MiR-221-3p drives the Shift of M2-Macrophage to a Pro-Inflammatory Function by Modulating JAK3/STAT3 Signaling and is Increased in Rheumatoid Arthritis
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ABSTRACT:
PROVIDER: PRJNA551688 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9621270.fastq.gz | Fastqsanger.gz | |||
| SRR9621280.fastq.gz | Fastqsanger.gz | |||
| SRR9621290.fastq.gz | Fastqsanger.gz | |||
| SRR9621300.fastq.gz | Fastqsanger.gz | |||
| SRR9621271.fastq.gz | Fastqsanger.gz |
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Project description:We performed a transcriptome analysis of M2 macrophages generated in vitro from monocytes isolated from peripheral blood of healthy donors, and studied the effect of miR-221-3p mimic transfection and stimulation with LPS.
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Project description:Objectives: Macrophages are conventionally classified as pro-inflammatory (M1) and anti-inflammatory (M2) functional types. There is evidence for a predominance of macrophages with an inflammatory phenotype (M1) in the rheumatoid arthritis (RA) synovium. MicroRNAs (miRs) play a pivotal role in regulating the inflammatory response in innate immune cells and are found at dysregulated levels in RA patients. Here we explored miRs that tune the inflammatory function of M2-macrophages. Methods: Expression profiles of miR-221-3p and miR-155-5p were analyzed in clinical samples from RA, other inflammatory arthritis (OIA), osteoarthritis (OA), and healthy donors (HD) by qPCR. In vitro generated macrophages were transfected with miR-mimics and inhibitors. Transcriptome profiling through RNA-sequencing was performed on M2-macrophages overexpressing miR-221-3p mimic with or without LPS treatment. Secretion of IL-6, IL-10, IL-12, IL-8, and CXCL13 was measured in M1- and M2-macrophages upon TLR2/TLR3/TLR4-stimulation using ELISA. Inflammatory pathways including NF-κB, IRF3, MAPKs, and JAK3/STAT3 were evaluated by immunoblotting. Direct target interaction of miR-221-3p and predicted target sites in 3'UTR of JAK3 were examined by luciferase reporter gene assay. Results: miR-221-3p in synovial tissue and fluid was increased in RA vs. OA or OIA. Endogenous expression levels of miR-221-3p and miR-155-5p were higher in M1- than M2-macrophages derived from RA patients or HD. TLR4-stimulation of M1- and M2-macrophages resulted in downregulation of miR-221-3p, but upregulation of miR-155-5p. M2-macrophages transfected with miR-221-3p mimics secreted less IL-10 and CXCL13 but more IL-6 and IL-8, exhibited downregulation of JAK3 protein and decreased pSTAT3 activation. JAK3 was identified as new direct target of miR-221-3p in macrophages. Co-transfection of miR-221-3p/miR-155-5p mimics in M2-macrophages increased M1-specific IL-12 secretion. Conclusions: miR-221-3p acts as a regulator of TLR4-induced inflammatory M2-macrophage function by directly targeting JAK3. Dysregulated miR-221-3p expression, as seen in synovium of RA patients, leads to a diminished anti-inflammatory response and drives M2-macrophages to exhibit a M1-cytokine profile.
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Project description:MicroRNAs (miRNAs) constitute fine tuners of gene expression and are implicated in a variety of diseases spanning from inflammation to cancer. miRNA expression is deregulated in rheumatoid arthritis (RA), however, their specific role in key arthritogenic cells such as the synovial fibroblast (SF) remains elusive. We have shown in the past that the expression of the miR-221/222 cluster is upregulated in RA SFs. Here, we demonstrate that miR-221/222 activation is downstream of major inflammatory cytokines, such as TNF and IL-1β, which promote miR-221/222 expression independently. miR-221/222 expression in SFs from the huTNFtg mouse model of arthritis correlates with disease progression. Targeted transgenic overexpression of miR-221/222 in SFs of the huTNFtg mouse model led to further expansion of synovial fibroblasts and disease exacerbation. miR-221/222 overexpression altered the transcriptional profile of SFs igniting pathways involved in cell cycle progression and ECM regulation. Validated targets of miR-221/222 included p27 and p57 cell cycle inhibitors, as well as Smarca1 (a chromatin remodeling component). In contrast, complete genetic ablation of miR-221/222 in arthritic mice led to decreased proliferation of fibroblasts, reduced synovial expansion and attenuated disease. scATAC-seq data analysis revealed increased miR-221/222 gene activity in the pathogenic and activated clusters of the intermediate and lining compartment. Taken together, our results establish an SF-specific pathogenic role of the miR-221/222 cluster in arthritis and suggest that its therapeutic targeting in specific subpopulations should inform the design of novel fibroblast-targeted therapies for human disease.
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Project description:Multiple cytokines play a pivotal role in the pathogenesis of Rheumatoid Arthritis by inducing intracellular signaling and it is known that the members of the Janus kinase (JAK) family are essential for such signal transduction. Janus kinase 3 is a tyrosine kinase that belongs to the Janus family of kinases. Drugs targeting JAK3 in the treatment of Rheumatoid arthritis is relevant. Therefore, it is of interest to design suitable inhibitors for JAK3 dimer using molecular docking with Molegro Virtual Docker. The compound possessing the highest affinity score is subjected to virtual screening to retrieve inhibitors. The compound SCHEMBL19100243 (PubChem CID- 76749591) displays a high affinity with the target protein. The affinity scores of this compound are more than known drugs. ADMET analysis and BOILED Egg plot provide insights into this compound as a potent inhibitor of JAK3.
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Project description:T-ALL is characterized by deregulation of transcriptional control and impairment of epigenetic homeostasis. We observed that T-ALL cases with mutations in the IL7R-JAK-STAT pathway frequently harbor inactivating mutations in the PRC2 complex, in particular SUZ12 mutations. PRC2 is an epigenetic complex responsible for writing the H3K27me3 mark associated with gene repression. To assess whether loss of SUZ12 cooperates with mutant JAK3 signaling during T-ALL development, we expressed the JAK3(M511I) mutant in mouse bone marrow cells in vivo and tested the effect of Cas9-mediated inactivation of Suz12 on leukemia development. The mice developed a T-cell leukemia that infiltrated most hematological organs (bone marrow, thymus, spleen, lymph nodes, blood). At time of sacrifice the majority of cells in the blood and hematopoietic organs was JAK3(M511I) positive and Suz12 negative. Reduced Suz12 protein and H3K27me3 levels were confirmed with flow cytometry. Moreover, JAK3(M511I) also cooperates with Suz12 gRNA-mediated inactivation in vitro. Pro-T cells carrying both JAK3(M511I) and Suz12 gRNA were able to transform to IL7 independent growth, whereas single mutant (JAK3(M511I) only or Suz12 gRNA only) pro-T cells could not. In order to get insight in the mechanism of cooperation between JAK3 mutant signaling and Suz12 inactivation, we performed and integrated RNA- and ChIP-sequencing (Suz12, H3K27me3, H3K27Ac, H3K4me1, H3K4me3) on leukemic cells from the mice. This showed a global upregulation of many genes, including known PRC2 target genes upon loss of Suz12. Moreover, Suz12 was bound to promoters of Wnt signaling genes (Fzd7, Lrp4) and Wnt target genes (Axin2, Jag2, Mmp9), concomitant with high H3K27me3 levels. Loss of Suz12 led to drastic reduction of H3K27me3 at these loci and upregulation of these Wnt genes. As Wnt signaling is ectopically activated, we wanted to test whether JAK3(M511I) + Suz12 gRNA cells are more sensitive to Wnt inhibitor. JAK3(M511I) + Suz12 pro-T cells and leukemic spleen cells exhibited increased sensitivity to Wnt inhibitor compared to JAK3 mutant control cells, indicating that Wnt signaling contributes to the cooperation between Suz12 loss and JAK3 mutant signaling. In conclusion, our data show that Suz12 inactivation cooperates with mutant JAK3 signaling to drive leukemia development in vivo and demonstrates the use of Cas9 transgenic mice to study the role of tumor suppressor genes in T-ALL mouse models. Moreover, Suz12 loss leads to ectopic activation of Wnt signaling, a pathway contributing to the cooperation of Suz12 loss and JAK3 mutant signaling that can also be targeted with inhibitors.
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