Macrophages exert homeostatic actions in pregnancy to protect against preterm birth and fetal inflammatory injury.

Macrophages are commonly thought to contribute to the pathophysiology of preterm labor by amplifying inflammation - but a protective role has not previously been considered to our knowledge. We hypothesized that given their antiinflammatory capability in early pregnancy, macrophages exert essential roles in maintenance of late gestation and that insufficient macrophages may predispose individuals to spontaneous preterm labor and adverse neonatal outcomes. Here, we showed that women with spontaneous preterm birth had reduced CD209+CD206+ expression in alternatively activated CD45+CD14+ICAM3- macrophages and increased TNF expression in proinflammatory CD45+CD14+CD80+HLA-DR+ macrophages in the uterine decidua at the materno-fetal interface. In Cd11bDTR/DTR mice, depletion of maternal CD11b+ myeloid cells caused preterm birth, neonatal death, and postnatal growth impairment, accompanied by uterine cytokine and leukocyte changes indicative of a proinflammatory response, while adoptive transfer of WT macrophages prevented preterm birth and partially rescued neonatal loss. In a model of intra-amniotic inflammation-induced preterm birth, macrophages polarized in vitro to an M2 phenotype showed superior capacity over nonpolarized macrophages to reduce uterine and fetal inflammation, prevent preterm birth, and improve neonatal survival. We conclude that macrophages exert a critical homeostatic regulatory role in late gestation and are implicated as a determinant of susceptibility to spontaneous preterm birth and fetal inflammatory injury.

Differential Response of Gestational Tissues to TLR3 Viral Priming Prior to Exposure to Bacterial TLR2 and TLR2/6 Agonists.

Background: Infection/inflammation is an important causal factor in spontaneous preterm birth (sPTB). Most mechanistic studies have concentrated on the role of bacteria, with limited focus on the role of viruses in sPTB. Murine studies support a potential multi-pathogen aetiology in which a double or sequential hit of both viral and bacterial pathogens leads to a higher risk preterm labour. This study aimed to determine the effect of viral priming on bacterial induced inflammation in human in vitro models of ascending and haematogenous infection. Methods: Vaginal epithelial cells, and primary amnion epithelial cells and myocytes were used to represent cell targets of ascending infection while interactions between peripheral blood mononuclear cells (PBMCs) and placental explants were used to model systemic infection. To model the effect of viral priming upon the subsequent response to bacterial stimuli, each cell type was stimulated first with a TLR3 viral agonist, and then with either a TLR2 or TLR2/6 agonist, and responses compared to those of each agonist alone. Immunoblotting was used to detect cellular NF-κB, AP-1, and IRF-3 activation. Cellular TLR3, TLR2, and TLR6 mRNA was quantified by RT-qPCR. Immunoassays were used to measure supernatant cytokine, chemokine and PGE2 concentrations. Results: TLR3 ("viral") priming prior to TLR2/6 agonist ("bacterial") exposure augmented the pro-inflammatory, pro-labour response in VECs, AECs, myocytes and PBMCs when compared to the effects of agonists alone. In contrast, enhanced anti-inflammatory cytokine production (IL-10) was observed in placental explants. Culturing placental explants in conditioned media derived from PBMCs primed with a TLR3 agonist enhanced TLR2/6 agonist stimulated production of IL-6 and IL-8, suggesting a differential response by the placenta to systemic inflammation compared to direct infection as a result of haematogenous spread. TLR3 agonism generally caused increased mRNA expression of TLR3 and TLR2 but not TLR6. Conclusion: This study provides human in vitro evidence that viral infection may increase the susceptibility of women to bacterial-induced sPTB. Improved understanding of interactions between viral and bacterial components of the maternal microbiome and host immune response may offer new therapeutic options, such as antivirals for the prevention of PTB.

Comparative Assessment of Leukocyte Infiltrate Composition in the Uterine and Vaginal Tissues in Rats with Experimental Endomyometritis Treated with Interferon-γ at Different Times of the Day.

The cell composition of leukocyte infiltrates in the endometrium, myometrium, and vaginal walls was studied in Wistar rats with modeled chronic endomyometritis after administration of IFNγ (0.1 µg/100 g body weight) in different daily regimens (10.00 or 20.00). Morning injections of this cytokine ameliorated inflammatory infiltration of the uterine wall and vagina, but increased the content of neutrophils in the endometrium. Evening cytokine injections reduced neutrophilic infiltration, enhanced mononuclear infiltration, and had no effect on plasmacytic infiltration of the uterine and vaginal walls. In the vaginal wall, both IFNγ administration schedules decreased neutrophil content. The data indicate the necessity to take into account the circadian rhythms in IFN therapy.

Inhibition of GPR91 Reduces Inflammatory Mediators Involved in Active Labor in Myometrium.

RESULTS: GPR91 mRNA expression was significantly higher in myometrium from women during term spontaneous labor compared to no labor. Likewise, in mice, GPR91 mRNA expression was significantly upregulated in myometrium during inflammation-induced preterm labor compared to preterm no labor. In myometrial cells, IL1B and TNF significantly increased GPR91 mRNA expression. Knockdown of GPR91 by siRNA in myometrial cells significantly suppressed the secretion and/or expression of IL1B- and TNF-induced proinflammatory cytokines (GM-CSF, IL1A, IL1B, and IL6) and chemokines (CXCL8 and CCL2), myometrial contractility (expression of the contraction-associated proteins PTGFR and CX43, secretion of the uterotonic PGF2α , and in situ collagen gel contraction), and the transcription factor NF-κB. CONCLUSION: Our findings demonstrate that GPR91 is involved in the genesis of proinflammatory and prolabor mediators induced by IL1B or TNF and collectively suggest that GPR91 may contribute to augmentation of the labor processes.

Distribution of dendritic cells in the septate uterus: An immunological perspective.

PROBLEM: Septate uterus is associated with spontaneous abortion. Surgical intervention of the uterine septa (US) is frequently performed following spontaneous abortion; however, immunological mechanisms for spontaneous abortion in patients with septate uterus remain completely unknown. METHOD OF STUDY: A total of 12 women with septate uterus who underwent hysteroscopic metroplasty and 10 women with uterine leiomyoma who underwent total hysterectomy were enrolled as the experimental and control groups, respectively. Immune cells, dendritic cells (DCs), macrophages, T cells, natural killer cells, invariant natural killer cells, and chemokine receptors in US and uterine myometrium tissue (UMT) were analyzed using flow cytometry and immunohistochemical staining. Additionally, the chemokine production of macrophage inflammatory protein 1 alpha (MIP-1α), regulated upon activation normal T-cell express sequence (RANTES), and macrophage inflammatory protein 3 beta (MIP-3ß) from the viable cells obtained from the US and UMT samples was evaluated in an ex vivo study. RESULTS: The percentage of CD141+ DCs in US was significantly lower than that in UMT. Both US and UMT showed CCR1 and CCR5 expression on CD141+ DCs; however, the production of chemokines, MIP-1α, RANTES, and MIP-3ß was abundant in UMT-obtained viable cells. CONCLUSION: The accumulation of CD141+ DCs was lower in US than that in UMT. This phenomenon may be caused by low chemokine productions in US. Our findings support the benefit of surgical intervention for septate uterus-that is, the elimination of inappropriate implantation sites.

Acute inflammation in the uterine isthmus coincides with postpartum acute myometritis in the uterine body involving refractory postpartum hemorrhage of unknown etiology after cesarean delivery.

Uterine atony is a major cause of postpartum hemorrhage. We recently proposed the new histological concept of postpartum acute myometritis (PAM) for the pathophysiology of refractory uterine atony of unknown etiology, which is characterized by the diffuse activation of mast cells and the complement system as well as the massive infiltration of macrophages and neutrophils into the uterine body. We herein focused on the uterine isthmus just adjacent to the body. The isthmus becomes significantly elongated throughout pregnancy. It is composed of myocytes and fibroblasts with an extracellular matrix that forms a passive lower segment during labor. The aim of this study was to histologically examine the uterine isthmus in cases of PAM in the uterine body. Under the amniotic fluid embolism-registry program in Japan, we selected PAM cases from uterine samples obtained by cesarean hysterectomy and delivered to us for analyses between 2011 and 2017. Control tissues were collected during elective cesarean section. We investigated the isthmus tissues of these cases and performed immunohistochemistry for inflammatory cell markers, i.e. neutrophil elastase, mast cell tryptase, CD68, CD3, and C5a receptor (C5aR). The numbers of tryptase-positive degranulating mast cells, elastase-positive neutrophils, CD68-positive macrophages, and C5aR-positive cells in the isthmus were significantly higher in uteri with PAM in the body than in controls without PAM. CD3 was negative in both groups. In conclusion, inflammation and an anaphylactoid reaction were histologically detected not only in the uterine body, but in the isthmus among cases of refractory PPH of unknown etiology after cesarean section.

Characterization of an Adapted Murine Model of Intrauterine Inflammation-Induced Preterm Birth.

Preterm birth (PTB) affects nearly 15 million infants each year. Of these PTBs, >25% are a result of inflammation or infection. Animal models have improved our understanding of the mechanisms leading to PTB. Prior work has described induction of intrauterine inflammation in mice with a single injection of lipopolysaccharide (LPS). Herein, we have improved the reproducibility and potency of LPS in the model using two injections distal to the cervix. An in vivo imaging system revealed more uniform distribution of Evans Blue Dye using a double distal injection (DDI) approach compared with a single proximal injection (SPI). Endotoxin concentrations in vaginal lavage fluid from SPI dams were significantly higher than from DDI dams. At equivalent LPS doses, DDI consistently induced more PTB than SPI, and DDI showed a linear dose-response, whereas SPI did not. Gene expression in myometrial tissue revealed increased levels of inflammatory markers in dams that received LPS DDI compared with LPS SPI. The SPI group showed more significant overexpression in cervical remodeling genes, likely due to the leakage of LPS from the uterine horns through the cervix. The more reliable PTB induction and uniform uterine exposure provided by this new model will be useful for further studying fetal outcomes and potential therapeutics for the prevention of inflammation-induced PTB.

Cathelicidins and the Onset of Labour.

Preterm birth, defined as delivery before 37 weeks of gestation, is the leading cause of neonatal mortality and morbidity. Infection and inflammation are frequent antecedents of spontaneous preterm birth. Cathelicidin, an antimicrobial host defence peptide, is induced by infection and inflammation and although expressed in the reproductive tract and fetal tissues, its role in the pathogenesis of spontaneous preterm birth is unknown. Here we demonstrate that cathelicidin expression is increased at RNA and protein level in the mouse uterus in a model of inflammation-induced labour, where ultrasound guided intrauterine injection of lipopolysaccharide (LPS) at E17 stimulates preterm delivery within 24 hours. Cathelicidin-deficient (Camp-/-) mice are less susceptible to preterm delivery than wild type mice following intrauterine injection of 1 µg of LPS, and this is accompanied by a decrease in circulating IL-6, an inflammatory mediator implicated in the onset of labour. We also show that the proportion of cathelicidin expressing cells in the myometrium is higher in samples obtained from women in labour at term than pre-labour. Together, these data suggest that cathelicidin has roles in mediating pro-inflammatory responses in a murine model of inflammation-induced labour, and in human term labour.

Novel anti-inflammatory actions of TIPE2 in human primary amnion and myometrial cells.

Inflammation plays a pivotal role in the terminal process of human labor and delivery, including myometrial contractions and membrane rupture. TNF-alpha-induced protein 8-like-2 (TIPE2) is a novel inflammation regulator; however, there are no studies on the role of TIPE2 in human labor. We report that in myometrium, there is decreased TIPE2 mRNA expression during late gestation which was further decreased in labor. In fetal membranes, TIPE2 mRNA expression was decreased with both term and preterm labor compared to no labor samples. Knockdown of TIPE2 by siRNA in primary myometrium and amnion cells was associated with an augmentation of IL1B and TNF-induced expression of pro-inflammatory cytokines and chemokines; expression of contraction-associated proteins and secretion of the uterotonic prostaglandin PGF2α and expression of extracellular matrix degrading enzymes. In TIPE2-deficient myometrial cells treated with inhibitors of NF-κB or ERK1/2, the secretion of pro-labor mediators was reduced back to control levels. In conclusion, these in vitro experiments indicate that loss of TIPE2 exacerbates the inflammatory response.

Expression and function of macrophage-inducible C-type lectin (Mincle) in inflammation driven parturition in fetal membranes and myometrium.

The pivotal role of inflammatory processes in human parturition is well known, but not completely understood. We have performed a study to examine the role of macrophage-inducible C-type lectin (Mincle) in inflammation-associated parturition. Using human samples, we show that spontaneous labour is associated with up-regulated Mincle expression in the myometrium and fetal membranes. Mincle expression was also increased in fetal membranes and myometrium in the presence of pro-labour mediators, the proinflammatory cytokines interleukin (IL)-1B and tumour necrosis factor (TNF), and Toll-like receptor (TLR) ligands fsl-1, poly(I:C), lipopolysaccharide (LPS) and flagellin. These clinical studies are supported by mouse studies, where an inflammatory challenge in a mouse model of preterm birth increased Mincle expression in the uterus. Importantly, elimination of Mincle decreased the effectiveness of proinflammatory cytokines and TLR ligands to induce the expression of pro-labour mediators; namely, proinflammatory cytokines and chemokines, contraction-associated proteins and prostaglandins, and extracellular matrix remodelling enzymes, matrix metalloproteinases. The data presented in this study suggest that Mincle is required when inflammatory activation precipitates parturition.

Toll-like receptor 9, maternal cell-free DNA and myometrial cell response to CpG oligodeoxynucleotide stimulation.

PROBLEM: Among mechanisms triggering onset of parturition, it has been recently postulated that Toll-Like Receptor (TLR)9 engagement by cell-free DNA (cfDNA) triggers inflammation, myometrial contractions, and labor in absence of infection. The current study evaluated whether direct (myometrial) or indirect (decidual) TLR9 engagement enhances human myometrial contractility. METHOD OF STUDY: Toll-like receptor 9 expression and cellular localization were surveyed by immunohistochemistry of placenta, fetal membranes, and myometrium in term (gestational age [GA]: >37 weeks) labor (TL, n = 7) or term non-labor (TNL, n = 7) tissues. Non-pregnant myometrium (n = 4) served as reference. TLR9 mRNA expression relative to other TLRs was evaluated through the mining of an RNA-seq dataset and confirmed by RT-PCR. Immortalized human myometrial cells (hTERT-HM) were treated with incremental concentrations of TLR9 agonist ODN2395, TNF-α, or LPS. Secreted cytokines were quantified by multiplex immunoassay, and contractility was assessed by an in-gel cell contraction assay (n = 9). Induction of hTERT-HM contractility was also evaluated indirectly following exposure to conditioned media from primary term decidual cells (n = 4) previously stimulated with ODN2395. RESULTS: Toll-like receptor 9 immunostaining in placenta and amniochorion was strongest in decidual cells, but unrelated to labor. TLR9 staining intensity was significantly decreased in TL compared with TNL myometrium (P = 0.002). Although total cfDNA in maternal circulation increased in TL (P = 0.025 vs TNL), difference in cffDNA was non-significant. Myometrial TLR9 mRNA levels were unaffected by contractile status and far less abundant than other pro-inflammatory TLRs. hTERT-HM contractility was enhanced by LPS (P = 0.002) and TNF-α (P = 0.003), but not by ODN2395 (P = 0.345) or supernatant of TLR9-stimulated decidual cells. CONCLUSION: Myometrial and decidual TLR9 are unlikely to directly regulate human parturition.

Clinicopathologic characteristics, tumor infiltrating lymphocytes and programed cell death ligand-1 expression in 162 endometrial carcinomas with deficient mismatch repair function.

OBJECTIVE: Endometrial carcinoma (EC) with deficient mismatch repair (dMMR) protein has been reported to have increased tumor infiltrating lymphocytes (TILs) and programed cell death ligand-1 (PD-L1) expression. TILs and PD-L1 expression are compared between two main types of dMMR ECs (epigenetic dMMR due to MLH1 promoter methylation vs mutated dMMR due to genetic mutation). METHODS: Immunohistochemistry for PD-L1 was performed in triplicate on tissue microarray sections. TILs were semi-quantitatively evaluated on whole-slide images of whole histologic sections. The clinicopathologic characteristics together with PD-L1 expression and TILs were analyzed between mutated and epigenetic dMMR ECs. RESULTS: Of the 162 dMMR ECs identified, 126 had epigenetic dMMR and 36 had mutated dMMR. Univariate analysis demonstrated mutated dMMR ECs showed younger age, less myometrium invasion of >50%, less lymphovascular invasion, and more TILs than epigenetic dMMR ECs. Multivariate analysis demonstrated significantly younger age and more TILs in mutated dMMR ECs than in epigenetic ECs. PD-L1 expression did not show any significant difference between these two groups. Seventeen (13.5%) patients with epigenetic dMMR EC had recurrence and 13 (10.3%) patients died of disease. In contrast, only one patient with mutated dMMR EC had recurrence (3%) and died of disease (3%). CONCLUSION: ECs with mutated dMMR demonstrated significantly increased TILs than ECs with epigenetic dMMR, suggesting a stronger immune reaction and potential response to immunotherapy in these tumors.

NOD-like receptor pyrin domain-containing-3 (NLRP3) regulates inflammation-induced pro-labor mediators in human myometrial cells.

PROBLEM: Inflammation plays a major role in preterm birth. Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) plays a role in inflammatory diseases. The aims of this study were to determine the effect of term labor on the expression of NLRP3 in human myometrium and the effect of NLRP3 silencing on pro-labor mediators in myometrial cells. METHOD OF STUDY: NLRP3 expression was assessed in myometrium from non-laboring and laboring women by qRT-PCR and Western blotting. Human primary myometrial cells were transfected with NLRP3 siRNA (siNLRP3), treated with pro-inflammatory cytokines and toll-like receptor (TLR) ligands, and assayed for pro-inflammatory mediators' expression. RESULTS: NLRP3 expression was higher in myometrium after term spontaneous labor and by TNF, IL1B, fsl-1, and flagellin. In siNLRP3-transfected cells, there was a significant decrease in the expression of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL8, CCL2), and adhesion molecules (ICAM1 and VCAM1) stimulated with IL1B, TNF, or TLR ligands; decrease in IL1B-stimulated PTGS2 and PTGFR mRNA expression and PGF2α release; and increase in TNF-stimulated myometrial gel shrinkage as assessed by an in vitro cell contraction assay. CONCLUSION: NLRP3 is increased with labor in myometrial, and knockdown of NLRP3 is associated with an attenuation of inflammation-induced expression of pro-inflammatory and pro-labor mediators in human myometrium.

Inhibition of Inflammatory Changes in Human Myometrial Cells by Cell Penetrating Peptide and Small Molecule Inhibitors of NFκB.

Complications arising from Preterm Birth are the leading causes of neonatal death globally. Current therapeutic strategies to prevent Preterm Birth are yet to demonstrate success in terms of reducing this neonatal disease burden. Upregulation of intracellular inflammatory pathways in uterine cells, including those involving nuclear factor kappa-B (NFκB), have been causally linked to both human term and preterm labor, but the barrier presented by the cell membrane presents an obstacle to interventions aimed at dampening these inflammatory responses. Cell penetrating peptides (CPPs) are novel vectors that can traverse cell membranes without the need for recognition by cell surface receptors and offer the ability to deliver therapeutic cargo internal to cell membranes. Using a human uterine cell culture inflammatory model, this study aimed to test the effectiveness of CPP-cargo delivery to inhibit inflammatory responses, comparing this effect with a small molecule inhibitor (Sc514) that has a similar intracellular target of action within the NFκB pathway (the IKK complex). The CPP Penetratin, conjugated to rhodamine, was able to enter uterine cells within a 60 min timeframe as assessed by live confocal microscopy, this phenomena was not observed with the use of a rhodamine-conjugated inert control peptide (GC(GS)4). Penetratin CPP conjugated to an IKK-inhibitory peptide (Pen-NBD) demonstrated ability to inhibit both the IL1ß-induced expression of the inflammatory protein COX2 and dampen the expression of a bespoke array of inflammatory genes. Truncation of the CPP vector rendered the CPP-cargo conjugate much less effective, demonstrating the importance of careful vector selection. The small molecule inhibitor Sc514 also demonstrated ability to inhibit COX2 protein responses and a broad down-regulatory effect on uterine cell inflammatory gene expression. These results support the further exploration of either CPP-based or small molecular treatment strategies to dampen gestational cell inflammatory responses in the context of preterm birth. The work underlines both the importance of careful selection of CPP vector-cargo combinations and basic testing over a broad time and concentration range to ensure effective responses. Further work should demonstrate the effectiveness of CPP-linked cargos to dampen alternative pathways of inflammation linked to Preterm Birth such as MAP Kinase or AP1.

Cardiac changes in apoptosis, inflammation, oxidative stress, and nitric oxide system induced by prenatal and postnatal zinc deficiency in male and female rats.

PURPOSE: Zinc restriction during fetal and postnatal development could program cardiovascular diseases in adulthood. The aim of this study was to determine the effects of zinc restriction during fetal life, lactation, and/or post-weaning growth on cardiac inflammation, apoptosis, oxidative stress, and nitric oxide system of male and female adult rats. METHODS: Wistar rats were fed a low- or a control zinc diet during pregnancy and up to weaning. Afterward, offspring were fed either a low- or a control zinc diet until 81 days of life. IL-6 and TNF-α levels, TUNEL assay, TGF-ß1 expression, thiobarbituric acid-reactive substances that determine lipoperoxidation damage, NADPH oxidase-dependent superoxide anion production, antioxidant and nitric oxide synthase activity, mRNA and protein expression of endothelial nitric oxide synthase, and serine1177 phosphorylation isoform were determined in left ventricle. RESULTS: Zinc deficiency activated apoptotic and inflammatory processes and decreased TGF-ß1 expression and nitric oxide synthase activity in cardiac tissue of both sexes. Male zinc-deficient rats showed no changes in endothelial nitric oxide synthase expression, but a lower serine1177 phosphorylation. Zinc deficiency induced an increase in antioxidant enzymes activity and no differences in lipoperoxidation products levels in males. Females were less sensitive to this deficiency exhibiting lower increase in apoptosis, lower decrease in expression of TGF-ß1, and higher antioxidant and nitric oxide enzymes activities. A zinc-adequate diet during postnatal life reversed most of these mechanisms. CONCLUSION: Prenatal and postnatal zinc deficiency induces alterations in cardiac apoptotic, inflammatory, oxidative, and nitric oxide pathways that could predispose the onset of cardiovascular diseases in adult life.

Progesterone, the maternal immune system and the onset of parturition in the mouse.

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.

Is myometrial inflammation a cause or a consequence of term human labour?

Myometrial inflammation is thought to have a pivotal role in the onset of term and some forms of preterm labour. This is based on the comparison of samples taken from women undergoing term elective CS prior to the onset of labour with those taken from women in established labour. Consequently, it is not clear whether myometrial inflammation is a cause or a consequence of labour. Our objective is to test the hypothesis that myometrial inflammation is a consequence of the onset of labour. To test this hypothesis, we have obtained myometrial samples from women at various stages of pregnancy and spontaneous labour and studied the activation of the AP-1 (c-Jun) and NFκB (p65) systems, cytokine mRNA expression and protein levels and inflammatory cell infiltration and activation. We found that the activation of p65 declined from preterm to term not in labour samples and thereafter increased in early and established labour. Cytokine mRNA expression and protein levels increased in established labour only. Using flow cytometry of myometrial tissue, we found that the number of neutrophils did increase with the onset of labour, but on tissue section, these were seen to be intravascular and not infiltrating into the myometrium. These data suggest that myometrial inflammation is a consequence rather than a cause of term labour.

TLR2, TLR3 and TLR5 regulation of pro-inflammatory and pro-labour mediators in human primary myometrial cells.

Preterm birth continues to be a significant global health care issue, due to our lack of understanding of the mechanisms that drive human labour and delivery. Toll-like receptors (TLRs) are essential in triggering an inflammatory response in human gestational tissues, leading to the production of pro-inflammatory and pro-labour mediators, and thus preterm birth. The aims of this study were to determine whether the adaptor molecules associated with TLR2, TLR3 and TLR5 signalling are involved in human myometrium. Primary human myometrial cells were transfected with siRNA against TIRAP, IRAK1, IRAK4, TAK1and stimulated with bacterial product fsl-1 (TLR2); TRIF, TRADD, TRAF6, RIP1, TAK1 and stimulated with dsRNA viral analogue poly(I:C) (TLR3); IRAK1, IRAK4, TAK1 and stimulated with bacterial product flagellin (TLR5), and assayed for production of pro-inflammatory and pro-labour mediators. Cells transfected with TIRAP, IRAK1, IRAK4 or TAK1 all showed a decrease in fsl-1-induced expression of cytokines (IL-1α, IL-1ß, IL-6), chemokines (GRO-α, IL-8, MCP-1), adhesion molecule ICAM-1, cyclooxygenase (COX)-2 mRNA and release of PGF2α and MMP-9 expression. Cells transfected with TRIF, TRAF6, RIP1 or TAK1 all decreased production of poly(I:C)-induced IL-1α, IL-1ß, IL-6, GRO-α, IL-8, MCP-1, ICAM-1 and MMP-9 expression. Cells transfected with IRAK1, IRAK4 or TAK1 all showed decreased expression of flagellin-induced cytokine and chemokine expression, ICAM-1 and MMP-9 expression. Lastly, transfection with these siRNAs decreased fsl-1, poly(I:C) and flagellin-induced NF-κB transcriptional activity. Our study signifies that these adaptor molecules are necessary for the proper production of cytokines, chemokines and pro-labour mediators after TLR ligation.

Optineurin suppression activates the mediators involved in the terminal effector pathways of human labour and delivery.

Spontaneous preterm birth remains the major cause of neonatal death and morbidity. Studies in non-gestational tissues report that optineurin (OPTN) is critical in the termination of NFKB1 activity and control of inflammation, central features of spontaneous preterm birth. The aims of the present study were to determine: (1) OPTN expression in fetal membranes and the myometrium during labour; (2) the effects of IL1B on OPTN expression in primary myometrial cells; and (3) the effects of OPTN short interference (si) RNA on IL1B-stimulated proinflammatory and prolabour mediators. OPTN mRNA and protein expression was significantly decreased with spontaneous term labour in fetal membranes and the myometrium. Although there was no effect of spontaneous preterm labour on OPTN expression in fetal membranes, there was decreased OPTN expression in membranes with chorioamnionitis and myometrial cells treated with 1ng mL-1 IL1B for 1 or 6h. In cells transfected with OPTN siRNA, significant increases were seen in IL1B-stimulated IL6, tumour necrosis factor, CXCL8 and monocyte chemoattractant protein-1 mRNA expression and release, cyclo-oxygenase-2 and prostanoid PTGFR receptor mRNA expression and the release of prostaglandin F2α. There was no change in IL1B-stimulated NFKBIA expression; however, there was increased NFKB1 p65 DNA-binding activity. The results of the present study suggest that OPTN is a negative regulator of inflammation-induced prolabour mediators.