Supplementary Material for: Micro Integral Membrane Protein (MIMP), a Newly Discovered Anti-Inflammatory Protein of Lactobacillus Plantarum, Enhances the Gut Barrier and Modulates Microbiota and Inflammatory Cytokines

<b><i>Background/Aims:</i></b> Recent studies have demonstrated that the manipulation of the gut microbiome represents a promising treatment for inflammatory bowel disease (IBD). We previously identified micro integral membrane protein (MIMP) as the smallest domain of surface layer protein from Lactobacillus Plantarum. However, the therapeutic relevance of MIMP in IBD remains unknown. <b><i>Methods:</i></b> We initially employed a dextran sodium sulphate (DSS)-induced colitis model and evaluated the effect of MIMP on the inflammation response, intestinal barrier and gut microbiota using histological examination, Fluorescein isothiocyanate-Dextran detection and pyrosequencing analysis respectively. We then established peripheral blood mononuclear cells (PBMCs) and an epithelial CaCO-2 co-culture model to investigate the regulatory role of MIMP in inflammatory cytokines. The level changes of inflammatory cytokines were detected using Enzyme-linked immunosorbent and real-time polymerase chain reaction assay. The involved regulatory mechanisms were investigated mainly using dual luciferase reporter and chromatin immunoprecipitation assay. <b><i>Results:</i></b> In the DSS-induced colitis model, we observed that MIMP intervention effectively improved the body weight loss, increased the colon length and decreased disease activity index. Consistently, the inflammation scores in the MIMP treatment group were significantly lower than those in the DSS treatment group. Furthermore, MIMP intervention was found to successfully neutralize DSS treatment by decreasing the expression of pro-inflammatory cytokines (IFN-γ, IL-17 and IL-23) and increasing the expression of anti-inflammatory cytokines (IL-4 and IL-10). Notably, the permeability assay demonstrated that the MIMP treatment group was remarkably lower than that in the DSS treatment group. We also showed that MIMP improved gut microbiota dysbiosis caused by DSS-induced inflammation. Additionally, in PBMCs and the CaCO-2 co-culture model, MIMP showed an obvious suppressive effect on lipopolysaccharide-induced inflammation in a time- and dose-dependent manner. Furthermore, we revealed that MIMP could modulate inflammatory cytokine expression through the toll-like receptor 4 pathway and histone acetylation. <b><i>Conclusions:</i></b> Our results suggested that MIMP showed a significant anti-inflammatory effect through regulating the gut barrier, microbiota and inflammatory cytokines. MIMP may have translational relevance as clinically relevant therapy for IBD patients.

<b><i>背景与目的：</i></b> 近期研究证实，调控肠道微生物组（gut microbiome）是治疗炎症性肠病（inflammatory bowel disease, IBD）的极具潜力的手段。本课题组此前从植物乳杆菌（Lactobacillus Plantarum）的表面层蛋白中鉴定出其最小功能结构域——微整合膜蛋白（micro integral membrane protein, MIMP），但MIMP在IBD中的治疗相关性仍未明确。<b><i>方法：</i></b> 本研究首先构建葡聚糖硫酸钠（dextran sodium sulphate, DSS）诱导的结肠炎模型，分别通过组织学检查、异硫氰酸荧光素-葡聚糖（Fluorescein isothiocyanate-Dextran）检测及焦磷酸测序分析，评估MIMP对炎症反应、肠屏障功能及肠道菌群的调控作用。随后构建外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）与上皮Caco-2细胞共培养模型，探究MIMP对炎症细胞因子的调控效应。采用酶联免疫吸附试验与实时聚合酶链反应检测炎症细胞因子的水平变化，并主要通过双荧光素酶报告基因试验及染色质免疫沉淀试验解析其潜在调控机制。<b><i>结果：</i></b> 在DSS诱导的结肠炎模型中，MIMP干预可有效缓解体质量下降、增加结肠长度并降低疾病活动指数（disease activity index, DAI）。与DSS模型组相比，MIMP治疗组的炎症评分显著降低。进一步研究发现，MIMP可通过下调促炎细胞因子（IFN-γ、IL-17及IL-23）的表达、上调抗炎细胞因子（IL-4及IL-10）的表达，逆转DSS诱导的炎症反应。值得注意的是，通透性实验结果显示，MIMP治疗组的肠屏障通透性显著低于DSS模型组。此外，MIMP可改善DSS诱导的炎症所致的肠道菌群失调。在外周血单个核细胞及Caco-2细胞共培养模型中，MIMP可呈时间与剂量依赖性地抑制脂多糖诱导的炎症反应。本研究还证实，MIMP可通过Toll样受体4（toll-like receptor 4, TLR4）通路及组蛋白乙酰化调控炎症细胞因子的表达。<b><i>结论：</i></b> 本研究结果表明，MIMP可通过调控肠屏障功能、肠道菌群及炎症细胞因子发挥显著的抗炎作用，有望成为治疗IBD患者的临床转化候选疗法。