[Anti-mouse CD(52) antibody ameliorates colitis through suppressing Th1/17 mediated inflammation and promoting regulatory T cell response in interleukin-10 deficient mice].

In this study, we evaluated the therapeutic effects and possible mechanisms of anti-CD(5)2 treatment on interleukin-10 (IL-10) deficient mice. Anti-mouse CD(52) monoclonal antibody was administrated to C3H. IL-10(-/-)mice. The disease activity index, histological grading of colitis, serum Th1/17 related cytokines, percentage of CD(25+)Foxp3(+) T cells in colon as well as CD(25), Foxp3 gene expression were measured. Our data suggested that anti-CD(52) treatment inhibited colitis in C3H.IL-10(-/-)mice and it might be related to the suppression of Th1/17 related inflammation and the promotion of regulatory T cell differentiation.

A mineral extract from red algae ameliorates chronic spontaneous colitis in IL-10 deficient mice in a mouse strain dependent manner.

Inflammatory bowel disease is an urgent public health problem with a high incidence in developed countries. Alterations of lifestyle or dietary interventions may attenuate the disease progression and increase the efficacy of current therapies. Here we tested the effect of chronic supplementation with a mineral extract from red marine algae - rich in calcium (34%), magnesium, phosphorus, selenium and other trace minerals - in a clinically relevant model of spontaneous enterocolitis, interleukin (IL)-10(-/-) mice. The mineral extract was administered in the drinking water of Il10(-/-) mice on C57BL/6 J and BALB/c strain backgrounds for 25 weeks commencing from 3 to 4 weeks of age. The mineral extract ameliorated the spontaneous development of colitis and severity of disease in Il10(-/-) mice on a C57BL/6 J background. Mineral extract-treated Il10(-/-) C57BL/6 J strain mice had significantly reduced mortality, circulating levels of serum Amyloid A and reduced colonic tissue damage. In contrast, comparable treatment of Il10(-/-) mice on a BALB/c background with the mineral extract did not alter the course of colitis. These data demonstrate that chronic supplementation with a natural mineral extract selectively ameliorates spontaneous mild-moderate colitis in Il10(-/-) mice on a C57BL/6 J, but does not attenuate more moderate-severe colitis in BALB/c strain animals.

Mouse Adaptation of Human Inflammatory Bowel Diseases Microbiota Enhances Colonization Efficiency and Alters Microbiome Aggressiveness Depending on Recipient Colonic Inflammatory Environment.

Understanding the cause vs consequence relationship of gut inflammation and microbial dysbiosis in inflammatory bowel diseases (IBD) requires a reproducible mouse model of human-microbiota-driven experimental colitis. Our study demonstrated that human fecal microbiota transplant (FMT) transfer efficiency is an underappreciated source of experimental variability in human microbiota associated (HMA) mice. Pooled human IBD patient fecal microbiota engrafted germ-free (GF) mice with low amplicon sequence variant (ASV)-level transfer efficiency, resulting in high recipient-to-recipient variation of microbiota composition and colitis severity in HMA Il-10-/- mice. In contrast, mouse-to-mouse transfer of mouse-adapted human IBD patient microbiota transferred with high efficiency and low compositional variability resulting in highly consistent and reproducible colitis phenotypes in recipient Il-10-/- mice. Human-to-mouse FMT caused a population bottleneck with reassembly of microbiota composition that was host inflammatory environment specific. Mouse-adaptation in the inflamed Il-10-/- host reassembled a more aggressive microbiota that induced more severe colitis in serial transplant to Il-10-/- mice than the distinct microbiota reassembled in non-inflamed WT hosts. Our findings support a model of IBD pathogenesis in which host inflammation promotes aggressive resident bacteria, which further drives a feed-forward process of dysbiosis exacerbated gut inflammation. This model implies that effective management of IBD requires treating both the dysregulated host immune response and aggressive inflammation-driven microbiota. We propose that our mouse-adapted human microbiota model is an optimized, reproducible, and rigorous system to study human microbiome-driven disease phenotypes, which may be generalized to mouse models of other human microbiota-modulated diseases, including metabolic syndrome/obesity, diabetes, autoimmune diseases, and cancer.

Mouse adaptation of human inflammatory bowel diseases microbiota enhances colonization efficiency and alters microbiome aggressiveness depending on the recipient colonic inflammatory environment.

BACKGROUND: Understanding the cause vs consequence relationship of gut inflammation and microbial dysbiosis in inflammatory bowel diseases (IBD) requires a reproducible mouse model of human-microbiota-driven experimental colitis. RESULTS: Our study demonstrated that human fecal microbiota transplant (FMT) transfer efficiency is an underappreciated source of experimental variability in human microbiota-associated (HMA) mice. Pooled human IBD patient fecal microbiota engrafted germ-free (GF) mice with low amplicon sequence variant (ASV)-level transfer efficiency, resulting in high recipient-to-recipient variation of microbiota composition and colitis severity in HMA Il-10-/- mice. In contrast, mouse-to-mouse transfer of mouse-adapted human IBD patient microbiota transferred with high efficiency and low compositional variability resulting in highly consistent and reproducible colitis phenotypes in recipient Il-10-/- mice. Engraftment of human-to-mouse FMT stochastically varied with individual transplantation events more than mouse-adapted FMT. Human-to-mouse FMT caused a population bottleneck with reassembly of microbiota composition that was host inflammatory environment specific. Mouse-adaptation in the inflamed Il-10-/- host reassembled a more aggressive microbiota that induced more severe colitis in serial transplant to Il-10-/- mice than the distinct microbiota reassembled in non-inflamed WT hosts. CONCLUSIONS: Our findings support a model of IBD pathogenesis in which host inflammation promotes aggressive resident bacteria, which further drives a feed-forward process of dysbiosis exacerbated by gut inflammation. This model implies that effective management of IBD requires treating both the dysregulated host immune response and aggressive inflammation-driven microbiota. We propose that our mouse-adapted human microbiota model is an optimized, reproducible, and rigorous system to study human microbiome-driven disease phenotypes, which may be generalized to mouse models of other human microbiota-modulated diseases, including metabolic syndrome/obesity, diabetes, autoimmune diseases, and cancer. Video Abstract.

A consortia of clinical E. coli strains with distinct in vitro adherent/invasive properties establish their own co-colonization niche and shape the intestinal microbiota in inflammation-susceptible mice.

BACKGROUND: Inflammatory bowel disease (IBD) patients experience recurrent episodes of intestinal inflammation and often follow an unpredictable disease course. Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation. However, it remains unclear if the 24-year-old AIEC in vitro definition fully predicts mucosal colonization in vivo. To fill this gap, we have developed a novel molecular barcoding approach to distinguish strain variants in the gut and have integrated this approach to explore mucosal colonization of distinct patient-derived E. coli isolates in gnotobiotic mouse models of colitis. RESULTS: Germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortium of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. 16S rRNA sequencing was used to evaluate the microbiome response to E. coli colonization. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner. In turn, specific microbes co-evolve in inflamed mice, potentially diversifying E. coli colonization patterns. We observed no selectivity in E. coli colonization patterns in the fecal contents, indicating minimal selective pressure in this niche from host-microbe and interbacterial interactions. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. CONCLUSIONS: Our findings establish the in vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in vivo colonization dynamics of patient-derived bacteria in murine models. Video Abstract.

Akkermansia deficiency and mucin depletion are implicated in intestinal barrier dysfunction as earlier event in the development of inflammation in interleukin-10-deficient mice.

Background: Dysbiosis and mucin depletion are related with intestinal barrier dysfunction and seems to be an early pathophysiological event in inflammatory bowel disease (IBD). The objective of this work is to study these parameters in the natural history of colitis in IL-10 deficient mice (IL-10-/-). Methods: Wild type (WT) and IL-10-/-. mice were followed until sacrifice at 3, 5, 10, 20, 57, and 70 weeks. Body weight, colonic weight/length ratio and in vivo intestinal permeability were registered. Expression of inflammatory and adhesion molecules in the colon was explored by qPCR as Mucin-2 (MUC-2) and molecules involved in goblet cell maturation Interleukin-18 (IL-18) and WAP Four-Disulfide Core Domain 2 (WFDC2), the endoplasmic reticulum stress markers X-box-binding protein (Xbp-1) and Reticulon-4B (RTN-4B). Bacterial composition in feces and colonic mucosa was determined by massive sequencing of the V3-V4 regions of 16S rDNA gene. Results: IL-10-/- mice showed histological inflammation at weeks 20 and 57, but most notably the intestinal permeability was significantly higher from week 10. Concordantly, the number of goblet cells and expression of MUC-2, IL-18, WFDC2 and Xbp-1 were significantly lower in KO from week 10. Nevertheless, no significant differences were found in the mRNA expression of MUC-2 or Xbp-1 between both groups-derived colon organoids. Significant bacterial differences began at week 5, being the Akkermansia deficiency in KO the most relevant result. Conclusion: Gut microbiota alterations and mucin depletion are associated with early intestinal barrier dysfunction and precede overt gut inflammation in this animal model of IBD.

Point-of-Care Monitoring of Colitis Using Intestinal Alkaline Phosphatase in Inflammatory Bowel Disease.

Intestinal Alkaline Phosphatase (IAP) was investigated as a potential biomarker to monitor colitis in a mouse model of Inflammatory Bowel Disease (IBD). We developed a Point-Of-Care (POC) assay to detect IAP with a glucose meter in 15 min. We synthesized a paracetamol-bearing compound specifically cleaved by IAP to release paracetamol, which can be detected with a personal glucometer. Interleukin 10 deficient (IL 10-/-) mouse model samples were used to compare the IAP level in mice with mild or severe colitis. The results showed that fecal IAP level was significantly lower in each mouse sample with severe colitis than with mild colitis. Mice treated with anti-Tumor Necrosis Factor-alpha (anti-TNF-α) to decrease inflammation exhibited a much higher level of IAP than those without treatment (IAP levels from anti-TNF-α treated vs nontreated = 2.80 U vs 0.11 U, P < 0.0001). Taken together, IAP can be considered as a potential biomarker to monitor colitis, and a rapid, user-friendly POC glucometer-based assay can be potentially used to monitor colitis levels and inflammation flareups in IBD.

Chronic Model of Inflammatory Bowel Disease in IL-10-/- Transgenic Mice: Evaluation with Ultrasound Molecular Imaging.

Objective: Acute mouse models of inflammatory bowel disease (IBD) fail to mirror the chronic nature of IBD in patients. We sought to develop a chronic mouse IBD model for assessing long-term anti-inflammatory effects with ultrasound molecular imaging (USMI) by using dual P- and E-selectin targeted microbubbles (MBSelectin). Materials and Methods: Interleukin 10 deficient (IL-10-/- on a C57BL/6 genetic background; n=55) and FVB (n=16) mice were used. In IL-10-/-mice, various experimental regimens including piroxicam, 2,4,6-trinitrobenzenesulfonic acid (TNBS) or dextran sulfate sodium (DSS), respectively were used for promoting colitis; colitis was induced with DSS in FVB mice. Using clinical and small animal ultrasound scanners, evolution of inflammation in proximal, middle and distal colon, was monitored with USMI by using MBSelectin at multiple time points. Imaged colon segments were analyzed ex vivo for inflammatory changes on H&E staining and for P-selectin expression on immunofluorescence staining. Results: Sustained colitis was not detected with USMI in IL-10-/- or FVB mice with various experimental regimens. USMI signals either gradually decreased after the colitis enhancing/inducing drug/agents were discontinued, or the mortality rate of mice was high. Inflammation was observed on H&E staining in IL-10-/- mice with piroxicam promotion, while stable overexpression of P-selectin was not found on immunofluorescence staining in the same mice. Conclusion: Sustained colitis in IL-10-/- mice induced with piroxicam, TNBS or DSS, and in FVB mice induced with DSS, was not detected with USMI using MBSelectin, and this was verified by immunofluorescence staining for inflammation marker P-selectin. Thus, these models may not be appropriate for long-term monitoring of chronic colitis and subsequent treatment response with dual-selectin targeted USMI.

Docosahexaenoic Acid Attenuated Experimental Chronic Colitis in Interleukin 10-Deficient Mice by Enhancing Autophagy Through Inhibition of the mTOR Pathway.

BACKGROUND: In the battle against Crohn's disease, autophagy stimulation is a promising therapeutic option-one both new and newly rediscovered. In experimental models, docosahexaenoic acid (DHA)-a long-chain polyunsaturated fatty acid-has been demonstrated to be useful in the treatment of inflammatory bowel disease through inhibition of the nuclear factor-κB pathway. However, the impact of DHA on autophagy in the colon remains unclear. METHODS: Mice were divided into 3 groups: wild type (placebo), the interleukin 10 knockout group (IL-10-/-, placebo), and the DHA group (IL-10-/-, DHA). DHA was administered to IL-10-/- mice by gavage at a dosage of 35.5 mg/kg/d for 2 weeks. The severity of colitis, expression of proinflammatory cytokines, expression/distribution of LC3B, and mTOR signaling pathway were evaluated in the proximal colon tissues collected from all mice at the end of the experiment. RESULTS: DHA administration ameliorated experimental colitis in the IL-10-/- mice, as demonstrated by decreased proinflammatory cytokines (TNF-α and IFN-γ), reduced infiltration of inflammatory cells, and lowered histologic scores of the proximal colon mucosa. Moreover, in the DHA-treated mice, enhanced autophagy was observed to be associated with (1) increased expression and restoration of the distribution integrity of LC3B in the colon and (2) inhibition of the mTOR signaling pathway. CONCLUSION: This study showed that DHA therapy could attenuate experimental chronic colitis in IL-10-/- mice by triggering autophagy via inhibition of the mTOR pathway.

Clostridium difficile infection aggravates colitis in interleukin 10-deficient mice.

AIM: To investigate the effect of Clostridium difficile (C. difficile) infection in an interleukin 10-deficient (IL-10(-/-)) mouse model of inflammatory bowel disease. METHODS: Bone marrow-derived dendritic cells isolated from wild type (WT) and IL-10(-/-)mice were stimulated for 4 h with C. difficile toxin A (200 µg/mL), and gene expression of interferon (IFN)-γ, IL-12 and IL-23 was determined by real-time reverse transcription polymerase chain reaction. WT and IL-10(-/-) mice (n = 20 each) were exposed to an antibiotic cocktail for three days and then were injected with clindamycin (i.p.). Mice (n = 10 WT, 10 IL-10(-/-)) were then challenged with oral administration of C. difficile (1 × 10(5) colony forming units of strain VPI 10463). Animals were monitored daily for 7 d for signs of colitis. Colonic tissue samples were evaluated for cytokine gene expression and histopathologic analysis. RESULTS: C. difficile toxin A treatment induced IFN-γ gene expression to a level that was significantly higher in BDMCs from IL-10(-/-) compared to those from WT mice (P < 0.05). However, expression of IL-12 and IL-23 was not different among the groups. Following C. difficile administration, mice developed diarrhea and lost weight within 2-3 d. Weight loss was significantly greater in IL-10(-/-) compared to WT mice (P < 0.05). C. difficile infection induced histopathologic features typical of colitis in both IL-10(-/-) and WT mice. The histopathologic severity score was significantly higher in the IL-10(-/-) than in WT mice (mean ± standard error; 5.50 ± 0.53 vs 2.44 ± 0.46; P < 0.05). This was accompanied by a significantly greater increase in IFN-γ gene expression in colonic tissues from IL-10(-/-) than from WT mice challenged with C. difficile (P < 0.05). CONCLUSION: These results indicate that colitis is more severe after C. difficile infection in IL-10(-/-)mice, and that IFN-γ expression is involved in this process.

Anti-mouse CD52antibody ameliorates colitis through suppressing Th1/17 mediated inflammation and promoting regulatory T cell response in interleukin-10 deficient mice / 中华内科杂志

In this study, we evaluated the therapeutic effects and possible mechanisms of anti-CD52 treatment on interleukin-10 (IL-10) deficient mice. Anti-mouse CD52 monoclonal antibody was administrated to C3H. IL-10-/-mice. The disease activity index, histological grading of colitis, serum Th1/17 related cytokines, percentage of CD25+Foxp3+T cells in colon as well as CD25, Foxp3 gene expression were measured. Our data suggested that anti-CD52 treatment inhibited colitis in C3H.IL-10-/-mice and it might be related to the suppression of Th1/17 related inflammation and the promotion of regulatory T cell differentiation.

Altered enteric microbiota ecology in interleukin 10-deficient mice during development and progression of intestinal inflammation.

Inflammatory bowel diseases (IBD) result from dysregulated immune responses toward microbial and perhaps other luminal antigens in a genetically susceptible host, and are associated with altered composition and diversity of the intestinal microbiota. The interleukin 10-deficient (IL-10 (-/-) ) mouse has been widely used to model human IBD; however the specific alterations that occur in the intestinal microbiota of this mouse model during the onset of colonic inflammation have not yet been defined. The aim of our study was to define the changes in diversity and composition that occur in the intestinal microbiota of IL-10 (-/-) mice during the onset and progression of colonic inflammation. We used high throughput sequencing of the 16S rRNA gene to characterize the diversity and composition of formerly germ-free, wild-type and IL-10 (-/-) mice associated with the same intestinal microbiota over time. Following two weeks of colonization with a specific pathogen-free (SPF) microbiota we observed a significant increase in the diversity and richness of the intestinal microbiota of wild-type mice. In contrast, a progressive decrease in diversity and richness was observed at three and four weeks in IL-10 (-/-) mice. This decrease in diversity and richness was mirrored by an increase in Proteobacteria and Escherichia coli in IL-10 (-/-) mice. An increase in E. coli was also observed in conventionally raised IL-10 (-/-) mice at the point of colonic inflammation. Our data reports the sequential changes in diversity and composition of the intestinal microbiota in an immune-mediated mouse model that may help provide insights into the primary vs. secondary role of dysbiosis in human IBD patients.