Sars-cov Spike Protein-pretreated Bronchial Epithelial Cells Step Up Inflammation In The Lung After Exposure To Lipopolysaccharide

Background and ObjectiveSevere respiratory diseases caused by SARS coronavirus (SARS-CoV),influenza A virus subtype H5N1( H5N1)pose a major public health threat. There is increasing evidence that the main cause of death in the patients infected with the respiratory virus was the secondary bacterial infection. However, the mechanism of secondary bacterial infection following virus infection is still unknown. Our preliminary data suggest that superinfection is a frequent, and the main cause of death due to acute damage of the immune system initiated by induction of interferon-gamma-inducible protein 10(IP-10)in response to SARS-CoV. We found that the S protein of SARS-CoV can induce IP-10 release through activation of JAK-STAT signaling pathway, which is related to SARS-CoV- induced immunopathology. We therefore suppose that the immune inflammation in the lung following virus infection might increase susceptibility to bacterial superinfection. The present study is aimed to investigate the link between a dys-regulated innate immunity in the pulmonary epithelial cells prior to the treatment of viral antigen protein and the inflammatory reaction induced by bacterial endotoxin. MethodsPart 1 Determination of activation of JAK-STAT signal pathway in the pulmonary epithelial cells challenged with S protein of SARS-CoV.Insect-baculovirus expression system was employed for expression of the recombinant spike protein (S) of SARS-CoV. The recombinant S protein was purified by Nickel affinity Magnet Beads and identified by western-blotting. Immunofluorescence was performed on determination of activation of JAK/STAT pathway in the cultured 16HBE cells following the treatment of S protein for different time periods.Part 2 Relationship between the impaired pulmonary epithelial cells prior to S protein treatment and lung inflammation induced by LPS.16HBE cells were treated in absence or presence of LPS(20ug/m l ) prior to S protein challenge, or treated with LPS alone; the treated–cells were divided into group A1 (S-LPS-), group A2(S+LPS-), group A3(S-LPS+,) and group A4(S+LPS+) respectively. Supernatants of the cultures were collected for detection of the levels of cytokine/chemokine using Luminex assay. After that, freshly isolated PBMCs were added to upper chamber in each well of the different groups(A1~A4), and then PBMCs counting was performed for determination of chemotactic activity. PBMCs were cultured with fresh medium containing 5% fetal bovine serum for 12h prior to treatment with the mix of culture(group A1~A4 separately) with fresh serum-free medium(1:2 ) for 12h, The levels of cytokine/chemokine were also detected by Luminex assay. Meanwhile, JAK3 inhibitor VI was subjected to evaluation of the role of targeting JAK3 in the management of dys-regulated anti-viral immunity associated with susceptibility to bacterial superinfection.ResultsPart 1 Effect of S protein of SARS coronavirus on JAK/STAT signaling in the 16HBE cells.By Immunofluorescence, we observed that S protein activated the molecules of JAK/STAT pathway including STAT1,STAT5,JAK2 and JAK3. The number of the cells with positive staining of STAT1 or STAT5 was increased significantly 30min after the treatment of S protein. The labeled STATs-fluorescence was observed to move from cytoplasm to nucleolus. The fluorescent staining for either JAK2 or JAK3 molecular was enhanced significantly 15min after S protein treatment.Part 2 Effect of the impaired pulmonary epithelial cells prior to S protein treatment on the susceptibility to inflammatory reaction after exposure to LPS .1.The level of IP-10 or RANTES was significantly higher in Group A2 (S+LPS-) than that of control(Group A1)(418.59±76.63 and 158.1±23.37 pg/ml vs 8.07±1.79 and 0.99±0.1 pg/ml),P<0.01;There was a slight elevation of level in IL-6 or IL-8 of group A2(87.17±7.8;22.25±0.44 pg/ml) and A3(S-LPS+)(63.16±1.07;43.82±4.38 pg/ml) compared to control, P <0.01 ; In Group A4( S+LPS+), the levels of the four cytokines/chemokines were increased further(IL-6 417.64±30.43;IL-8 128.75±6.55;IP-10 1305.45±35.17;RANTES 187.05±18.46pg/ml) compared to Group A2 or Group A3 ,P<0.01.2. The chemotactic activity of the PBMCs was enhanced in the well with the impaired 16HBE cells prior to S protein pretreatment group(50.66±13.93 PBMCs/vision field)and with the 16HBE cells of the treatment of LPS(44±17.1 PBMCs/vision field) compared to control group(3.2±4.81 PBMCs/vision field) . The chemotactic activity of the PBMCs was reinforced when co-cultured with the 16HBE cells of Group A4 (77.46±8.69 PBMCs/vision field),P<0.01.3. The levels of IFN-γ,MIP-1αand TNF-αproduced by PBMCs was increased after being cultured with the supernatants of the impaired 16HBE cells (group A2, S+LPS-)(57±26.09;171.18±0.2;29.8±4.76 pg/ml) compared to those cultured with normal control(group A1) (21.33±11.31;can not be detected; 22.3±1.9 pg/ml), P<0.05;The levels of the three cytokines above were elevated further in the PBMCs cultured with the supernatant of Group A4 (102.4±6.4;223.15±20.86;65.5±5.39 pg/ml),P<0.01. While we found that the JAK3 inhibitor VI can decrease the induction of IP-10(713.86±27.9 vs 1305.45±35.17 pg/ml) by the treatment of Group A4 (S+LPS+) , as well as down-regulate the increased levels of multi-cytokines(IL-8 71.72±3.2;rantes 77.94±1.53 vs 128.75±6.55;187.05±18.46 pg/ml), P<0.01.We also found that the increased level of IFN-γin the supernatant of the PBMCs triggered with the culture of Group A4 plus the pretreatment of the inhibitor was down-regulated as well, P<0.05.Conclusion1. The pretreatment of SARS coronavirus Spike protein can cause the significantly elevated inflammatory reaction in the pulmonary epithelial cells once upon lipopolysaccharide(LPS)challenge.2. The S protein-treated pulmonary epithelial cells display an ability of recruiting PBMCs, leading to a second wave of immune inflammation , which can be reinforced significantly when a small amount of LPS is added.3. A high level of IP-10 released from the pulmonary epithelial cells following the challenge of spike protein plays a crucial role in enhancement of the sensitivity of inflammatory reaction induced by LPS Selective JAK3 inhibitor could be used for prevention of the development of the inflammatory storm triggered by secondary bacterial infection.