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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., Competing Interests: AW, CW, KW, HS, RW, KP No competing interests declared, (© 2022, Wagner et al.)

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., (Copyright © 2021 The Authors.)

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 ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wagner, Scott, West, Vail, Fitzsimons, Coleman, Carter, Watson and Patrick.)

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-β 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., (Copyright © 2020 by The American Association of Immunologists, Inc.)

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., Competing Interests: CW, SB, KV, KW, KP, RW No competing interests declared, (© 2020, Weindel et al.)

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., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: Nonsteroidal anti-inflammatory drugs (NSAIDs) are assembled into two categories; cyclooxygenase (COX-1) sparing inhibitors of COX-2 and non-selective NSAIDs. Diclofenac (DICLO) is a non-selective NSAID that has been linked to serious side effects including gastric ulcers and renal injury. In this study, we examine the effect of poly(lactic-co-glycolic) acid nanoformulation on DICLO-associated adverse events and pharmacokinetics using a nanoparticle (NP) formulation previously developed in our laboratory., Materials and Methods: Rats were administered a single dose of methylcellulose (VEH), blank NP, DICLO (10 mg/kg), or a DICLO-NP suspension equivalent to the DICLO dose. Urinary and blood parameters were measured at baseline and following treatment. Duodenal and gastric prostaglandin E2 (PGE2) and duodenal myeloperoxidase (MPO) were collected to assess inflammation at 24 hrs post-treatment., Results: The mean percent change from baseline in sodium excretion rate (µmol/min/100 g body weight) differed significantly from VEH in the NP (p < 0.0001), DICLO (p < 0.0001), and DICLO-NP (p = 0.0001) groups. The differences among groups did not reach significance for plasma sodium or potassium concentrations, potassium excretion rate, gastric PGE2, or intestinal biomarker concentrations. Regarding renal histopathology, DICLO produced considerably more necrosis compared to VEH; while DICLO-NP did not elicit notable differences from VEH., Conclusions: Our results suggest that over the duration and dosage examined, DICLO-NP may reduce renal necrosis without influencing other side effects or drug characteristics.

Objective: Celecoxib (CEL) is a nonsteroidal anti-inflammatory drug (NSAID) showing selective cycloxygenase-2 inhibition. While effective as a pain reducer, CEL exerts some negative influence on renal and gastrointestinal parameters. This study examined CEL pharmacodynamics and pharmacokinetics following drug reformulation as a poly(lactic-co-glycolic) acid nanoparticle (NP)., Materials and Methods: Rats were administered either vehicle (VEH) (methylcellulose solution), blank NP, 40 mg/kg CEL in methylcellulose, or an equivalent NP dose (CEL-NP). Plasma and urine (over 12 hrs) samples were collected prior to and post-treatment. The mean percent change from baseline of urine flow rate along with electrolyte concentrations in plasma and urine were assessed based on 100 g body weight. Using tissues collected 24 hrs post-treatment, gastrointestinal inflammation was estimated through duodenal and gastric prostaglandin E2 (PGE2) and duodenal myeloperoxidase (MPO) levels; while kidney tissue was examined for dilatation and necrosis. CEL concentration was assayed in renal tissue and plasma utilizing high-performance liquid chromatography., Results: Although there were significant changes when comparing CEL and CEL-NP to VEH in plasma sodium concentration and potassium excretion rate, there was no significant variation between CEL and CEL-NP. There was a significant reduction of protective duodenal PGE2 in CEL compared to VEH (p = 0.0088) and CEL-NP (p = 0.02). In the CEL-NP formulation, t1/2, Cmax, AUC0-∞, and Vd/F increased significantly when compared to CEL., Conclusions: At the observed dosage and duration, CEL-NP may not affect CEL-associated electrolyte parameters in either plasma or urine; however, it does provide increased systemic exposure while potentially alleviating some gastrointestinal outcomes related to inflammation.