The Mechanisms Of Alveolar Macrophage Homeostasis During Influenza Virus Infection

Influenza virus usually causes worldwide pandemic respiratory diseases.The innate immune cells are the critical front line of defense during influenza A virus infection.Therefore,we need to better understand the mechanisms of how innate immune cells regulate the immune-mediated protection versus immune-driven tissue damage to develop efficient strategies against influenza virus infection.Among them,alveolar macrophages（AMs）are the most abundant lung tissue-resident macrophages in situ and are vital in pulmonary anti-microbial immunity.Impairment of mitochondrial function in AM is one of the primary cause of patients died of CDVID-19.But the roles of mitochondrial metabolism in anti-influenza viral responses of AMs are unknown.Here we explored the roles of mitochondrial transcription factor A（TFAM）-mediated mitochondrial fitness in AM homeostasis and anti-influenza viral responses.Next,Influenza virus infection of host facilitate neutrophil infiltration into the infection site to exert antiviral responses,but little is known about whether neutrophils are involved in AM homeostasis and anti-influenza viral responses.Therefore,we explored the effect of neutrophils on the homeostasis and anti-influenza viral responses of AMs.The main contents are as follows:1.TFAM expression and mitochondrial fitness of AMs were affected during influenza virus infectionFirst,influenza virus PR8 strain was used to infect AMs in vitro.The result showed that the expression of TFAM in AMs was decreased,and AM mitochondria were damaged.Similarly,we analyzed the data from RNA sequencing of influenza virus-infected human AMs,which showed that mitochondrial metabolism of AMs was disturbed.Therefore,these data suggested that TFAM-mediated mitochondrial metabolism might play a role in anti-influenza viral responses of AMs.2.TFAM deficiency resulted in diminished AM numbers and metabolic fitnessTo investigate the role of TFAM in AM development and function,we constructed conditional knockout(CD11c^creTfam^fl/fl)mice and full knockout(Ubc^{cre ER}Tfam^fl/fl)mice of Tfam gene.Experimental results exhibited that TFAM deficiency led to significant reduction in the percentages and cell numbers of mature AMs of bronchoalveolar lavage（BAL）and lungs from TFAM knockout mice by flow cytometry,and immature AMs were discovered.With the reduction of mature AMs,mitochondrial metabolism of AMs was disturbed,which resulted in abnormal lipid metabolism.Afterwards,analysis of bulk RNA sequencing data for TFAM-deleted AMs indicated that TFAM deletion down-regulated the expression of genes related to AM proliferation and self-renewal,and promoted the expression of inflammatory-associated genes in AMs.3.TFAM deficiency impaired the antiviral responses of AMsFurther study of BAL turbidity at wavelength of 590 nm（OD590）by spectrophotometry showed that TFAM-mediated AMs injury resulted in defective clearance of cellular debris and surfactant in the lung.Influenza virus PR8 infected CD11c^creTfam^fl/flmice,which caused the infiltration of massive inflammatory cytokines into infected tissues,and even death of the mice.These data illustrated the importance of TFAM-mediated mitochondrial metabolism in anti-influenza viral responses of AMs.4.BAL-neutrophils inhibited AM proliferationDue to the low number of neutrophils in the lungs in the steady state,we generated a model of neutrophils migrating into BAL and lung.The purified neutrophils of BAL（BAL-neutrophils）were co-cultured with AMs to detect Ki67expression of AMs,BAL-neutrophils downregulated Ki67 expression of AMs,suggesting that BAL-neutrophils inhibited the proliferation of AMs.Then experimental results showed that neutrophils of bone marrow did not affect AM proliferation.Based on the GSEA of published RNA sequencing data,we found that neutrophils had tissue heterogeneity.5.PR8-neutrophils did not affect AM proliferationNeutrophils of BAL were purified from influenza virus PR8-infected mice（PR8-neutrophils）,and then co-cultured with AMs.The result showed that the expression of Ki67 of AMs was not affected.Then we performed RNA sequencing which showed that AMs co-cultured with BAL-neutrophils expressed higher metabolism-and ROS-associated genes,and AMs co-cultured with PR8-neutrophils increased the expression of inflammatory genes,indicating that inflammatory factors promoted the activation of AM and which suppressed the function of inhibiting AM proliferation.6.neutrophil death regulated AM proliferationFinally,co-culture of AMs and BAL-neutrophils was treated with different inhibitors of different neutrophil death pathways.Experimental results revealed that BAL-neutrophil-mediated suppression on AM proliferation was abrogated via combination of different inhibitors for neutrophil multiple death pathways,indicating that multiple death pathways of neutrophils regulated AM proliferation and maintained AM homeostasis.These data suggested that TFAM and neutrophils influence AM homeostasis and anti-influenza viral responses.Those data presented the first genetic evidence on the roles of mitochondrial metabolism and neutrophil death in regulating AM homeostasis.Our studies provided novel insights into the interplay between mitochondrial metabolism and neutrophils and AM,the data offered potential target for development of new therapeutic or intervening directions for the influenza virus infection.