Development of a Macroarray To Specifically Analyze Immunological Gene Expression in Swine

Abstract

DNA arrays are useful tools for simultaneously studying the expressions of a large number of genes. Herein, we describe the construction and the optimization of conditions for a low-density DNA macroarray specific for the porcine immune system. This specific DNA macroarray contains 63 gene products, including 20 cytokines, 11 chemokines, and 12 immunologically relevant receptors. It was constructed by designing gene-specific oligonucleotide primers from porcine sequences available in the EMBL or TIGR expressed sequence tag data bank and using primers from conserved regions of aligned sequences from other species for sequences unavailable for swine. Amplicons produced by reverse transcription-PCR were cloned, sequenced, and spotted onto nylon filters. A trial DNA array was first produced to optimize the intensity, specificity, and variability of signals from amplicons amplified with either gene-specific or universal primers. The DNA macroarray was then validated by comparing the gene expression profile of nonstimulated peripheral blood mononuclear cells (PBMCs) to that of phorbol 12-myristate 13-acetate and ionomycin (PMA-Iono)-stimulated PBMCs from three different animals over a 48-h time period. As already described for more conventional techniques, we showed that certain genes, such as those for CD40, gamma interferon, interleukin 2 (IL-2), the IL-2 receptor, and tumor necrosis factor alpha, were upregulated in PMA-Iono-stimulated PBMCs. A detailed analysis also indicated a downregulation of several genes which are expressed mainly by macrophages (IL-1, IL-8, AMCF-1, natural-resistance-associated macrophage protein, neutrophil chemotactic protein, DAP-12, and monocyte chemoattractant protein) in samples stimulated for 24 h with PMA-Iono compared to their levels of expression in control samples. These results indicate that the DNA macroarray that we constructed can be a useful tool for simultaneously monitoring the mRNA expression of immunologically relevant genes in different porcine samples.

Porcine T-helper and regulatory T cells exhibit versatile mRNA expression capabilities for cytokines and co-stimulatory molecules

“…2) varied substantially from animal to animal as was also observed in other studies [9,22,32,33]. This might be due to the fact that in most studies no inbred pigs were used but animals with a different genetic, environmental, nutritional, and immunological background.…”

“…IL-10, IL-35 and TGF-b). Due to their important functions in immune regulation there have been numerous studies also in swine, which analysed the cytokine production of PBMC after polyclonal in vitro stimulation by various reagents like ConA [1], phytohemagglutinin (PHA) [6,9], phorbol myristate acetate (PMA)/Ionomycin [14,22], lipopolysaccharide (LPS) [10], and others [36]. However, in these studies cytokine expression was analysed in the entire cultured cell population (mostly PBMC), but the expression capacity of T-cell subsets was not addressed.…”

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).…”

Dose-Dependent Effects of Lactobacillus rhamnosus on Serum Interleukin-17 Production and Intestinal T-Cell Responses in Pigs Challenged with Escherichia coli

“…To quantify relative mRNA expression, the cycle threshold (C T ) values of the target genes were normalized to the C T value of the housekeeping gene coding for hypoxanthine phosphoribosyltransferase (HPRT) (27), and the results are presented as fold change using the 2 Ϫ⌬⌬CT method. The relative expression of target gene mRNA in each group was calculated using the following equations: ⌬C T ϭ C T target gene Ϫ C T HPRT and ⌬⌬C T ϭ ⌬C T treated group Ϫ ⌬C T control group .…”