Interleukin-6Receptor Mediates Expression Of Interleukin-32and Interleukin-6in Response To Influenza A Virus Infection

Influenza A virus (IAV) is a highly contagious single-stranded RNA virus that infects both the upper and lower respiratory tracts of humans, and is considered a major worldwide public health problem. Many proinflammatory cytokines, such as IL-6, IL-8, and TNF-α, are involved in the host inflammatory response to IAV infection. Furthermore, patients with severe acute infections most probably developed a "cytokine storm" response which leads to acute lung injury (ALI) or its more severe form, acute respiratory distress syndrome (ARDS). The intensity of the inflammatory response in the lungs reflects a balance between proinflammatory cytokines and their cognate soluble receptors or inhibitors. However, the mechanism regulating between cytokines is still unclear. To give a new sight into host immune response during IAV infection, our previous study found that the expression level of IL-6R changed significantly in response to viral infection using protein and antibody microarray chips. Thus, here we investigate the role of IL-6R involved in the inflammatory network during IAV infection.The pleiotropic cytokine IL-6has long been considered a marker of inflammation.In addition to immune cells, IL-6is produced by epithelial cells, endothelial cells, keratinocytes, and fibroblasts in response to specific stimuli. Central to the regulation of IL-6-mediated responses is the presence of a soluble IL-6receptor (sIL-6R), which undergoes trans-signaling to form a ligand-receptor complex that allows IL-6responsiveness in cell types that lack the cognate IL-6receptor. Studies have demonstrated that sIL-6R is agonistic with IL-6, indicating an enhanced cellular sensitivity to IL-6. Another proinflammatory cytokine, IL-32, has been shown to be induced by influenza virus infection in humans. Although IL-32and IL-6have been identified as vital factors in airway inflammation during influenza virus infection, the molecular mechanism by which these proinflammatory factors regulate each other during the infection remains to be elucidated. Moreover, the mechanism whereby IAV up-regulates IL-32expression remains unknown. Therefore, we investigated the role and mechanism of IL-6and sIL-6R signaling in the inflammatory response to IAV infection. Our data reveal a previously unidentified regulatory mechanism involving the IL-6R, IL-32, and IL-6during IAV infection.In this study, we sought to determine which cytokines interact with sIL-6R during IAV infection. According to the screening results, we concluded that, in human lung epithelial cells, sIL-6R exhibited a strong effect on the regulation of IL-32promoter activity in response to virus infection. These results were confirmed by sIL-6R overexpression and knockdown studies, which demonstrated an increase or decrease in IL-32promoter activity, respectively. Because sIL-6R had the same effect on IL-6promoter activity, we propose that the trans-signaling factor sIL-6R may react with IL-6through an alternative pathway involving IL-32. In addition, results from our screen indicated that the IL-6R may act as a multifunctional protein that regulates the promoter activity of other factors such as IL-18and IL-24in addition to IL-32and IL-6. These possibilities will be further investigated in future studies.Our data also show that the sIL-6R enhanced IL-32expression at the mRNA and protein levels. This effect was observed with endogenous and exogenous (i.e., recombinant human IL-6Ra protein) sIL-6R. Furthermore, using an RNAi approach, we found that IAV infection-induced IL-32expression was dependent on IL-6R. Since a positive regulatory relationship from IL-6R to IL-32was observed, we sought to determine whether a negative feedback mechanism exists from IL-32to the IL6R during the host inflammatory response to viral infection. Indeed, our results demonstrated that IL-32participates in a negative feedback loop to regulate expression of the IL-6R in response to IAV infection. We also verified that the IL-6R enhanced IL-6expression in response to IAV Infection.In addition, we demonstrated that IAV infection led to upregulation of the IL-6R, IL-32, and IL-6expression by a previous unknown mechanism. Based on these results, we propose a model as follows:IAV stimulates expression of the IL-6R, which upregulates IL-32production, which in turn attenuates IL-6R production in a negative feedback loop. Moreover, both IL-32and IL-6R enhance the IL-6pathway.Thus, we draw a conclusion that there exists a previously unrecognized pathway facilitating IL-6signaling. IL-6expression depends on both the IL-6R and IL-32during viral infection. IL-32, as a typical proinflammatory cytokine, also plays an important role in the inflammatory process. In the current study, we identified sIL-6R as a novel IL-32upstream factor that plays a critical role during IAV infection. These results extend our understanding from a previous clinical finding that IL-32is elevated in IAV-infected patients and provides new insight on how proinflammatory factors respond to viral infection.