Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/10/2004
Publication Date: 5/10/2004
Citation: Dawson, H.D., Royaee, A.R., Nishi, S., Kuhar, D.J., Schnitzlein, W.M., Zuckermann, F., Urban, J., Lunney, J.K. 2004. Identification of key imune mediators regulating t helper 1 reponses swine. Veterinary Immunology and Immunopathology 100:105-111. Interpretive Summary: Researchers have limited reagents to use to predict swine protective disease and vaccine responses. This manuscript presents data on a panel of new tools to measure T helper (Th) cell immune responses, particularly those that involve Th1 responses, i.e., those dominated by production of interferon-gamma (IFNG). Key upstream IFNG regulators (HLX1, ICSBP1, IRF1, MYD88, STAT1, TBX21) had been defined in other species. We then developed PCR primers to clone and sequence those for which no cDNA sequence had been reported. Additionally, genes that determine responding cell subsets (CD8A, CD8B, CD45) and their activation markers (CD28) were cloned. With this data we were then able to design real-time PCR assays to accurately measure expression of each of these genes, as well as a potential housekeeping gene (RPL32). Finally, proof of the importance of each of these genes was assayed using liver and mesenteric lymph node tissue cDNAs collected from control and Toxoplasma gondii infected pigs. Data presented in this paper show that IRF1, MYD88, and STAT1 are positive regulators of swine Th1 responses and were expressed prior to the simultaneous upregulation of IFNG, HLX1 and TBX21 gene expression. Thus, it is clear that we can now test how effectively a pig responds to infectious insults and activates the necessary pathway to develop a strong, and hopefully protective, Th1, IFNG dominated immune response. The data presented here is the first report of induced TBX21 expression during a Th1-associated response in an animal not of human or rodent origin. Likewise, a similar conclusion can be made regarding our assays of MYD88, ISCBP1 and HLX1 gene transcriptional regulation. Thus, these novel molecular reagents should prove valuable to evaluation of Th1 gene regulation in swine.
Technical Abstract: This publication describes the cloning of full or partial length sequences for pig TBX21 (T-bet), MYD88, ICSBP1, CD8A (CD8a), CD8B (CD8B), and CD28 cDNAs. Real-time PCR assays have been developed for these genes as well as for previously characterized transcripts that encode exon A-containing CD45, HLX1, IRF1, STAT1 and RPL32. When used for examining temporal immune gene expression in the liver of Toxoplasma gondii infected pigs, the positive regulators of Th1 responses, IRF1, MYD88, and STAT1, were found to be expressed prior to the simultaneous upregulation of interferon gamma (IFNG), HLX1 and TBX21 gene expression. Based on their demonstrated utility in establishing an immune response pathway, these PCR assays should be invaluable additions to our swine immune toolkit