Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, No.202, Gongyebei Road, Jinan, 250100, China.
Animal Genomics and Improvement Laboratory, BARC, USDA-ARS, Beltsville, MD, 20705, USA.
BMC Genomics. 2022 Apr 30;23(1):338. doi: 10.1186/s12864-022-08562-0.
Gram-negative bacteria are important pathogens in cattle, causing severe infectious diseases, including mastitis. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and crucial mediators of chronic inflammation in cattle. LPS modulations of bovine immune responses have been studied before. However, the single-cell transcriptomic and chromatin accessibility analyses of bovine peripheral blood mononuclear cells (PBMCs) and their responses to LPS stimulation were never reported.
We performed single-cell RNA sequencing (scRNA-seq) and single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) in bovine PBMCs before and after LPS treatment and demonstrated that seven major cell types, which included CD4 T cells, CD8 T cells, and B cells, monocytes, natural killer cells, innate lymphoid cells, and dendritic cells. Bioinformatic analyses indicated that LPS could increase PBMC cell cycle progression, cellular differentiation, and chromatin accessibility. Gene analyses further showed significant changes in differential expression, transcription factor binding site, gene ontology, and regulatory interactions during the PBMC responses to LPS. Consistent with the findings of previous studies, LPS induced activation of monocytes and dendritic cells, likely through their upregulated TLR4 receptor. NF-κB was observed to be activated by LPS and an increased transcription of an array of pro-inflammatory cytokines, in agreement that NF-κB is an LPS-responsive regulator of innate immune responses. In addition, by integrating LPS-induced differentially expressed genes (DEGs) with large-scale GWAS of 45 complex traits in Holstein, we detected trait-relevant cell types. We found that selected DEGs were significantly associated with immune-relevant health, milk production, and body conformation traits.
This study provided the first scRNAseq and scATAC-seq data for cattle PBMCs and their responses to the LPS stimulation to the best of our knowledge. These results should also serve as valuable resources for the future study of the bovine immune system and open the door for discoveries about immune cell roles in complex traits like mastitis at single-cell resolution.
革兰氏阴性菌是牛的重要病原体，可引起严重的传染病，包括乳腺炎。脂多糖（LPS）是革兰氏阴性菌外膜的组成部分，是牛慢性炎症的关键介质。已经研究了 LPS 对牛免疫反应的调节作用。然而，牛外周血单核细胞（PBMC）的单细胞转录组和染色质可及性分析及其对 LPS 刺激的反应从未被报道过。
我们在 LPS 处理前后对牛 PBMC 进行了单细胞 RNA 测序（scRNA-seq）和单细胞转座酶可及性染色质测序（scATAC-seq），并证明了七种主要细胞类型，包括 CD4 T 细胞、CD8 T 细胞和 B 细胞、单核细胞、自然杀伤细胞、固有淋巴细胞和树突状细胞。生物信息学分析表明，LPS 可以增加 PBMC 细胞周期进程、细胞分化和染色质可及性。基因分析进一步表明，在 PBMC 对 LPS 的反应过程中，差异表达、转录因子结合位点、基因本体和调控相互作用发生了显著变化。与先前研究的结果一致，LPS 诱导单核细胞和树突状细胞的激活，可能是通过其上调的 TLR4 受体。观察到 NF-κB 被 LPS 激活，并诱导一系列促炎细胞因子的转录增加，这表明 NF-κB 是 LPS 对固有免疫反应的响应调节剂。此外，通过将 LPS 诱导的差异表达基因（DEGs）与荷斯坦牛 45 个复杂性状的大规模 GWAS 整合，我们检测到与性状相关的细胞类型。我们发现，选定的 DEGs 与免疫相关的健康、产奶量和体型特征显著相关。
这项研究提供了牛 PBMC 及其对 LPS 刺激反应的首个 scRNAseq 和 scATAC-seq 数据，据我们所知，这是迄今为止的首次研究。这些结果也应该为牛免疫系统的未来研究提供有价值的资源，并为在单细胞分辨率下发现免疫细胞在乳腺炎等复杂性状中的作用开辟了道路。
