Background
Preterm birth is the leading cause of neonatal morbidity and mortality worldwide [
1‐
3], with two-thirds of all cases preceded by spontaneous preterm labor [
4]. The latter is a syndrome comprising multiple etiologies [
4,
5], with intra-amniotic infection and/or inflammation as the only well-established causal link to preterm birth [
6‐
21]. Intra-amniotic inflammation was thought to be exclusively initiated by the invasion of microbes into the amniotic cavity (i.e., intra-amniotic infection) [
22‐
25]. Yet, the advancement of molecular microbiological techniques has allowed for the discovery of a new entity, sterile intra-amniotic inflammation, in which elevated concentrations of cytokines [i.e., interleukin (IL)-6] occur in the absence of detectable microorganisms [
26‐
30]. From an immunological perspective, sterile inflammation is triggered by danger signals or damage-associated molecular patterns (DAMPs; also known as alarmins) released upon cellular stress, senescence, or necrosis [
31‐
34]. Therefore, we have proposed that elevated concentrations of alarmins in the amniotic cavity are responsible for activating the inflammatory cascade leading to preterm labor and birth [
27,
35]. In support of this concept, the concentrations of several classical alarmins, namely high-mobility group box-1 (HMGB1) [
27,
36], S100 calcium-binding protein-B (S100B) [
37], IL-1α [
38,
39], and heat-shock protein 70 (HSP70) [
40], are increased in women with intra-amniotic inflammation. Notably, sterile intra-amniotic inflammation is more prevalent than intra-amniotic infection in women with preterm labor and intact membranes [
27]. In addition, patients with both sterile intra-amniotic inflammation and an increased amniotic fluid concentration of HMGB1 deliver sooner than those with a lower concentration of this alarmin [
27], indicating that HMGB1 can serve as a predictor of preterm delivery. Indeed, we have mechanistically demonstrated that elevated concentrations of HMGB1 [
10,
17], as well as other alarmins [
14,
16,
21,
41], in the amniotic cavity induce sterile inflammation and cause preterm labor and birth. Therefore, we are actively engaged in finding strategies to prevent preterm labor and birth by inhibiting sterile intra-amniotic inflammation.
The mechanisms that lead to sterile intra-amniotic inflammation involve the activation of inflammatory pathways such as the NLR family pyrin domain-containing-3 (NLRP3) inflammasome [
14,
16,
21,
42‐
44]. Hence, we have proposed the use of inhibitors of NLRP3 inflammasome activation to treat sterile intra-amniotic inflammation and to prevent preterm labor and birth [
14]. However, a limitation of this approach is that a few cases of spontaneous preterm labor categorized as sterile intra-amniotic inflammation may be associated with undetectable microorganisms, thus the blockade of the NLRP3 inflammasome could limit the host response mechanisms required for clearance of such a pathogen. Alternatively, we have recently shown that treatment with betamethasone, a widely used corticosteroid, prevents preterm birth; nevertheless, such an approach did not rescue neonatal mortality induced by the intra-amniotic administration of HMGB1 [
17]. Therefore, a strategy that not only prevents preterm birth but also improves neonatal survival by dampening the intra-amniotic inflammatory response induced by alarmins is urgently needed.
Recent studies have shown that specific antibiotics, such as clarithromycin, not only display effective anti-microbial properties in women with intra-amniotic infection but also exert anti-inflammatory effects in the amniotic cavity [
45‐
49]. Therefore, we propose that clarithromycin, which is already approved for clinical use in pregnant women, could represent a viable treatment for women presenting with sterile intra-amniotic inflammation and risk of preterm delivery. Importantly, clarithromycin is the macrolide that most efficiently crosses the placenta [
50,
51]. Indeed, a recent clinical investigation showed a reduction in the severity of the intra-amniotic inflammatory response (as indicated by amniotic fluid IL-6 levels) in women who received treatment with clarithromycin [
48]. However, to date, there has been no mechanistic demonstration showing that treatment with clarithromycin prevents preterm birth and adverse neonatal outcomes. Furthermore, the anti-inflammatory effects exerted by clarithromycin in the maternal–fetal tissues have not been investigated.
In the current study, we utilized an established model of sterile intra-amniotic inflammation induced by the alarmin HMGB1 to evaluate whether treatment with clarithromycin prevents preterm birth and adverse neonatal outcomes. Moreover, we investigated the maternal and fetal inflammatory responses in mice treated with clarithromycin to elucidate the anti-inflammatory effects of this macrolide in the setting of sterile intra-amniotic inflammation.
Discussion
One of every four premature neonates is born to a woman with intra-amniotic inflammation [
27,
99,
100], which was largely attributed to cultivable and non-cultivable microbes invading the amniotic cavity (i.e., infection) [
22,
25‐
29]. Therefore, much research has been focused on exploring the appropriate antibiotic regimen for treating intra-amniotic infection [
45‐
49]. Such efforts have been fruitful by demonstrating that the correct antibiotics (including clarithromycin) can eradicate intra-amniotic infection [
45‐
49,
101], reduce intra-amniotic inflammation [
45‐
49], and, more importantly, extend gestational length and prevent adverse neonatal outcomes, including intra-ventricular hemorrhage, peri-ventricular leukomalacia, and cerebral palsy [
102]. Yet, recent research has provided solid evidence that intra-amniotic inflammation can occur in the absence of detectable and cultivable bacteria (i.e., sterile intra-amniotic inflammation) [
26‐
29]. In such a setting, we have proposed that the optimal treatment strategy includes the utilization of drugs with anti-inflammatory properties [
14,
17,
103], including antibiotics [
45‐
48]. Indeed, a recent report showed that the macrolide clarithromycin can be used to treat women with sterile intra-amniotic inflammation who were destined to deliver preterm [
48]. Herein, we put forth mechanistic evidence showing that clarithromycin can be utilized to prevent preterm birth and adverse neonatal outcomes in an animal model of sterile intra-amniotic inflammation.
In the current study, we explored the mechanisms whereby clarithromycin prevents preterm birth and adverse neonatal outcomes. First, we report that this macrolide interferes with the common pathway of labor by reducing the gene expression of contractility-associated proteins such as OXTR, CX43, and COX2, which are increased in preterm and term labor [
104]. This finding is in line with previous in vitro demonstrations showing that clarithromycin inhibits oxytocin-induced myometrial contractility [
105]. Furthermore, we have previously shown that treatment with clarithromycin prevented the premature onset of labor induced by
Ureaplasma parvum [
15]. Notably, in the current study, we showed that treatment with clarithromycin reduced the expression of several inflammatory mediators in the intra-uterine tissues (e.g., uterus, fetal membranes, decidua, and placenta) involved in the cascade of parturition. These data are consistent with previous demonstrations in non-reproductive tissues that clarithromycin suppresses the production and secretion of inflammatory cytokines by interfering with the AP-1 and NF-κB pathways [
106,
107]. Specifically, clarithromycin suppressed NF-κB-mediated pro-inflammatory cytokine production by interfering with the mitochondrial proteins 4-nitrophenylphosphatase domain and non-neuronal synaptosomal associated protein 25-like protein homolog (NIP-SNAP)-1 and -2 [
107]. Furthermore, clarithromycin can dampen inflammation by modulating the concentrations of chemokines such as IL-8 [
106], which results in reduced neutrophil infiltration at the site of injury [
108]. Alternatively, clarithromycin can foster an anti-inflammatory milieu by increasing suppressive cytokines such as IL-10 [
109], as shown in the decidua herein, and by expanding immunosuppressive innate immune cells [e.g., myeloid-derived suppressor cell (MDSC)-like cells] [
110]. Taken together, these data indicate that clarithromycin prevents preterm birth largely by interfering with the inflammatory cascade of labor in the intra-uterine maternal tissues (uterine decidua and cervix).
The strategy of using anti-inflammatory approaches to tackle prematurity has been utilized by us and others [
111‐
118]. For example, we have shown that anti-inflammatory drugs and peptides such as MCC950 (an NLRP3 inflammasome inhibitor [
119]), rosiglitazone (an anti-diabetic thiazolidinedione drug [
120]), and exendin-4 [an agonist of the glucagon-like protein-1 receptor (GLP1R) [
121]] prevent preterm birth induced by inflammation in mice [
12‐
14,
53‐
55,
122]. Yet, such strategies are not approved for use in pregnant women. To address this need, we and others have also utilized progesterone and betamethasone, drugs approved for pregnant women, to prevent inflammation-induced preterm birth in mice [
17,
52,
103,
123,
124]. Notably, betamethasone can prevent preterm birth in the animal model of sterile intra-amniotic inflammation used herein [
17]. Pertinent to the latter finding, clarithromycin has been shown to be as potent as corticosteroids (e.g., prednisolone) in inhibiting the production of pro-inflammatory cytokines [
125]. Therefore, the use of clarithromycin to treat inflammation-induced preterm birth is well-supported.
In the current study, we showed that treatment with clarithromycin reduced the expression of several inflammatory mediators in the placenta. This finding could be explained by previous pharmacokinetics studies showing that clarithromycin efficiently crosses the human placenta [
50,
51]. Indeed, clarithromycin is more efficient than other macrolides, such as azithromycin and erythromycin, at crossing the placenta [
50]. The mechanisms whereby clarithromycin reduces placental inflammation must involve the inhibition of the NF-κB pathway, as has been demonstrated in other systems [
106,
107] and the uterine decidua herein. Furthermore, clinical studies reported that the placentas of patients treated with clarithromycin displayed a lesser degree of histological funisitis (acute inflammation of the umbilical cord) [
102,
126]. However, we also found that treatment with clarithromycin did not reduce inflammation in the amniotic cavity, as was observed in humans [
45‐
48]; yet, a direct comparison between animal and human studies requires careful consideration. Regardless, it is tempting to suggest that the reason why clarithromycin did not fully prevent neonatal mortality induced by HMGB1 lies within its minimal effects in the amniotic cavity in our model. In the current study, another point to consider is that we utilized concentrations of clarithromycin similar to those utilized in humans [
57,
58], given that macrolides can exhibit cytotoxicity at high concentrations [
127]. Together, these findings also allow us to propose that the combination of approved approaches (e.g., betamethasone and clarithromycin) may be more effective for reducing the intra-amniotic inflammatory milieu compared to clarithromycin alone. Nevertheless, further research is required to test such a proposal.
A major finding of our study is that treatment with clarithromycin dampened inflammation in the fetal tissues, namely the lung, intestine, and liver. To our knowledge, this is the first evidence showing the anti-inflammatory effects of clarithromycin in a model of sterile intra-amniotic inflammation. Similarly, previous mechanistic studies in animal models have consistently shown that macrolides, such as azithromycin, reduce
Ureaplasma-induced inflammatory markers in the amniotic cavity, fetal lung, and/or fetal skin [
128‐
130]. Indeed, such a decrease in the local inflammatory response has been translated to improvements in physiological parameters, such as fetal cardiac output [
130]. Furthermore, clarithromycin has been successfully used to diminish inflammation in patients with lung pathologies (e.g., asthma [
108,
131], bronchiectasis [
132], and cystic fibrosis [
133]). The protective effects of clarithromycin have also been reported in gastrointestinal diseases (e.g.,
Helicobacter pylori infection [
134], intestinal mucositis [
135], and Crohn's disease [
136]). In line with the abovementioned studies, we also report that clarithromycin dampens inflammation in the fetal spleen, whose inflammatory status can serve as a marker (Doppler of the fetal splenic vein) of fetal damage induced by intra-amniotic inflammation [
137]. Collectively, these data suggest that the mechanisms whereby clarithromycin improves neonatal survival include dampening of inflammation in the fetal organs. However, further mechanistic demonstrations are required to investigate whether neonates born to women treated with clarithromycin are immunocompetent.
The current study has some limitations. There is a lack of information regarding the inflammatory effects induced by HMGB1 alone in gestational tissues utilizing in vivo models. Ongoing research in our lab is investigating such effects; yet, we have previously shown that the in vitro treatment of the chorioamniotic membranes with HMGB1 drives a similar inflammatory response [
42] to that observed in the current study. Thus, we surmise that the vehicle control group (HMGB1 + DMSO) would have similar results in mice injected by HMGB1 alone. Another limitation is that, for the mice utilized for tissue collection, we could not distinguish between those mice that would have delivered preterm from those that would have delivered at term. Ongoing research in our lab is also focused on establishing non-invasive approaches to monitor the progression of labor in mice and identify those that can benefit from treatments to prevent preterm birth.
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