Lipopolysaccharide-induced premature cervical ripening and inflammation in pregnant mice can be alleviated by nicotine


 Background

Clinical treatment of preterm birth (PTB) is usually dependent on inhibiting uterine contractions. Such therapy is only effective in the short-term and cannot fundamentally low the incidence of PTB. Premature cervical maturation is another important cause of PTB; it is associated with invasion of macrophages, which produce proinflammatory cytokines. We previously found that activation of the alpha-7 nicotinic acetylcholine receptor (α7nAChR) by nicotine alleviates the systemic inflammatory response in murine models of lipopolysaccharide (LPS)-induced PTB, but the underlying mechanisms remain unclear.
Methods

Cervical tissues were collected from women in preterm and term labor. An animal model of PTB was established by administering 25 µg/kg lipopolysaccharide (LPS) to normal pregnant C57BL/6 mice on gestational day 16 (GD16). PTB animals received 1 mg/kg of α7nAChR agonist nicotine or nicotine combined with 1 µg/kg α7nAChR antagonist α-bungarotoxin (α-BGT) from GD14 to GD17. The gestational age, the rates of preterm birth and the survival rate of pups were recorded. Immunofluorescence staining were utilized for the quantification of α7nAChR expression on cervical macrophages and changes in macrophage polarization, RT-PCR was used to determine the mRNA levels of M1 and M2 macrophage biomarkers. Elisa assay was used to detect changes in fetal inflammation. Immunofluorescence staining and western blotting were used to investigate the signaling pathways underlying nicotine’s promoting LPS-induced conversion of M1 macrophage to M2 in the cervix of PTB animals.
Results

α7nAChR expression on cervical macrophages and cervical collagen content was decreased in PTB patients and murine models of LPS-induced PTB. The number of M1 cervical macrophages, other pro-inflammatory mediators in the cervix and fetal inflammation was unregulated in pregnant mice following LPS administration. Nicotine treatment reduced the rate of PTB, decreased fetal inflammation, improved maternal and neonatal outcome; nicotine also increased polarization of cervical macrophages toward the anti-inflammatory M2 phenotype possibly by suppressing activation of the MAP kinases JNK and ERK1/2 and nuclear translocation of NF-κB and rescuing the inhibited JAK2/STAT3 and PI3K/AKT pathways. The effects of nicotine were reversed by the selective α7nAChR antagonist α-bungarotoxin (α-BGT).
Conclusions

Our findings indicate that nicotine prevents LPS-induced cervical ripening and inflammatory response by inducing M2 macrophage polarization, and nicotine may serve as potential anti-premature cervical maturation agents for treatment of PTB.

Immunofluorescence staining were utilized for the quantification of α7nAChR expression on cervical macrophages and changes in macrophage polarization, RT-PCR was used to determine the mRNA levels of M1 and M2 macrophage biomarkers. Elisa assay was used to detect changes in fetal inflammation. Immunofluorescence staining and western blotting were used to investigate the signaling pathways underlying nicotine's promoting LPS-induced conversion of M1 macrophage to M2 in the cervix of PTB animals. Results α7nAChR expression on cervical macrophages and cervical collagen content was decreased in PTB patients and murine models of LPS-induced PTB. The number of M1 cervical macrophages, other proinflammatory mediators in the cervix and fetal inflammation was unregulated in pregnant mice following LPS administration. Nicotine treatment reduced the rate of PTB, decreased fetal inflammation, improved maternal and neonatal outcome; nicotine also increased polarization of cervical macrophages toward the anti-inflammatory M2 phenotype possibly by suppressing activation 3 of the MAP kinases JNK and ERK1/2 and nuclear translocation of NF-κB and rescuing the inhibited JAK2/STAT3 and PI3K/AKT pathways. The effects of nicotine were reversed by the selective α7nAChR antagonist α-bungarotoxin (α-BGT).

Conclusions
Our findings indicate that nicotine prevents LPS-induced cervical ripening and inflammatory response by inducing M2 macrophage polarization, and nicotine may serve as potential anti-premature cervical maturation agents for treatment of PTB.

Background
Clinically, drugs that inhibit uterine contractions are the mainstay treatment for preterm birth (PTB) [1]. Although administration of tocolytic agents such as β2-adrenergic receptors, oxytocin-receptor antagonists, and prostaglandin synthetase inhibitors can inhibit uterine contraction within 48 hrs, they have not fundamentally decreased the incidence of PTB [2,3]. Consequently, there is an unmet need to further elucidate the pathophysiology of PTB and find more effective therapeutic targets.
Evidence suggests that premature cervical maturation is an important cause of PTB, and changes in collagen content reflect cervical maturity. Collagen level in the cervical tissue is 60% lower in pregnancy than in nonpregnancy and significantly lower in PTB patients and murine models of PTB than in normal pregnancy [4]. Increased secretion of collagenases (matrix metallopeptidase [MMP]-2, MMP-9) in the cervical tissues [5], cervicovaginal fluid [6], amnion and chorion [7] are responsible for cervical ripening and fetal membrane rupture at term birth, possibly by prompting breakdown of extracellular matrix (ECM) components consisting predominantly of collagens. An imbalance in the expression of MMPs and their inhibitors (TIMPs) is found in the preterm placenta and amniotic membranes [8,9]. Based on these findings, we propose that regulation of collagen content and collagenase level in the cervix represent key factors in the treatment of PTB.
Cervical ripening in normal pregnancy is an inflammatory process [10] that involves an increase in the expression of inflammatory cytokines. In PTB, the inflammatory response in the cervix is exaggerated [11]. Watari et al. reported that increased expression of inflammatory cytokines was associated with increased MMPs expression in smooth muscle cells from human cervix [12,13]. In 4 other studies, TNF-α and IL-33 induced monocytes/macrophages to secrete MMP-9 [14,15]. These data imply that excessive inflammation in the cervix has potential to upregulate MMP-9 expression, which promotes the degradation of collagens to cause premature cervical maturation.
Under different physiological and pathological environments, macrophages can be polarized into two states, M1 and M2 phenotypes, which have disparate roles in tissue remodeling [16]. M1 macrophages secrete inflammatory factors and MMPs to degrade the ECM. M2 macrophages secrete anti-inflammatory factors, including TGF-β and IGF-1, to promote the secretion of TIMPs and inhibit ECM degradation [17]. Numerous studies have demonstrated that increasing the proportion of M2/M1 macrophages improves the inflammatory microenvironment and inhibits collagen degradation to promote tissue repair after injury [18][19][20]. Cervical ripening is a process of ECM remodeling. A previous investigation showed a significant infiltration of macrophages into the cervix of normal pregnant women during cervical ripening in the third trimester, and that CD68-stained macrophages increased to an equivalent extent in cervical subepithelium and stroma from women in term labor, preterm labor, and not in labor at term [21]. Another report showed the number of activated macrophages involved in MMP induction peaked on the 18th day of gestation in the cervix of pregnant mice [22]. These data suggest that macrophage infiltration participates in the process of cervical ripening. However, changes in the polarization state of macrophages in preterm labor remain to be elucidated.
α7nAChR is widely expressed on macrophages. In clinical trials, activation of α7nAChR was used to treat inflammatory diseases such as enteritis and rheumatoid arthritis [23], and inhibition of α7nAChR was used to attenuate tissue fibrosis related to inflammation [24]. The regulatory role of α7nAChR in tissue fibrosis and tissue repair has been related to changes in collagen content [25]. Our previous studies showed that an α7nAChR agonist (nicotine) prolonged normal gestation and inhibited collagen degradation and cervical ripening in pregnant murine. Our further studies revealed that the serum levels of inflammatory cytokines, such as TNF-α and IL-6, were up-regulated in animal models of LPSinduced inflammation, and nicotine could reduce the serum levels of inflammatory cytokines [26].
However, the function and mechanisms of nicotine on macrophages in the cervix in PTB have not 5 been clearly defined.
In the present study, we examined the expression level of nicotine receptor (α7nAChR) on cervical macrophages in PTB patients and LPS-induced inflammatory animal models, investigated the role of nicotine in PTB in terms of collagen degradation, local inflammation of cervix, fetal inflammation, cervical macrophage polarization and the related downstream signaling pathway of α7nAChR. Our results show that the protective effects of nicotine in pregnant mice following LPS administration are related to promote cervical macrophage M2 polarization to reduce inflammation and collagen degradation.

Cervical Biopsies
Fresh cervical tissue specimens were obtained from women in preterm (n = 10) and term labor (n = 12). Biopsies were taken from the anterior cervical lip immediately after vaginal delivery or before caesarean section. Women with cervical dysfunction, premature rupture of fetal membranes, placenta previa, hydramnios, history of late abortion or uterine deformity were excluded from this study.
After biopsy, tissues were quickly washed with 0.9% physiological saline, and fixed with 4% paraformaldehyde in 0.01 M phosphate buffer (PBS) for 1-2 days at 4 °C. Cervical tissue was paraffinembedded, sectioned to a 4 µm thickness, and stained in Masson's trichrome and for immunohistochemistry analysis. Animal models of premature birth and treatment protocols According to methods described in studies from Nardhy et al. [27,28], LPS (strain Escherichia coli 0111:B4, cat# L4130) and nicotine hydrogen tartrate (α7nAchR agonist) (cat# BP822) were purchased from Sigma-Aldrich (St. Louis, MO, USA). LPS and nicotine were each dissolved in 0.9% physiological saline for a 1.0 mg/ml stock solution, and the pH of the nicotine solution was adjusted to 7.1-7.3 using NaOH. 1.0 mg/ml stock solutions were stored frozen in aliquots at -20 °C, and thawed and diluted for use. 1 mg α-BGT (cat# ZX0001, Shanghai BOYAO Biological Technology Co., Ltd; China) was dissolved in 10 ml 0.9% physiological saline, stored frozen in aliquots at -20 °C, and thawed and diluted to0.1 µg/ml for use.
On GD16, 25 µg/kg LPS was administered by intraperitoneal injection to pregnant mice to establish a model of PTB. From GD14 to GD17, nicotine exposed PTB mice were administered 1 mg/kg nicotine daily in two subcutaneous injections at a 6-hour interval; on GD16, nicotine was administered 40 minutes before LPS. From GD14 to GD17, nicotine exposed PTB mice treated with α-BGT were administered 1 µg/kg α-BGT daily in two intraperitoneal injections at a 6-hour interval; α-BGT was administered 30 minutes before nicotine.
Mice were scarified on GD 18. Cervical tissue was harvested immediately under anesthesia. Cervical tissue was paraffin-embedded, sectioned to a 4 µm thickness, and stained in Masson's trichrome and for immunohistochemistry analysis, or frozen in liquid nitrogen and stored at -80 °C.

Immunohistochemistry
Paraffin sections were dewaxed and rehydrated, antigen retrieval was performed, sections were Sections were photographed with a fluorescence microscope (DM4B, Leica, Wetzlar, Germany).
For single immunofluorescence staining, NIH Image J software was used to quantify fluorescence intensity (FD) of the stained areas (SA) relative to the background. A total of 28 sections (4 sections/animal, 7 animals/group) were analyzed. Images were obtained under the same light 8 exposure time and magnification (X10 and X20).
For double immunofluorescence staining, NIH ImageJ software was used to quantify the number of CD11b positive cells in a blinded fashion. A total of 36 sections (6 sections/animal, 6 animals/group) were analyzed. Values were expressed as percent change from the normal pregnant controls (P).

Real-time PCR
On GD18, the cervical tissues were dissected out from the anesthetized animals and stored at -80 °C .Total RNA was extracted from the cervical tissues using the RNeasy mini kit (Qiagen, cat#74104, Germany). A quantitect Reverse Transcription Kit (Qiagen, cat# 205310, Germany) was used to generate cDNA. The cDNA was used as template for quantitative RT-PCR, which was performed using the SYBR Green qPCR Millipore, Burlington, MA, USA) and exposed on a gel imaging system (ChemiDoc XRS; Bio-rad, Hercules, CA, USA). NIH ImageJ software was used for densitometric analysis.

Statistical Analysis
Statistical analyses were conducted with SPSS v17.0 (SPSS, Chicago, IL, USA). For the immunoreactivity data, Masson staining results and the protein expression by western blotting, between-group differences were compared using the one-way analysis of variance (ANOVA); a least significant difference (LSD) post hoc test when groups had the same variance or Dunnett's test when variances were not equal; data are expressed as the mean ± SEM. For rates of preterm labor, gestational age, pup survival rate, mRNA expression and chemokine/cytokine concentrations, between-group differences were compared using the Mann-Whitney U test. A p value < 0.05 was considered significant.

Nicotine significantly decreased the frequency of preterm labor and prolonged the gestation in a pregnant mouse model of LPS induced inflammation
Pregnant mice injected with LPS had a higher frequency of preterm labor (LPS vs. P, 66.7% vs. 0%, p=0.003, Figure 1A) and a shorter gestation than normal pregnant controls (LPS vs. P, 18.6 ± 0.16 vs. 18.6 ± 0.11 days, p=0.002, Figure 1B). Nicotine treatment decreased the PTB rate to 12.5% and prolonged gestation to 19.4± 0.11 days. α-BGT, an inhibitor of a7nAChR, reversed the beneficial effects of nicotine ( Figure 1A and 1B).

Nicotine significantly improved fetal outcomes
The live birth rate was decreased in pregnant mice injected with LPS compared to normal pregnant controls (LPS vs. P, 31.6% vs. 100%, p=0.001, Figure 1C). Nicotine treatment markedly increased the live birth rate to 77.5% in mice injected with LPS. α-BGT blocked the nicotine effect to some extent (LPS+N+A vs. LPS+N, 39.3% vs. 77.5%, p=0.029, Figure 1C).

α7nAchR expression on cervical macrophages was decreased in PTB patients and pregnant mice injected with LPS
Patient information was provided in Table 1. The gestational age at delivery in PTL group was lower than that in TL group (p<0.01); the number of previous preterm deliveries seemed more in PTL compared to TL groups (PTL vs. TL, 3 vs. 0). To investigate the possible role of α7nAchR on cervical macrophages in PTBwe analyzed the expression level of α7nAchR on cervical macrophages from PTB patients and pregnant mice with LPS injection by double immunofluorescence staining.CD68 and CD11b were used as markers of cervical macrophages for humans and mice, respectively. Findings showed α7nAchR immunoreactivity on cervical macrophages from PTB patients ( Figure 2A) and pregnant mice following LPS administration ( Figure 2B) were significantly decreased compared to normal pregnant controls. Treatment of PTB mice with nicotine rescued α7nAchR expression on cervical macrophages, but that protection was reversed by treatment with α-BGT ( Figure 2B).

α7nAchR activation by nicotine has the potential to inhibit premature cervical ripening in PTB mice
Changes in cervical collagen play a key role in the regulation of cervical ripening. To investigate the regulatory function of α7nAchR in cervical ripening, we analyzed the collagen content of cervical tissue from PTB patients and mice by Masson's trichrome and immunofluorescence staining.
Consistently, there was a substantial decrease in the number of blue stained collagenous fibers in cervical tissues from PTB patients ( Figure 3A) and mice ( Figure 3B), and collagen I and collagen III immunoreactivities were reduced in cervical tissues from PTB model compared to normal pregnant mice ( Figure 3C and 3D). Nicotine treatment significantly increased the number of blue stained fibers in cervical tissue from PTB ( Figure 3B) and the fluorescence intensity of collagen I and collagen III ( Figure 3C and 3D).These effects were blocked by treatment with α-BGT. These data suggest that α7nAchR activation attenuated the decrease in collagen content in the cervix of PTB mice, and imply that α7nAchR activation by nicotine has potential to inhibit premature cervical ripening.

Nicotine treatment inhibited MMP-9 expression in cervical tissues from PTB mice
Degradation of cervical collagens depends on the relative levels of MMP-9 and its inhibitor TIMP-1.
To investigate whether the increase in cervical collagen content following α7nAchR activation in PTB was related to decreased expression of MMP-9, we analyzed the immunoreactivity of MMP-9 and its inhibitor (TIMP-1) in cervical tissues from PTB mice. Findings showed that the MMP-9 immunoreactivity was significantly increased ( Figure 4A) while TIMP-1 immunoreactivity ( Figure 4B) was significantly decreased in cervical tissues from PTB mice compared to normal pregnant controls. Nicotine treatment remarkably reduced the immunoreactivity of MMP-9 but enhanced the immunoreactivity of TIMP-1; to some extent, these changes were reversed by treatment with α-BGT. These data indicate that collagen content in cervical tissues decreased in association with an increase in the expression of MMP-9 in PTB mice. Nicotine treatment attenuated the increase in MMP-9 expression and collagen degradation, and may prevent premature cervical ripening.

Macrophage M1 polarization in cervical tissues from PTB mice was inhibited by nicotine
We previously found that systemic inflammation had a role in promoting premature cervical ripening in PTB [26]. Macrophage infiltration into the cervix is significantly increased during PTB [21].
Macrophages are polarized into different inflammatory phenotypes, which have varying pathological consequences. To investigate whether changes in cervical collagen content and MMP-9 expression in PTB following α7nAchR activation are associated with reduced local inflammation in the cervix, we identified M1 and M2 macrophages in cervical tissues by immunofluorescence staining. M1 macrophages were identified using combinations of CD86, iNOS, inflammatory cytokine (TNF-α, IL-1β), and CD11b (macrophage marker) antibodies. M2 macrophages were identified using combinations of CD163, Arg-1, and CD11b antibodies. Findings showed significantly more M1 macrophages ( Figure   5A) and significantly less M2 macrophages ( Figure 5B) were present in cervical tissues from PTB mice compared to pregnant controls. Significantly more M1 surface (iNOS, Figure 6A) and phenotypic (inflammatory cytokines: TNF-α and IL-1β, Figure 7) markers and less M2 surface markers (Arg-1, Figure 6B) were expressed on cervical macrophages from PTB mice compared to normal pregnant controls. The expression of M1-associated molecules was significantly decreased and the expression of M2-associated molecules was significantly increased on cervical macrophages from nicotine treated compared to untreated PTB mice. The effects of nicotine were reversed by treatment with α-BGT. These data suggest that nicotine could induce polarization of cervical macrophages to an antiinflammatory M2 phenotype and create an anti-inflammatory environment in the cervix.

Nicotine modulates LPS-induced phenotype-associated cytokines in the maternal cervix and amniotic fluid
To further investigate whether changes in macrophage polarization can influence the inflammatory microenvironment at the maternal-fetal interface, we detected mRNA expression of some key proinflammatory and anti-inflammatory genes in the cervix. LPS stimulation strongly increased the expression of TNF-α, IL-1β, CCL-2, CXCL10, CD86 and iNOS in the cervix of pregnant mice (Figure 8).
In contrast, expression levels of anti-inflammatory genes, including IL-10, Arg-1, CD163 and CD206, were lower after LPS stimulation. Nicotine treatment significantly suppressed the LPS-induced increase in pro-inflammatory gene expression in the cervix, but enhanced the levels of IL-10, Arg-1, CD163 and CD206. The α7nAchR inhibitor (α-BGT) abolished the nicotine effects ( Figure 8).
Amniotic fluid concentrations of cytokines were determined to reflect the fetal inflammatory response, which is also associated with poor pregnancy outcomes. IFN-γ, IL-1β, IL-6, IL-13, CXCL10, CCL-2, CCL-5 and TNF-α levels were dramatically elevated in the amniotic fluid of pregnant mice injected with LPS; however, IL-10 and IL-4 levels were decreased (Figure 9). Nicotine significantly inhibited the LPS-induced changes. The beneficial effects of nicotine could be reversed by α-BGT.

Activation of α7nAchR by nicotine modulated down-stream signaling pathways related to cervical macrophage polarization in PTB mice
To elucidate the mechanisms by which α7nAchR regulates cervical macrophage polarization in PTB, we investigated changes in the expression of the MAP kinases that mediate macrophage polarization in cervical tissues from PTB animals by Western blotting and double immunofluorescence staining. Results showed that the JNK and ERK1/2 signaling pathways regulated M1 macrophage polarization. Expression levels of phosphorylated JNK and ERK1/2 were significantly increased in cervical tissues from PTB group compared to normal pregnant controls. Nicotine treatment effectively inhibited the phosphorylation of JNK and ERK1/2 in PTB group ( Figure 10A).
The pro-inflammatory NF-κB pathway mediates the anti-inflammatory effects of α7nAChR. To further examine the effects of α7nAchR activation on the NF-κB pathway, we detected its expression and nuclear translocation in macrophages by Western blotting and double immunofluorescence staining. The expression of NF-κB was profoundly increased in cervical tissues from PTB mice compared to normal pregnant controls. Immunofluorescence images showed a significant increase in NF-κB nuclear translocation in cervical macrophages from PTB mice. α7nAchR activation decreased NF-κB activity ( Figure 10B).
We also investigated the signaling pathways such as PI3K/AKT, which is responsible for the activation of M2 macrophages. Phosphorylation of JAK2, STAT3, and AKT was downregulated in cervical tissues from PTB mice compared to normal pregnant controls ( Figure 10A), suggesting that activation of JAK2/SAT3 and PI3K/AKT was inhibited in cervical tissues from PTB mice. Nicotine treatment increased the phosphorylation of JAK2, STAT3, and AKT in cervical tissues from PTB mice ( Figure 10A).

Discussion
We previously found that changes in α7nAChR activity participated in cervical ripening in normal pregnancy [31]. The present study further showed that α7nAChR expression on cervical macrophages α7nAChR is a primary receptor of the cholinergic anti-inflammatory pathway that exerts its antiinflammatory effects by decreasing the production of TNF-α and other cytokines from macrophages [32]. In normal pregnant murine, we have shown that α7nAChR expression on cervical macrophages and cervical collagen content decreased, and the number of M1 macrophages and expression of M1 macrophage biomarkers increased from mid-to late pregnancy [31], while nicotine exposure prolonged gestation and inhibited cervical ripening [33]. Further, in pregnant animals administered LPS, we found that nicotine exerted anti-inflammatory effects by decreasing LPS-induced increases in serum TNF-α and IL-6 concentrations, and rescued fetuses [26]. In pregnant women with threatened preterm labor, serum levels of inflammatory cytokines such as TNF-α and interleukin-10 (IL-10) are increased compared to controls without threatened preterm labor [34]. In the present study, the Evidence suggests that differential macrophage polarization is involved in tissue remodeling, as transformation of tissue macrophages from the M1 to M2 phenotype may decrease inflammation and inhibit collagen degradation. In a rat model of muscle injury, modulating macrophage phenotypes by decreasing the number of M1 macrophages and increasing the number of M2 macrophages potentiated an anti-inflammatory effect and promoted tissue repair [20]. In a mouse model of myocardial infarction, inducing M2 macrophage polarization reduced left ventricular adverse remodeling and improved cardiac function [19]. During pregnancy-related disorders such as miscarriages [35] and spontaneous preterm labor [36], the polarization pattern of decidua macrophages skewed toward M1. Such M1 polarization could result in an excessive pro-inflammatory response at the maternal-fetal interface, and interrupt the normal maintenance of pregnancy. In the present study, we showed a similar trend towards M1 polarization in macrophages in the cervix in PTB model. These findings corresponded to an increase in extracellular MMP-9 expression. Activation of α7nAchR impeded PTB-associated macrophage M1 polarization and inhibited the expression of MMP-9. These data suggest that M1 polarization promoted collagen degradation and premature cervical maturation in PTB. Regulating α7nAChR activity changed the macrophage polarization state and prevented cervical maturation.