Diversity in cytokine response to bacteria associated with preterm birth by fetal membranes

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Diversity in cytokine response to bacteria associated with preterm birth by fetal membranes Ramkumar Menon, PhD; Morgan R. Peltier, PhD; Judith Eckardt, MD; Stephen J. Fortunato, MD OBJECTIVE: This study compared cytokine and prostaglandin (PG) re-

RESULTS: LPS and E coli increased all cytokine and PG productions

sponses by fetal membranes stimulated with 4 different bacterial species associated with preterm birth (PTB).

compared with controls. Cytokine profiles were similar after G vaginalis and GBS stimulation. G vaginalis increased PGE2, whereas GBS increased PGF2␣. U parvum demonstrated the mildest response with only IL-10 and TNF-␣ concentrations being higher with no detectible effect on PGs.

STUDY DESIGN: Fetal membranes (n ⫽ 13 from normal term cesarean sections [not in labor]) in an organ explant system were stimulated with heat-killed Ureaplasma parvum, Gardanerella vaginalis, Escherichia coli, group B Streptococcus (GBS), or lipopolysaccharide (LPS). Cytokines (interleukin [IL]-1␤, IL-6, IL-8, IL-10, tumor necrosis factor [TNF]-␣, and interferon-␥) and PG (PGF2␣ and PGE2) concentrations were quantitated and compared.

CONCLUSION: Fetal membrane cytokine signatures of 4 different bac-

teria associated with PTB are distinct, suggesting that infection as a potential cause of PTB is not homogeneous in its presentation. Key words: amniochorion, inflammation, intraamniotic infection, preterm birth, prostaglandins

Cite this article as: Menon R, Peltier MR, Eckardt J, et al. Diversity in cytokine response to bacteria associated with preterm birth by fetal membranes. Am J Obstet Gynecol 2009;201:306.e1-6.

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pontaneous preterm birth (PTB; birth before 37 weeks’ gestation) is a major complication of pregnancy, and infection is associated with approximately 50% of cases.1-3 These infections are often asymptomatic and consist of bacteria that have ascended from the vagina through the cervix to colonize the tissues at the maternal-fetal interface 4-6 Microbial factors are thought to evoke a series of events that compromise the immunologic privileges that the fetus enjoys from conception until labor by stimulating the production of proinflammatory cytokines and chemokines. Cytokines and chemokines, in turn, upregulate the downstream effectors of specific clinical events of labor. For ex-

ample, prostaglandins (PGs) stimulate uterine contractions and cervical dilation and matrix metalloproteinases degrade extracellular matrix in fetal membranes, cervix, and placenta 7-11 Many preterm labors are resistant to tocolytics.11 This suggests that the biomolecular pathways of preterm labor resulting in PTB are complex and that patient-specific interventions targeted to a particular molecular mechanism of preterm birth may be necessary. Bacteria interact with host cells through toll-like receptors (TLRs) that can produce different but overlapping arrays of cytokines upon recognition of conserved biochemical motifs on various species of bacteria. For example, TLR-4 recognizes

From the Perinatal Research Center, Centennial Women’s Hospital (Drs Menon, Eckardt, and Fortunato), and Maternal-Fetal Group PediatriX (Dr Fortunato), Nashville, TN; the Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA, and the Department of Obstetrics and Gynecology and Reproductive Medicine, Yale University School of Medicine, New Haven, CT (Dr Menon); and the Departments of Obstetrics and Gynecology and Pediatrics, Winthrop University Hospital, Mineola, NY (Dr Peltier). Presented at the 29th Annual Meeting of the Society for Maternal-Fetal Medicine, San Diego, CA, Jan. 26-31, 2009. Received Feb. 18, 2009; revised May 11, 2009; accepted June 11, 2009. Reprints: Ramkumar Menon, PhD, Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA 30322. [email protected]/[email protected]. This study was supported in part by the Maternal-Fetal Group, Nashville, TN. 0002-9378/$36.00 • © 2009 Mosby, Inc. All rights reserved. • doi: 10.1016/j.ajog.2009.06.027

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the lipid A component of lipopolysaccharide from Gram-negative species; TLR-2 also recognizes peptidoglycan from Gram-positive bacteria and lipoproteins from different species of Mycoplasma.12,13 Previously we reported different in vitro inflammatory cytokine responses to lipopolysaccharide (LPS) from Gramnegative bacteria and peptidoglycan polysaccharide from Gram-positive bacteria by fetal membranes, suggesting that initiation of labor may depend on the type of immune response to the pathogen present.14 Other tissue and bacterial factor–specific cytokine response have also been reported.15-17 Not all cytokines, however, seem to activate the downstream mediators that cause labor and PTB.18,19 Nonhuman primate studies have shown the induction of preterm labor by interleukin (IL)-1␤ and tumor necrosis factor (TNF)-␣ but not IL-6 or IL-8.20 Therefore, we hypothesized that the immune response by fetal membranes to bacteria associated with preterm birth differs between specific organisms. We tested this hypothesis using an in vitro system in which clinically normal membranes were stimulated with heat-inactivated suspensions of Escherichia coli, Streptococcus agalactiae (group B Streptococcus [GBS]), Ureaplasma parvum,

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www.AJOG.org and Gardanerella vaginalis or an equivalent volume of culture medium and quantified the production of an array of proinflammatory cytokines and PGs associated with preterm birth.

M ATERIALS AND M ETHODS This protocol was approved by Tristar Nashville Institutional Review Board, and informed consent was obtained from all patients donating their tissues. The membrane explant culture system used for this study is standard for our laboratory. The validation and details of methods are reported elsewhere17,19-21 but are summarized here for the reader’s convenience.

Subjects and collection of fetal membranes Placental tissues were obtained from elective repeat cesarean sections at term (gestation of ⱖ37 weeks) without the onset of labor (n ⫽ 13) (mean gestational age, 39.5 weeks). All subjects included in this study were whites with singleton pregnancies. Maternal ages were between 22 and 34 years. Fetal membranes were dissected from placenta under sterile conditions. The membranes were cleaned from decidua and adhering blot clots using saline and sterile cotton gauze. These tissues were then placed in Hanks’ balanced salt solution (HBSS; Sigma Chemical Co., St. Louis, MO) containing penicillin/streptomycin (10 ␮L/mL) and 1 ␮L/mL amphotericin B (Sigma). Tissue culture of normal term fetal membranes Fetal membranes were washed twice with HBSS, and 6 mm circles were isolated using a biopsy punch. Tissue biopsies were washed with HBSS and placed in a Falcon cell culture insert (BectonDickinson Labware, Franklin Lakes, NJ) containing 200 ␮L Dulbecco’s modified Eagle’s medium–F12 Ham’s mixture supplemented with 15% heat-inactivated fetal bovine serum, 1% glutamine, 1% penicillin/streptomycin, and 1 ␮L/mL amphotericin B (culture medium). These inserts were placed in a Falcon cell culture plate with 500 ␮L culture medium. Cultures were incubated

at 37°C, 5% CO2 for 48 hours. Culture medium was changed every 24 hours.

Bacterial isolates Bacterial stocks used for this experiment were low-passage strains of clinical isolates that were purchased from the American Type Tissue Culture Collection (Manassas, VA). Bacteria were cultivated in nutrient broth (E coli; American Type Tissue Culture Collection #33908), New York City broth (G vaginalis; American Type Tissue Culture Collection #49145), brain heart infusion broth (S agalactiae [GBS]; American Type Tissue Culture Collection #BAA25), or heart infusion broth supplemented with 20% horse serum, 10% yeast extract, 2 g/L urea, 20 mg/L phenol red, and 1000 U/mL penicillin (U parvum, formerly classified as U urealyticum serotype 1; American Type Tissue Culture Collection #27813). Bacteria were harvested by centrifugation at 10,000 ⫻ g for 10 minutes, resuspended in culture media (RPMI 1640), and quantified by estimation of colonyforming units (CFU) or, for U parvum, color changing units (CCU, defined as the lowest 10-fold serial dilution to cause a color change in the broth). Organisms were then heat killed by heating to 80oC for 1 hour and stored at –70oC until use in membrane explant cultures. Stimulation of fetal membranes with LPS and heat-inactivated bacteria Membranes were maintained in tissue culture environment for a total of 72 hours. The first 48 hours consisted of a stabilization period during which the explants were cultured without any bacterial stimulation with a medium change after the first 24 hours of culture. Bacteria suspensions and LPS prepared in culture medium were then added to final concentrations of 107 CFU/CCU or 100 ng/mL, respectively. Additional control cultures received an equivalent volume of vehicle. Cultures were then returned to the humidified incubator and stimulated for 24 hours. Conditioned medium was harvested and stored at –20°C until analysis.

Multiplex analysis for cytokines A Luminex-based immunoassay was performed for a panel of proinflammatory cytokines frequently associated with preterm birth (IL-1␤, IL-6, IL-8, IL-10, TNF-␣ and interferon [IFN]-␥) as directed by the manufacturer. All samples and standards were assayed in duplicate. Samples that were above the standard curve were diluted with the manufacturer’s assay buffer and reassayed. Sensitivity of the assays was 1 pg/mL for IFN-␥, TNF-␣, and IL-10; 2 pg/mL for IL-1␤; 3 pg/mL for IL-6, and 15 pg/mL for IL-8. PGF2␣ and PGE2 measurements PGF2␣ and PGE2 were quantified in culture media using commercially available reagents as directed by the manufacturer (Biosource, Invitrogen, Carlsbad, CA). All samples were assayed in duplicate and the detection limits were 37.0 and 125 pg/mL for PGF2␣ and PGE2, respectively. Statistical analysis of data Because data were not normally distributed, nonparametric Kruskal-Wallis tests were performed to compare the differences between the stimulated groups and the unstimulated control group. All values are presented as medians and ranges. A P ⬍ .05 was considered significant.

R ESULTS A total of 13 different fetal membrane cultures were used for this study and assays were performed in duplicate. Cytokines and PGs were measurable in all experiments except IL-1␤ that was below the detection limits of the assay in U parvum-stimulated cultures. LPS was used to confirm that all of the fetal membranes used for this study (n ⫽ 13 for all treatments) were responsive to bacterial stimulation after 48 hours in culture. As expected, LPS stimulation significantly increased cytokine and PG concentrations compared with unstimulated cultures. Median concentrations of IL-1␤, TNF-␣, and IL-10 were severalfold higher in LPS-stimulated cultures than controls (Figure 1) with smaller increases observed for IL-8, IL-6, and IFN-␥. Both PG concentrations reached

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C OMMENT Intraamniotic infection and inflammation is 1 of the most common etiologic factors associated with preterm birth. Bacteria are thought to stimulate a host response that leads to increased production of PG and ultimately preterm delivery. Although animal models and detailed epidemiologic studies have demonstrated an independent and causal relationship between several pathogens and preterm birth, most cases are polymicrobial, and the individual contributions of these pathogens to the inflammatory environment is uncertain.1,22-25 In this study, we found that 306.e3

FIGURE 1

Concentration of cytokines 3000

P< 0.01

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highly significant levels after LPS stimulation. PGE2 and PGF2␣ were also significantly increased by stimulation with LPS (900.89 and 1982.28 pg/mL vs 337.59 and 728.25 pg/mL in controls, respectively; Figure 2). E coli stimulation mimicked that of LPS stimulation except that TNF-␣ was almost 2-fold higher after LPS than E coli whereas PGE2 was about 2-fold higher after E coli stimulation compared with LPS. IFN-␥ concentration was higher only in E coli-stimulated fetal membranes among bacterial stimulants (Figures 1 and 2). G vaginalis and GBS-stimulated tissues had similar cytokine responses. IL1␤, TNF-␣, IL-6, and IL-10 were significantly elevated compared with controls (Figure 1). No differences in the concentrations of IFN-␥ and IL-8 were seen after stimulation with either of these bacteria. The PG profile was different between these species; however, G vaginalis stimulation increased PGE2 production, whereas GBS increased PGF2␣ compared with controls (Figure 2). Cytokine concentrations in U parvum-stimulated fetal membranes were significantly increased for TNF-␣ and IL-10 (Figures 1 and 2) but not other cytokines. IL-1␤ was not detectible in most of the stimulated samples. Although median PGE2 concentrations were almost doubled after stimulation, the difference did not reach statistical significance. No effect of U parvum on PGE2␣ production was detected.

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300

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Concentration of cytokines (IL-1␤, IFN-␥, IL-8, TNF-␣, IL-6, and IL-10) in medium from cultured fetal membranes stimulated with heat-inactivated bacteria. Shown are medians with interquartile ranges and whiskers overlaid with individual patient values. Menon. Cytokine response to bacteria and preterm birth. Am J Obstet Gynecol 2009.

the host response of amniotic membranes varied between 4 pathogens commonly associated with preterm birth. E coli stimulated the most robust proinflammatory response causing the highest concentrations of both PGs and cytokines. In contrast, U parvum, a much more frequently isolated pathogen in preterm birth cases, was the least proinflammatory. G vaginalis, a bacterial isolate associated with bacterial vaginosis, and GBS, a common pathogen associated with chorioamnionitis and neonatal sepsis, showed a similar, intermediate cytokine footprint. E coli stimulation resulted in very high concentrations of IL1␤, IL-10, and TNF-␣ in conditioned medium, whereas these cytokines showed

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only a moderate increase with G vaginalis and GBS stimulation. This supports our earlier report in which similar cytokine production pattern was seen with LPS and PGPS, suggesting that inflammation is mediated mainly by cell wall components of these bacteria.14 Differential responses in PG production by G vaginalis (which increased PGF2␣) and GBS (which increased PGE2) despite similar cytokine profiles suggest that additional factors by these organisms or involvement of other biochemical factors may regulate PGs. In a clinical setting in which most infections are polymicrobial, it is possible that different bacterial species ini-

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FIGURE 2

Concentration of PGF2␣ and PGE2 4000

PGE2 (pg/mL)

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Concentration of PGF2␣ and PGE2 in media from cultured fetal membranes stimulated with heat-inactivated bacteria. Shown are medians with interquartile ranges and whiskers overlaid with individual patient values. Menon. Cytokine response to bacteria and preterm birth. Am J Obstet Gynecol 2009.

tiate different pathways and act synergistically to induce preterm birth. The pathways are likely dependent on a number of organisms for certain bacterial species, relative virulence of the strain, and robustness of the host response for a given patient (eg, cytokines, PGs) as seen in this study.

Ureaplasma is 1 of the most common amniotic fluid isolates in symptomatic and asymptomatic preterm births.25-29 Testing its proinflammatory properties on fetal membranes documented the least cytokine response in which only TNF-␣ and IL-10 were higher compared with controls. These data are supported

in recent reports in which similar cytokine footprinting was reported in in vitro choriodecidual models.30 PGE2 concentrations were higher from choriodecidual tissues; however, in amniochorion, in the absence of decidua, we did not see a change in PGE2 concentration. Similarly, other reports documented immune suppression in Ureaplasma colonizers and lower immune response in midtrimester amniotic fluid in subjects who were positive for Ureaplasma.31,32 The inability for Ureaplasma to stimulate PGE2 production by membrane explants in our study is similar to a previous report that also detected no effect of Ureaplasma on PGE2 production cultured amniotic cells.33 Another study, however, reported that low concentrations of Ureaplasma-stimulated PGE2 production but higher concentrations of bacteria inhibited PGE2 production by amniotic cells.34 The reasons for this discrepancy are unclear. It is possible that the concentrations of Ureaplasma used in our study were bioactive enough to be on the high end of this dose-response curve. Alternatively, amniochorion responds less efficiently to Ureaplasma than purified amniotic cells. Although the immune response to Ureaplasma the least proinflammatory of the organisms tested, previous clinical studies have reported a clear correlation with amniotic fluid inflammatory response, Ureaplasma isolation, and pregnancy outcome.25,35 One possibility is that other fetal and maternal cell types contribute to the immune response to Ureaplasma in vivo than are represented by our culture system. Alternatively, other bacterial species may be present in the amniotic fluid samples that test positive for Ureaplasma (some of which are uncultivable) and can stimulate cytokine responses by the fetal membranes. Recent reports documented presence of deoxyribonucleic acid from a greater diversity of microbes than previously suspected in the amniotic cavity of women in preterm labor including asyet uncultivated and previously uncharacterized.36 The ability for live organisms to form biofilms that can not

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SMFM Papers be represented by in vitro experiments with heat-killed bacteria may also contribute to the greater inflammatory response observed in clinical cases.37-40 In summary, we found that the inflammatory cytokine response associated with intrauterine infection is not generalizable and the pathways leading to PTB may be different in subjects based on pathogens present. Therefore, a better understanding of the molecular mechanisms of preterm labor based on the microbial diversity of the amniotic fluid may be necessary for developing better methods to diagnose and ultimately treat or prevent the infections that can cause preterm f birth. REFERENCES 1. Gonçalves LF, Chaiworapongsa T, Romero R. Intrauterine infection and prematurity. Ment Retard Dev Disabil Res Rev 2002;8:3-13. 2. Romero R, Espinoza J, Chaiworapongsa T, Kalache K. Infection and prematurity and the role of preventive strategies. Semin Neonatol 2002;7:259-74. 3. Goldenberg RL, Hauth JC, Andrews WW. Intrauterine infection and preterm delivery. N Engl J Med 2000;342:1500-7. 4. Goldenberg RL, Culhane JF. Infection as a cause of preterm birth. Clin Perinatol 2003; 30:677-700. 5. Casey ML, MacDonald PC. Biomolecular processes in the initiation of parturition: decidual activation. Clin Obstet Gynecol 1988; 31:533-52. 6. Bejar R, Curbelo V, Davis C, Gluck L. Premature labor. II. Bacterial sources of phospholipase. Obstet Gynecol 1981;57:479-82. 7. Bowen JM, Chamley L, Keelan JA, Mitchell MD. Cytokines of the placenta and extra-placental membranes: roles and regulation during human pregnancy and parturition. Placenta 2002;23:257-73. 8. Mohan AR, Loudon JA, Bennett PR. Molecular and biochemical mechanisms of preterm labour. Semin Fetal Neonatal Med 2004; 9:437-44. 9. Gibb W, Challis JR. Mechanisms of term and preterm birth. J Obstet Gynaecol Can 2002;24:855-60. 10. Olson DM, Ammann C. Role of the prostaglandins in labour and prostaglandin receptor inhibitors in the prevention of preterm labour. Front Biosci 2007;12:1329-43. 11. Menon R. Spontaneous preterm birth, a clinical dilemma: etiologic, pathophysiologic and genetic heterogeneities and racial disparity. Acta Obstet Gynecol Scand 2008;87:590-600. 12. Hirschfeld M, Weis JJ, Toshchakov V, et al. Signaling by toll-like receptor 2 and 4 agonists

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24. Pararas MV, Skevaki CL, Kafetzis DA. Preterm birth due to maternal infection: causative pathogens and modes of prevention. Eur J Clin Microbiol Infect Dis 2006;25:562-9. 25. Jacobsson B, Mattsby-Baltzer I, Hagberg H. Interleukin-6 and interleukin-8 in cervical and amniotic fluid: relationship to microbial invasion of the chorioamniotic membranes. BJOG 2005;112:719-24. 26. Witt A, Berger A, Gruber CJ, et al. Increased intrauterine frequency of Ureaplasma urealyticum in women with preterm labor and preterm premature rupture of the membranes and subsequent cesarean delivery. Am J Obstet Gynecol 2005;193:1663-9. 27. Cassell GH, Waites KB, Watson HL, Crouse DT, Harasawa R. Ureaplasma urealyticum intrauterine infection: role in prematurity and disease in newborns. Clin Microbiol Rev 1993;6:69-87. 28. Watts DH, Krohn MA, Hillier SI, Eschenbach DA. The association of amniotic fluid infection with gestational age and neonatal outcome among women in preterm labor. Obstet Gynecol 1992;79:351-7. 29. Yoon BH, Romero R, Lim JH, et al. The clinical significance of detecting Ureaplasma urealyticum by the polymerase chain reaction in the amniotic fluid of patients with preterm labor. Am J Obstet Gynecol 2003;189:919-24. 30. Aaltonen R, Heikkinen J, Vahlberg T, Jensen JS, Alanen A. Local inflammatory response in choriodecidua induced by Ureaplasma urealyticum. BJOG 2007;114:1432-5. 31. Doh K, Barton PT, Korneeva I, et al. Differential vaginal expression of interleukin-1 system cytokines in the presence of Mycoplasma hominis and Ureaplasma urealyticum in pregnant women. Infect Dis Obstet Gynecol 2004; 12:79-85. 32. Perni SC, Vardhana S, Korneeva I, et al. Mycoplasma hominis and Ureaplasma urealyticum in midtrimester amniotic fluid: association with amniotic fluid cytokine levels and pregnancy outcome. Am J Obstet Gynecol 2004;191:1382-6. 33. Lamont RF, Anthony F, Myatt L, Booth L, Furr PM, Taylor-Robinson D. Production of prostaglandin E2 by human amnion in vitro in response to addition of media conditioned by microorganisms associated with chorioamnionitis and preterm labor. Am J Obstet Gynecol 1990;162: 819-25. 34. Mitchell MD, Romero RJ, Avila C, Foster JT, Edwin SS. Prostaglandin production by amnion and decidual cells in response to bacterial products. Prostaglandins Leukot Essent Fatty Acids 1991;42:167-9. 35. Yoon BH, Romero R, Park JS, et al. Microbial invasion of the amniotic cavity with Ureaplasma urealyticum is associated with a robust host response in fetal, amniotic, and maternal compartments. Am J Obstet Gynecol 1998; 179:1254-60. 36. DiGiulio DB, Romero R, Amogan HP, et al Microbial prevalence, diversity and abundance in amniotic fluid during preterm labor: a molec-

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38. Espinoza J, Gonçalves LF, Romero R, et al. The prevalence and clinical significance of amniotic fluid “sludge” in patients with preterm labor and intact membranes. Ultrasound Obstet Gynecol 2005;25:346-52. 39. Romero R, Schaudinn C, Kusanovic JP, et al. Detection of a microbial biofilm in intraamni-

otic infection. Am J Obstet Gynecol 2008;198: 135.e1-5. 40. Romero R, Kusanovic JP, Espinoza J, et al. What is amniotic fluid “sludge”? Ultrasound Obstet Gynecol 2007;30:793-8.

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