Session Type: ACR Poster Session A
Session Time: 9:00AM-11:00AM
Background/Purpose: MicroRNAs (miRNAs) have been shown to contribute to the inflammatory response in rheumatoid arthritis (RA) and several of these miRNAs have been found to be dysregulated in synovial tissue, synovial fluid or plasma of RA patients. In addition, also inflammatory cells such as macrophages, which are infiltrating the synovium and promoting the pannus generation, exhibit an aberrant miRNA expression. As toll-like receptors (TLRs) play a pivotal role in RA, we conducted a study to explore the role of TLRs in regulating the expression of miRNAs in macrophages under conditions prone to RA. Based on a miRNA-screen in TLR-stimulated M1-and M2-macrophages we selected several potential candidates for further studies, among them miR-221-3p. We then compared the release of inflammatory versus anti-inflammatory cytokines upon miR-221-3p overexpression or inhibition in stimulated M1- and M2-macrophages.
Methods: Monocytes were isolated from buffy coats of 8 healthy donors by CD14 microbead separation and differentiated into M1 and M2 by culturing them in the presence of 50ng/ml GM-CSF and M-CSF, respectively, for 6 to 8 days. Subsequently, cells were stimulated for 8.5 or 24 hours with TLR2/3/4 ligands Pam3, PolyIC or LPS. Cytokine release was measured by ELISA and miRNA expression by qRT-PCR. For miRNA mimic and inhibition studies, cells were transfected with corresponding pre-miR or antagomir using lipofectamine for 72 hours prior to the stimulation of M1- and M2-macrophages with TLR ligands.
Results: The general outcome of the miRNA-screen showed that a higher proportion of the tested miRNAs were downregulated in M2 macrophages upon stimulation with TLR ligands Pam3 and LPS. The basal expression level of miR-221-3p was similar in M1- and M2-macrophages, however, stimulation with LPS and PolyIC resulted in a prominent downregulation of miR-221-3p only in M2-macrophages. In M1-macrophages LPS caused only a slight decrease of miR-221-3p expression. Since several recent studies detected higher miR-221-3p levels in serum of RA patients and also in synovium of an induced RA-mouse model, we checked how macrophages respond to aberrant miR-221-3p levels under inflammatory conditions. Overexpression or inhibition of miR-221-3p showed no effect in unchallenged M1- or M2-macrophages on the secretion of IL-6, IL-8 or IL-10. However, miR-221-3p overexpression in combination with LPS stimulation, but not Pam3 or PolyIC, significantly increased IL-6 and IL-8 cytokine secretion in M2 macrophages. In contrast, the same combinatorial treatment (LPS + miR-221-3p overexpression) significantly decreased the IL-10 secretion in M2 macrophages.
Conclusion: We herein demonstrate that aberrant miR-221-3p expression is significantly downregulating the anti-inflammatory activity of TLR4-stimulated M2-macrophages by decreasing the ratio of secreted IL-10/IL-6 and IL-10/IL-8. Thus, abundant miR-221-3p might contribute to promote and sustain the chronically inflammatory conditions apparent in disease settings such as RA synovial tissue by deregulating the pro-/anti-inflammatory cytokine profile of synovial macrophage subpopulations.
To cite this abstract in AMA style:Quero L, Tiaden A, Kyburz D. MiR-221-3p Overexpression Impairs Anti-Inflammatory Activity of TLR4-Stimulated M2-Macrophages [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/mir-221-3p-overexpression-impairs-anti-inflammatory-activity-of-tlr4-stimulated-m2-macrophages/. Accessed September 25, 2021.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mir-221-3p-overexpression-impairs-anti-inflammatory-activity-of-tlr4-stimulated-m2-macrophages/