Transcription factor T-bet regulates skin sclerosis through its function in innate immunity and via IL-13

Abstract

Tissue remodeling with fibrosis is a predominant pathophysiological mechanism of many human diseases. Systemic sclerosis is a rare, often lethal, disorder of unknown etiology manifested by dermal fibrosis (scleroderma) and excessive connective tissue deposition in internal organs. Currently, there are no available antifibrotic therapeutics, a reflection of our lack of understanding of this process. Animal models of scleroderma are useful tools to dissect the transcription factors and cytokines that govern fibrosis. A disproportionate increase of type 2 cytokines, like TGF-beta and IL-4, more than type 1 cytokines, like IFN-gamma, is thought to underlie the pathogenesis of scleroderma. In this study, we show that mice deficient in the transcription factor T-box expressed in T cells (T-bet), a master regulator of type 1 immunity, display increased sensitivity to bleomycin-induced dermal sclerosis. Despite the well-established role of T-bet in adaptive immunity, we also show that RAG2(-/-) mice, which lack T and B cells, are vulnerable to bleomycin-induced scleroderma and that RAG2/T-bet double-deficient mice maintain the increased sensitivity to bleomycin observed in T-bet(-/-) mice. Furthermore, overexpression of T-bet in T cells does not affect the induction of skin sclerosis in this model. Lastly, we show that IL-13 is the profibrotic cytokine regulated by T-bet in this model. Together, we conclude that T-bet serves as a repressor of dermal sclerosis through an IL-13-dependent pathway in innate immune cells. T-bet, and its transcriptional network, represent an attractive target for the treatment of systemic sclerosis and other fibrosing disorders.

Fig. 1.

Bleomycin-induced skin sclerosis does not require the adaptive immune system. BALB/c (WT) or RAG2^−/− mice on BALB/c background (RAG2 KO) received s.c. injections of PBS (□) or bleomycin (■) for 4 weeks to induce skin sclerosis. The degree of collagen deposition was measured by a hydroxyproline assay. Statistically significant increases were observed in the hydroxyproline content in the skin of bleomycin-treated WT and RAG2^−/− mice compared with PBS-treated control mice (P = 0.020 and 0.019, respectively). No statistical difference was observed in the hydroxyproline content in the skin of bleomycin-treated WT or RAG2^−/− mice (∗, P > 0.05).

Fig. 2.

Histopathological analysis of bleomycin-treated T-bet KO mice reveals increased dermal sclerosis compared with WT mice. Skin sections from BALB/c (WT) and T-bet^−/− (Tbet KO) mice injected daily for 4 weeks with bleomycin or PBS were stained with H&E (A–D) or Masson trichrome (E–H). Note the increased skin thickness and more robust deposition of collagen observed in bleomycin-treated TbetKO mice compared with similarly treated WT mice.

Fig. 3.

The transcription factor T-bet functions within the innate immune system to augment bleomycin-induced skin sclerosis. (A) BALB/c (WT), T-bet^−/− mice (Tbet KO), and RAG2^−/− × T-bet^−/− DKO mice (Tbet/RAG2 DKO) received s.c. injections of PBS (□) or bleomycin (■) for 4 weeks to induce skin sclerosis. The degree of collagen deposition was measured by a hydroxyproline assay (∗, P < 0.002; ∗∗, P < 0.05). The results are representative of four independent experiments. (B) BALB/c (WT) and transgenic mice that overexpress T-bet under the control of the CD2 promoter were treated with bleomycin and analyzed as in (A) (∗, P > 0.05). The data represent the average values of each group of mice (n = 3–6) with standard deviation. Asterisks denote statistical significance. The results are representative of two independent experiments.

Fig. 4.

T-bet regulate dermal sclerosis through IL-13. Seven- to 8-week-old BALB/c (WT), IL-13^−/− mice on BALB/c background (IL-13 KO), and T-bet^−/− × IL-13^−/− mice (Tbet/IL-13 DKO) received s.c. bleomycin injections for 4 weeks to induce skin sclerosis. The degree of collagen deposition was measured by a hydroxyproline assay (∗, P < 0.05; ∗∗, P < 0.05). The data represent the average values of each group of mice (n = 3–5) with standard deviation. Asterisks denote statistical significance. The results shown are representative of three separate experiments.