HnRNPA2B1 tunes antimycobacterial immune responses in macrophages through alternative splicing of Irgm1

Onset and progression of active tuberculosis disease result from upsetting the delicate balance between Mtb virulence and host defenses. Because it dynamically tunes the functional output of protein expression in cells, alternative splicing, a process by which different mRNAs can be gen-erated from a single gene, is positioned to play a critical role in maintaining an equilibrated Mtb-macrophage host-pathogen interface. To gain insight into how alternative splicing shapes anti-mycobacterial immune responses, we used RNA-sequencing and splicing-aware computational pipelines to quantify alternative splicing in Mtb-infected bone marrow-derived murine macro-phages. We found that [~]5% of expressed macrophage genes exhibit one or more splicing changes at 8h post-Mtb infection, highlighting alternative splicing as a key regulatory node in the macrophage response to Mtb. We next sought to identify RNA binding proteins that play an out-sized role in shaping the macrophage transcriptome during Mtb infection. We discovered that the splicing factor heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) promotes the early induction of inflammatory genes while dampening several type I interferon-stimulated genes in response to Mtb. HnRNPA2B1 also controls alternative splicing of many genes during Mtb infection, including Irgm1, a critical immunity-related GTPase. The balance of Irgm1-long vs. -short is differentially regulated in response to diverse inflammatory cues and macrophages overexpressing Irgm1-short are defective in autophagosomal targeting, lysosomal homeostasis, and restriction of Mtb replication. These data highlight a key role for AS in shaping the macro-phage transcriptome and pinpoint hnRNPA2B1 as a novel restriction factor in the cell-intrinsic response to Mtb. IMPORTANCEAlthough the process of making proteins from RNAs requires many steps (transcription, cap-ping/polyadenylation, pre-mRNA splicing, mRNA export, mRNA modifications, etc.), we know very little about how post-transcriptional steps contribute to host immune defenses. Here, we show that alternative splicing, the process of making different mature RNAs from a single pre-cursor RNA, is a prominent and dynamic feature of macrophage infection with the bacterial pathogen Mycobacterium tuberculosis (Mtb). We identify the splicing regulator hnRNPA2B1 as a key coordinator of early gene expression during Mtb infection, influencing pathways that pro-mote inflammation and help restrict bacterial growth. Notably, we report that hnRNPA2B1 con-trols the splicing of the antimycobacterial protein Irgm1 to generate different flavors of the pro-tein. Since only one Irgm1 flavor can restrict Mtb growth inside macrophages, maintaining the balance of these proteins in response to diverse inflammatory cues is important. By revealing how RNA processing shapes the macrophage response to Mtb, our work highlights an often-overlooked layer of immune regulation and opens new avenues for splicing-targeted therapies designed to boost Mtb killing in macrophages.