Aryl hydrocarbon receptor deficiency in T cells suppresses the development of collagen-induced arthritis

Abstract

The contributions of aryl hydrocarbon receptor (Ahr) to the pathogenesis of rheumatoid arthritis have not been elucidated. Here, we show that Ahr deficiency ameliorated collagen-induced arthritis, a mouse model of RA. Collagen-immunized Ahr KO mice showed decreased serum levels of such proinflammatory cytokines as IL-1β and IL-6. The Th17 and Th1 cell populations in lymph nodes from these mice decreased and increased, respectively, whereas the percentage of regulatory T cells was unchanged. Interestingly, a lack of Ahr specifically in T cells significantly suppressed collagen-induced arthritis development, whereas Ahr deficiency in macrophages had no effect. These finding indicate that the development of experimental autoimmune arthritis depends on the presence of Ahr in T cells, and that Th1/Th17 balance may be particularly important for this process.

Fig. 1.

Ahr promotes CIA development. Mice were immunized with chicken type II collagen emulsified with CFA. Intradermal injections were made at several sites in the base of the tail on days 0 and 21. The mean clinical score (A), CIA incidence (B), and maximum clinical score (C) were recorded until day 60. Data are presented as the mean clinical and maximum scores ± SEM (WT, n = 14; Ahr KO, n = 12). P values were obtained with Student t tests. (D) Hind joints obtained 14 d after the second immunization. (E) Sera were collected from collagen-immunized mice on day 60 and serum levels of collagen-specific IgG were determined by ELISA (WT, n = 12; Ahr KO, n = 11; *P < 0.05; **P < 0.01).

Fig. 2.

Ahr deficiency inhibits CIA-induced joint inflammation. (A and B) Representative sections of ankle joints from WT (a and b) and Ahr KO (c and d) mice 60 d after the second immunization were stained with H&E (A) or Safranin O (B). (a) Severe pathologic features were observed in the WT ankle joint, including synovial hyperplasia (black arrowheads) and infiltrating cells (M, macrophages; N, neutrophils). (b) Pannus formation (black arrowhead) and cartilage destruction (yellow arrowhead) were also observed in the WT sample. (c and d) Ankle joints from Ahr KO mice appeared normal. (C) Fourteen days after the second immunization, sera were collected and MMP-3 levels were measured by using a specific ELISA (WT, n = 8; Ahr KO, n = 6; **P < 0.01).

Fig. 3.

Ahr deficiency inhibits Th17 cell differentiation in response to collagen immunization. (A and B) Fourteen days after the second immunization, sera were collected and levels of the proinflammatory cytokines IL-1β (A) and IL-6 (B) were measured by using specific ELISAs (WT, n = 8; Ahr KO, n = 6). (C) CD4⁺ T cells selected from inguinal lymph node cells by magnetic-activated cell sorting (MACS) obtained from mice 14 d after the second immunization were counted after intracellular staining of IFN-γ, IL-17, or Foxp3 (WT, n = 3; Ahr KO, n = 4; *P < 0.05; **P < 0.01).

Fig. 4.

CIA is significantly suppressed in Lck-Cre Ahr^flox/flox mice. Ahr^flox/flox and Lck-Cre Ahr^flox/flox mice were immunized with collagen. Mice were assessed for the clinical arthritis score (A), disease incidence (B), and maximum CIA score (C) until day 60. Data are presented as the mean clinical and maximum scores ± SEM (Ahr^flox/flox, n = 12; Lck-Cre Ahr^flox/flox, n = 11). Fourteen days after the second immunization, sera were collected and levels of the proinflammatory cytokines IL-1β (D) and IL-6 (E) were measured using specific ELISAs (Ahr^flox/flox, n = 6; Lck-Cre Ahr^flox/flox, n = 6). (F) CD4⁺ T cells selected from inguinal lymph node cells by MACS obtained from mice 14 d after the second immunization were counted after IFN-γ, IL-17, or Foxp3 were intracellularly stained (Ahr^flox/flox, n = 7; Lck-Cre Ahr^flox/flox, n = 7; *P < 0.05; **P < 0.01).

Fig. 5.

CIA is not suppressed in LysM-Cre Ahr^flox/flox mice. CIA was induced in Ahr^flox/flox and LysM-Cre Ahr^flox/flox mice as described in Fig. 1. The clinical arthritis score (A), disease incidence (B), and maximum CIA score (C) were recorded until day 60. Data are presented as the mean clinical and maximum scores ± SEM (Ahr^flox/flox, n = 10; LysM-Cre Ahr^flox/flox, n = 17). Fourteen days after the second immunization, sera were collected and levels of the proinflammatory cytokines IL-1β (D) and IL-6 (E) were measured by using specific ELISAs (Ahr^flox/flox, n = 5; LysM-Cre Ahr^flox/flox, n = 5). (F) CD4⁺ T cells selected from inguinal lymph node cells by MACS obtained from mice 14 d after the second immunization were counted after intracellular staining of IFN-γ, IL-17, or Foxp3 (Ahr^flox/flox, n = 4; LysM-Cre Ahr^flox/flox, n = 4).