技术资料

When inhaled,ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung lining fluid,generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols,epoxycholesterol-α and β (α-EpCh,β-EpCh) and Secosterol A and B (Seco A,SecoB),in cell lysates and apical washes. Similarly,bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected,O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly,expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1),which is regulated by activation of the liver X receptor (LXR),was suppressed in epithelial cells exposed to O3. Additionally,exposure of LXR knockout mice to O3 enhanced pro-inflammatory cytokine production in the lung,suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols,our data demonstrate adduction of LXR with Seco A. Similarly,supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared to stimulation with T09 alone,indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall,these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR,thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects. View Publication

Human rhinovirus (HRV) is the most common virus contributing to acute exacerbations of chronic obstructive pulmonary disease (COPD) nearly year-round,but the mechanisms have not been well elucidated. Recent clinical studies suggest that high levels of growth differentiation factor 15 (GDF15) protein in the blood are associated with an increased yearly rate of all-cause COPD exacerbations. Therefore,in the current study,we investigated whether GDF15 promotes HRV infection and virus-induced lung inflammation. We first examined the role of GDF15 in regulating host defense and HRV-induced inflammation using human GDF15 transgenic mice and cultured human GDF15 transgenic mouse tracheal epithelial cells. Next,we determined the effect of GDF15 on viral replication,antiviral responses,and inflammation in human airway epithelial cells with GDF15 knockdown and HRV infection. Finally,we explored the signaling pathways involved in airway epithelial responses to HRV infection in the context of GDF15. Human GDF15 protein over-expression in mice led to exaggerated inflammatory responses to HRV,increased infectious particle release,and decreased IFN-λ2/3 (IL-28A/B) mRNA expression in the lung. Moreover,GDF15 facilitated HRV replication and inflammation via inhibiting IFN-λ1/IL-29 protein production in human airway epithelial cells. Lastly,Smad1 cooperated with interferon regulatory factor 7 (IRF7) to regulate airway epithelial responses to HRV infection partly via GDF15 signaling. Our results reveal a novel function of GDF15 in promoting lung HRV infection and virus-induced inflammation,which may be a new mechanism for the increased susceptibility and severity of respiratory viral (i.e.,HRV) infection in cigarette smoke-exposed airways with GDF15 over-production. View Publication

Rotavirus,a leading cause of severe gastroenteritis and diarrhoea in young children,accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling,raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo,especially by NOD-like receptor (NLR) inflammasomes,is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that,via RNA helicase Dhx9,Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens. View Publication

BACKGROUND & AIMS Clostridium difficile induces intestinal inflammation by releasing toxins A and B. The antimicrobial compound cationic steroid antimicrobial 13 (CSA13) has been developed for treating gastrointestinal infections. The CSA13-Eudragit formulation can be given orally and releases CSA13 in the terminal ileum and colon. We investigated whether this form of CSA13 reduces C difficile infection (CDI) in mice. METHODS C57BL/6J mice were infected with C difficile on day 0,followed by subcutaneous administration of pure CSA13 or oral administration of CSA13-Eudragit (10 mg/kg/d for 10 days). Some mice were given intraperitoneal vancomycin (50 mg/kg daily) on days 0-4 and relapse was measured after antibiotic withdrawal. The mice were monitored until day 20; colon and fecal samples were collected on day 3 for analysis. Blood samples were collected for flow cytometry analyses. Fecal pellets were collected each day from mice injected with CSA13 and analyzed by high-performance liquid chromatography or 16S sequencing; feces were also homogenized in phosphate-buffered saline and fed to mice with CDI via gavage. RESULTS CDI of mice caused 60{\%} mortality,significant bodyweight loss,and colonic damage 3 days after infection; these events were prevented by subcutaneous injection of CSA13 or oral administration CSA13-Eudragit. There was reduced relapse of CDI after administration of CSA13 was stopped. Levels of CSA13 in feces from mice given CSA13-Eudragit were significantly higher than those of mice given subcutaneous CSA13. Subcutaneous and oral CSA13 each significantly increased the abundance of Peptostreptococcaceae bacteria and reduced the abundance of C difficile in fecal samples of mice. When feces from mice with CDI and given CSA13 were fed to mice with CDI that had not received CSA13,the recipient mice had significantly increased rates of survival. CSA13 reduced fecal levels of inflammatory metabolites (endocannabinoids) and increased fecal levels of 4 protective metabolites (ie,citrulline,3-aminoisobutyric acid,retinol,and ursodeoxycholic acid) in mice with CDI. Oral administration of these CSA13-dependent protective metabolites reduced the severity of CDI. CONCLUSIONS In studies of mice,we found the CSA13-Eudragit formulation to be effective in eradicating CDI by modulating the intestinal microbiota and metabolites. View Publication