Toll-like receptor 5 (TLR5), IL-1ß secretion, and asparagine endopeptidase are critical factors for alveolar macrophage phagocytosis and bacterial killing.

A deficit in early clearance of Pseudomonas aeruginosa (P. aeruginosa) is crucial in nosocomial pneumonia and in chronic lung infections. Few studies have addressed the role of Toll-like receptors (TLRs), which are early pathogen associated molecular pattern receptors, in pathogen uptake and clearance by alveolar macrophages (AMs). Here, we report that TLR5 engagement is crucial for bacterial clearance by AMs in vitro and in vivo because unflagellated P. aeruginosa or different mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. In addition, the clearance of PAK (a wild-type P. aeruginosa strain) by primary AMs was causally associated with increased IL-1ß release, which was dramatically reduced with PAK mutants or in WT PAK-infected primary TLR5(-/-) AMs, demonstrating the dependence of IL-1ß production on TLR5. We showed that this IL-1ß production was important in endosomal pH acidification and in inducing the killing of bacteria by AMs through asparagine endopeptidase (AEP), a key endosomal cysteine protease. In agreement, AMs from IL-1R1(-/-) and AEP(-/-) mice were unable to kill P. aeruginosa. Altogether, these findings demonstrate that TLR5 engagement plays a major role in P. aeruginosa internalization and in triggering IL-1ß formation.

Overexpressing mouse model demonstrates the protective role of Muc5ac in the lungs.

MUC5AC, a major gel-forming mucin expressed in the lungs, is secreted at increased rates in response to infectious agents, implying that mucins exert a protective role against inhaled pathogens. However, epidemiological and pathological studies suggest that excessive mucin secretion causes airways obstruction and inflammation. To determine whether increased MUC5AC secretion alone produces airway obstruction and/or inflammation, we generated a mouse model overexpressing Muc5ac mRNA ~20-fold in the lungs, using the rCCSP promoter. The Muc5ac cDNA was cloned from mouse lungs and tagged internally with GFP. Bronchoalveolar lavage fluid (BALF) analysis demonstrated an approximate 18-fold increase in Muc5ac protein, which formed high-molecular-weight polymers. Histopathological studies and cell counts revealed no airway mucus obstruction or inflammation in the lungs of Muc5ac-transgenic (Muc5ac-Tg) mice. Mucus clearance was preserved, implying that the excess Muc5ac secretion produced an "expanded" rather than more concentrated mucus layer, a prediction confirmed by electron microscopy. To test whether the larger mucus barrier conferred increased protection against pathogens, Muc5ac-Tg animals were challenged with PR8/H1N1 influenza viruses and showed significant decreases in infection and neutrophilic responses. Plaque assay experiments demonstrated that Muc5ac-Tg BALF and purified Muc5ac reduced infection, likely via binding to α2,3-linked sialic acids, consistent with influenza protection in vivo. In conclusion, the normal mucus transport and absence of a pulmonary phenotype in Muc5ac-Tg mice suggests that mucin hypersecretion alone is not sufficient to trigger luminal mucus plugging or airways inflammation/goblet cell hyperplasia. In contrast, increased Muc5ac secretion appears to exhibit a protective role against influenza infection.

Influenza A induces the major secreted airway mucin MUC5AC in a protease-EGFR-extracellular regulated kinase-Sp1-dependent pathway.

Mucins, the main glycoproteins present within mucus, modulate the rheologic properties of airways and participate in lung defense. They are thought to be able to trap and eliminate microorganisms from the lung. Among the mucins secreted in the lung, MUC5AC is the most prominent factor secreted by surface epithelial cells. Although much is known about the signaling pathways involved in the regulation of MUC5AC by host factors such as cytokines or proteases, less is known about the pathways triggered by microorganisms and, specifically, by influenza A virus (IAV). We therefore set up experiments to dissect the molecular mechanisms responsible for the potential modulation of MUC5AC by IAV. Using epithelial cells, C57/Bl6 mice, and IAV strains, we measured MUC5AC expression at the RNA and protein levels, specificity protein 1 (Sp1) activation, and protease activity. Intermediate molecular partners were confirmed using pharmacological inhibitors, blocking antibodies, and small interfering (si)RNAs. We showed in vitro and in vivo that IAV up-regulates epithelial cell-derived MUC5AC and Muc5ac expression in mice, both at transcriptional (through the induction of Sp1) and translational levels. In addition, we determined that this induction was dependent on a protease-epithelial growth factor receptor-extracellular regulated kinase-Sp1 signaling cascade, involving in particular the human airway trypsin. Our data point to MUC5AC as a potential modulatory mechanism by which the lung epithelia respond to IAV infection, and we dissect, for the first time to the best of our knowledge, the molecular partners involved. Future experiments using MUC5AC-targeted strategies should help further unravel the pathophysiological consequences of IAV-induced MUC5AC expression for lung homeostasis.