The Role Of Chemotaxis CCL2 And Long Noncoding RNA AVAN In The Anti-influenza Viral Responses

Influenza,one of the most common respiratory infection diseases with significant negative impact on human health,it has caused several pandemics globally and took millions of lives away.In here,we aimed to focus on a specific type of influenza virus,influenza A Virus(IAV).Most IAV infections resulted in acute respiratory distress syndrome(ARDS),which is a severe form of acute lung injury(ALI)that contributes to morbidity and mortality.The majority of infected patients have exhibited excessive cytokine and chemokine production,which could result in hypercytokinemia and is associated with disease severity.Hypercytokinemia,also known as a “cytokine storm”,is an early indicator of IAV induced ALI.Previous studies have shown that many important cytokines and chemokines played crucial roles in IAV infection;however,the functionality of cytokines and chemokines on the pathogenesis of infection are controversial.In addition,there is limited information about the role of cytokines and chemokines play during avian-origin IAV infection,especially the H7N9-induced ALI.As the advancement and application of the second generation of sequencing became more readily available,more and more long noncoding RNAs(lnc RNA)have been discovered and recognized,and they played a significant part in many life processes,such as cancer biology,ontogentic development,and neuroscience.However,the role of lnc RNAs in antiviral innate immune responses remained largely unknown.In addition,there is no report on the lnc RNA profile about neutrophils.And that is why we planned to discover one lnc RNA that participated in antiviral innate immune responses.Based on these presumptions,we divided our research into two parts:Part I: C-C motif chemokine ligand 2(CCL2)mediates the acute lung injury induced by lethal influenza H7N9 virusTo evaluate the pathogenicity of influenza H7N9 virus Ah01 and Hb01,C57BL/6 mice were inoculated intranasally(i.n.)with Ah01 and Hb01.The results of weight loss and survival rate indicated that Hb01 is more virulent in mice compared to Ah01.In addition,we evaluated the histological changes in lung tissues and the lung edemas and found that mice infected with influenza H7N9 virus Hb01 developed severe ALI.Notably,the H7N9-infected cases usually died about 1 week after infection.Thus,the innate immune responses may be crucial for influenza H7N9 virus infection-related pathogenesis.In order to confirm this hypothesis,we inoculated i.n.Rag1-/-mice that have no matured functional T and B cells with Hb01.There are no differences in survival rate and body weight changes between the Rag1-/-mice and wild-type mice following Hb01 infection,which indicated that innate immune response is crucial for influenza H7N9 virus infection.To study the cytokine and chemokine responses in mice infected with influenza H7N9 Hb01 virus,the levels of cytokines and chemokines in mice BALF and serum at 5 day post infection(DPI)are investigated.The results showed that the most of the proinflammation cytokines and chemokines were significantly upregulated upon the H7N9 challenge.Strikingly,CCL2 levels in both BALF and serum remained high following H7N9 infection.We next explored the dynamic expression manner of CCL2 upon H7N9 challenge and found that CCL2 was significantly upregulated beginning at 1 DPI in mice BALF and serum,and remained high in BALF up to 9 DPI.This clearly attested to the fact that CCL2 remains high in mice serum following Hb01 infection.Furthermore,the lung tissues of mice exhibited a high level of CCL2 at 5 DPI.Moreover,the plasma concentrations of CCL2 of the confirmed H7N9-infected patients were significantly elevated compared to the control group,confirming previous reports.Thus,we hypothesized that CCL2 might play a critical role in the host's response to lethal influenza H7N9 virus infection.To elucidate the functional role of CCL2 in ALI,CCL2-deficient mice were infected with lethal influenza H7N9 virus Hb01;survival rate has significantly improved compared to infected wild-type mice.Rapid and irreversible weight loss(>20%)occurred in infected wild-type control mice but not in CCL2-/-mice following infection.In addition,lung edema was significantly ameliorated in infected CCL2-/-mice.Moreover,lung histopathology was significantly improved and leukocyte infiltration of lung tissue was significantly ameliorated in CCL2-/-mice.Furthermore,we found that the infiltration of macrophages was reduced while the neutrophils were significantly increased in CCL2-/-mice BALF compared to wild-type mice.There were no difference in lung virus loads between infected CCL2-/-mice and wild-type mice.These results confirmed the important role of CCL2 in H7N9-induced ALI,and suggested that this factor is a potential therapeutic target.Together,we reported a role for the chemokine CCL2 in the pathogenesis of avian-origin influenza H7N9.By infecting wild-type mice with lethal doses of H7N9,we showed that elevated levels of proinflammatory cytokines/chemokines in serum and BALF samples resulted in cell infiltration to lung tissues,which resulted in subsequent lung injury and the eventual animal death.CCL2 levels in particular were elevated throughout the course of the infection.In mice deficient of CCL2,infection with H7N9 was not as lethal compared to the wild-type mice,and lung injury and cell infiltration had diminished relative to the wild-type mice.These results combined with patient data that indicated elevated CCL2 levels in their serum upon infection with H7N9 provided a reasonable conclusion for the role of CCL2 in overactivation of the immune response that results in H7N9 pathogenesis.Part II:The role of lnc RNA AVAN in anti-IAV innate immune responseTo investigate the molecular pathogenesis in IAV infection,we searched for whole transcriptional alterations using RNA-seq in neutrophils samples from patients infected with IAV at acute stage and their matched recovery stage samples.A total of 433 novel lnc RNAs were detected in influenza patients compared to their recovery stage(FC>2,p<0.05).Within all that we decided to classify a novel lnc RNA that was significantly up-regulated in IAV-infected patients and the experiment model,and we named it,AVAN.Only one transcript variant(approximately 500nt)of AVAN was found upon IAV infection using northern blot analysis and the exact length transcript(517nt)of AVAN was confirm by 5' and 3' RACE studies.We resolved cytosolic and nuclear extracts via subcellular fractionation and found that AVAN was localized to both cytoplasm and nuclear.The observation was confirmed by RNA fluorescence in situ hybridization(RNA-FISH)with AVAN probes.Moreover,the expression of AVAN was significantly up-regulated upon IAV challenging in a time-and dose-dependent manner.To evaluate the potential function of AVAN,we next analyzed the related gene coexpression networks from the sequencing data and gene set enrichment analysis(GSEA)and found the influenza viral RNA transcription and immune cell activation were tightly related to the AVAN.Functionally,AVAN enhanced the antiviral responses by positively regulated type I interferons induction.In addition,AVAN can trigger inflammation cytokine expression and activate neutrophils and enhance the chemotaxis of neutrophils.To investigate the molecular mechanism by which AVAN regulates type I IFN expression,the RNA pull-down assay with biotin-labeled AVAN or AVAN antisense control was implemented,then mass spectrometry(MS)analysis were performed.We identified that the E3 ubiquitin ligase TRIM25,a RNA-binding protein,was identified to bind AVAN upon IAV challegening compared to AVAN antisense control.The result was confirmed by AVAN RNA pull-down western blot experiments and TRIM25 RIP assays.Furthermore,the IP assay demonstrated that AVAN enhance the interaction between TRIM25 and RIG-I and promoted the poly-ubiquitylation of RIG-I,which will activate the TRIM25-and RIG-I-mediated antiviral innate immune signaling.Recent studies showed that divergent lnc RNA can positively regulate the transcription of nearby gene through chromatin remodeling.The genomic location of AVAN suggested that it was divergently transcribed from upstream of FOXO3 a gene.To investigate the role of AVAN in FOXO3 a expression,we constructed the FOXO3 a promoter luciferase reporter and found that AVAN can significantly enhance the activity of the luciferase.In addition,AVAN can up-regulate the transcription of FOXO3 a,consistent with its role of enhancing the expression of FOXO3 a at protein level.The association of AVAN in FOXO3 a expression and subcellular localization of AVAN in both cytoplasm and nucleus prompted us to test whether AVAN could interact with FOXO3 a promoter DNA.To that end,we conducted chromatin isolation by RNA purification(Ch IRP)using antisense oligoes of AVAN followed by q RT-PCR and found that AVAN could bind to the segments of FOXO3 a upstream approximately-1000 to-600 bp.We further verified the potential role of AVAN in modulation the chromatin modification of FOXO3 promoter region using Ch IP-q RT-PCR and found that AVAN increased the level of Pol II binding and H3K4me3,H3K27 ac chromatin modification in the AVAN coated FOXO3 promoter region.These data indicated that AVAN plays a role in regulation FOXO3 expression in cis through association with the latter's promoter DNA and performing chromatin remodeling.To evaluate the global influences of AVAN during virus infection,we performed the cellular transcriptome profiling using c DNA microarray on A549 cells and found that the overexpression of AVAN altered the expression genes were enriched with the antiviral innate immune responses and inflammation diseases.To date,the gene that associated with interferon signaling and cytokine signaling in the immune system were markedly up-regulated and were confirmed.These data again revealed that AVAN strongly participated in antiviral innate immune process.In order to investigate the significance of AVAN in antiviral responses in vivo,we constructed an AAV2/9 vector containing AVAN and then delivered into the C57BL/6 mice intranasally(i.n.).After IAV infection,the body weight loss,survival rate,lung edema,histopathology,and virus load of mice were observed.We found that AVAN could ameliorate the IAV-induced acute lung injury and protect mice from IAV infection.Together,we were the first to successfully landscape the lnc RNAs profile of IAV-patient neutrophils and identified the function of AVAN,a novel lnc RNA,in innate antiviral immune response.AVAN served as a positive regulator in RIG-I signaling activation through direct conjugation of TRIM25 and enhanced the interaction between TRIM25 and RIG-I.At the same time,AVAN also played a role in the positive regulation of FOXO3 a expression in cis through association with the latter's promoter DNA and performed chromatin remodeling to maintain the activation and enhanced the chemotaxis of neutrophils.Moreover,our research demonstrated that AVAN protects mice from influenza A infection in vivo.In conclusion,our research first identified the chemokine CCL2 as a pivotal mediator in H7N9 infection and should be considered as a promising therapeutic target;in addition,we revealed a novel lnc RNA,AVAN,as a key positive regulator of antiviral innate immune response that might have important clinical implications for virus diseases.