Fig. 4: GHRH-R is required for Th17 cell differentiation in vitro.

a–d Naïve CD4⁺ T cells from WT and Ghrhr^lit/lit mice were cultured in Th0, Th1, Th2, Th17, or iTreg polarizing conditions for 3 days (n = 8) and evaluated by flow cytometry for the expression of cytokines IFN-γ, IL-4, and IL-17A for Th1, Th2, and Th17, respectively; and transcription factor T-bet, GATA3, RORγt and Foxp3 for Th1, Th2, Th17, and iTreg respectively. MFI mean fluorescence intensity. The exact p value (Th17) was 2.09 × 10⁻⁸. d The F value of the one-way ANOVA test is 55.46, and the corresponding p value is less than 0.0001. e Naïve CD4⁺ T cells from WT and Ghrhr^lit/lit mice were cultured for 3 days in non-pathogenic Th17 polarizing condition (IL-6 and TGF-β), or pathogenic Th17 polarizing condition: (1) IL-6, TGF-β, and IL-23; (2) IL-6, IL-1β, and IL-23. f Frequency of IL-17A⁺CD4⁺ T cells was determined by flow cytometry (n = 6). The F value of the one-way ANOVA test is 67.21, and the corresponding p value is less than 0.0001. The exact p values (IL-6 + TGFβ WT vs Ghrhr^lit/lit and IL-6 + TGFβ + IL23 WT vs Ghrhr^lit/lit) were 2.03 × 10⁻¹¹ and 4.54 × 10⁻⁷ respectively. g, h Naïve CD4⁺ T cells from WT and Ghrhr^lit/lit were cultured in non-pathogenic (IL-6 and TGF-β) or pathogenic Th17 cell differentiation condition (IL-6, IL-1β, and IL-23) for 3 days. Expression of Gp130, Il1r1, Il21r, and Il23r was quantified and normalized to Gapdh (n = 6). Fold change is relative to the Ghrhr^lit/lit group. Data were the representation of at least two independent experiments. Data were presented as mean ± SEM. Two-sided student’s t-test with Bonferroni correction (b, g, h) and one-way ANOVA followed by Bonferroni post hoc test (d, f). *, ***, and **** represent \(\widetilde{{{{{{\rm{P}}}}}}}\) < 0.05, \(\widetilde{{{{{{\rm{P}}}}}}}\) < 0.001, and \(\widetilde{{{{{{\rm{P}}}}}}}\) < 0.0001 respectively. ns represents no significant difference.