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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.

SUBMITTER: Wagner AR

PROVIDER: S-EPMC8299563 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Purpose: The goal of this study is to measure how steady state RNA and alternatively spliced transcript abundance levels are impacted by loss of specific RNA-binding proteins as part of macrophage transcriptional activation upon pathogen sensing. Methods: SRSF 1, 2, 6, 7, 9, and hnRNP C, F, K, U were knockdown using shRNA in RAW 264.7 macrophages. Macrophages were collected as uninfected (resting) cells and infected with Salmonella Typhimuirum for 4HR. mRNA profiles of uninfected (UN) and Salmonella infected (SAL) hnRNP C, F, K, U and SRSF 1, 2, 6, 7, 9 were generated by deep sequencing, in triplicate, using Illumina. The sequence reads that passed quality filters were analyzed at the gene expression and transcript expression level with CLC Genomics Workbench. qRT–PCR validation was performed using SYBR Green assays. Results: We identified thousands of transcripts whose abundance is increased or decreased by SR/hnRNP knockdown in macrophages. We observed that different SR/hnRNPs control the ex-pression of distinct gene regulons in uninfected and Salmonella-infected macrophages, with sev-eral key innate immune genes (Nos2, Mx1, Il1a) relying heavily on multiple SR/hnRNPs to main-tain proper induction and/or repression. Knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for a number of key immune sensors and signaling molecules and many of these splicing changes were distinct in uninfected and Salmonella-infected macrophages. After observ-ing a surprising degree of similarity between the differentially expressed genes (DEGs) and DIUs in hnRNP K and U knockdown macrophages, we found that these cells are better able to restrict vesicular stomatitis virus repli-cation than control cells, supporting a role for these hnRNPs in controlling infection outcomes in macrophages ex vivo. Based on these findings, we conclude that many innate immune genes have evolved to rely on one or more splicing regulatory factors to ensure the proper timing and magnitude of their induction, bolstering a model wherein pre-mRNA splicing is a critical regulatory node in the innate immune response. Conclusions: SR and hnRNP proteins have profound impacts on the abundance of innate immune transcripts and seem to contribute to macrophage gene expression via distinct mechanisms

Project description:BackgroundMonocytes and macrophages contribute to the dysfunction of immune responses in human filariasis. During patent infection monocytes encounter microfilariae in the blood, an event that occurs in asymptomatically infected filariasis patients that are immunologically hyporeactive.AimTo determine whether blood microfilariae directly act on blood monocytes and in vitro generated macrophages to induce a regulatory phenotype that interferes with innate and adaptive responses.Methodology and principal findingsMonocytes and in vitro generated macrophages from filaria non-endemic normal donors were stimulated in vitro with Brugia malayi microfilarial (Mf) lysate. We could show that monocytes stimulated with Mf lysate develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype was recapitulated in monocytes from Wuchereria bancrofti asymptomatically infected patients but not patients with pathology or endemic normals. Monocytes from non-endemic donors stimulated with Mf lysate directly inhibited CD4+ T cell proliferation and cytokine production (IFN-?, IL-13 and IL-10). IFN-? responses were restored by neutralising IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally Mf lysate applied during the differentiation of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner.Conclusions and significanceConclusively, our study demonstrates that Mf lysate stimulation of monocytes from healthy donors in vitro induces a regulatory phenotype, characterized by expression of PD-L1 and IL-10. This phenotype is directly reflected in monocytes from filarial patients with asymptomatic infection but not patients with pathology or endemic normals. We suggest that suppression of T cell functions typically seen in lymphatic filariasis is caused by microfilaria-modulated monocytes in an IL-10-dependent manner. Together with suppression of macrophage innate responses, this may contribute to the overall down-regulation of immune responses observed in asymptomatically infected patients.

Project description:Purpose:To decipher the transcriptional signature of macrophages of the human vitreous, also known as hyalocytes, and compare it to the profiles of other myeloid cell populations including human blood-derived monocytes, macrophages, and brain microglia. Methods:This study involves a total of 13 patients of advanced age with disorders of the vitreoretinal interface undergoing vitrectomy at the University Eye Hospital Freiburg between 2018 and 2019. Vitreal hyalocytes were analyzed by fluorescence-activated cell sorting (FACS) and isolated as CD45+CD11b+CX3CR1+Mat-Mac+ cells using a FACS-based sorting protocol. RNA extraction, library preparation and RNA sequencing were performed and the sequencing data was analyzed using the Galaxy web platform. The transcriptome of human hyalocytes was compared to the transcriptional profile of human blood-derived monocytes, macrophages and brain microglia obtained from public databases. Protein validation for selected factors was performed by immunohistochemistry on paraffin sections from three human donor eyes. Results:On average, 383 ± 233 hyalocytes were isolated per patient, resulting in 128 pg/?l ± 76 pg/?l total RNA per sample. RNA sequencing revealed that SPP1, FTL, CD74, and HLA-DRA are among the most abundantly expressed genes in hyalocytes, which was confirmed by immunofluorescence for CD74, FTL, and HLA-DRA. Gene ontology (GO) enrichment analysis showed that biological processes such as "humoral immune response," "leukocyte migration," and "antigen processing and presentation of peptide antigen" (adjusted p < 0.001) are dominating in vitreal hyalocytes. While the comparison of the gene expression profiles of hyalocytes and other myeloid cell populations showed an overall strong similarity (R 2 > 0.637, p < 0.001), hyalocytes demonstrated significant differences with respect to common leukocyte-associated factors. In particular, transcripts involved in the immune privilege of the eye, such as POMC, CD46, and CD86, were significantly increased in hyalocytes compared to other myeloid cell subsets. Conclusion:Human hyalocytes represent a unique and distinct innate immune cell population specialized and adapted for the tissue-specific needs in the human vitreous. Vitreal hyalocytes are characterized by a strong expression of genes related to antigen processing and presentation as well as immune modulation. Thus, hyalocytes may represent an underestimated mediator in vitreoretinal disease and for the immune privilege of the eye.

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

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