Role of SIRT1 in Streptococcus pneumoniae-induced human ß-defensin-2 and interleukin-8 expression in A549 cell.

Streptococcus pneumoniae is an important pathogen of pneumonia in human. Human alveolar epithelium acts as an effective barrier and is an active participant in host defense against invasion of bacterial by production of various mediators. Sirtuin 1 (SIRT1), the prototypic class III histone deacetylase, is involved in the molecular control of lifespans and immune responses. This study aimed at examining the role of SIRT1 in mediating S. pneumoniae-induced human ß-defensin-2 (hBD2) and interleukin-8(IL-8) expression in the alveolar epithelial cell line A549 and the underlying mechanisms involved. A549 cells were infected with S. pneumoniae for indicated times. Exposure of A549 cells to S. pneumoniae increased the expressions of SIRT1 protein, hBD2 and IL-8 mRNA, and protein. The SIRT1 activator resveratrol enhanced S. pneumoniae-induced gene expression of hBD2 but decreased IL-8 mRNA levels. Blockade of SIRT1 activity by the SIRT1 inhibitors nicotinamide reduced S. pneumoniae-induced hBD2 mRNA expression but increased its stimulatory effects on IL-8 mRNA. S. pneumoniae-induced activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SIRT1 expression was attenuated by selective inhibitors of ERK and p38 MAPK. The hBD2 mRNA production was decreased by pretreatment with p38 MAPK inhibitor but not with ERK inhibitor, whereas the IL-8 mRNA expression was controlled by phosphorylation of ERK. These results suggest that SIRT1 mediates the induction of hBD2 and IL-8 gene expression levels in A549 cell by S. pneumoniae. SIRT1 may play a key role in host immune and defense response in A549.

The clinical application of flexible bronchoscopy in a neonatal intensive care unit.

Objective: Flexible bronchoscopy is widely used in infants and it plays a crucial role. The aim of this study was to investigate the value and clinical safety of flexible bronchoscopy in a neonatal intensive care unit. Methods: A retrospective analysis was performed on the clinical data of 116 neonates who underwent flexible bronchoscopy and the outcomes of 147 procedures. A correlation analysis was performed on the relationship between flexible bronchoscopy findings, microscopic indications, and clinical disease. Results: The 147 procedures performed were due to the following reasons: problems related to artificial airways, 58 cases (39.45%); upper respiratory problems, 60 cases (40.81%) (recurrent dyspnea, 23 cases; upper airway obstruction, 17 cases; recurrent stridor, 14 cases; and hoarseness, six cases), lower respiratory problems, 51 cases (34.69%) (persistent pneumonia, 21 cases; suspicious airway anatomical disease, 21 cases; recurrent atelectasis, eight cases; and pneumorrhagia, one case), feeding difficulty three cases (2.04%). The 147 endoscopic examinations were performed for the following reasons: pathological changes, 141 cases (95.92%); laryngomalacia, 78 cases (53.06%); mucosal inflammation/secretions, 64 cases (43.54%); vocal cord paralysis, 29 cases (19.72%); trachea/bronchus stenosis, 17 cases (11.56%) [five cases of congenital annular constriction of the trachea, seven cases of left main tracheal stenosis, one case of the right middle bronchial stenosis, two cases of tracheal compression, and two cases of congenital tracheal stenosis]; subglottic lesions, 15 cases (10.20%) [eight cases of subglottic granulation tissue, six cases of subglottic stenosis, one cases of subglottic hemangioma]; tracheomalacia, 14 cases (9.52%); laryngeal edema, five cases (3.40%); tracheoesophageal fistula, four cases (2.72%); rhinostenosis, three cases (2.04%); tracheal bronchus, three cases (2.04%); glossoptosis, two cases (1.36%); laryngeal cyst, two cases (1.36%); laryngeal cleft, two cases (1.36%); tongue base cysts, one case (0.68%); and pneumorrhagia, one case (0.68%). Complications were rare and mild. Conclusion: Flexible bronchoscopy is safe and effective for diagnosing and differentiating neonatal respiratory disorders in neonatal intensive care units.

TNF-α increases Staphylococcus aureus-induced death of human alveolar epithelial cell line A549 associated with RIP3-mediated necroptosis.

AIM: To explore the role of tumor necrosis factor-alpha (TNF-α) on Staphylococcus aureus-induced necroptosis in alveolar epithelial cells. MAIN METHODS: The A549 alveolar epithelial cell line was pretreated with small interfering RNA (siRNA) against receptor interacting protein-3 (RIP3) and then stimulated by S. aureus, where some cells were pretreated with TNF-α or TNF-α with anti-TNF-α antibody simultaneously. A549 cell death was assessed using lactate dehydrogenase (LDH) leakage and flow cytometry analyses. The protein expressions of RIP1, RIP3, cleaved caspase-1, and cleaved caspase-8 were analyzed by western blot. KEY FINDINGS: S. aureus-induced LDH release was increased significantly by TNF-α. In addition, flow cytometry showed that TNF-α increased A549 cell apoptosis and necrosis in S. aureus-infected cell cultures. Levels of RIP3 and cleaved caspase-1 protein in A549 cells infected with S. aureus increased at 12 h post-infection, as shown by western blot. Significant additional increases in RIP3 expression were observed following the addition of TNF-α. Decreasing RIP3 levels by siRNA significantly suppressed the release of LDH induced by TNF-α and S. aureus. RIP3 siRNA also significantly suppressed A549 cell necrosis induced by S. aureus and TNF-α at 6 and 12 h post-infection as shown by flow cytometry analysis. SIGNIFICANCE: TNF-α enhances the damage of S. aureus on lung epithelial cells, and its mechanism is associated with RIP3 mediated necroptosis.

Adjunctive dexamethasone therapy improves lung injury by inhibiting inflammation and reducing RIP3 expression during Staphylococcus aureus pneumonia in mice.

Antibiotic-induced immunopathology associated with the release of bacterial cell wall components has been suggested to contribute to poor outcomes in bacterial pneumonia. Adjunctive systemic glucocorticoid steroid (GC) therapy for pneumonia has been a controversial issue. In the present study, we first found that dexamethasone (2.5 mg/kg/day) in combination with oxacillin was beneficial for improving lung injury in mice inoculated intratracheally with live Staphylococcus aureus, and did not interfere with bacterial clearance. Alleviation of lung injury was evidenced by attenuated lung pathology, reduced total protein levels, soluble receptor for advanced glycation end-products (sRAGE), tumor necrosis factor alpha (TNF-α), and keratinocyte chemoattractant (KC) and interleukin (IL)-6 in bronchoalveolar lavage fluid (BALF). It was further confirmed by inhibition of receptor interacting protein-3 (RIP3) expression in pulmonary tissues. As in the live S. aureus experiments, dexamethasone (2.5 mg/kg/day) improved lung injury in mice challenged with heat-killed S. aureus (HKSA). In conclusion, our results demonstrated that an appropriate dose of adjunctive dexamethasone (2.5 mg/kg/day) with oxacillin alleviated experimental S. aureus-induced lung injury via its inhibition of inflammatory cytokine release and RIP3 expression.