Early life exposure of pigs to topsoil alters miRNA and mRNA expression in peripheral blood mononuclear cells

Exposure to less hygienic conditions during early childhood has been associated with stimulation and development of the immune system. A recent study indicated exposure of piglets to soil-borne microbes during lactation was related with modulation of gut microbiota and immune function. To identify potential molecular mechanisms and pathways impacted by early life topsoil exposure, we analyzed gene expression (mRNA and miRNA) in peripheral blood mononuclear cells (PBMC) from these piglets. Total RNA was extracted from PBMC of piglets exposed to topsoil only from d 4-d 21 of life (mRNA n=6; miRNA n=5) or unexposed control pigs (mRNA n=6; miRNA n=8) at 11, 20, and 56 days of age. Small RNA and mRNA were sequenced with 50-bp single-end reads using Illumina chemistry. Differential expression (DE) analysis was performed using PROC Glimmix of SAS to evaluate changes in expression due to topsoil exposure over time with genes declared DE at a false discovery rate (FDR) of q<0.10. A total of 138 mRNA and 21 miRNAs were identified as DE for the treatment by age interaction. Ontology enrichment analysis of DE mRNA revealed GO terms directly involved in the connection between T-cell and antigen-presenting cells that are associated with T-cell activation. Key regulatory genes identified include PTPRJ, ITGB3, TRBV30, CD3D, mir-143, mir-29, and mir-148a. While these results require validation, this study provides data supporting the hypothesis that less hygienic environments during early life may contribute to the development of the immune system RNA-Seq mRNA and miRNA. Piglets exposed to topsoil from days 4 to 21 of life (mRNA n=6; miRNA n=5) or unexposed control pigs (mRNA n=6; miRNA n=8). Samples were collected at 11, 20, and 56 days of age.

幼年时期暴露于低卫生条件环境与免疫系统的激活及发育密切相关。近期一项研究表明，哺乳仔猪接触土壤源微生物可调节肠道菌群与免疫功能。为探明生命早期表层土壤暴露所影响的潜在分子机制与通路，本研究对该批次仔猪外周血单个核细胞（peripheral blood mononuclear cells，PBMC）中的基因表达（mRNA和miRNA）进行了分析。研究对象为仅在出生后第4~21天接触表层土壤的仔猪（mRNA测序样本量n=6；miRNA测序样本量n=5），以及未接触土壤的对照组仔猪（mRNA测序样本量n=6；miRNA测序样本量n=8），分别于仔猪11、20和56日龄时采集样本。采用Illumina测序平台，以50bp单端读长对小RNA及mRNA进行测序。使用SAS软件的PROC Glimmix程序进行差异表达（differential expression，DE）分析，以评估表层土壤暴露随时间推移对基因表达的影响，以错误发现率（false discovery rate，FDR）q<0.10作为基因差异表达的判定阈值。共鉴定出138个mRNA和21个miRNA在处理组与日龄的交互作用下呈现差异表达。对差异表达mRNA进行基因本体（Gene Ontology，GO）富集分析，结果显示其富集的GO条目直接关联T细胞与抗原呈递细胞之间的连接通路，该通路与T细胞活化相关。鉴定得到的关键调控基因包括PTPRJ、ITGB3、TRBV30、CD3D、miR-143、miR-29及miR-148a。尽管本研究结果尚需进一步验证，但本研究数据支持“生命早期低卫生环境可促进免疫系统发育”这一假说。