Rapid clearance of heavy chain-modified hyaluronan during resolving acute lung injury.

BACKGROUND: Several inflammatory lung diseases display abundant presence of hyaluronic acid (HA) bound to heavy chains (HC) of serum protein inter-alpha-inhibitor (IαI) in the extracellular matrix. The HC-HA modification is critical to neutrophil sequestration in liver sinusoids and to survival during experimental lipopolysaccharide (LPS)-induced sepsis. Therefore, the covalent HC-HA binding, which is exclusively mediated by tumor necrosis factor α (TNFα)-stimulated-gene-6 (TSG-6), may play an important role in the onset or the resolution of lung inflammation in acute lung injury (ALI) induced by respiratory infection. METHODS: Reversible ALI was induced by a single intratracheal instillation of LPS or Pseudomonas aeruginosa in mice and outcomes were studied for up to six days. We measured in the lung or the bronchoalveolar fluid HC-HA formation, HA immunostaining localization and roughness, HA fragment abundance, and markers of lung inflammation and lung injury. We also assessed TSG-6 secretion by TNFα- or LPS-stimulated human alveolar macrophages, lung fibroblast Wi38, and bronchial epithelial BEAS-2B cells. RESULTS: Extensive HC-modification of lung HA, localized predominantly in the peri-broncho-vascular extracellular matrix, was notable early during the onset of inflammation and was markedly decreased during its resolution. Whereas human alveolar macrophages secreted functional TSG-6 following both TNFα and LPS stimulation, fibroblasts and bronchial epithelial cells responded to only TNFα. Compared to wild type, TSG-6-KO mice, which lacked HC-modified HA, exhibited modest increases in inflammatory cells in the lung, but no significant differences in markers of lung inflammation or injury, including histopathological lung injury scores. CONCLUSIONS: Respiratory infection induces rapid HC modification of HA followed by fragmentation and clearance, with kinetics that parallel the onset and resolution phase of ALI, respectively. Alveolar macrophages may be an important source of pulmonary TSG-6 required for HA remodeling. The formation of HC-modified HA had a minor role in the onset, severity, or resolution of experimental reversible ALI induced by respiratory infection with gram-negative bacteria.

Visualization of mucosal homeostasis via single- and multiphoton intravital fluorescence microscopy.

FIVM has provided many insights into the regulation of immunity. We report the validation of an approach for visualizing murine small bowel via single- and multiphoton FIVM. Tissue damage is limited to ∼200 µm, immediately adjacent to the incision, as confirmed by intravital PI staining. Treatment with 10 KDa dextran-FITC and 70 KDa dextran-TR confirms that perfusion is intact. Selective filtration of 10 KDa but not 70 KDa dextran from the blood indicated that kidney function is also intact. Interestingly, lamina propria vasculature is semipermeable to 10 KDa dextran. Next, reporter mice expressing egfp from the CX3CR1 locus, egfp from the FoxP3 locus, or RFP from the IL-17F locus were used to track DC subsets, FoxP3(+) Tregs, or Th17f cells, respectively. Resident cx3cr1(+/egfp) cells were sessile but actively probed the surrounding microenvironment. Both T cell populations patrol the lamina propria, but the Th17f cells migrate more rapidly than Tregs. Together, these data demonstrate intact vascular perfusion, while intravitally visualizing the mucosal surface of the small bowel. Lastly, the cx3cr1(+) DCs and T cells display activity similar to that found in steady-state, secondary lymphoid organs.