Understanding Haemophilus Parasuis Infection In Porcine Spleen Through A Tanscriptomics Approach And Gene Pathways/Networks Analysis

Haemophilus parasuis (HPS) is a small, pleomorphic, Gram-negative bacillus that causes Glasser’s disease, which is characterized by fibrinous polyserositis, meningitis and arthritis. It has become an increasing threat in pig herds worldwide and causes devastating losses to the pig industry. Commercial vaccines against HPS have been developed, but none of them offer effective protection against all heterologous strains. One of the most important reasons is that little is known about the pathogenesis, especially, the host immune responses (IR) to the infection. In the long run, it will be the best to shift our focus in breeding for resistance to Glasser’s disease. Research on host genes is not only helpful for our understanding of the pathogenesis but also very useful to enhance porcine genetic abilities to combat HPS. May be, there is still a long way to go, but much progress will be achived sooner or later. Because of these, in this article:1. For the first time, we analyzed the global change following HPS infection in porcine spleen (6 days post infection) based on the Affmetrix Porcine GeneChip(?) technology. Histology detections of porcine main organs show that HPS infection result in significant responses in spleen, lung, and liver; Raw data of the GeneChip(?) was normalized by GCRMA method, following significant analysis using SAM. After gene annotations, a total of 286 genes (Fold change>2, False discovery rate<10%) are differentially expressed, in which 157 and 129 genes are up-regulated and down-regulated, respectively. No evidence was obtained that expression levels had changed as a result of significant migration of cells, which could result in masking of the gene expression levels (Quantitative real-time PCR results).10 genes were selectively validated using Quantitative real-time PCR (qRT-PCR)2. Ingenuity(?) Pathway Analysis (IPA) of the microarray result show that HPS infection result in a serial significant changes of host biology functions, including those involved in innate IR, e.g., Inflammatory Response, Immune Cell Trafficking, and adaptive IR, e.g., Humoral Immune Response, Cell-mediated Immune Response, and Antimicrobial Response; Infection also result in several kinds of infectious diseases such as respiratory disease; Additionally, tissue or organ abnormals and metabolisms such as those in lipids, amino acids, vitamins, and minerals are significantly affected too. These data indicate a molecular connection between IRs and organismal development.3. Host IRs are the main issue during HPS infection. Our results indicate that the MYD88 (myeloid differentiation primary response gene 88) dependent TLR2 (Toll-like Receptor 2) signaling plays a crucial role in initiating host IRs following HPS infection. CEBPB (Ccaat/enhancer binding protein beta) is the core transcription factor in this signaling that connect to a wide range of cascades like inflammatory responses and acute phase reactions. Gene networks on respiratory disease, genetic disorder, arthritis, etc., are identified too; Canonical pathway analysis indicate that HPS employes several strategies for immune evasion, including inhibiting leukocyte migrations, phagocytosis, and communations between immune cells in innate IR and activation of T/B lymphocytes in adaptive IR and so on. Key down-regulated genes in these dead pathways are CCL5 (chemokine (C-C motif) ligand 5), PRKCH(protein kinase C, eta), ACTA1 (actin, alpha 1, smooth muscle, aorta), ACTC1 (actin, alpha, cardiac muscle 1), ACTG2 (actin, gamma 2, smooth muscle, enteric), TRD@ (T cell receptor delta locus), and CD3.4. All of the three genes encoding porcine calgranulins:S100A8 (S100 calcium binding protein A8), S100A9 (S100 calcium binding protein A9), and S100A12 (S100 calcium binding protein A12), which are closely related to HPS infection are cloned. Our results show that they share the same gene structures with human/mouse homologs. The genome (1000 bp、2817 bp、1440 bp in order) comprises 3 exons dividing by 2 introns; Gene mapping using SCHP (Somatic Cell Hybrid Panel) and IMpRH (INRA-UMN porcine Radiation Hybrid panel) show that they are located on SSC4 q21-q23 and closely linked with SW512; Under normal condition, most abundant mRNA levels are seen in the organs of the immune system, e.g., bone marrow, spleen, and lymph nodes, and other organs that have important functions during IR such as lung and liver; Immunostimulations in PK-15 (porcine kidney-15) cells and porcine whole blood cultures mimicking bacterial or viral infections using LPS and Poly (I:C) significantly enhanced their mRNA levels. These results further confirmed their immunological characterizations.5. Promoter analysis of porcine S100A8, S100A9, and S100A12 were performed by bioinformatics predictions in combine with promoter serial deletion experiments. We show that CEBPB is required for their up-regulations during HPS infection. The up-regulation is also closely related to p38 MAPK pathway and maybe IL10 dependent. Importantly, there are many key amino acid sites in these molecues such as Met42 in porcine S100A8 and Zn2+-binding sites that have been proven to play key antimicrobial roles in human and/or mouse. All together, we herein presume that porcine calgranulins might protect host from HPS infection (or many other kinds of pathogens in pig) through regulating inflammations.6. At last but not least, we show a meta-analysis between differentially expressed genes during HPS infection and the up-to-date pig QTLs (quantitative trait locus) of Immune Capacity. Many interesting candiadate genes are identified, indicating that important relationships exist between these genes and the pig general resistance. The method we use provide a new way for high-throughout data analysis and may have a "bright" usage in the future for a deep understanding of candidate genes in pig breeding. In conclusion, we herein fill the "blank" on host responses to HPS infection. Genes, pathways, and gene networks underlying the infection are identified. These results will lead to therapies for HPS and candidate genes for HPS resistance as well as provide fundamental information regarding porcine immune response mechanisms.