Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza.

Hemolytic uremic syndrome (HUS) is a potentially life-threatening condition. It often occurs after gastro-intestinal infection with E. coli O157:H7, which produces Shiga toxins (Stx) that cause hemolytic anemia, thrombocytopenia, and renal injury. Stx-mediated changes in endothelial phenotype have been linked to the pathogenesis of HUS. Here we report our studies investigating Stx-induced changes in gene expression and their contribution to the pathogenesis of HUS. Stx function by inactivating host ribosomes but can also alter gene expression at concentrations that minimally affect global protein synthesis. Gene expression profiling of human microvascular endothelium treated with Stx implicated a role for activation of CXCR4 and CXCR7 by their shared cognate chemokine ligand (stromal cell-derived factor-1 [SDF-1]) in Stx-mediated pathophysiology. The changes in gene expression required a catalytically active Stx A subunit and were mediated by enhanced transcription and mRNA stability. Stx also enhanced the association of CXCR4, CXCR7, and SDF1 mRNAs with ribosomes. In a mouse model of Stx-mediated pathology, we noted changes in plasma and tissue content of CXCR4, CXCR7, and SDF-1 after Stx exposure. Furthermore, inhibition of the CXCR4/SDF-1 interaction decreased endothelial activation and organ injury and improved animal survival. Finally, in children infected with E. coli O157:H7, plasma SDF-1 levels were elevated in individuals who progressed to HUS. Collectively, these data implicate the CXCR4/CXCR7/SDF-1 pathway in Stx-mediated pathogenesis and suggest novel therapeutic strategies for prevention and/or treatment of complications associated with E. coli O157:H7 infection.

Increased permeability of the microvascular endothelium to fluids and proteins is the hallmark of inflammatory conditions such as sepsis. Leakage can occur between (paracellular) or through (transcytosis) endothelial cells, yet little is known about whether these pathways are linked. Understanding the regulation of microvascular permeability is essential for the identification of novel therapies to combat inflammation. We investigated whether transcytosis and paracellular leakage are co-regulated. Using molecular and pharmacologic approaches, we inhibited transcytosis of albumin in primary human microvascular endothelium and measured paracellular permeability. Blockade of transcytosis induced a rapid increase in paracellular leakage that was not explained by decreases in caveolin-1 or increases in activity of nitric oxide synthase. The effect required caveolin-1 but was observed in cells depleted of clathrin, indicating that it was not due to the general inhibition of endocytosis. Inhibiting transcytosis by dynamin blockade increased paracellular leakage concomitantly with the loss of cortical actin from the plasma membrane and the displacement of active Rac from the plasmalemma. Importantly, inhibition of paracellular leakage by sphingosine-1-phosphate, which activates Rac and induces cortical actin, caused a significant increase in transcytosis of albumin in vitro and in an ex vivo whole-lung model. In addition, dominant-negative Rac significantly diminished albumin uptake by endothelia. Our findings indicate that transcytosis and paracellular permeability are co-regulated through a signaling pathway linking dynamin, Rac, and actin.

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Increased permeability of the endothelium is the hallmark of inflammation. Leakage can occur between (paracellular) or through (transcytosis) endothelial cells, yet little is known about whether these pathways are linked. We investigated whether transcytosis and paracellular leakage are co‐regulated. Using molecular and pharmacologic approaches, we inhibited transcytosis of albumin in primary human microvascular endothelium and measured paracellular permeability. Blockade of transcytosis induced an increase in paracellular leak that was not explained by decreases in caveolin‐1 or by increased activity of nitric oxide synthase. The effect required caveolin‐1 but occurred in cells depleted of clathrin, indicating that it was not due to the general inhibition of endocytosis. Inhibiting transcytosis by blocking dynamin increased paracellular leak concomitantly with the loss of cortical actin from the plasma membrane and the displacement of active Rac from the plasmalemma. Importantly, inhibition of paracellular leak by sphingosine‐1‐phosphate, which activates Rac and induces cortical actin, caused a significant increase in transcytosis of albumin in vitro and in an ex vivo whole lung model. Dominant‐negative Rac diminished albumin uptake by endothelia. Thus, transcytosis and paracellular permeability are co‐regulated through a signalling pathway linking dynamin, Rac and actin.