SHP2Positively Regulates Interferon Alpha-Induced Jak-Stat1Signaling Pathway And Contributes To The Anti-RSV Activity

BackgroundRespiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in infants and young children. About40-50%of bronchiolitis and25%of pneumonia in hospitalized infants is caused by RSV infection. Approximately160,000deaths per year worldwide are caused by RSV infection. RSV infection in non-high-risk groups could only cause the cold, but there still are a small part of people (0.5-2%) will develop to severe bronchiolitis and pneumonia. Severe RSV infection in infancy is closely related to asthma in adolescence. Till now, there are no effective vaccines since the pathogenesis of RSV has not been clearly elucidated. The difficulties in the prevention and treatment of diseases caused by RSV infection is that the virus has evolved various immune evasion mechanisms to antagonize the immunity, such as, inhibition of T cell proliferation, inhibition of the activation and function of DC, NS1and NS2proteins of RSV could inhibit the production and the antiviral activity of IFN-a.Phosphorylation of tyrosine kinase and dephosphorylation of tyrosine phosphatase is a ubiquitous process in regulating of cellular signal transductions. SHP2(Src homology phospho-tyrosyl phosphatase2) is a non-transmembrane protein tyrosine phosphatase. SHP2is one member of PTPs family, it is encoded by PTPN11and widely expressed in various tissues and cells. SHP2contains two N-terminal SH2domains by which SHP2binding with and then phosphorylates the tyrosyl acid, thus activating the structure protein-tyrosine phosphatase (PTP enzyme), and plays the dephosphorylation role in the intracellular signaling pathways. SHP2plays an important role in the intracellular signaling pathways.Interferon (IFN) is a cytokine secreted by host cells infected with viruses or stimulated by IFN inducers which has a broad-spectrum antiviral activity. IFN first binds to their corresponding receptors, then activates the Jak/Stat (Janus kinase, Jak; signal transducer and activator of transcription, Stat) signaling pathway. This signal cascade is amplified through intracellular signal transduction, and ultimately leads to the transcription of different kinds of antiviral protein, and thus plays its antiviral role. The study on RSV and IFN show that RSV could inhibit the production of IFN through its non-structural proteins (NS). IFN-induced signaling pathways are regulated by three important protein families. They are protein tyrosine phosphatases (PTPs), suppressor of cytokine signaling,(SOCSs) and protein inhibitor of activated STAT (PIAS). The main role of these three protein families is its negative feedback regulation of intracellular signaling pathways to prevent excessive activation. Src homology phospho-tyrosyl phosphatase2(SHP2) is a member of PTPs. In the IFN induced signaling pathway, SHP2has been shown to be a dual-function protein, which can directly negative regulate the signaling pathway by playing the negative feedback role, also play the positive role by interfering with other regulatory proteins in the signaling pathway. However, the role of SHP2in RSV infection and its associated mechanisms have not yet to be studied.ObjectiveThe role of SHP2in RSV infection and its associated mechanisms; provide evidences for the pathogenesis and immune escape mechanism of RSV.Methods1. Cell Experiment:1.1RSV infection of human alveolar epithelial cell line A549, the gene and protein expression of SHP2were detected by RT-qPCR and Western blot; inhibition of the function of SHP2by PHPS1(phenyl hydrazono pyrazolone sulfonate1), the RSV-F protein expression and RSV titer were detected by RT-qPCR and plaque assay, respectively; IFN-a production was detected by RT-qPCR; cells were pretreated with IFN-a to activate the Jak/Stat signaling pathway, then treated with PHPS1to inhibit the function of SHP2before infection with RSV, the expression of RSV-F and2’5’-OAS1was detected by RT-qPCR; the phosphorylation of Jakl and Statl was determined by Western blot.1.2peritoneal macrophages and bone marrow-derived macrophages from WT and monocyte SHP2-specific knockout mice were infected with RSV, the expression of RSV-F protein was determined by RT-qPCR.2. Animal experiments:Wild C57BL/6mice and monocyte SHP2-specific knockout mice were intranasally infected with RSV, RSV-F protein was detected by RT-qPCR; the expression of inflammatory cytokines were determined by RT-qPCR; pathological change of lung tissues were detected by HE staining.Results1.12hours post infection of RSV, SHP2protein expression in A549cells was significantly increased.2. RSV infection with PHPS1treated cells, there was no significant differences of RSV-F expression and IFN-a expression, it shows SHP2has no direct effect on RSV replication and IFN-a production in epithelial cells.3. A549cells pre-treated with IFN-a and PHPS1before infection with RSV, the replication of RSV was increased; correspondingly, the expression of IFN-a induced antiviral protein2’5’-OAS1was significantly decreased; the phosphorylation of Statl was suppressed.4. The replication of RSV in BMM/PM from monocyte Shp2-specific knockout mice was elevated.5. There were no significant differences in RSV replication, inflammatory cytokines expression and pathological changes in lung in monocyte SHP2-specific knockout mice infected with RSV. Conclusions:1. In the RSV-infected epithelial cells, SHP2has no effect on IFN-α production, but plays the positive role in the IFN-α induced Jak/Statl signaling pathway. SHP2can promote α-interferon-induced Jak/Stat1signaling pathway and inhibit the replication of RSV.2. In the RSV-infected macrophages, SHP2can directly inhibit the replication of RSV. SHP2specific knockout in monocytes has no effect on RSV replication in vivo. It suggests that SHP2plays the different role in macrophages cells and epithelial cells and the related mechanism will be clarified in future.