Steroidogenic enzyme Cyp11a1 regulates Type 2 CD8+ T cell skewing in allergic lung disease

Abstract

Allergic asthma is a heterogeneous inflammatory disorder of the airways characterized by chronic airway inflammation and airway hyperresponsiveness. Numbers of CD8(+)IL-13(+) T cells are increased in asthmatics and during the development of experimental asthma in mice. In an atopic environment rich in IL-4, these CD8(+) T cells mediate asthmatic responses, but the mechanisms regulating the conversion of CD8(+) effector T cells from IFN-γ- to pathogenic IL-13-producing effector cells that contribute to an asthma phenotype have not been defined. Here, we show that cholesterol side-chain cleavage P450 enzyme, Cyp11a1, is a key regulator of CD8(+) T-cell conversion. Expression of the gene, protein, and enzymatic activity of Cyp11a1 were markedly increased in CD8(+) T cells differentiated in the presence of IL-2 plus IL-4 compared with cells differentiated in IL-2 alone. Inhibition of Cyp11a1 enzymatic activity with aminoglutethimide or reduction in the expression of Cyp11a1 using short hairpin RNA prevented the IL-4-induced conversion of IFN-γ- to IL-13-producing cells without affecting expression of the lineage-specific transcription factors T-box expressed in T cells (T-bet) or GATA binding protein 3 (GATA3). Adoptive transfer of aminoglutethimide-treated CD8(+) T cells into sensitized and challenged CD8-deficient recipients failed to restore airway hyperresponsiveness and inflammation. We demonstrate that Cyp11a1 controls the phenotypic conversion of CD8(+) T cells from IFN-γ to IL-13 production, linking steroidogenesis in CD8(+) T cells, a nonclassical steroidogenic tissue, to a proallergic differentiation pathway.

Fig. 1.

Cyp11a1 expression in CD8⁺ T cells generated in the presence of IL-2 or IL-2+IL-4. (A) Protocol for differentiation of CD8⁺ T cells in IL-2 or IL-2+IL-4 in vitro. (B) Cyp11a1 mRNA expression as detected by quantitative RT-PCR in CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4. (C) Cyp11a1 protein levels as detected by immunoblot analysis and densitometry of autoradiographs in CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4. (D) Immunohistochemical staining for Cyp11a1 in CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 in vitro. (Magnification: ×200.) Quantitative analysis was performed by counting Cyp11a1-positive cells under the microscope. Data (means ± SEM) are from at least three independent experiments. **P < 0.01 compared with the IL-2 group.

Fig. 2.

Cyp11a1 enzymatic activity regulates the functional conversion of CD8⁺ T cells from IFN-γ– to IL-13–producing cells. (A) Pregnenolone levels determined by ELISA in supernatants from CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 in the presence or absence of AMG (500 μM). **P < 0.01 compared with the IL-2 group; ^##P < 0.01 compared with the IL-2+IL-4 group. (B) Cyp11a1 protein levels detected by immunoblot analysis and densitometry of autoradiographs in CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 with 500 μM AMG. **P < 0.01 compared with the IL-2 group; ^##P < 0.01 compared with the IL-2 plus SIINFEKL group. (C) Flow-cytometric analysis of cytokine expression in CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 and treatment with different concentrations of AMG. Data are from at least seven independent experiments.

Fig. 3.

shRNA specific for Cyp11a1 prevents the conversion of CD8⁺ T cells from IFN-γ– to IL-13–producing cells. (A) Representative flow-cytometric analysis of Cyp11a1 expression after transfection with plasmids encoding a Cyp11a1 shRNA or a scrambled control shRNA. For quantitative analysis of Cyp11a1-positive cells, data are from at least four independent experiments. **P < 0.01 compared with scramble shRNA group. (B) Representative flow-cytometric analysis of cytokine expression in CD8⁺ T cells after transfection. For quantitative analysis of Cyp11a1-positive cells, data are from at least three independent experiments. **P < 0.01 compared with scramble shRNA group.

Fig. 4.

Lineage-specific transcription factor expression in CD8⁺ T cells. T-bet and GATA3 expression was detected by quantitative RT-PCR in CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 with or without SIINFEKL in the presence or absence of 500 μM AMG. Data (means ± SEM) are from at least eight independent experiments. **P < 0.01 compared with the IL-2 group; ^##P < 0.01 compared with the IL-2 plus SIINFEKL group.

Fig. 5.

Treatment of CD8-deficient recipients with CD8⁺ T cells differentiated in IL-2 and AMG (500 μM) fails to restore AHR and inflammation. (A) Experimental protocol. (B) Changes in RL. (C) Cell composition in BAL fluid. (D) Cytokine levels in BAL fluid. (E) Representative photomicrographs of lung histology. (Magnification: ×200.) Quantitative analysis of goblet cells was as described in Materials and Methods. (F) Number of Cyp11a1-positive cells in the lung. Data (means ± SEM) were from at least 6–10 mice. *P < 0.05 and **P < 0.01 compared with secondary challenged CD8-deficient recipients; ^#P < 0.05 and ^##P < 0.01 compared with secondary challenged CD8-deficient recipients of 5 × 10⁶ IL-2differentiated CD8⁺ T cells. 2i.p., two intraperitoneal injections; 4 nebs, four nebulized allergen challenges.