Activation of AMPK in human fetal membranes alleviates infection-induced expression of pro-inflammatory and pro-labour mediators

Lim, Ratana; Barker, Gillian; Lappas, Martha

doi:10.1016/j.placenta.2015.01.007

Cited by 28 publications

(26 citation statements)

References 68 publications

(85 reference statements)

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“…To determine if IRF5 regulates pro-labour mediators, transfection of primary myometrial cells with siRNA was performed as previously described (Lim et al 2015). Briefly, myometrial cells at approximately 50% confluence were transfected using Lipofectamine 3000 according to manufacturer's guidelines (Life Technologies).…”

Section: Irf5 Sirna Transfection In Primary Myometrial Cellsmentioning

confidence: 99%

“…Immunohistochemistry (IHC) was performed on paraffin sections as described previously (Lim et al 2015) using the IHC Select HRP Detection Set (Merck Millipore). Briefly, sections were deparaffinised followed by an antigen retrieval step (boiled in 10 mM Tris, 1 mM EDTA, pH 9.0 for 10 min followed by 20-min incubation) and then endogenous peroxidases were inactivated by adding 3% hydrogen peroxide for 10 min.…”

Section: Immunohistochemistrymentioning

confidence: 99%

“…RNA extractions and qRT-PCR was performed as previously described (Lim et al 2015). RNA concentration and purity were measured using a NanoDrop ND1000 (Thermo Fisher Scientific).…”

Section: Rna Extraction and Qrt-pcrmentioning

confidence: 99%

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

2018

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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. 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, PGF and 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.

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Section: Irf5 Sirna Transfection In Primary Myometrial Cellsmentioning

confidence: 99%

Section: Immunohistochemistrymentioning

confidence: 99%

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

2018

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“…Fresh amnion (obtained 2 cm from the periplacental edge) and myometrium were obtained from women who delivered healthy, singleton infants at term (37-41 wk gestation) undergoing elective cesarean section in the absence of labor. Primary amnion and myometrial cells were prepared as we have previously described [38,40]. Cells at approximately 70% confluence were transfected with 150 ng IRF1 reporter construct (Qiagen) using FuGENE HD transfection reagent (Promega) for 48 h. The medium was then replaced with Dulbeccomodified Eagle medium (DMEM)/F-12 (containing 0.5% BSA for myometrial cells or 2% heat-inactivated fetal calf serum for amnion cells) with or without 1 ng/ml IL1B, 5 lg/ml poly (I:C), or 250 ng/ml fsl-1, and the cells incubated at 378C for an additional 24 h. The cells were harvested in lysis buffer, and luminescence activity was measured using a Luciferase Reporter Assay Kit (Life Research) and Renilla Luciferase Flash Assay kit (Thermo Fisher Scientific) as instructed by the manufacturers.…”

Section: Western Blot Analysismentioning

confidence: 99%

The Transcription Factor Interferon Regulatory Factor-1 (IRF1) Plays a Key Role in the Terminal Effector Pathways of Human Preterm Labor1

Lim

Tran

Liong

et al. 2016

Biology of Reproduction

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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 infectionassociated preterm birth. IRF1 inhibitors as therapeutics for the management of spontaneous preterm birth warrants further investigation. fetal membranes, human labor, inflammation, IRF1, myometrium

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“…A lower concentration of poly(I:C) was used for cells (5 μg/mL) compared to tissue explants (50 μg/mL) as this concentration is sufficient to elicit an inflammatory response without any adverse effects on cell toxicity. 24 Cells were collected and stored at −80°C until assayed for mRNA expression by qRT-PCR and protein expression by Western blotting as detailed below. Media was collected and stored at −80°C until assayed for cytokine release as detailed below.…”

Section: Gene Silencing Of Havcr2 With Sirnamentioning

confidence: 99%