Effect Of CSFV And Its Non-structural Proteins On TLR-mediated Innate Immune Response In Swine Macrophages

Toll-like receptors(TLRs) are fundamental sensor molecules of the host innate immune system, which detect conserved structures of virus and initiate innate immune responses via distinct signaling pathways. Paradoxically, viruses are obligate parasites that depend on host cells for survival and, throughout evolution, they have developed strategies to evade the immune response. Currently, the mechanism of immune response to classical swine fever virus(CSFV) infection is not completely clear, therefore, the mechanism of CSFV to evade the innate immune response in the early stage of infection is still no answer. Since, the present study takes the TLR-mediated innate immune response as entry point to analyze the influence of CSFV on the expression of TLR-dependent cytokine responses and the activation of TLR signaling elements. On this basis, the macrophages expressing CSFV non-structural proteins are established, for determining the “strategic proteins” that affecting TLRs gene expression, and analyzing the impact on downstream of TLRs signaling pathways. The results will indicate the effect of CSFV non-structural proteins on TLRs-mediated innate immune response in swine macrophages. The main research results are as follows:(1)Comparison of the impact of Shimen strain and C strain of CSFV on toll-like receptor expression, the results show that the m RNA and protein levels of TLR2, TLR4 and TLR7 are upregulated in response to CSFV infection, but TLR3 remains unchanged and is downregulated after infection with the C strain and the Shimen virus, respectively. Furthermore, TLR3-mediated innate immune responses are inhibited in Shimen-strain-infected p MDMs by stimulation with poly(I:C). Accordingly, comprehensive analyses are performed to detect TLR-dependent cytokine responses and the activation of TLR signaling elements. CSFV infection induces MAPKs activation but do not elicit NF-κB activation, thereby affecting the production of pro-inflammatory cytokines. The Shimen strain infection results in a significant activation of IRF7 and suppression of IRF3.(2)The expression patterns of TLR2, TLR3, TLR4 and TLR7 in the CSFV infected tissues(heart, liver, spleen, lung, kidney and lymph node) are first revealed. The results validate that TLR2, TLR3, TLR4, and TLR7 are differentially expressed in various porcine tissues infected with CSFV. Compared with the control group, the m RNA and protein levels of TLR7 are significantly up-regulated in the infected groups; For TLR2 and TLR4 expression, are significantly increased in the lung and kidney while inhibited in the spleen and lymph nodes; The expression of TLR3 is significantly decreased in the lymph nodes. IHC staining reveals that TLRs are not only expressed in both macrophages and lymphocytes but also in many ‘non-immune’ cell types, including cardiac fibroblasts, smooth muscle cells, and bronchial and renal tubular epithelial cells. The intensities of TLR expression in these cells from CSFV-infected tissues differed from the control tissues, suggesting that the expression of TLRs is diversified rather than specific in response to pathogens. The observed TLR expression may provide insights into the underlying immunological recognition and pathological changes that occur in swine following CSFV infection.(3)Though lentiviral packaging, Npro, NS2, NS3, NS3/4A, NS4 B, NS5 A and NS5 B are overexpressed in swine macrophages, and the effect of these proteins on TLR signaling pathway are analyzed. The results indicate that the expression of Npro protein to downregulation of TLR2 and upregulation of TLR4; NS2 can promote the high expression of TLR2, TLR4 and TLR7; TLR3 is significantly downregulated by NS3, NS3/4A, NS4 B, and NS5 A. Npro, NS2, NS3, NS3/4A, NS4 B and NS5 A do not elicit the phosphorylation of NF-κB p65; the expression of NS3, NS3/4A, NS4 B and NS5 A result in a significant suppression of TRIF and IRF3, thereby inhibition of IL-6 and IFN-β. Moreover, TLR3-mediated innate immune responses are inhibited in stable cell lines expressing NS3, NS3/4A, NS4 B and NS5 A by stimulation with poly(I:C). In a word, the “strategic proteins” that affecting TLRs signaling pathways are preliminarily determined.(4)The successful construction of the stable cell lines expressing Npro, NS2, NS3, NS3/4A, NS4 B, NS5 A and NS5 B, providing a material basis for the later research on the interaction and co-localization between non-structural proteins and the key proteins of the TLR signaling pathway and for elucidating the mechanism of CSFV regulating TLR-mediated innate immune response.In summary, in this study, a preliminary study on the effects of CSFV and its non-structural proteins on TLR-mediated innate immune response, and explore a fundamental problem that the role of CSFV and its non-structural proteins in which TLRs and TLRs signaling pathway in which key sites and to modulate the immune response. Meanwhile, the expression pattern of TLRs in CSFV infected tissues are added, and preliminarily build up technological platform to study the underlying mechanism of CSFV non-structural proteins on TLRs-mediated innate immune response in swine macrophages.