Myeloid sirtuin 6 deficiency accelerates experimental rheumatoid arthritis by enhancing macrophage activation and infiltration into synovium

Abstract

Background: We recently reported that myeloid sirtuin 6 (Sirt6) is a critical determinant of phenotypic switching and the migratory responses of macrophages. Given the prominent role of macrophages in the pathogenesis of rheumatoid arthritis (RA), we tested whether myeloid Sirt6 deficiency affects the development and exacerbation of RA.

Methods: Arthritis was induced in wild type and myeloid Sirt6 knockout (mS6KO) mice using collagen-induced and K/BxN serum transfer models. Sirt6 expression (or activity) and inflammatory activities were compared in peripheral blood mononuclear cells (PBMCs) and monocytes/macrophages obtained from patients with RA or osteoarthritis.

Findings: Based on clinical score, ankle thickness, pathology, and radiology, arthritis was more severe in mS6KO mice relative to wild type, with a greater accumulation of macrophages in the synovium. Consistent with these findings, myeloid Sirt6 deficiency increased the migration potential of macrophages toward synoviocyte-derived chemoattractants. Mechanistically, Sirt6 deficiency in macrophages caused an inflammation with increases in acetylation and protein stability of forkhead box protein O1. Conversely, ectopic overexpression of Sirt6 in knockout cells reduced the inflammatory responses. Lastly, PBMCs and monocytes/macrophages from RA patients exhibited lower expression of Sirt6 than those from patients with osteoarthritis, and their Sirt6 activity was inversely correlated with disease severity.

Interpretation: Our data identify a role of myeloid Sirt6 in clinical and experimental RA and suggest that myeloid Sirt6 may be an intriguing therapeutic target. FUND: Medical Research Center Program and Basic Science Research Program through the National Research Foundation of Korea.

Keywords: FoxO1; Inflammation; Macrophage; RA; Sirt6.

Graphical abstract

Fig. 1

Worsening of arthritis in mS6KO mice with CIA. (a) Mean clinical score (n = 10–12), (b) ankle thickness change (n = 10–12), and (c) gross and micro-CT images and score for bone volume (BV) and ratio of bone surface to bone volume (BS/BV) (n = 7) of CIA mice. (d) Pathological staining with H&E, Safranin-O, and TRAP (bar = 1 mm), and scores for synovial inflammation, cartilage damage, and bone erosion (n = 9). (e) Staining with CD68 (for macrophages) or Ly6G (for neutrophils) and mean number of CD68⁺ macrophages or Ly6G⁺ neutrophils in the ankle joints (bar = 50 μm) (n = 5). (f) Staining with CD68 or Sirt6 and mean number of CD68- or Sirt6-positive cells in the synovial lining and sub-lining of ankle joints (bar = 100 μm) (n = 3). (g) Serum concentrations of IL-6 and RANTES (n = 10–12). Values are mean ± SEM. ^⁎, p < 0.05 and ^⁎⁎, p < 0.01 vs. WT.

Fig. 2

Worsening of arthritis in mS6KO mice with K/BxN serum transfer arthritis. (a) Mean clinical score (n = 14), (b) ankle thickness change (n = 14), and (c) pathological staining with H&E, Safranin-O, and TRAP (bar = 1 mm) and scores for synovial inflammation, cartilage damage, and bone erosion (n = 4–6). (d) Serum concentrations of IL-6 and RANTES (n = 5–6). Values are mean ± SEM. ^⁎, p < 0.05 and ^⁎⁎, p < 0.01 vs. WT.

Fig. 3

Regulation of macrophage migration by Sirt6. (a) FLS were cultured with 10 ng/ml LPS or 20 ng/ml TNF-α for 24 h, and chemokine concentrations in the culture supernatants were analyzed by ELISA (n = 4–5). (b, c) BMMs from WT or mS6KO mice were allowed to migrate through porous membranes for 3 h for cell migration toward 10 ng/ml MCP-1, 20 ng/ml RANTES or fibroblast-like synoviocyte-conditioned medium (FLS-CM). Representative microphotographs (original magnification ×40). Mean number of migratory cells in trans-well chamber were counted (n = 3). (d–f) BMMs from WT or mS6KO mice were treated with 10 ng/ml LPS for 3 h and subjected to analysis of CCR3 expression (n = 4–5) and confocal microscopic analysis for subcellular localization of CCR3. Values are mean ± SEM. ^⁎, p < 0.05 and ^⁎⁎p < 0.01 vs. WT; ^#, p < 0.05 and ^##, p < 0.01 vs. VEH. VEH, Vehicle.

Fig. 4

Regulation of macrophage survival and cytokine excretion by Sirt6. (a, b) BMMs (2 × 10⁵) from WT or mS6KO mice were treated with 10 ng/ml LPS as indicated (a) or for 24 h (b), followed by washing twice with PBS. Proliferation rate and excretion of TNF-α and IL-6 into culture medium were determined by BrdU incorporation assay and ELISA analysis, respectively. (c) BMMs (2 × 10⁵) were cultured in serum-starved conditions as indicated, and cell viability was determined by MTT assay. Values are mean ± SEM (n = 5–7). ^⁎, p < 0.05 and ^⁎⁎, p < 0.01 vs. WT.

Fig. 5

Total- and acetylated-FoxO1 in mice with CIA (a-c) or K/BxN serum transfer arthritis (d, e). All experimental procedures were same as described in Fig. 1, Fig. 2 legends. (a) Staining of FoxO1, Ac-FoxO1, and CD68 in ankle joints (bar = 100 μm), and mean number of CD68 and Ac-FoxO1 double-positive cells (n = 3). (b, c) mRNA and protein levels of total- and Ac-FoxO1 in the ankle joints of CIA mice (n = 9–11). (d, e) mRNA (n = 5–6) and protein levels (n = 3–5) of total- and Ac-FoxO1 in the ankle joints of K/BxN serum transfer arthritis. Values are mean ± SEM. ^⁎, p < 0.05 and ^⁎⁎, p < 0.01 vs. WT.

Fig. 6

Regulation of acetylation and degradation of FoxO1 by Sirt6. (a) BMMs were prepared from WT mice, and the interaction between Sirt6 and FoxO1 was analyzed. (b, c) Protein levels of FoxO1 and acetylated FoxO1 in BMMs from WT and mS6KO mice. (d) BMMs from WT or mS6KO mice were treated with 10 μM MG132 for 3 h, and protein levels of FoxO1 in cytosolic extract (CE) and nuclear extract (NE) were determined. (e) Total lysates of BMMs were immunoprecipitated with anti–FoxO1-antibodies and immunoblotted with anti-ubiquitin antibodies. (f, g) Protein levels of total and acetylated FoxO1 in BMMs whole lysates following transduction with AdLacZ or AdSirt6. (h, i) Following transduction of BMMs with AdLacZ or AdSirt6, cells were treated with 10 ng/ml LPS for 3 h and protein levels of Sirt6 and the expression of cytokines were determined. Values are mean ± SEM (n = 6). ^⁎, p < 0.05 and ^⁎⁎, p < 0.01 vs. WT; ^#, p < 0.05 and ^##, p < 0.01 vs. AdLacZ.

Fig. 7

Expression and activity of Sirt6 in RA patients and relationship with disease severity. (a) Staining with CD68 and Sirt6 in the synovial tissue from osteoarthritis (OA) and RA patients (bar = 100 μm). (b) Protein levels of Sirt6 in blood monocytes and synovial fluid monocytes from OA patients and RA patients (n = 4–6). (c) Sirt6 enzyme activity in PBMCs from patients with OA (n = 23), mild RA (DAS 28 ≤ 5.0, n = 7), and severe RA (DAS28 > 5.1, n = 13). (d) Correlation analysis of Sirt6 enzyme activity in PBMCs with RA severity (DAS28-CRP, n = 20), (e) DAS28-ESR (n = 20), and (f) IL-6 concentration (n = 10) in serum. (g–j) Synovial fluid monocytes/macrophages were transduced with AdSirt6 or AdmSirt6 and then incubated with 10 ng/ml LPS for 24 h (G) or as indicated (H, I). Cell migration (n = 4), cell proliferation (n = 3), IL-6 excretion (n = 4), and cell viability under serum-starved condition (n = 4) were determined. Values are mean ± SEM. ^⁎, p < 0.05 and ^⁎⁎, p < 0.01 vs. OA or AdLacZ. ^#, p < 0.05 and ^##, p < 0.01 vs. AdSirt6. Blood MC, blood monocytes; SF MC, synovial fluid monocytes; DAS, disease activity score.

Fig. 8

Proposed scheme. A healthy joint (left side) contains 1–2 layers of FLS with a few macrophages (Mφ) in the synovium. FoxO1 is deacetylated by Sirt6, exported from the nucleus to the cytosol, and targeted for degradation through the ubiquitination-proteasome pathway. In arthritic joint tissues of mS6KO mice (right side), the synovial lining is thickened, the vascularity is increased, and the hyperplastic synovium is infiltrated by inflammatory cells such as macrophages, dendritic cells, and T cells. Sirt6-mediated FoxO1 deacetylation in macrophages is impaired, which recruits more inflammatory cells into synovium. This inflamed condition damages cartilage, bone, and surrounding tissue, causing joint deformity.