The Galectin-9/Tim-3 pathway is involved in the regulation of NK cell function at the maternal-fetal interface in early pregnancy

Abstract

Decidual natural killer (dNK) cells actively participate in the establishment and maintenance of maternal-fetal immune tolerance and act as local guardians against infection. However, how dNK cells maintain the immune balance between tolerance and anti-infection immune responses during pregnancy remains unknown. Here, we demonstrated that the inhibitory molecule T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) are expressed on over 60% of dNK cells. Tim-3(+) dNK cells display higher interleukin (IL)-4 and lower tumor necrosis factor (TNF)-α and perforin production. Human trophoblast cells can induce the transformation of peripheral NK cells into a dNK-like phenotype via the secretion of galectin-9 (Gal-9) and the interaction between Gal-9 and Tim-3. In addition, trophoblasts inhibit lipopolysaccharide (LPS)-induced pro-inflammatory cytokine and perforin production by dNK cells, which can be attenuated by Tim-3 neutralizing antibodies. Interestingly, a decreased percentage of Tim-3-expressing dNK cells were observed in human miscarriages and murine abortion-prone models. Moreover, T helper (Th)2-type cytokines were decreased and Th1-type cytokines were increased in Tim-3(+) but not Tim-3(-) dNK cells from human and mouse miscarriages. Therefore, our results suggest that the Gal-9/Tim-3 signal is important for the regulation of dNK cell function, which is beneficial for the maintenance of a normal pregnancy.

Figure 1

Tim-3⁺ NK cells are present in human deciduas during the first trimester. (a) Representative density plots showing the analysis of Tim-3 expression in decidual and peripheral blood CD3⁻CD56⁺ NK cells during the first trimester (Anti–Tim-3: open graph; isotype control: filled graph). (b) Relative number of Tim-3⁺ NK cells in gated CD3⁻CD56⁺ dNK and pNK cells (n=30). The data in b are presented as the mean±s.e.m. dNK, decidual NK cells; pNK, peripheral blood NK cells.

Figure 2

Decidual Tim-3⁺ NK cells display a Th2 shift with low cytotoxicity. After treatment of the purified dNK cells with PMA, ionomycin and BFA for 4 h, cells were harvested and subjected to FCM analysis. The relative amount of Th2-type cytokines (a and b), Th1-type cytokines (c, d) and perforin (e, f) in gated CD3⁻CD56⁺ dNK cells, Tim-3⁺CD3⁻CD56⁺ dNK cells and Tim-3⁻CD3⁻CD56⁺ dNK cells, respectively, are shown. Data in b, d and f are presented as the mean±s.e.m. of 10 different samples. Representative density plots are shown in a, c and e. **P<0.01; ***P<0.001. dNK, decidual NK cells; FCM, flow cytometry.

Figure 3

Trophoblast cells instruct pNKs to exhibit dNK cell-like phenotypes via interactions between Tim-3 and Gal-9. (a) FCM was performed to analyze intracellular expression of Gal-9 in trophoblasts. (b) An ELISA assay was used to assess the secretion of Gal-9 from human primary trophoblasts cultured for different periods of time. (c–h) Purified pNK cells from the first trimester were cultured with trophoblasts in the presence or absence of anti-Tim-3 neutralizing Abs for 72 h. FCM analysis was used to determine the levels of (c, d) Th1-type cytokines, (e, f) Th2-type cytokines and (g, h) cytotoxicity of pNK cells. The results are representative of 9 independent experiments. Data are shown as the mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001. FCM, flow cytometry; pNK, peripheral NK cells; Tro, trophoblast; α-Tim-3, anti-Tim-3 neutralizing antibodies.

Figure 4

Tim-3 signaling inhibits inflammatory cytokine production by LPS-induced dNK cells. Purified dNK cells alone or cocultured with trophoblasts were treated with control or LPS for 48 h. Some cells were pretreated with rhGalectin-9 or anti-Tim-3 neutralizing Abs. The percentages of cells producing the inflammatory cytokine TNF-α (a), cytotoxic perforin (b) and IL-4 (c) in gated CD3⁻CD56⁺ dNK cells were determined using FCM after pre-stimulation with PMA, ionomycin and BFA for 4 h. The results are representative of nine independent experiments. Data are shown as the mean±s.e.m. **P<0.01, compared with dNK cells cultured alone; ^# P<0.05, ^## P<0.01, ^### P<0.001 compared with dNK cells treated with LPS; ^▴▴ P<0.01, ^▴▴▴ P<0.001 compared with dNK cells treated with LPS and rhGal-9; ^▵▵▵ P<0.001 compared with dNK cells cocultured with trophoblasts and treated with LPS; dNK, decidual NK cells; FCM, flow cytometry; LPS, Lipopolysaccharide; Tro, trophoblast; α-Tim-3, anti-Tim-3 neutralizing antibodies.

Figure 5

Decreased numbers of Tim-3⁺ dNK cells is accompanied by dysregulated Th2 predominance and higher cytotoxicity in human miscarriages. (a) FCM was used to analyze the relative number of Tim-3⁺ dNK cells in normal pregnancies and miscarriages. (b–f) dNK cells from normal pregnancies and miscarriages were treated with PMA, ionomycin and BFA for 4 h, and intracellular cytokines and perforin were detected using FCM. Histograms show Th2 cytokine (IL-4 and IL-10) (b, c), Th1 cytokine (TNF-α and IFN-γ) (d, e) and perforin (f) production by Tim-3⁺ and Tim-3⁻ dNK cells from normal pregnancies and miscarriages. (g) Tim-3 expression on NK cells from decidual tissues of normal pregnant mice and abortion-prone mice during the first trimester. (h) FCM analysis of cytokine production by NK cells from decidua of normal pregnant mice and abortion-prone mice. Data are shown as the mean±s.e.m. of 10 different samples. *P<0.05; **P<0.01; ***P<0.001. NP, normal pregnancy. CBA/J×Balb/c, normal pregnancy mouse model; CBA/J×DBA/2, abortion-prone mouse model; dNK, decidual NK cells; FCM, flow cytometry.