Regulation Of NLRP3-mediated Inflammatory Responses During Porcine Reproductive And Respiratory Syndrome Virus(PRRSV) Infection

Porcine reproductive and respiratory syndrome(PRRS) is an important viral infectious disease in swine industry worldwide. The causative agent is porcine reproductive and respiratory syndrome virus(PRRSV), which can inhibit host innate and adaptive immune response, cause immunosuppression, and lead to persistent infection. So it is difficult to control and eradicate PRRS. PRRSV can cause a strong interstitial pneumonia in infected pigs, and induce proinflammatory cytokines in lung. Recent studies have shown that PRRSV activated the NLRP3 inflammsome and induced IL-1β secretion. The inflammatory response is the solid line in host anti-viral immunity; however, how PRRSV regulate the inflammatory response to escape the immunologic surveillance remains unknown. Therefore, this study focused on the molecular mechanism of how PRRSV regulated the NLRP3-mediated the inflammatory response. The results of this study were as follows:1. The dynamic changes of NLRP3 inflammasome during PRRSV infection. PAMs were infected with the PRRSV strain BJ-4 at different times(12 h, 24 h, 48 h, 72 h), then, NLRP3, ASC, procaspase-1 and pro-IL-1β mRNA expression and IL-1β production were detected by qPCR and ELISA. The obtained results showed that PRRSV could induce NLRP3, ASC, procaspase-1 and pro-IL-1β mRNA expression and secretion of IL-1β in early infection in porcine alveolar macrophages(PAMs), but the levels of pro-IL-1β mRNA and IL-1β protein decreased to a degree that was similar to the level of the mock-infected group in later infection. And different dose of PRRSV infection induced procaspase-1 and pro-IL-1β mRNA expression and secretion of IL-1β at a dose dependent manner. The experiment results showed that PRRSV promoted early inflammatory response through up-regulating NLRP3 signaling pathway proteins, but inhibited NLRP3-mediated inflammatory response through some unknown mechanisms in later infection. So the following studies first confirmed whether the inflammatory response induced by PRRSV was dependent on the NLRP3 signaling pathway, and then screened the components of PRRSV which inhibited the inflammatory response. Finally, this study explored the molecular mechanism on how PRRSV promoted the inflammatory response in early infection.2. The secretion of IL-1β induced by PRRSV is dependent on the NLRP3 inflammasome pathway. NLRP3 inhibitor-glyburide and specific siRNA targeting NLRP3 or ASC were used to inhibit the NLRP3 inflammasome activation. The results showed that glyburide could inhibit the secretion of IL-1β induced by PRRSV significantly. And the siRNA targeting NLRP3 or ASC could down-regulated the gene expression and the IL-1β induction.3. PRRSV nsp11 and nsp1α are the components to inhibit NLRP3 inflammasome mediated IL-1β secretion. PAMs were transfected with PRRSV nsp11 and the inactivated endoribonuclease mutants or nsp1α and the deletion or mutation of Zinc-Finger(ZF) domain mutants. The results showed that nsp11 and nsp1α could inhibit the expression of pro-IL-1β mRNA induced by lipopolysaccharide(LPS) and the secretion of IL-1β induced by LPS plus nigericin. Furthermore, the mutation studies showed that the endoribonuclease activity and ZF domain were essential for nsp11 and nsp1α to inhibit the secretion of IL-1β. Then, the NLRP3 inflammasome was reconstructed in HEK293 T cells which were then transfected with expression plasmid encoding nsp11, nsp1α and the mutants. The results were consistent with that in PAMs, which further determined the inhibition of nsp11 and nsp1α to NLRP3 inflammasome mediated IL-1β secretion.4. MiR-373 is up-regulated by PRRSV and it can promote the inflammatory response. Our previous study showed that PRRSV could up-regulate mi R-373 expression in MARC-145 cells. And further study found overexpression of miR-373 in PAMs stimulated by LPS plus nigericin could increase secretion of IL-1β and NLRP3, procaspase-1 and pro-IL-1β mRNA expression, which could promote the inflammatory response. In addition, PRRSV infection could down-regulated the expression of target gene TGFBR2(immuno-inhibitory receptor) of mi R-373 significantly.In conclusion, PRRSV infection could activate the NLRP3 inflammasome and induced the proinflammatory cytokine IL-1β secretion which can protect host from the virus invasion. Meanwhile, PRRSV could encode nsp11 and nsp1α to antagonize the activation of NLRP3 inflammasome and inhibit the inflammatory response, which were in favor of the virus proliferation. In addition, we for the first time found that miR-373 could promote the inflammatory response and regulate the innate immune response induced by PRRSV. PRRSV infection could up-regulated miR-373 and down-regulated TGFBR2 expression significantly, which promoted the inflammatory response in early infection. Our study reveals a new mechanism that PRRSV antagonize host innate immune responses and may provide some insights into the research on molecular targets of anti-PRRSV drugs and prevention of PRRS.