Fig. 5: GHRH-R signaling promotes Th17 cell differentiation via the JAK-STAT3 pathway.

a, b Naïve CD4⁺ T cells from WT and Ghrhr^lit/lit mice were cultured in Th17 polarizing condition for 3 days. a 10 μg/ml of anti-GHRH, anti-GH or anti-IGF-1 antibodies were added to cells for 3 days. The frequency of IL-17A⁺CD4⁺ T cells was determined by flow cytometry (n = 6 independent experiments). The F value of one-way ANOVA test is 36.15, and the corresponding p value is less than 0.0001. The exact p values (IL-6 + TGFβ WT vs Ghrhr^lit/lit, anti-GH WT vs Ghrhr^lit/lit, and anti-IGF-1 WT vs Ghrhr^lit/lit) were 1.43 × 10⁻⁷, 8.24 × 10⁻¹¹, and 7.07 × 10⁻⁹ respectively. b DMSO, hGHRH, MR-409, or MIA-602 were added to cells for 3 days. The frequency of IL-17A⁺CD4⁺ T cells was determined by flow cytometry (n = 6 independent experiments). The F value of the one-way ANOVA test is 79.74, and the corresponding p value is less than 0.0001. The exact p values (DMSO WT vs Ghrhr^lit/lit, hGHRH WT vs Ghrhr^lit/lit, and MR-409 WT vs Ghrhr^lit/lit) were 2.35 × 10⁻⁷, 1.63 × 10⁻¹², and 4.71 × 10⁻¹³, respectively. c Naïve CD4⁺ T cells from WT and Ghrhr^lit/lit mice were activated for 3 days in Th17 polarizing condition, washed, and serum-starved for 4 h and then re-stimulated with MR-409 for 60 min. Expression of Il17a, Il17f, Il22, Il23r, Rorc, Rora, Hif1a, Il10, Irf4, and Batf was quantified (n = 8 independent experiments). Data were normalized to Gapdh and fold change is relative to Ghrhr^lit/lit. d Naïve CD4⁺ T cells from WT and Ghrhr^lit/lit mice were labeled with CellTrace dye (CFSE). Cells were cultured in Th17 polarizing condition with or without MR-409 for 3 days (n = 6 independent experiments). e Then cells were treated with or without ruxolitinib (1 μM) for 4 h. MR-409 and MIA-602 were then added to cells for 30 min. Expression of phosphorylation of STAT3 Y705 in CD4⁺IL-17⁺ T cells was quantified (n = 6 independent experiments). The F value of the one-way ANOVA test is 17.77, and the corresponding p value is less than 0.0001. f MR-409 was treated to cells for 0, 15, 30, and 60 min. Expression of phosphorylation of STAT3 Y705 in CD4⁺IL-17⁺ T cells was quantified (n = 6 independent experiments). The F value of the one-way ANOVA test is 17.38, and the corresponding p value is less than 0.0001. g Naïve CD4⁺ T cells from WT and Ghrhr^lit/lit mice were cultured for 3 days in Th17 polarizing condition in the presence of MR-409 with or without ruxolitinib (1 μM) or Stattic (1 μM) for 3 days. The frequency of IL-17A⁺CD4⁺ T cells was determined by flow cytometry (n = 6 independent experiments). The F value of the one-way ANOVA test is 67.11, and the corresponding p value is less than 0.0001. The exact p values (WT + DMSO vs WT + MR-409, WT + MR-409 vs WT + MR-409+Rux, and WT + MR-409 vs WT + MR-409+Stattic) were 7.62 × 10⁻⁶, 1 × 10⁻¹⁵, and 1 × 10⁻¹⁵ respectively. 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 (c) and one-way ANOVA followed by Bonferroni post hoc test (a, b, e–g). *, **, and **** represent \(\widetilde{{{{{{\rm{P}}}}}}}\) < 0.05, \(\widetilde{{{{{{\rm{P}}}}}}}\) < 0.01, and \(\widetilde{{{{{{\rm{P}}}}}}}\) < 0.0001, respectively. ns represents no significant difference.