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Macrophages are the primary host cell that supports the survival and replication of the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). We are interested in how macrophage proteins are functionalized during Mtb infection. Global proteomics analysis of Mtb-infected primary macrophages found that many RNA binding proteins of the Serine/Arginine rich splicing family (SRSF) can be differentially phosphorylated, suggesting that these factors regulate splicing decisions during Mtb infection. Knockdown of one such SRSF protein, called SR6, results in basal upregulation of interferon stimulated genes (ISGs) in macrophages. We hypothesized that this phenotype was driven by mitochondrial damage that leaks mitochondria DNA into the cytosol and activates cGAS. Consistent with this idea, we measured increased mitochondrial stress and increased cell death in SR6 KD macrophages. Transcriptomics analysis of these cells revealed upregulation of an alternatively spliced (AS) isoform of the apoptotic factor BAX, which contributes to mitochondrial dysfunction, mtDNA release, and basal ISG expression. While BAX isoforms are known to regulate cell death, this is the first report of BAX AS regulating the type I IFN response. Lastly, we found that SR6 KD macrophages are permissive to Mtb replication, suggesting that controlling cell death at the level of BAX isoform abundance is an important but unappreciated component of the macrophage antibacterial repertoire. Together, our data support a model whereby SR6-dependent AS of Bax triggers mitochondria stress causing mtDNA leakage and type I IFN expression, metabolic dysfunction that primes cells to undergo cell death, and increased susceptibility to Mtb infection.

To defend against invading pathogens, the macrophage innate immune response requires rapid, robust gene expression reprogramming. Innate immune genes that are induced as part of this response are regulated at many steps. While transcriptional activation of innate immune genes has been extensively studied, the mechanisms driving their post-transcriptional regulation (i.e. alternative splicing) needs further investigation. To begin learning how pre-mRNA splicing changes impact the innate immune response in macrophages, we examined how loss of a splicing regulatory protein called SRSF6 (serine arginine rich splicing factor 6) impacts gene expression. We discovered a novel phenotype whereby loss of SRSF6 increased basal expression of many interferon stimulated genes (ISGs). Consistent with mitochondrial dysfunction, a phenotype that we and others have previously associated high basal ISG expression, we measured an increase in apoptosis and macrophage depolarization in SRSF6 knockdown macrophages compared to scramble controls. Subsequent bioinformatic analysis of RNA-seq data from SRSF6 knockdown macrophages identified an alternative splicing event in the apoptosis activation factor BAX. While Bax isoforms have previously been linked to cell death regulation, no one has reported a role for Bax isoforms in modulating mtDNA-driven innate immune responses. Our data is consistent with a model whereby upregulation of a Bax isoform promotes pore formation in mitochondria which 1) causes leakage of mtDNA into the cytosol where it is detected by cGAS, eliciting type I interferon production and 2) primes macrophages to undergo programmed cell death.