Myeloid deletion of SIRT1 aggravates serum transfer arthritis in mice via nuclear factor-κB activation

Abstract

Objective: SIRT1 modulates the acetylation of the p65 subunit of nuclear factor-κB (NF-κB) and plays a pivotal role in the inflammatory response. This study sought to assess the role of SIRT1 in rheumatoid arthritis (RA) using a myeloid cell-specific SIRT1 knockout (mSIRT1 KO) mouse.

Methods: mSIRT1 KO mice were generated using the loxP/Cre recombinase system. K/BxN serum transfer arthritis was induced in mSIRT1 KO mice and age-matched littermate loxP control mice. Arthritis severity was assessed by clinical and pathological scoring. The levels of inflammatory cytokines in the serum and joints were measured by ELISA. Migration, M1 polarization, cytokine production, osteoclastogenesis, and p65 acetylation were assessed in bone marrow-derived monocytes/macrophages (BMMs).

Results: mSIRT1 KO mice showed more severe inflammatory arthritis and aggravated pathological findings than control mice. These effects were paralleled by increases in IL-1, TNF-α, TRAP-positive osteoclasts, and F4/80⁺ macrophages in the ankles of mSIRT1 KO mice. In addition, BMMs from mSIRT1 KO mice displayed hyperacetylated p65 and increased NF-κB binding activity when compared to control mice, which resulted in increased M1 polarization, migration, pro-inflammatory cytokine production, and osteoclastogenesis.

Conclusion: Our study provides in vivo evidence that myeloid cell-specific deletion of SIRT1 exacerbates inflammatory arthritis via the hyperactivation of NF-κB signaling, which suggests that SIRT1 activation may be beneficial in the treatment of inflammatory arthritis.

Figure 1. Myeloid cell-specific deletion of SIRT1.

(A) Breeding strategy. (B) PCR genotyping. PCR analysis of several tissues from a SIRT1^loxP/loxP;LysM-Cre^+/+ mouse was performed using 3 primers designed against sequences upstream and downstream of the loxP-flanked sirt1 exon 4: 5′ KO primer, 5′-GGTTGACTTAGGTCTTGTCTG; 5′ KO primer, 5′-AGGCGGATTTCTGAGTTCGA; and 3′ common primer, 5′-CGTCCCTTGTAATGTTTCCC. The use of these primers enabled discrimination between the loxP-flanked (upper band) and the Cre-deleted (lower band) Sirt1 alleles. (C) Western blot analysis for SIRT1 in peritoneal macrophages (PM), BMMs, osteoclasts, and various tissues from SIRT1^loxP/loxP; LysM-Cre^+/+ mice.

Figure 2. Myeloid cell-specific SIRT1 deficiency aggravates synovial inflammation and bone destruction in K/BxN serum transfer arthritis.

Male (8- to 10-week-old) mSIRT1 KO and WT mice were injected with K/BxN serum (50 µL) intraperitoneally on days 0 and 2. (A) The severity of arthritis was defined according to semi-quantitative clinical scoring and the change in ankle thickness. (B) ELISA results for inflammatory cytokines levels in tissue extracts from mouse ankles. (C) Representative sections of the ankle joints stained with H&E, TRAP, and F4/80. The original magnifications are ×100, ×400, and ×100, respectively. (D) Mean pathologic scores for synovial inflammation and bone erosion. (E) F4/80⁺ macrophages in ankle joints and (F) the numbers of TRAP-positive cells. The results of two independent experiments with 10 mice per group are expressed as mean ± SEM, *p<0.05, **p<0.01 vs. WT.

Figure 3. SIRT1 deficiency increases the migration, cytokine release, and osteoclastogenesis of BMMs.

(A) Regulation of macrophage polarization by SIRT1. SIRT1-deficient or WT BMMs were treated with either 50 U/ml of IFN-γ (for M1 polarization) or 10 ng/ml of IL-4 (for M2 polarization) for 48 h. (B) Effect of recombinant MCP-1 on the migration of BMMs. (C) Increased release of IL-1β from SIRT1-deficient BMMs following 12-h TNF-α treatment. (D) Microscopic view of TRAP-positive cells from BMM precursors cultured with M-CSF and RANKL. Original magnification, ×100 (E) The TRAP-positive multinucleated osteoclasts in each well were counted. Values  =  mean ± SE, n = 4–5 mice for each group, *p<0.05 and ^** p<0.01 vs. WT.

Figure 4. SIRT1 deficiency induces hyperactivation of NF-κB in BMMs.

BMMs were cultured from mSIRT1 or WT mice and stimulated with TNF-α (10 ng/ml) for 1 h. (A) Immunostaining for acetylated p65 showed that loss of SIRT1 resulted in higher and sustained levels of acetylated p65 in the nuclei of macrophages following TNF-α treatment. (B) The levels of acetylated p65 were analyzed by Western blotting. Representative blot shown is from one of the three independent experiments with similar results. Values  =  mean ± SE, *p<0.05 vs. WT. (C) The DNA binding activity of NF-κB was analyzed by EMSA.