Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Loss of Tumour Suppressor TMEM127 Drives RET-mediated Transformation Through Disrupted Membrane Dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTK) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumour pheochromocytoma (PCC) can be caused by activating mutations of the RET receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumour suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability, and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Quantitative modeling of EGF receptor ligand discrimination via internalization proofreading.

The epidermal growth factor receptor (EGFR) is a central regulator of cell physiology that is stimulated by multiple distinct ligands. Although ligands bind to EGFR while the receptor is exposed on the plasma membrane, EGFR incorporation into endosomes following receptor internalization is an important aspect of EGFR signaling, with EGFR internalization behavior dependent upon the type of ligand bound. We develop quantitative modeling for EGFR recruitment to and internalization from clathrin domains, focusing on how internalization competes with ligand unbinding from EGFR. We develop two model versions: a kinetic model with EGFR behavior described as transitions between discrete states and a spatial model with EGFR diffusion to circular clathrin domains. We find that a combination of spatial and kinetic proofreading leads to enhanced EGFR internalization ratios in comparison to unbinding differences between ligand types. Various stages of the EGFR internalization process, including recruitment to and internalization from clathrin domains, modulate the internalization differences between receptors bound to different ligands. Our results indicate that following ligand binding, EGFR may encounter multiple clathrin domains before successful recruitment and internalization. The quantitative modeling we have developed describes competition between EGFR internalization and ligand unbinding and the resulting proofreading.

Improved imaging and preservation of lysosome dynamics using silver nanoparticle-enhanced fluorescence.

The dynamics of living cells can be studied by live-cell fluorescence microscopy. However, this requires the use of excessive light energy to obtain good signal-to-noise ratio, which can then photobleach fluorochromes, and more worrisomely, lead to phototoxicity. Upon light excitation, noble metal nanoparticles such as silver nanoparticles (AgNPs) generate plasmons, which can then amplify excitation in direct proximity of the nanoparticle's surface and couple to the oscillating dipole of nearby radiating fluorophores, modifying their rate of emission and thus, enhancing their fluorescence. Here, we show that AgNPs fed to cells to accumulate within lysosomes enhanced the fluorescence of lysosome-targeted Alexa488-conjugated dextran, BODIPY-cholesterol, and DQ-BSA. Moreover, AgNP increased the fluorescence of GFP fused to the cytosolic tail of LAMP1, showing that metal enhanced fluorescence can occur across the lysosomal membrane. The inclusion of AgNPs in lysosomes did not disturb lysosomal properties such as lysosomal pH, degradative capacity, autophagy and autophagic flux, and membrane integrity, though AgNP seemed to increase basal lysosome tubulation. Importantly, by using AgNP, we could track lysosome motility with reduced laser power without damaging and altering lysosome dynamics. Overall, AgNP-enhanced fluorescence may be a useful tool to study the dynamics of the endo-lysosomal pathway while minimizing phototoxicity.

Confinement of unliganded EGFR by tetraspanin nanodomains gates EGFR ligand binding and signaling.

The epidermal growth factor receptor (EGFR) is a central regulator of cell physiology. EGFR is activated by ligand binding, triggering receptor dimerization, activation of kinase activity, and intracellular signaling. EGFR is transiently confined within various plasma membrane nanodomains, yet how this may contribute to regulation of EGFR ligand binding is poorly understood. To resolve how EGFR nanoscale compartmentalization gates ligand binding, we developed single-particle tracking methods to track the mobility of ligand-bound and total EGFR, in combination with modeling of EGFR ligand binding. In comparison to unliganded EGFR, ligand-bound EGFR is more confined and distinctly regulated by clathrin and tetraspanin nanodomains. Ligand binding to unliganded EGFR occurs preferentially in tetraspanin nanodomains, and disruption of tetraspanin nanodomains impairs EGFR ligand binding and alters the conformation of the receptor's ectodomain. We thus reveal a mechanism by which EGFR confinement within tetraspanin nanodomains regulates receptor signaling at the level of ligand binding.

FASN inhibitor TVB-3166 prevents S-acylation of the spike protein of human coronaviruses.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses mediates host cell entry and is S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes have been reported to post-translationally modify spike proteins; however, strategies to exploit this modification are lacking. Using resin-assisted capture MS, we demonstrate that the spike protein is S-acylated in SARS-CoV-2-infected human and monkey epithelial cells. We further show that increased abundance of the acyltransferase ZDHHC5 associates with increased S-acylation of the spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also found that the S-acylation of the spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also labelled the protein. Yet, we observed that ZDHHC5 was only modified when incubated with alkyne-palmitate, suggesting it has specificity for this acyl-CoA, and that other ZDHHC enzymes may use additional fatty acids to modify the spike protein. Since multiple ZDHHC isoforms may modify the spike protein, we also examined the ability of the FASN inhibitor TVB-3166 to prevent S-acylation of the spike proteins of SARS-CoV-2 and human CoV-229E. We show that treating cells with TVB-3166 inhibited S-acylation of expressed spike proteins and attenuated the ability of SARS-CoV-2 and human CoV-229E to spread in vitro. Our findings further substantiate the necessity of CoV spike protein S-acylation and demonstrate that de novo fatty acid synthesis is critical for the proper S-acylation of the spike protein.

Fyn and TOM1L1 are recruited to clathrin-coated pits and regulate Akt signaling.

The epidermal growth factor (EGF) receptor (EGFR) controls many aspects of cell physiology. EGF binding to EGFR elicits the membrane recruitment and activation of phosphatidylinositol-3-kinase, leading to Akt phosphorylation and activation. Concomitantly, EGFR is recruited to clathrin-coated pits (CCPs), eventually leading to receptor endocytosis. Previous work uncovered that clathrin, but not receptor endocytosis, is required for EGF-stimulated Akt activation, and that some EGFR signals are enriched in CCPs. Here, we examine how CCPs control EGFR signaling. The signaling adaptor TOM1L1 and the Src-family kinase Fyn are enriched within a subset of CCPs with unique lifetimes and protein composition. Perturbation of TOM1L1 or Fyn impairs EGF-stimulated phosphorylation of Akt2 but not Akt1. EGF stimulation also triggered the TOM1L1- and Fyn-dependent recruitment of the phosphoinositide 5-phosphatase SHIP2 to CCPs. Thus, the recruitment of TOM1L1 and Fyn to a subset of CCPs underlies a role for these structures in the support of EGFR signaling leading to Akt activation.

Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease.

The COVID-19 pandemic has highlighted the urgent need for the identification of new antiviral drug therapies for a variety of diseases. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2, while other related human coronaviruses cause diseases ranging from severe respiratory infections to the common cold. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of a range of human coronavirus diseases. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cell-based experimental assessment reveals several clinically-relevant drug repurposing candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation.

Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR clustering within detergent-insoluble plasma mebrane-associated tubules. Sym004 induced lysosomal degradation independently of EGFR ubiquitylation but dependent upon Hrs/Tsg101 that are required for the formation of intraluminal vesicles (ILVs) within late endosomes. We propose Sym004 cross-links EGFR physically triggering EGFR endocytosis and incorporation onto ILVs and so Sym004 sensitivity correlates with EGFR numbers available for binding, rather than specific signalling events. Consistently Sym004 efficacy and potentiation of cisplatin responses correlated with EGFR surface expression in head and neck cancer cells. These findings will have implications in understanding the mode of action of this new class of cancer therapeutics.

Caveolin-1 Regulates Atherogenesis by Attenuating Low-Density Lipoprotein Transcytosis and Vascular Inflammation Independently of Endothelial Nitric Oxide Synthase Activation.

BACKGROUND: Atherosclerosis is driven by synergistic interactions between pathological, biomechanical, inflammatory, and lipid metabolic factors. Our previous studies demonstrated that absence of caveolin-1 (Cav1)/caveolae in hyperlipidemic mice strongly inhibits atherosclerosis, which was attributed to activation of endothelial nitric oxide (NO) synthase (eNOS) and increased production of NO and reduced inflammation and low-density lipoprotein trafficking. However, the contribution of eNOS activation and NO production in the athero-protection of Cav1 and the exact mechanisms by which Cav1/caveolae control the pathogenesis of diet-induced atherosclerosis are still not clear. METHODS: Triple-knockout mouse lacking expression of eNOS, Cav1, and Ldlr were generated to explore the role of NO production in Cav1-dependent athero-protective function. The effects of Cav1 on lipid trafficking, extracellular matrix remodeling, and vascular inflammation were studied both in vitro and in vivo with a mouse model of diet-induced atherosclerosis. The expression of Cav1 and distribution of caveolae regulated by flow were analyzed by immunofluorescence staining and transmission electron microscopy. RESULTS: We found that absence of Cav1 significantly suppressed atherogenesis in Ldlr-/-eNOS-/- mice, demonstrating that athero-suppression is independent of increased NO production. Instead, we find that the absence of Cav1/caveolae inhibited low-density lipoprotein transport across the endothelium and proatherogenic fibronectin deposition and disturbed flow-mediated endothelial cell inflammation. Consistent with the idea that Cav1/caveolae may play a role in early flow-dependent inflammatory priming, distinct patterns of Cav1 expression and caveolae distribution were observed in athero-prone and athero-resistant areas of the aortic arch even in wild-type mice. CONCLUSIONS: These findings support a role for Cav1/caveolae as a central regulator of atherosclerosis that links biomechanical, metabolic, and inflammatory pathways independently of endothelial eNOS activation and NO production.

Akt-ing Up Just About Everywhere: Compartment-Specific Akt Activation and Function in Receptor Tyrosine Kinase Signaling.

The serine/threonine kinase Akt is a master regulator of many diverse cellular functions, including survival, growth, metabolism, migration, and differentiation. Receptor tyrosine kinases are critical regulators of Akt, as a result of activation of phosphatidylinositol-3-kinase (PI3K) signaling leading to Akt activation upon receptor stimulation. The signaling axis formed by receptor tyrosine kinases, PI3K and Akt, as well as the vast range of downstream substrates is thus central to control of cell physiology in many different contexts and tissues. This axis must be tightly regulated, as disruption of PI3K-Akt signaling underlies the pathology of many diseases such as cancer and diabetes. This sophisticated regulation of PI3K-Akt signaling is due in part to the spatial and temporal compartmentalization of Akt activation and function, including in specific nanoscale domains of the plasma membrane as well as in specific intracellular membrane compartments. Here, we review the evidence for localized activation of PI3K-Akt signaling by receptor tyrosine kinases in various specific cellular compartments, as well as that of compartment-specific functions of Akt leading to control of several fundamental cellular processes. This spatial and temporal control of Akt activation and function occurs by a large number of parallel molecular mechanisms that are central to regulation of cell physiology.

Staphylococcus aureus Leukocidins Target Endothelial DARC to Cause Lethality in Mice.

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.

Estrogen Inhibits LDL (Low-Density Lipoprotein) Transcytosis by Human Coronary Artery Endothelial Cells via GPER (G-Protein-Coupled Estrogen Receptor) and SR-BI (Scavenger Receptor Class B Type 1).

Objective- The atheroprotective effects of estrogen are independent of circulating lipid levels. Whether estrogen regulates transcytosis of LDL (low-density lipoprotein) across the coronary endothelium is unknown. Approach and Results- Using total internal reflection fluorescence microscopy, we quantified transcytosis of LDL across human coronary artery endothelial cells from multiple donors. LDL transcytosis was significantly higher in cells from men compared with premenopausal women. Estrogen significantly attenuated LDL transcytosis by endothelial cells from male but not female donors; transcytosis of albumin was not affected. Estrogen caused downregulation of endothelial SR-BI (scavenger receptor class B type 1), and overexpression of SR-BI was sufficient to restore LDL transcytosis. Similarly, depletion of SR-BI by siRNA attenuated endothelial LDL transcytosis and prevented any further effect of estrogen. In contrast, treatment with estrogen had no effect on SR-BI expression by liver cells. Inhibition of estrogen receptors α and ß had no effect on estrogen-mediated attenuation of LDL transcytosis. However, estrogen's effect on LDL transcytosis was blocked by depletion of the GPER (G-protein-coupled estrogen receptor). GPER was found to be enriched in endothelial cells compared with hepatocytes and is reported to signal via transactivation of the EGFR (epidermal growth factor receptor); inhibition of EGFR prevented the effect of estrogen on LDL transcytosis and SR-BI mRNA. Last, SR-BI expression was significantly higher in human coronary artery endothelial cells from male compared with premenopausal female donors. Conclusions- Estrogen significantly inhibits LDL transcytosis by downregulating endothelial SR-BI; this effect requires GPER.

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

Influenza Virus Infection Induces Platelet-Endothelial Adhesion Which Contributes to Lung Injury.

Lung injury after influenza infection is characterized by increased permeability of the lung microvasculature, culminating in acute respiratory failure. Platelets interact with activated endothelial cells and have been implicated in the pathogenesis of some forms of acute lung injury. Autopsy studies have revealed pulmonary microthrombi after influenza infection, and epidemiological studies suggest that influenza vaccination is protective against pulmonary thromboembolism; however, the effect of influenza infection on platelet-endothelial interactions is unclear. We demonstrate that endothelial infection with both laboratory and clinical strains of influenza virus increased the adhesion of human platelets to primary human lung microvascular endothelial cells. Platelets adhered to infected cells as well as to neighboring cells, suggesting a paracrine effect. Influenza infection caused the upregulation of von Willebrand factor and ICAM-1, but blocking these receptors did not prevent platelet-endothelial adhesion. Instead, platelet adhesion was inhibited by both RGDS peptide and a blocking antibody to platelet integrin α5ß1, implicating endothelial fibronectin. Concordantly, lung histology from infected mice revealed viral dose-dependent colocalization of viral nucleoprotein and the endothelial marker PECAM-1, while platelet adhesion and fibronectin deposition also were observed in the lungs of influenza-infected mice. Inhibition of platelets using acetylsalicylic acid significantly improved survival, a finding confirmed using a second antiplatelet agent. Thus, influenza infection induces platelet-lung endothelial adhesion via fibronectin, contributing to mortality from acute lung injury. The inhibition of platelets may constitute a practical adjunctive strategy to the treatment of severe infections with influenza.IMPORTANCE There is growing appreciation of the involvement of the lung endothelium in the pathogenesis of severe infections with influenza virus. We have recently shown that the virus can infect human lung endothelial cells, but the functional consequences of this infection are unknown (S. M. Armstrong, C. Wang, J. Tigdi, X. Si, C. Dumpit, S. Charles, A. Gamage, T. J. Moraes, and W. L. Lee, PLoS One 7:e47323, 2012, http://dx.doi.org/10.1371/journal.pone.0047323). Here, we show that this infection causes platelets to adhere to the lung endothelium. Importantly, blocking platelets using two distinct antiplatelet drugs improved survival in a mouse model of severe influenza infection. Thus, platelet inhibition may constitute a novel therapeutic strategy to improve the host response to severe infections with influenza.

A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis.

AIMS: Retention of low-density lipoprotein (LDL) cholesterol beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. Since the overlying endothelium is healthy and intact early on, it is likely that LDL passes through endothelial cells by transcytosis. However, technical challenges have made confirming this notion and elucidating the mechanisms of transcytosis difficult. We developed a novel assay for measuring LDL transcytosis in real time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved. METHODS AND RESULTS: Murine aortas were perfused ex vivo with LDL and dextran of a smaller molecular radius. LDL (but not dextran) accumulated under the endothelium, indicating that LDL transcytosis occurs in intact vessels. We then confirmed that LDL transcytosis occurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosis of DiI-LDL in real time using total internal reflection fluorescence microscopy. DiI-LDL transcytosis was inhibited by excess unlabelled LDL, while degradation of the LDL receptor by PCSK9 had no effect. Instead, LDL colocalized partially with the scavenger receptor SR-BI and overexpression of SR-BI increased LDL transcytosis; knockdown by siRNA significantly reduced it. Excess HDL, the canonical SR-BI ligand, significantly decreased LDL transcytosis. Aortas from SR-BI-deficient mice were perfused ex vivo with LDL and accumulated significantly less sub-endothelial LDL compared with wild-type littermates. CONCLUSION: We developed an assay to quantify LDL transcytosis across endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosis may identify novel targets for the prevention or therapy of atherosclerosis.

The Tie2-agonist Vasculotide rescues mice from influenza virus infection.

Seasonal influenza virus infections cause hundreds of thousands of deaths annually while viral mutation raises the threat of a novel pandemic strain. Antiviral drugs exhibit limited efficacy unless administered early and may induce viral resistance. Thus, targeting the host response directly has been proposed as a novel therapeutic strategy with the added potential benefit of not eliciting viral resistance. Severe influenza virus infections are complicated by respiratory failure due to the development of lung microvascular leak and acute lung injury. We hypothesized that enhancing lung endothelial barrier integrity could improve the outcome. Here we demonstrate that the Tie2-agonist tetrameric peptide Vasculotide improves survival in murine models of severe influenza, even if administered as late as 72 hours after infection; the benefit was observed using three strains of the virus and two strains of mice. The effect required Tie2, was independent of viral replication and did not impair lung neutrophil recruitment. Administration of the drug decreased lung edema, arterial hypoxemia and lung endothelial apoptosis; importantly, Vasculotide is inexpensive to produce, is chemically stable and is unrelated to any Tie2 ligands. Thus, Vasculotide may represent a novel and practical therapy for severe infections with influenza.

Influenza-Induced Priming and Leak of Human Lung Microvascular Endothelium upon Exposure to Staphylococcus aureus.

A major cause of death after influenza virus infection is lung injury due to a bacterial superinfection, yet the mechanism is unknown. Death has been attributed to virus-induced immunosuppression and bacterial overgrowth, but this hypothesis is based on data from the preantibiotic era and animal models that omit antimicrobial therapy. Because of diagnostic uncertainty, most patients with influenza receive antibiotics, making bacterial overgrowth unlikely. Respiratory failure after superinfection presents as acute respiratory distress syndrome, a disorder characterized by lung microvascular leak and edema. The objective of this study was to determine whether the influenza virus sensitizes the lung endothelium to leak upon exposure to circulating bacterial-derived molecular patterns from Staphylococcus aureus. In vitro as well as in vivo models of influenza followed by S. aureus superinfection were used. Molecular mechanisms were explored using molecular biology, knockout mice, and human autopsy specimens. Influenza virus infection sensitized human lung endothelium to leak when challenged with S. aureus, even at low doses of influenza and even when the pathogens were given days apart. Influenza virus increased endothelial expression of TNFR1 both in vitro and in intact lungs, a finding corroborated by human autopsy specimens of patients with influenza. Leak was recapitulated with protein A, a TNFR1 ligand, and sequential infection caused protein A-dependent loss of IκB, cleavage of caspases 8 and 3, and lung endothelial apoptosis. Mice infected sequentially with influenza virus and S. aureus developed significantly increased lung edema that was protein A and TNFR1 dependent. Influenza virus primes the lung endothelium to leak, predisposing patients to acute respiratory distress syndrome upon exposure to S. aureus.

The ileal lipid binding protein is required for efficient absorption and transport of bile acids in the distal portion of the murine small intestine.

The ileal lipid binding protein (ilbp) is a cytoplasmic protein that binds bile acids with high affinity. However evidence demonstrating the role of this protein in bile acid transport and homeostasis is missing. We created a mouse strain lacking ilbp (Fabp6(-/-) mice) and assessed the impact of ilbp deficiency on bile acid homeostasis and transport in vivo. Elimination of ilbp increased fecal bile acid excretion (54.2%, P<0.05) in female but not male Fabp6(-/-) mice. The activity of cholesterol 7α-hydroxylase (cyp7a1), the rate-controlling enzyme of the classical bile acid biosynthetic pathway, was significantly increased in female (63.5%, P<0.05) but not in male Fabp6(-/-) mice. The amount of [(3)H]taurocholic acid (TCA) excreted by 24 h after oral administration was 102% (P<0.025) higher for female Fabp6(-/-) mice whereas it was 57.3% (P<0.01) lower for male Fabp6(-/-) mice, compared to wild-type mice. The retained fraction of the [(3)H]TCA localized in the small and large intestines was increased by 22% (P<0.02) and decreased by 62.7% (P<0.01), respectively, in male Fabp6(-/-) mice relative wild-type mice, whereas no changes were seen in female Fabp6(-/-) mice. Mucosal to serosal bile acid transport using everted distal gut sacs was decreased by 74% (P<0.03) in both sexes of Fabp6(-/-) mice as compared to wild-type mice. The results demonstrate that ilbp is involved in the apical to basolateral transport of bile acids in ileal enterocytes, and is vital for the maintenance of bile acid homeostasis in the enterohepatic circulation (EHC) in mice.