Transcriptome analysis of porcine PMBCs after stimulation by LPS or PMA/ionomycin using SLA-RI/NRSP8-13K chip. Sus scrofa

Background: During the past 25 years, selection of production traits highly increased pig production but diseases have emerged that may cause economic loss of extreme importance. Designing sustainable animal production that better balances productivity with resistance to disease is a major concern and challenge for the next decade. In order to address questions related to immunity and resistance to disease, it is necessary to increase knowledge on the pig immune system and to produce efficient tools dedicated to this species. In this context we produced a generic array enriched in immunity genes and validated its relevance by studying innate immune response of pigs by stimulating porcine mononuclear cells with lipopolysaccharide (LPS) or a mixture or phorboml myristate acetae (PMA) and ionomycin for 24 hours. Results: A long oligonucleotide-based chip was produced by combining a generic set of 13K probes targeting 8541 genes to a newly designed SLA-RI set that targets all genes and pseudogenes of the pig major histocompatibility complex region (SLA complex) in both orientations (906 probes) and immunity genes outside the SLA complex (2957 porbes). The porcine chip was referred to as SLA-RI/NRSP8-13K. Transcripotme analysis of PBMCs stimulated by LPS or PMA/ionomycin was carried out. Ten times more genes were up regulated after PMA/ionomycin stimulation by comparison to LPS stimulation. The LPS response was more related to the catalog Diseases and Disorders and the PMA/ionomycin response to the catalog Molecular and Cellular Function. LPS stimulation induced a general inflammation response with over-expression of SAA1, pro-inflammatory chemokines IL8, CCL2, CXCL5, CXCL3, CXCL2 and CCL8 as well as genes related to oxidative processes (SOD2) and calcium pathways (S100A9 and S100A12). PMA/ionomycin stimulation induced a stronger up-regulation of T cell activation than B cell activation with dominance toward a Th1 response, including IL2, CD69 and TNFRSF9 genes. In addition, a very intense repression of THBS1 was observed, suggesting a predominant role of this matricellular glycoprotein during T/B cell stimulation by tumor inducers. Repression of MHC class I genes was observed after PMA/ionomycin stimulation despite an up-regulation of the gene cascade involved in peptide processing and repression of MHC class II genes was observed after both stimulations. A significant reduction of antigen presentation to T cells was shared by the two types of responses that is likely to be due to separate mechanisms that need further elucidation. In addition, our results provided preliminary data suggesting a role of antisense transcripts mapping to the SLA complex during immune response. Conclusion: The SLA-RI/NRSP8-13K was highly relevant to. On the one hand the SLA-RI/NRSP8-13K was shown to be relevant and accurate to decipher two distinct innate immune responses by PBMCs indicating that this chip will constitute a valuable tool to further study immunity and resistance to disease in pig. On the other hand, the transcriptom analysis revealed specific and common features of the innate immune response according to stimulation that increase knowledge on pig immunity. Keywords: immune response activation in pig PBMCs Overall design: Two-condition experiment, LPS stimulated PBMCs vs. mock-stimulated PBMCs, PMA_ionomycine stimulated PBMCs vs.mock-stimulated PBMCs. Biological replicates: 1 control, 1 LPS stimulated and 1 PMA_ionomycine stimulated from 7 animals independently grown and harvested. Dye-swap design. One replicate per array. 28 slides.

背景：过去25年间，生产性状的高强度选育大幅提升了生猪生产性能，但同时也催生了可造成重大经济损失的各类疾病。构建可持续的动物生产体系，在生产力与疾病抗性之间实现更好平衡，是未来十年畜牧领域的核心关切与重大挑战。为解答免疫与疾病抗性相关的科学问题，亟需加深对猪免疫系统的认知，并开发针对该物种的高效研究工具。为此，本研究构建了一套富集免疫基因的通用基因芯片，并通过用脂多糖（lipopolysaccharide, LPS）或佛波醇肉豆蔻酸乙酸酯（phorbol myristate acetate, PMA）与离子霉素（ionomycin）混合物刺激猪单核细胞，研究猪的先天免疫应答，以此验证该芯片的应用价值。

结果：本研究将靶向8541个基因的13K通用探针集，与新设计的SLA-RI探针集相结合，制备了长寡核苷酸芯片。其中SLA-RI探针集包含两类探针：一是靶向猪主要组织相容性复合体（major histocompatibility complex, 简称SLA复合体）所有基因及假基因的906条双向探针；二是靶向SLA复合体外免疫基因的2957条探针。该猪用芯片被命名为SLA-RI/NRSP8-13K。本研究对脂多糖或PMA/离子霉素刺激的外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）开展了转录组分析。相较于脂多糖刺激，PMA/离子霉素刺激后上调的基因数量多出10倍。脂多糖诱导的免疫应答更富集于「疾病与生理功能」功能类别，而PMA/离子霉素诱导的应答则更偏向「分子与细胞功能」类别。脂多糖刺激可引发全身性炎症应答，表现为SAA1、促炎趋化因子IL8、CCL2、CXCL5、CXCL3、CXCL2、CCL8，以及与氧化过程相关的SOD2、钙通路相关基因S100A9和S100A12的过表达。PMA/离子霉素刺激诱导的T细胞活化上调程度显著强于B细胞，且应答偏向Th1型，涉及IL2、CD69及TNFRSF9等基因。此外，本研究观察到THBS1的显著下调，提示该基质细胞糖蛋白在肿瘤诱导剂刺激T/B细胞的过程中发挥主导作用。PMA/离子霉素刺激后可检测到MHC I类基因的下调，尽管此时肽加工相关的基因级联反应出现上调；而两种刺激均导致了MHC II类基因的下调。两类刺激诱导的免疫应答均伴随T细胞抗原呈递能力的显著降低，其潜在分子机制尚需进一步阐明。此外，本研究的结果还提供了初步数据，提示定位于SLA复合体的反义转录本在免疫应答过程中发挥一定作用。

结论：SLA-RI/NRSP8-13K芯片具有极高的研究应用价值。一方面，该芯片可有效且准确地解析PBMC介导的两种不同先天免疫应答，表明其可作为后续研究猪免疫功能与疾病抗性的可靠工具。另一方面，转录组分析揭示了不同刺激条件下先天免疫应答的特异性与共性特征，进一步加深了学界对猪免疫系统的认知。

关键词：猪外周血单个核细胞免疫应答激活

整体实验设计：双条件对照实验，分别为脂多糖刺激PBMCs组 vs 空白对照刺激PBMCs组、PMA/离子霉素刺激PBMCs组 vs 空白对照刺激PBMCs组。生物学重复：从7只独立饲养、采样的实验猪中，分别获取1份对照样本、1份脂多糖刺激样本及1份PMA/离子霉素刺激样本。实验采用染料交换（dye-swap）设计，每张芯片对应1个重复样本，共使用28张芯片。