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

Abstract

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.

A Bioinformatics Approach to Identifying Potential Biomarkers for Cryptosporidium parvum: A Coccidian Parasite Associated with Fetal Diarrhea

“…In mice, a virulent dam mutant Salmonella vs. WT Salmonella infection revealed ISG role in host response [59]. Although IFN-/ does not directly activate the MX1 gene, evidence demonstrates that it is involved in the host response to Salmonella infection [59,60]. In uninfected and Salmonella-infected macrophages, serine-arginine regulates the expression of numerous gene regulations, with several essential innate immunity genes (Nos2, MX1) relying on several SR/hnRNPs to maintain repression [60].…”

“…Although IFN-/ does not directly activate the MX1 gene, evidence demonstrates that it is involved in the host response to Salmonella infection [59,60]. In uninfected and Salmonella-infected macrophages, serine-arginine regulates the expression of numerous gene regulations, with several essential innate immunity genes (Nos2, MX1) relying on several SR/hnRNPs to maintain repression [60]. At the host-parasite interface, C. parvum and intestinal epithelial cells interact by transmitting effector molecules from the host cell and the parasite [61].…”

Shaping the Innate Immune Response Through Post-Transcriptional Regulation of Gene Expression Mediated by RNA-Binding Proteins

“…RNA splicing is one way among others to ensure the proper timing and intensity of ISG expression. Splicing regulation involves one or more splicing factors acting like a regulatory node, as illustrated by heterogeneous nuclear RiboNucleoProteins (hnRNPs) ( 177 ). hnRNPs are complexes comprising typical RNA-binding and modular proteins mostly present in the nucleus ( 178 ).…”

The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferon by engaging cytosolic DNA sensing in macrophages

“…Proinflammatory cytokines, particularly TNF-α and IFN-γ have been shown to be important in controlling R. equi infection in foals. To begin to define the nature of the macrophage innate immune response to R. equiinfection, we set out to compare innate immune responses in primary macrophages derived from both foals and mice to those elicited in RAW 264.7 cells, a genetically tractable macrophage-like cell line in which our lab has developed tools to knockdown or -out key components of the innate immune system [27,[41][42][43][44]. In past studies using RAW 264.7 cells with Mtb, Salmonella, and Listeria, we have found that activation of both TLR and cytosolic nucleic acid sensing pathways induce transcriptional responses comparable to primary macrophages [27,41,[43][44][45].…”

“…To begin to define the nature of the macrophage innate immune response to R. equiinfection, we set out to compare innate immune responses in primary macrophages derived from both foals and mice to those elicited in RAW 264.7 cells, a genetically tractable macrophage-like cell line in which our lab has developed tools to knockdown or -out key components of the innate immune system [27,[41][42][43][44]. In past studies using RAW 264.7 cells with Mtb, Salmonella, and Listeria, we have found that activation of both TLR and cytosolic nucleic acid sensing pathways induce transcriptional responses comparable to primary macrophages [27,41,[43][44][45]. To determine whether R. equi infection followed a similar paradigm, we first compared pro-inflammatory cytokine induction at the transcript level in both primary murine bone marrow derived macrophages (BMDMs) and equine bronchoalveolar cells (consisting of 85% macrophages) (S1A Fig) . Primary cells were infected with virulent R. equi (ATCC 330701+) at a multiplicity of infection (MOI) of 5.…”