Figure 6

Effect of HFD and immunosuppression with tacrolimus on the peri-conceptional phenotypic frequencies and proportions of circulating maternal CD4⁺IFNγ⁺, CD4⁺IL4⁺ and CD4⁺IL17A⁺ Tregs and alterations to the Th1:Th2 and Th17:Th2 cell ratios in the HFD-dNONcNZO mice. (A–C) Graphic representations of mean ± SDM of percentages (%) of CD4⁺ T cells gated for their fluorescence activated intracellular staining for IFNγ (A), IL4 (B) and IL17A (C) in lymphocytes of HFD-dNONcNZO mice (n = 17), their normative control NFD-NONcNZO (n = 17) and those receiving metformin (n = 17) or tacrolimus (n = 17). (D,E) depict, respectively, ratios of circulating Th1 (CD4⁺IFNγ⁺): Th2 (CD4⁺IL4⁺) (D) and Th17 (CD4⁺IL17A⁺): Th2 (CD4⁺IL4⁺) (E) cells at gd 4.5 among experimental groups. Compared to control values, the Th1:Th2 cell ratio (D) was significantly elevated in the untreated HFD-dNONcNZO mice (mean difference = 0.698, p < 0.05, t = 7.721; 95% confidence interval = 0.438–0.959) vs the tacrolimus- (mean difference = 0.752, p < 0.001, t = 8.304; 95% confidence interval = 0.491–1.012) or the metformin-treated HFD-dNONcNZO mice (mean difference = 0.728, p < 0.001, t = 8.054; 95% confidence interval = 0.468–0.989). Constitutively, the use of both treatment modalities significantly inhibited an aberrant peri-conceptional Th17:Th2 cell ratio (E) in the HFD-dNONcNZO mice (mean difference = 1.147, p < 0.0001, t = 5.855; 95% confidence interval = 0.584–1.712 compared to the tacrolimus-treated mice vs a mean difference of 1.044, p = 0.0001, t = 5.324; 95% confidence interval = 0.479–1.607 in the metformin-treated mice).