Study On The Mechanism Of C Fiber Regulation Action Of Antiviral Response Post RSV Infection

Part Ⅰ.MULTI-ORGAN RNA SEQUENCING STUDY OF C FIBER DEGENERATED BALB/C MICEObjective: In 1977,Jancsó et al.first reported that subcutaneous administration of 50 mg/kg capsaicin to neonatal rats at 2 days after birth selectively and irreversibly degenerated nociceptive primary sensory neurons.Subsequently,they further observed that treatment of neonatal rats with capsaicin selectively degenerated the unmyelinated C fiber but had no effect on the myelinated A fiber by electron microscopy.In the following half century,C fiber degenerated(KCF)animal models have provided the possibility to study the regulatory role of neuro-immune,especially C fiber,in different diseases.However,the establishment and validation of KCF models are mostly based on rat models,and a comprehensive understanding of KCF mice is lacking.In this study,we intend to comprehensively explore the gene expression profiles and possible biological functional changes in four important organs of KCF mice including lung,intestine,brain and spleen.Methods: KCF mouse model was established by subcutaneous injection of 50 mg/kg capsaicin into newborn BALB/c mice 2 days after birth.The lungs,intestines,brains and spleens of 6-8W KCF mice and untreated BALB/c mice(Intact mice)were dehydrated with sucrose and then frozen sectioned.The changes of C fiber expression in each organ of KCF and Intact mice were observed by immunofluorescence with CGRP.RNA sequencing(RNA-Seq)was performed on the lung,intestine,brain and spleen of KCF and Intact mice,and the immune cell infiltration status of different organs was analyzed by CIBERSORT algorithm to investigate the alterations of immune cell infiltration in different organs of KCF mice.Differential analysis was performed by DESeq2 for each organ of KCF and Intact mice to find differential genes,and GO function annotation and KEGG pathway enrichment analysis were performed for differential genes in each organ.Results: Immunofluorescence of C fibers revealed significantly lower CGRP fluorescence intensity in KCF mice compared with intact mice in lung,intestine,brain and spleen,suggesting complete degeneration of C fibers.Immune infiltration analysis based on RNA-Seq data revealed that the proportion of resting mast cells in the lung and intestine was reduced in KCF mice and the proportion of spleen monocytes was reduced compared with Intact mice.Analysis of RNA-Seq data also revealed significant alterations at the transcriptional level in all four organs of KCF mice compared to Intact mice,with the intestine being the most significant.Gene expression profiles of KCF mice were most similar in lung and intestine as revealed by clustering heat map.GO functional enrichment of differential genes suggested significant enrichment of antiviral pathways in all organs,and KEGG pathway enrichment analysis revealed altered immune,endocrine and metabolic pathways in four organs.Conclusion: Subcutaneous capsaicin injection could effectively degenerate C fiber in BALB/c mice.C fiber degeneration had significant effects on the lung,intestine,brain and spleen at the transcriptional level,and endocrine,metabolic,and immune-related pathways were altered in all organs.Notably,the antiviral pathways were significantly altered in all organs of KCF mice,suggesting that C fiber regulated host antiviral responses.Part Ⅱ C FIBER REGULATES HOST ANTIVIRAL RESPONSE POST RSV INFECTIONObjective: The first part of the study showed that C fiber affects antiviral responses in the lung,intestine,brain and spleen.Our research group explored the role of C fiber in the host antiviral effects post respiratory syncytial virus(RSV)infection in the early stage and found that RSV-infected KCF mice showed significantly lower RSV titers and reduced lung inflammation compared to RSV-infected intact mice,suggesting that C fiber was involved in regulating the host anti-RSV effects.TRPV1(Transient receptor potential vanilloid 1)is a nociceptive receptor specifically expressed on C fiber,and increased TRPV1 expression after RSV infection has been reported.In this section we will further explore the role of C fiber in regulating the host antiviral effect after RSV infection by RNA-Seq,inhibition or activation of TRPV1.Methods: KCF and intact mice were given nasal drops of RSV,and lung tissues were taken 12 h post RSV infection for RNA-Seq.Differential analysis was performed by DESeq2 to obtain differential genes.GSEA pathway enrichment analysis was performed,and qPCR was performed to verify the expression of key genes.Intact mice were treated with TRPV1 inhibitor CPZ(Capzepine)and TRPV1 agonist CAP(Capsaicin),respectively,then given nasal drops of RSV solution within 30 min after treatment.BALF(bronchoalveolar lavage fluid)at 12 h post-infection,and lung tissues were collected 3 days post-infection.Plaque assay,qPCR,and histochemical staining were performed to observe the replication level of RSV in mouse lung tissues.HE staining was performed to observe the pathological changes in mouse lungs,and ELISA was performed to detect the changes of IFN-α/β signaling pathway.Results: RNA-Seq results suggested that pathways associated with IFN-α/β production after RSV infection was significantly enriched in KCF mice compared with intact mice.qPCR further verified that KCF mice had a significantly higher level of IFN-α/β expression than intact mice.CPZ inhibited viral replication in intact mice,attenuated pathological inflammation in the lungs after RSV infection,and promoted IFN-α/βproduction in the lungs.However,CAP treatment exerted opposite effects in intact mice after RSV infection.Conclusion: Degeneration and inhibition of C fiber promotes IFN-α/βproduction after RSV infection to enhance host antiviral response.Activation of C fiber reduces IFN-α/β production after RSV infection to impair the antiviral response of the host.Part Ⅲ STUDY ON THE ROLE OF VIP IN C FIBER-MEDIATED HOST ANTIVIRAL RESPONSE POST RSV INFECTIONObjective: In the previous part,we verified that C fiber regulates anti-RSV effects by RNA-Seq and activation or inhibition of TRPV1.Our previous study found that the level of vasoactive intestinal peptide(VIP)in BALF of KCF mice was significantly higher than that of Intact mice,and VIP receptor inhibitor(VIPhyb)promoted viral replication in KCF mice,while VIP inhibited viral replication in intact mice,suggesting that C fiber can regulate the antiviral effect of RSV infection via VIP.In this part,we intend to:(1)clarify whether C fiber regulates the antiviral effect after RSV infection through VIP;(2)verify that VIP can promote the antiviral effect after RSV infection both in vivo and in vitro;(3)further explore the regulatory relationship between C fiber and VIP in BALF through RNA-Seq.Methods: VIPhyb was used to observe the pathological changes of lung,virus replication and lung IFN-α/β levels after RSV infection in KCF mice.VIP was used to observe the changes of expression levels of IFN-α/β,viral replication and lung pathology after RSV infection in intact mice.In vitro,VIP was used to observe IFN-α/β changes after RSV infection and to explore the role of PKA and PKC in the antiviral effect in alveolar macrophage cell line MH-S.VIP m RNA expression levels in four organs of intact and KCF mice were analyzed by RNA-Seq,and protein levels of VIP in BALF and intestine in intact and KCF mice were detected by ELISA.Intact and KCF mice were nasal dripped with RSV solution,and the intestinal tissues of intact and KCF mice were taken for RNA-Seq 12 h after infection.Differential analysis was performed by DESeq2 to find differentially expressed genes,and GO functional annotation analysis was performed for differentially expressed genes.The correlation analysis between intestinal VIP and IFN-α/β expression of lung tissue after RSV infection was further performed based on RNA-Seq data.Results: VIPhyb inhibited IFN-α/β production in the lungs of RSV-infected KCF mice,promoted RSV replication,and aggravated lung inflammation.VIP promoted IFN-α/β production in the lungs of RSV-infected intact mice,inhibited RSV replication,and reduced lung inflammation.VIP promoted IFN-α/β expression in MH-S in vitro,and this effect is counteracted by PKA inhibitor but not PKC inhibitor.RNA-Seq showed that intact and KCF mice had high expression of VIP in intestine and brain,while lung and spleen barely expressed VIP.Moreover,the intestinal VIP expression level of KCF mice was significantly higher than that of intact mice.Further ELISA experiments revealed that the expression levels of VIP in BALF and intestine were higher in KCF mice than in intact mice.The intestinal RNA-Seq results showed that the intestinal peptide secretion and transport pathways were significantly enriched in KCF mice after RSV infection and a significant positive correlation between intestinal VIP levels and lung IFN-α/β secretion levels after RSV infection.In summary,these results suppoted that C fiber may affect VIP levels in BALF by regulating intestinal VIP levels.Conclusion: C fiber regulates host anti-RSV effects via VIP;VIP enhances host anti-RSV effects by promoting IFN-α/β expression through PKA.Part Ⅳ STUDY ON THE ROLE OF ALVEOLAR MACROPHAGE IN VIP ENHANCING ANTIVIRAL RESPONSE POST RSV INFECTIONObjective: C fiber enhances host antiviral ability by promoting IFN-α/βproduction after RSV infection via VIP,but the exact mechanism is unknown.Studies have shown that alveolar macrophages(AMs)are the most important source of IFN-α/β production after RSV infection.Deletion of AMs significantly reduced IFN-β production in RSV-infected mice and promoted RSV replication.While airway treatment of mice with alveolar-like macrophages increased IFN-β production in RSV-infected mice and inhibited RSV replication.These researches suggest that number of AMs affects IFN-α/β production after RSV infection,thereby modulating the host anti-RSV effect.This part will further investigate:(1)whether AMs are the target cells of C fiber to regulate the host anti-RSV effect after RSV infection via VIP;(2)whether VIP can increase the number of AMs after RSV infection;and(3)the specific mechanism of the change in the number of AMs after RSV infection regulated by VIP.Methods: RNA-Seq data of lung tissues from KCF and Intact mice at 12 h after RSV infection were analyzed by bioinformatics,immune cell infiltration was predicted by Cibersort,and the number of AMs was detected by flow cytometry.AMs were removed from KCF mice by liposomes,and the successful removal of AMs in mice was verified by flow cytometry.KCF and AMs removed KCF mice were given nasal drops of RSV viral solution,and BALF was collected 12 h after RSV infection,and the levels of IFN-α/β in BALF were measured by ELISA.The number of AMs was detected by flow cytometry 12 h after RSV infection in KCF mice treated with VIPhyb and in intact mice treated with VIP,respectively.The alveolar macrophage line MH-S was treated with different concentrations of VIP in vitro,and the death and proliferation of MH-S were detected by flow cytometry at different time points after RSV infection.Results: Immuno-infiltration analysis and flow cytometry results displayed a higher number AMs in lung tissue 12 h after RSV infection in KCF mice compared to intact mice.Depletion of AMs in KCF mice significantly decreased IFN-α/β levels in BALF after RSV infection.VIPhyb decreased the number of AMs after RSV infection in KCF mice,while VIP increased the number of AMs in RSV-infected intact mice.Different concentrations of VIP in vitro were able to inhibit MH-S apoptosis and necrosis and promote their proliferation after RSV infection to different degrees.Conclusion: VIP increased the number of AMs after RSV infection and enhanced the host antiviral effect by promoting the proliferation and inhibiting the apoptosis of AMs after RSV infection.