Viral Infection Sensitizes Human Fetal Membranes to Bacterial Lipopolysaccharide by MERTK Inhibition and Inflammasome Activation This information is current as of September 24, 2021. Sarah N. Cross, Julie A. Potter, Paulomi Aldo, Ja Young Kwon, Mary Pitruzzello, Mancy Tong, Seth Guller, Carla V. Rothlin, Gil Mor and Vikki M. Abrahams J Immunol published online 15 September 2017 http://www.jimmunol.org/content/early/2017/09/15/jimmun Downloaded from ol.1700870 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published September 15, 2017, doi:10.4049/jimmunol.1700870 The Journal of Immunology Viral Infection Sensitizes Human Fetal Membranes to Bacterial Lipopolysaccharide by MERTK Inhibition and Inflammasome Activation Sarah N. Cross,*,1 Julie A. Potter,*,1 Paulomi Aldo,* Ja Young Kwon,*,2 Mary Pitruzzello,* Mancy Tong,* Seth Guller,* Carla V. Rothlin,† Gil Mor,* and Vikki M. Abrahams* Chorioamnionitis, premature rupture of fetal membranes (FMs), and subsequent preterm birth are associated with local infection and inflammation, particularly IL-1b production. Although bacterial infections are commonly identified, other microorganisms may play a role in the pathogenesis. Because viral pandemics, such as influenza, Ebola, and Zika, are becoming more common, and pregnant women are at increased risk for associated complications, this study evaluated the impact that viral infection had on human FM innate immune responses. This study shows that a herpes viral infection of FMs sensitizes the tissue to low levels of Downloaded from bacterial LPS, giving rise to an exaggerated IL-1b response. Using an ex vivo human FM explant system and an in vivo mouse model of pregnancy, we report that the mechanism by which this aggravated inflammation arises is through the inhibition of the TAM receptor, MERTK, and activation of the inflammasome. The TAM receptor ligand, growth arrest specific 6, re-establishes the normal FM response to LPS by restoring and augmenting TAM receptor and ligand expression, as well as by preventing the exacerbated IL-1b processing and secretion. These findings indicate a novel mechanism by which viruses alter normal FM immune responses to bacteria, potentially giving rise to adverse pregnancy outcomes. The Journal of Immunology, 2017, 199: 000–000. http://www.jimmunol.org/ horioamnionitis, preterm premature rupture of mem- attributed to preterm birth (17), and identifiable bacteria associ- branes (PPROM) and preterm birth resulting from in- ated with chorioamnionitis, PPROM, and preterm birth are often C fection are thought to be initiated by bacteria ascending common to the genital tract and the placenta (18). Moreover, al- from the lower genital tract, gaining access to the fetal membranes though the FMs are likely the first tissue colonized by the normal (FMs), and activating innate immune responses (1). The proin- flora of the lower genital tract or by an ascending pathogen (19), flammatory cytokine IL-1b is an important mediator of PPROM most FMs from normal deliveries also have bacteria present (20). and preterm birth (2–5). Normal human FMs express a range of Thus, bacterial stimulation of the FMs may, in and of itself, be by guest on September 24, 2021 innate immune pattern recognition receptors, such as TLRs, Nod- insufficient to trigger chorioamnionitis and preterm birth. like receptors, and inflammasome family members, and can generate A number of diseases are caused by polymicrobial infections, inflammatory responses following their activation by infectious including disorders of the urogenital tract, like vaginosis (21). components (6–8). Although clinical and experimental studies have Thus, one potential risk factor that could contribute to bacterial- correlated bacterial infection and inflammation at the maternal–fetal associated preterm birth may be another type of infection, such as interface with prematurity (9–16), no single bacterium has been a virus. Although not all women with a viral infection during pregnancy will have complications, some viruses that are detected *Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University in the amniotic fluid or gestational tissues have been linked to an School of Medicine, New Haven, CT 06510; and †Department of Immunobiology increased risk for chorioamnionitis and preterm birth. These in- and Pharmacology, Yale University School of Medicine, New Haven, CT 06510 clude adenovirus and herpes viruses, such as CMV, EBV, and HSV 1S.N.C. and J.A.P. contributed equally to this work. (22–31). If a virus, which can infect the placenta and FMs increases 2Current address: Department of Obstetrics and Gynecology, Yonsei University Col- a woman’s risk for preterm birth by altering local responses to lege of Medicine, Seoul, Korea. bacterial components, then the mechanisms likely involve modu- ORCIDs: 0000-0002-5019-7038 (S.N.C.); 0000-0003-3009-6325 (J.Y.K.); 0000- lation of innate immune receptors and their regulators. TLR-driven 0002-5693-5572 (C.V.R.); 0000-0002-5499-3912 (G.M.). immune responses are tightly controlled by inhibitors, including the Received for publication June 15, 2017. Accepted for publication August 21, 2017. TAM tyrosine kinase receptors (32, 33). Three TAM receptors, This work was supported in part by Grants R01AI121183 (to V.M.A.) and R56AI124356 (to G.M.) from the National Institute of Allergy and Infectious Dis- TYRO3, AXL, and MERTK, are activated by two endogenous eases, National Institutes of Health and by the McKern Scholar Award for Perinatal ligands: growth arrest specific 6 (GAS6) and protein S1 (PROS1) Research (to V.M.A.). (33). GAS6 activates all three TAM receptors, whereas PROS1 Address correspondence and reprint requests to Dr. Vikki M. Abrahams, Department activates TYRO3 and MERTK (33). Upon ligand binding, TAM of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, LSOG 305C, New Haven, CT 06510. E-mail address: receptors trigger STAT1 phosphorylation, inducing SOCS1 and [email protected] SOCS3, which broadly inhibit TLR signaling (33, 34). In this Abbreviations used in this article: E, embryonic day; FM, fetal membrane; GAS6, study, we investigated how a polymicrobial infection could impact growth arrest specific 6; GRO-a, growth regulated oncogene-a; IP-10, IFN-g– induced protein 10; MHV-68, murine g herpes virus 68; MNS, 3,4-methylenedioxy- human FM innate immune responses and, thus, pregnancy out- b-nitrostyrene; NT, not treated; Poly(I:C), polyinosinic-polycytidylic acid; PPROM, come. Using an ex vivo human FM explant system and an in vivo preterm premature rupture of membranes; PROS1, protein S1; r, recombinant; s, mouse model of pregnancy, we examined the effect that a herpes soluble; VEGF, vascular endothelial growth factor. virus infection had on FM responses to low levels of bacterial Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 LPS, as well as the role of the regulatory TAM receptors. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700870 2 VIRUS SENSITIZES FETAL MEMBRANES TO BACTERIAL LPS Materials and Methods from R&D Systems). IL-1b levels were evaluated using the anti-human Human FM collection and preparation primary Abs against pro–IL-1b (product number 12703) and the active form (product number 2022; both from Cell Signaling Technology). Human FMs were collected from planned uncomplicated term (37–41-wk) b-Actin was used as a loading control (Sigma-Aldrich). Images were cesarean deliveries without labor or known infection/inflammation, as pre- recorded, and semiquantitative densitometry was performed using the viously described (7, 8). Tissue collection was approved by Yale University’s Gel Logic 100 system and Carestream software (Carestream Molecular Human Research Protection Program. After washing the FMs with sterile Imaging, Woodbridge, CT). ELISA was used to measure the tissue PBS supplemented with penicillin (100 U/ml) and streptomycin (100 mg/ml) levels of GAS6 (R&D Systems) and total PROS1 (Innovative Research, (Life Technologies, Grand Island, NY), adherent blood clots were removed, Novi, MI). and explants in which the chorion and amnion were intact were obtained using a 6-mm biopsy punch. The FM explants were placed in 0.4-mmpore Statistical analysis cell culture inserts (BD Falcon, Franklin Lakes, NJ) with 500 mlofDMEM Each in vitro FM treatment experiment was performed in triplicate and (Life Technologies) supplemented with 10% FBS (HyClone, Logan, UT), repeated at least three times. For in vivo studies, all FMs from each pregnant and these were placed in a 24-well plate containing 500 mlofthesame animal were pooled. All data are reported as mean 6 SEM