Synergistic Regulation of Inflammatory Responses by Monocyte Tim-3 and PD-L1 in Th17/Treg Imbalance in Preeclampsia

Blood samples were obtained from PE patients and normal pregnant controls. The expression of Tim-3, PD-L1 and TLR4 on CD14⁺ monocytes as well as the proportions of Th17 and Treg cells, were determined by flow cytometry. Plasma levels of TNF-α, IL-6, IL-12 and IL-10 were measured by ELISA.

CD14⁺ monocytes from PE patients were assigned to five groups: blank control, LPS/R848 stimulation, Gal-9 treatment, PD-1-Fc treatment, and combined treatment groups. Following culture, TLR4 expression on monocytes and inflammatory cytokine levels in supernatant were measured. Monocytes from each treatment group were co-cultured with CD4⁺ T cells from normal pregnancies to evaluate Th17/Treg differentiation.

Synergistic effects were evaluated using the Bliss model.Results Compared with normal pregnancy group, the positive rates of Tim-3 and PD-L1 on peripheral blood CD14⁺ monocytes in PE patients were significantly decreased (both P<0.01), while TLR4 positive rate was significantly increased (P<0.01). Tim-3 and PD-L1 levels on monocytes were negatively correlated with TLR4 expression and Th17/Treg ratio (all P<0.01).

Plasma levels of TNF-α, IL-6 and IL-12 in PE group were significantly elevated, while IL-10 was decreased (all P<0.01). TLR4 expression was positively correlated with pro-inflammatory cytokines and negatively correlated with IL-10 (all P<0.01). In vitro, combined Gal-9 and PD-1-Fc treatment showed significantly stronger inhibitory effects on TLR4 expression and TNF-α, IL-6, IL-12 secretion from PE-derived monocytes compared with either agonist alone (all P<0.01), with Bliss synergy (cooperation) index (CI) of 1.13, 1.83, 3.13 and 1.37, respectively (all >1); IL-10 level was markedly elevated (CI=3.38).

In co-culture experiments, the combined treatment group further decreased the Th17 proportion and increased the Treg proportion compared with each single treatment group (all P<0.01), and the Th17/Treg ratio was restored to a level comparable to that of the normal control co-culture group (P=0.5440).Conclusion Coordinated low expression of Tim-3 and PD-L1 on monocytes from PE patients leads to TLR4 pathway de-inhibition, resulting in uncontrolled inflammatory cytokine secretion and subsequent Th17/Treg imbalance.

Combined activation of Tim-3/PD-L1 signaling synergistically rescues this pathological process, providing novel targets for immunological intervention in PE.