Fetal Membrane Inflammasome Activation by Choriodecidual Ureaplasma parvum Infection without Intra-Amniotic Infection in an NHP Model

Abstract

Intrauterine infection is a significant cause of preterm labor and neonatal morbidity and mortality. Ureaplasma parvum is the micro-organism most commonly isolated from cases of preterm birth and preterm premature rupture of membranes (pPROM). However, the mechanisms during the early stages of ascending reproductive tract infection that initiate maternal-fetal inflammatory pathways, preterm birth and pPROM remain poorly understood. To examine inflammation in fetal (chorioamnionic) membranes in response to Ureaplasma parvum infection, we utilized a novel in vivo non-human primate model of early choriodecidual infection. Eight chronically catheterized pregnant rhesus macaques underwent maternal-fetal catheterization surgery at 105-112 days gestation and choriodecidual inoculation with Ureaplasma parvum (105cfu/mL of a low passaged clinical isolate, serovar 1; n=4) or saline/sterile media (Controls; n=4) starting at 115-119 days gestation, repeated every 5 days until scheduled cesarean-section at 136-140d gestation (term=167d). The average inoculation to delivery interval was 21 days and Ureaplasma infection of the amniotic fluid was undetectable by culture and PCR in all animals. Inflammatory mediators in amniotic fluid (AF) were assessed by Luminex, ELISA and multiplex assays. RNA was extracted from the chorion and amnionic membranes for single gene analysis (qRT-PCR) and protein expression was determined by Western blot and immunohistochemistry. Our NHP model of choriodecidual Ureaplasma infection, representing an early-stage ascending reproductive tract infection without microbial invasion of the amniotic cavity, resulted in increased fetal membrane protein and gene expression of MMP-9 and PTGS2, but did not result in preterm labor (no increase in uterine contractility) or increased concentrations of amniotic fluid pro-inflammatory cytokines (IL-1{beta}, IL-6, IL-8, IL-18, TNF-). However, membrane expression of inflammasome sensor molecules, NLRP3, NLRC4, AIM2 and NOD2, and the adaptor protein ASC (PYCARD) gene expression were significantly increased in the Ureaplasma group when compared to non-infected controls. Gene expression of IL-1{beta}, IL-18, the IL-18R1 receptor, CASPASE-1 and pro-CASPASE-1 protein were also increased in the fetal membranes with Ureaplasma infection. Downstream inflammatory signaling genes MYD88 was also significantly upregulated in both the amnion and chorion, along with a significant increase in NFKB in the chorion. These results demonstrate that even at the early stages of ascending reproductive tract Ureaplasma infection, activation of inflammasome complexes and pathways associated with degradation of chorioamnionic membrane integrity are present. This study therefore provides experimental evidence for the importance of the early stages of ascending Ureaplasma infection in initiating processes of pPROM and preterm labor. These findings have implications for the identification of intrauterine inflammation before microbes are detectable in the amniotic fluid (sterile inflammation) and the timing of potential treatments for preterm labor and fetal injury caused by intrauterine infection.