Transcriptome analysis of porcine PBMCs after in vitro stimulation by LPS or PMA/ionomycin using an expression array targeting the pig immune response

Abstract

BackgroundDesigning sustainable animal production systems that better balance productivity and resistance to disease is a major concern. In order to address questions related to immunity and resistance to disease in pig, it is necessary to increase knowledge on its immune system and to produce efficient tools dedicated to this species.ResultsA long-oligonucleotide-based chip referred to as SLA-RI/NRSP8-13K was produced by combining a generic set with a newly designed SLA-RI set that targets all annotated loci of the pig major histocompatibility complex (MHC) region (SLA complex) in both orientations as well as immunity genes outside the SLA complex.The chip was used to study the immune response of pigs following stimulation of porcine peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) or a mixture of phorbol myristate acetate (PMA) and ionomycin for 24 hours. Transcriptome analysis revealed that ten times more genes were differentially expressed after PMA/ionomycin stimulation than after LPS stimulation. 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 of B cell activation with dominance toward a Th1 response, including IL2, CD69 and TNFRSF9 (tumor necrosis factor receptor superfamily, member 9) genes. In addition, a very intense repression of THBS1 (thrombospondin 1) was observed. Repression of MHC class I genes was observed after PMA/ionomycin stimulation despite an up-regulation of the gene cascade involved in peptide processing. Repression of MHC class II genes was observed after both stimulations. Our results provide preliminary data suggesting that antisense transcripts mapping to the SLA complex may have a role during immune response.ConclusionThe SLA-RI/NRSP8-13K chip was found to accurately decipher two distinct immune response activations of PBMCs indicating that it constitutes a valuable tool to further study immunity and resistance to disease in pig. The transcriptome analysis revealed specific and common features of the immune responses depending on the stimulation agent that increase knowledge on pig immunity.

Lactobacillus paracasei and Lactobacillus plantarum strains downregulate proinflammatory genes in an ex vivo system of cultured human colonic mucosa

“…Cluster 6 grouped a significant number of genes coding for proinflammatory cytokines, chemokines and other inflammatory mediators, including IL-2, IFN-c, IRF-1, IL-17A, CXCL9, CXCL11, TNFRSF4 or GZMB, that were upregulated by PMA/IO, but markedly reduced by all the lactobacilli, LP, LP(A -) and BL23. Among them, a number of cytokine genes were upregulated by PMA/IO, which suggested T cell proliferation (IL-2, IFN-c and IL-17A) (Murphy et al 2007) and T cell activation (IL2RA) (Gao et al 2010;Ledger et al 2004). The reduction in the expression of those genes after treatment with the lactobacilli suggests that all three strains probably counteract the molecular events leading to T cell activation, as shown before for the probiotic strain L. casei DN 114-001 (Carol et al 2006).…”

Transcriptomic and nuclear architecture of immune cells after LPS activation

“…The CyDye-labelled cDNAs were resuspended at a final concentration of 2.5 pmol/μl in DNA/long-oligonucleotide hybridization buffer. A total of ten SLA-RI/NRSP8-13K chips (Gao et al 2010) were used in our study. Chip hybridization was performed using the Corning hybridization system.…”

“…The analysis of gene expression profiles of control and activated macrophages was carried out using a dedicated microarray, the porcine SLA-RI/NRSP8-13K chip which includes a dedicated SLA-RI oligonucleotide set (3,773 probes targeting Swine Leukocyte Antigens and Immune Response), the Qiagen-NRSP8 microarray oligonucleotide set and a series of positive and negative controls, totalling 19,200 probes (Gao et al 2010). The experimental model was designed to compare samples from control macrophages with LPS/IFNγ-activated macrophages using a common reference.…”

“…Some transcriptomic studies are now available for porcine alveolar macrophages infected by viruses (Zhang et al 2006;Xiao et al 2010). Today, progress in gene annotations (Archibald et al 2010) and DNA chips targeting immunity (Gao et al 2010) makes it possible to carry the transcriptional analysis of porcine macrophages further. A recent analysis was conducted on peripheral blood mononuclear cells (PBMC) (Gao et al 2010) using the same dedicated microarray, but our study focuses on swine monocytederived macrophages under LPS/IFNγ stimulation.…”

“…Today, progress in gene annotations (Archibald et al 2010) and DNA chips targeting immunity (Gao et al 2010) makes it possible to carry the transcriptional analysis of porcine macrophages further. A recent analysis was conducted on peripheral blood mononuclear cells (PBMC) (Gao et al 2010) using the same dedicated microarray, but our study focuses on swine monocytederived macrophages under LPS/IFNγ stimulation. Indeed, LPS, which interacts with a large number of proteins including LPS-binding protein, CD14 and TLR4, has been extensively used to study innate immune response in different species (Boldrick et al 2002;Nau et al 2002;Wells et al 2003) and is particularly appropriate for activating monocytes and macrophages that express the cell surface receptor CD14.…”