Inhibition of the influenza A virus-induced, tubulin-dependent apical mislocalization of the Na+,K+-ATPase in infected cells: Improving vectorial water transport and pulmonary edema clearance
One of the fatal complication of influenza A virus (IAV) infection is the acute respiratory distress syndrome (ARDS) associated with sever formation of alveolar edema. Impaired resolution of pulmonary edema is a result of a direct destruction of the alveolar epithelium induced by IAV replication and an infringement of the osmotic gradient in the alveolar microenvironment, which is the main driving force of alveolar fluid clearance. IAV infection down-regulates the amount and function of several membrane ion-channels and pumps that are needed to establish the osmotic gradient.In the present work a novel mechanism of IAV pathogenicity affecting the basolateral membrane-located Na+,K+-ATPase (NKA), the major regulator of fluid homeostasis, is described. IAV infection did not reduce the overall amount of membrane-associated NKA, but induced a mislocalization of the enzyme to the apical site of infected polarized human bronchial epithelial cells (Calu3), as well as canine kidney epithelial cells (MDCK II). The mislocalization of NKA was not dependent on the IAV subtype or viral replication efficiency, but seems to be induced by IAV in general. The results of the present work indicate that the source of apically localized NKA is the vesicular intracellular NKA depot, since no decrease of NKA in the basolateral cell membrane was observed during IAV-infection, as well as inhibition of newly synthesized NKA maturation did not prevent its apical distribution. Application of the actin polymerization inhibitor cytochalasin D, the actin polymerization enhancer jasplakinolide, the inhibitor of microtubule polymerization nocodazole or the stabilizer of microtubule polymer paclitaxel, indicated that NKA mistargeting to the apical cell membrane depends on the integrity of the tubulin network. Moreover, a post-translational modification of alpha-tubulin (acetylation of residue K40), is needed for IAV-induced NKA mistargeting. This modification seems to be is indirectly regulated by Rho-kinase (ROCK) as NKA mislocalization in IAV- infected cells can be prevented by ROCK inhibition, which impairs IAV-induced caspase-3- dependent degradation of histone deacetylase 6 (HDAC6), resulting in a reduced amount of acetylated alpha-tubulin needed for apical NKA transport. In addition, ROCK inhibition not only prevented mistargeting of NKA to the apical membrane, but also reduced virus titer and restored vectorial water transport through the monolayer of highly polarized infected Calu3 cells. Application of Fasudil HCl to IAV-infected mice improved pulmonary edema clearance and reduced the virus titer and immune cell infiltration in the lungs. Taking together, an administration of ROCK inhibitors might be a potential treatment scenario for the patients with IAV-induced ARDS.
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