COPD-Related Modification to the Airway Epithelium Permits Intracellular Residence of Nontypeable Haemophilus influenzae and May Be Potentiated by Macrolide Arrest of Autophagy.

Introduction: COPD is an inflammatory airway pathology associated with recurrent infection by nontypeable Haemophilus influenzae (NTHi) that is not effectively managed by macrolide antibiotic therapy. We hypothesised that NTHi is able to reside intracellularly within COPD-derived airway epithelial cells (AEC), and that the factors contained in cigarette smoke when coupled with exposure to erythromycin or azithromycin arrest autophagy, the principle mechanism responsible for clearing intracellular bacteria (called "xenophagy"). Methods: Cultures of bronchial airway epithelial cells derived from control and COPD participants were differentiated at an air-liquid interface and exposed to macrolide antibiotics, 10% cigarette smoke-extract (CSE) and NTHi. Markers of autophagic flux and intracellular NTHi were assessed using Western blot analysis and transmission electron microscopy. Results: AEC treated with macrolide antibiotics or 10% CSE exhibited a block in autophagic flux as evidenced by a concomitant increase in LC3-II and Sequestosome abundance (vs control; both P < 0.01). While control AEC showed no clear evidence of intracellular NTHi, COPD-derived cultures exhibited abundant NTHi within the cytoplasm. Further, intracellular NTHi that were encapsulated within vesicles propagated from the apical epithelial layer to the basal cell layer. Discussion: Taken together, our findings indicate that COPD, cigarette smoke and macrolide antibiotics potentiate the susceptibility to persistent intracellular NTHi. A major mechanism for this is arresting normal autophagic flux in airway epithelial cells. Hence, structural modifications that mitigate this off-target effect of macrolides have significant potential to clear intracellular NTHi and thereby reduce the influence of this pathogen in the airways afflicted by COPD.

Timing of excision after a non-severe burn has a significant impact on the subsequent immune response in a murine model.

BACKGROUND: Burn excision has emerged as the dominant clinical paradigm in treatment of deep burns. Surgical intervention is common but the timing of wound excision is a balance between wound depth assessment, avoidance of infection and unnecessary intervention. However the physiological impact of timing of excision and consequences for the immune response are not well understood. METHODS: Mice were subject to full-thickness burn (<8% TBSA) followed by early (day 1) or late (day 8) surgical excision. Draining lymph nodes, wound tissue and sera were collected longitudinally at day 2 and day 6 after excision and analyzed for cytokine, dendritic cell and T cell profiles using FACS and multiplex ELISA assays. RESULTS: Delayed excision after injury initiated acute and severe inflammatory responses, with high levels of inflammatory cytokines, increased chemokine responses, and elevated Th2 promoting cytokines compared to early excision. Cellular inflammation in the wound was exacerbated with elevated neutrophils, eosinophil and monocytes. Wound cellular innate immune response decreased after late excision with a loss of inflammatory dendritic cells (DC), decreased NKT cells, and inhibition of NK cell activation. Systemically late excision increased trafficking conventional CD8α(-) DC to the lymph node, but there was no apparent DC activation. This was reflected in the induction of CD4T regulatory (Treg) cells and suppression of CD8T cell proliferation after late excision. No suppression was observed with early excision. CONCLUSION: This data suggests early excision of the wound, during the phase of immune down-regulation initiated by the burn, maintains an innate and adaptive immune cell response. In contrast, late wound excision induced a severe inflammatory response, with subsequent down-regulation of innate and adaptive immune cell responses. Therefore timing of excision is critical in affecting the immune response to burn.

Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis.

Complications of acute respiratory distress syndrome (ARDS) are common among critically ill patients infected with highly pathogenic influenza viruses. Macrophages and neutrophils constitute the majority of cells recruited into infected lungs, and are associated with immunopathology in influenza pneumonia. We examined pathological manifestations in models of macrophage- or neutrophil-depleted mice challenged with sublethal doses of influenza A virus H1N1 strain PR8. Infected mice depleted of macrophages displayed excessive neutrophilic infiltration, alveolar damage, and increased viral load, later progressing into ARDS-like pathological signs with diffuse alveolar damage, pulmonary edema, hemorrhage, and hypoxemia. In contrast, neutrophil-depleted animals showed mild pathology in lungs. The brochoalveolar lavage fluid of infected macrophage-depleted mice exhibited elevated protein content, T1-α, thrombomodulin, matrix metalloproteinase-9, and myeloperoxidase activities indicating augmented alveolar-capillary damage, compared to neutrophil-depleted animals. We provide evidence for the formation of neutrophil extracellular traps (NETs), entangled with alveoli in areas of tissue injury, suggesting their potential link with lung damage. When co-incubated with infected alveolar epithelial cells in vitro, neutrophils from infected lungs strongly induced NETs generation, and augmented endothelial damage. NETs induction was abrogated by anti-myeloperoxidase antibody and an inhibitor of superoxide dismutase, thus implying that NETs generation is induced by redox enzymes in influenza pneumonia. These findings support the pathogenic effects of excessive neutrophils in acute lung injury of influenza pneumonia by instigating alveolar-capillary damage.