DataSheet_1_Chitosan Protects Immunosuppressed Mice Against Cryptosporidium parvum Infection Through TLR4/STAT1 Signaling Pathways and Gut Microbiota Modulation.docx

Cryptosporidium parvum infection is very common in infants, immunocompromised patients, or in young ruminants, and chitosan supplementation exhibits beneficial effects against the infection caused by C. parvum. This study investigated whether chitosan supplementation modulates the gut microbiota and mediates the TLR4/STAT1 signaling pathways and related cytokines to attenuate C. parvum infection in immunosuppressed mice. Immunosuppressed C57BL/6 mice were divided into five treatment groups. The unchallenged mice received a basal diet (control), and three groups of mice challenged with 1 × 106 C. parvum received a basal diet, a diet supplemented with 50 mg/kg/day paromomycin, and 1 mg/kg/day chitosan, and unchallenged mice treated with 1 mg/kg/day chitosan. Chitosan supplementation regulated serum biochemical indices and significantly (p < 0.01) reduced C. parvum oocyst excretion in infected mice treated with chitosan compared with the infected mice that received no treatment. Chitosan-fed infected mice showed significantly (p < 0.01) decreased mRNA expression levels of interferon-gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) compared to infected mice that received no treatment. Chitosan significantly inhibited TLR4 and upregulated STAT1 protein expression (p < 0.01) in C. parvum-infected mice. 16S rRNA sequencing analysis revealed that chitosan supplementation increased the relative abundance of Bacteroidetes/Bacteroides, while that of Proteobacteria, Tenericutes, Defferribacteres, and Firmicutes decreased (p < 0.05). Overall, the findings revealed that chitosan supplementation can ameliorate C. parvum infection by remodeling the composition of the gut microbiota of mice, leading to mediated STAT1/TLR4 up- and downregulation and decreased production of IFN-γ and TNF-α, and these changes resulted in better resolution and control of C. parvum infection.

Cryptosporidium parvum感染在婴幼儿、免疫受损患者或幼龄反刍动物中极为常见，而壳聚糖的补充剂对由C. parvum引起的感染具有显著的益处。本研究旨在探究壳聚糖的补充是否能够调节肠道菌群，并通过TLR4/STAT1信号通路及其相关细胞因子来减轻免疫抑制小鼠中的C. parvum感染。免疫抑制的C57BL/6小鼠被分为五个治疗组。未经挑战的小鼠接受基础饮食（对照组），而接受1 × 10^6 C. parvum挑战的三个小组小鼠分别接受基础饮食、含50 mg/kg/天的帕罗霉素饮食和1 mg/kg/天的壳聚糖饮食，以及未经挑战但接受1 mg/kg/天壳聚糖治疗的小鼠。壳聚糖的补充调节了血清生化指标，并且与未接受治疗的小鼠相比，显著（p < 0.01）减少了接受壳聚糖治疗的小鼠中C. parvum卵囊的排出。与未接受治疗的小鼠相比，壳聚糖喂养的感染小鼠显著（p < 0.01）降低了干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）的mRNA表达水平。壳聚糖在C. parvum感染小鼠中显著抑制TLR4并上调STAT1蛋白表达（p < 0.01）。16S rRNA测序分析显示，壳聚糖的补充增加了拟杆菌门/拟杆菌的相对丰度，而变形菌门、放线菌门、厚壁菌门和纤维菌门的相对丰度则显著降低（p < 0.05）。总体而言，研究结果揭示了壳聚糖的补充可以通过重塑小鼠的肠道菌群组成，进而介导STAT1/TLR4的上调和下调，减少IFN-γ和TNF-α的产生，这些变化最终导致了C. parvum感染的更好解决和控制。