Evidence of the involvement of caspase-1 under physiologic and pathologic cellular stress during human pregnancy: A link between the inflammasome and parturition

Abstract

Objective. Caspase-1 is a component of the NALP3 inflammasome, a cytosolic multiprotein complex that mediates the processing of pro-inflammatory caspases and cytokines. The inflammasome represents the first line of defense against cellular stress and is a crucial component of innate immunity. Caspase-1 is the enzyme responsible for the cleavage and activation of interleukin (IL)-1β, which is a potent pro-inflammatory cytokine, and plays a central role in the mechanisms leading to labor (preterm and term) particularly in the context of intrauterine infection/inflammation. In addition, caspase-1 cleaves IL-18 and IL-33. The objectives of this study were to determine whether there is a relationship between amniotic fluid concentrations of caspase-1 and gestational age, parturition (term and preterm), and intra-amniotic infection/inflammation (IAI).

Study design. A cross-sectional study was conducted including 143 pregnant women in the following groups: (1) mid-trimester of pregnancy (n = 18); (2) term not in labor (n = 25); (3) term in labor (n = 28); (4) preterm labor (PTL) who delivered at term (n = 23); (5) PTL without IAI who delivered preterm (n = 32); (6) PTL with IAI who delivered preterm neonates (n = 17). Caspase-1 concentrations in amniotic fluid were determined by a specific and sensitive immunoassay. Non-parametric statistics were used for analysis.

Results. (1) Caspase-1 was detected in amniotic fluid of women at term, but in none of the mid-trimester samples. (2) Patients in labor at term had a significantly higher median amniotic fluid concentration of caspase-1 than women at term not in labor (term in labor: 10.5 pg/mL, range 0.0–666.0 vs. term not in labor: 5.99 pg/mL, range 0.0–237.4; p < 0.05). (3) Among patients with spontaneous PTL, those with IAI (median 41.4 pg/mL, range 0.0–515.0) had a significantly higher median amniotic fluid caspase-1 concentration than those without IAI who delivered preterm (median 0.0 pg/mL, range 0.0–78.4) and than those who delivered at term (median 0.0 pg/mL, range 0.0–199.5); p < 0.001 for both comparisons.

Conclusions. (1) The presence and concentration of caspase-1 in the amniotic fluid varies as a function of gestational age. (2) Women with spontaneous labor at term had a higher median caspase-1 amniotic fluid concentration than women at term without labor. This suggests that the inflammasome may be activated in spontaneous parturition at term. Since most women with labor do not have intra-amniotic infection, we propose that cellular stress during labor accounts for activation of the inflammasome. (3) Preterm labor associated with infection/inflammation was also associated with a high concentration of caspase-1, suggesting that infection may induce caspase-1 production and activation of the inflammasome. (4) The sequential activation of the inflammasome and caspase-1, leading to interleukin-1β processing and secretion, is a candidate pathway leading to the activation of the common pathway of parturition.

Keywords:

• Interleukin-1 converting enzyme
• labor
• delivery
• intra-amniotic infection
• intra-amniotic inflammation
• chorioamnionitis
• preterm birth
• inflammasome

Introduction

Caspase-1 [1],[2], also known as interleukin-1β converting enzyme (ICE) [3],[4], is a cysteine-aspartic protease[5], promoting the processing, secretion, and release of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-18, and IL-33 [6-8]. Caspase-1 is the prototype of the inflammatory caspases, and a key component of the inflammasome [8-10], which has been recently defined as a first line of the immune response to cell stress [10], as well as a ‘master switch of inflammation’[11]. The inflammasome, indeed, is an inducible multiprotein cytosolic complex [8], assembling upon recognition of ‘danger signals’ by intracellular sensors, including the NOD-like receptors [12], or pathogen-associated compounds [10]. The inflammasome, in turn, activates inflammatory caspases [8], of which caspase-1 is the prototype, and promotes the processing, secretion, and release of pro-inflammatory cytokines, including IL-1β, IL-18, and IL-33 [6-8],[10].

Term and preterm labor are inflammatory processes [13-21], in which inflammatory mediators including cytokines such as IL-1 [22-26], IL-6 [22],[27-30], tumor necrosis factor [27],[31-36], IL-18 [37], IL-16 [38], and chemokines [39-45] play a central role. Of note, labor is associated with increased concentrations of IL-1β in both gestational tissues [46-48] and biological fluids [13],[49], and intra-amniotic and/or systemic administration of IL-1β to mice and monkeys induces preterm labor [24],[50],[51].

Considering the pivotal role of caspase-1 as a link between the activation of the inflammasome and the processing of IL-1β, we conducted this study to determine whether there is a relationship between amniotic fluid concentrations of caspase-1 and gestational age, parturition (term and preterm), and intra-amniotic infection/inflammation (IAI).

Materials and methods

Study design

This cross-sectional study was designed to examine the relationship among amniotic fluid concentrations of caspase-1 and gestational age, spontaneous labor at term, preterm parturition, and IAI.

Amniotic fluid was collected by transabdominal amniocentesis from 143 singleton pregnancies in the following groups: (1) Patients undergoing mid-trimester amniocentesis for clinical indications who delivered at term (n = 18); (2) women at term not in labor (n = 25); (3) patients at term in labor (n = 28); (4) women with preterm labor (PTL) and intact membranes who delivered at term (n = 23); (5) women in PTL without IAI who delivered preterm (n = 32); (6) women with PTL and IAI who delivered preterm (n = 17).

Clinical definitions

Patients were considered to have a normal pregnancy if they did not have an obstetrical, medical, or surgical complication of pregnancy and delivered a term (≥37 weeks) neonate. PTL was defined in the presence of regular uterine contractions occurring at a frequency of at least two every 10 minutes, combined with documented cervical changes prior to 37 weeks of gestation. Intra-amniotic infection was defined by a positive amniotic fluid culture for microorganisms. Intra-amniotic inflammation was defined in the presence of a white blood cell (WBC) count of > 100 cells/mm³.

Amniotic fluid sample collection and analysis

Amniotic fluid was collected by transabdominal amniocentesis. Fluid not required for clinical purposes was centrifuged to remove cellular and particulate matter and aliquots stored at −70°C until analysis. Amniotic fluid WBC count, Gram stain, and glucose concentrations were used in the management of patients with preterm labor. Amniotic fluid samples from the term and preterm groups were cultured for aerobic and anaerobic species and genital mycoplasmas (Ureaplasma urealyticum and Mycoplasma hominis). Pregnant patients were enrolled at Hutzel Hospital, Detroit, MI, USA, Pennsylvania Hospital, Philadelphia, Pennsylvania, USA, and Sotero del Rio Hospital, Puente Alto, Chile. The collection of samples for research was approved by the institutional review boards of Wayne State University, Pennsylvania Hospital, and Sotero del Rio Hospital, as well as the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services (NIH/DHHS). All participants provided written informed consent for collection of clinical data and biological materials. Many of these samples have been previously employed to study the biology of inflammation, hemostasis, angiogenesis regulation, and growth factor concentrations in non-pregnant women, normal pregnant women, and those with complications.

Human caspase-1 immunoassays in amniotic fluid

A specific and sensitive immunoassay was used to determine the concentration of caspase-1 in amniotic fluid samples. Human caspase-1 immunoassay kits (Cistron Biotechnology, Pine Brook, NJ, USA) were validated for human amniotic fluid in our laboratory prior to the conduction of this study. Validation included spike and recovery experiments, which produced parallel curves indicating that amniotic fluid constituents did not interfere with antigen–antibody binding in this assay system. The inter-assay and intra-assay coefficients of variation for the caspase-1 immunoassay were 7.9% and 4.7%, respectively. The sensitivity was 3.54 pg/mL.

Statistical analysis

Data were tested for normality using the Kolmogorov–Smirnov test. Kruskal–Wallis was used for analysis of variance. Comparisons between two groups were performed using Mann–Whitney rank sum tests. A p value < 0.05 was considered statistically significant. We used SPSS v.14.0 (SPSS Inc., Chicago, IL, USA) for analysis.

Results

One hundred and forty-three patients were included in this study. The demographic and clinical characteristics of patients with PTL are shown in Table I.

Table I.  Demographic and clinical characteristics of patients with preterm labor.

	Gestational age at amniocentesis (weeks)	Gestational age at delivery (weeks)	Birth weight (grams)	Maternal age (years)
PTL delivered at term	31.8 (28.6–33.8)	38.7 (37.9–40.1)	3020 (2640–3317)	20 (18–22)
PTL delivered preterm, no IAI	28.4 (23.2–31.3)	34.2 (31–35.7)	1956 (1730–2570)	24 (22–28)
PTL delivered preterm with IAI	26.6 (24.4–32.2)	26.9 (25.5–32.8)	960 (795–1964)	26 (23–32.5)

PTL, preterm labor; IAI, intra-amniotic infection/inflammation. Results are median (inter-quartile range).

Caspase-1 in amniotic fluid

Caspase-1 was detectable in 56.6% (81/143) of the amniotic fluid samples. Of interest, caspase-1 was detected in 76% (19/25) of women at term not in labor, but in none of the mid-trimester samples.

Spontaneous labor at term is associated with higher amniotic fluid concentrations of caspase-1

The median amniotic fluid caspase-1 concentration was significantly higher in women in spontaneous labor at term than in those not in labor at term (term in labor: median 10.5 pg/mL, range 0.0–666 vs. term not in labor: median 5.99 pg/mL, range 0.0–237.4; p < 0.05; Figure 1).

Figure 1. Amniotic fluid concentrations of caspase-1 in patients at term. The median amniotic fluid caspase-1 concentration was significantly higher in women in spontaneous labor at term than in those not in labor (term in labor: median 10.5 pg/mL, range 0.0–666.0 vs. term not in labor: median 5.99 pg/mL, range 0.0–237.4; p < 0.05).

Caspase-1 is increased in the amniotic fluid of women with preterm labor in the presence of intra-amniotic infection/inflammation

Among patients with spontaneous PTL, those with IAI (median 41.4 pg/mL, range 0.0–515) had a significantly higher median amniotic fluid caspase-1 concentration than those without IAI who delivered preterm (median 0.0 pg/mL, range 0.0–78.4) and than those who delivered at term (median 0.0 pg/mL, range 0.0–199.5); p < 0.001 for both comparisons; (Figure 2).

Figure 2. Amniotic fluid concentrations of caspase-1 in women with preterm labor. Among patients with spontaneous PTL, those with intra-amniotic infection/inflammation (median 41.4 pg/mL, range 0.0–515) had a significantly higher median amniotic fluid caspase-1 concentration than those without intra-amniotic infection/inflammation who delivered preterm (median 0.0 pg/mL, range 0.0–78.4) and than those who delivered at term (median 0.0 pg/mL, range 0.0–199.5); p < 0.001 for both comparisons.

Discussion

Principal findings of the study

(1) Caspase-1 is detectable in amniotic fluid at term but not in the midtrimester. (2) Women in spontaneous labor at term have a higher median amniotic fluid concentration of caspase-1 than patients at term not in labor. (3) Among patients with preterm labor, those with IAI had a significantly higher median amniotic fluid caspase-1 concentration than those without IAI. These observations suggest that caspase-1 participates in the mechanisms leading to spontaneous term parturition and preterm labor associated with infection/inflammation.

Biology of caspase-1

Caspase-1 [1],[2], also known as interleukin-1β converting enzyme (ICE) [3],[4], is a member of the cysteine-aspartic proteases family (‘c-asp-ases’) [5], enzymes involved in inflammation, apoptosis, cellular proliferation, and terminal differentiation [52]. Caspases have been classified as ‘inflammatory’ (caspase-1, -4, and -5 and murine caspase-11 and -12) and ‘apoptotic’ (‘initiators’: caspase-2, -8, -9, and -10 and ‘executors’: caspase-3, -6, and -7) [7],[53], according to their predominant pathway of involvement. However, this distinction is not absolute [54], and differences between the in vivo and in vitro activity of these enzymes have been proposed [7]. Caspases process several proteins that are critical to major changes in cellular state, including nuclear proteins, transcription factors, cell cycle regulators, and kinases, as well as cytoskeletal proteins [52]. Caspase-1 is the prototype of the inflammatory caspases, and a key component of the inflammasome [8],[11]. Its central role in inflammation and the potentially harmful consequences of an inappropriate and/or unbalanced activation, justify the existence of tight regulatory mechanisms operating both before and after its synthesis [55]. Despite the existence of alternative pathways [56],[57], the major pathway leading to caspase-1 activation requires the assembly of the inflammasome [8], and the active form of caspase-1 has only been detected in extracts of IL-1β producing cells [58]. With high-resolution immunoelectron microscopy performed on lipopolysaccharide (LPS)-stimulated human monocytes, both the active and the precursor domains of caspase-1 have been localized in the cytoplasmatic matrix and on the cell surface [59]. Of interest, only small amounts of the active form of caspase-1 are detectable in the intracellular milieu, even when IL-1β actively producing monocytes are tested [60],[61]. It has been proposed that the caspase-1 mediated processing of IL-1β occurs during the transport of this cytokine across the plasma membrane, followed by rapid secretion in the extracellular milieu [59].

Caspase-1 is initially synthesized as a 45-kDa catalytically inactive zymogen (pro-caspase) [62]. Meager amounts of this inactive precursor protein are constitutively produced by monocytic cells [60], and mainly localized in the cytoplasmatic compartment [59]. The active form of caspase-1 is heterodimeric in structure, and composed by the association of two polypeptides, p20 and p10 [2],[3],[55],[63],[64].

Monocytes constitutively express very low levels of caspase-1 mRNA [60], and its transcripts are also detected in other cells of the immune system, including T and B lymphocytes, and cells of monocytic origin, such as microglia [65]. The transcription of caspase-1 gene, which maps on chromosome 11 [66], is increased upon LPS and interferon (IFN)-γ stimulation; however, there is no evidence that higher intracellular mRNA levels result in increased pro-enzyme production [67],[68]. In addition, there is recent evidence that leptin induces mRNA expression of caspase-1 in monocytes/macrophages [69], and that also the pro-inflammatory response mediated by statins in mitogen-activated peripheral blood mononuclear cells is mediated by caspase-1 activation [70].

Caspase-1 processes and activates pro-IL-1β

IL-1β is a pro-inflammatory cytokine whose inactive precursor, pro-IL-1β, is localized in the cytoplasm of monocytes [71]. Caspase-1 [1],[3-5],[72] catalyzes the proteolytic cleavage of pro-IL-1β, between Asp116 and Ala117, which is necessary for functional activity [73],[74]. Maturation and secretion of IL-1β occur in a coordinated fashion, with a unique kinetic [73]. Indeed, caspase-1 participates in both IL-1β processing and secretion [58],[59],[75]. Evidence in support of a role of caspase-1 in IL-1β cleavage and activation includes: (1) production of IL-1β and IL-18 is concordantly blocked in caspase-1 null mice [76],[77] and (2) a selective caspase-1 inhibitor blocks the LPS-induced IL-1β production from human mononuclear cells [78]. The critical role of IL-1β in the control of infections, and the harmful and potentially lethal consequences of an excessive cytokine production and release (i.e., endotoxic shock) [79], deserves the existence of tight regulatory mechanisms controlling its transcription, processing, and secretion, as well as the presence of competitive inhibitors of its signaling [80],[81].

IL-1β is a potent pleiotropic pro-inflammatory cytokine, which participates in the activation of nuclear factor-kB (NF-kB) and mitogen-activated protein kinase (MAPK) pathways [82], and is involved in the amplification of the inflammatory cascade [53]. Evidence has been accumulating in the past years on the role of IL-1β in the mechanisms leading to implantation [83],[84] and term [13],[46],[47],[49] and preterm labor [23],[50].

Caspase-1 processes IL-18

IL-18, also known as IFN-γ-inducing factor, is a recently described cytokine sharing structural homology with the IL-1 family of proteins and functional properties with IL-12 [85-87]. IL-18, like IL-1β, is synthesized as a 24-kDa inactive precursor lacking a signal peptide (pro-IL-18), which is cleaved after Asp35 by caspase-1 to yield an active 8-kD molecule [78],[88]. Evidence in support of a role of caspase-1 in IL-18 cleavage and activation includes: (1) caspase-1 deficient mice are defective in LPS-induced IL-18 production [78]; (2) a selective caspase-1 inhibitor blocks LPS-induced IL-18 production from human peripheral blood mononuclear cells [78]; (3) Kupffer cells deficient in caspase-1, upon LPS stimulation synthesize the IL-18 precursor but fail to process it into its active form [88]; (4) caspase-1 inhibitors abolish the secretion of mature IL-18 by human granulocyte macrophages infected with influenza or Sendai virus [89]. However, caspase-1 cleavage of pro-IL-18 is not exclusive and other proteases, such as proteinase-3, have been recently reported to generate biological activity from IL-18 [90].

IL-18 is an activator of the T helper (Th)-1 response [91], mainly through the induction of IFN-γ production in both T cells and natural killer (NK) cells [86]. IL-18 enhances T and NK cells maturation and activation [92], cytokine production [93],[94], and cytotoxicity [85],[95-99], as well as neutrophil activation, reactive oxygen intermediate synthesis, cytokine release, and degranulation [100,101]. IL-18 is involved in the immune defense against bacteria (Chlamydias [102,103], Mycoplasmas [104-106], Mycobacterium tuberculosis[107-109] and Mycobacterium leprae[110], Salmonellas [111], Shigella [112], and Yersinia [113]), fungi [114-116], protozoa [117,118] and viruses [89],[119]. Of note, the roles of IL-18 in implantation [120-123] and in the context of microbial invasion of the amniotic fluid [124], intra-amniotic inflammation [124], and preterm delivery [124,125] have been subject of research.

Amniotic fluid caspase-1 in normal pregnancy

This is the first study reporting that caspase-1 is detectable in the amniotic fluid of normal pregnant women. The sources of the intra-amniotic caspase-1 are still unclear. Caspase-1 may participate in the processing and activation of IL-1β and IL-18, which take place at the maternal–fetal interface, where these cytokines play an autocrine–paracrine role for successful human implantation, pregnancy maintenance [84],[120-122],[126], and labor [13],[46-49],[127].

Sources of IL-1β during pregnancy include cyto-, syncytio-, and intermediate trophoblasts [47],[84],[128], macrophages (Hofbauer cells) [47], amniochorionic membranes [129], and decidual stromal [84],[128] and NK cells [128]. IL-1β regulates the activity of the matrix metalloproteinase (MMP)- 9, which has been linked to cytotrophoblast invasion [83].

IL-18 (both gene and protein) is constitutively expressed in human chorion and decidua, but not in the amniotic epithelium [130]. Evidence supports a role for IL-18 in modulating uterine NK cells, including their cytokine production and cytolytic activity, as well as in local vascular remodeling, through the induction of IFN-γ production [123].

Our finding that caspase-1 was not detectable in any of the mid-trimester samples in contrast to 76% of amniotic fluid from women at term not in labor is unexpected. Indeed, previous studies have reported that placental IL-1β release, in which caspase-1 may play a role, is high in the first trimester and declines as term gestation approaches, reflecting the invasiveness of the cytotrophoblasts [83]. There are several explanations for this result. First, recent gene array studies have elucidated that during decidualization there is an up-regulation of the gene, mRNA, and protein expression of IL-1β, but this is not accompanied by a coincidental increase in the secretion of the bioactive form of this cytokine [131], a step requiring caspase-1. Second, it is possible that sources other than cytotrophoblast contribute to the amniotic fluid concentrations of caspase-1. Desquamated fetal epithelia (urinary tract, gastrointestinal, or other cells found in normal amniotic fluid) have been proposed to be the major contributors to the amniotic fluid IL-1 [49]. If this is true, fetal epithelial cells may contribute to the pool of caspase-1 present in amniotic fluid in greater amounts as term approaches. Third, the detection of caspase-1 in the amniotic fluid at term, but not in mid-gestation, is in agreement with the previous report of cell-associated IL-1 activity in the amniotic fluid collected in the third but not in the second trimester [49], as well as with the presence of higher IL-18 amniotic fluid concentrations as term approaches [37].

Amniotic fluid caspase-1 in spontaneous labor at term

Women in spontaneous labor at term had a higher median amniotic fluid concentration of caspase-1 than patients at term not in labor. This finding is consistent with the view that labor at term is an event characterized by activation of the inflammatory cascade [18],[20],[132]. The pathways leading to the activation of the inflammasome in the context of labor require further investigation. Of note, the higher concentrations of caspase-1 during labor at term mirror the increase in IL-1β concentrations reported in gestational tissues (placenta [47], chorion [48], amniotic membranes [48], decidua [46],[48]) and biological fluids (amniotic fluid [13],[22],[49]), as well as the higher maternal serum [127] and amniotic fluid concentrations [37] of IL-18, when compared to normal term controls. In turn, IL-1β stimulates myometrial contractility by several mechanisms, including: (1) induction of prostaglandin (PG) synthesis (PGE2 [133-135], PGI2 [134], and PGF2 [136]) in myometrial cells [133,134], amnion [135,136], and choriodecidua [136]; (2) activation of the transcription factor NF-kB, which is functionally linked to the expression of cyclo-oxygenase 2 messenger ribonucleic acid, protein, and PG synthesis [137]; (3) potentiation of the forskolin-promoted cAMP (cyclic-adenosine monophosphate) production, resulting in cAMP accumulation [134].

Amniotic fluid caspase-1 in preterm labor

Among patients with preterm labor, those with IAI had a significantly higher median amniotic fluid caspase-1 concentration than those without IAI. This finding is in agreement with previous reports supporting a role for two of the cytokines processed by caspase-1, IL-1β and IL-18, in the molecular mechanisms leading to the onset of preterm parturition [23],[37],[49],[50],[124],[125].

Evidence of the involvement of IL-1 in preterm labor

A solid body of evidence suggests that IL-1β is implicated in the mechanisms leading to preterm parturition, particularly in the context of IAI. There is, indeed, an epidemiologic/causal link between the presence of intra-amniotic infection, higher activity and/or concentrations of IL-1β in biological fluids, and higher concentrations of prostaglandins, as supported by the following observations: (1) Our group previously reported that the systemic administration (by subcutaneous injections) of recombinant human IL-1 to timed-pregnant C3H/HeJ inbred mice was followed by parturition within 24 hours in all of the treated mice, and that a premature delivery occurred in all IL-1-injected animals [50]. (2) An immunoradiometric assay, validated for amniotic fluid and with a sensitivity of 50 pg/mL, was capable of detecting IL-1β in the amniotic fluid of all (15/15) pregnant women with preterm labor and intra-amniotic infection, versus 47.3% (10/21) of women in preterm labor, negative amniotic fluid cultures, and delivering prematurely, versus 47.2% (17/36) of samples from women with negative amniotic fluid cultures responding to tocolysis. All patients with histopathological signs of chorioamnionitis had detectable IL-1β in the amniotic fluid. Of interest, both IL-1α and IL-1β were significantly higher in women with microbial invasion of the amniotic cavity than in women who delivered prematurely but had negative amniotic fluid cultures (p = 0.0001 and p = 0.0006, respectively) [23]. (3) Patients in preterm labor with intra-amniotic infection had a fluid phase IL-1β activity that strongly correlated with the PGE2 and PGF2α amniotic fluid concentrations [49]. An increase in the fluid phase IL-1 activity is detectable also in amniotic fluid from women with spontaneous labor at term, but in this context it is predominantly attributable to IL-1α and not to IL-1β[49], and only modest elevation in the amniotic fluid concentrations of prostaglandins and their metabolites [138],[139].

In summary, a microbial colonization of the amniotic cavity and the subsequent intrauterine inflammation may lead to an increase in local production of IL-1β, followed by PG-mediated myometrial contractility. The fetus, the mother, and the placenta may contribute to the higher amniotic fluid concentrations of IL-1β and caspase-1 in the context of preterm labor. A transmembranous passage of IL-1 has been demonstrated in our laboratory (unpublished observation). In addition, experimental evidence indicates that supernatants from endotoxin-stimulated human decidua contain a significantly higher IL-1 bioactivity (attributable both to IL-1α and IL-1β) than supernatants from unstimulated decidua [140]. Furthermore, production of IL-1 from placentas of women in labor is 17-fold higher in the presence of chorioamnionitis [47].

Evidence of the involvement of IL-18 in preterm labor

Previous investigations on the role of IL-18 in the context of preterm labor have yielded conflicting results. We have previously reported that: (1) both term and preterm parturition are associated with a modest increase in amniotic fluid IL-18 concentrations, although this trend did not reach statistical significance, and that (2) the microbial invasion of the amniotic cavity, in either preterm or term parturition, is associated with a significant increase in the amniotic fluid concentration of IL-18 [37]. These findings are in agreement with another previous study in which it was reported that: (1) the median concentrations of IL-18 in cervical mucus and amniotic fluid are higher in women with preterm labor than in women not in labor at term [124]. (2) Among women with preterm labor, the median amniotic fluid concentrations of IL-18 are significantly higher in the presence of microbial invasion of the amniotic cavity and correlate with the indices of intra-amniotic inflammation (defined as increased levels of IL-6 (≥1.5 ng/mL) and/or IL-8 (≥1.3 ng/mL)) [124]. (3) Women with preterm labor and prompt delivery (delivery within seven days or <34 weeks of gestation) have significantly higher amniotic fluid concentrations of IL-18 than those delivering after several days or > 34 weeks [124]. Of note, the IL-18 gene is constitutively expressed (RT-PCR, in situ hybridization, and immunohistochemistry) in human chorion and decidua, whereas the amniotic epithelium is devoid of IL-18 mRNA and protein [130]. However, whether a higher expression of IL-18 is associated with preterm labor is not clear yet, as mice lacking IL-18 (by disruption of the IL-18 gene and/or IL-18 neutralization with intraperitoneal administration of a specific inhibitor of IL-18) have been shown to be more sensitive to LPS-induced preterm delivery and preterm fetal losses than the wild-type counterparts [125]. The role of IL-18 as a negative regulator of TNF-α during sepsis (LPS challenge induces higher serum TNF-α concentrations in the IL-18 −/− mice than in the wild type) [141], may contribute to this unexpected finding [125].

Caspase-1 inhibitors: Implications for further research

Mice and rats deficient in caspase-1 (following administration of a caspase-1 inhibitor or gene deletion) display deficient maturation of pro-IL-1β[77],[142-144] and pro-IL-18 [142,143]. Of interest, there is evidence that these animals, which are three- to four-fold more sensitive to bacterial infection than wild-type controls [145], have significantly lower serum concentrations of TNF-α[144], are resistant to the lethal effects of endotoxins [77], have a longer survival than controls during experimental pancreatitis [144], and are less likely to die from Escherichia coli intraperitoneal injection-induced sepsis [146]. Thus, administration of caspase-1 inhibitors has been proposed as a therapeutic strategy in the treatment of local and systemic inflammatory processes, particularly in the context of sepsis [142],[147]. Caspase-1 inhibitors are the first orally active agents targeting cytokines, preventing the processing and release of the active forms of IL-1β and IL-18 [142]. A blockage of lymphocyte apoptosis [146], an event associated with the development of sepsis [148-150], has been proposed as a further mechanism of action of caspase inhibitors, when tested in a model of cecal ligation and puncture [147].

Of note, it has been proposed that caspase-1 inhibitors may be beneficial in the context of inflammation-related neurological damage. Administration of caspase-1 inhibitors to neonatally infected (E. coli) rats followed by subsequent LPS challenge prevented the expected LPS-induced memory impairment. These data suggest that IL-1β participates in the set of immune/inflammatory events resulting from neonatal infection and which likely contribute to cognitive alterations [151]. In addition, caspase-1 inhibitors have been shown to reduce apoptosis, increase proliferation, and enhance survival of transplanted stem cells [152]. This has considerable implications for the treatment of infection/inflammation-induced perinatal brain injury.

This study focuses on the immunoreactive caspase-1 concentrations in amniotic fluid. Further studies investigating the functional or biological activity of caspase-1 in the same milieu or in other maternal/fetal biological fluids, will contribute to clarifying the role of this enzyme in normal pregnancy, as well as in term and preterm parturition.

Acknowledgment

This research was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, DHHS.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.