Transcriptomic profiling of bovine blood dendritic cells and monocytes following toll-like receptor stimulation

Dendritic cells (DC) and monocytes are vital for the initiation of innate and adaptive immune responses. Recently, we identified bona fide DC subsets in blood of cattle, revealing subset- and species-specific transcription of toll-like receptors (TLR). In the present study, we analyzed phenotypic and transcriptional responses of bovine DC subsets and monocytes to in-vitro stimulation with 4-6 different TLR ligands. Bovine DC subsets, especially plasmacytoid DC (pDC), showed a clear increase of CCR7, CD25, CD40, CD80, CD86 and MHC-II expression both on mRNA and protein level. Flow cytometric detection of p38 MAPK phosphorylation 15 minutes after stimulation confirmed activation of DC subsets and monocytes in accordance with TLR gene expression. Whole-transcriptome sequencing of sorted and TLR-stimulated subsets revealed clear subset- and ligand-specific regulation of genes associated with inflammation, T-cell stimulation, migration, metabolic reprogramming and antiviral activity. Gardiquimod was found to evoke strong responses both in DC subsets and monocytes, while Poly(I:C) and CpG preferentially triggered responses in cDC1 and pDC, respectively. This in-depth analysis of ligand responsiveness is essential for the rational design of vaccine adjuvants in cattle, and provides a solid basis for comparative studies on DC and monocyte biology across species.

树突状细胞（Dendritic cells, DC）与单核细胞在先天免疫与适应性免疫应答的启动过程中发挥核心作用。此前本团队已在牛血液中鉴定出确凿的DC亚群，揭示了Toll样受体（toll-like receptors, TLR）的亚群特异性与物种特异性转录特征。本研究中，我们分析了牛DC亚群与单核细胞经4~6种不同TLR配体体外刺激后的表型与转录应答特征。牛DC亚群，尤其是浆细胞样DC（plasmacytoid DC, pDC），在mRNA与蛋白水平均表现出CCR7、CD25、CD40、CD80、CD86以及MHC-II表达量的显著上调。刺激15分钟后通过流式细胞术检测p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase, p38 MAPK）的磷酸化水平，结果证实DC亚群与单核细胞的活化情况与TLR基因表达模式一致。对分选后经TLR配体刺激的细胞亚群进行全转录组测序，结果显示炎症、T细胞活化、细胞迁移、代谢重编程以及抗病毒活性相关基因均呈现显著的亚群特异性与配体特异性调控模式。研究发现，Gardiquimod可在DC亚群与单核细胞中诱导强烈应答；而Poly(I:C)与CpG则分别优先激活经典DC1（classical DC1, cDC1）与pDC。本次针对配体应答特征的深度分析，可为牛用疫苗佐剂的合理设计提供关键依据，同时也为跨物种DC与单核细胞生物学的比较研究奠定了坚实基础。