SRSF6 balances mitochondrial-driven innate immune outcomes through alternative splicing of BAX.

To mount a protective response to infection while preventing hyperinflammation, gene expression in innate immune cells must be tightly regulated. Despite the importance of pre-mRNA splicing in shaping the proteome, its role in balancing immune outcomes remains understudied. Transcriptomic analysis of murine macrophage cell lines identified Serine/Arginine Rich Splicing factor 6 (SRSF6) as a gatekeeper of mitochondrial homeostasis. SRSF6-dependent orchestration of mitochondrial health is directed in large part by alternative splicing of the pro-apoptosis pore-forming protein BAX. Loss of SRSF6 promotes accumulation of BAX-κ, a variant that sensitizes macrophages to undergo cell death and triggers upregulation of interferon stimulated genes through cGAS sensing of cytosolic mitochondrial DNA. Upon pathogen sensing, macrophages regulate SRSF6 expression to control the liberation of immunogenic mtDNA and adjust the threshold for entry into programmed cell death. This work defines BAX alternative splicing by SRSF6 as a critical node not only in mitochondrial homeostasis but also in the macrophage's response to pathogens.

PRMT5 Promotes Symmetric Dimethylation of RNA Processing Proteins and Modulates Activated T Cell Alternative Splicing and Ca2+/NFAT Signaling.

Protein arginine methyltransferase (PRMT) 5 is the type 2 methyltransferase catalyzing symmetric dimethylation of arginine. PRMT5 inhibition or deletion in CD4 Th cells reduces TCR engagement-induced IL-2 production and Th cell expansion and confers protection against experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. However, the mechanisms by which PRMT5 modulates Th cell proliferation are still not completely understood, and neither are the methylation targets in T cells. In this manuscript, we uncover the role of PRMT5 on alternative splicing in activated mouse T cells and identify several targets of PRMT5 symmetric dimethylation involved in splicing. In addition, we find a possible link between PRMT5-mediated alternative splicing of transient receptor potential cation channel subfamily M member 4 (Trpm4) and TCR/NFAT signaling/IL-2 production. This understanding may guide development of drugs targeting these processes to benefit patients with T cell-mediated diseases.

Global Transcriptomics Uncovers Distinct Contributions From Splicing Regulatory Proteins to the Macrophage Innate Immune Response.

Pathogen sensing via pattern recognition receptors triggers massive reprogramming of macrophage gene expression. While the signaling cascades and transcription factors that activate these responses are well-known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Given their crucial role in regulating pre-mRNA splicing and other RNA processing steps, we hypothesized that members of the SR/hnRNP protein families regulate innate immune gene expression in distinct ways. We analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage-like cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We identified thousands of transcripts whose abundance is increased or decreased by SR/hnRNP knockdown in macrophages. Notably, we observed that SR and hnRNP proteins influence expression of different genes in uninfected versus Salmonella-infected macrophages, suggesting functionalization of these proteins upon pathogen sensing. Likewise, we found that knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for thousands of macrophage transcripts and that these alternative splicing changes were distinct in uninfected and Salmonella-infected macrophages. Finally, having observed a surprising degree of similarity between the differentially expressed genes (DEGs) and DIUs in hnRNP K and U knockdown macrophages, we found that hnRNP K and U knockdown macrophages are both more restrictive to Vesicular Stomatitis Virus (VSV), while hnRNP K knockdown macrophages are more permissive to Salmonella Typhimurium. Based on these findings, we conclude that many innate immune genes evolved to rely on one or more SR/hnRNPs to ensure the proper magnitude of their induction, supporting a model wherein pre-mRNA splicing is critical for regulating innate immune gene expression and controlling infection outcomes in macrophages ex vivo.

TRIM14 Is a Key Regulator of the Type I IFN Response during Mycobacterium tuberculosis Infection.

Tripartite motif-containing proteins (TRIMs) play a variety of recently described roles in innate immunity. Although many TRIMs regulate type I IFN expression following cytosolic nucleic acid sensing of viruses, their contribution to innate immune signaling and gene expression during bacterial infection remains largely unknown. Because Mycobacterium tuberculosis is an activator of cGAS-dependent cytosolic DNA sensing, we set out to investigate a role for TRIM proteins in regulating macrophage responses to M. tuberculosis In this study, we demonstrate that TRIM14, a noncanonical TRIM that lacks an E3 ubiquitin ligase RING domain, is a critical negative regulator of the type I IFN response in Mus musculus macrophages. We show that TRIM14 interacts with both cGAS and TBK1 and that macrophages lacking TRIM14 dramatically hyperinduce IFN stimulated gene (ISG) expression following M. tuberculosis infection, cytosolic nucleic acid transfection, and IFN-ß treatment. Consistent with a defect in resolution of the type I IFN response, Trim14 knockout macrophages have more phospho-Ser754 STAT3 relative to phospho-Ser727 and fail to upregulate the STAT3 target Socs3, which is required to turn off IFNAR signaling. These data support a model whereby TRIM14 acts as a scaffold between TBK1 and STAT3 to promote phosphorylation of STAT3 at Ser727 and resolve ISG expression. Remarkably, Trim14 knockout macrophages hyperinduce expression of antimicrobial genes like Nos2 and are significantly better than control cells at limiting M. tuberculosis replication. Collectively, these data reveal an unappreciated role for TRIM14 in resolving type I IFN responses and controlling M. tuberculosis infection.

LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis.

The Parkinson's disease (PD)-associated gene leucine-rich repeat kinase 2 (LRRK2) has been studied extensively in the brain. However, several studies have established that mutations in LRRK2 confer susceptibility to mycobacterial infection, suggesting LRRK2 also controls immunity. We demonstrate that loss of LRRK2 in macrophages induces elevated basal levels of type I interferon (IFN) and interferon stimulated genes (ISGs) and causes blunted interferon responses to mycobacterial pathogens and cytosolic nucleic acid agonists. Altered innate immune gene expression in Lrrk2 knockout (KO) macrophages is driven by a combination of mitochondrial stresses, including oxidative stress from low levels of purine metabolites and DRP1-dependent mitochondrial fragmentation. Together, these defects promote mtDNA leakage into the cytosol and chronic cGAS engagement. While Lrrk2 KO mice can control Mycobacterium tuberculosis (Mtb) replication, they have exacerbated inflammation and lower ISG expression in the lungs. These results demonstrate previously unappreciated consequences of LRRK2-dependent mitochondrial defects in controlling innate immune outcomes.

Parkinson's disease is a progressive nervous system disorder that causes tremors, slow movements, and stiff and inflexible muscles. The symptoms are caused by the loss of cells known as neurons in a specific part of the brain that helps to regulate how the body moves. Researchers have identified mutations in several genes that are associated with an increased risk of developing Parkinson's. The most common of these mutations occur in a gene called LRRK2. This gene produces a protein that has been shown to be important for maintaining cellular compartments known as mitochondria, which play a crucial role in generating energy. It remains unclear how these mutations lead to the death of neurons. Mutations in LRRK2 have also been shown to make individuals more susceptible to bacterial infections, suggesting that the protein that LRRK2 codes for may help our immune system. Weindel, Bell et al. set out to understand how this protein works in immune cells called macrophages, which 'eat' invading bacteria and produce type I interferons, molecules that promote immune responses. Mouse cells were used to measure the ability of normal macrophages and macrophages that lack the mouse equivalent to LRRK2 (referred to as Lrrk2 knockout macrophages) to make type I interferons. The experiments showed that the Lrrk2 knockout macrophages made type I interferons even when they were not infected with bacteria, suggesting they are subject to stress that triggers immune responses. It was possible to correct the behavior of the Lrrk2 knockout macrophages by repairing their mitochondria. When mice missing the gene equivalent to LRRK2 were infected with the bacterium that causes tuberculosis, they experienced more severe disease. The protein encoded by the LRRK2 gene is considered a potential target for therapies to treat Parkinson's disease, and several drugs that inhibit this protein are being tested in clinical trials. The findings of Weindel, Bell et al. suggest that these drugs may have unintended negative effects on a patient's ability to fight infection. This work also indicates that LRRK2 mutations may disrupt immune responses in the brain, where macrophage-like cells called microglia play a crucial role in maintaining healthy neurons. Future studies that examine how mutations in LRRK2 affect microglia may help us understand how Parkinson's disease develops.

The Splicing Factor hnRNP M Is a Critical Regulator of Innate Immune Gene Expression in Macrophages.

While transcriptional control of innate immune gene expression is well characterized, almost nothing is known about how pre-mRNA splicing decisions influence, or are influenced by, macrophage activation. Here, we demonstrate that the splicing factor hnRNP M is a critical repressor of innate immune gene expression and that its function is regulated by pathogen sensing cascades. Loss of hnRNP M led to hyperinduction of a unique regulon of inflammatory and antimicrobial genes following diverse innate immune stimuli. While mutating specific serines on hnRNP M had little effect on its ability to control pre-mRNA splicing or transcript levels of housekeeping genes in resting macrophages, it greatly impacted the protein's ability to dampen induction of specific innate immune transcripts following pathogen sensing. These data reveal a previously unappreciated role for pattern recognition receptor signaling in controlling splicing factor phosphorylation and establish pre-mRNA splicing as a critical regulatory node in defining innate immune outcomes.