Differential cytokine and metabolite production by cervicovaginal epithelial cells infected with Lactobacillus crispatus and Ureaplasma urealyticum.

INTRODUCTION: We sought to quantify targeted metabolites (d-lactate, pyruvate, urea, ammonia) and the cytokine IL-8 produced by human cervicovaginal epithelial cells co-cultured with Ureaplasma urealyticum (a preterm birth-associated bacterium) or Lactobacillus crispatus (a healthy vaginal commensal associated with term birth). METHODS: Concentrations of d-lactate, pyruvate, urea and ammonia measured by enzyme-based spectrophotometry and IL-8 by ELISA were determined and compared between monolayer-cultured HeLa cells (ATCC 35241) infected with strains of U. urealyticum (ATCC 27618, 0.5 mL = 3640 CFU/mL, U. urealyticum) or L. crispatus (ATCC 33820, MOI = 10,000, 1000 and 100, L. crispatus) and incubated in 5% CO2 at 37 °C for 24 h. Uninfected HeLa cells (Hc) were used as controls and cytotoxicity was determined by the amount (optical density) of lactate dehydrogenase (LDH) released by the dead HeLa cells. RESULTS: The amount of LDH released by untreated Hc (P = 0.002) and U. urealyticum-infected cells (P < 0.0001) was higher than those of L. crispatus-infected cells, with U. urealyticum-infected cells recording the highest % cytotoxicity and L. crispatus-infected cells MOI 10,000 (Lc10,000) the least (P < 0.0001). Though there was no significant difference in the concentration of urea between the samples, U. urealyticum-infected cells showed higher ammonia compared to other samples (p = 0.03). In contrast, all L. crispatus samples had higher d-lactate than untreated Hc (p = 0.01) and U. urealyticum-infected cells (P = 0.01). Also, Lc10,000 had the highest d-lactate (p = 0.001) and lowest pyruvate (P = 0.04, excluding UU) compared to other samples. Furthermore, U. urealyticum-infected cells produced the highest IL-8 (P = 0.01) compared to other samples, with Lc10,000 producing undetectable levels. CONCLUSION: Infection of cervicovaginal epithelial cells by U. urealyticum stimulates production of ammonia from urea and induces elevated IL-8 production possibly leading to significantly higher cytotoxicity. In contrast, L. crispatus appeared protective against HeLa cell inflammation and death, producing more d-lactate and less IL-8, consistent with a role for L. crispatus in promoting vaginal floral health and reducing infection/inflammation-associated preterm birth.

Synergic activation of toll-like receptor (TLR) 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells.

Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), preterm labour (PL) pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to "sense" pathogens. One such family of PRRs are the Toll like receptor family (TLR). In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK) transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA), trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly.

Role of biofilm formation in Ureaplasma antibiotic susceptibility and development of bronchopulmonary dysplasia in preterm neonates.

BACKGROUND: Ureaplasma respiratory tract colonization is a risk factor for bronchopulmonary dysplasia (BPD) in preterm infants, but whether Ureaplasma isolates from colonized infants can form biofilms is unknown. We hypothesized that Ureaplasma isolates vary in capacity to form biofilms that contribute to their antibiotic resistance and ability to evade host immune responses. Study objectives were to (1) determine the ability of Ureaplasma isolates from preterm neonates to form biofilms in vitro; (2) compare the susceptibility of the sessile and planktonic organisms to azithromycin (AZI) and erythromycin; and (3) determine the relationship of biofilm-forming capacity in Ureaplasma isolates and the risk for BPD. METHODS: Forty-three clinical isolates from preterm neonates and 5 American Tissue Culture Collection strains were characterized for their capacity to form biofilms in vitro, and antibiotic susceptibility was performed on each isolate prebiofilm and postbiofilm formation. RESULTS: Forty-one (95%) clinical and 4 of 5 (80%) American Tissue Culture Collection isolates formed biofilms. All isolates were more susceptible to AZI (minimum inhibitory concentration, MIC50 2 µg/mL) than erythromycin (MIC50 4 µg/mL), and biofilm formation did not significantly affect antibiotic susceptibility for the 2 tested antibiotics. The MIC50 and minimum biofilm inhibitory concentrations (MBIC50) for Ureaplasma urealyticum clinical isolates for AZI were higher than for MIC50 and MBIC50 for Ureaplasma parvum isolates. There were no differences in MIC or MBICs among isolates from BPD infants and non-BPD infants. CONCLUSIONS: Capacity to form biofilms is common among Ureaplasma spp. isolates, but biofilm formation did not impact MICs for AZI or erythromycin.

Amniotic fluid interleukin-1 beta and interleukin-6, but not interleukin-8 correlate with microbial invasion of the amniotic cavity in preterm labor.

PROBLEM: We compared the frequency of intra-amniotic infection in preterm labor (PL) with women not in labor, and correlated infection with amniotic fluid (AF) cytokines. Detailed identification of species, especially mycoplasmata, was tried to improve our understanding of the pathogenesis of PL. METHOD OF STUDY: AF from 20 women with PL and 20 controls were evaluated. Infection was detected by PCR for Mycoplasma hominis, Ureaplasma urealyticum and 16S rRNA bacterial gene, which was cloned and sequenced for bacterial identification. Interleukin (IL)-1ß, IL-6, IL-8 and tumor necrosis factor (TNF)-α levels were measured by ELISA. RESULTS: Frequency of intra-amniotic infection is higher in PL (40.0%). Sequencing-based method identified Bacteroides fragilis, Prevotella bivia and Leptotrichia amnionii, in addition to Mycoplasma species detected by PCR. AF infection correlated with increased IL-1ß and IL-6 levels. CONCLUSION: The frequency of intra-amniotic infection, especially M. hominis, in PL women who delivered with 7 days, is high and correlates with high IL-1ß and IL-6 levels, but not IL-8.

Interaction between pathogenic bacteria and intrauterine leukocytes triggers alternative molecular signaling cascades leading to labor in women.

Increased risk of preterm labor has been linked to cervicovaginal infection with Ureaplasma urealyticum and group B streptococci. Although various experimental models have been developed to study the role of amniochorion infection in preterm labor, they typically exclude the initial interaction between intrauterine leukocytes (recruited from decidual vessels into the avascular fetal membranes) and infecting bacteria. In this work, we ascertained whether inflammatory molecules secreted by bacterium-activated intrauterine leukocytes stimulate the amniochorion production of mediators involved in human labor. Using a two-step process beginning with placental circulating leukocytes as a proxy for intrauterine leukocytes, we found that coincubation of amniochorion explants with plasma from placental whole blood preincubated with group B streptococci resulted in a significant increase in tumor necrosis factor alpha (TNF-α) and matrix metalloproteinase 9 (MMP-9) levels in tissue. Extensive changes in the connective tissue arrangement and a decrease in collagen content demonstrated the degradation of the extracellular matrix following this treatment. In contrast, plasma from blood preconditioned with U. urealyticum induced a highly significant secretion of interleukin-1ß (IL-1ß) and prostaglandin E(2) (PGE(2)) by the amniochorion without changes in the extracellular matrix organization or content. These data demonstrate that group B streptococci induce degradation of the amniochorion as a result of MMP-9 production, probably via TNF-α, whereas U. urealyticum stimulates the secretion of PGE(2), probably via IL-1ß, potentially stimulating myometrial contraction. Our study provides novel evidence that the immunological cells circulating within the uterine microenvironment respond differentially to an infectious agent, triggering alternative molecular signaling pathways leading to human labor.

Expression of IL-1alpha, IL-6, TGF-beta, FasL and ZNF265 during sertoli cell infection by ureaplasma urealyticum.

To investigate immunoregulatory mechanisms of Sertoli cells in the testis in vitro and in vivo, we utilized our well-characterized Ureaplasma Urealyticum (UU)-induced model. We investigated the expressions of IL-1alpha, IL-6, TGF-beta, FasL and ZNF265 at the first, second and third weeks post-infection. During recovery from inflammation and with the help of negative regulators TGF-beta and FasL, the high levels of IL-1alpha and IL-6 expressions were observed in the early stages of the infection, and decreased gradually in the later weeks both in vitro and in vivo. The trend of varied expression of ZNF265 was similar to those of TGF-beta and FasL in vitro and in vivo for Sertoli cells infected with UU.

[Expression of inducible nitric oxide synthase was regulated by activating nuclear factor kappaB in mouse macrophages stimulated with ureaplasma urealyticum lipid-associated membrane proteins].

The aim was to investigate the molecular mechanisms responsible for the inducible nitric oxide synthase (iNOS) gene expression stimulated by lipid associated membrane proteins (LAMPs) of Ureaplasma urealyticum (Uu). Mouse macrophages were stimulated by Ureaplasma urealyticum LAMPs to analyze the production of nitric oxide (NO) and the expression of iNOS detected by RT-PCR and Western blot. The activation of NF-kappaB was examined in mouse macrophages treated with LAMPs by indirect immunofluorescence (IFA), immunocytochemistry and Western blot. The effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB and of cycloheximide (CHX), a protein synthase inhibitor, on the expression of iNOS and on the activation of NF-kappaB were also investigated in mouse macrophages treated with LAMPs. Results showed Ureaplasma urealyticum LAMPs stimulated mouse macrophages to express iNOS and thus produce NO in dose- and time-dependent manners by activating nuclear factor kappaB. The activation of NF-kappaB and the expression of iNOS were inhibited by LAMPs combination with PDTC or CHX. In conclusion, these findings suggested Ureaplasma urealyticum may be an important pathogenic factor due to the ability of LAMPs to stimulate the expression of iNOS, which is probably medicated by the activation of NF-kappaB.

Induction of human macrophage vascular endothelial growth factor and intercellular adhesion molecule-1 by Ureaplasma urealyticum and downregulation by steroids.

Chronic lung disease (CLD) remains a major cause of morbidity for the prematurely born infant. The pathogenesis of CLD is complex and has not been defined entirely. Infection and lung inflammatory events have been thought to play a key role in the development of CLD. However, the contribution of Ureaplasma urealyticum to the development of CLD is debated and steroids produce some improvement in neonates with this disease. The aim of this study was to investigate if U. urealyticum could stimulate macrophages to produce vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) in vitro, which are potentially associated with both early and later pathological changes in the lung during the development of CLD. In addition, the impact of dexamethasone and budesonide on these processes was examined. We found that U. urealyticum antigen (>/=4 x 10(7) color-changing units/ml) stimulated human macrophages (phorbol 12-myristate 13-acetate-differentiated THP-1 cell line) to produce VEGF and soluble ICAM-1 in a dose-dependent manner (p < 0.05) measured by ELISA. Likewise, cell surface ICAM-1 (CD54) measured by flow cytometry was increased after stimulation with U. urealyticum. This effect was attenuated by budesonide and dexamethasone (p < 0.05). The mRNA expressions of VEGF and ICAM-1 detected by a semi-quantitative reverse transcriptase polymerase chain reaction were also induced in response to U. urealyticum and inhibited by the steroids (p < 0.05). The expression of ICAM-1 was reduced by 85.5% when the TNF-alpha production was neutralized with an anti-TNF-alpha antibody. Our findings imply that U. urealyticum might be involved in the development of CLD of prematurity.

Activation of nuclear factor kappaB and induction of inducible nitric oxide synthase by Ureaplasma urealyticum in macrophages.

Chronic lung disease (CLD) of prematurity is an inflammatory disease with a multifactorial etiology. The importance of Ureaplasma urealyticum in the development of CLD is debated, and steroids produce some improvement in neonates with this disease. In the present study, the capability of U. urealyticum to stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS), and activate nuclear factor kappaB (NF-kappaB) in vitro was characterized. The effect of NO on the growth of U. urealyticum was also investigated. In addition, the impact of dexamethasone and budesonide on these processes was examined. We found that U. urealyticum antigen (> or =4 x 10(7) color-changing units/ml) stimulated alveolar macrophages to produce NO in a dose- and time-dependent manner (P<0.05). This effect was further enhanced by gamma interferon (100 IU/ml; P<0.05) but was attenuated by budesonide and dexamethasone (10(-4) to 10(-6) M) (P<0.05). The mRNA and protein levels of iNOS were also induced in response to U. urealyticum and inhibited by steroids. U. urealyticum antigen triggered NF-kappaB activation, a possible mechanism for the induced iNOS expression, which also was inhibited by steroids. NO induced by U. urealyticum caused a sixfold reduction of its own growth after infection for 10 h. Our findings imply that U. urealyticum may be an important factor in the development of CLD. The host defense response against U. urealyticum infection may also be influenced by NO. The down-regulatory effect of steroids on NF-kappaB activation, iNOS expression, and NO production might partly explain the beneficial effect of steroids in neonates with CLD.

Ureaplasma urealyticum-induced production of proinflammatory cytokines by macrophages.

Ureaplasma urealyticum is relatively common in the respiratory tract of very low birth weight infants and has been hypothesized to be involved in the development of chronic lung disease. The purpose of this study was to investigate whether U. urealyticum could stimulate macrophages to produce proinflammatory cytokines in vitro, which are early pathologic changes in the lung during the development of chronic lung disease. A human monocytic cell line (THP-1) differentiated to macrophages, a rat alveolar macrophage cell line (Nr8383), and human lung macrophages from tracheobronchial aspirate fluid in preterm infants were exposed to U. urealyticum antigen for 24 h. The protein levels of human IL-6, tumor necrosis factor-alpha (TNF-alpha), and rat TNF-alpha were measured with ELISA. Rat IL-6 was analyzed with a specific bioassay. The mRNA levels of these cytokines were detected by reverse transcriptase-PCR. The production of TNF-alpha and IL-6 increased after stimulation with U. urealyticum in both the human and rat macrophage cell lines. In tracheobronchial aspirate fluid macrophages, U. urealyticum increased the production of TNF-alpha from 14 to 84% and IL-6 from 46 to 268% above control levels. U. urealyticum also induced gene expression of TNF-alpha and IL-6. In conclusion, U. urealyticum could be an important factor in the development of chronic lung disease because of its ability to induce alveolar macrophage proinflammatory cytokine production.