miR-221-3p Drives the Shift of M2-Macrophages to a Pro-Inflammatory Function by Suppressing JAK3/STAT3 Activation.

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.

Quero L ，Tiaden AN ，Hanser E ，Roux J ，Laski A ，Hall J ，Kyburz D ... -  《Frontiers in Immunology》

TLR2 stimulation impairs anti-inflammatory activity of M2-like macrophages, generating a chimeric M1/M2 phenotype.

Quero L ，Hanser E ，Manigold T ，Tiaden AN ，Kyburz D ... -  《-》

Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1ß, and TNF-α, and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells.

Wu J ，Niu P ，Zhao Y ，Cheng Y ，Chen W ，Lin L ，Lu J ，Cheng X ，Xu Z ... -  《-》

Antiphospholipid antibody-induced miR-146a-3p drives trophoblast interleukin-8 secretion through activation of Toll-like receptor 8.

What is the role of microRNAs (miRs) in antiphospholipid antibody (aPL)-induced trophoblast inflammation? aPL-induced up-regulation of trophoblast miR-146a-3p is mediated by Toll-like receptor 4 (TLR4), and miR-146a-3p in turn drives the cells to secrete interleukin (IL)-8 by activating the RNA sensor, TLR8. Obstetric antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating aPL and an increased risk of pregnancy complications. We previously showed that aPL recognizing beta2 glycoprotein I (β2GPI) elicit human first trimester trophoblast secretion of IL-8 by activating TLR4. Since some miRs control TLR responses, their regulation in trophoblast cells by aPL and functional role in the aPL-mediated inflammatory response was investigated. miRs can be released from cells via exosomes, and therefore, miR exosome expression was also examined. A panel of miRs was selected based on their involvement with TLR signaling: miR-9; miR-146a-5p and its isomiR, miR-146a-3p; miR-155, miR-210; and Let-7c. Since certain miRs can activate the RNA sensor, TLR8, this was also investigated. For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR8 was studied. HTR8 cells transfected to express a TLR8 dominant negative (DN) were also used. Plasma was evaluated from pregnant women who have aPL, either with or without systemic lupus erythematous (SLE) (n = 39); SLE patients without aPL (n = 30); and healthy pregnant controls (n = 20). Trophoblast HTR8 wildtype and TLR8-DN cells were incubated with or without aPL (mouse anti-human β2GPI mAb) for 48-72 h. HTR8 cells were also treated with or without aPL in the presence and the absence of a TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides; LPS-RS), specific miR inhibitors or specific miR mimics. miR expression levels in trophoblast cells, trophoblast-derived exosomes and exosomes isolated from patient plasma were measured by qPCR. Trophoblast IL-8 secretion was measured by ELISA. aPL significantly increased trophoblast cellular and exosome expression of miR-146a-5p, miR-146a-3p, miR-155 and miR-210. aPL-induced up-regulation of trophoblast miR-146a-5p, miR-146a-3p and miR-210, but not miR-155, was inhibited by the TLR4 antagonist, LPS-RS. While inhibition or overexpression of miR-146a-5p had no effect on aPL-induced trophoblast IL-8 secretion, miR-146a-3p inhibition significantly reduced this response. aPL-induced trophoblast IL-8 secretion was inhibited by the presence of the TLR8-DN. In the absence of aPL, transfection of trophoblast cells with a miR-146a-3p mimic significantly increased IL-8 secretion and this was inhibited by the presence of the TLR8-DN. Patients with aPL and adverse pregnancy outcomes (APOs) expressed significantly higher levels of circulating miR-146a-3p compared with healthy pregnant controls with no pregnancy complications (P < 0.05). While the enrichment of miR-146a-3p in trophoblast-derived exosomes support the role of this miR acting in a paracrine or endocrine manner through exosome delivery, this has not been demonstrated. However, miR-146a-3p may also exert its pro-inflammatory effect intracellularly within the same trophoblast cell targeted by aPL. These findings provide a novel mechanism of trophoblast inflammation through miRs activating RNA-sensing receptors. Furthermore, circulating exosomal-associated miR-146a-3p in APS patients may serve clinically as a biomarker for related APOs. This study was supported in part by grants from the American Heart Association (#10GRNT3640032 to V.M.A.), the March of Dimes Foundation (Gene Discovery and Translational Research Grant #6-FY12-255 to V.M.A.), NICHD, NIH (R01HD049446 to V.M.A.), the Gina M. Finzi Memorial Student Summer Fellowship from the Lupus Foundation of America (to S.M.G.), and the Yale University School of Medicine Medical Student Fellowship (to S.M.G.). The authors declare no competing financial interests. N/A.

Gysler SM ，Mulla MJ ，Guerra M ，Brosens JJ ，Salmon JE ，Chamley LW ，Abrahams VM ... -  《-》

MiR-140-5p inhibits synovial fibroblasts proliferation and inflammatory cytokines secretion through targeting TLR4.

This study was aimed to investigate the effects of miR-140-5p on the proliferation and inflammatory cytokines secretion of rheumatoid arthritis synovial fibroblasts (RASFs). Synovial tissue samples from 23 rheumatoid arthritis (RA) patients and 18 normal synovial tissue samples were collected. The RASFs were isolated and cultured. Then, miR-140-5p and TLR4 expression in both synovial tissue and RASFs were detected using the quantitative real-time PCR (qPCR) and western blot. Dual luciferase reporter gene assay was employed to evaluate the interaction between miR-140-5p and 3'UTR of TLR4. Western blotting and qPCR were used to examine TLR2 expression after upregulation or downregulation of miR-140-5p in RASFs. After RASFs co-infected with TLR4 overexpression lentivirus and lentivirus containing miR-140-5p or miR-control respectively, the cellular proliferation and secretion of IL-6 and IL-8 level were detected through the MTS assay and enzyme-linked immunosorbent assay, respectively. MiR-140-5p was significantly down-regulated, and TLR4 was significantly up-regulated in synovial tissue samples from 23 RA patients and RASFs. Dual luciferase activity assay showed that miR-140-5p could specifically bind to the 3'UTR of TLR4. Down-regulation or up-regulation of miR-140-5p not only significantly increased or decreased the expression of TLR4, but also could promote or inhibit RASF proliferation and secretion of IL-6, and IL-8 in RASFs. Furthermore, overexpression of TLR4 can reverse the inhibitory effects of miR-140-5p on proliferation and inflammatory cytokines release of RASFs. MiR-140-5p could inhibit the proliferation and secretion of IL-6 and IL-8 through regulation of TLR4 expression.

Li H ，Guan SB ，Lu Y ，Wang F ... -  《-》