IFN-γ modulates Ly-49 receptors on NK cells in IFN-γ-induced pregnancy failure

Abstract

We have previously shown that interferon gamma (IFN-γ) induces aberrant CD49b(+) natural killer (NK) cell recruitment by regulating CX3CL1 and eventually provokes foetal loss. In this study, we show that IFN-γ also modulates Ly-49 receptors on NK cells during pregnancy failure. The percentages of Ly-49A(+) and Ly-49G2(+) NK cells in the uteri of the IFN-γ-treated group were significantly lower than those observed in the control group. Moreover, the median fluorescence intensity (MFI) values of Ly-49A and Ly-49G2 expression on NK cells in the uteri of the IFN-γ-treated group were significantly lower than those of the control group. Using isolated spleen leucocytes, we further found that IFN-γ significantly reduced the percentage of Ly-49A(+) NK cells in vitro. However, CX3CL1 was not involved in the modulation of Ly-49 receptors, and the expression of CX3CR1 was not regulated by IFN-γ in spleen leucocytes. Collectively, our data indicate that IFN-γ can modulate Ly-49 receptors on NK cells and this process may play a role in IFN-γ-induced pregnancy failure. Thus, we provide a new line of evidence correlating the deleterious effects of IFN-γ with its role in regulating NK cell Ly-49 receptors during pregnancy failure.

Figure 1. IFN-γ altered the percentages of Ly-49⁺ NK cells in the blood in an IFN-γ-induced pregnancy failure model.

(a–i) Syngeneically mated BALB/c females were injected with placebo or IFN-γ intraperitoneally on GD6 and sacrificed on GD8. (a) Representative flow cytometric analysis of Ly-49A and Ly-49G2 expression on gated CD3⁻CD49b⁺ NK cells in the blood and spleen. See Supplementary Fig. S1b, c for the gating strategy; the numbers in the dot plots indicate the percentages of the Ly-49⁺ NK cells (gated on CD3⁻CD49b⁺ NK)/MFI values of Ly-49 receptor expression. Data summary of the percentages of Ly-49⁺ NK cells (b,c,f,g) and MFI values of Ly-49 receptor expression (d,e,h,i) in the blood (b–e) and spleen (f–i). Data show the mean ± SEM of three (blood) or four (spleen) independent experiments and were obtained from three (blood) or four (spleen) mice per group, respectively. ^*P < 0.05, ^**P < 0.01 by independent sample T-test.

Figure 2. IFN-γ altered the expression of Ly-49 receptors on NK cells in the uterus in an IFN-γ-induced pregnancy failure model.

(a–e) Syngeneically mated BALB/c females were injected with placebo or IFN-γ intraperitoneally on GD6 and sacrificed on GD8. (a) Representative flow cytometric analysis of Ly-49A and Ly-49G2 expression on gated CD3⁻CD49b⁺ NK cells in the uterus. See Supplementary Fig. S2 for the gating strategy; the numbers in the dot plots indicate the percentages of the Ly-49⁺ NK cells (gated on CD3⁻CD49b⁺ NK)/MFI values of Ly-49 receptor expression. Data summary of the percentages of Ly-49⁺ NK cells (b,d) and MFI values of Ly-49 receptor expression (c,e). Data show the mean ± SEM of three independent experiments and were obtained from three mice per group. ^*P < 0.05, ^**P < 0.01 by independent sample T-test.

Figure 3. IFN-γ but not CX3CL1 modulated the percentages of Ly-49⁺ NK cells in vitro.

(a–i) Splenic leucocytes were cultured with IFN-γ (250 U/ml) or CX3CL1 (250 ng/ml) for 24 h. (a) Representative flow cytometric analysis of Ly-49A and Ly-49G2 expression on gated CD3⁻CD49b⁺ NK cells; the numbers in the dot plots indicate the percentages of Ly-49⁺ NK cells (gated on CD3⁻CD49b⁺ NK)/MFI values of Ly-49 receptor expression. Data summary of the percentages of Ly-49⁺ NK cells (b,c,f,g) and MFI values of Ly-49 receptor expression (d,e,h,i) after IFN-γ (b–e) and CX3CL1 (f–i) treatment. Data show the mean ± SEM of four (IFN-γ) or three (CX3CL1) independent experiments, respectively. ^*P < 0.05, by independent sample T-test.

Figure 4. CX3CR1 mRNA levels were not regulated by IFN-γ in splenic leucocytes.

(a–c) Splenic leucocytes were cultured with IFN-γ at a dose of 250 U/ml. At the indicated times, cells were harvested to determine IRF-1 (a,b) and CX3CR1 (c) expression by quantitative PCR. Data show the mean ± SEM of four independent experiments. ^**P < 0.01 by independent sample T-test.

Figure 5. CXCL12 did not modulate the percentages of Ly-49⁺ NK cells in vitro.

(a) Syngeneically mated BALB/c females were injected with placebo or IFN-γ intraperitoneally on GD6 and sacrificed on GD8. CXCL12 concentration in the serum was determined by ELISA. Data show the mean ± SEM of four independent experiments and were obtained from four mice per group. (b–f) Splenic leucocytes were cultured with CXCL12 at a dose of 500 ng/ml for 24 h. (b) Representative flow cytometric analysis of Ly-49A and Ly-49G2 expression on gated CD3⁻CD49b⁺ NK cells; the numbers in the dot plots indicate the percentages of the Ly-49⁺ NK cells (gated on CD3⁻CD49b⁺ NK)/MFI values of Ly-49 receptors expression. Data summary of the percentages of Ly-49⁺ NK cells (c,d) and MFI values of Ly-49 receptor expression (e,f). Data show the mean ± SEM of three independent experiments. (g) Splenic leucocytes were cultured with IFN-γ at a dose of 250 U/ml and at the indicated times, splenic leucocytes were harvested to determine CXCR4 expression by quantitative PCR. Data show the mean ± SEM of four independent experiments.