Abomasal RNA-seq reveals a strong local cell response of suckling lambs with resistance against Haemonchus contortus

Santa Ines (SI) and Ile de France (IF) sheep are known to be resistant and susceptible to H. contortus infection, respectively. Several studies have shown some genes as potential biological markers for sheep resistance against gastrointestinal nematodes using molecular tools, including transcriptomic analysis. In this study, we sequenced the polyadenylated RNA of the abomasal tissue of SI and IF suckling lambs to identify mucosa-specific transcript alterations between breeds artificially infected with H. contortus. Naïve SI (n=4) and IF (n=4) lambs were artificially infected every other day, over a period of 52 days, from 14 to 66 days old, with a total of 5,400 H. contortus infective larvae. Fundic abomasal tissue samples were collected at 68 days old, and submitted to high-throughput RNA sequencing. Differential expression analysis (P value &lt; 0.001 and FDR &lt; 0.05) between SI and IF samples identified 292 genes, most of which showed greater expression in SI lambs. To help annotate and assign possible function to differentially expressed genes (DEGs), we used previously available single-cell RNAseq (scRNAseq) data from ovine abomasal mucosa to putatively identify cell types and possible mechanisms involved in resistance to H. contortus. In particular, genes associated with endothelial and tuft cells showed most increase in expression in SI relative to IF lambs. Santa Ines lambs had higher percentual of tuft cells than IF lambs in the fundic abomasal mucosa. Although we found innate immunity (cell mediated in mucosa) acting as protagonist for impairing H. contortus infection, a stronger acquired immune response was being modulated at an earlier stage by SI lambs. We suggest that the complex connection between innate and adaptive immunities are via cellular antigen processing and presentation (APP). Based on comparison with scRNAseq data, SI lambs showed a robust APP mechanism characterized mainly by greater T cell APP, macrophage differentiation, and cytokine signalling. We identified potential mechanisms and markers to advance knowledge for selection of H. contortus resistance at a very early age, in SI as well as in other commercial sheep breeds.已知圣塔伊内斯羊（Santa Ines, SI）与法兰西岛羊（Ile de France, IF）分别对捻转血矛线虫（H. contortus）感染具有抗性与易感性。已有多项研究借助包括转录组学分析在内的分子生物学技术，筛选出若干可作为绵羊抗胃肠道线虫感染潜在生物标志物的基因。本研究对SI与IF品系哺乳羔羊的皱胃组织聚腺苷酸RNA进行测序，旨在鉴定人工感染捻转血矛线虫后不同品种间黏膜特异性转录组差异。将未感染过病原体的SI组（n=4）与IF组（n=4）羔羊，在14至66日龄期间每隔一日进行人工感染，总感染周期为52天，每只羔羊共接种5400条捻转血矛线虫感染性幼虫。于68日龄采集其胃底皱胃组织样本，开展高通量RNA测序。对SI与IF样本进行差异表达分析（P值<0.001且错误发现率FDR<0.05），共鉴定得到292个差异表达基因，其中多数在SI羔羊中呈现高表达水平。为辅助注释差异表达基因（Differentially Expressed Genes, DEGs）并推定其功能，我们使用已公开的绵羊皱胃黏膜单细胞RNA测序（single-cell RNA sequencing, scRNAseq）数据，来推测与捻转血矛线虫抗性相关的细胞类型及潜在调控机制。具体而言，与内皮细胞及簇细胞相关的基因在SI羔羊中的表达上调幅度显著高于IF羔羊。同时，SI羔羊的胃底皱胃黏膜中簇细胞占比高于IF羔羊。尽管我们发现黏膜固有免疫（细胞介导免疫）在抑制捻转血矛线虫感染中发挥核心作用，但SI羔羊在感染早期即已启动更强的适应性免疫应答调控。我们推测，固有免疫与适应性免疫之间的复杂互作通过细胞抗原加工与呈递（Antigen Processing and Presentation, APP）通路实现。通过与单细胞RNA测序数据的比对分析，我们发现SI羔羊具备更为活跃的细胞抗原加工与呈递机制，具体表现为T细胞抗原加工与呈递、巨噬细胞分化及细胞因子信号通路的显著增强。本研究鉴定出若干潜在调控机制与生物标志物，可为早期选育捻转血矛线虫抗性羊只（包括圣塔伊内斯羊及其他商用绵羊品种）提供理论依据。