*2.1. Pattern Recognition Receptors (PRRs)*

The potential for a cell to promote inflammation is evident by its expression of a repertoire of PRRs. Two major families of PRRs are the Toll-like receptors (TLRs) and the nucleotide-binding domain leucine-rich repeat containing receptors [37], also known as Nod-like receptors (NLRs) (Figure 3). TLRs are transmembrane receptors, which allow for the sensing of PAMPs or DAMPs either at the cell surface or within endosomal compartments. There are 10 human TLRs that have been identified, each with distinct specificities to activate downstream cascade of events in response to Gram-positive/Gram-negative bacterial infection or viral RNA [43,44]. NLRs are cytoplasmic-based PRRs, which provides an intracellular recognition system for sensing PAMPs or DAMPs. Endogenous danger signals initiate and maintain inflammatory responses through activation of NLRs (Figure 3).

Multiple NLRs and NLR-dependent inflammasomes have been identified including pyrin-domain containing the initiator proteins including NLRP-1, NLRP3, NLR family caspase activation, and recruitment domain (CARD) domain-containing protein-4 (NLRC4) and the adaptor protein called ASC (apoptosis-associated speck-like protein containing a CARD) [45]. Additionally, NLR-independent inflammasomes that are driven by sensor molecules such as absent in melanoma-2 (AIMS2) and pyrin have also been described [46]. In the absence of TLRs or reduced signaling of TLRs, NLRs synergize with TLRs for a greater response or provide a compensatory system [46]. Several of the sensor molecules of the inflammasomes such as NLRP1, NLRP3, NLRP7, NLRC4, AIM2, and pyrin have been well characterized for their specific ligands, mechanisms of action, and roles in disease pathogenesis

(reviewed in Strowig et al. [47]). However, inflammasomes such as NLRP6, NLRP12, retinoic-acid inducible gene-1 (RIG-I), and interferon-γ inducible protein-16 (INFI16) are ye<sup>t</sup> to be fully characterized in health and disease.

**Figure 3.** Depicts the role of inflammasomes in placental inflammation in preeclampsia. Cholesterol or uric acid crystals (alarmins) and extracellular vesicles/microparticles can trigger NLRP (1/3/7) inflammasomes in the placenta, which releases active caspase-1 and mature IL-1β and increases inflammation.

#### *2.2. Inflammasome Components in the Gestational Tissues during Normal Pregnancy*

An inflammatory environment is mandatory in order to ensure an adequate reconstruction of the uterine epithelium, elimination of cellular debris, and tissue remodeling during implantation, placentation, maintenance of pregnancy throughout gestation and in parturition [48–50]. Inflammasome components have been identified in both maternal and fetal compartments throughout gestation. The mRNA transcripts, as well as the protein expression of NLRP1-4; the adaptor protein ASC (or PYCARD) and the caspases (1 and 4) have all been detected in the human placental trophoblasts, myometrium, and in the amniotic membranes throughout gestation [51–57]. Hyperactivity of inflammasomes has also been reported in the choriodecidua, myometrium, and cervix during term parturition (reviewed in [36]). Specifically, several studies indicate that the activation of NLRP3 inflammasome leads to the pyroptosis as part of the sterile inflammatory milieu during physiological labour in term pregnancies [58]. Emerging studies describing inflammasome expression and activation in physiological inflammation associated with uncomplicated human pregnancies provide important knowledge on their role in the pathophysiology of pregnancy complications such as PE [35] and FGR [59].

#### *2.3. Activation of Inflammasomes in Preeclampsia-A*ff*ected Pregnancies*

Activation of inflammasomes are an essential element of the innate immune system and disturbances in these processes have been implicated in various inflammatory diseases including placental inflammation associated with PE [35], FGR [59], and gestational diabetes mellitus [60]. Inflammasome hyperactivity has been reported in placental tissues from pregnan<sup>t</sup> women with PE [35,41,61]. Mulla et al. [62] reported that inflammasome activation in STB could be a possible mechanism of induction of inflammation at the maternal–fetal interface that causes adverse pregnancy outcomes, including PE. To further support this, elevated levels of TLR2, TLR4, NLRP3, and IL-1β have been reported in the neutrophils of women with PE, when compared to normal pregnan<sup>t</sup> women [44]. Furthermore, Pontillo et al. [52] reported enhanced gene transcripts for NLRP1, NLRP3, NLRC4, ASC, caspase-1, and IL-1β following stimulation with lipopolysaccharide (LPS) in human first-trimester cytotrophoblasts, decidual stromal cells, and endothelial cells in vitro. Anti-angiogenic factor (sFlt-1) in the syncytial knots and TNF α release from the STB were also implicated in inducing higher inflammasome activation in the placental tissues from PE pregnancies [35,41]. Further to this, an association between higher levels of TNF α and NLRP3 activation in peripheral blood monocytes from PE pregnancies demonstrated a direct involvement of TNF α in inflammasome activation [63].

Recent studies by Nunes et al. reported that alterations in STB functions as a consequence of the imbalance between pro- and antioxidant properties may also cause cellular stress and injury to activate inflammasomes [64]. Although the molecular mechanism involved in placental inflammasome activation in PE is largely unknown, it is possible that the inappropriate inflammatory response observed in PE may have its origin in the placenta. Potential mechanisms may include shedding of STB-derived micro or nanovesicles (that can act as DAMPs) into the maternal circulation, which are known to exert pro-inflammatory, procoagulant, and anti-endothelial activity in vitro [40,65]. Recent studies [41,65] also demonstrated that oxidative stress induced increase in the release of high-mobility group box 1 protein (HMGB1) from STB that may contribute to the pathogenesis of PE. Furthermore, Ivernsen (2013) [66] reported that the inflammatory molecules such as heat shock protein 70 (Hsp70), HMGB1, Galectin 3, and Synctin 1 carried by microvesicles in PE pregnancies may also act as DAMPS in the placenta and in the peripheral blood mononuclear cells (PBMC) in patients with PE. Thus, as illustrated in Figure 3, placental milieu resultant from the STB activation by inflammatory cytokines/or release of microparticles from injured or necrotic cells or complement primed and endogenous uric acid accumulation can activate the inflammasome machinery [35,61–63,67].

Although PE has been considered the disease of the primigravid for many years, a robust evidence for this important observation has never been reported. Several studies have also reported that there is a role for the immune system during pregnancy reacting against and/or tolerating the paternal antigens of the conceptus [68–74]. It was suggested that that increased exposure to the father's semen assists this immunological tolerance [68]. In addition to these benefits, although semen is not sterile, microbial tolerance mechanisms may exist [75]. Recent reports [68,75] have shown evidence that semen may be responsible for inoculating the developing conceptus, including the placenta with microbes, not all of which are infectious. It was suggested that when they are infectious, it may cause PE [68]. Furthermore, a variety of epidemiological and other evidence is entirely consistent with this, not least correlations between semen infection and PE [71,76]. Overall, these studies strongly sugges<sup>t</sup> a significant paternal role in the development of PE through microbial infection of the mother via insemination.

Taken together, the above studies sugges<sup>t</sup> that inflammasome activation may play a central role in the placental inflammatory processes that are associated with the pathophysiology of pregnancy complications including PE. However, further studies are required to investigate whether the inhibition of inflammasomes can be considered as a potential therapeutic strategy to prevent placental inflammation-induced development of PE.

#### **3. Inflammasome-Mediated Downstream Molecular Pathways in Long-Term Vascular Functions**

There is evidence that women who have PE and pregnancy-induced hypertension or who deliver a preterm baby have an increased risk of developing cardiovascular disease later in life [10,77]. Several systematic reviews and meta-analyses have determined that after a diagnosis of PE the relative risks for developing hypertension, cardiometabolic disorders are significantly increased in both mother and in child [78–80]. Follow-up studies of children who were born prematurely show evidence of an increased risk of high blood pressure and insulin resistance in their adulthood [81]. Thus, these reports sugges<sup>t</sup> that the pregnancies associated with PE, pregnancy hypertension; as well as pregnancies associated with preterm delivery show evidence of significant changes in vascular function (detailed below) at the time of the pregnancy. These changes greatly impact the cardiovascular health of both the mother and child later in life. The cascade of events downstream of inflammasomes may play a critical role in changes associated with vascular functions in hypertensive disorders [82] including PE; however, the molecular mechanisms by which inflammasomes promote pathogenesis of PE is ye<sup>t</sup> to be investigated. In the following section, we provide possible mechanisms of inflammasome-mediated alterations observed in hypertensive disorders, which may provide a foundation for developing improved managemen<sup>t</sup> and treatment strategies to reduce pregnancy specific burden of vascular dysfunction associated with PE.

#### *3.1. Activation of Inflammasomes in Hypertensive Disorders*

Hypertension, defined as individuals presenting with blood pressure greater than 140/90 mmHg [83], represents a worldwide-spread cardiovascular abnormality and is a major cause of subsequent end-organ damage observed in a ffected patients. Numerous studies have detailed the pathogenesis of hypertensive disorders and reported that the molecular inflammasome platform represents a central pathogenic mechanism in initiating and promoting organ damage attributed to hypertension. Eight-week-old male Dahl salt-sensitive rats fed with a high-salt diet (8% NaCl) for six weeks were found to have higher levels of NLRP3 and IL-1β in the hypothalamic paraventricular nucleus when compared to rats fed a normal diet (0.3% NaCl) [84]. Similarly, bilateral hypothalamic paraventricular nucleus injection of an IL-1β inhibitor, gevokizumab (1 μL of 10 μg), reduced the mean arterial pressure, heart rate, and levels of plasma norepinephrine, as well as, attenuated the levels of oxidative stress (NOX-2 and NOX-4) and restored the levels of NLRP3, IL-1β, and IL-10 [84]. Additionally, via inhibiting NLRP3-induced inflammation and idiopathic pulmonary fibrosis, the clinically used TGF- β blocker, pirfenidone protected against thoracic aortic constriction (TAC)-induced hypertension and left ventricular hypertrophy, collectively contributing to myocardial fibrosis, via blocking NLRP3-mediated inflammation and fibrosis [85].

Furthermore, the ASC adaptor protein of the NLRP3 inflammasome, was shown to be critical in hypoxia-induced pulmonary hypertension and right ventricular remodeling which was associated with increased protein levels of caspase-1, IL-18, and IL-1β [86]. Moreover, Asc−/− mice demonstrated reduced collagen deposition and muscularization around arteries [86]. Collectively, the findings from this study indicate that hypoxia promotion of right ventricular pressure and remodeling were attenuated in mice lacking Asc, but not in mice lacking Nlrp3, indicating that the inflammasome molecular platform plays a critical role in the pathogenesis of pulmonary hypertension [86]. Another study reported that 1K/DOCA/salt-induced hypertensive mice demonstrated increased expression of renal Nlrp3, Asc, and pro-caspase-1, as well as IL-1β and IL-18 mRNA [87]. Additionally, Asc−/− mice in the same model were protected from an increase in the renal inflammatory profile (IL-6, IL-17A, CCL2, ICAM-1, and VCAM-1) and accumulation of macrophages and collagen [87]. These studies suggested that the cascade of events downstream of inflammasomes play a critical role in disease progression; their mechanism of actions include both a central nervous and a peripheral modulation of the inflammatory pathways.

#### *3.2. Inflammasomes: A Potential Molecular Link for Long-Term Vascular Dysfunction and End-Organ Failure in Preeclampsia*

The villous stroma of the placenta provides the microenvironment for placental vascular development where immune cells reside and serve as a barrier to induce inflammatory (inflammasome)- mediated responses [88]. PE involves the excessive activation of inflammatory immune cells [63], including monocytes, fibroblasts, and granulocytes and their exacerbated production

of pro-inflammatory cytokines, IL-1β, IL-6, and IL-8 [89,90], and reduced production of regulatory cytokines such as IL-10 and TGF-α [91]. In this setting, TGF-β-promoted extracellular matrix (ECM) proteins, such as collagens, laminins, and fibronectin, play a key modulatory role in tissue remodeling [88,92,93]. Placental fibroblasts modulate the expression of ECM proteins (collagens I and IV, fibronectin, and fibrillin I) more prominently in the first trimester and term tissue [88]. Placental ischemia primes aberrant vascular and uteroplacental remodeling via the release of pro-inflammatory factors cytokines such as TNF-α in the maternal circulation [94–97]. Li et al. quantified the levels and distribution of MMPs measured in the aorta, uterus, and placenta of normal versus pregnan<sup>t</sup> rats with reduced uterine perfusion pressure (RUPP) [94]. Gelatin zymography showed marked levels of uterine MMP-2 and MMP-9, whereas casein zymography demonstrated upregulated MMP-1 and MMP-7 in the aorta, uterus, and placenta of pregnan<sup>t</sup> rats with reduced uterine perfusion pressure, compared with that from normal pregnan<sup>t</sup> rats. Supplementary organ culture work in the same study demonstrated that TNF-α stimulation upregulated the levels of MMP-1 and MMP-7 in the aorta, uterus, and placenta of normal pregnan<sup>t</sup> rats, whereas a TNF-α inhibitor antagonized the increased tissue MMP levels in rats with RUPP [94].

Collectively, these findings sugges<sup>t</sup> that placenta ischemia, via TNF-α mediated signal transduction and potentially through priming of the inflammasome platform, could lead to inadequate uteroplacental and aberrant vascular remodeling in pregnancies associated with hypertension and PE. Targeting MMP-1 and MMP-7, and/or the TNF receptor upstream of that, may also present a novel avenue in the therapeutic modulation of inflammasome priming that promotes hypertension and PE [94]. As previously discussed, women with PE also demonstrate an elevated hyperuricemia profile associated with proteinuria, suggesting that increased levels of uric acid promote the disease severity and pathogenesis associated with PE, via inducers of the NLRP3 inflammasome [90]. Uric acid is known to promote inflammation and endothelial dysfunction [98] and its crystals, monosodium urate (MSU) promote the release of IL-1β via activation of the NLRP3 inflammasome (Figure 2) [35,38,63,87]. Monocytes from PE women were activated and hence released higher levels of TNF-<sup>α</sup>, superoxide anion (O2−), and H2O2 compared to monocytes derived from normotensive pregnan<sup>t</sup> women [99]. These findings indicated that monocytes from the maternal peripheral blood are a key source of reactive oxygen species, free radicals, and pro-inflammatory cytokines. Collectively, these studies sugges<sup>t</sup> that the production of IL-1β, via activation of the inflammasome cascade, is key to driving the pathogenesis of PE. To date, research is trying to design inflammasome antagonists or equivalent inhibition strategies.

#### **4. Therapeutic Targeting for the Components of Inflammasomes**

Due to the wide range of hypertension-driven inflammatory diseases, a number of targeted therapies have been investigated for antagonizing the effects of the inflammatory (inflammasome)- pathway. Pharmacological blockade of human cord blood leukocytes demonstrated that ATP released as a result of tissue injury can further promote the secretion of IL-1β from laboring and nonlaboring women [100], indicating that inhibition of the P2X7 receptor may protect from inflammation induced vascular injury during pregnancy. A separate study evaluated pharmacological blockade using a P2X7 receptor inhibitor and pannexin-1 blocker carbenoxolone, was shown to attenuate the LPS-induced increase in the levels of secreted IL-1β [101]. Silibinin (SB) is a flavonoid complex medicinal herb with anti-inflammatory, hepatoprotective, antioxidant, and antifibrotic properties. The antioxidant and anti-inflammatory properties of SB were assessed via dose-dependent inhibition of H2O2 release, production of TNF-<sup>α</sup>, IL-10, TGF-β, and prostaglandin E2 (PGE2) following LPS stimulation of peripheral blood monocytes from healthy individuals. Monocytes were treated with SB to determine whether SB can modulate the NLRP1 and NLRP3 inflammasomes, as well as influence upstream TLR-4/NF-κB activation [99]. Administration of SB to MSU-stimulated monocytes reduced the degree of NLRP1 and NLRP3 inflammasome activation, as well as TLR-4/NF-kB activation [99]. Furthermore, administration of SB to pregnan<sup>t</sup> rats in an experimental model of PE,

induced by nitric oxide synthase inhibition (with N-omega-nitro-l-arginine methyl (l-NAME)) protected reproductive outcomes, normalized blood pressure, reduced proteinuria, and also serum levels of pro-inflammatory cytokines [102]. Additional studies have examined targeting of the NLRP3 sensor itself, via administration of the small and highly selective NLRP3 inflammasome inhibitor, MCC950, a diarylsulfonylurea-containing compound [103]. The mechanism of action of MCC950 is via its ability to inhibit NLRP3-induced ASC oligomerization to subsequently block the secretion of IL-1β [103], where MCC950 was recently shown to attenuate the high deoxycorticosterone (DOCA)-induced hypertensive e ffects [87,104]. Krishnan et al. [87] demonstrated an increased DOCA/salt-induced renal inflammatory profile (IL-6, IL-17A, CCL2, ICAM-1, and VCAM-1), fibrosis (assessed via extent of renal collagen accumulation), via an inflammasome/IL-1β-dependent mechanism.

As a follow-up study, Krishnan et al. [104], showed that pharmacological inhibition of the NLRP3 inflammasome, with MCC950, significantly lowered the 1K/DOCA/salt-induced increase in blood pressure, renal expression of inflammasome markers (NLRP3, ASC, pro-caspase-1, pro-IL-1β and pro-IL-18), and markers associated with renal inflammation and injury (IL-17A, TNFα, osteopontin, ICAM-1, VCAM-1, CCL2, and vimentin). AMCC950-induced reduction in these 1K/DOCA/salt-induced measures was accompanied by an additional marked attenuation in the levels of renal interstitial collagen and renal albuminuria (by up to 25%) in C57Bl/6 mice [104]. Similarly, by blocking the ability of the NLRP3 inflammasome components to assemble and oligomerize, as well as inhibiting K<sup>+</sup> efflux, β-hydroxybutyrate (BHB) was shown to reduce the production of both IL-1β and IL-18 [105]. A separate study also demonstrated that treatment with EMD638683, a specific glucocorticoid-inducible kinase (SGK1) inhibitor, significantly reduced hypertension induced cardiac damage [106]. Taken together, these studies provide proof-of-concept that pharmacological inhibition of upstream, as well as downstream targets of the NLRP3 inflammasome signaling cascade; and the inflammasome platform, present a viable anti-hypertensive strategy in attenuating the pathogenesis of PE with an underpinning inflammatory component.
