Role and regulation of alveolar protein clearance in influenza virus-induced lung injury
Acute respiratory distress syndrome (ARDS) is a common complication of influenza virus (IV) infection. During ARDS, alveolar protein concentrations often reach 40-90% of plasma levels, causing severe impairment of gas exchange and promoting deleterious alveolar remodeling. Protein clearance from the alveolar space is initiated by ... clathrin-mediated endocytosis, in part by the multi-ligand receptor megalin. Here, we aimed to investigate whether IV infection impairs alveolar protein clearance. Infection with IV A/Puerto Rico/8/1934 H1N1 seasonal, mouse-adapted strain caused a marked downregulation of albumin uptake in MLE-12 cells and in alveolar epithelial cells (AEC) from PCLS, as assessed by flow cytometry and confocal microscopy, which was associated with decreased plasma membrane abundance, total protein levels and mRNA expression of megalin. Previous work from our group showed that megalin downregulation can be triggered by the binding of TGF-β1 to its receptor, which leads to the dephosphorylation and activation of glycogen synthase kinase -3β(GSK3β). The activity of this kinase phosphorylates the cytoplasmic domain of megalin, signal that internalizes the receptor and impairs protein uptake. Contrary to our expectations, we found no signs of activation of the TGF-β1/GSK3β/megalin axis in our in vitro models. To further dissect the effects of IV infection on endocytic pathways, we infected mice with IV in vivo and subjected isolated AEC and cells obtained from bronchoalveolar lavage (BAL) to bulk RNA sequencing. Of note, downregulation of megalin and other endocytosis related receptors like the scavenger receptor CD36 and Lrp6 was evident in AEC. Moreover, we observed that downstream targets of TGF-β1, such as Itgb6 and Tgfbi were markedly upregulated in AEC upon IV infection, suggesting the presence of the active form of the cytokine which is of relevance as our group had previously shown that activation of TGF-β1 is sufficient to reduce cell-surface abundance of megalin. We also identified a significant upregulation of MMP-14 in BAL cells. Moreover, the specific inhibition of this protease partially recovered the levels of total megalin and albumin uptake. This suggests that the previously described MMP-driven shedding mechanisms are potentially involved in downregulation of megalin and thus clearance of excess alveolar protein. Importantly, it highlights the therapeutic potential of timely MMPs inhibition in the treatment of IV-induced ARDS. As lower alveolar edema protein concentrations are associated with better outcomes in ARDS, identifying targetable mechanisms of impaired alveolar protein clearance after IV infection may hold a therapeutic promise.