The Mechanism Of S1PR1 Regulates Exudative Inflammation Induced By Different Virulence Of NDV Strains

Newcastle disease virus(NDV) is the causative agent of Newcastle disease(ND),which characterized by inflammatory pathological changes in organs of chicken. Newcastle disease is a highly contagious avian disease that causes economic losses to the poultry industry. Birds infected NDV exhibited different degrees of inflammatory responses in various organs, resulted in tissue lesions and dysfunction. NDV pathogenicity depends on multiple factors, and it is mainly determined by the extent to which the virulence of the infecting strain and host cytokine responses. Up to now, the inflammatory response in NDV infection has not been well characterized.Sphingosine-1-phosphate-1 receptor(S1PR1), as a member of G protein- coupled receptors, is a major factor to active inflammatory response and its expression is crucial to inflammation initial. Previous reports showed that S1PR1 as an immune modulator in the context of influenza virus and respiratory syncytial virus(RSV) infections. While there are few reports about the function of chicken S1PR1 in NDV induced inflammation. Based on the immune regulation of S1PR1 in viral infection, we analyzed the dynamic expression of S1PR1 and pro-inflammatory cytokines during different NDV strains infection in vivo and in vitro. Based on these results, a further study was continued to discover how S1PR1 affects the inflammatory response and NDV pathogenicity, which provide a new ideas to control NDV iindeced inflammation and provide theoretical guidance of immune regulatory drugs application to prevent ND.The main content of this research are as follows:1. Inflammatory response and S1PR1 expression in chickens infected with NDVChickens are the most susceptible to NDV, different NDV strains infection causeddifferent degrees of inflammation. To better understand the viral pathogenesis and host inflammatory response, the inflammatory responses and cytokines transcription of SPF chickens which infected with NDV strain La Sota or velogenic strain GM. Chickens showed different clinical signs and pathological alterations after infected with this two NDV strains, GM infection caused severe inflammatory responses, and led to substantial death, while La Sota exhibited no overt disease. There are more bleeding spots and inflammatory cell infiltration observed in tissue sections of chickens’ brain, lung, glandular stomach and spleen after GM infection than La Sota infected tissues. Compared to uninfected chickens, virus replication of GM-infected chickens showed upregulation in small intestine(27.5-fold), brain(14-fold), bursa of fabricius(5.6-fold), stomach(3.8-fold),and caecum tonsil(3.5-fold). Whereas it showed low expression level in liver and kidney,and no significant difference compared with uninfected and La Sota group. Virus mRNA experssion level upregulated only in the brain(6.8-fold) of La Sota infected chickens, and there are no differences in other organizations compare with uninfected group. Consistent with this, higher levels pro-inflammatory cytokines expression were tested in GM infected chickens than La Sota. IL-1β showed upregulation in GM-infected tissus(peaked at8.7-fold) and La Sota(peaked at 6.4-fold). The loealization and distribution of the S1PR1 gene in chickens’ tissues from the infected goslings were investigated by using qPCR,S1PR1 could be detected in multiple organs. The results showed that S1PR1 mRNA expression level was upregulated in the brains(6-fold), livers, glandular stomach and bursa of fabricius(2-fold). In the kidney, pancreas and small intestine tissues of NDV-infected chickens exhibited less S1PR1 expression than in uninfected group. Furthermore, we cloned S1PR1 in frame with eGFP to pCI-neo and transfected this construct into DF-1 cells,S1PR1 expression was observed by using fluorescence microscopy and by Western blotting.2. S1PR1 involved in regulating inflammatory responses induced by NDV infectionTo better understand S1PR1 and NDV induced inflammatory response, we analyzed the dynamic expression of S1PR1 during NDV infection. Here, we observed more rapidand robust pro-inflammatory cytokine expression induced by highly virulent GM than by the low virulence La Sota strain. GM infected DF-1 showed higher expression level of pro-inflammatory cytokines, including IL-1β(22-fold), IL-6(41-fold) and IL-18(5.5-fold),comparing with La Sota infection and control, accompanied by up grated S1PR1 level expression(2.5-fold). Further results showed when intrinsic S1PR1 blocked by specific antagonist W146, IL-1β(40-fold) and IL-6(10-fold) production declined. And overexpressed S1PR1 raised virus-induced IL-1β 5-fold transcriptiion and 250 pg/mL production. Virus titers tested in status of inhibited/overexpressed S1PR1 treatment and the control showed that S1PR1 did not alter NDV replication.3. Signaling pathways of S1PR1 regulated NDV inflammationBased on previous research results, we focus on the nuclear factor kappa B(NF-κB),which may be a correlative signaling pathway between S1PR1 and IL-1β in regulating inflammatory responses during NDV infection. RelA/p65 mRNA(6.7-fold) and protein level were increased and detected by qPCR and western blotting after GM-infected DF-1 3hours. Inhibiting S1PR1 with W146 reduced RelA/p65 mRNA level(10-fold) and luciferase activity(1.5-fold), not protein expression or nuclear localization. The activation of NF-κB need IκB phosphorylation and other process, therefore, this study cannot provide definite conclusions of S1PR1 regulate NF-κB signaling pathway. Either by NF-κB or by another signaling pathway in the process of S1PR1 regulated NDV inflammation, specific pathways need to be researched.4. Inflammatory response in chickens dendritic cells infected with NDVDendritic cells(DCs) are the most potent antigen-presenting cells and acquire cellular antigens and danger signals from dying cells to initiate antitumor immune responses via direct cell-to-cell interaction and cytokine production. In this study, mononuclear cells were isolated from the bone marrow of chickens, and differentiated into mature DCs with GM-CSF and IL-4 in vitro to test the inflammatory responses and S1PR1 expression induced by NDV strains GM and La Sota infection. The result revealed NDV could infect DCs and replicate as it in DF-1 cells, the infected DCs showed significant cytopathic effect(CPE). Virus titer was detected in GM-infected cells of 107.2 TCID50/100μL, 10 000 times higher than La Sota group. Compared to the control group, different mRNA level of IL-1β(10.2-fold), IFN-β(7.6-fold) and CCL5(35.4-fold) were detected in GM infected DCs;IL-10(7.5-fold) and CCL5(14.3-fold) upregulation in La Sota group. NDV infection decreased S1PR1 expression in DCs, especially by GM strain.In conclusion, this study focused on the molecular mechanism of S1PR1 regulated inflammation induced by NDV infection in chicken, and explored the interaction of S1PR1 in NDV infection, which act as an immune modulator during NDV infection. Our data support the hypothesis that NDV infection activates inflammatory responses in CEF cells in a mechanism that is at least partially dependent upon S1PR1. Whether modulating S1PR1 activity could be leveraged to reduce excessive inflammatory responses to NDV infection and thus mitigate tissue damage remains to be determined. These findings shed new light on the crucial role played by S1PR1 as a key molecular to cellular inflammatory responses in NDV infection, and provided a new idea to control NDV induced inflammation and provide theoretical guidance of immune regulatory drugs application to prevent ND.