Abstract
The nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome plays a critical role in various inflammatory diseases. We sought to investigate the role of NLRP3 inflammasome in uterine activation for labor at term and preterm. We found that NLRP3 inflammasome was activated in the myometrium tissues obtained from the pregnant women undergoing labor at term (TL) compared with those not undergoing labor (TNL) at term. NLRP3 inflammasome was also activated in amnion and chorion-deciduas in TL and preterm labor (PTL) groups. In the mouse model, uterine NLRP3 inflammasome and nuclear factor kappaB (NF-κB) were activated toward term and during labor. Treatment of pregnant mice with lipopolysaccharide (LPS) and RU38486 induced preterm birth (PTB) and also promoted uterine NLRP3 inflammasome and NF-κB activation. Treatment of pregnant mice with NLRP3 inflammasome inhibitor BAY11-7082 and MCC950 delayed the onset of labor and suppressed NLRP3 inflammasome and NF-κB activation in uterus. MCC950 postponed labor onset of the mice with LPS and RU38486 treatment and inhibited NLRP3 inflammasome activation in uterus. Our data provide the evidence that NLRP3 inflammasome is involved in uterine activation for labor onset in term and PTB in humans and mouse model.
Introduction
Preterm birth (PTB), which occurs in 5–18% of all pregnancies, is the leading cause of perinatal mortality and morbidity worldwide. PTB can cause long-term disorders including cerebral palsy, cognitive impairment, blindness, deafness, and respiratory illness (Goldenberg et al. 2008, Romero et al. 2014). So far, the pathogenesis of preterm labor (PTL) remains largely unknown, which is largely attributed to our ignorance of fundamental mechanisms underlying the initiation of term labor (TL).
In recent years, it has been recognized that parturition is an inflammatory event, which is characterized by an influx of inflammatory cells into the myometrium and cervix, with concomitant increases in vascular and leukocyte adhesion molecule expression and increased production of chemokines and pro-inflammatory cytokines within uterus (Romero et al. 2007, 2014, Di Renzo et al. 2018, Gilman-Sachs et al. 2018, Green & Arck 2020). Pro-inflammatory cytokines initiate a cascade of inflammatory mediator production, including matrix metalloproteinases and prostaglandins, which in turn leads to cervical dilation, rupture of membranes, and uterine contractions (Hua et al. 2012, Romero et al. 2014, Lappas 2016, Green & Arck 2020). Early initiation of the inflammatory pathway by infection accounts for 25–40% of all human premature deliveries (Higgins et al. 2016, Yeh et al. 2017, Helmo et al. 2018). Intrauterine infections can arise systemically, or commensal bacteria can ascend from the female genital tract. Therefore, several mouse models of infection-induced PTB have been developed (Agrawal & Hirsch 2012, McCarthy et al. 2018), most commonly using Escherichia coli or the toxic component on the surface of gram-negative bacteria, lipopolysaccharide (LPS).
Steroid hormone progesterone is the key hormone that maintains the quiescent state of uterus during pregnancy. Progesterone levels remain high throughout human pregnancy, but women undergo a 'functional' progesterone withdrawal at the end of pregnancy (Patel et al. 2015, Vannuccini et al. 2016). Spontaneous labor is likely mediated by a concerted series of biochemical events that negatively impact the ability of progesterone to regulate target genes that maintain myometrial quiescence (Conley & Ball 2019). Thus, PTB induced by progesterone receptor antagonist RU38486 in rodents is a frequently used model for studying PTL (Dudley et al. 1996, Yellon et al. 2013, McCarthy et al. 2018b). Emerging evidence indicates that progesterone maintains the quiescent state of uterus is associated with its suppression of the generation of inflammatory mediators including inflammatory cytokines and prostaglandins (Hardy et al. 2006, Shynlova et al. 2013, Lei et al. 2015). For instance, Hardy et al. (2006) have demonstrated that progesterone inhibits interleukin (IL)-1-induced recruitment of nuclear factor kappaB (NF-κB) p65 to NF-κB elements within the cyclooxygenase-2 (COX-2) gene promoter and upregulates the expression of inhibitor α of nuclear factor kappaB (IκBα), the inhibitor of NF-κB. Shynlova and coworkers (Shynlova et al. 2013) reported that treatment of pregnant mice with RU38486 could increase the levels of pro-inflammatory cytokines and leukocytes infiltration in myometrium, suggesting that RU38486-induced PTB is associated with inflammation.
The nucleotide binding and oligomerization domain-like (Nod) receptor family pyrin domain-containing 3 (NLRP3) inflammasome, the well-characterized inflammasome, plays a critical role in various inflammatory diseases (Kelley et al. 2019, Yang et al. 2019, Wang et al. 2020). The NLRP3 inflammasome is activated by a wide range of the molecules, including pathogen-associated molecular patterns and both endogenous and exogenous danger-associated molecular patterns or alarmins, and subsequently results in activation of caspase-1 by which inactive forms of IL-1β and IL-18 (i.e. pro-IL-1β and pro-IL-18) are processed to mature IL-1β and IL-18 (Kelley et al. 2019, Yang et al. 2019). Several studies have shown that NLRP3 inflammasome is activated in the gestational tissues of term and PTB in humans (Gomez-Lopez et al. 2017, 2019a, Romero et al. 2018).
Since NLRP3-driven inflammation is a pathological event in the development of many diseases including cryopyrin-associated periodic syndromes, Alzheimer’s Disease, type 2 diabetes, atherosclerosis, non-alcoholic fatty liver disease, asthma, and silicosis (Kelley et al. 2019, Yang et al. 2019, Wang et al. 2020), inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases (Zahid et al. 2019). A number of small molecules have been developed to inhibit NLRP3 inflammasome activation. For instance, BAY11-7082, a NF-κB inhibitor, can alkylate cysteines in the ATPase domain of NLRP3 and subsequently inhibit NLRP3 ATPase activity (Juliana et al. 2010, Zahid et al. 2019). MCC950, a small molecular inhibitor developed in 2015, can block the ATPase domain of NLRP3, thereby resulting in inhibition of canonical and noncanonical NLRP3 inflammasome activation (Coll et al. 2015, 2019). More recently, a study of Gomez-Lopez’s group has shown that MCC950 prevents PTB induced by sterile intra-amniotic inflammation in mice (Gomez-Lopez et al. 2019b).
In this study, we examined the NLRP3 inflammasome activation in the gestational tissues from pregnant patients with term birth and PTB and explored the role of NLRP3 inflammasome in the initiation of term labor and LPS- and RU38486-induced PTL in mice. We found that NLRP3 inflammasome was activated in human myometrium after the onset of TL. In fetal membranes, NLRP3 inflammasome activation not only occurred in the patients with onset of TL but also in those with PTL. In mouse model, we revealed that uterine NLRP3 inflammasome was activated in term birth and LPS- and RU38486-induced PTB. Furthermore, we investigated the effects of NLRP3 inhibitors on initiation of parturition in term birth and PTB. It was found that BAY11-7082 and MCC950 postponed the timing of term birth and inhibited RU38486-induced PTB. MCC950 could postpone LPS-induced PTB. Our study indicates that NLRP3 inflammasome plays an important role in the initiation of term and PTL and highlights that NLRP3 inflammasome within uterus could be a potential therapeutic target.
Materials and methods
Tissue collection
Tissue collections were performed with the approval of the specialty committee on ethics of biomedicine research, Navy Medical University, Shanghai, China. Written informed consent was obtained from all patients. Human myometrium and fetal membranes were obtained from the Changhai Hospital, the affiliated Hospital of Navy Military Medical University, Shanghai. Human myometrial tissues were collected from women who underwent elective (term no labor (TNL), n = 12) and emergency cesarean section (TL, n = 12) with singleton pregnancy. Biopsies of myometrium were excised from the middle portion of upper edge of the incision line in the lower uterine segment at cesarean section. Fetal membranes were collected from the following groups of women with singleton pregnancy: TNL (n = 12), TL (n = 12), and PTL (n = 23). TNL tissues were collected at elective cesarean section. The fetal membranes of TL and PTL were collected from women with uncomplicated pregnancies, who went into natural labor, and delivered vaginally. PTL group was divided into the PTL with chorioamnionitis (n = 11) and without chorioamnionitis (n = 12). The chorioamnionitis was determined by a professional pathologist. The patients who had the evidence of underlying disease (e.g. hypertension, diabetes, preeclampsia, intrauterine growth restriction, etc.) were not included in this study. The characteristics of the patients were listed in Table 1. The amnion and chorion laeve (including decidua) were separated. Collected samples were frozen immediately in liquid nitrogen and then stored at −80°C.
Characteristics of study participants in term no labor (TNL), term labor (TL), and preterm labor (PTL) groups. Data were expressed as mean ± s.e.m.
| Clinical features | TNL (n = 12) | TL (n = 12) | PTL | |
|---|---|---|---|---|
| Without CAM (n = 12) | CAM (n = 11) | |||
| Weeks of gestation | 38.93 ± 0.48 | 39.82 ± 1.27 | 34.09 ± 0.36** | 34.31 ± 0.21** |
| Labor status | ||||
| Spontaneous | 0 | 100% | 100% | 100% |
| Induced | 0 | 0 | 0 | 0 |
| Delivery mode | ||||
| Vaginal | 0 | 100% | 100% | 87.5% (7/8) |
| C-section | 100% (24/24) | 12.5% (1/8) | ||
| Membrane rupture | ||||
| SPOM | 66.7% (8/12) | |||
| PROM | 83.3% (10/12) | 100% | ||
| ARM | 100% (24/24) | 33.3% (4/12) | 16.7% (2/12) | |
| Fetal weight (g) | 3321.38 ± 78.71 | 3409.38 ± 82.27 | 2417.78 ± 120.15** | 2371.47 ± 167.38** |
| Maternal age (years) | 26.46 ± 1.01 | 28.87 ± 0.93 | 28.01 ± 0.89 | 29.31 ± 1.21 |
**P < 0.01 vs (TL+TNL).
ARM, artificial rupture of membranes; C-section, caesarean section; CAM, chorioamnionitis; PROM, prelabor rupture of membranes; SROM, spontaneous rupture of membranes with labor.
Animal protocols
Institute of Cancer Research (ICR) mice (20–25 g body weight, 6–8 weeks old) were obtained from Shanghai SLAC Laboratory Animal Co (Shanghai, China) and then housed in social groups of three to five in a cage with regular light–darkness cycles (7:00 h light:19:00 h darkness) under controlled temperature (22 ± 2°C) and humidity (50 ± 10%) and were given standard diet and water ad libitum. All animal procedures were carried out in accordance with the guidelines for the use of laboratory animals published by the People’s Republic of China Ministry of Health (May 2016), with the approval of the Scientific Investigation Board of Experimental Animals of Navy Medical University as well as ethic committee of medical research of Xiangya Hospital. Breeding females were handled daily for 1 week. One to three virgin female mice were housed with a male mouse overnight beginning at 20:00 h. Female mice were found to have vaginal plugs at 8:00 h, and the following day was designated to be gestational day (gd) 0.5. There were five animal experiments in this study. In the first experiment, timed pregnant mice at gd 15, 17, 19, and labor (delivery of the first pup) were sacrificed by deep anaesthetization (urethane and alpha-chloralose, i.p.). Then uterus tissues were collected, and myometrium was gently scraped to remove the endometrial layer, frozen immediately in liquid nitrogen and then stored at −80°C. In the second experiment, pregnant mice were randomly divided into four groups: BAY11-7082 group and its corresponding control group, MCC950 group and its corresponding control group. The timed mice of BAY11-7082 group were injected (s.c.) with BAY11-7082 (Sigma-Aldrich) at 5 mg/kg in 100 μL sterile saline once at 8:30 h at gd 18.5 and received second injection 12 h later. Corresponding control timed pregnant mice received injection (s.c.) of 100 μL saline. The timed mice of MCC950 groups were injected (i.p.) with NLRP3 inflammasome inhibitor MCC950 (Sigma-Aldrich) at 200 mg/kg (i.p.) in 100 μL sterile saline at 8:30 h at gd18.5. They received second injection 12 h later. Control timed pregnant mice received 100 μL saline (i.p.). Uterine samples were collected under deep anaesthetization when the mice delivered first pup. In the third experiment, the pregnant mice at gd 15.5 were divided into four groups: control group, RU38486 group, RU38486 with BAY11-7082 group, and RU38486 with saline group. The reagent of RU38486 was dissolved in absolute alcohol and then diluted in corn oil (Sigma-Aldrich). The final concentration of alcohol was 0.4%. The mice of RU38486 with BAY11-7082 group were injected with RU38486 (s.c.) at a spot, and they were also injected with BAY11-7082 at 5 mg/kg (s.c.) at another spot. Twelve hours later, the animals were injected with same dosage of BAY11-7082. The mice in RU38486 group were injected with RU38486 (Sigma-Aldrich) at 7.5 mg/kg (s.c.). RU38486 with saline group were injected with RU38486 (s.c.) at one spot and injected with 100 μL sterile saline (s.c.) at another spot. Control mice were injected (s.c.) with 100 μL corn oil containing 0.4% alcohol. The time of labor was recorded when the first pup was delivered. The uterus samples of mice with RU38486 and BAY11-7082 treatment were collected at time of parturition under deep anaesthetization. The uterus samples of control mice were collected at the time of corresponding RU38486 group. In the fourth experiment, the pregnant mice at gd 15.5 were divided into four groups: control group, RU38486 group, RU38486 with MCC950 group, and RU38486 with saline group. The mice of RU38486 with MCC950 group were injected with RU38486 (s.c.) and injected with MCC950 at 200 mg/kg (i.p.) at the meantime. Twelve hours later, the animals were injected with same dosage of MCC950. The mice in RU38486 group were injected with RU38486 at 7.5 mg/kg (s.c.) and received administration with 100 μL saline (i.p.) at same time. The mice of RU38486 with saline group were injected with RU38486 (s.c.) and injected with saline (i.p.). Control mice were injected with 100 μL corn oil containing 0.4% alcohol (s.c.). The labor time was then recorded. The uterus samples were collected from these mice. In the fifth experiment, pregnant mice at gd 15 were randomly divided into four groups: control group, LPS group, LPS with MCC950 group, and LPS with saline group. Timed mice on gd 15 were injected with Escherichia coli 0111:B4 LPS (Sigma-Aldrich) at 0.5 mg/kg (i.p.) or in combination with two injections of MCC950 at 200 mg/kg. Control timed pregnant mice received injection of 100 μL saline. Control mice received injection of same volume of saline. Uterus samples were then collected from these mice after deep anesthetization. The dosages of BAY11-7082 and MCC950 were chosen according to the literature (Juliana et al. 2010, Coll et al. 2015) and our preliminary study.
Immunohistochemistry
Paraffin sections (5 μm) were rehydrated and microwaved in citric acid buffer to retrieve antigens. Endogenous peroxidases were inhibited by incubating with 3% H2O2. Subsequently, unspecific antibody binding was blocked with 10% rabbit serum. The sections were then incubated with NLRP3 antibodies (Cell Signaling Technology, 15101) at the dilution 1:200 overnight at 4°C. The bound antibodies were detected with the biotin–streptavidin–peroxidase system (UltraSensitive-SP-kit, MaiXin Biotechnology, Fuzhou, China) using diaminobenzidine (Sigma-Aldrich) as chromogen. Counterstaining was performed with hematoxylin. Negative controls were performed by substituting primary antibody with IgG.
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IL-1β and IL-18 in the uterus of pregnant mice and human myometrium obtained from pregnant women were determined by using specific ELISA kit (Westang Biotechnology, Shanghai, China). Briefly, the tissues were homogenized on ice, then centrifuged at 4°C. The supernatants were harvested for determination for IL-1β and IL-18 concentration according to the manufacturer’s instructions.
Western blotting analysis
Fifty milligrams of tissues were homogenized in cold strong radio immunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail tablet (Beyotime Biotechnology, Shanghai, China). The tissue homogenates were centrifuged at 12,000×g for 15 min at 4°C, and the supernatants were collected. The samples with 35–50 µg proteins were denatured and separated by SDS (10%)-PAGE and subsequently transferred to nitrocellulose membranes by electroblotting. Following transfer, membranes were incubated in blocking buffer, then with specific antibodies (Santa Cruz Biotechnology) that recognize NLRP3 (Cell Signaling Technology, 15101), P65 (Santa Cruz, sc-8008), phospho-P65 Ser536 (p-P65) (Santa Cruz. sc-136548), COX-2 (Cell Signaling Technology, 12282s), oxytocin receptor (OTR) (Abcam, ab87312), FP receptor (FP) (Beijing Biosynthesis Biotechnology, Beijing, China, bs-11411R), IL-1β (ImmunoWay Biotechnology, Plano, TX, YT5201), IL-18 (ImmunoWay, YN1926), and GAPDH (Abcam, ab8245) overnight at 4°C. Membranes were then washed and incubated with a secondary horseradish peroxidase-conjugated antibody, and immunoreactive proteins were visualized using enhanced chemiluminescence (Santa Cruz). The intensities of light-emitting bands were detected and quantified using Tanon 4600SF Image system (Shanghai Tanon. Ltd, Shanghai, China). To control sampling errors, the ratio of band intensities to GAPDH was obtained to quantify the relative protein expression level.
Measurement of caspase-1 activity
The caspase-1 enzyme activity was measured with a Caspase-1 Activity Assay Kit (Beyotime, Shanghai, China) following the manufacturer’s recommendations. Briefly, the uterus tissues were lysed at 4°C. The supernatants were incubated with a substrate of caspase-1 (Ac-YVAD-pNA) to produce the yellow formazan product p-nitroaniline (pNA) at 37°C for 2 h. The pNA levels were detected at 405 nm by an Epoch 2 microplate spectrophotometer (Biotek).
Statistical analysis
Data are presented as mean ± s.e.m. Statistical analyses were performed using IBM SPSS Statistics 20. Normal distribution was assessed by Shapiro–Wilk test. Statistical significance was determined according to sample distribution and homogeneity of variance. Statistical comparisons between two groups were determined by two-tailed Student’s t-test. One-way ANOVA following by Bonferroni’s post hoc test was performed for comparisons among multiple groups. Comparison of incidence between two groups was analyzed by a two-sided Fisher’s exact test. P < 0.05 was considered statistically significant.
Results
Localization of NLRP3 expression in pregnant mouse uterus and myometrium of pregnant women
Mouse uterine tissues were obtained from the mice with term birth. Immunocytochemistry analysis showed that NLRP3 positive staining was found in endometrium and myometrium and localized in epithelial cells of endometrium and smooth muscle cells of myometrium (Fig. 1A). In myometrium of pregnant women at term, NLRP3 expression was predominantly localized in smooth muscle cells (Fig. 1B).
Localization of NLRP3 expression in pregnant mouse uterus and pregnant human myometrium. (A) The representative sections of NLRP3 expression in mouse uterus. Uterus tissues were obtained from pregnant mice at term birth. The sections of uterus were used for immunocytochemistry analysis of NLRP3. (B) The representative sections of NLRP3 expression in human myomentrium. Myometrium samples were obtained from healthy pregnant women at term. The tissue sections were used for NLRP3 immunocytochemistry analysis. Arrows indicate the positive staining of NLRP3.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
Localization of NLRP3 expression in pregnant mouse uterus and pregnant human myometrium. (A) The representative sections of NLRP3 expression in mouse uterus. Uterus tissues were obtained from pregnant mice at term birth. The sections of uterus were used for immunocytochemistry analysis of NLRP3. (B) The representative sections of NLRP3 expression in human myomentrium. Myometrium samples were obtained from healthy pregnant women at term. The tissue sections were used for NLRP3 immunocytochemistry analysis. Arrows indicate the positive staining of NLRP3.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
Localization of NLRP3 expression in pregnant mouse uterus and pregnant human myometrium. (A) The representative sections of NLRP3 expression in mouse uterus. Uterus tissues were obtained from pregnant mice at term birth. The sections of uterus were used for immunocytochemistry analysis of NLRP3. (B) The representative sections of NLRP3 expression in human myomentrium. Myometrium samples were obtained from healthy pregnant women at term. The tissue sections were used for NLRP3 immunocytochemistry analysis. Arrows indicate the positive staining of NLRP3.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
NLPR3 inflammasome activation in myometrium and fetal membranes of patients with term and preterm labor
As shown in Fig. 2A, B, C, and D, NLPR3 expression level was significantly increased in myometrium of TL group compared with that of TNL group (P < 0.05). Consistently, caspase-1 activity and IL-1β and IL-18 concentration were also significantly increased in TL myometrial tissues (P < 0.05).
NLRP3 expression, caspase-1 activity, and IL-1 and IL-18 concentration in myometrium and fetal membranes of pregnant women before and after the onset of labor at term and preterm. (A, B, C, and D) NLRP3 expression (A), caspase-1 activity (B) and IL-1 (C) and IL-18 (D) concentration in myometrium of TNL and TL groups. Myometrial samples were collected from pregnant women with or without labor at term. (E, F, G, and H) NLRP3 expression (E), caspase-1 activity (F) and IL-1 (G) and IL-18 (H) concentration in amnion of TNL,TL, and PTL with and without chorioamnionitis. Amnion samples were collected from patients not undergoing labor at term or undergoing labor at term and preterm. (J, K, L, and M) NLRP3 expression (J), caspase-1 activity (K) and IL-1 (L) and IL-18 (M) concentration in chorion-decidua of TNL,TL, and PTL with and without chorioamnionitis. Chorion-decidua samples were collected from patients not undergoing labor at term or undergoing labor at term and preterm. Western blotting analysis was performed to determine NLRP3 protein level. The levels of IL-1 and IL-18 were determined by ELISA. Caspase-1 activity was measured by the commercially available kit. Data were expressed as mean ± s.e.m. TNL: n = 12; TL: n = 12; PTL(+): PTL with chorioamnionitis, n = 11; PTL(−): PTL without chorioamnionitis, n = 12. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
NLRP3 expression, caspase-1 activity, and IL-1 and IL-18 concentration in myometrium and fetal membranes of pregnant women before and after the onset of labor at term and preterm. (A, B, C, and D) NLRP3 expression (A), caspase-1 activity (B) and IL-1 (C) and IL-18 (D) concentration in myometrium of TNL and TL groups. Myometrial samples were collected from pregnant women with or without labor at term. (E, F, G, and H) NLRP3 expression (E), caspase-1 activity (F) and IL-1 (G) and IL-18 (H) concentration in amnion of TNL,TL, and PTL with and without chorioamnionitis. Amnion samples were collected from patients not undergoing labor at term or undergoing labor at term and preterm. (J, K, L, and M) NLRP3 expression (J), caspase-1 activity (K) and IL-1 (L) and IL-18 (M) concentration in chorion-decidua of TNL,TL, and PTL with and without chorioamnionitis. Chorion-decidua samples were collected from patients not undergoing labor at term or undergoing labor at term and preterm. Western blotting analysis was performed to determine NLRP3 protein level. The levels of IL-1 and IL-18 were determined by ELISA. Caspase-1 activity was measured by the commercially available kit. Data were expressed as mean ± s.e.m. TNL: n = 12; TL: n = 12; PTL(+): PTL with chorioamnionitis, n = 11; PTL(−): PTL without chorioamnionitis, n = 12. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
NLRP3 expression, caspase-1 activity, and IL-1 and IL-18 concentration in myometrium and fetal membranes of pregnant women before and after the onset of labor at term and preterm. (A, B, C, and D) NLRP3 expression (A), caspase-1 activity (B) and IL-1 (C) and IL-18 (D) concentration in myometrium of TNL and TL groups. Myometrial samples were collected from pregnant women with or without labor at term. (E, F, G, and H) NLRP3 expression (E), caspase-1 activity (F) and IL-1 (G) and IL-18 (H) concentration in amnion of TNL,TL, and PTL with and without chorioamnionitis. Amnion samples were collected from patients not undergoing labor at term or undergoing labor at term and preterm. (J, K, L, and M) NLRP3 expression (J), caspase-1 activity (K) and IL-1 (L) and IL-18 (M) concentration in chorion-decidua of TNL,TL, and PTL with and without chorioamnionitis. Chorion-decidua samples were collected from patients not undergoing labor at term or undergoing labor at term and preterm. Western blotting analysis was performed to determine NLRP3 protein level. The levels of IL-1 and IL-18 were determined by ELISA. Caspase-1 activity was measured by the commercially available kit. Data were expressed as mean ± s.e.m. TNL: n = 12; TL: n = 12; PTL(+): PTL with chorioamnionitis, n = 11; PTL(−): PTL without chorioamnionitis, n = 12. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
We also determined NLPR3 activation in fetal membranes (Fig. 2E, F, G, H, I, J, K, and L). In amnion, NLPR3 expression level and caspase-1 activity were significantly increased in TL group compared with TNL group (P < 0.01). The levels of IL-1β and IL-18 were also increased compared with those of TNL group (P < 0.01). Given that some patients with PTB have chorioamnionitis, we divided PTL group into chorioamnionitis and non-chorioamnionitis. It was found that NLRP3 protein level and caspase-1 activity were significantly higher in PTL groups either with or without chorioamnionitis compared with those in TNL group (P < 0.01). IL-1β level was higher in PTL groups than those in TNL group (P < 0.05), and IL-18 level was also higher in PTL group compared with TNL group (P < 0.01). In chorion-decidua, NLPR3 expression level (P < 0.01), caspase-1 activity (P < 0.05), and IL-1β and IL-18 levels (P < 0.01) were significantly increased in TL group compared with TNL group. In the PTL groups, it was found that NLPR3 expression level and caspase1 activity were higher in patients either with or without chorioamnionitis than those in TNL group (P < 0.01). However, IL-1β and IL-18 levels were higher in PTL with chorioamnionitis (P < 0.05 and P < 0.01), but not in PTL without chorioamnionitis compared with TNL group.
Uterine NLPR3 inflammasome is activated in mice with term birth and RU38486- and LPS-induced preterm birth
At first, we examined NLRP3 activation in uterus in different timed pregnant mice. As shown in Fig. 3A, B and C, NLPR3 protein expression was increased by 43.84% at gd 19 compared with that at gd 15 (P < 0.0001) and reached the highest level at the time of onset of labor (P < 0.05 vs gd 15). Caspase-1 activity was increased by 34.21% at gd19 compared with that at gd 15 (P < 0.0001). It remained at high level at the time of onset of labor. Consistently, mature IL-1β and IL-18 levels showed a 4.84-fold increase (P < 0.01) and a 1.55-fold (P < 0.0001) increase at gd 19 compared with gd 15 respectively. They remained at high level at the time of onset of labor. We also examined NF-κB activation in uterus. Phospho-P65 Ser536 (p-P65) is the activated form of P65. As shown in Fig. 3D, p-P65 level was significantly increased by 91.12% at gd 19 compared with that at gd 15 (P < 0.05). At the time of onset of labor, it displayed an 8.15-fold increase (P < 0.0001 vs gd15).
Characterization of NLPR3 inflammasome and NK-κB activation as well as expression of UAPs in uterus of pregnant mice from gd 15 to labor. The pregnant mice were sacrificed at gd 15, 17, and 19 and at labor. Uterine tissues were obtained for determination of NLRP3 and NF-κB activation and the expression of UAPs. (A) The protein level of NLRP3 was determined by Western blotting analysis. Representative bands were listed on top of histogram. (B) Caspase-1 activity was determined by a commercial kit. (C, D, and E) IL-1β and IL-18 levels (C), p-P65 and P-65 levels (D), and the levels of UAPs were determined by Western blotting analysis. Representative bands were placed on the left of corresponding histogram. Data were expressed as mean ± s.e.m (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
Characterization of NLPR3 inflammasome and NK-κB activation as well as expression of UAPs in uterus of pregnant mice from gd 15 to labor. The pregnant mice were sacrificed at gd 15, 17, and 19 and at labor. Uterine tissues were obtained for determination of NLRP3 and NF-κB activation and the expression of UAPs. (A) The protein level of NLRP3 was determined by Western blotting analysis. Representative bands were listed on top of histogram. (B) Caspase-1 activity was determined by a commercial kit. (C, D, and E) IL-1β and IL-18 levels (C), p-P65 and P-65 levels (D), and the levels of UAPs were determined by Western blotting analysis. Representative bands were placed on the left of corresponding histogram. Data were expressed as mean ± s.e.m (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
Characterization of NLPR3 inflammasome and NK-κB activation as well as expression of UAPs in uterus of pregnant mice from gd 15 to labor. The pregnant mice were sacrificed at gd 15, 17, and 19 and at labor. Uterine tissues were obtained for determination of NLRP3 and NF-κB activation and the expression of UAPs. (A) The protein level of NLRP3 was determined by Western blotting analysis. Representative bands were listed on top of histogram. (B) Caspase-1 activity was determined by a commercial kit. (C, D, and E) IL-1β and IL-18 levels (C), p-P65 and P-65 levels (D), and the levels of UAPs were determined by Western blotting analysis. Representative bands were placed on the left of corresponding histogram. Data were expressed as mean ± s.e.m (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
The levels of uterine activated proteins (UAPs) including COX-2, OTR, and FP receptor (FP) are increased at term and during labor (Dudley et al. 1996, Lappas 2016). As shown in Fig. 3E, COX-2 and FP levels at gd 19 were elevated by 4.42- and 3.87-fold compared with those at gd 15 respectively (P < 0.0001). It remained at high level at the time of labor onset. OTR level was significantly increased by 3.38-fold at the time of labor onset compared with gd 15 (P < 0.001).
The pregnant mice received injection of RU38486 displayed onset of labor in 15.80 ± 0.46 h after injection (Fig. 4A). The NLPR3 expression level was increased by 6.16-fold (P < 0.0001) and caspase-1 activity was enhanced by 87.52% (P < 0.01) in mice with RU38486 treatment compared with control mice (Fig. 4B and C). Mature IL-1β level was increased by 3.87-fold (P < 0.0001) and IL-18 level was increased by 59.54% (P < 0.01) in these mice compared with control group (Fig. 4D). A 2.66-fold increase in p-P65 level (P < 0.001), a 3.68-fold increase in COX-2 level (P < 0.0001), a 3.15-fold increase in OTR (P < 0.001), and a 4.26-fold increase in FP expression (P < 0.0001) occurred in RU38486 group compared with control group (Fig. 4E and F).
RU38486 induces preterm birth and uterine NLPR3 inflammasome and NK-κB activation and increases uterine UAPs expression in pregnant mice. (A) Pregnant mice were administrated with RU38486 or vehicle at gd 15.5. The time of labor onset was determined when the first pup was delivered. (B, C, D, E, and F) Pregnant mice were administrated with RU38486 at gd 15.5. The mice with RU38486 were sacrificed after the onset of labor. The control mice were sacrificed at the time of corresponding RU38486 group. Uterine tissues were then collected. Uterine NLRP3 expression (B) was determined by Western blotting analysis. Representative bands were listed on top of histogram. Caspase-1 activity (C) was measured by a commercial kit. IL-1β and IL-18 levels (D), the levels of p-P65 and P-65 (E) and, the levels of UAPs (F) were determined by Western blotting analysis. Representative bands were placed on the left of corresponding histogram. Data were expressed as mean ± s.e.m (n = 13). **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
RU38486 induces preterm birth and uterine NLPR3 inflammasome and NK-κB activation and increases uterine UAPs expression in pregnant mice. (A) Pregnant mice were administrated with RU38486 or vehicle at gd 15.5. The time of labor onset was determined when the first pup was delivered. (B, C, D, E, and F) Pregnant mice were administrated with RU38486 at gd 15.5. The mice with RU38486 were sacrificed after the onset of labor. The control mice were sacrificed at the time of corresponding RU38486 group. Uterine tissues were then collected. Uterine NLRP3 expression (B) was determined by Western blotting analysis. Representative bands were listed on top of histogram. Caspase-1 activity (C) was measured by a commercial kit. IL-1β and IL-18 levels (D), the levels of p-P65 and P-65 (E) and, the levels of UAPs (F) were determined by Western blotting analysis. Representative bands were placed on the left of corresponding histogram. Data were expressed as mean ± s.e.m (n = 13). **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
RU38486 induces preterm birth and uterine NLPR3 inflammasome and NK-κB activation and increases uterine UAPs expression in pregnant mice. (A) Pregnant mice were administrated with RU38486 or vehicle at gd 15.5. The time of labor onset was determined when the first pup was delivered. (B, C, D, E, and F) Pregnant mice were administrated with RU38486 at gd 15.5. The mice with RU38486 were sacrificed after the onset of labor. The control mice were sacrificed at the time of corresponding RU38486 group. Uterine tissues were then collected. Uterine NLRP3 expression (B) was determined by Western blotting analysis. Representative bands were listed on top of histogram. Caspase-1 activity (C) was measured by a commercial kit. IL-1β and IL-18 levels (D), the levels of p-P65 and P-65 (E) and, the levels of UAPs (F) were determined by Western blotting analysis. Representative bands were placed on the left of corresponding histogram. Data were expressed as mean ± s.e.m (n = 13). **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
As expected, LPS treatment induced preterm delivery in 10.56 ± 0.43 h after injection (Fig. 5A). LPS treatment resulted in 7.25-fold increase in NLPR3 expression compared with vehicle treatment (P < 0.0001, Fig. 5B). Mature IL-1β level was increased by 6.29-fold (P < 0.0001), and IL-18 was increased by 43.07% (P < 0.001) (Fig. 5C). The levels of p-P65, COX-2, OTR, and FP were increased by 6.61-, 12.87-, 8.26-, and 7.22-fold, respectively (P < 0.0001, Fig. 5D and E).
LPS induces preterm birth and uterine NLPR3 inflammasome and NK-κB activation and stimulates uterine UAPs expression in pregnant mice. (A) Pregnant mice were administrated with LPS or saline at gd 15. The time of labor onset was determined when the first pup was delivered. (B, C, D, and E) Pregnant mice were administrated with LPS at gd 15. The mice with LPS were sacrificed after the onset of labor. The control mice were administrated same volume of saline and sacrificed at the time of corresponding LPS group. Myometrium tissues were collected. Uterine NLRP3 expression (B), IL-1β and IL-18 levels (C), p-P65 and P-65 levels (D), and expression of UAPs (E) were determined by Western blotting analysis. Representative bands were placed on the top of histogram or the left of the corresponding histogram. Data were expressed as mean ± s.e.m (n = 10). ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
LPS induces preterm birth and uterine NLPR3 inflammasome and NK-κB activation and stimulates uterine UAPs expression in pregnant mice. (A) Pregnant mice were administrated with LPS or saline at gd 15. The time of labor onset was determined when the first pup was delivered. (B, C, D, and E) Pregnant mice were administrated with LPS at gd 15. The mice with LPS were sacrificed after the onset of labor. The control mice were administrated same volume of saline and sacrificed at the time of corresponding LPS group. Myometrium tissues were collected. Uterine NLRP3 expression (B), IL-1β and IL-18 levels (C), p-P65 and P-65 levels (D), and expression of UAPs (E) were determined by Western blotting analysis. Representative bands were placed on the top of histogram or the left of the corresponding histogram. Data were expressed as mean ± s.e.m (n = 10). ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
LPS induces preterm birth and uterine NLPR3 inflammasome and NK-κB activation and stimulates uterine UAPs expression in pregnant mice. (A) Pregnant mice were administrated with LPS or saline at gd 15. The time of labor onset was determined when the first pup was delivered. (B, C, D, and E) Pregnant mice were administrated with LPS at gd 15. The mice with LPS were sacrificed after the onset of labor. The control mice were administrated same volume of saline and sacrificed at the time of corresponding LPS group. Myometrium tissues were collected. Uterine NLRP3 expression (B), IL-1β and IL-18 levels (C), p-P65 and P-65 levels (D), and expression of UAPs (E) were determined by Western blotting analysis. Representative bands were placed on the top of histogram or the left of the corresponding histogram. Data were expressed as mean ± s.e.m (n = 10). ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
BAY11-7082 and MCC950 treatment delays term birth and inhibits uterine NLRP3 inflammasome activation in mice
BAY11-7082, a NF-κB inhibitor, can act as an inhibitor of NLPR3 inflammasome (Juliana et al. 2010). As shown in Fig. 6, BAY11-7082 treatment significantly delayed the time of term birth from 19.23 ± 0.01992 days to 19.54 ± 0.04543 days (P < 0.01). Among them, 30.7% (4/13) mice showed delayed labor onset over 12 h above the mean value. NLRP3 level, caspase-1 activity, and IL-1β and IL-18 concentration were significantly decreased in the mice with BAY11-7082 treatment compared with those with vehicle treatment (P < 0.05).
The effects of BAY11-7082 on gestational length and uterine NLRP3 inflammasome activation in pregnant mice. (A) Pregnant mice were administrated with BAY11-7082 (i.p.) on gd 18.5. Control mice were injected with same volume of saline. The time of labor onset was determined when the first pup was delivered. (B, C, D, and E) Pregnant mice were administrated with BAY11-7082 (s.c.) or saline at gd18.5. The mice were sacrificed after the onset of labor. The NLRP3 expression was determined by Western blotting analysis (B). Caspase-1 activity was measured by the commercially available kit (C). The levels of IL-1 (D) and IL-18 (E) were determined by ELISA. Data were expressed as mean ± s.e.m (n = 13). *P < 0.05, ****P < 0.0001. BAY: BAY11-7082.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
The effects of BAY11-7082 on gestational length and uterine NLRP3 inflammasome activation in pregnant mice. (A) Pregnant mice were administrated with BAY11-7082 (i.p.) on gd 18.5. Control mice were injected with same volume of saline. The time of labor onset was determined when the first pup was delivered. (B, C, D, and E) Pregnant mice were administrated with BAY11-7082 (s.c.) or saline at gd18.5. The mice were sacrificed after the onset of labor. The NLRP3 expression was determined by Western blotting analysis (B). Caspase-1 activity was measured by the commercially available kit (C). The levels of IL-1 (D) and IL-18 (E) were determined by ELISA. Data were expressed as mean ± s.e.m (n = 13). *P < 0.05, ****P < 0.0001. BAY: BAY11-7082.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
The effects of BAY11-7082 on gestational length and uterine NLRP3 inflammasome activation in pregnant mice. (A) Pregnant mice were administrated with BAY11-7082 (i.p.) on gd 18.5. Control mice were injected with same volume of saline. The time of labor onset was determined when the first pup was delivered. (B, C, D, and E) Pregnant mice were administrated with BAY11-7082 (s.c.) or saline at gd18.5. The mice were sacrificed after the onset of labor. The NLRP3 expression was determined by Western blotting analysis (B). Caspase-1 activity was measured by the commercially available kit (C). The levels of IL-1 (D) and IL-18 (E) were determined by ELISA. Data were expressed as mean ± s.e.m (n = 13). *P < 0.05, ****P < 0.0001. BAY: BAY11-7082.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
We then examined the effect of specific NLRP3 inflammasome inhibitor MCC950 on term birth. As shown in Fig. 7, the mice received MCC950 administration at gd 18.5 exhibited delayed onset of parturition from 19.22 ± 0.02637 days to 19.98 ± 0.2117 days (P < 0.01). Among them, 40% (4/10) mice exhibited delayed timing of birth over 12 h above the mean value, and 30% (3/10) dam had dystocia. Compared with vehicle treatment, MCC950 treatment led to a 47.59% decrease in NLRP3 level (P < 0.001), a 37.07% decrease in caspase-1 activity (P < 0.05), and a 54.07% decrease in mature IL-1β level (P < 0.01) and a 32.86% decrease in mature IL-18 level (P < 0.05). It was also found that p-P65, COX-2, OTR, and FP levels were suppressed by 45.85 (P < 0.05), 80.57 (P < 0.0001), 65.64 (P < 0.001), 77.62% (P < 0.001) respectively.
The effects of MCC950 on gestational length, NLRP3 inflammasome and NF-κB activation, and UAPs expression in pregnant mice. (A) Pregnant mice were administrated with MCC950 (i.p.) at gd 18.5. Control mice were injected with same volume of saline. The time of labor onset was determined when the first pup was delivered. (B, C, D, E, and F) Pregnant mice were administrated with MCC950 (i.p.) or saline at gd 18.5. The mice were sacrificed after the onset of labor. Uterine tissues were obtained. NLRP3 expression was determined by Western blotting analysis (B). Representative bands were listed on top of histogram. Caspase-1 activity was determined by a commercial kit (C). IL-1β and IL-18 levels (D), p-P65 and P-65 levels (E), and UAPs expression (F) were determined by Western blotting analysis. Representative bands were placed on the left of corresponding histogram. Data were expressed as mean ± s.e.m (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
The effects of MCC950 on gestational length, NLRP3 inflammasome and NF-κB activation, and UAPs expression in pregnant mice. (A) Pregnant mice were administrated with MCC950 (i.p.) at gd 18.5. Control mice were injected with same volume of saline. The time of labor onset was determined when the first pup was delivered. (B, C, D, E, and F) Pregnant mice were administrated with MCC950 (i.p.) or saline at gd 18.5. The mice were sacrificed after the onset of labor. Uterine tissues were obtained. NLRP3 expression was determined by Western blotting analysis (B). Representative bands were listed on top of histogram. Caspase-1 activity was determined by a commercial kit (C). IL-1β and IL-18 levels (D), p-P65 and P-65 levels (E), and UAPs expression (F) were determined by Western blotting analysis. Representative bands were placed on the left of corresponding histogram. Data were expressed as mean ± s.e.m (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
The effects of MCC950 on gestational length, NLRP3 inflammasome and NF-κB activation, and UAPs expression in pregnant mice. (A) Pregnant mice were administrated with MCC950 (i.p.) at gd 18.5. Control mice were injected with same volume of saline. The time of labor onset was determined when the first pup was delivered. (B, C, D, E, and F) Pregnant mice were administrated with MCC950 (i.p.) or saline at gd 18.5. The mice were sacrificed after the onset of labor. Uterine tissues were obtained. NLRP3 expression was determined by Western blotting analysis (B). Representative bands were listed on top of histogram. Caspase-1 activity was determined by a commercial kit (C). IL-1β and IL-18 levels (D), p-P65 and P-65 levels (E), and UAPs expression (F) were determined by Western blotting analysis. Representative bands were placed on the left of corresponding histogram. Data were expressed as mean ± s.e.m (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
MCC950 delays RU38486- and LPS-induced preterm birth and inhibits uterine NLRP3 inflammasome and NF-κB activation in mice
The pregnant mice received RU38486 in combination with BAY11-7082 or MCC950 treatment at gd15.5. As shown in Fig. 8A, BAY11-7082 treatment could postpone birth time by 7.95 ± 1.43 h in RU38486 mice (P < 0.05). Among them, 46.15% mice (6/13) exhibited delayed birth time over 8 h above the mean value of RU38486 group (P < 0.05), and 15.38 % mice (2/13) showed delayed birth time over 12 h. NLRP3 level, caspase-1 activity, and IL-1β and IL-18 concentration were significantly decreased by 73.59, 56.08, 43.82 and 45.19% respectively (P < 0.01, Fig. 8B, C, D and E).
The effects of BAY11-7082 on RU38486-induced preterm birth and uterine NLRP3 inflammasome activation in pregnant mice. Pregnant mice were administrated with RU38486 plus BAY11-7082 at gd 15.5. The control mice were administered with RU38486 plus saline. The time of labor onset was determined when the first pup was delivered (A). The mice were sacrificed after the onset of labor. Uterine tissues were collected. NLRP3 expression was determined by Western blotting analysis (B). Representative bands were placed on the top of histogram. Caspase-1 activity was measured by a commercially available kit (C). The levels of IL-1 (D) and IL-18 (E) were determined by ELISA. Data were expressed as mean ± s.e.m (n = 13). **P < 0.01, ***P < 0.001, ****P < 0.0001. BAY: BAY11-7082.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
The effects of BAY11-7082 on RU38486-induced preterm birth and uterine NLRP3 inflammasome activation in pregnant mice. Pregnant mice were administrated with RU38486 plus BAY11-7082 at gd 15.5. The control mice were administered with RU38486 plus saline. The time of labor onset was determined when the first pup was delivered (A). The mice were sacrificed after the onset of labor. Uterine tissues were collected. NLRP3 expression was determined by Western blotting analysis (B). Representative bands were placed on the top of histogram. Caspase-1 activity was measured by a commercially available kit (C). The levels of IL-1 (D) and IL-18 (E) were determined by ELISA. Data were expressed as mean ± s.e.m (n = 13). **P < 0.01, ***P < 0.001, ****P < 0.0001. BAY: BAY11-7082.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
The effects of BAY11-7082 on RU38486-induced preterm birth and uterine NLRP3 inflammasome activation in pregnant mice. Pregnant mice were administrated with RU38486 plus BAY11-7082 at gd 15.5. The control mice were administered with RU38486 plus saline. The time of labor onset was determined when the first pup was delivered (A). The mice were sacrificed after the onset of labor. Uterine tissues were collected. NLRP3 expression was determined by Western blotting analysis (B). Representative bands were placed on the top of histogram. Caspase-1 activity was measured by a commercially available kit (C). The levels of IL-1 (D) and IL-18 (E) were determined by ELISA. Data were expressed as mean ± s.e.m (n = 13). **P < 0.01, ***P < 0.001, ****P < 0.0001. BAY: BAY11-7082.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
MCC950 treatment could also delay birth time by 7.09 ± 2.41 h compared with saline treatment in RU38486 treatment (P < 0.01, Fig. 9A). Among the mice with MCC950 treatment, 28.57% mice (4/14) exhibited delayed birth time over 8 h (P < 0.05), and 21.43% mice (3/14) were delayed over 12 h above the mean value of RU38486 group. Three mice had dystocia. Uterine NLRP3 level, caspase-1 activity, and mature IL-1β and IL-18 levels were decreased by 73.59 (P < 0.01), 56.08, (P < 0.05), 43.82 (P < 0.001), and 45.19 (P < 0.01) respectively (Fig. 9B, C and D). The levels of p-P65, COX-2, OTR, and FP were reduced by 59.12 (P < 0.01), 71.48 (P < 0.0001), 71.48 (P < 0.001), 61.40 (P < 0.001), and 74.8 (P < 0.0001) respectively (Fig. 9E and F).
The effects of MCC950 on RU38486-induced preterm birth, uterine NLRP3 inflammasome and NF-κB activation, and uterine UAPs expression in pregnant mice. Pregnant mice were administrated with RU38486 plus saline (n = 15) or RU38486 plus MCC950 (n = 14) at gd15.5. The time of labor onset was determined when the first pup was delivered (A). The mice were sacrificed after the onset of labor. Uterine tissues were then obtained. NLRP3 expression was determined by Western blotting analysis (B). Representative bands were placed on the top of histogram. Caspase-1 activity was measured by a specific kit (C). IL-1β and IL-18 levels (D), p-P65 and P-65 levels (E), and UAPs expression (F) were determined by Western blotting analysis. Representative bands were placed on the left of the corresponding histogram. Data were expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
The effects of MCC950 on RU38486-induced preterm birth, uterine NLRP3 inflammasome and NF-κB activation, and uterine UAPs expression in pregnant mice. Pregnant mice were administrated with RU38486 plus saline (n = 15) or RU38486 plus MCC950 (n = 14) at gd15.5. The time of labor onset was determined when the first pup was delivered (A). The mice were sacrificed after the onset of labor. Uterine tissues were then obtained. NLRP3 expression was determined by Western blotting analysis (B). Representative bands were placed on the top of histogram. Caspase-1 activity was measured by a specific kit (C). IL-1β and IL-18 levels (D), p-P65 and P-65 levels (E), and UAPs expression (F) were determined by Western blotting analysis. Representative bands were placed on the left of the corresponding histogram. Data were expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
The effects of MCC950 on RU38486-induced preterm birth, uterine NLRP3 inflammasome and NF-κB activation, and uterine UAPs expression in pregnant mice. Pregnant mice were administrated with RU38486 plus saline (n = 15) or RU38486 plus MCC950 (n = 14) at gd15.5. The time of labor onset was determined when the first pup was delivered (A). The mice were sacrificed after the onset of labor. Uterine tissues were then obtained. NLRP3 expression was determined by Western blotting analysis (B). Representative bands were placed on the top of histogram. Caspase-1 activity was measured by a specific kit (C). IL-1β and IL-18 levels (D), p-P65 and P-65 levels (E), and UAPs expression (F) were determined by Western blotting analysis. Representative bands were placed on the left of the corresponding histogram. Data were expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
MCC950 treatment postponed LPS-induced PTB by 8.01 ± 3.7 h (P < 0.05, Fig. 10A). Among them, 40% mice (4/10) exhibited delayed birth time over 8 h, and 30% mice (3/10) exhibited delayed birth time over 12 h above the mean value of LPS group. Two dam exhibited dystocia. MCC950 treatment significantly suppressed NLRP3 expression by 27.75% (P < 0.01) and the levels of COX-2, OTR, and FP by 60.97 (P < 0.001) ,15.47 (P < 0.05) , and 57.4% (P < 0.01) respectively (Fig. 10B, C, D, and E). In contrast, IL-1β, IL-18, and p-P65 levels were not significantly changed.
The effects of MCC950 on LPS-induced preterm birth, uterine NLRP3 inflammasome and NF-κB activation, and uterine UAPs expression in pregnant mice. Pregnant mice were administrated with LPS plus saline or LPS plus MCC950 at gd 15. The time of labor onset was determined when the first pup was delivered (A). The mice were sacrificed after the onset of labor. Uterine tissues were obtained. NLRP3 expression (B), IL-1β and IL-18 levels (C), p-P65 and P-65 levels (D), and expression of COX2, OTR, and FP (E) were determined by Western blotting analysis. Representative bands were placed on the top of histogram or the left of the corresponding histogram. Data were expressed as mean ± s.e.m (n =10). *P < 0.05, **P < 0.01, ***P < 0.001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
The effects of MCC950 on LPS-induced preterm birth, uterine NLRP3 inflammasome and NF-κB activation, and uterine UAPs expression in pregnant mice. Pregnant mice were administrated with LPS plus saline or LPS plus MCC950 at gd 15. The time of labor onset was determined when the first pup was delivered (A). The mice were sacrificed after the onset of labor. Uterine tissues were obtained. NLRP3 expression (B), IL-1β and IL-18 levels (C), p-P65 and P-65 levels (D), and expression of COX2, OTR, and FP (E) were determined by Western blotting analysis. Representative bands were placed on the top of histogram or the left of the corresponding histogram. Data were expressed as mean ± s.e.m (n =10). *P < 0.05, **P < 0.01, ***P < 0.001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
The effects of MCC950 on LPS-induced preterm birth, uterine NLRP3 inflammasome and NF-κB activation, and uterine UAPs expression in pregnant mice. Pregnant mice were administrated with LPS plus saline or LPS plus MCC950 at gd 15. The time of labor onset was determined when the first pup was delivered (A). The mice were sacrificed after the onset of labor. Uterine tissues were obtained. NLRP3 expression (B), IL-1β and IL-18 levels (C), p-P65 and P-65 levels (D), and expression of COX2, OTR, and FP (E) were determined by Western blotting analysis. Representative bands were placed on the top of histogram or the left of the corresponding histogram. Data were expressed as mean ± s.e.m (n =10). *P < 0.05, **P < 0.01, ***P < 0.001.
Citation: Reproduction 162, 6; 10.1530/REP-21-0047
Discussion
In this study, we have provided the evidence that NLRP3 inflammasome plays an important role in the initiation of term and PTB in the mouse model. Moreover, we have demonstrated that NLRP3 inflammasome is activated in myometrium and fetal membranes from the pregnant women with onset of labor at term and preterm.
As mentioned, the parturition is an event of inflammation in the gestational tissues. Many studies have demonstrated that the levels of pro-inflammatory cytokines are increased in myometrium and fetal membranes in humans (Gilman-Sachs et al. 2018, Romero et al. 2007, Green & Arck 2020, Young et al. 2002, Leimert et al. 2019). Consistently, we also found that pro-inflammatory cytokine IL-1β and IL-18 concentration was significantly increased in myometrium of TL group compared with that of TNL group. Similar data were obtained in amnion and chorion-decidua. Furthermore, we revealed that IL-1β and IL-18 level in fetal membranes was also elevated in the patients with PTB no matter with or without chorioamnionitis. Given that chorioamnionitis is usually associated with uterine infection, these data suggest that uterine inflammation is existed in PTB with and without infection. In the animal model, the concentration of IL-1β and IL-18 protein and p-P65 level were increased in term birth and RU38486-induced PTB. Altogether, our data support the concept that inflammatory responses occur in the gestational tissues in the processes of parturition.
NLRP3 inflammasome is involved in various physiological and pathophysiological processes because it is one of key players in inflammatory cascades (Wang et al. 2020). Prior studies have demonstrated that NLRP3 inflammasome is activated in gestational tissues, such as fetal membrane, after onset of TL and PTL in humans (Gomez-Lopez et al. 2017, 2018, Romero et al. 2018). In this study, we have shown that NLRP3 inflammasome activation occurred in amnion and chorion-decidua at preterm and term birth in humans. Of note, we showed that NLRP3 inflammasome in fetal membranes is activated in the patients either with or without histological chorioamnionitis. These data are consistent with prior studies by Gomez-Lopez’s group (Gomez-Lopez et al. 2017, 2018, Romero et al. 2018, Faro et al. 2019), suggesting that NLPR3 inflammasome is involved in intrauterine inflammatory response in PTB with and without infection. We also found that NLPR3 inflammasome was activated in the myometrial tissues after the onset of labor in human pregnancy at term. Using the approach of transcriptomics, Migale et al. have shown that human labor is most relevant to LPS-induced PTB in mice, and progesterone withdrawal initiates labor without inflammatory gene activation (Migale et al. 2016). In this study, we did find that uterine NLRP3 inflammasome was activated in the mice of LPS-induced PTB, which is consistent to the findings in human myometrium. However, we also found that NLRP3 inflammasome as well as NF-κB are activated in uterus of the mice with RU38486-induced PTB.
Recently, Gomez-Lopez et al. reported that MCC950, a specific NLRP3 inflammasome inhibitor, could prevent the PTB induced by intraamniotic administration of S100B, indicating that NLRP3 inflammasome is involved in PTB caused by sterile intraamniotic inflammation (Gomez-Lopez et al. 2019b). In this study, we revealed that MCC950 treatment inhibited RU38486- and LPS-induced PTB in mice and suppressed uterine NLRP3 inflammasome activation. Together, the above data provide the evidence that NLRP3 inflammasome play a key role in the initiation of labor by various stimuli. Moreover, this study also showed that BAY11-7082 and MCC950 could also postpone the time of onset of labor at term, and concomitantly decreased NLRP3 activation in uterus, suggesting that NLRP3 inflammasome also play an important role in the onset of term birth.
Activated NF-κB is one of the key indicators of inflammatory responses. In consistence with prior studies, it was found that uterine p-P65 level was increased at term birth and LPS- and RU38486-induced PTB. NF-κB can be activated by various signaling pathways, such as toll-like receptor signaling and pro-inflammatory cytokines (Kawai & Akira 2007, Oeckinghaus et al. 2011). As a result of transcriptional signaling, NF-κB can induce NLRP3 expression (Kelley et al. 2019). Thus, positive feedback loop existed between NLRP3 inflammasome and NF-κB activation, that is, NF-κB activation can lead to NLRP3 inflammasome activation and subsequently promote IL-1β and Il-18 secretion. In turn, IL-1β further leads to NF-κB activation. Thus, it is easily understood that both of BAY11-7082 and MCC950 treatment suppress both of NLRP3 inflammasome and NF-κB activation in uterus.
We found that both of BAY 11-7082 and MCC950 treatment could only partly postpone the birth time at term in mice, that is, BAY 11-7082 could postpone birth time over 12 h in 30.7% mice, while MCC950 postponed birth time over 12 h in 40% mice of term birth. Although BAY11-7082 and MCC950 treatment postponed the onset of labor by more than 7 h in RU38486-induced PTB, 46.15 and 28.57% mice showed delayed birth time over 8 h. Similarly, MCC950 treatment could postpone birth time by 8 h in LPS-induced PTB, but 40% mice exhibited delayed birth time more than 8 h. Thus, it seems that NLRP3 inflammasome inhibitors do not fully block the initiation of labor onset in term and PTB. Of note, MCC950 treatment led to suppression of uterine NLRP3 expression but not IL-1β, IL-18, and p-P65 levels in LPS-treated mice, suggesting that uterine inflammation is not suppressed upon MCC950 treatment at current dosage. Thus, the inhibitory effects of NLRP3 inflammasome inhibitors on onset of parturition might be attributed to that the amount of BAY11-7082 and MCC950 within uterine tissues is not sufficient although BAY11-7082 and MCC950 treatment could result in suppression of uterine NLRP3 inflammasome. However, these data might also indicate that other inflammatory mechanisms also contribute to the parturition processes. Given that inflammation mainly occurs in intrauterine tissues during pregnancy, the experiments of specific inhibition of NLRP3 inflammasome activation in intrauterine tissues should be done in the future studies.
There are a number of limitations in this study. For instance, cell types responsible for NLRP3 inflammasome activation in uterus remain to be elucidated. Many studies have shown that uterine smooth muscle cells are able to secrete pro-inflammatory cytokines (You et al. 2014, Shynlova et al. 2020). As NLRP3 is identified in smooth muscle cells, NLRP3 inflammasome in smooth muscle cells would contribute to uterine activation for labor. However, other cells such as immune cells in uterus cannot be excluded. As immune cells recruited are critical for inflammatory cascades in uterus in onset of parturition (Gomez-Lopez et al. 2014), the link of immune cell recruitment in the uterus and the NLRP3 inflammasome should also be clarified.
In conclusion, this study has provided the evidence that NLRP3 inflammasome is involved in the initiation of parturition in term birth and PTB in humans and mouse model.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by Natural Science Foundation of China (Nos. 81620108013, 31971892, 31771667, and 31800988), Sate Key Research and Development Program of China (2017YFC1001404 and 2018YFC1002802), Science and Technology Commission of Shanghai Municipals (1814090300), and Hunan Provincial Science and Technology Department (2018RS3030).
Author contribution statement
Xin Ni conceived and designed the study. Zixi Chen and Yali Shan performed the majority of the experiments. Xingji You, Chen Xu, and Jing Long participated in some experiments. Hang Gu performed the collections of human tissues.
Acknowledgement
The authors wish to thank all the patients participated in this study.
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