Promotion of Inflammatory Arthritis by Interferon Regulatory Factor 5 in a Mouse Model

Abstract

Objective: Polymorphisms in the transcription factor interferon regulatory factor 5 (IRF5) are associated with an increased risk of developing rheumatoid arthritis (RA). This study was undertaken to determine the role of IRF5 in a mouse model of arthritis development.

Methods: K/BxN serum-transfer arthritis was induced in mice deficient in IRF5, or lacking IRF5 only in myeloid cells, and arthritis severity was evaluated. K/BxN arthritis was also induced in mice deficient in TRIF, Toll-like receptor 2 (TLR2), TLR3, TLR4, and TLR7 to determine the pathways through which IRF5 might promote arthritis. In vitro studies were performed to determine the role of IRF5 in interleukin-1 (IL-1) receptor and TLR signaling.

Results: Arthritis severity was reduced in IRF5-deficient, TRIF-deficient, TLR3-deficient, and TLR7-deficient mice. The expression of multiple genes regulating neutrophil recruitment or function and bioactive IL-1β formation was reduced in the joints during active arthritis in IRF5-deficient mice. In vitro studies showed that TLR7 and the TRIF-dependent TLR3 pathway induce proinflammatory cytokine production in disease-relevant cell types in an IRF5-dependent manner.

Conclusion: Our findings indicate that IRF5 contributes to disease pathogenesis in inflammatory arthritis. This is likely due at least in part to the role of IRF5 in mediating proinflammatory cytokine production downstream of TLR7 and TLR3. Since TLR7 and TLR3 are both RNA-sensing TLRs, this suggests that endogenous RNA ligands present in the inflamed joint promote arthritis development. These findings may be relevant to human RA, since RNA capable of activating TLR7 and TLR3 is present in synovial fluid and TLR7 and TLR3 are up-regulated in the joints of RA patients.

Figure 1

Arthritis is ameliorated in IRF5-deficient mice following transfer of K/BxN serum. C57BL/6 wildtype (WT; n = 10) and IRF5-deficient (IRF5^−/−; n = 10) mice were injected intraperitoneally (i.p) with 150 μl of K/BxN serum on day 0 and day 2. (A) Change in ankle thickness (left) and clinical score (right) were measured on days 0, 2, 4 and 7. (B) Ankles (2 per mouse) isolated on day 7 after serum transfer were prepared as described in Materials and Methods, after which ankle sections were scored for various histologic features (left panel). Representative images are shown in the panels on the right; B, bone C, cartilage S, synovium ; long arrow indicates a bone erosion and arrowhead indicates inflammatory cells. Data are expressed as mean ± SEM. * = P < 0.05; ** = P < 0. 005; *** = P < 0. 0001.

Figure 2

IRF5 deficiency does not impair IL-1 receptor signaling, but IRF5-deficient mice have reduced serum IL-1β and chemokine levels after K/BxN serum transfer. (A) Mouse embryonic fibroblasts (MEFs), (B) M-CSF bone marrow-derived macrophages and (C) synovial fibroblasts from C57BL/6 wildtype (WT) and IRF5-deficient (IRF5^−/−) mice were stimulated, or not stimulated (media), with TNF-α (10 ng/ml) and IL-1β (10 ng/ml). Supernatants were collected after 24 h and IL-6 and KC measured by ELISA and nitric oxide (NO) measured by the Griess method. Data represent mean ± SEM of 5 independent experiments (A and C) and 4 independent experiments (B). (D) Serum cytokines were measured on day 7 after K/BxN serum injection in the C57BL/6 WT (n = 10) and IRF5^−/− (n = 10) mice shown in Figure 1. Data represent mean ± SEM. * = P < 0.05; ** = P < 0. 01; *** = P < 0. 001. (E) C57BL/6 WT ( n = 8) and IFNAR1-deficient (IFNAR1^−/−(n = 7)) mice were injected i.p. with 150 μl of K/BxN serum on day 0 and day 2. Change in ankle thickness (left) and clinical score (right) were measured on days 0, 2, 4 and 7.

Figure 3

TLR7-deficient ,TRIF-mutant and TLR3-deficient mice develop less severe arthritis following transfer of K/BxN serum. (A) C57BL/6 wildtype (WT; n = 10), TLR7-deficient (TLR7^−/−; n = 10), TRIF-mutant (TRIF^Lps2/Lps2; n = 10), and TLR2 and 4 double-deficient (TLR2^−/−4^−/−; n = 5) mice were injected i.p with 150 μl of K/BxN serum on day 0 and day 2. Change in ankle thickness (left) and clinical score (right) were measured on days 0, 2, 4 and 7. (B) C57BL/6 wildtype (WT; n = 10) and TLR3-deficient (TLR3^−/−; n =8) mice were injected i.p with 75 μl of K/BxN serum on day 0. Change in ankle thickness (left) and clinical score (right) were measured on days 0, 2, 4, 7, 9, 11 and 14. (C) IRF5 expression levels were measured by RT-PCR in wrist joints isolated 7 days after K/BxN serum administration from C57BL/6 wildtype (WT; n = 4), TLR7-deficient (TLR7^−/−; n = 5) and TRIF-mutant (TRIF^Lps2/Lps2; n = 5) mice described in (A). * = P < 0.05; ** = P < 0.01; *** = P < 0. 001 versus WT.

Figure 4

TLR7 and TLR3 promote inflammatory cytokine and chemokine production through IRF5 and TLR3 synergizes with TLR7 to enhance these effects. (A) Synovial fibroblasts from C57BL/6 WT and IRF5^−/− mice were stimulated, or not stimulated (media), with the TLR2 ligand Pam3Cys (100 ng/ml), the TLR3 ligand Poly(I:C) (10 ug/ml), the TLR4 ligand LPS (100 ng/ml) and the TLR7 ligand R837 (1 ug/ml). Supernatants were collected after 24 h and KC and IL-6 measured by ELISA. Data represent mean ± SEM of 5 independent experiments. Bone marrow-derived M-CSF macrophages (B) and GM-CSF macrophages (C) from C57BL/6 WT and IRF5^−/− mice were stimulated, or not stimulated (media), with Pam3Cys (100 ng/ml), Poly(I:C) (10 ug/ml), LPS (100 ng/ml), R837 (0.3 ug/ml) or R837 (0.3 ug/ml) plus Poly(I:C) (10 ug /ml). On the TNF-α plate, supernatants were collected 24 h after the addition of stimuli. On the IL-1β plate, nigericin (40 μM) was added 16 h after addition of the stimuli and supernatants collected 2 h thereafter. TNF-α and IL-1β were measured by ELISA. Data represent mean ± SEM of 4 independent experiments (B) and 5 independent experiments (C). * = P < 0.05.

Figure 5

Mice deficient in IRF5 or TLR7 develop less severe arthritis but no additional protection is seen in mice deficient in both TLR7 and TLR3. C57BL/6 wildtype (WT; n = 20), IRF5-deficient (IRF5^−/−; n = 12), TLR7-deficient (TLR7^−/−; n = 13), and TLR3 and 7 double-deficient (TLR3^−/−7^−/−; n = 12) mice were injected i.p with 150 μl of K/BxN serum on day 0 and day 2. Change in ankle thickness (A) and clinical score (B) were measured on days 0, 2, 4, 7, 9, 11 and 15. The IRF5^−/− data are shown in the left hand panels and the TLR7^−/− and TLR3^−/−7^−/− data are shown in the right hand panels for clarity. There were no statistically significant differences between the IRF5^−/−, TLR7^−/− and the TLR3^−/−7^−/− cohorts except for day 15 where the change in ankle thickness and clinical score were lower in the IRF5^−/− cohort than in the other 2 cohorts (P < 0.05). (C) IRF5^flox/flox mice (wild-type; n = 5) and LyzM^cre IRF5^flox/flox mice (n = 5) were injected i.p with 75 μl of K/BxN serum on day 0. * = P < 0.05; ** = P < 0.01; *** = P < 0. 001 versus WT.

Figure 6

Gene expression in the joints of wildtype and IRF5-deficient mice during active arthritis. Gene expression was analyzed using Nanostring technology on RNA isolated from the joints of C57BL/6 wildtype (WT; n = 4) and IRF5-deficient (IRF5^−/−; n = 5) mice 7 days after the i.p. injection of 150 μl of K/BxN serum on day 0 and day 2. Genes that differed between WT and IRF5^−/− mice using a threshold statistical significance of p ≤ 0.02 are shown. Samples are arranged by hierarchical clustering and are displayed as a heat map.