Lung-brain Axis: Pathological Changes Of Lung And Brain In Mice Infected With Respiratory Syncytial Virus And Related Mechanisms

Background and Objective:In the emerging biomedical sciences,biological pathways ranging from gut microbiota-brain axis to lung-brain axis have attracted strong scientific interest.Lung and lung pathology has been shown to be associated with the brain.Considering the large number of evidence-based medicines,this discipline is still in its infancy and therefore the need to approach this concept in a multi-dimesional and pronged study.In this regard,we developed the concept of the lung-brain axis.In the lung-brain axis,pathological imprints in the lung cavity are thought to be associated with brain pathology on time and dose depedent effects.Taking RSV infection as a model,some neurological alterations such as seizures and ataxia were observed in infected RSV patients.In the hippocampus of RSV-infected mice,RNA was detected in a separate study and this provided evidence of the virus spreading through the airways to the central nervous system(CNS).Since these biological pathways are not widely explored,this study hypothesized that the lung-brain axis of the viral propagation model will confer functional alterations to the brain.For example,in one study,CNS manifestations were observed in at least 2% of patients with RSV associated with bronchiolitis.Cerebrospinal fluid(CSF)abnormalities,muscular tone or strabismus abnormalities,seizures,central apnea,feeding or swallowing difficulties,encephalopathy,and lethargy were important symptoms studied with RSV-associated bronchiolitis.Although the detrimental effects of hyperoxia on the immature retina become apparent,the lung-brain axis has only come into focus in the last decade.Hyperoxia causes excessive production of reactive oxygen species(ROS),this can promote oxidative stress and inflammation.It also contribute to pulmonary growth restriction and abnormal neurodevelopment,including myelination deficits.Recent studies on the mechanisms of hyperoxia-induced neonatal lung and brain injury are scanty.However novel studies is required to explicitly explain the pathophysiological pathways that potentially link the lung and brain.There is an urgent need to understand the mechanisms underlying complications since there are limited therapeutic options.Once these mechanisms are explored,it will provide therapeutic options and hence pave the way for the development of novel multimodal therapies,ideally targeting CNS alterations induced by lung infection.It is also important to investigate whether severe neurological dysfunction and injury are associated with lung injury.For example,in severe acute respiratory syndrome Covid-19 virus 2(SARS-Co V-2)lung damage,virus-induced inflammation and oxidative stress may be common mechanisms responsible for SARS-Co V-2 neurological symptoms.To this end,this study aims to elucidate the pathological changes in lung and brain tissue of RSV-infected mice compared with normal mice,and to explore the relationship between these changes and nervous system function.The study hopes to find Pathophysiology pathways in the lung-brain axis,these include: 1)whether the virus enters the brain directly,2)whether the lung microbiome affects the function of the lung-brain axis,and 3)whether the RSV-induced metabolites cause functional changes in the nervous system.Experimental Materials and Methods(1)Pathological changes of lung and brain in RSV infected mice:Balb/C female mice(16-20g)aged 6-8 weeks were randomly divided into control group(n = 6)and RSV group(n = 6).After RSV infection was established in mice,the inflammatory reaction of lung and brain was detected by HE staining.The expression of IL-1β,IL-6 and TNFɑ in lung and brain were detected by TRIZOL reagent.The expression of RSV protein and m RNA in lung and brain,the expression of microglia marker IBA-1 and neuron marker Neu N in brain were detected by IFA and q RT-PCR.(2)RSV infection disrupts the respiratory tract microbiota and causes microglia phenotype shift: the model of RSV infection was established in one-week-old littermates,mice were randomly divided into control group,Ampicillin group,RSV Group and RSV+Ampicillin Group(6 mice in each group).HE staining,immunofluorescence and q RT-PCR were used to detect RSV infection,and biochemical methods were used to detect the contents of malondialdehyde(MDA)and Superoxide dismutase(SOD).The expressions of IL-1β,IL-6,IL-10 and IFN-γ were detected by ELISA kit,and the expressions of RORrt,GATA3,Foxp3 and T-bet were detected by q RT-PCR.16 s DNA technique was used to detect the microbiome and metabolic changes in the lung.The expressions of IL-1β,i NOS,IL-10 and Arg1 were detected by q RT-PCR and ELISA,and the polarization of microglia M1M2 was detected by fluorescence double staining.The titers of RSV in the brain were measured by q RT-PCR,and the contents of MDA and SOD in the brain were measured by biochemical Kits.(2)Regulation of mitochondrial heterogeneity by RSV-induced metabolites: the metabolites in lung tissues of RSV-infected and non-infected mice were detected by non-targeted metabolomics.The effects of 12 up-regulated metabolites on cell proliferation(CCK-8)and IL-1β(El ISA)secretion in LPS-induced neuronal injury model were observed.We selected three metabolites,propanoic acid,spermine and glutaric acid,to observe their effects on proliferation,ROS production and IL-1β secretion of HT-22 cells.q RT-PCR and Western blot were used to analyze the effects of these three metabolites on Dynamin-related protein 1(Drp1)and mitochondrial fusion protein-2(Mfn2).Mdivi-1 was used to inhibit the function of Drp1.The effects of three metabolites on the expression of Drp1 and Mfn2 genes,the proliferation of neural cells and the release of IL-1β were further examined.Results(1)From this study,we found that in the lung,RSV infection may promote epithelial shedding and also infiltration of inflammatory cells.RSV immunofluorescence and titers were significantly increased.Moreover,interleukin(IL)-1,IL-6 and tumor necrosis factor-α(TNF-α)were also significantly increased after RSV infection.In the brain,we observe the cellular structure of the hippocampus CA1 region was loose and disordered.Inflammatory cytokines IL-6 and IL-1β expression in the brain were also increased.However,TNF-α expression showed no differences among the control and RSV group.Increased expression of microglia biomarker IBA-1 and decreased neuronal biomarker Neu N were observed.In addition,RSV m RNA expression levels were also increased in the brains.(2)RSV infection disrupts respiratory microbiota and causes microglia phenotype shift: RSV infection causes pulmonary inflammation,elevated oxidative stress and decreased antioxidant capacity.HE staining showed that Ampicillin had no effect on pulmonary inflammatory injury under RSV infection.By measuring the release of inflammatory mediators,we found that IL-1β,IL-4,IL-6,and IFN-γ were significantly increased in RSV-infected group compared with normal group.Compared with RSV group,IFN-γ was significantly reduced in RSV+Ampicillin treatment group.Moreover,the expression of RORrt,GATA3,Fox P3 and T-bet were increased after RSV infection.Compared with RSV group,the expression of Fox P3 and T-bet in RSV+Ampicillin treatment group was significantly decreased.Then we observed the effects of RSV and RSV+ampicillin on M1/M2 microglia phenotypic shift.The results of q RT-PCR showed that the expression levels of IL-1βand i NOS of M1 phenotypic markers in the brain after RSV infection were significantly higher than those in the control group,while the m RNA levels of IL-10 and Arg1 of M2 phenotypic markers were not different.i NOS and Arg1 m RNA in RSV+ampicillin group were further increased.We then used ELISA to detect the secretion levels of M1 and M2 phenotypic markers.The results showed that the expression levels of IL-1β and i NOS were significantly increased after RSV infection,and further increased in RSV+Ampicillin group.The expression levels of IL-10 and Arg1 also increased after RSV infection,and further increased in RSV+Ampicillin group.The phenotypic shift of microglia cells was further detected by confocal dichroic immunofluorescence.The results showed that IBA-1(+)/i NOS(+)and IBA-1(+)/CD206(+)were significantly increased after RSV infection,and IBA-1(+)/CD206(+)was further increased in RSV+Ampicillin treatment.These results suggest that dysbiosis of the lung microbiota promotes the microglia phenotype to M2.RSV m RNA was also detected in murine brains,but there was no difference between RSV and RSV+ampicillin groups.Moreover,RSV infection increased MDA level and decreased SOD level in brain.RSV+ampicillin further promoted the secretion of MDA and inhibited SOD in brain tissue.16 s DNA sequencing showed that the number of Escherichia coli,Acinetobacter,Streptococcus,Pseudomonas and Treptococcus in RSV infected group was significantly increased,while the number of Bifidobacterium,Fibrobacter,Selenoselomonas and Butyrivibrio was significantly decreased.Compared with RSV infection group,RSV+Ampicillin Group had more obvious dysbacteriosis and less probiotics.(3)Regulation of mitochondrial heterogeneity by RSV-induced metabolites: using non-targeted metabolomic assays,we observed that RSV infection induced up-regulation of 16 metabolites in the lung.We further selected 12 up-regulated metabolites and observed their effects on cell proliferation and IL-1β secretion in LPS-induced neuronal injury model.The results showed that propanoic acid promoted the proliferation of LPS-treated neurons and inhibited the production of ROS and the secretion of IL-1β and IL-4.Spermine inhibited the proliferation of LPS-treated neurons and the secretion of IL-1β.Glutaric acid inhibited the proliferation of LPS-treated neurons and promoted the production of ROS and the secretion of IL-1β,IL-6 and IFN-γ.Moreover,propanoic acid inhibited the expression of Drp1 protein and m RNA,and promoted the expression of Mfn2 protein and m RNA.Spermine and glutaric acid promoted the expression of Drp1 m RNA and protein,and inhibited the expression of Mfn2 m RNA and protein.MDIVI-1 treatment inhibited Drp1 expression and promoted Mfn2 expression,thus reversing spermine-and glutaric acid-induced effects.Conclusions(1)RSV infection induced microglia activation and down-regulation of neuronal expression in the brain.RSV can also enter the brain directly.(2)RSV infection can induce dysbacteriosis in the lung,which is characterized by an increase in the number of common clinical pathogenic bacteria and a decrease in the number of probiotics,and antibiotic treatment can further decrease the number of probiotics.Antibiotic treatment weakened the Th1-dominated immune response in the lungs and promoted the microglia to an M2 phenotype.(3)RSV-induced spermine and glutaric acid could induce neuronal damage and mitochondrial heterogeneity,while propanoic acid had protective effect.In this study,a mouse model of RSV infection was established to explore the pathological changes of lung and brain,and many biological pathways between lung and brain were observed,including the direct translocation of RSV,immune and metabolic pathways.Since similar central nervous system sequelae have been observed in infections with various pathogens,therefore,the establishment of a biological pathway connecting these two organs will provide a promising strategy for the prevention and treatment of viral infectious diseases.