Gut Microbiota Inhibits Influenza Virus-induced N6-Methyladenosine: Role of Microbiota-mediated Epigenetic Modification in Antiviral Immunity

Abstract Rationale: N6-methyladenosine (m6A), a prevalent post-transcriptional methylation of RNA in eukaryotes, plays a crucial role in the modulation of antiviral immunity. However, the mechanisms regulating m6A modification during virus infection remain largely unclear. Gut microbiota that is indispensable in priming host's defense to influenza virus infection was recently reported to be involved in driving m6A modification in extrapulmonary organs. We hypothesized that gut microbiota-mediated m6A modification in the lung was linked to its anti-influenza effect. Methods: Specific pathogen-free (SPF) mice were intranasally challenged with an influenza A virus (H1N1) strain (A/PR8) or PBS. Depletion of gut microbiota was performed by a cocktail of antibiotics (ampicillin, metronidazole, neomycin sulfate, and vancomycin) in drinking water for 4 weeks. Level of m6A methylation in lung tissue was quantified by mRNA dot blot assay and immunohistochemistry. Molecules involved in m6A modification were measured by WB and qPCR. Bronchoalveolar lavage fluid was collected for the quantification of inflammatory cells and virus-associated interferons (IFN-α and IFN-β). Protein and mRNA level of IFN-α and IFN-β in lung tissue were also measured. Viral load was quantified by detection of viral nucleoprotein in lung tissue. Results: Level of m6A methylation in the lung was significantly elevated in SPF mice after infection with H1N1. Consistently, a key m6A writer protein (METTL3) that drives methylation was highly expressed after viral infection. Of note, two m6A erasers (ALKBH5 and FTO) that mediate demethylation were also elevated, which potentially represents protective feedback that limits the overmethylation of lung mRNAs. Microbiota depletion amplified m6A modification in mice lungs as evidenced by higher level of m6A methylation in antibiotic-treated mice compared to naïve ones. Strikingly, depletion of microbiota not only enhanced the expression of METTL3, but suppressed the expression of ALKBH5 and FTO. Concomitantly, mice devoid of microbiota showed impaired antiviral immunity characterized by decreased secretion of IFN-α and IFN-β and increased viral load. Microbiota depletion was also associated with worse infection-related consequences including heightened airway inflammation, more weight-loss, and increased mortality. Conclusion: Gut microbiota inhibits influenza virus-induced m6A methylation in the lung which is linked to its antiviral immunity. Precise mechanisms underlying this microbiota-mediated epigenetic modification in viral infection deserve further investigation.