MicroRNA-155 as a proinflammatory regulator in clinical and experimental arthritis

Abstract

MicroRNA (miRNA) species (miR) regulate mRNA translation and are implicated as mediators of disease pathology via coordinated regulation of molecular effector pathways. Unraveling miR disease-related activities will facilitate future therapeutic interventions. miR-155 recently has been identified with critical immune regulatory functions. Although detected in articular tissues, the functional role of miR-155 in inflammatory arthritis has not been defined. We report here that miR-155 is up-regulated in synovial membrane and synovial fluid (SF) macrophages from patients with rheumatoid arthritis (RA). The increased expression of miR-155 in SF CD14(+) cells was associated with lower expression of the miR-155 target, Src homology 2-containing inositol phosphatase-1 (SHIP-1), an inhibitor of inflammation. Similarly, SHIP-1 expression was decreased in CD68(+) cells in the synovial lining layer in RA patients as compared with osteoarthritis patients. Overexpression of miR-155 in PB CD14(+) cells led to down-regulation of SHIP-1 and an increase in the production of proinflammatory cytokines. Conversely, inhibition of miR-155 in RA synovial CD14(+) cells reduced TNF-α production. Finally, miR-155-deficient mice are resistant to collagen-induced arthritis, with profound suppression of antigen-specific Th17 cell and autoantibody responses and markedly reduced articular inflammation. Our data therefore identify a role of miR-155 in clinical and experimental arthritis and suggest that miR-155 may be an intriguing therapeutic target.

Fig. 1.

MiR-155 is up-regulated in RA synovial macrophages and monocytes and promotes production of proinflammatory cytokines. (A and B) Expression of miR-155 in RA and OA biopsies. (A) Representative staining. (B) Quantitative evaluation of miR-155 expression, *P < 0.05 RA vs. OA. (C) Representative photograph of double staining for miR-155 (green) and macrophage marker CD68 (red) in RA lining layer. Double-positive cells are orange or yellow. (D) Expression of miR-155 in paired PB and SF CD14⁺ cells from RA patients (n = 4), *P < 0.05 PB vs. SF. (E) TLR ligands and RA SF up-regulate miR-155 expression in PB CD14⁺ cells (n = 3). Cells were stimulated with TLR ligands or PAR2 agonist or 25% SF for 24 h as described in Materials and Methods. *P < 0.05 stimulated vs nonstimulated. (F) PB CD14⁺ cells overexpressing miR-155 produce proinflammatory cytokines. CD14⁺ cells were transfected with control or miR-155 mimic (20 nM). After 24 h, LPS (100 ng/mL) was added to some wells for a further 16 h (n = 5). (G and H) PB CD14⁺ cells and RA SF CD14⁺ cells (n = 2 and 3, respectively) were transfected with control or miR-155 antagomir. After 24 h cells were exposed to SF (F) or LPS (G) for a further 24 h. *P < 0.05 as indicated. Data are means ± SEM or box-and-whisker diagrams. Cm, control mimic; miR155ant, miR-155 antagomir; miR155m, miR-155 mimic, C ant, control antagomir; PAR2, PAR2 agonist, (SLIGKV-NH2). The in situ scoring system is described in SI Materials and Methods.

Fig. 2.

The miR-155 target SHIP-1 is down-regulated in RA SF CD14⁺ cells and in RA synovial tissue macrophages. (A) miR-155 regulates expression of SHIP-1 in RA PB and SF CD14⁺ cells. Cells were transfected with miR-155 mimic or antagomir or with appropriate controls as described in Fig. 1. Quantitative analysis of SHIP-1 mRNA expression is shown (n = 3). (B) miR-155 binds directly to the 3′ UTR of human SHIP-1 mRNA. pMir-hSHIP1 (sense) and pMir-hSHIP1 (antisense) 3′ UTR luciferase plasmids were cotransfected with control or miR-155 mimic (40 nM) in HEK293 cells. Luciferase activity was analyzed at 24 h. (C) Expression of SHIP-1 in paired PB and SF CD14⁺ cells from RA patients (n = 6). Data are means ± SEM. *P < 0.05. (D and E) Synovial specimens from RA (n = 5) and OA (n = 5) patients stained with anti-human SHIP-1 (D, brown) and counterstained with anti-CD68 antibody (E). Representative staining of one of five specimens is shown; blue, DAPI; red, CD68+; green, SHIP-1+; yellow, CD68+/SHIP-1+. (Magnification: 63×.) Enlargements in E show a SHIP-1⁺ cell (green). Cont, control mimic; hSHIP-1, human SHIP-1; miR155antg, miR-155 antagomir; miR155m, miR-155 mimic; Cont antg, control antagomir. Relative luciferase activity is the maximal activity in the presence of pMiR plasmid alone.

Fig. 3.

miR-155 is crucial for the development of CIA. On day 0, miR-155^−/− and WT mice (n = 15–17 per group) were injected intradermally with either a type II chicken collagen/Freund's complete adjuvant emulsion (CII/CFA) (200 μg) or PBS. On day 21, type II chicken collagen in PBS (200 μg) was injected i.p. Mice were killed on day 34. (A) miR-155 is up-regulated in the articular tissue of WT mice with CIA. (B and C) The incidence (B) and mean clinical score (C) are shown. In contrast to WT littermates, miR-155^−/− mice do not develop signs of CIA. (D) In contrast to WT mice, miR-155^−/− mice did not develop articular inflammation and degradation of cartilage and bone. (Magnification: ×10.) Data are means ± SEM or box-and-whisker diagrams. (n = 15–17); *P < 0.05, miR-155^−/− vs. WT mice (CIA protocol).

Fig. 4.

miR-155^−/− mice exhibit reduced development of T- and B-cell responses during CIA. Groups are as in Fig. 3. CII/miR-155^−/− mice show (A) reduced systemic cytokine and chemokine concentrations and (B) reduced expression of TNF-α mRNA in articular tissue. (C and D) CII-miR-155^−/− mice show a reduced differentiation of Th17 and Th1 cells in DLN. Representative staining (C) and quantitative evaluation (D) are shown. (E and F) miR-155 is required for the production of collagen-specific antibodies. Quantitative evaluation of antibody activity in isotypes IgG2a (E) and IgG1 (F) is shown. Data are means ± SEM, n = 15–17; *P < 0.05, miR-155^−/− vs. WT mice (CIA protocol). CII, type II chicken collagen.