Investigation On The Role And Mechanism Of Progranulin In Influenza A Virus And Streptococcus Pneumoniae Coinfection

Background:Influenza A virus?IAV?is a respiratory RNA virus that causes influenza in animal and human.IAV infection results in about 0.5million deaths annually and periodic pandemics worldwide.Investigation of clinical cases and autopsy showed that major severe cases and deaths in influenza pandemics were complicated by bacterial coinfection,and Streptococcus pneumoniae?S.pneumoniae/S.pn?was one of the most detected coinfecting bacteria.Study of coinfection in mouse models has identified a range of possible mechanisms for IAV–S.pn coinfection,suggesting that multiple factors including virus,bacteria and host contribute to bacterial susceptibility.Impairment of the antibacterial immune response caused by IAV infection is especially important for the increased susceptibility.However,there is no unified conclusion regarding the viral regulation of immune response at present.And there is still a lack of immune intervention for the prevention and treatment of coinfection.Progranulin?PGRN?is an important immune regulatory factor and exerts anti-inflammatory effect by its affinity and binding to tumor necrosis factor receptors?TNFR?,which blocks TNF-?signaling pathway and suppresses inflammation response.Besides,our group previously reported that PGRN protected mice from excessive inflammatory injury in sepsis.PGRN also showed the similar protective role in pneumonia caused by Staphylococcus aureus infection.The immune regulatory property of PGRN implies its potential role in viral and bacterial coinfection.Objective:Our study aims to investigate on the role and mechanism of PGRN in IAV-S.pn coinfection.It will provide experimental evidence for further understanding the mechanism of increased susceptibility to post-influenza bacterial infection and exploring new immune intervention targets for the prevention and treatment of coinfection.Methods:Serum samples were collected from pediatric patients with single IAV/S.pn infection,IAV-S.pn coinfection and healthy subjects.All subjects were enrolled in Children's Hospital of Chongqing Medical University during the winter flu season.PGRN levels in clinical samples were detected by ELISA assays.Laboratory standard strains IAV?H1N1?PR8??and S.pn?19F?were used in our study to infect mice intranasally at different time points to establish single PR8/19F infection and PR8+19F coinfection pneumonia models.PGRN levels in lung and serum samples of infected mice were detected again by ELISA assays.The role of PGRN in coinfection was defined by comparing the survival rate,viral loads,bacterial loads and pathological features of lung sections between WT and PGRN^-/-mice in different infectious scenarios.To determine the immune regulatory effect of PGRN in coinfection,the classification and counting of inflammatory cells collected from lungs were analyzed by flow cytomertry?FCM?,while the concentrations of cytokines and chemokines in lungs were quantified with ELISA assays.Mouse neutrophils and macrophages were isolated and cultured in vitro,the effect of PGRN on the intrinsic antibacterial functions of these cells were analyzed by phagocytosis and killing capacity assays.FCM,fluorescence TUNEL,immunohistochemical method and western blotting were adopted to explore the influence of PGRN on apoptosis and signaling pathways that mediate apoptosis in infected mice.In the end,the survival rate,bacterial loads and apoptosis were observed in coinfected PGRN^-/-mice after rPGRN treatment to verify the role of PGRN in coinfection.Results:1.Patients with IAV/S.pn single infection showed significantly elevated PGRN levels compared to healthy controls?p<0.001,p<0.01?,while patients with IAV-S.pn coinfection exhibited remarkably higher PGRN levels than patients with IAV/S.pn single infection?p<0.001?.In accordance with the PGRN variation of clinical samples,coinfected mice also showed the highest PGRN concentrations among single infections and control groups.Immunohistochemical detection of PGRN antigen revealed that the increased production of PGRN was mainly distributed in lung epithelial cells and the infiltrated inflammatory cells.2.Pathogen loads analyses further revealed that there was no difference in viral loads between PR8 infected or coinfected WT and PGRN^-/-mice?p>0.05?,indicating that PGRN deficiency has no impact on virus replication.While in 19F infection,WT and PGRN^-/-mice exhibited similar bacterial loads in lungs?p>0.05?,which implied that PGRN was not required for bacterial clearance in mice without preceding viral infection.However,coinfected PGRN^-/-mice displayed significantly higher lung bacterial loads than those of WT mice?p<0.05?.Therefore,PGRN could decrease the susceptibility to bacterial infection in mice infected with influenza virus.3.WT and PGRN^-/-mice inoculated with single PR8/19F showed similar weight loss,both with 100%survival.In PR8+19F coinfection,PGRN deficiency significantly decreased the survival rate,with 11%survival observed in PGRN^-/-mice,whereas the survival rate among WT mice was 50%?p<0.05?.Moreover,coinfected PGRN^-/-mice showed significantly increased weight loss compared to WT mice?p<0.001?.Administration of rPGRN increased the survival of coinfected PGRN^-/-mice to a level equivalent to WT mice,which was significantly higher than that of PBS treated PGRN^-/-mice.These results suggest that PGRN protects mice against lethal coinfection.4.PGRN played an anti-inflammatory role in coinfection and protected lungs from overwhelming inflammatory injury.1)Hematoxylin and eosin?H&E?staining of lung tissues revealed the pathological manifestations and inflammation in infected mice.PR8infected mice showed severe lung edema and mild inflammatory cell infiltration,while 19F infected mice showed thickening of the bronchial epithelium and moderate inflammatory cell infiltration.No difference was found in PR8/19F singly infected WT and PGRN^-/-mice.However,coinfected PGRN^-/-mice exhibited more severe lung injury including multifocal necrosis,massive inflammatory cells aggregation in the alveolar spaces,vacuolation and hemorrhage compared to WT mice,which demonstrated that PGRN protected mice from lung injury in coinfection.2)By quantification of the lung injury associated markers,we observed that coinfected PGRN^-/-mice showed significantly increased lactate dehydrogenase?LDH?activity,total proteins,and lung edema compared with WT mice?p<0.05?.Besides,the pro-inflammatory cytokines TNF-?and IL-1?levels in lungs of coinfected PGRN^-/-mice were significantly increased compared with WT mice?p<0.05,p<0.01?.These data suggest that PGRN played an anti-inflammatory role in coinfection and protected lungs from uncontrolled inflammatory injury.5.Furthermore,FCM analyses showed that there was no difference in the percentages of neutrophils?CD11b⁺Ly6G⁺?and macrophages?CD11b⁺F4/80⁺?in WT and PGRN^-/-mice in different infection scenarios.However,coinfected PGRN^-/-mice showed obviously increased inflammatory cells,macrophages and neutrophils numbers in lungs compared to WT mice?p<0.05,p<0.01,p<0.001?.The levels of corresponding chemokines CXCL1 and CCL2 were also obviously elevated in lungs of coinfected PGRN^-/-mice?p<0.05?.These results confirmed the anti-inflammatory effect of PGRN in coinfection.In addition,coinfected WT and PGRN^-/-mice showed similar numbers and percentages of CD8⁺IFN-?⁺T cells in lungs?p>0.05?,suggesting that the deficiency of PGRN did not influence the viral clearance in mice.6.PGRN inhibited apoptosis of epithelial and inflammatory cells from mice in influenza and coinfection.1)WT and PGRN^-/-macrophages and neutrophils were isolated and cultured in vitro.The phagocytosis and killing assays showed no difference in the phagocytosis and killing capacity between WT and PGRN^-/-inflammatory cells in single 19F infection?without virus infection??p>0.05?.In other words,PGRN did not influence the intrinsic antibacterial function of inflammatory cells.2)By analyzing the apoptosis with FCM,we observed that apoptosis of inflammatory cells of PGRN^-/-mice was significantly higher than that of WT mice in single PR8 infection and coinfection?p<0.01,p<0.001?.WT and PGRN^-/-mice showed no difference in apoptosis of inflammatory cells in single 19F infection?p>0.05?.The results of fluorescence TUNEL and immunohistochemical detection of cleaved caspase-3 further showed that apoptosis of lung epithelial and inflammatory cells were both remarkably increased in PGRN^-/-mice in PR8 infection and coinfection.Thus,PGRN suppressed apoptosis of lung epithelial and inflammatory cells in influenza and coinfection.7.PGRN decreased the susceptibility to bacterial infection through suppressing ER stress-mediated apoptosis of inflammatory and epithelial cells in influenza.1)Western blotting analyses showed that the activation of ER stress and the downstream apoptosis signaling pathways?XBP1s,CHOP,JNK and Caspase-3?were markedly enhanced in PGRN^-/-mice,implying the involvement of ER stress in the regulation of apoptosis by PGRN.2)Apoptosis and the activation of ER stress were analyzed again in mice administrated with rPGRN or PBA?ER stress inhibitor?.We observed that WT and PGRN^-/-mice showed obvious decline in apoptosis of inflammatory cells in PR8 infection and coinfection after the inhibition of ER stress by PBA?p<0.05,p<0.01?.The expressions of XBP1s,CHOP,phosphor-JNK and cleaved caspase-3 in PR8 infected and coinfected PGRN^-/-lungs were decreased after treatment with PBA or rPGRN.Additionally,the protein levels and bacterial loads in coinfected PGRN^-/-mice were significantly decreased after PBA or rPGRN treatment?p<0.05?.Above all,ER stress-mediated apoptosis is involved in primary influenza infection and coinfection.Inhibition of ER stress or rPGRN treatment could rescue the increased apoptosis and susceptibility to bacterial coinfection caused by deficiency of PGRN.3)Human rPGRN or PBA pretreated human lung epithelial cells A549were infected with PR8 and 19F.The apoptosis and ER stress-mediated apoptosis signaling pathways were evaluated with FCM and western blotting,respectively.PR8 infected and coinfected A549 cells exhibited significant reduction in apoptosis with the pretreatment of rPGRN or PBA?p<0.05,p<0.01,p<0.001?.Western blotting revealed that the activation of ER stress-mediated apoptosis signaling pathways XBP1s,CHOP,phosphor-JNK and cleaved caspase-3 were significantly decreased in A549cells pretreated with rPGRN or PBA.The in vitro experiments confirmed that PGRN suppresses apoptosis though inhibiting ER stress.Conclusion:PGRN levels are significantly elevated in influenza A virus and S.pneumoniae coinfected clinical and mice samples.PGRN could decrease susceptibility to post-influenza S.pneumoniae infection and protect mice from lethal coinfection.During early influenza virus infection,PGRN attenuates ER stress-mediated apoptosis signaling pathways to reduce apoptosis in inflammatory and epithelial cells in lungs.By relieving the stress from viral proteins synthesis and accumulation and reducing apoptosis caused by over-activation of ER stress,PGRN improves the intactness of epithelium and survive of inflammatory cells in lungs.Thus,PGRN decreases the susceptibility to S.pneumoniae coinfection in influenza.When bacterial coinfection occurs,PGRN protects against lung injury through decreasing bacterial loads and suppressing inflammation response,which increases the survival of mice.The immune intervention of decreasing susceptibility to post-influenza bacterial infection and protecting mice from overwhelming inflammatory injury mediated by PGRN provides new ideas for prevention and treatment of coinfection.