Contents lists available at ScienceDirect Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jri Polybacterial stimulation suggests discrete IL-6/IL-6R signaling in human fetal membranes: Potential implications on IL-6 bioactivity Nathalia Mayumi Noda-Nicolaua,b, Jossimara Polettinia,b,c, Márcia Guimarães da Silvab, Morgan R. Peltierd,e, Ramkumar Menona,⁎ a Division of Maternal-Fetal Medicine and Perinatal Research, Department of Obstetrics and Gynecology, The University of Texas Medical Branch at Galveston, Galveston, TX, United States bDepartment of Pathology, Botucatu Medical School, UNESP – Univ. Estadual Paulista, Botucatu, São Paulo, Brazil cMaster’s Course in Health Sciences, University of Western São Paulo, UNOESTE, Presidente Prudente, São Paulo, Brazil d Department of Biomedical Research, NYU-Winthrop University Hospital, Mineola, NY, United States e Department of Obstetrics and Gynecology, NYU-Winthrop University Hospital, Mineola, NY, United States A R T I C L E I N F O Keywords: Fetal membranes in vitro Genital mycoplasmas Interleukin-6 Polybacterial infection A B S T R A C T The polybacterial invasion of the amniotic cavity and risk of preterm birth is often due to cervicovaginal bacteria such as genital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum) and Gardnerella vaginalis. The most studied biomarker associated with preterm birth is interleukin-6 (IL-6), a pleiotropic cytokine that performs different functions based on classical or trans-signaling mechanisms. This study evaluated the changes in IL-6 and IL-6 function associated accessory molecules by human fetal membranes to determine the functional availability of IL-6 assessment in an in vitro model of polybacterial infection. Fetal membranes were treated with LPS or heat-inactivated genital mycoplasmas and G. vaginalis alone or in combination. IL-6 and its soluble re- ceptors (sgp130, sIL-6R) were assessed in conditioned medium by immunoassays and membrane-bound re- ceptors were evaluated in the tissue using immunohistochemistry and RT-PCR. Data from protein and gene expression were evaluated using linear mixed effects models. Data from immunohistochemistry were evaluated using one-way analysis of variance followed by the Tukey test. Genital mycoplasmas alone, or in combination, inhibited IL-6 trans-signaling with increased sgp130 production. G. vaginalis activated the classical IL-6 signaling pathway, as did LPS. Polybacterial treatment resulted in a balanced response with neither pathway being fa- vored. The increase in IL-6 production by fetal membranes in response to infection is likely a non-specific innate response and not an indicator of a functional mediator of any labor-inducing pathways. This suggests that correlating the risk of adverse pregnancy outcomes and designing interventions based on IL-6 levels without considering soluble receptors may be an ineffective strategy. 1. Introduction Intraamniotic infection and inflammation are associated with spontaneous preterm birth (PTB) (Goldenberg et al., 2000; Helmo et al., 2017). These infections are generally due to cervicovaginal colonizers (DiGiulio, 2012; Mendz et al., 2013) such as genital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum) and Gardnerella va- ginalis (Menard et al., 2010), a pathogen frequently associated with bacterial vaginosis (Santos-Greatti et al., 2016) that ascends from the lower genital tract. M. hominis and U. urealyticum are the most com- monly isolated bacteria in the amniotic fluid in both PTB with intact membranes and in preterm premature rupture of membranes (pPROM) (DiGiulio, 2012). Both species, along with G. vaginalis, are present at higher frequencies in human fetal membranes from women with pre- term labor (Doyle et al., 2014). Although the relative roles of bacterial diversity, bacterial load and the host response to infection in the in- duction of PTB remain unclear, polybacterial invasion of the amniotic cavity (PIAC) is thought to trigger an inflammatory response in fetal membranes (Romero et al., 2006), which, in turn, can increase pros- taglandin production and lead to preterm contractions that result in preterm labor and ultimately preterm delivery. Inflammation by microbial pathogens is heterogeneous, and there are no predictive biomarkers for predicting PTB. In a recent study published by our group (Noda-Nicolau et al., 2016), different https://doi.org/10.1016/j.jri.2018.02.007 Received 11 September 2017; Received in revised form 4 December 2017; Accepted 26 February 2018 ⁎ Corresponding author at: Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine & Perinatal Research, The University of Texas Medical Branch at Galveston, 301 University Blvd, MRB 11-158, Galveston, TX 77555, United States. E-mail address: ram.menon@utmb.edu (R. Menon). Journal of Reproductive Immunology 126 (2018) 60–68 0165-0378/ © 2018 Elsevier B.V. All rights reserved. T http://www.sciencedirect.com/science/journal/01650378 https://www.elsevier.com/locate/jri https://doi.org/10.1016/j.jri.2018.02.007 https://doi.org/10.1016/j.jri.2018.02.007 mailto:ram.menon@utmb.edu https://doi.org/10.1016/j.jri.2018.02.007 http://crossmark.crossref.org/dialog/?doi=10.1016/j.jri.2018.02.007&domain=pdf combinations and loads of bacterial species had distinct effects on the array of cytokines produced in membrane host immune response. Therefore, using a single inflammatory biomarker to evaluate the health of asymptomatic pregnancies may not be feasible. The most studied biomarker associated with PTB is interleukin-6 (IL-6) (Menon et al., 2011). High levels of IL-6 in cervicovaginal fluid (Taylor et al., 2013) and amniotic fluid (Kim et al., 2013) are associated with infection and PTB. IL-6 is a pleiotropic cytokine that performs its functions either through classical or trans-signaling mechanisms, which determine its biological activities (Fig. 1). Classical signaling, through the membrane-bound IL-6 receptor (mIL-6R), produces an anti-in- flammatory effect; conversely, trans-signaling through the soluble IL-6 receptor (sIL-6R) generates a proinflammatory effect. In both cases, the binding between IL-6 and mIL-6R or sIL-6R is followed by homo- dimerization of β-subunit glycoprotein 130 (gp130), the signal trans- duction subunit. However, soluble gp130 (sgp130) functions as a nat- ural antagonist of IL6/sIL6R trans-signaling, neutralizing IL-6 bioactivity by blocking the IL-6/sIL-6R complex binding to gp130 (Lee et al., 2011; Rose-John, 2012) and suppressing inflammation. To better understand how polybacterial infections may affect IL-6 signaling and bioactivity, we evaluated the expression of IL-6 and its various IL-6 receptors (mIL-6R and sIL-6R) and signaling modifiers (gp130 and sgp130) in conditioned medium and tissues from a polybacterial in- fection tissue culture model of fetal membranes. 2. Materials and methods 2.1. Patient and collection of fetal membranes Fetal membranes were obtained from elective repeat cesarean sec- tions at term (>37 weeks of gestation) prior to the onset of labor (n=6). The decidua and adhering blot clots were removed from the membranes using saline and sterile cotton gauze. Segments (6mm disks isolated with a skin biopsy punch), were placed in Hanks balanced salt solution (HBSS; Sigma Chemical Co., St. Louis, MO, USA) containing penicillin/streptomycin (10 IU/mL) and amphotericin B (1 IU/mL) and incubated at 37 °C, 5% CO2 for 48 h. Culture medium was replaced every 24 h, as previously described (Fortunato et al., 1994; Menon et al., 2009). Women with a prior history of pPROM or spontaneous PTB were excluded, as were patients with obstetrical and medical complications and infections (subclinical infections as indicated by high C-reactive protein levels, foul smelling vaginal discharge, fever, and those un- dergoing antibiotic treatment). The Institutional Review Board at The University of Texas Medical Branch (UTMB), John Sealy Hospital, Galveston, TX, approved this study on October 7, 2011 until April 15, 2016 (IRB #: 11-251). 2.2. Stimulation of fetal membranes The preparation of heat-killed stocks of U. urealyticum (ATCC #27816), M. hominis (ATCC #14027) and G. vaginalis (ATCC #49145) is described in our previous report (Noda-Nicolau et al., 2016). Con- sidering that the usual load used in the most of in vitro studies is 106 CFU(Flores-Herrera et al., 2012; Noda-Nicolau et al., 2016; Zaga- Clavellina et al., 2007), we employed this load and 103 CFU (an arbi- trary value) of heat-inactivated bacteria to stimulated tissue cultures for 24 h performing different treatments as decribed in Table 1. The ne- gative control consisted of sterile tissue medium, and purified lipopo- lysaccharide (LPS) from Escherichia coli (O55:B5) at 100 ng/mL was used as a positive control. Supernatants and tissues were then harvested and stored at −80 °C until biochemical analysis, as described below. Additional tissue samples were fixed in 10% formalin for paraffin em- bedding and immunohistochemistry. 2.3. Protein biomarker assays A customized cytokine panel (EMD, Millipore, Billerica, MA, USA) based on Luminex xMAP™ technology (Luminex, Austin TX) was used to quantify IL-6 and sIL-6R concentrations in conditioned medium. A commercially available ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantify sgp130 levels in these samples. All assays were performed according to the manufacturer’s instructions. Samples in which cytokine levels were estimated to be below the sensitivity of the assay were set equal to the sensitivity of the assay and those with Fig. 1. Representative model of classical, trans-signaling and inhibition of IL-6 activity. Classical signaling produces an anti-inflammatory effect when IL-6 binds to mIL-6R along with gp130. Conversely, trans-signaling generates a proinflammatory effect when IL-6 binds to sIL-6R, which binds to gp130. The inhibition of trans-signaling occurs when sgp130 neutralizes IL-6 bioactivity by blocking the IL-6/sIL-6R complex binding to gp130, acting as a natural antagonist of IL6/sIL6R trans-signaling. Modified with permission from I. Buhimschi and C. Buhimschi13. Table 1 Treatments used to stimulate fetal membranes in vitro. MH(6): Mycoplasma hominis (106 CFU) UU(6): Ureaplasma urealyticum (106 CFU) GV(6): Gardnerella vaginalis (106 CFU) MH(6)+UU(6):M. hominis+U. urealyticum (106 CFU) MH(6)+UU(6)+GV(6):M. hominis+U. urealyticum (106 CFU)+ G. vaginalis (106 CFU) MH(6)+UU(6)+GV(3):M. hominis+U. urealyticum (106 CFU) + G. vaginalis (103 CFU) MH(3)+UU(3)+GV(6):M. hominis+U. urealyticum (103 CFU) + G. vaginalis (106 CFU) CFU=colony forming unit. N.M. Noda-Nicolau et al. Journal of Reproductive Immunology 126 (2018) 60–68 61 concentrations at levels above standard curve were diluted and re-as- sayed. 2.4. Gene expression Frozen fetal membranes were homogenized in 1mL of Trizol (Invitrogen, Grand Island, NY, USA), and extracted using Direct-zol RNA Mini Prep kits (Zymo-Research, Irvine, CA, USA), according to the manufacturer’s instructions. The quality and concentration of extracted total RNA were measured by UV-spectroscopy. Aliquots of the RNA samples (0.1 mg/mL) were then reverse transcribed using the First- Strand cDNA Synthesis SuperScript II reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA). Relative mRNA expression levels of gp130 (hs00174360m1) and mIL-6R (hs00169842m1) and the housekeeping gene, GAPDH, were measured using TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA, USA). The ab- sence of DNA contamination was confirmed by amplifying mock con- trols of RNA that were not reverse transcribed. 2.5. Immunohistochemistry analysis Fetal membranes embedded in paraffin blocks were sectioned (6 μm thick) and subject to deparaffinization, rehydration and antigen re- trieval by boiling in Trilogy™ solution (Cell Marque, Hot Springs, AR, USA) for 30min. Sections were then treated with Peroxidazed 1™ (Biocare Medical, Pacheco, CA, USA) and Protein block™ (Spring Bioscience, Pleasanton, CA, USA) followed by Mach 4™ Universal HRP Polymer Kit with DAB (Biocare Medical, Pacheco, CA, USA). The pri- mary antibodies used were: rabbit anti-human mIL-6R (sc-661, dilution 1:150, 3 h of incubation at 4 °C, Santa Cruz Biotechnology, Dallas, TX, USA) and mouse anti-human gp130 (sc-9994, dilution 1:25, overnight incubation at 4 °C, Santa Cruz Biotechnology, Dallas, TX, USA). Sections were counterstained with Harris hematoxylin. In this system, a brown color indicates positive staining for immunolocalization in the slide. Images from three different areas from each slide were analyzed using Image J software and applying hematoxylin-diaminobenzidine (H-DAB) specific color deconvolution in the RBG (red-green-blue) image of the amnion and chorion layers. Median intensity was calculated using the measure function. Since the maximum intensity value of RGB image analyzed in the program is 255, and the amount of chromogen present is inversely proportional the intensity value, in order to facilitate the interpretation of results, we deducted the value of 255 from median intensities represents in the graph, making the results directly propor- tional. 2.6. Statistical analysis Data from protein and gene expression were evaluated using linear mixed effects models using the lme4 library of the R statistical lan- guage. Effects due to tissue donor were considered random and all other effects were fixed. Differences between individual treatments were as- certained using contrast procedures and results are expressed as dif- ferences from control with 95% confidence intervals (CI). The residuals of all fit models were analyzed for compliance with the assumptions of parametric statistical techniques (normality, independence and equality of errors). Parametric bootstrapping was performed (on 10,000 re- plicates) and data were reanalyzed whenever violations of these as- sumptions were detected. Immunohistochemistry results were eval- uated using one-way analysis of variance (ANOVA) followed by Tukey’s test with GraphPad Prism, in which a p value < 0.05 was considered to be statistically significant. 3. Results The combination of different doses of genital mycoplasmas and G. vaginalis are defined here as polybacterial treatments, and the results of these treatments, were compared with cultures from unstimulated samples (negative control). G. vaginalis, alone or in combination with genital mycoplasmas, significantly increased IL-6 production. Although other treatment groups, except for M. hominis, also showed increased IL-6 concentra- tions (Fig. 2), the increase was not as great as that produced by G. vaginalis. Soluble IL-6R concentrations did not change in response to poly- bacterial treatment, LPS, M. hominis or G. vaginalis. However, U. ur- ealyticum significantly increased sIL-6R production alone, or in com- bined treatment with M. hominis (Fig. 2). In addition, the sgp130 concentration was increased by LPS as well as M. hominis and U. ur- ealyticum, and in the polybacterial treatments, except for the combi- nation of the same load of all the studied bacteria. G. vaginalis also had no effect on sgp130 production (Fig. 2). The increase in IL-6, sIL-6R and sgp130 during genital mycoplasma stimulation indicates the binding of Fig. 2. Differential signature of IL-6 cytokine and receptors (soluble receptor (sIL-6R) and soluble β-subunit glycoprotein 130 (sgp130)) secreted by fetal membranes maintained in organ explants system. Control= unstimulated, MH(6)=Mycoplasma hominis at 106 CFU, UU(6)=Ureaplasma urealyticum at 106 CFU, GV(6)=Gardnerella vaginalis at 106 CFU, MH(6)+UU (6)=Mycoplasma hominis and Ureaplasma urealyticum at 106 CFU, MH(6)+UU(6)+GV(6)=Mycoplasma hominis and Ureaplasma urealyticum and Gardnerella vaginalis at 106 CFU, MH (6)+UU(6)+GV(3)=Mycoplasma hominis and Ureaplasma urealyticum at 106 CFU and Gardnerella vaginalis at 103 CFU, MH(3)+UU(3)+GV(6)=Mycoplasma hominis and Ureaplasma urealyticum at 103 CFU and Gardnerella vaginalis at 106 CFU, LPS-lipopolysaccharide. N.M. Noda-Nicolau et al. Journal of Reproductive Immunology 126 (2018) 60–68 62 these mediators; thus, there was no interaction with membrane-bound receptors, thereby inhibiting the proinflammatory action of IL-6 (trans- signaling). Similarly, in the polybacterial stimulation with different loads of bacteria, sgp130 was increased but without sIL-6R, suggesting the inhibition of trans-signaling. The mRNA levels of membrane-bound receptors, i.e. mIL-6R and gp130, were reduced by G. vaginalis, but LPS was only effective at re- ducing mIL-6R expression. No other effects of bacterial stimulation were detected (Fig. 3). Immunohistochemistry analysis suggested that neither polybacterial stimulation nor treatment with M. hominis or U. urealyticum affected mIL-6R levels in amnion or chorion cells (Fig. 4), and only LPS and G. vaginalis treatments increased mIL-6R expression in amnion cells (Fig. 4; p < 0.001). Moreover, gp130 was not changed following treatment with genital mycoplasmas, but it was increased in both amnion and chorion cells by LPS, G. vaginalis and all polybacterial treatments (Fig. 5; p < 0.001), suggesting the activation of classical signaling in the presence of G. vaginalis or LPS. 4. Discussion Polybacterial infections are commonly associated with spontaneous PTB and pPROM (DiGiulio, 2012; DiGiulio et al., 2010; Marconi et al., 2011). However, the host inflammatory response varies with the bac- terial load, the combination of bacteria, and the tissues affected (Menon et al., 2009; Noda-Nicolau et al., 2016; Zaga-Clavellina et al., 2007). This non-homogenous immune profile complicates the search for bio- markers of intrauterine infections and the development of interventions (Menon et al., 2007; Ryckman et al., 2009). Genital mycoplasmas are one of the most common isolates from pregnancies with intra-amniotic infection (IAI) or PIAC (DiGiulio, 2012). However, recent findings question the contribution of genital mycoplasmas to adverse outcomes (Choi et al., 2012; Govender et al., 2009; Lee et al., 2009). Our prior work (Noda-Nicolau et al., 2016), showed that genital mycoplasmas are not highly immunogenic in fetal tissues; in fact, they tend to buffer overwhelming inflammation generated by other pathogens by in- creasing the anti-inflammatory cytokine response (increasing IL-10, IL- 13 and sTNFR2). In this study, we further tested the role of polybacterial infections on the innate immune response by fetal mem- branes. Since IL-6 is one of the most studied cytokines as a marker of IAI and inflammation (Menon et al., 2011) and it is used clinically in multiple point of care (POC) test kits to diagnose these conditions (Chaemsaithong et al., 2015a,b), we studied how the host response may modulate IL-6 and its signaling molecules during PIAC. In Fig. 6, our data were used to build a representative model of classical, trans-signaling and inhibition of IL-6 activity after 24 h of fetal membrane treatment. In summary, we observed that: 1) our positive control (LPS) activated the IL-6 classical signaling pathway, since the expression of membrane-bound receptors was increased, along with IL- 6. 2) G. vaginalis activated IL-6 classical signaling, since IL-6 was in- creased along with gp130 and mIL-6R, similar to the LPS response. Of note, the effect of LPS and G. vaginalis treatment induced an anti-in- flammatory response, although they both increased IL-6 significantly compared to the control treatment. 3) M. hominis has no detectible ef- fect on altering either IL-6 or IL-6 receptor levels; therefore, M. hominis is unlikely to have any effect on IL-6 signaling. 4) The increase in IL-6 and sIL-6R expression by U. urealyticum alone or in combination with M. hominis (genital mycoplasmas) suggests that an upregulation of these molecules needed for trans-signaling activation; however, the con- current increase in sgp130, which inhibits trans-signaling, suggests that this response is either balanced or tightly regulated. Classical signaling is also unlikely with Mycoplasma stimulation, as membrane bound-re- ceptors were not increased upon exposure to these pathogens. 5) Polybacterial stimulation with similar or different loads of pathogens increased IL-6 and gp130 levels, but had no effect on mIL-6R. This suggests an impairment of the biological activity of IL-6 via classical signaling. Similarly, no changes in sIL-6R were observed in response to polybacterial treatment, which suggests that any effect was triggered by trans-signaling. Finally, polybacterial treatment did not show potential for an IL-6-mediated effect, as all markers tested showed a balanced response regardless of treatment (combination and load). Our results confirm that the type of bacteria and bacterial load play major roles in determining IL-6 production, the expression of its receptors, and likely its bioactivity. The high prevalence of vaginal, cervical and intra-amniotic Fig. 3. The relative mRNA expression of the membrane-bound receptor (mIL-6R) and gp130 receptor by fetal membranes maintained in an organ explants system, in the studied groups: Control = unstimulated, MH(6)=Mycoplasma hominis at 106 CFU, UU(6)=Ureaplasma urealyticum at 106 CFU, GV(6)=Gardnerella vaginalis at 106 CFU, MH(6)+UU(6)=Mycoplasma hominis and Ureaplasma urealyticum at 106 CFU, MH(6)+UU(6)+GV(6)=Mycoplasma hominis and Ureaplasma urealyticum and Gardnerella vaginalis at 106 CFU, MH(6)+UU(6)+GV (3)=Mycoplasma hominis and Ureaplasma urealyticum at 106 CFU and Gardnerella vaginalis at 103 CFU, MH(3)+UU(3)+GV(6)=Mycoplasma hominis and Ureaplasma urealyticum at 103 CFU and Gardnerella vaginalis at 106 CFU, LPS= lipopolysaccharide. N.M. Noda-Nicolau et al. Journal of Reproductive Immunology 126 (2018) 60–68 63 colonization by genital mycoplasmas have been extensively demon- strated in pregnancy complications, such as PTB and pPROM (Capoccia et al., 2013; Kacerovský et al., 2009; Kwak et al., 2014). Additional studies have shown that IAI is associated with mycoplasmas and increased IL-6 concentrations in the amniotic fluid (Romero et al., 2014; Yoon et al., 2003). These observations have suggested that IL-6 may be a valuable biomarker, and resulted in the development of POC tests (Chaemsaithong et al., 2015a,b; Musilova et al., 2016) designed Fig. 4. A) Immunohistochemical staining for mIL- 6R in the amnion (ac) and chorion (cc) maintained in an organ explants system for each treatment: a) Control= unstimulated, b) MH(6)=Mycoplasma hominis at 106 CFU, c) UU(6)=Ureaplasma ur- ealyticum at 106 CFU, d) GV(6)=Gardnerella vagi- nalis at 106 CFU, e) MH(6)+UU(6)=Mycoplasma hominis and Ureaplasma urealyticum at 106 CFU, f) MH(6)+UU(6)+GV(6)=Mycoplasma hominis and Ureaplasma urealyticum and Gardnerella vaginalis at 106 CFU, g) MH(6)+UU(6)+GV (3)=Mycoplasma hominis and Ureaplasma ur- ealyticum at 106 CFU and Gardnerella vaginalis at 103 CFU, h) MH(3)+UU(3)+GV(6)=Mycoplasma hominis and Ureaplasma urealyticum at 103 CFU and Gardnerella vaginalis at 106 CFU, i) LPS= lipopolysaccharide. B) Differential intensity of gp130 in amnion cells by immunohistochemistry in the studied treatments compared to control. * p < 0.001. C) Differential intensity of gp130 in chorion cells by immunohistochemistry in the stu- died treatments compared to control. N.M. Noda-Nicolau et al. Journal of Reproductive Immunology 126 (2018) 60–68 64 for clinical use. However, information on quantitative change alone is not sufficient to understand the functional downstream effects of IL-6 that activate or inhibit labor pathways. Therefore, we tested changes in other molecules that IL-6 interacts with to exert its biological effects in response to polybacterial pathogens associated with PTB. It is possible that increased circulating sgp130 levels is part of a convalescent re- sponse to minimize IL-6 production hours after the initial exposure. Quantification of sIL-6R and sgp130 may also be useful for identifying Fig. 5. A) Immunohistochemical staining for gp130 in the amnion (ac) and chorion (cc) maintained in an organ explants system for each treatment: a) Control= unstimulated, b) MH(6)=Mycoplasma ho- minis at 106 CFU, c) UU(6)=Ureaplasma urealyticum at 106 CFU, d) GV(6)=Gardnerella vaginalis at 106 CFU, e) MH(6)+UU(6)=Mycoplasma hominis and Ureaplasma urealyticum at 106 CFU, f) MH(6)+UU (6)+GV(6)=Mycoplasma hominis and Ureaplasma ur- ealyticum and Gardnerella vaginalis at 106 CFU, g) MH (6)+UU(6)+GV(3)=Mycoplasma hominis and Ureaplasma urealyticum at 106 CFU and Gardnerella va- ginalis at 103 CFU, h) MH(3)+UU(3)+GV (6)=Mycoplasma hominis and Ureaplasma urealyticum at 103 CFU and Gardnerella vaginalis at 106 CFU, i) LPS= lipopolysaccharide. B) Differential intensity of gp130 in amnion cells by immunohistochemistry in the studied treatments compared to control. ANOVA fol- lowed by Tukey’s test, * p < 0.0001. C) Differential intensity of gp130 in chorion cells by im- munohistochemistry in the studied treatments com- pared to control. ANOVA followed by Tukey’s test, * p < 0.001. N.M. Noda-Nicolau et al. Journal of Reproductive Immunology 126 (2018) 60–68 65 Fig. 6. Conceptual model for how IL-6 signaling may occur in fetal membranes stimulated with different bacterial combinations in vitro based on the expression of IL-6 and factors that affect its biological activities. N.M. Noda-Nicolau et al. Journal of Reproductive Immunology 126 (2018) 60–68 66 patients most likely to benefit from sgp130Fc therapy due to excessive IL-6 trans-signaling (Jones et al., 2011). Our results regarding the in- hibition of IL-6 trans-signaling through increased sgp130 could also help explain why many pregnancies that test positive for genital my- coplasmas and other microorganisms do not result an adverse preg- nancy outcomes (Aagaard et al., 2014; Govender et al., 2009; Lee et al., 2009; Naessens et al., 1989; Steel et al., 2005). Inhibition of IL-6 trans- signaling results in the downregulation of inflammatory signaling, which could also favor the survival of Ureaplasma species that live off the urea-rich (Shepard and Lunceford, 1967) amniotic fluid and need to evade the maternal immune system. Similarly, polybacterial stimula- tion with different loads of genital mycoplasma and G. vaginalis do not suggest a functional role for IL-6 through either the classical or trans- signaling pathways. Therefore, although IL-6 may increase in quantity in response to polybacterial infection, the presence of genital myco- plasmas may mitigate the function of IL-6 as an anti-inflammatory mediator. A disturbance or immune weakness in the uterine cavity may attract mycoplasma due to an underlying pathology, forcing them to ascend from the genital tract. Based on data from this report and our prior report (Noda-Nicolau et al., 2016), we postulate that this ascen- sion is likely to provide a probiotic environment to assure fetal well- being. A stronger anti-inflammatory response (Noda-Nicolau et al., 2016) and the absence of IL-6 trans-signaling support this hypothesis. However, access of these microbes to fetal organs can contribute to adverse neonatal outcomes (Waites et al., 2005). G. vaginalis increases the expression levels of IL-6 and the mem- brane-bound receptor proteins mIL-6R and gp130; however, gene ex- pression of these receptors was downregulated. Considering that, the turnover times for proteins and mRNA of the same gene are not similar, i.e., the protein may stay longer than the mRNA(Eden et al., 2011; Yen et al., 2008), and the level of mRNA in steady state may be low, but protein translated can accumulate over time(Vogel and Marcotte, 2013). Moreover, since a protein is expressed from its corresponding mRNA, mRNA is prone to degradation. We firstly suggested that low concentration of mRNA and high mIL-6R protein occur because after translation, that particular mRNA gets degraded while its protein has higher half-life and remains in the cell. Secondly, considering that the expression is dynamics and we performed assays in one time point (24 h), this controversial result could be a transient state where func- tionally viable protein expression is already effective and could be identified, while mRNA of mIL-6R was not in steady state and could not be detected. This hypothesis becomes relevant since IL-6 production in amnion cells is normally increased by 12 h of bacterial stimulation (Reisenberger et al., 1997). Lastly, since mRNA are regulated by several factors such as microRNAs(Wu and Brewer, 2012), we suggested that the decreased genic expression of mIL-6R could be a regulation me- chanism, once there is mIL-6R protein sufficient in the tissue after 24 h and the inflammatory response needs to be regulated. Although the signal transducing chain gp130 is shared by members of the IL-6 family of cytokines, mIL-6R is a unique binding receptor for IL-6 (Tanaka et al., 2014). Therefore, it is likely that the fetal membrane may try to mini- mize the induction of an intense inflammatory response by G. vaginalis (as seen by increased IL-6 and all other associated signaling markers) by minimizing membrane receptors after 24 h. Other factors that could contribute to the function of IL-6 include IL- 6 and IL-6R polymorphisms and their interactions with infection, which can strongly influence IL-6 production and signaling (Velez et al., 2008). Further studies will be needed to determine how they may im- pact the host response to infection of the fetal membranes. The observed IL-6 increase by various PIAC-associated bacteria suggest its potential as a biomarker of infection, irrespective of the bacteria involved. However, an increase in this cytokine alone may not be indicative of any functional role, as its biological activities are controlled by additional molecules whose expression also changes in response to infection. IL-6 change alone (increase or decrease) may not have functional relevance in fetal cells as shown in our study; however, we do not rule out the possibility of an impact of IL-6 change in other tissues such as decidua, which is an extremely immunocompetent compartment. Additional information will be valuable to determine the possible downstream effects of increased IL-6 levels and/or to design an interventional target to modulate IL-6 function. As shown here, not all bacteria or combinations of them produce a molecular arrangement for IL-6 to exert a function. IL-6 expression by membranes is likely a non- specific innate response and not a functional mediator of any labor- inducing pathways. Indeed, previous studies have demonstrated that IL- 6 is neither necessary nor sufficient to include PTB in mouse models (Sadowsky et al., 2006; Yoshimura and Hirsch, 2003). Conversely, Robertson’s group (Robertson et al., 2010) have shown that IL-6 null mutant mice have an extended gestational period after LPS treatment, suggesting yet another role for IL-6 in pregnancy maintenance. 5. Conclusions Therefore, additional consideration of all the factors that influence IL-6 bioactivity need to be considered if it is to be used as a biomarker for adverse pregnancy outcomes or a target for designing drugs to prevent them. Conflict of interest The authors report no conflict of interest. Funding This study was supported by: developmental funds granted to Dr. R Menon from the Department of OBGYN, the University of Texas Medical Branch; Galveston, TX, USA; by São Paulo Research Foundation − FAPESP (Grant 2012/17234-1) granted to Dr. Márcia Guimarães da Silva from the Department of Pathology, Botucatu Medical School, UNESP− Univ. Estadual Paulista, Botucatu, São Paulo, Brazil; and by Coordination for the Improvement of Higher Education Personnel − CAPES (BEX 3511/13-8) granted to Nathália Mayumi Noda Nicolau, PhD student in the Post-graduate Program in Pathology, Botucatu Medical School, UNESP − Univ. Estadual Paulista, Botucatu, São Paulo, Brazil. 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