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In our earlier work, we found that intrauterine (i.u.) and intraperitoneal (i.p.) injection of LPS (10-μg serotype 0111:B4) induced preterm labor (PTL) with high pup mortality, marked systemic inflammatory response and hypotension. Here, we used both i.u. and i.p. LPS models in pregnant wild-type (wt) and CCR2 knockout (CCR2-/-) mice on E16 to investigate the role played by the CCL2/CCR2 system in the response to LPS. Basally, lower numbers of monocytes and macrophages and higher numbers of neutrophils were found in the myometrium, placenta, and blood of CCR2-/- vs. wt mice. After i.u. LPS, parturition occurred at 14 h in both groups of mice. At 7 h post-injection, 70% of wt pups were dead vs. 10% of CCR2-/- pups, but at delivery 100% of wt and 90% of CCR2-/- pups were dead. Myometrial and placental monocytes and macrophages were generally lower in CCR2-/- mice, but this was less consistent in the circulation, lung, and liver. At 7 h post-LPS, myometrial ERK activation was greater and JNK and p65 lower and the mRNA levels of chemokines were higher and of inflammatory cytokines lower in CCR2-/- vs. wt mice. Pup brain and placental inflammation were similar. Using the IP LPS model, we found that all measures of arterial pressure increased in CCR2-/- but declined in wt mice. These data suggest that the CCL2/CCR2 system plays a critical role in the cardiovascular response to LPS and contributes to pup death but does not influence the onset of inflammation-induced PTL., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Although progesterone (P4) supplementation is the most widely used therapy for the prevention of preterm labor (PTL), reports of its clinical efficacy have been conflicting. We have previously shown that the anti-inflammatory effects of P4 can be enhanced by increasing intracellular cyclic adenosine monophosphate (cAMP) levels in primary human myometrial cells. Here, we have examined whether adding aminophylline (Am), a non-specific phosphodiesterase inhibitor that increases intracellular cAMP levels, to P4 might improve its efficacy using in vivo and in vitro models of PTL. In a mouse model of lipopolysaccharide (LPS)-induced PTL, we found that the combination of P4 and Am delayed the onset of LPS-induced PTL, while the same dose of P4 and Am alone had no effect. Pup survival was not improved by either agent alone or in combination. Myometrial prolabor and inflammatory cytokine gene expression was reduced, but the reduction was similar in P4 and P4/Am treated mice. There was no effect of the combination of P4 and Am on an ex vivo assessment of myometrial contractility. In human myometrial cells and myometrial tissue explants, we found that the combination had marked anti-inflammatory effects, reducing cytokine and COX-2 mRNA and protein levels to a greater extent than either agent alone. These data suggest that the combination of P4 and Am has a more potent anti-inflammatory effect than either agent alone and may be an effective combination in women at high-risk of PTL., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The role of progesterone (P4) in the regulation of the local (uterine) and systemic innate immune system, myometrial expression of connexin 43 (Cx-43) and cyclooxygenase 2 (COX-2), and the onset of parturition was examined in (i) naïve mice delivering at term; (ii) E16 mice treated with RU486 (P4-antagonist) to induce preterm parturition; and (iii) in mice treated with P4 to prevent term parturition. In naïve mice, myometrial neutrophil and monocyte numbers peaked at E18 and declined with the onset of parturition. In contrast, circulating monocytes did not change and although neutrophils were increased with pregnancy, they did not change across gestation. The myometrial mRNA and protein levels of most chemokines/cytokines, Cx-43, and COX-2 increased with, but not before, parturition. With RU486-induced parturition, myometrial and systemic neutrophil numbers increased before and myometrial monocyte numbers increased with parturition only. Myometrial chemokine/cytokine mRNA abundance increased with parturition, but protein levels peaked earlier at between 4.5 and 9 h post-RU486. Cx-43, but not COX-2, mRNA expression and protein levels increased prior to the onset of parturition. In mice treated with P4, the gestation-linked increase in myometrial monocyte, but not neutrophil, numbers was prevented, and expression of Cx-43 and COX-2 was reduced. On E20 of P4 supplementation, myometrial chemokine/cytokine and leukocyte numbers, but not Cx-43 and COX-2 expression, increased. These data show that during pregnancy P4 controls myometrial monocyte infiltration, cytokine and prolabor factor synthesis via mRNA-dependent and independent mechanisms and, with prolonged P4 supplementation, P4 action is repressed resulting in increased myometrial inflammation.

Inflammation plays a key role in human term and preterm labor (PTL). Intrauterine LPS has been widely used to model inflammation-induced complications of pregnancy, including PTL. It has been shown to induce an intense myometrial inflammatory cell infiltration, but the role of LPS-induced inflammatory cell activation in labor onset and fetal demise is unclear. We investigated this using a mouse model of PTL, where an intrauterine injection of 10 μg of LPS (serotype 0111:B4) was given at E16 of CD1 mouse pregnancy. This dose induced PTL at an average of 12.7 h postinjection in association with 85% fetal demise. Flow cytometry showed that LPS induced a dramatic systemic inflammatory response provoking a rapid and marked leucocyte infiltration into the maternal lung and liver in association with increased cytokine levels. Although there was acute placental inflammatory gene expression, there was no corresponding increase in fetal brain inflammatory gene expression until after fetal demise. There was marked myometrial activation of NFκB and MAPK/AP-1 systems in association with increased chemokine and cytokine levels, both of which peaked with the onset of parturition. Myometrial macrophage and neutrophil numbers were greater in the LPS-injected mice with labor onset only; prior to labor, myometrial neutrophils and monocytes numbers were greater in PBS-injected mice, but this was not associated with an earlier onset of labor. These data suggest that intrauterine LPS induces parturition directly, independent of myometrial inflammatory cell infiltration, and that fetal demise occurs without fetal inflammation. Intrauterine LPS provokes a marked local and systemic inflammatory response but with limited inflammatory cell infiltration into the myometrium or placenta., (© 2016 by the Society for the Study of Reproduction, Inc.)