Ureaplasma urealyticum-derived lipid-associated membrane proteins introduce IL-6, IL-8, and TNF-α cytokines into human amniotic epithelial cells via Toll-like receptor 2

Ureaplasma urealyticum- derived lipid-associated membrane proteins introduce IL-6, IL-8, and TNF-α cytokines into human amniotic epithelial cells via Toll-like receptor 2 解脲脲原体脂质相关膜蛋白经Toll样受体2信号通路调控人羊膜上皮细胞诱导IL-6、IL-8和TNF-α的产生 1 corresp first-author ‡Corresponding Author ‡Corresponding Author †E-mail:ygy20518158@zju.edu.cn †E-mail:ygy20518158@zju.edu.cn ygy20518158@zju.edu.cn 2 3 1 Women’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China Women’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China Central Laboratory, Hangzhou First People’s Hospital, Hangzhou 310006, China Central Laboratory, Hangzhou First People’s Hospital, Hangzhou 310006, China Department of Clinical Laboratory, Shaanxi Provincial People’s Hospital, Xi’an 710068, China Department of Clinical Laboratory, Shaanxi Provincial People’s Hospital, Xi’an 710068, China Objective: 2 The purpose of this study was to determine the role of Ureaplasma urealyticum- derived lipid-associated membrane proteins (LAMPs) in the host innate immune system, specifically their effect on Toll-like receptors (TLRs). Methods: 2 LAMPs were derived from U. urealyticum strains, and human amniotic epithelial cells (HAECs) were isolated from healthy full-term placentas. Cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA) and TLR2 mRNA by real-time PCR. Expression of TLR2 was confirmed by Western blotting and immunohistochemistry. Results: 2 LAMPs induced HAECs to produce inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α. Cytokine production was reduced after blocking TLR2 using TLR2 inhibitor (anti-hTLR2-IgA). Conclusions: 2 LAMPs isolated from U. urealyticum induced TLR2-dependent up-regulation of inflammatory genes and cytokines in HAECs. 目的 2 探讨解脲脲原体( Ureaplasma urealyticum )及其脂质相关膜蛋白(LAMPs)作用于人羊膜上皮细胞(HAECs)过程中白介素6(IL-6)、IL-8和肿瘤坏死因子α(TNF-α)的变化情况,阐明Toll样受体2(TLR2)的调控机制,明确解脲脲原体潜在的致病性. 创新点 2 从解脲脲原体诱导炎症反应的分子机制入手,提出TLR2信号通路在其中的关键作用. 方法 2 经TX-114处理萃取解脲脲原体获得LAMPs,将LAMPs和解脲脲原体分别感染HAECs,用酶联免疫吸附试验(ELISA)测定炎症细胞因子 (IL-6、IL-8和TNF-α);采用实时聚合酶链反应(real-time PCR)测定TLR2 mRNA水平,用蛋白质印迹(Western blot)检测TLR2的表达量;经TLR2阻断剂(anti-hTLR2-IgA)处理后,测定相应炎症细胞因子. 结论 2 解脲脲原体LAMPs能诱导HAECs的TLR2表达上调和炎症因子增加,从而发生炎症反应;TLR2受阻断后,炎症因子表达减少,炎症水平下降.TLR2在解脲脲原体LAMPs感染HAECs过程起关键作用. Ureaplasma urealyticum Lipid-associated membrane protein Human amniotic epithelial cell Toll-like receptor 2 解脲脲原体 脂质相关膜蛋白 人羊膜上皮细胞 Toll样受体2 Introduction 1 Intra-amniotic infection is a primary cause of preterm birth. Studies have shown that Mycoplasma infection is an important risk factor for adverse pregnancy outcomes and low birth weight in infants (Viscardi, 2010 2010 ; Capoccia et al., 2013 2013 ). Ureaplasma spp., belonging to the Mycoplasmataceae family, are the most common organisms isolated from the urogenital tracts of women who present with preterm labor (Viscardi, 2010 2010 ). Ureaplasma has 14 known serotypes and is divided into two biovars: Ureaplasma parvum and Ureaplasma urealyticum . Gerber et al. ( 2003 2003 ) estimated that preterm birth occurs in 58.6% of U. urealyticum -positive women compared with only 4.4% in U. urealyticum -negative women. Infection-induced preterm labor is an inflammatory process, where pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), sense microorganisms and their products. Chemokines (e.g. interleukin (IL)-1, IL-8, chemokine (C-C motif) ligand 2 (CCL-2)), cytokines (e.g. IL-1β, tumor necrosis factor (TNF)-α), prostaglandins, and proteases are produced, which lead to activation of the common pathway of parturition (Romero et al., 2001 2001 ; Kannan et al., 2012 2012 ). Triantafilou et al. ( 2013 2013 ) demonstrated that TLR2, TLR6, and TLR9 are involved in U . urealyticum -induced inflammation of human amniotic epithelial cells (HAECs). Mycoplasma lipid-associated membrane proteins (LAMPs) are cell-surface lipoproteins which induce potent inflammatory responses (He et al., 2009 2009 ). Several reports have confirmed that Mycoplasma- derived LAMPs can induce nuclear transcription factor κB (NF-κB) activation in human acute monocytic leukemia cell lines (Shimizu et al., 2008 2008 ) and embryonic bovine lung (EBL) cells (He et al., 2009 2009 ). Studies have revealed that it is possible to demonstrate the initial interaction between Mycoplasma membrane properties and host cells, such as leukocytes and epithelial cells (Shimizu et al., 2008 2008 ; He et al., 2009 2009 ; Choi et al., 2012 2012 ). We hypothesized that U. urealyticum ligate TLRs can initiate inflammatory responses in HAECs. To test this hypothesis, we used U. urealyticum -derived LAMPs incubated with HAECs in culture ex vivo to examine inflammatory responses and to determine if the responses are mediated via TLR2 ligation. Materials and methods 1 Preparation and culture of HAECs 2 Healthy full-term placentas were obtained with informed consent from women who had undergone a cesarean section at the Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, China. The study was approved by the local research ethics committee. HAECs were isolated from the placentas and cultured as follows (Liu et al., 2012 2012 ). Briefly, the membrane was placed in Dulbecco’s modified Eagle medium (DMEM; HyClone, USA) and cut with a razor to yield 0.5–1.0 cm 2 segments. The segments were digested with 0.25% (2.5 g/L) trypsin-ethylenediaminetetraacetic acid (EDTA) at 37 °C for 45 min. The resulting cell suspensions were cultured in RPMI 1640 (HyClone, USA) containing 10% heat-inactivated fetal bovine serum (FBS; HyClone, USA), 2 mmol/L L -glutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin at 37 °C in a 5% CO 2 humid atmosphere. Sample collection 2 The cultured HAEC cells were transferred to 12-well culture plates after the cell concentration was adjusted to 4×10 5 cells/well. The group of HAECs treated with LAMPs was regarded as the LAMP test group, and the HAECs treated with U. urealyticum were regarded as the U. urealyticum test group. Aqueous phase from the preparation of LAMPs (see below) was regarded as the negative control group. Preparation and culture of U. urealyticum 2 U. urealyticum (ATCC 27618) was cultivated in modified Mycoplasma liquid broth (Oxoid, England) at 37 °C in a 5% CO 2 humid atmosphere. Cell density was measured and a concentration of 1×10 8 color forming units (CCU) per milliliter was used. CCU was used for measuring U. urealyticum concentration. According to the metabolic activity of microbes in the culture medium, the relative content of microbes is counted. The method is described briefly: (1) 1.8 ml of the liquid Mycoplasma medium was added to each of 12 aseptic small tubes; (2) a 10-time gradient dilution was based on 1×10 0 –1×10 −11 ; (3) all tubes were cultured at 37 °C for 14 d. The highest dilution showing a color change in the culture medium, which corresponds to the maximum metabolic activity of Mycoplasma , was used as the CCU of the Mycoplasma to be tested. Preparation of LAMPs 2 LAMPs were prepared as described previously (He et al., 2009 2009 ). U. urealyticum was cultured in modified Mycoplasma solid medium (Oxoid, England) at 5% CO 2 , at 37 °C for 3–7 d until “fried egg” colonies were visible at low magnification. A single “fried egg” colony was then cultured in 500 ml of modified Mycoplasma liquid medium (Oxoid, England) at 5% CO 2 , at 37 °C for 3–7 d. When the medium color changed from red to yellow and was clear, it was regarded as indicating the logarithmic phase of U. urealyticum . Cells of U. urealyticum in the logarithmic phase were centrifuged at 12 000 g for 10 min and then the sediment was resuspended in 5 ml TBSE solution (50 mmol/L Tris (pH 8.0), 0.15 mmol/L NaCl, 1.0 mmol/L EDTA). A final concentration of 2% of Triton X-114 (TX-114) was added to lyse U . urealyticum at 4 °C for 1 h. Phase separation of the lysates of U. urealyticum occurred at 37 °C for 10 min. The upper aqueous phase separation, which was regarded as the negative control, was stored at −80 °C after 12 000 g centrifugation for 20 min. The TBSE solution (4 °C) with an equal volume of TX-114 phase was added and mixed with the TX-114 phase at 4 °C for 10 min. The phase separation process was carried out three times. The TX-114 phase was suspended with TBSE (4 °C) to the initial volume. After adding 2.5 times the volume of anhydrous alcohol, the whole suspension was left overnight at −20 °C to precipitate the membrane protein components. The precipitate was suspended in phosphate-buffered saline (PBS) after centrifugation. LAMPs were obtained after ultrasonic treatment. The protein concentration of the suspension was measured by the Bradford method (Coomassie blue protein assay). HAECs stimulated with U. urealyticum or LAMPs 2 HAECs were seeded into 12-well plates at a concentration of 4×10 5 cells/well until the cells reached about 70%–80% confluence. HAECs were stimulated for 2, 6, or 24 h with the different concentrations (1.0–7.0 µg/ml) of LAMP or with U. urealyticum (1×10 8 CCU/ml). The supernatants were collected and frozen until the cytokine assays were performed. The expression of TLR2 mRNA was detected by real-time PCR after the cells were collected. Immunohistochemistry was used to detect the location of TLR2 in the cell. TLR2 expression blocked by TLR2 inhibitor in HAECs 2 Anti-hTLR2-IgA (InvivoGen, USA), a TLR2 inhibitor (10 µg/ml), was added to the culture medium of HAECs for culture at 37 °C for 2 h, and then U. urealyticum (1×10 8 CCU/ml) or LAMPs (4 µg/ml) were added for 24 h. The culture solution was collected to detect cytokines. Immunohistochemistry was used to detect the location of TLR2 in the cell and the expression of TLR2 was detected by Western blotting. Cytokine assays 2 Concentrations of cytokines in culture supernatants after stimulation were measured using enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, USA) according to the manufacturer’s instructions. High-sensitivity ELISA kits were used to determine IL-6, IL-8, and TNF-α concentrations (the limits of detection were 0.10 , 0.05, and 0.13  pg/ml, respectively). Real-time PCR assay 2 Total mRNA was extracted from infected and non-infected HAECs using TRIzol reagent (Invitrogen Carlsbad, CA, USA), according to the manufacturer’s instructions. The RNA concentration was measured using a NanoDrop ND-2000 spectrophotometer (Life Technologies, USA). TLR2 mRNA in HAECs was determined by conducting quantitative SYBR®Green-based real-time quantitative reverse transcription PCR (real-time qRT-PCR). All treatments were performed in triplicate. β-Actin was used as an internal control; two negative controls were also used in all RT-PCR experiments. The sequences of the primers used for real-time PCR were as follows: TLR2 sense, 5'-GGCCAGCAAATTACCTGTGTG-3'; TLR2 antisense, 5'-AGGCGGACATCCTGAACC-3'; TNF-α sense, 5'-TTCTCCTTCCTGATCGTGGC-3'; TNF-α antisense, 5'-TTCTCCTTCCTGATCGTGGC-3'; IL-8 sense, 5'-CTGGCCGTGGCTCTCTTG-3'; IL-8 antisense, 5'-CCTTGGCAAAACTGCACCTT-3'; IL-6 sense, 5'-CCACTCACCTCTTCAGAACG-3'; IL-6 antisense, 5'-CATCTTTGGAAGGTTCAGGTTG-3'; β-actin sense, 5'-GCTCTGGCTCCTAGCACCAT-3'; β-actin antisense, 5'-GCCACCGATCCACACAGA GT-3'. Western blotting 2 Compared with control β-actin, the amount of TLR2 protein in HAECs was detected both by antibodies against TLR2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and by Western blotting using enhanced chemiluminescence as specified by the manufacturer’s instructions (GE Healthcare, Piscataway, NJ, USA). Immunohistochemistry 2 HAECs infected with U. urealyticum or LAMPs were mounted on glass slides for 24 h. The cells were routinely processed and stained overnight with an appropriate concentration of a primary monoclonal antibody, followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Statistical analysis 2 Statistical comparisons were made using the t- test. The results are reported as mean±standard deviation (SD). P- values of < 0.05 were considered statistically significant. All statistical analyses were performed with SPSS 20.0 software (IBM, USA). Results 1 TLR2 mRNA expression in HAECs 2 TLR2 mRNA expression in HAECs varied depending on the concentration of LAMPs (1.0–7.0 µg/ml). Expression reached a maximum at a concentration of 4.0 µg/ml, and then remained largely unchanged at higher concentrations (Fig. 1 1 ). To determine whether TLR2 mRNA expression in HAECs stimulated with U. urealyticum (1×10 8 CCU/ml) or LAMPs (4.0 µg/ml) varied in relation to incubation time, HAECs were collected after incubation for 2, 6, or 24 h and tested by the following methods. Compared with the control group, expression of TLR2 mRNA was higher in HAECs infected with U. urealyticum or LAMPs for 6 or 24 h ( P < 0.01). After 2 h, TLR2 mRNA expression was higher in HAECs incubated with U. urealyticum than in the control or in HAECs incubated with LAMPs ( P < 0.05; Fig. 2 2 ). Data are expressed as mean±SD ( n =6). * P < 0.05, ** P < 0.01 vs. control; # P < 0.05 vs. LAMPs TLR2 expression in HAECs induced by U. urealyticum or LAMPs 2 Immunohistochemical analysis showed that expression of TLR2 occurred after stimulation with U . urealyticum or LAMPs, and was repressed following treatment with TLR2 inhibitor, suggesting that expression of TLR2 was associated with stimulation by U. urealyticum or LAMPs (Fig. 3 3 ). (a) Negative control groups (as infection control). (b) Negative control groups (as inhibitor control). (c) TLR2 expression after U. urealyticum infection for 24 h (arrow). (d) TLR2 expression after LAMP treatment for 24 h (arrow). (e) TLR2 expression after U. urealyticum infection for 24 h (pretreatment with TLR2 inhibitor, arrow). (f) TLR2 expression after LAMP infection for 24 h (pretreatment with TLR2 inhibitor, arrow) Compared to the control, TLR2 expression was significantly higher in HAECs after infection with U. urealyticum or treatment with LAMPs for 24 h ( P < 0.01). TLR2 expression in HAECs was inhibited by TLR2 inhibitor in the U. urealyticum group ( P < 0.05) and the LAMP group ( P < 0.01) (Fig. 4 4 ). Data are expressed as mean±SD ( n =6). ** P < 0.01 vs. control; # P < 0.05, ## P < 0.01 vs. U. urealyticum Cytokine production induced by U. urealyticum and LAMPs in HAECs 2 As HAECs are commonly used as models for the human amnion, we initially tested the ability of U . urealyticum and LAMPs to stimulate the production of various cytokines in HAECs. HAECs were incubated with U. urealyticum (1×10 8 CCU/ml) or LAMPs (4.0 µg/ml) for up to 24 h. Compared with the control group, HAECs produced significantly more cytokines (IL-6, IL-8, TNF-α) after stimulation with U. urealyticum or LAMPs for 24 h ( P < 0.01; Fig. 5 5 ). Data are expressed as mean±SD ( n =6). ** P < 0.01 vs. control U. urealyticum- or LAMP-mediated production of cytokines inhibited by TLR2 inhibitor 2 HAECs were treated with TLR2 inhibitor for 2 h before incubation with U. urealyticum or LAMPs, and cytokines in the supernatants were measured after incubation for 24 h (Fig. 6 6 ). The control group of HAECs was treated with PBS or TLR2 inhibitor. The results showed that after treatment with TLR2 inhibitor, cytokine production by HAECs infected with U. urealyticum or treated with LAMPs was significantly lower than that by HAECs treated with PBS ( P < 0.01). (a) Mean production of IL-6. (b) Mean production of IL-8. (c) Mean production of TNF-α. Data are expressed as mean±SD ( n =6). ** P < 0.01 vs. HAECs treated with PBS Discussion 1 To our knowledge, this is the first report of U. urealyticum -derived LAMPs stimulating HAECs to express TLR2. A growing body of evidence suggests that the presence of U. urealyticum in the amniotic cavity correlates with an adverse effect on pregnancy outcomes and complications of preterm birth. Previous studies (Abele-Horn et al., 2000 2000 ; Kwak et al., 2014 2014 ) have demonstrated that the incidence of preterm delivery, low Apgar scores, neonatal intensive care unit (NICU) admission, and histologic chorioamnionitis are directly correlated with the level of U. urealyticum colonization. High levels of cytokines in the amniotic fluid may serve as an indicator of bacterial infection and can predict subsequent pregnancy loss (Daskalakis et al., 2009 2009 ). A shift in signaling from anti-inflammatory to pro-inflammatory pathways plays an important role in adverse outcomes of preterm birth (Wenstrom et al., 1996 1996 ). In this study, U. urealyticum -derived LAMPs and U. urealyticum were capable of inducing secretion of pro-inflammatory cytokines IL-6, IL-8, and TNF-α after the host cells were treated for 24 h. These results are similar to those of Wang et al. ( 2016 2016 ) who found that Mycoplasma bovis -derived LAMPs can activate IL-1β production via TLR2. He et al. ( 2009 2009 ) reported that Mycoplasma genitalium -derived LAMPs can activate the immune system of the cell. This study built on previous research which determined that U. urealyticum or Ureaplasma spp. trigger the secretion of pro-inflammatory cytokines (Triantafilou et al., 2013 2013 ). Since LAMPs are the major surface proteins of U. urealyticum , it seemed likely that they may play a role in the induction of cytokine expression. We confirmed that U. urealyticum -derived LAMPs can induce cytokine expression in HAECs and that TLR2 was involved in triggering pro-inflammatory responses in response to them. There is accumulating evidence (Aalaei-Andabili and Rezaei, 2013 2013 ) that intrauterine infection-induced preterm labor is the result of an inflammatory cascade initiated by bacterial interaction with host PRRs such as the TLRs 2, 6, and 9. Following engagement with PRRs, there is a sequential rise in inflammatory cytokines such as IL-1β, TNF-α, IL-6, and IL-8 followed by leukocyte recruitment, and increases in prostaglandins such as prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) and matrix metalloproteinases. It is increasingly reported that non-immune cells, including HAECs, can regulate the secretion of inflammatory cytokines via the TLR signaling pathway (Triantafilou et al., 2013 2013 ). Our experiments confirmed that TLR2 is involved in activation of the inflammatory cascade. While LAMPs did not induce cytokine expression as strongly as whole cell U. urealyticum , which stimulated HAECs to express TLR2 within 2 h, they activated TLR2 after 6 h (Fig. 2 2 ). TLR2 has been identified as a receptor that is important to the innate immune response and transmits signaling pathways via IL-1 receptor (IL-1R)-associated signal molecules, such as myeloid differentiation factor 88 (MyD88), mitogen-activated protein kinases (Sweet and Schorey, 2006 2006 ; Lyu et al., 2017 2017 ). Further research is needed to confirm which intracellular pathway is involved in this interaction. Inhibition of the interaction between TLRs and lipoproteins may result in a reduction of the inflammatory response induced by LAMPs. After HAECs were treated with TLR2 inhibitor, cytokine production was significantly reduced in HAECs treated with either LAMPs or U. urealyticum (Fig. 2 2 ). Pre-treatment with inhibitors offers promise as a protective means to lower the activity of cytokines. Glushkova et al. ( 2013 2013 ) found that inhibitors of TLR4 signaling reduced the toxic effect of lipopolysaccharide. The results of our study might help in the search for new TLR-based therapeutic targets for LAMPs and U. urealyticum- induced chorioamnionitis. Our data showed that TLR2 mRNA expression continued to rise to some extent as the concentration of LAMPs increased. The fact that LAMPs and U. urealyticum engage TLR2 might reveal the reasons why U. urealyticum can cause the development of chronic, low-level inflammation of amniotic epithelial cells leading to irreversible injury of fetal membranes. Activation via TLR2 results in a soft inflammatory response allowing the LAMPs to establish a chronic foothold in amniotic epithelial cells. Taken together, our data indicate that U . urealyticum -derived LAMPs have potent inflammatory properties and can mediate changes in gene expression in HAECs. Our studies suggest that using the inhibitor anti-hTLR2-IgA to inhibit the signaling pathway can result in a reduction in the downstream expression of several pro-inflammatory cytokines. Our data also sustain the notion that U. urealyticum may have additional mechanisms for inducing inflammatory responses in HAECs beyond those mediated by LAMPs. There were several limitations inherent in this study. First, we detected only three kinds of cytokines; more chemokines should be investigated in further studies. Second, although we confirmed that cytokine expression was reduced after blocking TLR2, other TLRs may play a similar role and should be investigated. Conclusions 1 This is the first investigation into the effects of U. urealyticum -derived LAMPs on the expression of cytokines IL-6, IL-8, and TNF-α in HAECs via TLR2. References SH Aalaei-Andabili , N Rezaei . Toll like receptor (TLR)-induced differential expression of microRNAs (MiRs) and immune response against infection: a systematic review . J Infect , 2013 . 67 ( 4 ): 251 - 264 . 23850616 DOI: 10.1016/j.jinf.2013.07.016 http://doi.org/10.1016/j.jinf.2013.07.016 . Toll like receptor (TLR)-induced differential expression of microRNAs (MiRs) and immune response against infection: a systematic review M Abele-Horn , M Scholz , C Wolff , 等 . High-density vaginal Ureaplasma urealyticum colonization as a risk factor for chorioamnionitis and preterm delivery . Acta Obstet Gynecol Scand , 2000 . 79 ( 11 ): 973 - 978 . 11081683 DOI: 10.1034/j.1600-0412.2000.079011973.x http://doi.org/10.1034/j.1600-0412.2000.079011973.x . High-density vaginal Ureaplasma urealyticum colonization as a risk factor for chorioamnionitis and preterm delivery R Capoccia , G Greub , D Baud . Ureaplasma urealyticum , Mycoplasma hominis and adverse pregnancy outcomes . Curr Opin Infect Dis , 2013 . 26 ( 3 ): 231 - 240 . 23587772 DOI: 10.1097/QCO.0b013e328360db58 http://doi.org/10.1097/QCO.0b013e328360db58 . Ureaplasma urealyticum, Mycoplasma hominis and adverse pregnancy outcomes SY Choi , JW Lim , T Shimizu , 等 . Reactive oxygen species mediate Jak2/Stat3 activation and IL-8 expression in pulmonary epithelial cells stimulated with lipid-associated membrane proteins from Mycoplasma pneumoniae . Inflamm Res , 2012 . 61 ( 5 ): 493 - 501 . 22270622 DOI: 10.1007/s00011-012-0437-7 http://doi.org/10.1007/s00011-012-0437-7 . Reactive oxygen species mediate Jak2/Stat3 activation and IL-8 expression in pulmonary epithelial cells stimulated with lipid-associated membrane proteins from Mycoplasma pneumoniae G Daskalakis , N Thomakos , A Papapanagiotou , 等 . Amniotic fluid interleukin-18 at mid-trimester genetic amniocentesis: relationship to intraamniotic microbial invasion and preterm delivery . BJOG , 2009 . 116 ( 13 ): 1743 - 1748 . 19906019 DOI: 10.1111/j.1471-0528.2009.02364.x http://doi.org/10.1111/j.1471-0528.2009.02364.x . Amniotic fluid interleukin-18 at mid-trimester genetic amniocentesis: relationship to intraamniotic microbial invasion and preterm delivery S Gerber , Y Vial , P Hohlfeld , 等 . Detection of Ureaplasma urealyticum in second-trimester amniotic fluid by polymerase chain reaction correlates with subsequent preterm labor and delivery . J Infect Dis , 2003 . 187 ( 3 ): 518 - 521 . 12552439 DOI: 10.1086/368205 http://doi.org/10.1086/368205 . Detection of Ureaplasma urealyticum in second-trimester amniotic fluid by polymerase chain reaction correlates with subsequent preterm labor and delivery OV Glushkova , SB Parfenyuk , MO Khrenov , 等 . Inhibitors of TLR-4, NF-κB, and SAPK/JNK signaling reduce the toxic effect of lipopolysaccharide on RAW 264.7 cells . J Immunotoxicol , 2013 . 10 ( 2 ): 133 - 140 . 22830990 DOI: 10.3109/1547691X.2012.700652 http://doi.org/10.3109/1547691X.2012.700652 . Inhibitors of TLR-4, NF-κB, and SAPK/JNK signaling reduce the toxic effect of lipopolysaccharide on RAW 264.7 cells J He , XX You , YH Zeng , 等 . Mycoplasma genitalium -derived lipid-associated membrane proteins activate NF-κB through Toll-like receptors 1, 2, and 6 and CD14 in a MyD88-dependent pathway . Clin Vaccine Immunol , 2009 . 16 ( 12 ): 1750 - 1757 . 19793902 DOI: 10.1128/CVI.00281-09 http://doi.org/10.1128/CVI.00281-09 . Mycoplasma genitalium-derived lipid-associated membrane proteins activate NF-κB through Toll-like receptors 1, 2, and 6 and CD14 in a MyD88-dependent pathway S Kannan , H Dai , RS Navath , 等 . Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a rabbit model . Sci Transl Med , 2012 . 4 ( 130 ): 130ra46 22517883 DOI: 10.1126/scitranslmed.3003162 http://doi.org/10.1126/scitranslmed.3003162 . Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a rabbit model DW Kwak , HS Hwang , JY Kwon , 等 . Co-infection with vaginal Ureaplasma urealyticum and Mycoplasma hominis increases adverse pregnancy outcomes in patients with preterm labor or preterm premature rupture of membranes . J Matern Fetal Neonatal Med , 2014 . 27 ( 4 ): 333 - 337 . 23796000 DOI: 10.3109/14767058.2013.818124 http://doi.org/10.3109/14767058.2013.818124 . Co-infection with vaginal Ureaplasma urealyticum and Mycoplasma hominis increases adverse pregnancy outcomes in patients with preterm labor or preterm premature rupture of membranes T Liu , WW Cheng , YY Huang , 等 . Human amniotic epithelial cell feeder layers maintain human IPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression . Exp Cell Res , 2012 . 318 ( 4 ): 424 - 434 . 22200372 DOI: 10.1016/j.yexcr.2011.12.004 http://doi.org/10.1016/j.yexcr.2011.12.004 . Human amniotic epithelial cell feeder layers maintain human IPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression A Lyu , JJ Chen , HC Wang , 等 . Punicalagin protects bovine endometrial epithelial cells against lipopolysaccharide-induced inflammatory injury . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 2017 . 18 ( 6 ): 481 - 491 . DOI: 10.1631/jzus.B1600224 http://doi.org/10.1631/jzus.B1600224 . Punicalagin protects bovine endometrial epithelial cells against lipopolysaccharide-induced inflammatory injury R Romero , R Gómez , T Chaiworapongsa , 等 . The role of infection in preterm labour and delivery . Paediatr Perinat Epidemiol , 2001 . 15 ( S2 ): 41 - 56 . 11520399 DOI: 10.1046/j.1365-3016.2001.00007.x http://doi.org/10.1046/j.1365-3016.2001.00007.x . The role of infection in preterm labour and delivery T Shimizu , Y Kida , K Kuwano . A triacylated lipoprotein from Mycoplasma genitalium activates NF-κB through Toll-like receptor 1 (TLR1) and TLR2 . Infect Immun , 2008 . 76 ( 8 ): 3672 - 3678 . 18474641 DOI: 10.1128/IAI.00257-08 http://doi.org/10.1128/IAI.00257-08 . A triacylated lipoprotein from Mycoplasma genitalium activates NF-κB through Toll-like receptor 1 (TLR1) and TLR2 L Sweet , JS Schorey . Glycopeptidolipids from Mycobacterium avium promote macrophage activation in a TLR2-and MyD88-dependent manner . J Leukoc Biol , 2006 . 80 ( 2 ): 415 - 423 . 16760377 DOI: 10.1189/jlb.1205702 http://doi.org/10.1189/jlb.1205702 . Glycopeptidolipids from Mycobacterium avium promote macrophage activation in a TLR2-and MyD88-dependent manner M Triantafilou , B de Glanville , AF Aboklaish , 等 . Synergic activation of Toll-Like Receptor (TLR) 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells . PLoS ONE , 2013 . 8 ( 4 ): e61199 23593431 DOI: 10.1371/journal.pone.0061199 http://doi.org/10.1371/journal.pone.0061199 . Synergic activation of Toll-Like Receptor (TLR) 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells RM Viscardi . Ureaplasma species: role in diseases of prematurity . Clin Perinatol , 2010 . 37 ( 2 ): 393 - 409 . 20569814 DOI: 10.1016/j.clp.2009.12.003 http://doi.org/10.1016/j.clp.2009.12.003 . Ureaplasma species: role in diseases of prematurity Y Wang , SL Liu , Y Li , 等 . Mycoplasma bovis -derived lipid-associated membrane proteins activate IL-1β production through the NF-κB pathway via Toll-like receptor 2 and MyD88 . Dev Comp Immunol , 2016 . 55 111 - 118 . 26499291 DOI: 10.1016/j.dci.2015.10.017 http://doi.org/10.1016/j.dci.2015.10.017 . Mycoplasma bovis-derived lipid-associated membrane proteins activate IL-1β production through the NF-κB pathway via Toll-like receptor 2 and MyD88 KD Wenstrom , WW Andrews , T Tamura , 等 . Elevated amniotic fluid interleukin-6 levels at genetic amniocentesis predict subsequent pregnancy loss . Am J Obstet Gynecol , 1996 . 175 ( 4 ): 830 - 833 . 8885730 DOI: 10.1016/S0002-9378(96)80007-X http://doi.org/10.1016/S0002-9378(96)80007-X . Elevated amniotic fluid interleukin-6 levels at genetic amniocentesis predict subsequent pregnancy loss Compliance with ethics guidelines Guang-yong YE, Ke-yi WANG, Qiao-di GUI, and Min WANG declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. 10.1631/jzus.B1800005 Project supported by the Zhejiang Provincial Natural Science Foundation of China (Nos. LY18H040001 and LY16H040003), the Department of Education of Zhejiang Province (No. Y201534723), and the Science and Technology Development Project in Hangzhou (No. 20160533B13), China 2018-01-04 2018-02-09 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2018 2018 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 19