Lipoxin A4 suppresses angiotensin II type 1 receptor autoantibody in preeclampsia via modulating caspase-1

Abstract

Preeclampsia (PE) remains a leading cause of maternal and neonatal morbidity and mortality. Numerous studies have shown that women with PE develop autoantibody, termed angiotensin II type 1 receptor autoantibody (AT1-AA), and key features of the disease result from it. Emerging evidence has indicated that inflammatory cell necrosis, such as pyroptosis, could lead to autoantigen exposure and stimulate autoantibody production. Caspase-1, the central enzyme of inflammasome and key target of pyroptosis, may play roles in AT1R exposure and AT1-AA production. Exploring endogenous regulator that could inhibit AT1-AA production by targeting pyroptosis will be essential for treating PE. Lipoxin A₄ (LXA₄), endogenous dual anti-inflammatory and proresolving lipid mediator, may inhibit AT1-AA production via modulating caspase-1. Thus, we explore whether caspase-1 is essential for AT1-AA production and LXA₄ inhibits AT1-AA via modulating caspase-1. PE patients and mice developed AT1-AA associated with caspase-1 activation. Caspase-1 deletion leaded to AT1-AA decrease in PE mice. Consistent with these findings, we confirmed caspase-1 activation, trophoblast pyroptosis and AT1R exposure in PE mice and trophoblast model, while caspase-1 deficiency showed decreased trophoblast pyroptosis and AT1R exposure in vitro and in vivo. Interestingly, LXA₄ could suppress AT1-AA production via regulating caspase-1 as well as enhancing phagocytosis of dead trophoblasts by macrophages. These results suggest that caspase-1 promotes AT1-AA production via inducing trophoblast pyroptosis and AT1R exposure, while LXA₄ suppresses AT1-AA production via modulating caspase-1, supporting caspase-1 serving as a therapeutic target for attenuating AT1-AA and LXA₄ protecting patients from AT1-AA and PE.

Fig. 1. AT1-AA expression, caspase-1 activation and trophoblast pyroptosis in PE patients.

a Comparison of serum AT1-AA levels between PE patients and healthy controls. b, c Comparison of placental caspase-1 expressions between PE patients and healthy controls. IHC (b) and WB (c) analysis of caspase-1 in placenta were shown. Bar = 25 μm. d Comparison of placental caspase-1 activity between PE patients and healthy controls. e Comparison of placental IL-1β and IL-18 levels between PE patients and healthy controls. IL-1β and IL-18 were detected by ELISA. f Comparison of trophoblast death between PE patients and healthy controls. TUNEL analysis in placenta was shown. Bar = 25 μm. g Comparison of placental LDH activity between PE patients and healthy controls. h–k The correlation between AT1-AA and caspase-1 activity, IL-1β, IL-18 and LDH activity in PE women and normal pregnancies. Results are expressed as means ± SEM (n = 16 in each group). *p < 0.05 and **p < 0.01 versus control group, two-tailed Student’s t test.

Fig. 2. Caspase-1 activation, pyroptosis and AT1R exposure in PE mice and trophoblast model.

a Comparison of trophoblast death between control and PE mice. TUNEL analysis in placenta was shown. Bar = 50 μm. b, c Comparison of placental caspase-1 expression between control and PE mice. WB (b) and IHC (c) analysis of caspase-1 in placenta were shown. The histogram represents means ± SEM of the densitometric scans for protein bands (n = 7 mice in each group), normalized by comparison with β-actin and expressed as a percentage of Control. Bar = 50 μm. d Comparison of placental caspase-1 activity between control and PE mice. Comparison of IL-1β and IL-18 levels in serum (e) and placenta (f) between control and PE mice. IL-1β and IL-18 were detected by ELISA. Results are expressed as means ± SEM (n = 7 mice in each group). **p < 0.01 and ***p < 0.001 versus control group, two-tailed Student’s t test. g Effect of LPS/ATP on trophoblast apoptosis. Human first-trimester trophoblast cell line HTR-8/SVneo was treated with LPS and ATP. Cell apoptosis was detected by using flow cytometry. h Effect of LPS/ATP on trophoblast caspase-1 expression. Caspase-1 was detected by WB. i Effect of LPS/ATP on trophoblast caspase-1 activity. j Effect of LPS/ATP on trophoblast IL-1β and IL-18 levels. IL-1β and IL-18 were detected by ELISA. k Effect of LPS/ATP on trophoblast AT1R exposure. AT1R was detected by WB. Results are expressed as means ± SEM from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 versus control group, two-tailed Student’s t test.

Fig. 3. AT1-AA production in PE mice model.

a Comparison of serum AT1-AA levels between control and PE mice. b Comparison of spleen size between control and PE mice. c Comparison of spleen pathological features between control and PE mice. Representative H&E staining images of spleen are showed. The bar is 250 μm. d, e IHC analysis of spleen and lymph node. Sections of spleen (d) and lymph node (e) were prepared from control and PE mice, and stained with antibody against CD68 (red) and TUNEL (green); staining profiles were merged in the third column. Bar = 100 μm. f, g IHC analysis of spleen and lymph node. Sections of spleen (f) and lymph node (g) were prepared from control and PE mice, and stained with antibody against IgG (red). Bar = 200 μm. h Comparison of peripheral AT1-AA-producing CD19⁺CD5⁺ B cells between control and PE mice. CD19⁺CD5⁺ B cells in peripheral blood of mice were stained with APC-conjugated Rat Anti-Mouse CD19 and PE-conjugated Rat Anti-Mouse CD5, and subjected to flow cytometry analysis. Results are expressed as means ± SEM (n = 7 mice in each group). *p < 0.05 and **p < 0.01 versus control group, two-tailed Student’s t test.

Fig. 4. Caspase-1 deficiency inhibits trophoblast pyroptosis and AT1R antigen exposure in PE mice and trophoblast model.

a Effect of caspase-1 knockout on trophoblast death in PE mice. TUNEL analysis in placenta was shown. Bar = 50 μm. Effect of caspase-1 knockout on IL-1β and IL-18 levels in serum (b) and placenta (c) of PE mice. IL-1β and IL-18 were detected by ELISA. Results are expressed as means ± SEM (n = 7 mice in each group). **P < 0.01 and ***P < 0.001 versus control group, ^††P < 0.01 and ^†††P < 0.001 versus LPS group, one-way ANOVA with S-N-K posttest. d Effect of caspase-1 knockdown on trophoblast apoptosis. Cell apoptosis was detected by using flow cytometry. e Effect of caspase-1 knockdown on trophoblast IL-1β and IL-18 levels. IL-1β and IL-18 were detected by ELISA. f Effect of caspase-1 knockdown on trophoblast AT1R exposure. AT1R was detected by WB. Results are expressed as means ± SEM from three independent experiments. **P < 0.01 and ***P < 0.001 versus control group, ^†P < 0.05 and ^††P < 0.01 versus LPS group, one-way ANOVA with S-N-K posttest.

Fig. 5. Caspase-1 knockout suppresses AT1-AA production in PE mice.

a Effect of caspase-1 knockout on AT1-AA expressions in PE mice. b Effect of caspase-1 knockout on spleen size in PE mice. c Effect of caspase-1 knockout on spleen pathological features in PE mice. Representative H&E staining images of spleen are showed. The bar is 250 μm. d, e IHC analysis of spleen and lymph node. Sections of spleen (d) and lymph node (e) were prepared from control, PE and Casp1^−/− mice, and stained with antibody against CD68 (red) and TUNEL (green); staining profiles were merged in the third column. Bar = 100 μm. f, g IHC analysis of spleen and lymph node. Sections of spleen (f) and lymph node (g) were prepared from control, PE and Casp1^−/− mice, and stained with antibody against IgG (red). Bar = 200 μm. h Effect of caspase-1 knockout on peripheral AT1-AA-producing CD19⁺CD5⁺ B cells in PE mice. CD19⁺CD5⁺ B cells in peripheral blood of mice were stained with APC-conjugated Rat Anti-Mouse CD19 and PE-conjugated Rat Anti-Mouse CD5, and subjected to flow cytometry analysis. Results are expressed as means ± SEM (n = 7 mice in each group). **P < 0.01 and ***P < 0.001 versus control group, ^†P < 0.05, ^††P < 0.01 and ^†††P < 0.001 versus LPS group, one-way ANOVA with S-N-K posttest.

Fig. 6. The relationship between LXA₄ and AT1-AA and caspase-1 in PE patients.

a Comparison of serum LXA₄ levels between PE patients and healthy controls. b, c Comparison of LXA₄-synthesizing enzymes expressions between PE patients and healthy controls. b WB analysis of ALOX5, ALOX12, ALOX15 and ALOX15B in placenta. β-actin from Fig. 1c was reused in Fig. 6b for ease of reference. In Fig. 1c and Fig. 6b, three gels were run, and six blots were performed for detecting ALOX12, ALOX15, ALOX15B and caspase-1. In detail, ALOX12 (76 kDa) and β-actin (43 kDa) proteins were cut from the first gel, electroblotted onto PVDF membrane, and then probed with ALOX12 and β-actin antibodies, respectively. ALOX15 (75 kDa), caspase-1 (45 kDa) and caspase-1 (20 kDa) proteins were cut from the second gel, electroblotted onto PVDF membrane, and then probed with ALOX15 and caspase-1 antibodies, respectively. ALOX15B (75 kDa) protein was cut from the third gel, electroblotted onto PVDF membrane, and then probed with ALOX15B antibody. c IHC analysis of ALOX5, ALOX12, ALOX15 and ALOX15B in placenta. Bar = 50 μm. d, e The correlation between LXA₄ and AT1-AA and caspase-1 in PE patients. Results are expressed as means ± SEM (n = 16 in each group). **p < 0.01 versus control group, two-tailed Student’s t test.

Fig. 7. LXA₄ inhibits caspase-1 activation, trophoblast pyroptosis and AT1R exposure as well as enhance phagocytosis of apoptotic trophoblasts by macrophages.

a Effect of LXA₄ on trophoblast death in PE mice. TUNEL analysis in placenta was shown. Bar = 50 μm. b, c Effect of LXA₄ on placental caspase-1 expression in PE mice. IHC (b) and WB (c) analysis of caspase-1 in placenta were shown. The histogram represents means ± SEM of the densitometric scans for protein bands (n = 7 mice in each group), normalized by comparison with β-actin and expressed as a percentage of Control. Bar = 50 μm. d Effect of LXA₄ on placental caspase-1 activity in PE mice. Effect of LXA₄ on IL-1β and IL-18 levels in serum (e) and placenta (f) of PE mice. IL-1β and IL-18 were detected by ELISA. Results are expressed as means ± SEM (n = 7 mice in each group). ***P < 0.001 versus control group, ^†P < 0.05 and ^††P < 0.01 versus LPS group, one-way ANOVA with S-N-K posttest. g Effect of LXA₄ on trophoblast apoptosis. Cell apoptosis was detected by using flow cytometry. h Effect of LXA₄ on trophoblast caspase-1 expression. Caspase-1 expression was detected by WB. i Effect of LXA₄ on trophoblast caspase-1 activity_. j Effect of LXA₄ on trophoblast IL-1β and IL-18 levels. IL-1β and IL-18 were detected by ELISA. k Effect of LXA₄ on trophoblast AT1R exposure. AT1R was detected by WB. Results are expressed as means ± SEM from three independent experiments. **P < 0.01 and ***P < 0.001 versus control group, ^†P < 0.05, ^††P < 0.01 and ^†††P < 0.001 versus LPS group, one^-way ANOVA with S-N-K posttest. l, m Effect of LXA₄ on phagocytosis of apoptotic trophoblasts by macrophages. IF analysis of phagocytosis of apoptotic trophoblasts (l) and AT1R (m) by macrophages were shown. Slides of macrophages phagocytosis of apoptotic trophoblasts were prepared from control and LXA₄ group, and stained with antibody against CD68 (red) and TUNEL (green), or antibody against CD68 (red) and AT1R (green); staining profiles were merged in the third column. Bar = 50 μm.

Fig. 8. LXA₄ suppresses AT1-AA production in PE mice.

a Effect of LXA₄ on AT1-AA expressions in PE mice. b Effect of LXA₄ on spleen size in PE mice. c Effect of LXA₄ on spleen pathological features in PE mice. Representative H&E staining images of spleen are showed. The bar is 250 μm. d, e IHC analysis of spleen and lymph node. Sections of spleen (d) and lymph node (e) were prepared from control, PE and LXA₄ mice, and stained with antibody against CD68 (red) and TUNEL (green); staining profiles were merged in the third column. Bar = 100 μm. f, g IHC analysis of spleen and lymph node. Sections of spleen (f) and lymph node (g) were prepared from control, PE and LXA₄ mice, and stained with antibody against IgG (red). Bar = 200 μm. h Effect of LXA₄ on peripheral CD19⁺CD5⁺ B cells in PE mice. CD19⁺CD5⁺ B cells in peripheral blood of mice were stained with APC-conjugated Rat Anti-Mouse CD19 and PE-conjugated Rat Anti-Mouse CD5, and subjected to flow cytometry analysis. Results are expressed as means ± SEM (n = 7 mice in each group). **P < 0.01 and ***P < 0.001 versus control group, ^†P < 0.05 and ^††P < 0.01 versus LPS group, one-way ANOVA with S-N-K posttest.