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).
Table 1. Patient characteristics for immunohistochemistry and Masson staining study in Cervix Biopsies from women who were PTL and TL.
|
Age(years)
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Previous PTL
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Previous cervical surgery
|
Gestational age(weeks)
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TL (n=12)
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29(20-38)
|
0
|
0
|
39(38-40)
|
PTL (n=10)
|
28(20-36)
|
3
|
0
|
32 (28-35) **
|
|
|
|
|
|
|
|
PTL, spontaneous preterm labor through vaginal delivery; TL, term labor through vaginal delivery. The data are expressed as median and analyzed with the nonparametric two-tailed t test. **P<0.01, compared to TL.
To investigate the possible role of α7nAchR on cervical macrophages in PTB,we 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 anti-inflammatory 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 pro-inflammatory 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).
The nicotine-induced modulation of JNK, ERK1/2, NF-κB nuclear translocation in cervical macrophages, JAK2/STAT3 and PI3K/AKT was rescued by treatment with α-BGT, which inhibited α7nAChR activity in cervical macrophages (Figure 10A and 10B).