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Transcriptional analysis of transcription factor c-Maf as a molecular switch that controls immune suppression by programming macrophages in lung cancer

ABSTRACT: To investigate c-Maf-controlled gene expression, M2-like bone marrow-derived macrophages (BMM) were transfected with c-Maf or control siRNA. Knockdown of c-Maf significantly decreased c-Maf protein and mRNA expression levels. In addition, IL-10 and arginase mRNA levels were significantly decreased while IL-12 was increased. In contrast, ectopic expression of c-Maf in the M1-like BMM significantly upregulated IL-10 and arginase mRNA expression levels while downregulated IL-12 expression level, suggesting an M2-like phenotype. These data indicate that c-Maf may be a critical controller in regulating M2-related gene expression. To further determine which part of the M2 macrophage transcriptomic profile is controlled by c-Maf, we performed microarray analysis using polarized M2-like BMM from WT or c-Maf KO fetal liver chimeric mice. Notably, many M2 genes were differentially regulated by c-Maf. Further real-time PCR (qPCR) analysis confirmed that the mRNA expression levels of IL-12, IL-1β, IL-6, arginase, IL-10, VEGF, TGF-β, IRF-4, and CCR2 were significantly altered in c-Maf-deficient M2 BMM. Since M2-like macrophages have a potent immunosuppressive function on T cell activation, we next determined whether deficiency of c-Maf in M2 BMM would reverse such effect. M2 BMM from WT mice exhibited potent immunosuppressive activity as IFN-γ production from antigen (Ag)-specific CD4 and CD8 T cells was significantly diminished. In contrast, c-Maf deficiency in M2 BMM significantly increased IFN-γ production by CD4 and CD8 T cells compared to WT M2 BMM. These data suggest that c-Maf not only controls many M2-related gene expression but also is critical in regulating M2-like macrophage-mediated T cell immunosuppression.

ORGANISM(S): Mus musculus

PROVIDER: GSE139541 | GEO | 2020/04/17

REPOSITORIES: GEO

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Project description:A favourable immunotherapeutic strategy in the treatment of chronic inflammatory disease, is to dampen the pro-inflammatory macrophage response and upregulate the anti-inflammatory macrophage response. Previous studies have shown that Arginase-2 (Arg2), a mitochondrial enzyme, is a key regulator of the macrophage anti-inflammatory response with an essential role in IL-10 signalling. Here we show that IL-10 in the presence of LPS increased Arg2 expression in human macrophages and peripheral blood mononuclear cells. Target site blockers (TSBs) (locked nucleic acid antisense oligonucleotides) specifically designed to target the 3’ UTR of Arg2 messenger RNA were used to inhibit binding of specific microRNAs thus enhancing Arg2 expression. Eleven Arg2-TSBs were screened and the TSB targeting miR-155 (TSB-155) and the TSB targeting miR-3202 (TSB-3202) were found to increase Arg2 expression in human macrophages resulting in decreased gene expression and cytokine production of TNF-α and CCL2. In addition, following TSB-3202 stimulation, there were statistically significant increases in the ‘M2-like’ macrophage marker, CD206; and CD16, associated with an IL-10 response, and a decrease in the ‘M1-like’ macrophage marker, HLA-DR, indicating a shift in the macrophage phenotype. Proteomic analysis demonstrated that multiple pro-inflammatory responsive proteins including Signal Transducer and Activator of Transcription 1 (STAT-1), Toll-like receptors, Signalling Lymphocytic Activation Molecule F7 (SLAMF7) and Interferon Induced Protein with Tetratricopeptide Repeats 3 (IFIT3), were downregulated by TSB-155 and TSB-3202 while Sequestomsome-1 (SQSTM1) was increased after treatment. In silico analysis of significantly dysregulated proteins predicted that TSB-3202 supressed several upstream pro-inflammatory regulators including STAT-1 and Interferon α2 (IFNA2) while enhancing anti-inflammatory associated interleukin 1 receptor antagonist (IL1RN) and STAT-3. Proteomic data was validated by confirming increased levels of SQSTM1, decreased levels of phosphoylated-STAT-1 and STAT-1, and downregulation of Ifit3 and Slamf7 by TSB treatment. In conclusion, upregulation of Arg2 protein by TSBs inhibits several pro-inflammatory signalling proteins resulting in reduced pro-inflammatory cytokine secretion, reduced ‘M1-like’ marker expression and enhanced ‘M2-like’ macrophage marker expression. Arg2 is a promising novel therapeutic target to modulate inflammatory signalling in macrophages.

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Transcriptional analysis of transcription factor c-Maf as a molecular switch that controls immune suppression by programming macrophages in lung cancer

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