Unknown,Transcriptomics,Genomics,Proteomics

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Control of the inflammatory macrophage transcriptional signature by miR-155

ABSTRACT: Classically activated (M1) macrophages protect from infection but can cause inflammatory disease and tissue damage while alternatively activated (M2) macrophages reduce inflammation and promote tissue repair. Modulation of macrophage phenotype may be therapeutically beneficial and requires further understanding of the molecular programs that control macrophage differentiation. A potential mechanism by which macrophages differentiate may be through microRNA (miRNA), which bind to messenger RNA and post-transcriptionally modify gene expression, cell phenotype and function. The inflammation-associated miRNA, miR-155, was rapidly up-regulated over 100-fold in M1, but not M2, macrophages. Inflammatory M1 genes and proteins iNOS, IL-1b and TNF-a were reduced up to 72% in miR-155 knockout mouse macrophages, but miR-155 deficiency did not affect expression of genes associated with M2 macrophages (e.g., Arginase-1). Additionally, a miR-155 oligonucleotide inhibitor efficiently suppressed iNOS and TNF-a gene expression in wild-type M1 macrophages. Comparative transcriptional profiling of unactivated (M0) and M1 macrophages derived from wild-type and miR-155 knockout (KO) mice revealed an M1 signature of approximately 1300 genes, half of which were dependent on miR-155. Real-Time PCR of independent datasets validated miR-155's contribution to induction of iNOS, IL-1b, TNF-a, IL-6 and IL-12, as well as suppression of miR-155 targets Inpp5d, Tspan14, Ptprj and Mafb. Overall, these data indicate that miR-155 plays an essential role in driving the differentiation and effector potential of inflammatory M1 macrophages. Total RNA was prepared from bone marrow-derived macrophages of miR-155 knockout mice (n=2 independent mice) treated in M0, M1 or M2 conditions (n=2 replicates per condition originating from different mice)

ORGANISM(S): Mus musculus

SUBMITTER: Mireia Guerau

PROVIDER: E-GEOD-77425 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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Control of the inflammatory macrophage transcriptional signature by miR-155

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