Figure 4

Immunosuppression with tacrolimus inhibited an adversary pro-inflammatory ovarian milieu in the HFD-dNONcNZO mice. Using a membrane-based antibody array for the parallel determination of the relative levels of selected mouse ovarian cytokines and chemokines, bar graphs in A–F are representations of mean ± SDM of three replicates measuring the expression of ovarian cytokines and chemokine receptors and their ligands at postcoital/gd 4.5 among mated untreated and treated HFD-dNONcNZO mice. A 1.5-fold change in the protein expression of the cytokine relative to that of the NFD-NONcNZO mice was considered significant (p < 0.01 at 95% confidence, n = 3/phenotype). Of the most upregulated peri-conceptional ovarian cytokines and chemokines in the HFD-dNONcNZO mice which were significantly inhibited using tacrolimus were the pro-inflammatory mediators IL1β and IFNγ (A), IL12p70, IL17, IL23 and IL27 (B), IL2, TNFα and its downstream targets TREM1 (Triggering Receptor Expressed On Myeloid Cells 1) and TARC (CCL17: Thymus and Activation Regulated Cytokine) (C), IL6 (D), GM-CSF and the CXC ligands 9, 10 and 11 (E) and the monocytes-macrophage regulatory chemokine MIP-1α (CCL3) (F). Except for suppressing IL6 (p < 0.01) and a significant trend in downregulating IL4 by use of tacrolimus (mean difference between treated and untreated = 0.91, p = 0.031, t = 2.798; 95% confidence interval = 0.027–1.786), there has been no significant downregulation of the anti-inflammatory cytokines IL10 and IL1r α (p > 0.05) (D). Constitutively, no significant pan-cytokine suppression was observed in the metformin-treated mice apart from a significant downregulation of IFNγ (A), IL12/IL17 family members (B), IL2 (C) and IL6 (D). Due to the gestational age examined and the low dose of tacrolimus (0.1 mg/kg), the monocytes and macrophage chemoattractant and regulatory chemokine ligands MCP1 (CCL2), MCP5 (CCL12) and MIP-1β (CCL4) (F) were the least affected chemokines in the tacrolimus- and the metformin-treated mice.