Newborn and child-like molecular signatures in older adults stem from TCR shifts across human lifespan.

CD8+ T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8+ T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M158-66 (A2/M158) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M158+CD8+ T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8+ T cells, which was linked to highly functional public T cell receptor (TCR)αß signatures. Suboptimal TCRαß signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8+ T cell responses toward viral infections.

The short-chain fatty acids butyrate and propionate protect against inflammation-induced activation of mediators involved in active labor: implications for preterm birth.

Spontaneous preterm birth is a global health issue affecting up to 20% of pregnancies and leaves a legacy of neurodevelopmental complications. Inflammation has been implicated in a significant proportion of preterm births, where pro-inflammatory insults trigger production of additional pro-inflammatory and pro-labor mediators. Thus, novel therapeutics that can target inflammation may be a novel avenue for preventing preterm birth and improving adverse fetal outcomes. Short-chain fatty acids (SCFAs), such as butyrate and propionate, are dietary metabolites produced by bacterial fermentation of fiber in the gut. SCFAs are known to possess anti-inflammatory properties and have been found to function through G-coupled-receptors and histone deacetylases. Therefore, this study aimed to investigate the effect of SCFAs on pro-inflammatory and pro-labor mediators in an in vitro model of preterm birth. Primary human cells isolated from myometrium and fetal membranes (decidua, amnion mesenchymal and amnion epithelial cells) were stimulated with the pro-inflammatory cytokines tumor necrosis factor alpha (TNF) or interleukin 1B (IL1B). The SCFAs butyrate and propionate suppressed inflammation-induced expression of pro-inflammatory cytokines and chemokines, adhesion molecules, the uterotonic prostaglandin PGF2alpha and enzymes involved in remodeling of myometrium and degradation of the fetal membranes. Notably, propionate and butyrate also suppressed inflammation-induced prostaglandin signaling and myometrial cell contraction. These effects appear to be mediated through suppression of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) activation. These results suggest that the SCFAs may be able to prevent myometrial contractions and rupture of membranes. Further in vivo studies are warranted to identify the efficacy of SCFAs as a novel anti-inflammatory therapeutic to prevent inflammation-induced spontaneous preterm birth.

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.

The immunoproteasome inhibitor ONX-0914 regulates inflammation and expression of contraction associated proteins in myometrium.

There are currently no effective treatments to prevent spontaneous preterm labor. The precise upstream biochemical pathways that regulate the transition between uterine quiescence during pregnancy and contractility during labor remain unclear. It is well known however that intrauterine inflammation, including infection, is commonly associated with preterm labor. In this study, we identified the immunoproteasome subunit low-molecular-mass protein (LMP)7 mRNA expression to be significantly upregulated in laboring human myometrium. Silencing LMP7 using siRNA-targeted knockdown of LMP7 and its inhibitor ONX-0914 in human myometrial cells and tissues decreased proinflammatory cytokines (IL-6), cell chemotaxis (CXCL8, CCL2 expression, and THP-1 migration), cell to cell adhesion (ICAM1 expression and myometrial adhesion), contraction-associated proteins (PTGS2, FP, PGE2, and PGF2α), as well as suppressing contractions in myometrial cells and in myometrial tissues obtained from laboring women. In addition, LMP7 silencing reduced NF-κB RelA activity. ONX-0914 alleviated inflammation (CCL3, CXCL1, PTGS2, and IL-6) in myometrium, placenta, fetal brain, amniotic fluid, and maternal serum induced by LPS in pregnant mice. Collectively, our data suggest a novel role for ONX-014 to suppress uterine activation and contractility associated with preterm labor.

GIT2 deficiency attenuates inflammation-induced expression of pro-labor mediators in human amnion and myometrial cells†.

Untimely activation of the inflammatory response by sterile or infective insults in uterine tissues can result in preterm birth. Pro-inflammatory cytokines and pathogenic activation of toll-like receptors (TLRs) initiate a biochemical cascade of events leading to myometrial activation and contractility, cervical dilatation, and rupture of the chorioamniotic membranes. GIT2 is a signaling protein known to play a role in innate and adaptive immunity; however, its role in the inflammatory pathways of human labor is not known. In this article, we report that GIT2 expression is lower in human myometrium and fetal membranes with term labor, and in preterm amnion with histological chorioamnionitis. GIT2 knockdown by siRNA in primary myometrial and amnion cells exhibited reduced expression of pro-inflammatory cytokines and chemokines in response to inflammatory challenge by cytokines or TLR ligands. In addition, the pro-inflammatory cytokines IL1B and TNF could not induce the expression of extracellular matrix degrading enzymes in GIT2-deficient amnion cells. Myometrial activation in response to pro-inflammatory cytokines was also significantly suppressed in GIT2-deficient cells as evidenced by decreased prostaglandin release and expression of contraction-associated proteins. Further to this, collagen gel assays demonstrated that TNF had a reduced ability to induce myometrial contractility in situ in GIT2-deficient myometrial cells compared to control-transfected cells. In summary, the loss of GIT2 diminishes the effects inflammatory mediators have in promoting myometrial contraction and fetal membrane rupture in vitro, suggesting that GIT2 could be a possible target for preterm birth therapies.

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.

Targeting bromodomain-containing proteins to prevent spontaneous preterm birth.

Preterm birth is a global healthcare challenge. Spontaneous preterm birth (sPTB) is commonly caused by inflammation, yet there are currently no effective therapies available. The Bromodomain and Extra-Terminal motif (BET) proteins, Bromodomain-containing protein (Brd) 2 (Brd2), Brd3 and Brd4 regulate inflammation in non-gestational tissues. The roles of Brd2-4 in human pregnancy are unknown. Using human and mouse models, the present study has identified the Brd proteins part of the process by which inflammation induces parturition. Using human clinical samples, we demonstrate that labor and infection increase the expression of Brds in the uterus and fetal membranes. In primary human myometrial, amnion and decidual cells, we found that global Brd protein inhibition, as well as selective inhibition of Brds, suppressed inflammation-induced expression of mediators involved in myometrial contractions and rupture of fetal membranes. Importantly, studies in the mouse model demonstrate that the pan-Brd inhibitor JQ1 reduced intrauterine inflammation induced by bacterial endotoxin LPS as well as decreasing the effectiveness of LPS to induce parturition. These results implicate BET proteins as novel therapeutic targets for reducing inflammation associated with spontaneous preterm labor.

Formyl peptide receptor-2 is decreased in foetal growth restriction and contributes to placental dysfunction.

STUDY QUESTION: What is the association between placental formyl peptide receptor 2 (FPR2) and trophoblast and endothelial functions in pregnancies affected by foetal growth restriction (FGR)? SUMMARY ANSWER: Reduced FPR2 placental expression in idiopathic FGR results in significantly altered trophoblast differentiation and endothelial function in vitro. WHAT IS KNOWN ALREADY: FGR is associated with placental insufficiency, where defective trophoblast and endothelial functions contribute to reduced feto-placental growth. STUDY DESIGN, SIZE, DURATION: The expression of FPR2 in placental tissues from human pregnancies complicated with FGR was compared to that in gestation-matched uncomplicated control pregnancies (n = 25 from each group). Fpr2 expression was also determined in placental tissues obtained from a murine model of FGR (n = 4). The functional role of FPR2 in primary trophoblasts and endothelial cells in vitro was assessed in diverse assays in a time-dependent manner. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placentae from third-trimester pregnancies complicated by idiopathic FGR (n = 25) and those from gestation-matched pregnancies with appropriately grown infants as controls (n = 25) were collected at gestation 27-40 weeks. Placental tissues were also collected from a spontaneous CBA/CaH × DBA/2 J murine model of FGR. Placental FPR2/Fpr2 mRNA expression was determined by real-time PCR, while protein expression was examined by immunoblotting and immunohistochemistry. siRNA transfection was used to silence FPR2 expression in primary trophoblasts and in human umbilical vein endothelial cells (HUVEC), and the quantitation of cytokines, chemokines and apoptosis was performed following a cDNA array analyses. Functional effects of trophoblast differentiation were measured using HCGB/ß-hCG and syncytin-2 expression as well as markers of apoptosis, tumour protein 53 (TP53), caspase 8, B cell lymphoma 2 (BCL2) and BCL associated X (BAX). Endothelial function was assessed by proliferation, network formation and permeability assays. MAIN RESULTS AND THE ROLE OF CHANCE: Placental FPR2/Fpr2 expression was significantly decreased in FGR placentae (n = 25, P < 0.05) as well as in murine FGR placentae compared to controls (n = 4, P < 0.05). FPR2 siRNA (siFPR2) in term trophoblasts significantly increased differentiation markers, HCGB and syncytin-2; cytokines, interleukin (IL)-6, CXCL8; and apoptotic markers, TP53, caspase 8 and BAX, but significantly reduced the expression of the chemokines CXCL12 and its receptors CXCR4 and CXCR7; CXCL16 and its receptor, CXCR6; and cytokine, IL-10, compared with control siRNA (siCONT). Treatment of HUVECs with siFPR2 significantly reduced proliferation and endothelial tube formation, but significantly increased permeability of HUVECs. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Reduced expression of placental FPR2/Fpr2 was observed in the third trimester at delivery after development of FGR, suggesting that FPR2 is associated with FGR pregnancies. However, there is a possibility that the decreased placental FPR2 observed in FGR may be a consequence rather than a cause of FGR, although our in vitro functional analyses using primary trophoblasts and endothelial cells suggest that FPR2 may have a direct or indirect regulatory role on trophoblast differentiation and endothelial function in FGR. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study linking placental FPR2 expression with changes in the trophoblast and endothelial functions that may explain the placental insufficiency observed in FGR. STUDY FUNDING/COMPETING INTERESTS: P.M. and P.R.E. received funding from the Australian Institute of Musculoskeletal Science, Western Health, St. Albans, Victoria 3021, Australia. M.L. is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; Grant no. 1047025). Monash Health is supported by the Victorian Government's Operational Infrastructure Support Programme. The authors declare that there is no conflict of interest in publishing this work.

IRF5 is increased in labouring myometrium and regulates pro-labour mediators.

Preterm birth continues to be the leading cause of neonatal mortality and morbidities that can extend into adult life. Few treatment options stem from our incomplete understanding of the mechanisms of human labour and delivery. Activation of the inflammatory response in gestational tissues by inflammation and/or infection leads to the production of pro-inflammatory and pro-labour mediators, thus preterm birth. Interferon regulatory factor 5 (IRF5) has recently emerged as an important pro-inflammatory transcription factor involved in acute and chronic inflammation. The aims of this study were to determine the expression of IRF5 in human myometrium from labouring and non-labouring women, and whether IRF5 is involved in the genesis of pro-inflammatory and pro-labour mediators induced by pro-inflammatory cytokines or toll-like receptor (TLR) ligands. IRF5 mRNA and protein expression was significantly higher in human myometrium after spontaneous term labour, compared to non-labouring tissues. IRF5 mRNA expression was also significantly higher in primary myometrial cells treated with the pro-inflammatory cytokines IL1B or TNF. In primary myometrial cells, IRF5 knockdown by siRNA (siIRF5) was associated with significantly decreased expression and or secretion of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL8, CCL2), adhesion molecules (ICAM1, VCAM1) and contraction-associated proteins PTGS2, PGF2α and PTGFR when in the presence of IL1B, TNF, fsl-1 (TLR2/6 ligand) or flagellin (TLR5 ligand). siIRF5-transfected cells also displayed decreased NF-κB RELA transcriptional activity in the presence of these preterm birth mediators. Our study suggests a novel role for IRF5 in the regulation of the inflammatory response in human myometrium.

PARK7 regulates inflammation-induced pro-labour mediators in myometrial and amnion cells.

Preterm birth is a prevalent cause of neonatal deaths worldwide. Inflammation has been implicated in spontaneous preterm birth involved in the processes of uterine contractility and membrane rupture. Parkinson protein 7 (PARK7) has been found to play an inflammatory role in non-gestational tissues. The aims of this study were to determine the expression of PARK7 in myometrium and fetal membranes with respect to term labour onset and to elucidate the effect of PARK7 silencing in primary myometrium and amnion cells on pro-inflammatory and pro-labour mediators. PARK7 mRNA expression was higher in term myometrium and fetal membranes from women in labour compared to non-labouring samples and in amnion from preterm deliveries with chorioamnionitis. In human primary myometrial cells transfected with PARK7 siRNA (siPARK7), there was a significant decrease in IL1B, TNF, fsl-1 and poly(I:C)-induced expression of pro-inflammatory cytokine IL6, chemokines (CXCL8, CCL2), adhesion molecule ICAM1, prostaglandin PGF2α and its receptor PTGFR. Similarly, amnion cells transfected with siPARK7 displayed a decrease in IL1B-induced expression of IL6, CXCL8 and ICAM1. In myometrial cells transfected with siPARK7, there was a significant reduction of NF-κB RELA transcriptional activity when stimulated with fsl-1, flagellin and poly(I:C), but not with IL1B or TNF. Collectively, our novel data describe a role for PARK7 in regulating inflammation-induced pro-inflammatory and pro-labour mediators in human myometrial and amnion cells.

SMAD7 regulates proinflammatory and prolabor mediators in amnion and myometrium.

Preterm birth continues to be a significant public health problem. Infection (bacterial and or viral) and inflammation, by stimulating proinflammatory cytokines, adhesion molecules, and matrix metalloproteinase 9 (MMP9), play a central role in the rupture of membranes and myometrial contractions. SMAD7 has been implicated in regulating the inflammatory response; however, no studies have been performed with regard to human labor. In this study, we determined the effect of spontaneous human labor and prolabor mediators on SMAD7 expression in myometrium and fetal membranes. Functional studies were employed to investigate the effect of siRNA knockdown of SMAD7 (siSMAD7) in regulating infection and inflammation-induced prolabor mediators. SMAD7 mRNA and protein expression were significantly higher with spontaneous term labor, compared to no labor, in myometrium and fetal membranes. SMAD7 expression was also significantly higher in amnion from women with preterm chorioamnionitis. The proinflammatory cytokines IL1B and TNF, the bacterial product fsl-1, and the viral dsRNA analog poly(I:C) significantly increased SMAD7 in myometrial cells and amnion cells. In myometrial cells, siSMAD7 cells significantly decreased cytokine (IL6) and chemokine (CXCL1, CXCL8, CCL2 are also known as GRO-alpha, interleukin (IL)-8 and monocyte chemotactic protein-1 (MCP-1)) production induced by IL1B, TNF, and fsl-1. There was also a decrease in the expression of adhesion molecules intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) in siSMAD7 cells, and MMP9 expression. In amnion, siSMAD7 cells treated with IL1B also decreased cytokine and chemokine production, ICAM1 and MMP9 expression. In conclusion, we report a proinflammatory role for SMAD7 in human gestational tissues, with SMAD7 silencing attenuating the inflammatory response.

Inhibition of PIM1 kinase attenuates inflammation-induced pro-labour mediators in human foetal membranes in vitro.

STUDY QUESTION: Does proviral integration site for Moloney murine leukaemic virus (PIM)1 kinase play a role in regulating the inflammatory processes of human labour and delivery? SUMMARY ANSWER: PIM1 kinase plays a critical role in foetal membranes in regulating pro-inflammatory and pro-labour mediators. WHAT IS KNOWN ALREADY: Infection and inflammation have strong causal links to preterm delivery by stimulating pro-inflammatory cytokines and collagen degrading enzymes, which can lead to rupture of membranes. PIM1 has been shown to have a role in immune regulation and inflammation in non-gestational tissues; however, its role has not been explored in the field of human labour. STUDY DESIGN, SIZE, DURATION: PIM1 expression was analysed in myometrium and/or foetal membranes obtained at term and preterm (n = 8-9 patients per group). Foetal membranes, freshly isolated amnion cells and primary myometrial cells were used to investigate the effect of PIM1 inhibition on pro-labour mediators (n = 5 patients per treatment group). PARTICIPANTS/MATERIALS, SETTING AND METHODS: Foetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and from preterm pre-labour rupture of membranes (PPROM) (n = 9 per group). Amnion was collected from women with and without preterm chorioamnionitis (n = 8 per group). Expression of PIM1 kinase was determined by qRT-PCR and western blotting. To determine the effect of PIM1 kinase inhibition on the expression of pro-inflammatory and pro-labour mediators induced by bacterial products lipopolysaccharide (LPS) (10 µg/ml) and flagellin (1 µg/ml) and pro-inflammatory cytokine tumour necrosis factor (TNF) (10 ng/ml), chemical inhibitors SMI-4a (20 µM) and AZD1208 (50 µM) were used in foetal membrane explants and siRNA against PIM1 was used in primary amnion cells. Statistical significance was set at P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: PIM1 expression was significantly increased in foetal membranes after spontaneous term labour compared to no labour at term and in amnion with preterm chorioamnionitis compared to preterm with no chorioamnionitis. There was no change in PIM1 expression with preterm labour or PPROM compared to preterm with no labour or PPROM. In human foetal membranes, PIM1 inhibitors SMI-4a and AZD1208 significantly decreased the expression of pro-inflammatory cytokine interleukin-6 (IL6) and chemokines CXCL8 and CCL2 mRNA and release, prostaglandin prostaglandin F2α (PGF2α) release, adhesion molecule intercellular adhesion molecule 1 mRNA expression and release, and oxidative stress marker 8-isoprostane release after stimulation with either LPS or flagellin. Primary amnion cells transfected with PIM1 siRNA also showed decreased expression of IL6, CXCL8 and CCL2, PTGS2 mRNA and PGF2α release, and matrix metalloproteinase-9 (MMP9) expression, when stimulated with TNF. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The conclusions were drawn from in vitro experiments using foetal membrane explants and primary cells isolated from amnion. Animal models are necessary to determine whether PIM1 kinase inhibitors can prevent spontaneous preterm birth in vivo. WIDER IMPLICATIONS OF THE FINDINGS: PIM1 kinase inhibitors may provide a novel therapeutic approach for preventing spontaneous preterm birth. STUDY FUNDING/COMPETING INTEREST(S): Associate Professor Martha Lappas is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; grant no. 1047025). Funding for this study was provided by the NHMRC (grant no. 1058786), Norman Beischer Medical Research Foundation and the Mercy Research Foundation. The authors have no conflict of interest.

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.

A Novel Role for SIRT3 in Regulating Mediators Involved in the Terminal Pathways of Human Labor and Delivery.

Preterm birth remains the major cause of neonatal mortality and morbidity, mediated largely by an inflammatory process. The sirtuin (SIRT) family of cellular regulators has been implicated as key inhibitors of inflammation. We have previously reported a role for SIRT1, SIRT2, and SIRT6 in regulating inflammation-induced prolabor mediators. In this study, we determined the effect of term labor and pro-inflammatory cytokines on SIRT3, SIRT4, SIRT5, and SIRT7 expression in human myometrium. Functional studies were also used to investigate the effect of small interfering RNA (siRNA) knockdown of SIRTs in regulating inflammation-induced prolabor mediators. Western blot analysis and qRT-PCR were used to determine SIRT3, SIRT4, SIRT5, and SIRT7 mRNA and protein expression in human myometrium. Small interfering RNA knockdown of SIRT3 in myometrial primary cells determined its role in response to inflammatory stimuli IL1B and TNF. SIRT3 mRNA and protein expression levels were significantly lower in term laboring myometrium compared with term nonlaboring myometrium. There was no effect of labor on SIRT4, SIRT5 or SIRT7 protein expression. The pro-inflammatory cytokines IL1B and TNF significantly decreased levels of SIRT3 mRNA and protein expression. SIRT3 knockdown by siRNA significantly augmented IL1B- and TNF-stimulated IL6, CXCL8, and CCL2 mRNA expression and release; PTGS2 mRNA expression and subsequent PGF2alpha release; the mRNA expression and secretion of the adhesion molecule ICAM1 and the extracellular matrix remodeling enzyme MMP9; and nuclear factor kappa B1 (NFkappaB1) transcriptional activity. In human myometrium, SIRT3 expression decreases with term labor and regulates the mediators involved in the terminal effector pathways of human labor and delivery through the NFkappaB1 pathway.

The Transcription Factor Interferon Regulatory Factor-1 (IRF1) Plays a Key Role in the Terminal Effector Pathways of Human Preterm Labor.

Preterm birth is the largest single cause of neonatal death and morbidity. By activating cytokine- and Toll-like receptor (TLR)-signaling pathways, infection and/or inflammation are strongly associated with preterm delivery. Interferon regulatory factor-1 (IRF1) is an important regulator of the inflammatory response. The aims of this study were to establish the effect of 1) labor on IRF1 expression in human fetal membranes and myometrium, 2) prolabor mediators on IRF1 expression and activity, and 3) IRF1 small interfering RNA on the expression of prolabor mediators. IRF1 expression was higher in fetal membranes and myometrium after spontaneous term labor and in preterm fetal membranes with infection. The proinflammatory cytokine IL1B, the bacterial product fsl-1, and viral analog polyinosinic:polycytidylic acid (poly [I:C]) significantly increased IRF1 mRNA expression and transcriptional activity in human primary myometrial cells. In addition, IL1B increased IRF1 activity in primary amnion cells. IRF1 silencing in myometrial cells decreased IL1B-, fsl-1-, and poly (I:C)-induced cytokine (IL6, TNF, IL1B) and chemokine (CXCL8, CCL2) mRNA expression and IL6, CXCL8, and CCL2 release. IL1B-, fsl-1-, and poly (I:C)-induced PTGS2 mRNA expression and IL1B-induced prostaglandin release was also decreased by IRF1 silencing. In conclusion, IRF1 upregulation in fetal membranes and myometrium after term labor indicates a proinflammatory role for IRF1 in human parturition. IRF1 is involved in TLR- and cytokine-mediated signaling in human myometrium. These data provide new insights into the mechanisms associated with inflammation- and infection-associated preterm birth. IRF1 inhibitors as therapeutics for the management of spontaneous preterm birth warrants further investigation.

A novel role for GSK3 in the regulation of the processes of human labour.

Preterm birth remains the largest single cause of neonatal death and morbidity. Infection and/or inflammation are strongly associated with preterm delivery. Glycogen synthase kinase 3 (GSK3) is known to be a crucial mediator of inflammation homeostasis. The aims of this study were to determine the effect of spontaneous human labour in foetal membranes and myometrium on GSK3α/ß expression, and the effect of inhibition of GSK3α/ß on pro-labour mediators in foetal membranes and myometrium stimulated with Toll-like receptor (TLR) ligands and pro-inflammatory cytokines. Term and preterm labour in foetal membranes was associated with significantly decreased serine phosphorylated GSK3α and ß expression, and thus increased GSK3 activity. There was no effect of term labour on serine phosphorylated GSK3ß expression in myometrium. The specific GSK3α/ß inhibitor CHIR99021 significantly decreased lipopolysaccharide (ligand to TLR4)-stimulated pro-inflammatory cytokine gene expression and release; COX2 gene expression and prostaglandin release; and MMP9 gene expression and pro MMP9 release in foetal membranes and/or myometrium. CHIR99021 also decreased FSL1 (TLR2 ligand) and flagellin (TLR5 ligand)-induced pro-inflammatory cytokine gene expression and release and COX2 mRNA expression and prostaglandin release. GSK3ß siRNA knockdown in primary myometrial cells was associated with a significant decrease in IL1ß and TNFα-induced pro-inflammatory cytokine and prostaglandin release. In conclusion, GSK3α/ß activity is increased in foetal membranes after term and preterm labour. Pharmacological blockade of the kinase GSK3 markedly reduced pro-inflammatory and pro-labour mediators in human foetal membranes and myometrium, providing a possible therapeutics for the management of preterm labour.

FOXM1 is lower in human fetal membranes after spontaneous preterm labour and delivery.

Spontaneous preterm birth is usually associated with infection, inflammation or both. Forkhead box (FOX) M1 (FOXM1), a member of the FOX family of transcription factors, has been associated with inflammation. The aim of this study was to determine whether FOXM1 regulates the expression and release of pro-labour mediators in human gestational tissues. FOXM1 mRNA and protein expression were determined in fetal membranes from women at (1) preterm no labour: Caesarean section with no labour and (2) preterm labour: after spontaneous labour and delivery. Primary amnion cells were utilised to investigate the effect of small interfering RNA (siRNA)-mediated gene silencing of FOXM1 on pro-labour mediators. Spontaneous preterm labour decreased FOXM1 gene and nuclear protein expression. FOXM1 silencing in primary amnion cells increased interleukin (IL)-1ß-induced pro-inflammatory cytokines (IL-6 and IL-8 mRNA expression and secretion), cyclooxygenase (COX)-2 expression and subsequent prostaglandin (PG)E2 and PGF2α release as well as gene expression and secretion of the matrix-degrading enzyme matrix metalloproteinase 9 (MMP-9). In conclusion, spontaneous preterm labour is associated with decreased FOXM1 expression in fetal membranes.

SIRT6 is decreased with preterm labor and regulates key terminal effector pathways of human labor in fetal membranes.

Preterm birth is a major determinant of neonatal mortality and morbidity, affecting approximately one-third of preterm births as a result of prelabor rupturing of membranes. Infection and inflammation have strong causal links to preterm delivery, resulting in the activation of nuclear factor-kappaB (NFKB) and its downstream targets. Human sirtuin (SIRT) 6, which has ADP-ribosyl transferase and deacetylase activity, exhibits anti-inflammatory actions. The aims of this study were to determine the effect of 1) human preterm labor on SIRT6 expression in human gestational tissue and 2) the effect in primary amnion cells of SIRT6 inhibition, using small interfering RNA (siRNA) on prolabor mediators. To determine the effect of human preterm labor on SIRT6 expression, human fetal membranes were collected from women at preterm at the time of Cesarean section (no labor; n = 9) and from women after spontaneous labor and delivery (n = 9). SIRT6 mRNA and protein expression were significantly lower in fetal membranes after spontaneous preterm labor. Transfection of primary amnion cells with SIRT6 siRNA was associated with an increase in IL-1beta-induced proinflammatory cytokine gene expression and release (IL6, IL8, TNF [TNF-alpha]), cyclooxygenase ([COX]-2; official symbol PTGS2) expression and subsequent prostaglandin (PGE(2) and PGF(2alpha)) release, and MMP9 gene expression and release of pro-MMP9. To determine whether SIRT6 affects NFKB transcriptional activity, primary amnion cells were transfected with NFKB tagged with luciferase and stimulated with IL1B. As expected, IL1B induced NFKB transcriptional activity. However, when cells were also cotransfected with a vector expressing SIRT6, there was a decrease in NFKB transcriptional activity. In conclusion, SIRT6 plays a role in regulating the terminal effector pathways of human labor and delivery via the NFKB pathway.

A novel role for FOXO3 in human labor: increased expression in laboring myometrium, and regulation of proinflammatory and prolabor mediators in pregnant human myometrial cells.

Preterm birth is the leading factor causing neonatal mortality and morbidity. Inflammation plays a central role in stimulating uterine contractility, which is responsible for approximately one-third of all preterm births. Recent studies have shown that the transcription factor Forkhead box O3 (FOXO3) regulates inflammation in nongestational tissues such as adipocytes and hepatocytes. Thus, in this study, we sought to determine the effect of 1) human term labor on myometrial FOXO3 expression and 2) FOXO3 inhibition and FOXO3 overexpression on proinflammatory and prolabor mediators in human myometrial cells. Higher FOXO3 gene and protein expression were detected in myometrium obtained from women in labor when compared to samples taken from nonlaboring women. Myometrial cells were isolated from pregnant human myometrium, and FOXO3 silencing was achieved using siRNA and overexpression using a cDNA clone. We found that the loss of FOXO3 in myometrial cells was associated with a significant decrease in IL1B-induced IL6 and IL8 expression and production, cyclooxygenase ([COX]-2, official symbol PTGS2) expression and subsequent prostaglandin (PGE2 and PGF2alpha) release, and matrix metalloproteinase 9 (MMP9) and mRNA expression and activity. Conversely, FOXO3 overexpression increased cytokine expression and secretion, prostaglandin production, and MMP9 expression in myometrial cells treated with IL1B. In summary, we have identified FOXO3 as an upstream mediator of inflammation in human myometrium. Thus, FOXO3 may present an alternative therapeutic target for preventing preterm birth and its associated morbidity and mortality.

Dietary phytophenols curcumin, naringenin and apigenin reduce infection-induced inflammatory and contractile pathways in human placenta, foetal membranes and myometrium.

A tenet of contemporary obstetrics is that a significant proportion of preterm births involve bacterial infection. Bacterial endotoxin induces pro-inflammatory cytokines, prostaglandins and proteases via the pro-inflammatory pathway nuclear factor-κB (NF-κB), which plays a key role in initiating uterine contractions and rupture of foetal membranes. In non-gestational tissues, the phytophenols curcumin, naringenin and apigenin exert anti-inflammatory properties via inhibition of NF-κB. The aim of this study was to determine whether these treatments regulate pro-inflammatory and pro-labour mediators in human gestational tissues. Placenta, foetal membranes and myometrium were treated with curcumin, naringenin and apigenin in the presence of lipopolysaccharide (LPS) or interleukin (IL)-1ß. In placenta and foetal membranes, all treatments significantly reduced LPS-stimulated release and gene expression of pro-inflammatory cytokines IL-6 and IL-8; placenta decreased cyclooxygenase (COX-2) mRNA expression, subsequent release of prostaglandins PGE2 and PGF2α and expression and activity of matrix-degrading enzyme matrix metalloproteinase (MMP)-9. In myometrial cells, all treatments attenuated IL-1ß-induced COX-2 expression, release of PGE2 and PGF2α and expression and activity of MMP-9. Although naringenin significantly attenuated IL-1ß-induced IL-6 and IL-8 mRNA expression and release, there was no effect of curcumin and apigenin. LPS-stimulated release of 8-isoprostane, a marker of oxidative stress, was attenuated by all treatments. NF-κB p65 DNA-binding activity was also decreased using these treatments. In conclusion, curcumin, naringenin and apigenin exert anti-inflammatory properties in human gestational tissues by inhibiting the transcriptional activity of NF-κB. Further studies should be undertaken to define a possible implication of these natural spices in the management of preterm labour and delivery.