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

Abstract

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

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

“…Cells were isolated and cultured as previously described [41]. Briefly, myometrium was minced and digested for 1 h in Dulbecco modified Eagle Medium/Nutrient Mixture F-12 Ham medium (DMEM/F-12) with 3 mg/ml type I collagenase (Worthington Biochemical) and 80 μg/ml DNase 1 (Roche Diagnostics) at 37°C.…”

RAF1 is increased in labouring myometrium and modulates inflammation-induced pro-labour mediators

“…A luciferase assay was utilised to determine possible interactions between RAF1 and NF-kB, as described previously (Lim et al 2016). Primary myometrial cells, prepared as described above, at w70% confluence, were transfected with 0.75 ng NF-kB reporter construct (Qiagen, Chadstone Centre) using FuGENE HD transfection reagent (Promega).…”

“…RNA extraction and qRT-PCR was performed, as described previously (Lim et al 2016). Briefly, total RNA was extracted from cells using TRIsure reagent, according to manufacturer's instructions (Bioline; Alexandria, Australia).…”

“…Western blotting was performed, as described previously (Lim et al 2016). Blots were incubated in 1 mg/ml rabbit polyclonal anti-RAF1 (GTX107763; GeneTex, CA, USA), 1 mg/ml rabbit polyclonal anti-MAPK1 (sc-93; Santa Cruz Biotechnology) or 1 mg/ml mouse monoclonal anti-phosphorylated MAPK1 (sc-7383; Santa Cruz Biotechnology) prepared in blocking buffer (5% skim milk in TBS with 0.05% Tween-20) for 16 h at 4 8C.…”

“…Semi-quantitative analysis of the relative density of the bands in western blots was performed using Quantity One 4.2.1 Image Analysis Software (Bio-Rad Laboratories). For the labour studies, RAF1 protein expression was normalised to Ponceau S stain, as described previously (Lim et al 2016); a section of the Ponceau S stained membrane was chosen which did not show variation with labour status.…”