Table_8_Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection.csv

IntroductionCryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum, with a high prevalence in livestock (cattle, sheep, and goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and particularly of type 1 conventional dendritic cells (cDC1) among mononuclear phagocytes (MPs) in controlling the acute phase of C. parvum infection. These immune populations are well described in mice and humans, but their fine characterization in the intestine of young ruminants remained to be further explored.
MethodsImmune cells of the small intestinal Peyer’s patches and of the distal jejunum were isolated from naive lambs and calves at different ages. This was followed by their fine characterization by flow cytometry and transcriptomic analyses (q-RT-PCR and single cell RNAseq (lamb cells)). Newborn animals were infected with C. parvum, clinical signs and parasite burden were quantified, and isolated MP cells were characterized by flow cytometry in comparison with age matched control animals.
ResultsHere, we identified one population of macrophages and three subsets of cDC (cDC1, cDC2, and a minor cDC subset with migratory properties) in the intestine of lamb and calf by phenotypic and targeted gene expression analyses. Unsupervised single-cell transcriptomic analysis confirmed the identification of these four intestinal MP subpopulations in lamb, while highlighting a deeper diversity of cell subsets among monocytic and dendritic cells. We demonstrated a weak proportion of cDC1 in the intestine of highly susceptible newborn lambs together with an increase of these cells within the first days of life and in response to the infection.
DiscussionConsidering cDC1 importance for efficient parasite control in the mouse model, one may speculate that the cDC1/cDC2 ratio plays also a key role for the efficient control of C. parvum in young ruminants. In this study, we established the first fine characterization of intestinal MP subsets in young lambs and calves providing new insights for comparative immunology of the intestinal MP system across species and for future investigations on host–Cryptosporidium interactions in target species.

引言：隐孢子虫病（Cryptosporidiosis）是一种难以有效防控的人兽共患病，由肠道寄生虫微小隐孢子虫（Cryptosporidium parvum）引发，在牛、绵羊、山羊等家畜中具有较高流行率。幼畜因肠道免疫系统尚未发育成熟，对该感染尤为易感。此前我们在新生小鼠模型中证实，单核吞噬细胞（mononuclear phagocytes, MPs）中的天然免疫，尤其是1型常规树突状细胞（type 1 conventional dendritic cells, cDC1），在控制微小隐孢子虫感染急性期的过程中发挥关键作用。目前小鼠与人类体内的此类免疫细胞群已得到充分研究，但幼龄反刍动物肠道内这类细胞的精细特征仍有待进一步探索。

材料与方法：从不同日龄的未感染羔羊和犊牛体内分离小肠派尔集合淋巴结（Peyer’s patches）及远端空肠的免疫细胞，随后通过流式细胞术（flow cytometry）与转录组学分析（实时定量反转录聚合酶链反应q-RT-PCR及羔羊细胞单细胞RNA测序single cell RNAseq）对这些细胞进行精细表征。对新生动物接种微小隐孢子虫，量化其临床症状与寄生虫载量，并通过流式细胞术对分离得到的单核吞噬细胞进行表征，同时与同龄对照组动物进行对比。

结果：本研究通过表型分析及靶向基因表达分析，在羔羊和犊牛肠道内鉴定出1个巨噬细胞群与3个常规树突状细胞亚群：cDC1、cDC2，以及1个具备迁移特性的小型cDC亚群。无监督单细胞转录组分析证实了羔羊肠道内上述4种单核吞噬细胞亚群的存在，同时揭示了单核细胞与树突状细胞中更为丰富的细胞亚群多样性。研究发现，在易感性极高的新生羔羊肠道内，cDC1占比极低，且该细胞群在出生后初期及感染应答过程中占比会有所升高。

讨论：鉴于小鼠模型中cDC1对寄生虫高效防控的重要性，我们可推测，cDC1/cDC2比值在幼龄反刍动物对微小隐孢子虫的高效防控中同样发挥关键作用。本研究首次完成了幼龄羔羊与犊牛肠道单核吞噬细胞亚群的精细表征，为跨物种肠道单核吞噬细胞系统的比较免疫学研究，以及后续针对靶物种宿主-隐孢子虫互作的相关探索提供了全新的研究视角。