GO: BP terms upregulated in Stressed calves.

The development of Bovine Respiratory Disease (BRD) in beef cattle is associated with stressful events, including auction and transport. In addition to the effects of commingling on pathogen exposure, stress also impacts immune function and has classically been associated with an immunosuppressed state. Much of the research on cattle immunity in stress has focused on peripheral blood immune functionality rather than mucosal immune responses. To characterize immunity at the primary site of pathogen colonization in BRD, we evaluated stress responses in lung cells from beef stocker cattle to understand mucosal immune changes. Stocker calves were sampled via bronchoalveolar lavage fluid (BALF) collection within 24 hours of auction and transport to a new facility (Stressed, n = 12), or allowed to acclimate for 2 months at the new facility before sampling (Acclimated, n = 7). Lavage cellular RNA was extracted and sequenced for bulk RNA-seq gene expression. Differential gene expression analysis of RNA sequencing data demonstrated a profound upregulation of inflammatory genes in Stressed calves compared to Acclimated calves, including expression of CXCL8, CSF3R, IL1B, and CCL22. Top pathway upregulation in Stressed calves involved neutrophil migration and chemotaxis, and cytokine signaling. To predict cellular population proportions from the data, CIBERSORTx was used to deconvolute bulk RNA-seq gene counts. This analysis showed that Stressed calves had significantly increased BALF neutrophils compared to Acclimated calves (p = 0.003). Neutrophilic infiltration occurred in the absence of pathogen colonization of the lungs in most calves, as demonstrated by a multi-pathogen respiratory qPCR screen. As such, the stress induced an inflammatory response in lungs not explained by pathogen exposure. This study provides strong evidence that shipping stress in beef stocker calves can trigger increased inflammatory pulmonary mucosal immune responses, which has important implications for the pathogenesis of BRD.肉牛呼吸道疾病（Bovine Respiratory Disease, BRD）的发生与拍卖、运输等应激事件密切相关。除混群行为会增加病原体暴露风险外，应激还会损害免疫功能，经典上与免疫抑制状态存在关联。既往针对牛应激免疫的研究多聚焦于外周血免疫功能，而非黏膜免疫应答。为了表征BRD病原体定植主要部位的免疫状态，我们对肉牛育成牛的肺细胞应激反应进行了评估，以明确其黏膜免疫变化。 试验将犊牛分为两组：应激组（Stressed, n=12）在完成拍卖并转运至新养殖场所后的24小时内，通过支气管肺泡灌洗液（bronchoalveolar lavage fluid, BALF）采集样本；适应组（Acclimated, n=7）则在新养殖场适应2个月后再进行采样。我们提取灌洗液细胞的RNA，开展批量RNA测序（bulk RNA-seq）以分析基因表达。 对RNA测序数据进行差异基因表达分析后发现，相较于适应组犊牛，应激组犊牛的炎症基因出现显著上调，其中包括CXCL8、CSF3R、IL1B及CCL22。应激组上调的核心通路涉及中性粒细胞迁移趋化与细胞因子信号传导。为了从测序数据中预测细胞群体比例，我们使用CIBERSORTx对批量RNA测序的基因计数进行反卷积分析。结果显示，应激组犊牛支气管肺泡灌洗液中的中性粒细胞比例显著高于适应组（p=0.003）。 多数犊牛的肺部未检测到病原体定植（经多病原体呼吸道qPCR筛查证实），但仍出现了中性粒细胞浸润。由此可见，应激可在无病原体暴露的情况下诱发肺部炎症反应。本研究提供了充分证据，证明肉牛育成犊牛的运输应激可触发肺部黏膜免疫炎症反应增强，这对BRD的发病机制研究具有重要意义。