Transcriptomic Analysis For Lymphocyte Count Variation In Poly I:C-Induced Peripheral Blood Of Duroc-Erhualian F2 Pigs And Construction Of Pocine Gene Expression Database

The recurring virus infections, which restrict the development of pig industry, can be addressed fundamentally by anti-disease breeding strategy, an approach that can serve to genetically improve disease resistance ability of pigs, which can potentially lower the costs of using drugs. Indeed, it is still unclear about the genetic mechanisms for general disease susceptibility in pigs and variations in their anti-disease abilities, so it is crucial to identify vital disease-resistant genes for disease resistance breeding and unraveling the potential mechanisms attributed to general anti-disease abilities.Lymphocyte is a major component of immune cells, and lymphocyte count is often seen as an important phenotypic metric for the individual anti-virus capacity. Polyriboinosinic:polyribocytidylic acid (Poly I:C), a synthetic analog of double-stranded RNA viruses, has been demonstrated to trigger an immune response similar to that induced by viral dsRNAs, therefore, it has been commonly used to model viral infections for investigating the immune response. The transcriptomic analysis for lymphocyte count variation in Poly I:C-Induced peripheral blood could reveal the crucial differentially expressed genes and pathways for porcine differential immune response, which will provide a molecular base for investigating genetic mechanism of the porcine general disease resistance capacity. In addition, despite a wide variety of the bioinformatics database, there is also a lack of specialized databases on the porcine immune genes. Establishing a database specially archiving porcine immune genes data will offer investigators a good resource for porcine immune gene expression information.In the present study, we first stimulated Duroc-Erhualian F2 Pigs with Poly I:C, and selected respectively 10 pigs with larger and smaller differences of lymphocyte counts before and after Poly I:C stimulation (designated as LOW pigs and HIGH pigs, respectively), and designed a Affymetrix microarray hybridization experiment with a total of 40 peripheral blood samples. Then we analyzed the differential expression data of 40 micrarrays and performed funcional annotations, cluster and pathway analysis for selected differentially expressed genes. Also, we constucted a database for storing and displaying the immune gene information of pigs. The major results of the present work are as follows:(1) A significant reduction of total lymphocyte counts in peripheral blood was observed after poly I:C stimulation for most Duroc-Erhualian F2 Pigs. The maximum absolute difference of the lymphocyte counts in LOW pigs was over 6-fold higher than that of the minimum difference in HIGH pigs, which suggest that the dsRNA (poly I:C)-induced lymphocyte count decrease was highly variable in the pig population.(2) Differentially expressed genes (DEGs) were identified,981 DEGs in HIGH pigs and 904 DEGs in LOW pigs(P<0.01). Most of these DEGs are related to immune responses and lymphocyte count decrease, which played a role in the innate and adaptive immune response. There were more down-regulated than up-regulated genes in the two groups, which inferred that the predominance of down-regulation might be due to the suppression of a large proportion of host mRNAs by the virus-induced activation of cell death or apoptosis.(3) According to the GO annotation results, the term number for biological processes in both groups was much higher than the term numbers for cellular components and molecular functions. As far as the biological process were concerned, both the HIGH and LOW groups had DEGs involved in immune responses, but the LOW group had more DEGs participating in cell death, apoptosis, immune response and the inflammatory response, from which we hypothesized that the LOW pigs might have a less immune capacity against the viral infections than the HIGH pigs.(4) Some commmon essential pathways are activated to immun responses in HIGH and LOW pigs, which is necessary to rapidly prevent viral invasion at the early stage of Poly I:C infection. The pathways such as cell death, cytotoxicity and apoptosis were found to directly or indirectly mediate lymphocyte number variation. There are also different pathways involved DEGs in HIGH and LOW pigs. Furthermore, different genes in the same pathway play different roles in regulating the poly I:C-induced decrease of lymphocyte count, which showed that the regulatory landscape varies individually among different pigs.(5) In the LOW group, some chemokines were identified among the DEGs, including CCL8, CCR1, CCR5,CXCL14, CXCL16, CXCL9, CXCR4 and CXCR7; whereas only three chemokines were identified in the HIGH group, namely, CCR1, CXCL16 and CXCR4. The differential expression of chemokines between the two groups might suggest that lymphocyte migration through chemokines is likely more effective in LOW pigs, resulting in a greater degree of decrease of lymphocytes in LOW pigs and less in HIGH pigs.(6)FAS, an important candidate that could affect the degree of decrease in the lymphocyte count, was up-regulated only in the LOW group. The result indicated that FAS might play a crucial role in affecting the degree of decrease in the lymphocyte count and that its differential expression might contribute to the degree of the difference in the lymphocyte count decrease between LOW and HIGH pigs.(7) We also constructed an open-source web database for porcine gene expression data. The Porcine Gene Expression Database (PGED) collected porcine gene expression data in different experiments and associated functional annotation and allows users to browse, retrieve, analyze and visualize data, which will be useful to easier and faster analysis of porcine functional genomics.