Ratana Lim1,2, Gillian Barker1,2, Martha Lappas1,2. 1. Obstetrics, Nutrition and Endocrinology Group, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, Victoria 3084, Australia. 2. Mercy Perinatal Research Centre, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, Victoria 3084, Australia.
Abstract
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.
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, prostaglandinprostaglandin 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.