TGFβ1 signaling sustains aryl hydrocarbon receptor (AHR) expression and restrains the pathogenic potential of TH17 cells by an AHR-independent mechanism

Abstract

The aryl hydrocarbon receptor (AHR) is a transcription factor activated by ligand highly expressed on T_H17 cells, and AHR-deficient CD4⁺ T cells have impaired production of IL-17A and IL-22. Although AHR activation can exacerbate in vivo T_H17 cell-mediated autoimmunity, accumulating data indicate that AHR is a nonpathogenic T_H17 marker. Thus it remains unclear how AHR activation is regulated and impacts on the generation of T_H17 subsets. Here we demonstrated that AHR pathway is activated during in vitro pathogenic T_H17 polarization, but it is quickly downregulated. Under these conditions, additional AHR activation promoted IL-22 but not IL-17A. Interestingly, AHR high sustained expression and IL-17A promotion were only achieved when TGFβ1 was present in the culture. In addition to the effect on AHR regulation, TGFβ1 presented a dual role by simultaneously suppressing the T_H17 pathogenic phenotype acquisition. This latter effect was independent of AHR stimulation, since its activation did not confer a T_H17 anti-inflammatory profile and Ahr^-/- cells did not upregulate any T_H17 pathogenic marker. Through the use of EAE model, we demonstrated that AHR is still functional in encephalitogenic CD4⁺ T cells and the adoptive transfer of Ahr^-/- T_H17 cells to recipient mice resulted in milder EAE development when compared to their WT counterparts. Altogether, our data demonstrated that although AHR is highly expressed on in vitro-generated nonpathogenic T_H17 cells, its ligation does not shift T_H17 cells to an anti-inflammatory phenotype. Further studies investigating the role of AHR beyond T_H17 differentiation may provide a useful understanding of the physiopathology of autoimmune diseases.

Fig. 1. AHR is activated during in vitro pathogenic T_H17 cell differentiation, and it regulates IL-17A production in a TGFβ1-dependent manner.

a Heatmap of Ahr, Cyp1a1, Ahrr, Il22, and Il17a mRNA expression in CD4⁺CD44^loCD62L^hi naive T cells differentiated for 12, 24, 36, 48, 60, and 72 h under nonpathogenic (TGFβ1 plus IL-6) and pathogenic (IL-1β, IL-6, and IL-23) T_H17 conditions. b qPCR analysis of Ahr, Cyp1a1, and Ahrr mRNA expression of naive CD4⁺ T cells (white bars) activated 24 h under different combinations of IL-6, TGFβ1, IL-1β, and IL-23 (as indicated). c Frequency of IL-17A⁺ and IL-22⁺ cells from pathogenic T_H17 cells differentiated with TGFβ1, IL-6 and IL-23 (pT_H17 (TGFβ1)) or IL-1β, IL-6 plus IL-23 (pT_H17 (IL-1β)) in the presence of FICZ. d Frequency of IL-17A⁺ and IL-22⁺ cells from pT_H17 cells (TGFβ1, IL-6, and IL-23) differentiated under different concentrations of TGFβ1. b P < 0.05 when compared to ^amedium; ^bIL-6; ^cTGFβ1, and ^dTGFβ1+IL-6 groups (two-way ANOVA). NS not significant; *P < 0.05, *P < 0.01, and ***P < 0.001 (c, unpaired, two-tailed Student’s t test and d two-way ANOVA). Data are representative of more than three independent experiments with similar results

Fig. 2. TGFβ1 signaling maintains Ahr expression in T_H17 cells, while it opposes IL-23-driven conversion into pathogenic T_H17 cells.

a Naive CD4⁺ T cells were cultured 3 days under nonpathogenic T_H17-polarizing conditions (TGFβ1+IL-6) in the absence or presence of FICZ and then stained intracellularly for IL-17A and IL-22 (first culture). A fraction of recovered cells cultured without FICZ was re-stimulated for additional 3 days with TGFβ1, IL-6 plus IL-23, or only IL-23, with or without FICZ stimulation (second culture). b Frequencies of IL-17A⁺ and IL-22⁺ cells after the second culture shown in a. c qPCR analysis of Ahr, Cyp1a1, Ahrr, Il17a, and Tbx21 of naive CD4⁺ T cells cultured under nonpathogenic T_H17-polarizing conditions (TGFβ1+IL-6) for 3 days (first culture, white bars) and re-stimulated with TGFβ1, IL-6, and IL-23 combinations for 24 h (second culture). NS not significant; *P < 0.05, **P < 0.01, and ***P < 0.001 (two-way ANOVA)

Fig. 3. AHR signaling does not drive anti-inflammatory properties to T_H17 cells.

Quantitative RT-PCR analysis of Rorc, Tbx21, Maf, Il17a, Il22, Csf2, and l10 from WT (white bars) or Ahr-deficient (green bars) cells differentiated for 72 h under nonpathogenic (TGFβ1 and IL-6) and pathogenic T_H17 cell conditions (TGFβ1, IL-6 plus IL-23 or IL-1β, IL-6 and IL-23). b Frequency of IL-17A⁺, IL-10⁺, and GM-CSF⁺ cells from WT and Ahr^−/− naive CD4⁺ T cells differentiated for 72 h with TGFβ1+IL-6+IL-23 under FICZ stimulation. NS not significant; *P < 0.05 and ***P < 0.001 (two-way ANOVA)

Fig. 4. AHR is still highly expressed and functional on CNS-infiltrating pathogenic CD4⁺ T cells.

a qPCR analysis of Ahr, Cyp1a1, Il17a, Tbx21, Rorc, and Il22 transcripts of CD4⁺ T cells isolated from draining lymph nodes (dLNs) after 3, 6, or 14 days after EAE induction. Spinal cord (SC) CD4⁺ T cells were isolated 14 days after MOG immunization. b IL-22 production of purified dLNs cells from WT mice 7 and 14 days after EAE induction and CNS-infiltrating cells 14 days after MOG-CFA immunization re-stimulated for 72 h with MOG (50 µg/mL) with or without FICZ (300 nM). NS not significant; *P < 0.05 (unpaired, two-tailed Student’s t test)

Fig. 5. AHR activation promotes effector functions to encephalitogenic T_H17 cells.

a EAE clinical score of WT mice treated with AHR antagonist for 10 consecutive days (CH223191, i.p. 10 mg/kg). b Representative plots of IL-17A⁺, IFN-γ, and GM-CSF⁺ CNS-derived CD4⁺ T cells from vehicle and CH223191-treated EAE animals. c Frequency of cytokine-producer cells shown in b. d EAE clinical score of Rag1^−/− mice recipients of WT or Ahr^−/− MOG-specific T_H17 cells. NS not significant; ***P < 0.001 (unpaired, two-tailed Student’s t test)