Abstract
Although accepted agents in chorioamnionitis and preterm birth, the role of Ureaplasma species (spp.) in inflammation-driven morbidities of prematurity, including the development of bronchopulmonary dysplasia, remains controversial. To add to scarce in vitro data addressing the pro-inflammatory capacity of Ureaplasma spp., pulmonary epithelial-like A549 cells and human pulmonary microvascular endothelial cells (HPMEC) were incubated with Ureaplasma (U.) urealyticum, U. parvum, and Escherichia coli lipopolysaccharide (LPS). Ureaplasma isolates down-regulated caspase mRNA levels in A549 cells (caspase 8: p<0.001, 9: p<0.001, vs. broth), while increasing caspase protein expression, enzyme activity, and cell death in HPMEC (active caspase 3: p<0.05, caspase 8: p<0.05, active caspase 9: p<0.05, viability: p<0.05). LPS, contrarily, induced caspase mRNA expression in HPMEC (caspase 3: p<0.01, 4: p<0.001, 5: p<0.001, 8: p<0.001, vs. control), but not in A549 cells, and did not affect enzyme activity or protein levels in either cell line. LPS, but neither Ureaplasma isolate, enhanced mRNA expression of pro-inflammatory interleukin (IL)-6 in both A549 (p<0.05, vs. control) and HPMEC (p<0.001) as well as tumor necrosis factor-α (p<0.01), IL-1β (p<0.001), and IL-8 (p<0.05) in HPMEC. We are therefore the first to demonstrate a differential modulation of pulmonary caspases by Ureaplasma spp. in vitro. Ureaplasma-driven enhanced protein expression and activity of caspases in pulmonary endothelial cells result in cell death and may cause structural damage. Down-regulated caspase mRNA in pulmonary epithelial cells, contrarily, may indicate Ureaplasma-induced inhibition of apoptosis and prevent effective immune responses. Both may ultimately contribute to chronic Ureaplasma colonization and long-term pulmonary inflammation.
Highlights
MethodsUreaplasma (U.) urealyticum serovar 8 and U. parvum serovar 3 were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA; serovar 8: ATCC 27618, serovar 3: ATCC 27815)
We addressed Ureaplasma-induced caspase expression as well as cytokine responses in the well described epithelial type II cell-like line A549 and human pulmonary microvascular endothelial cell line human pulmonary microvascular endothelial cells (HPMEC)-ST1.6R (HPMEC) [19, 29,30,31]
In A549 cells, we generally counted low numbers of dead cells, without any significant influences on cell viability yielded by Ureaplasma spp. (Fig 1A)
Summary
Ureaplasma (U.) urealyticum serovar 8 and U. parvum serovar 3 were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA; serovar 8: ATCC 27618, serovar 3: ATCC 27815). Isolates were cultured in in-house modified 10-B medium [32] (referred to as “broth”), containing 82% pleuropneumonia-like organism medium (Becton, Dickinson & Company, Franklin Lakes, NJ), 10% heat-inactivated horse serum (v/v), 1% urea (w/v) and 0.002% phenol red (w/v) (all: Sigma-Aldrich, St. Louis, CA), adjusted to pH 6.5. An endotoxin level < 0.06 EU/mL broth was verified using ToxinSensor Endotoxin Detection System (GenScript, Piscataway, NJ). Serial 10-fold dilutions were incubated for 18–20 h to achieve titers of 1×109−1×1010 color-changing units (CCU)/mL of viable cells.
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