Notch signaling drives IL-22 secretion in CD4+ T cells by stimulating the aryl hydrocarbon receptor

Abstract

CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses.

Fig. 1.

Enforced expression of Notch in T cells induces IL-22. (A) Total spleen cells from C57BL/6 mice were stimulated with soluble anti-CD3 mAb (1 μg/mL) for 24 h and transduced with a retrovirus carrying N1ICD, N2ICD, N3ICD, or a control virus. Cells were further stimulated with anti-CD3 mAb (1 μg/mL) for 48 h. Then, the expression of Il22 in MACS-enriched CD4⁺ T cells was analyzed by real-time PCR. (B) Total spleen cells were stimulated with anti-CD3 mAb in Th0, Th1, Th2, and Th17 conditions for 24 h and then transduced with N2ICD or control virus. After 48 h of further culture under the same conditions, Il22 and Il17a expression in MACS-enriched CD4⁺ T cells was measured by real-time PCR. (C) ELISA for the detection of IL-22 secretion from naive CD4⁺ T cells (CD4⁺CD62L⁺) isolated from OT-II TCR transgenic mice stimulated with OVA peptide-pulsed Cont-DC (open) or DL1-DC (filled) for 3 days. (D) ELISA for the detection of IL-22 secretion from naive CD4⁺ T cells isolated from STAT3^F/F or STAT3^F/F-Cre transgenic mice stimulated with allogenic Cont-DC (open) or DL1-DC (filled) prepared from BALB/c mice. *, P < 0.05, indicates a statistically significant difference. Data are representative of at least four independent experiments. N.D., not detected; N.S., not significant.

Fig. 2.

Deficiency of Notch signaling in CD4⁺ T cell impairs IL-22 expression. (A) Naive CD4⁺ T cells isolated from RBP-J^+/+-Cre OT-II (filled) or RBP-J^F/F-Cre OT-II (open) mice were stimulated with OVA peptide-pulsed BMDCs for 3 days. IL-22 concentrations in the supernatants were evaluated by ELISA. (B) CD4⁺ T cells were isolated from RBP-J^+/+-Cre (filled) and RBP-J^F/F-Cre (open) mice 7 days after immunization with OVA emulsified in CFA. As a control, CD4⁺ T cells from unimmunized RBP-J^F/F-Cre mice (dot) were isolated. Il22 expression in CD4⁺ T cells was evaluated by real-time PCR. (C) CFSE-labeled splenic T cells from RBP-J^+/+-Cre OT-II or RBP-J^F/F-Cre OT-II mice were transferred into C57BL/6 Thy1.1 mice that were immunized with OVA protein emulsified in CFA soon after T cell transfer. CFSE dilution was evaluated after gating on CD4⁺ Thy1.2⁺ cells 7 days after OVA immunization. *, P < 0.05, indicates a statistically significant difference. Data in Fig. 2 are representative of at least four independent experiments.

Fig. 3.

Notch deficiency in T cells results in susceptibility to ConA-induced hepatitis. The RBP-J^+/+-Cre or RBP-J^F/F-Cre mice received ConA (10 μg/g) and/or IL-22 (2 μg per mouse). As a control, RBP-J^F/F-Cre mice received PBS. (A) Liver sections 48 h after ConA injection were stained with hematoxylin and eosin. Red arrows indicate bleeding. (B) The ALT and AST levels in the serum of each mouse were measured. (C) CFSE-labeled total spleen cells from RBP-J^+/+-Cre or RBP-J^F/F-Cre mice were transferred into C57BL/6 Thy 1.1 mice, and transferred mice received ConA. Lymphocytes were purified from liver and spleen by Lympholyte M 48 h after ConA injection, and CFSE dilution was plotted after gating on CD4⁺Thy1.2⁺ cells. (D) RBP-J^+/+-Cre or RBP-J^F/F-Cre mice received ConA (10 μg/g). CD4⁺ T cells were purified from livers and spleens 48 h after ConA injection by Lympholyte M and MACS beads and Il22 mRNA was measured by real-time PCR. (E) RBP-J^+/+-Cre and RBP-J^F/F-Cre mice received ConA (10 μg/g) with IL-22 (2 μg) or PBS. Liver sections 48 h after ConA and IL-22 injection were stained with hematoxylin and eosin. Red arrows indicate bleeding, and black arrow indicates cell infiltration. (F) The ALT and AST levels in the serum of each mouse were measured. *, P < 0.05, indicates a statistically significant difference. Data in Fig. 3 are representative of at least four independent experiments.

Fig. 4.

Notch signaling induces AhR stimulators. Total spleen cells were stimulated with anti-CD3 mAb for 24 h and transduced with a retrovirus carrying N2ICD or a control vector. After infection, cells were restimulated with anti-CD3 mAb for 48 h in the presence (filled) or absence (open) of an AhR antagoinst. Then Il22 and Il17a (A) or AhR expression in CD4⁺ T cells (B) was measured by real-time PCR. (C) The expression of Il22 after retroviral transduction of N2ICD (filled or dot) or EV (open) in DO11.10 T cells hybridoma in the presence (filled) or absence (dot) of the AhR antagonist was examined by real-time PCR. (D) The supernatant from total spleen cells transduced with N2ICD (dot or shaded) or EV (open or filled) was collected 72 h after initial stimulation. CD4⁺ T cells from C57BL/6 mice were stimulated with anti-CD3 mAb in the presence of different concentrations of supernatant and in the presence (open, shaded) or absence (filled, dot) of an AhR antagonist. The expression of Il22 (Left) and Cyp1a1 (Right) in mature CD4⁺ T cells was tested 48 h after initial stimulation. (E) The RBP-J^+/+-Cre or RBP-J^F/F-Cre mice received ConA (10 μg/g) and the expression of Cyp1a1 in liver CD4⁺ T cells purified by MACS beads 48 h after ConA injection was measured by real-time PCR. *, P < 0.05, indicates a statistically significant difference. Data in Fig. 4 are representative of at least three independent experiments.