages exhibit a mature phenotype and are derived from blood monocytes. In the steady-state, replenishment of these cells is reduced in the absence of the chemokine receptor CCR2. Within the intestine of mice with colitis, there is a marked increase in the accumulation of immature macrophages that demonstrate an inflammatory phenotype. Here, we asked whether CCR2 is necessary for the development of colitis in mice lacking the receptor for IL10. We compared the development of intestinal inflammation in mice lacking IL10RA or both IL10RA and CCR2. The absence of CCR2 interfered with the accumulation of immature macrophages in IL10R-deficient mice, including a novel population of rounded submucosal Iba1+ cells, and reduced the severity of colitis in these mice. In contrast, the absence of CCR2 did not reduce the augmented inflammatory gene expression observed in mature intestinal macrophages isolated from mice lacking IL10RA. These data suggest that both newly recruited CCR2-dependent immature macrophages and CCR2-independent residual mature macrophages contribute to the development of intestinal inflammation observed in IL10R-deficient mice.

Covalent drug discovery targeting G protein-coupled receptors

G protein-coupled receptors (GPCRs) play pivotal roles in converting physicochemical stimuli due to environmental changes to intracellular responses. After ligand stimulation, many GPCRs are desensitized and then recycled or degraded through phosphorylation and β-arrestin-dependent internalization, an important process to maintain protein quality control of GPCRs. However, it is unknown how GPCRs with low β-arrestin sensitivity are controlled. Here we unmasked a β-arrestin-independent GPCR internalization, named Redox-dependent Alternative Internalization (REDAI), focusing on β-arrestin-resistant purinergic P2Y6 receptor (P2Y6R). P2Y6R is highly expressed in macrophage and pathologically contributes to the development of colitis in mice. Natural electrophiles including in functional foods induce REDAI-mediated P2Y6R degradation leading to anti-inflammation in macrophages. Prevention of Cys220 modification on P2Y6R resulted in aggravation of the colitis. These results strongly suggest that targeting REDAI on GPCRs will be a breakthrough strategy for the prevention and treatment of inflammatory diseases.

Inhibitor of Differentiation-2 Protein Ameliorates DSS-Induced Ulcerative Colitis by Inhibiting NF-κB Activation in Neutrophils.

The loss of inhibitor of differentiation-2 (ID2) could lead to the development of colitis in mice, supplementation with exogenous ID2 protein might be a potential strategy to ameliorate colitis. In this study, the effects of ID2 protein supplementation on Dextran sodium sulfate (DSS)-induced colitis were investigated. Firstly, we confirmed that the expression of ID2 was reduced in the colon tissues of DSS-induced colitis mice and patients with ulcerative colitis (UC). Then, we constructed a recombinant plasmid containing the human Id2 gene and expressed it in Escherichia coli (E. coli) successfully. After purification and identification, purified hID2 could ameliorate DSS-induced colitis efficiently in mice by improving disease symptoms, decreasing the levels of proinflammatory cytokines in colon tissues, maintaining the integrity of intestinal barrier and reducing the infiltration of neutrophils and macrophages in the colon. Further study showed that hID2 could be endocytosed efficiently by neutrophils and macrophages, and hID2 lost its protection function against colitis when neutrophils were depleted with an anti-Gr-1 antibody. hID2 decreased the mRNA levels of IL-6, IL-1β and TNF-α in lipopolysaccharides (LPS)-stimulated neutrophils and efficiently inhibited the activation of NF-κB signalling pathway in neutrophils. Interestingly, hID2 showed a synergistic role in inhibition of NF-κB activation with pyrrolidine dithiocarbamic acid (PDTC), an inhibitor of NF-κB activation. Therefore, this study demonstrated the potential use of hID2 to treat UC, and hID2 protein might be a promising anti-inflammatory agent that targets the NF-κB signalling pathway in neutrophils.

Food colorants metabolized by commensal bacteria promote colitis in mice with dysregulated expression of interleukin-23

Both genetic predisposition and environmental factors appear to play a role in inflammatory bowel disease (IBD) development. Genetic studies in humans have linked the interleukin (IL)-23 signaling pathway with IBD, but the environmental factors contributing to disease have remained elusive. Here, we show that the azo dyes Red 40 and Yellow 6, the most abundant food colorants in the world, can trigger an IBD-like colitis in mice conditionally expressing IL-23, or in two additional animal models in which IL-23 expression was augmented. Increased IL-23 expression led to generation of activated CD4+ Tcells that expressed interferon-γ and transferred disease to mice exposed to Red 40. Colitis induction was dependent on the commensal microbiota promoting the azo reduction of Red 40 and generation of a metabolite, 1-amino-2-naphthol-6-sulfonate sodium salt. Together these findings suggest that specific food colorants represent novel risk factors for development of colitis in mice with increased IL-23 signaling.

Intestinal Phospholipid Disequilibrium Initiates an ER Stress Response That Drives Goblet Cell Necroptosis and Spontaneous Colitis in Mice.

Background & AimsPatients with ulcerative colitis have low concentrations of the major membrane lipid phosphatidylcholine (PC) in gastrointestinal mucus, suggesting that defects in colonic PC metabolism might be involved in the development of colitis. To determine the precise role that PC plays in colonic barrier function, we examined mice with intestinal epithelial cell (IEC)-specific deletion of the rate-limiting enzyme in the major pathway for PC synthesis: cytidine triphosphate:phosphocholine cytidylyltransferase-α (CTαIKO mice).MethodsColonic tissue of CTαIKO mice and control mice was analyzed by histology, immunofluorescence, electron microscopy, quantitative polymerase chain reaction, Western blot, and thin-layer chromatography. Histopathologic colitis scores were assigned by a pathologist blinded to the experimental groupings. Intestinal permeability was assessed by fluorescein isothiocyanate–dextran gavage and fecal microbial composition was analyzed by sequencing 16s ribosomal RNA amplicons. Subsets of CTαIKO mice and control mice were treated with dietary PC supplementation, antibiotics, or 4-phenylbutyrate.ResultsInducible loss of CTα in the intestinal epithelium reduced colonic PC concentrations and resulted in rapid and spontaneous colitis with 100% penetrance in adult mice. Colitis development in CTαIKO mice was traced to a severe and unresolving endoplasmic reticulum stress response in IECs with altered membrane phospholipid composition. This endoplasmic reticulum stress response was linked to the necroptotic death of IECs, leading to excessive loss of goblet cells, formation of a thin mucus barrier, increased intestinal permeability, and infiltration of the epithelium by microbes.ConclusionsMaintaining the PC content of IEC membranes protects against colitis development in mice, showing a crucial role for IEC phospholipid equilibrium in colonic homeostasis. SRA accession number: PRJNA562603.

Bacteroides thetaiotaomicron and Lactobacillus johnsonii modulate intestinal inflammation and eliminate fungi via enzymatic hydrolysis of the fungal cell wall

Alterations to the gut microbiota can cause an amplification of the inflammatory response to intestinal pathogens. We assessed the effect of Bacteroides thetaiotaomicron and Lactobacillus johnsonii on the elimination of Candida species and whether restoration of these two anaerobic bacteria could attenuate the development of colitis in mice. In this study, L. johnsonii and B. thetaiotaomicron interacted directly with Candida species and induced a degradation of the fungal cell wall, mediated via chitinase-like and mannosidase-like activities, which promoted the inhibition of Candida species growth. In the DSS-induced colitis model, oral administration of L. johnsonii and B. thetaiotaomicron to mice reduced the overgrowth of Escherichia coli, Enterococcus faecalis and Candida glabrata populations and resulted in a significant reduction in inflammatory parameters. L. johnsonii and B. thetaiotaomicron decreased pro-inflammatory mediators and enhanced the anti-inflammatory cytokine response with high TLR9 expression and chitinase-like protein-1 activation, which promoted the elimination of C. glabrata from the gut. Overall, these findings provide evidence that L. johnsonii and B. thetaiotaomicron decrease the development of colitis mediated by TLR9 and promote the elimination of C. glabrata from the gut via chitinase-like and mannosidase-like activities.

Inhibiting PGGT1B Disrupts Function of RHOA, Resulting in T-cell Expression of Integrin α4β7 and Development of Colitis in Mice

It is not clear how regulation of T-cell function is altered during development of inflammatory bowel diseases (IBD). We studied the mechanisms by which geranylgeranyltransferase-mediated prenylation controls T-cell localization to the intestine and chronic inflammation. We generated mice with T-cell-specific disruption of the geranylgeranyltransferase type I, beta subunit gene (Pggt1b), called Pggt1bΔCD4 mice, or the ras homolog family member A gene (Rhoa), called RhoaΔCD4 mice. We also studied mice with knockout of CDC42 or RAC1 and wild-type mice (controls). Intestinal tissues were analyzed by histology, multiphoton and confocal microscopy, and real-time polymerase chain reaction. Activation of CDC42, RAC1, and RHOA were measured with G-LISA, cell fractionation, and immunoblots. T cells and lamina propria mononuclear cells from mice were analyzed by flow cytometry or transferred to Rag1-/- mice. Mice were given injections of antibodies against integrin alpha4beta7 or gavaged with the RORC antagonist GSK805. We obtained peripheral blood and intestinal tissue samples from patients with and without IBD and analyzed them by flow cytometry. Pggt1bΔCD4 mice developed spontaneous colitis, characterized by thickening of the intestinal wall, edema, fibrosis, accumulation of T cells in the colon, and increased expression of inflammatory cytokines. Compared with control CD4+ T cells, PGGT1B-deficient CD4+ T cells expressed significantly higher levels of integrin alpha4beta7, which regulates their localization to the intestine. Inflammation induced by transfer of PGGT1B-deficient CD4+ T cells to Rag1-/- mice was blocked by injection of an antibody against integrin alpha4beta7. Lamina propria of Pggt1bΔCD4 mice had increased numbers of CD4+ T cells that expressed RORC and higher levels of cytokines produced by T-helper 17 cells (granulocyte-macrophage colony-stimulating factor, interleukin [IL]17A, IL17F, IL22, and tumor necrosis factor [TNF]). The RORC inverse agonist GSK805, but not antibodies against IL17A or IL17F, prevented colitis in Pggt1bΔCD4 mice. PGGT1B-deficient CD4+ T cells had decreased activation of RHOA. RhoAΔCD4 mice had a similar phenotype to Pggt1bΔCD4 mice, including development of colitis, increased numbers of CD4+ T cells in colon, increased expression of integrin alpha4beta7 by CD4+ T cells, and increased levels of IL17A and other inflammatory cytokines in lamina propria. T cells isolated from intestinal tissues from patients with IBD had significantly lower levels ofPGGT1B than tissues from individuals without IBD. Loss of PGGT1B from T cells in mice impairs RHOA function, increasing CD4+ T-cell expression of integrin alpha4beta7 and localization to colon, resulting in increased expression of inflammatory cytokines and colitis. T cells isolated from gut tissues from patients with IBD have lower levels of PGGT1B than tissues from patients without IBD.

Nitric Oxide Is Involved in Activation of Toll-Like Receptor 4 Signaling through Tyrosine Nitration of Src Homology Protein Tyrosine Phosphatase 2 in Murine Dextran Sulfate-Induced Colitis.

Ulcerative colitis is characterized by colonic mucosal bleeding and ulceration, often with repeated active and remission stages. One factor in ulcerative colitis development is increased susceptibility to commensal bacteria and lipopolysaccharide (LPS). LPS activates macrophages to release nitric oxide (NO) through Toll-like receptor 4 (TLR4) signaling. However, whether NO is beneficial or detrimental to colitis remains controversial. In this study, we investigated whether NO enhances the development of colitis in mice treated with dextran sulfate sodium (DSS) and inflammation in cells treated with low-dose LPS. An NO donor, NOC18, induced colitis and increased CD14 protein and nitrotyrosine levels in colonic macrophages from mice treated with DSS for 7 d (molecular weight: 5000). In the mouse peritoneal macrophage cell line RAW264.7 stimulated with 3 ng/mL LPS, NO activated the CD14-TLR4-nuclear factor kappa B (NF-κB) axis. Low-dose LPS stimulation did not change the levels of signal transducer and activator of transcription (STAT) 3 phosphorylation, CD14, inducible NO synthase, interleukin (IL)-6, or NF-κB. In addition, low-dose LPS increased phosphorylation of src homology protein tyrosine phosphatase 2 (SHP2), a negative regulator of STAT3 phosphorylation. However, NO decreased SHP2 phosphorylation and significantly activated the downstream signaling molecules. NO increased SHP2 nitration in LPS-stimulated RAW264.7 cells and DSS-treated mice. These results indicate that SHP2 nitration in macrophages might be involved in activation of the CD14-TLR4-NF-κB axis through STAT3 signaling in mice with DSS-induced colitis.

Increased Anticolitic Effects in C57BL/6 Mice Based on Functional Ingredients of Ramyeon Noodles and Soup.

We investigated the preventive effects of Ramyeon made from brown rice-sorghum noodles and doenjang-bamboo salt soup (BS+DB) on colitis induced by dextran sulfate sodium (DSS) in C57BL/6 mice. Noodles were prepared with 10% brown rice and 10% sorghum powders added to wheat flour and potato powder, and soup was made using starter fermented (SF) doenjang powder (32%) and bamboo salt (13.5%). The experimental animals were divided into five groups: Normal, Control, BS (brown rice and sorghum noodles)+DB (doenjang and bamboo salt soup) (BS+DB Ramyeon), W (white flour noodles, commercial one)+DB, W+dC (W+doenjang commercial soup), and W + D (W+SF doenjang powder [100%]). The BS+DB and W + D groups showed significant reduction of DSS-induced colitis symptoms (P < .05). Doenjang soup (100%) (W+D) also showed a strong anticolitic effect even though the noodles were prepared with W. Histological observation of the colon revealed that BS+DB Ramyeon markedly alleviated colitis development in mice. Serum protein and mRNA levels of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) were significantly suppressed in colon tissue of the BS+DB group compared with those of the W+DB and W+dC groups. BS+DB Ramyeon also reduced colon mRNA levels of inducible nitric oxide synthase and cyclooxygenase-2 compared with those of other groups (P < .05). Our results show that modification of noodle ingredients using brown rice and sorghum as well as alteration of soup composition using doenjang and bamboo salt improved the health benefits of Ramyeon.

Enteric Delivery of Regenerating Family Member 3 alpha Alters the Intestinal Microbiota and Controls Inflammation in Mice With Colitis

Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic (TG) expression of the human regenerating family member 3 alpha gene (REG3A) alters the fecal microbiota and affects development of colitis in mice. We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulfate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S ribosomal RNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expressing hREG3A were monitored for DSS-induced colitis after cohousing or feeding feces from control mice. Colitis was induced in another set of control and hREG3A-TG mice by administration of trinitrobenzene sulfonic acid; some mice were given intrarectal injections of the hREG3A protein. Colon tissues were collected from mice and analyzed by histology and immunohistochemistry to detect mucin 2, as well as by 16S ribosomal RNA fluorescence in situ hybridization, transcriptional analyses, and quantitative polymerase chain reaction. We measured levels of reactive oxygen species (ROS) in bacterial cultures and fecal microbiota using 2',7'-dichlorofluorescein diacetate and flow cytometry. The fecal microbiota of mice that express hREG3A had a significant shift in composition, compared with control mice, with enrichment of Clostridiales (Ruminococcaceae, Lachnospiraceae) and depletion of Bacteroidetes (Prevotellaceae); the TG mice developed less-severe colitis following administration of DSS than control mice, associated with preserved gut barrier integrity and reduced bacterial translocation, epithelial inflammation, and oxidative damage. A similar shift in the composition of the fecal microbiota occurred after a few months in TG mice heterozygous for REG3A that harbored a wild-type maternal microbiota at birth; these mice developed less-severe forms of colitis following DSS administration. Cohoused and germ-free mice fed feces from REG3A-TG mice and given DSS developed less-severe forms of colitis and had reduced lipopolysaccharide activation of the toll-like receptor 4 and increased survival times compared with mice not fed feces from REG3A-TG mice. REG3A TG mice developed only mild colonic inflammation after exposure to 2,4,6-trinitrobenzene sulfonic acid, compared with control mice. Control mice given intrarectal hREG3A and exposed to 2,4,6-trinitrobenzene sulfonic acid showed less colon damage and inflammation than mice not given intrarectal hREG3A. Fecal samples from REG3A-TG mice had lower levels of ROS than feces from control mice during DSS administration. Addition of hREG3A to bacterial cultures reduced levels of ROS and increased survival of oxygen-sensitive commensal bacteria (Faecalibacterium prausnitzii and Roseburia intestinalis). Mice with hepatocytes that express hREG3A, which travels to the intestinal lumen, are less sensitive to colitis than control mice. We found hREG3A to alter the colonic microbiota by decreasing levels of ROS. Fecal microbiota from REG3A-TG mice protect non-TG mice from induction of colitis. These findings indicate a role for reduction of oxidative stress in preserving the gut microbiota and its ability to prevent inflammation.

Inhibitory Effects of Nobiletin on Colitis were Associated with Altered Gut Microbiota

Understanding how dietary compounds such as nobiletin alter the colonic environment is critical in studying their roles in modulating gut health and gut associated diseases. In this study, we determined the effects of dietary nobiletin treatment on dextran sodium sulfate (DSS)‐induced colitis as well as the composition of gut microbiota in mice. The results revealed that nobiletin treatment significantly decreased the severity of colonic inflammation in DSS‐treated mice, evidenced by the reduced production of pro‐inflammatory cytokines (i.e., GM‐CSF, INF‐γ, IL‐1β, IL‐2, IL‐6, KC/GRO, and TNF‐α) in the colonic mucosa, increased colon length, and decreased disease activity index and histologic score of inflammation (p&lt;0.05) by the nobiletin treatment. Results from next generation sequencing of the fecal microbiota showed that DSS treatment significantly altered the microbial structure of fecal microbiota in mice, and dietary nobiletin treatment tend to reverse these alterations. It was also demonstrated that DSS treatment decreased the abundance of Bifidobacterium and Lactobacillus compared to the negative control (healthy) mice. Nobiletin treatment partially reversed the DSS‐induced decrease in these two genera in DSS treated mice. It is noteworthy that nobiletin had no effect on Bifidobacterium, but increased the abundance of Lactobacillus in the healthy mice (non‐DSS treated). DSS treatment significantly increased the abundance of Sutterella and Bilophila, while nobiletin effectively reversed DSS‐induced increases in these two genera. Overall, our results for the first time suggested that modification of gut microbiota by dietary nobiletin might contribute to its inhibitory effects against the development of colitis in mice.

Rectal Delivery of a DNAzyme That Specifically Blocks the Transcription Factor GATA3 and Reduces Colitis in Mice

GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice. We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n= 61) or ulcerative colitis (UC, n=74) or from patients without inflammatory bowel diseases (n= 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated andanalyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by invivo imaging (endoscopy) of mice. Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA, compared with mice given a control DNAzyme. Mice given hgd40 did not develop colitis after administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid; lamina propria cells from these mice expressed lower levels of IL6, IL9, and IL13 than cells from mice given the control DNAzyme. Mini-endoscopic images revealed that hgd40 and anti-TNF reduced colon inflammation over 3 days; hgd40 reduced colitis in TNFR double-knockout mice. Levels of GATA3 are increased in patients with UC and correlate with production of inflammatory cytokines in mice and humans. A DNAzyme that prevents expression of GATA3 reduces colitis in mice, independently of TNF, and reduces levels of cytokines in the colon. This DNAzyme might be developed for treatment of patients withUC.

Different tissue distribution properties for glycosylation variants of fusion proteins containing the p40 subunit of murine interleukin-12.

Antibody-based fusion proteins are gaining increasing importance for therapeutic applications, but the impact of glycosylation on in vivo biopharmaceutical performance is not always completely understood. In this article, we have analyzed biochemical and pharmaceutical properties of fusion proteins, consisting of the F8 antibody (specific to the EDA domain of fibronectin, a marker of tissue remodeling and of angiogenesis) and of the p40 subunit of interleukin-12, an inhibitor of inflammation. The corresponding fusion protein (F8-IL12p40), which inhibits colitis development in mice, is a glycosylated protein with suboptimal disease targeting properties in vivo Since the protein was extensively glycosylated, as evidenced by PNGase F treatment and mass spectrometric analysis, we mutated four asparagine residues in various combinations. The corresponding proteins exhibited similar biochemical and antigen-binding properties, but differences in thermal stability and bioactivity. Asparagine mutations did not lead to recovery of disease targeting performance in vivo, as evidenced by quantitative biodistribution studies with radioiodinated protein preparations in tumor-bearing mice. By contrast, an almost complete recovery of targeting was achieved with an enzymatically deglycosylated protein preparation. These findings reinforce the concept that different glycostructures can have an impact on tissue distribution properties.

NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-κB-Dependent and -Independent Functions

SummaryIntestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor κB (NF-κB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-κB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.

Osteopontin Deficiency Accelerates Spontaneous Colitis in Mice with Disrupted Gut Microbiota and Macrophage Phagocytic Activity.

BackgroundOsteopontin (OPN) is a multifunctional protein expressed in a variety of tissues and cells. Recent studies revealed increased OPN expression in the inflamed intestinal tissues of patients with inflammatory bowel disease (IBD). The role of OPN in the pathophysiology of IBD, however, remains unclear.AimsTo investigate the role of OPN in the development of intestinal inflammation using a murine model of IBD, interleukin-10 knock out (IL-10 KO) mice.MethodsWe compared the development of colitis between IL-10 KO and OPN/IL-10 double KO (DKO) mice. OPN expression in the colonic tissues of IL-10 KO mice was examined by fluorescence in situ hybridization (FISH) analysis. Enteric microbiota were compared between IL-10 KO and OPN/IL-10 DKO mice by terminal restriction fragment length polymorphism analysis. The effect of OPN on macrophage phagocytic function was evaluated by phagocytosis assay.ResultsOPN/IL-10 DKO mice had an accelerated onset of colitis compared to IL-10 KO mice. FISH analysis revealed enhanced OPN synthesis in the colonic epithelial cells of IL-10 KO mice. OPN/IL-10 DKO mice had a distinctly different enteric bacterial profile with a significantly lower abundance of Clostridium subcluster XIVa and a greater abundance of Clostridium cluster XVIII compared to IL-10 KO mice. Intracellular OPN deletion in macrophages impaired phagocytosis of fluorescence particle-conjugated Escherichia coli in vitro. Exogenous OPN enhanced phagocytosis by OPN-deleted macrophages when administered at doses of 1 to 100 ng/ml, but not 1000 ng/ml.ConclusionsOPN deficiency accelerated the spontaneous development of colitis in mice with disrupted gut microbiota and macrophage phagocytic activity.