MicroRNA-146a governs fibroblast activation and joint pathology in arthritis

Abstract

Synovial fibroblasts are key cells orchestrating the inflammatory response in arthritis. Here we demonstrate that loss of miR-146a, a key epigenetic regulator of the innate immune response, leads to increased joint destruction in a TNF-driven model of arthritis by specifically regulating the behavior of synovial fibroblasts. Absence of miR-146a in synovial fibroblasts display a highly deregulated gene expression pattern and enhanced proliferation in vitro and in vivo. Deficiency of miR-146a induces deregulation of tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) in synovial fibroblasts, leading to increased proliferation. In addition, loss of miR-146a shifts the metabolic state of fibroblasts towards glycolysis and augments the ability of synovial fibroblasts to support the generation of osteoclasts by controlling the balance of osteoclastogenic regulatory factors receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). Bone marrow transplantation experiments confirmed the importance of miR-146a in the radioresistant mesenchymal compartment for the control of arthritis severity, in particular for inflammatory joint destruction. This study therefore identifies microRNA-146a as an important local epigenetic regulator of the inflammatory response in arthritis. It is a central element of an anti-inflammatory feedback loop in resident synovial fibroblasts, who are orchestrating the inflammatory response in chronic arthritis. MiR-146a restricts their activation, thereby preventing excessive tissue damage during arthritis.

Fig. 1.. Human TNF transgenic mice lacking miR-146a (miR-146a^−/−/hTNFtg) show more severe bone destruction compared to hTNFtg mice.

Histological sections of the hind paws of hTNFtg and miR-146a^−/−/hTNFtg mice obtained at 10 weeks of age stained with A, Hematoxylin and eosin (H&E) B, Tartrate-resistant acid phosphatase (TRAP) (bars 500 μm, magnification x 5). Osteoclasts are displayed as purple-stained cells. C-F, Histomorphometric analysis of the extent of inflammation, bone erosion, number of osteoclasts and number of osteoclasts per inflammation area; in the tarsal area of the hind paws of hTNFtg (n = 16) and miR-146a^−/−/hTNFtg (n = 15) mice. G and H, FACS analysis of CD11c⁺ (G) and CD11b⁺ (H) blood cells from hTNFtg (n = 19) and miR-146a^−/−/hTNFtg (n = 20) mice at the indicated age, using anti-CD11c and -CD11b antibodies. I, Quantification of F4/80⁺ cells among total synovial cells, from immunohistochemically stained sections of hind paws from hTNFtg (n = 12) and miR-146a^−/−/hTNFtg (n = 14) mice using anti-F4/80 antibody. *p < 0,05, **p < 0,01 Results are shown as mean ± SEM.

Fig. 2.. Loss of miR-146a in hTNFtg mice does not have an impact on the development of osteoporosis.

A-F, Quantitative histological analysis of tartrate-resistant acid phosphatase (TRAP) stained sections of the tibiae from wt (n = 11), miR-146a^−/− (n = 14), hTNFtg (n = 14) and miR-146a^−/−/hTNFtg (n = 16) mice. A, Bone volume per tissue volume B, Trabecular number C, Trabecular thickness D, Trabecular separation E, Number of Osteoclasts per bone perimeter and F, Number of Osteoblasts per bone perimeter (n = 11).

Fig. 3.. Gene expression vary significantly in miR-146a deficient fibroblasts but not in bone marrow cells.

A, Bone marrow isolated from wt or miR-146a^−/− mice was stimulated with MCSF (d0) and MCSF + RANKL (d3) to induce osteoclastogenesis. The number of OCs/well among TRAP positive stained multinucleated cells in wt and miR-146a^−/− mice (d7) was analyzed (data shown are representative of 3 independent experiments). B and C, Volcano plots showing differentially expressed genes from wt versus miR-146a^−/− bone marrow cells (n = 3) (B) and synovial fibroblasts (n = 2) (C). D, Pathways affected by loss of miR-146a in synovial fibroblasts as detected by ingenuity pathway analysis. Results are shown as mean ± SEM.

Fig. 4.. MiR-146a deficiency leads to increased proliferation of synovial fibroblasts.

A, In vitro proliferation of wt and miR-146a^−/− synovial fibroblasts, measured by ³[H] thymidine incorporation (data shown are representative of 3 independent experiments). B, Hematoxylin and eosin staining from histological sections of wt and miR-146a^−/− synovial fibroblasts cultured in micromasses (bars 500 μm, magnification x 5). C, Quantification of Ki-67⁺ synovial fibroblast cells among total synovial lining cells, from immunohistochemically stained sections of micromasses from wt (n = 7) and miR-146a^−/− (n = 7) mice using anti Ki-67 antibody. D, Expression level of TRAF6 in synovial fibroblasts from wt (n = 9) and miR-146a^−/− (n = 10) animals was analyzed using quantitative real time PCR. E and F, Synovial fibroblasts from wt or miR-146a deficient animals were transfected with either control (Ctrl) or TRAF6 siRNA, proliferation analysis was performed using ³[H]thymidine incorporation (data shown are mean values of 2 independent experiments). *p < 0,05, **p < 0,01, ***p < 0,001 Results are shown as mean ± SEM.

Fig. 5.. TRAF6 and consequential RANKL expression is controlled by miR-146a in synovial fibroblasts.

A, MRNA expression of TRAF6 in hind paws of hTNFtg (n = 10) and miR-146a^−/−/hTNFtg (n = 6) animals, measured using Q-PCR. B, Synovial tissue of hTNFtg and miR-146a^−/−/hTNFtg mice analyzed histologically for Ki-67 expression (bars 100 μm, magnification x 20). C, Ki-67 positive cells among total synovial cells in the inflamed tissue (n = 5). D and E, ECAR and OCR from wt and miR-146a deficient SF, analyzed using seahorse flux analyser (representative of 2 independent experiments). F, Schematic illustration of coculture assay from wt and miR-146a^−/− SF and wt BMDM supplemented with IL-1 β. G, Number of osteoclasts from these cocultures was analyzed (representative of 3 independent experiments). H, Number of osteoclasts after transfection of wt or miR-146a^−/− SF with control or TRAF6 siRNA following coculture with wt BMDM supplemented with IL-1 β (representative of 2 independent experiments). I, RANKL production of SF from wt and miR-146a deficient animals cultured with IL-1 β, analyzed by Elisa (mean values of 4 independent experiments). J and K, Expression level of RANKL and OPG mRNA in hind paws of hTNFtg (n = 4) and miR-146a^−/−/hTNFtg (n = 6) animals measured using Q-PCR. *p < 0,05 Results are shown as mean ± SEM.

Fig. 6.. Chimeric mice reveal an important regulatory function of miR-146a in mesenchymal cells, by controlling osteoclast development and bone destruction in arthritis.

A-D, Human TNFtg or miR-146a^−/−/hTNFtg recipient mice were irradiated and challenged with either wt or miR-146a^−/− bone marrow cells. A, TRAP stained sections of the hind paws of recipient mice obtained at 10 weeks of age (bars 500 μm, magnification x 5). B-D, Histomorphometric analysis of the extent of B, Inflammation C, Bone erosion and D, Number of Osteoclasts in the tarsal area of the hind paws of recipient mice (n = 5). *p < 0,05, **p < 0,01, ***p < 0,001 Results are shown as mean ± SEM.