Rattiyaporn Kanlaya

Vascular Leakage in Severe Dengue Virus Infections: A Potential Role for the Nonstructural Viral Protein NS1 and Complement

BACKGROUND Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown. METHODS The major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patients with DSS were also analyzed. RESULTS Soluble NS1 activated complement to completion, and activation was enhanced by polyclonal and monoclonal antibodies against NS1. Complement was also activated by cell-associated NS1 in the presence of specific antibodies. Plasma levels of NS1 and terminal SC5b-9 complexes correlated with disease severity. Large amounts of NS1, complement anaphylatoxin C5a, and the terminal complement complex SC5b-9 were present in pleural fluids from patients with DSS. CONCLUSIONS Complement activation mediated by NS1 leads to local and systemic generation of anaphylatoxins and SC5b-9, which may contribute to the pathogenesis of the vascular leakage that occurs in patients with DHF/DSS.

Alterations in actin cytoskeletal assembly and junctional protein complexes in human endothelial cells induced by dengue virus infection and mimicry of leukocyte transendothelial migration.

Vascular leakage is a hallmark of severe dengue infection. Although extensive studies have been conducted during the past several decades, the molecular mechanisms underlying vascular leakage in dengue shock syndrome (DSS) remain unclear. We thus performed a proteomics study to characterize responses in human endothelial cells (EA.hy926) after DEN-2 virus infection (MOI=10). Comparative 2-D PAGE analysis revealed significantly altered abundance levels of 15 proteins, which were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS). These altered proteins were involved in several biological processes, for example, mRNA stability/processing, transcription and translation regulation, molecular chaperoning, oxidative stress response/regulation, cytoskeletal assembly, protein degradation, and cellular metabolisms. We also performed functional analyses of alterations in actin cytoskeletal assembly and endothelial integrity focusing on adherens junction (VE-cadherin), tight junction (ZO-1) and adhesive molecule (PECAM-1) after 24-h of DEN-2 infection and simulation of transendothelial migration by PECAM-1 cross-linking. Decreased expression and disorganization of the actin-cytoskeleton were observed in the infected cells, whereas the increase in actin stress fibers was found in adjacent noninfected cells. Additionally, a decrease in adhesive protein PECAM-1 was observed. Furthermore, DEN-2 infection caused decreased expression and redistribution of both VE-cadherin and ZO-1, whose changes were enhanced by PECAM-1 engagement. These alterations may potentially be a molecular basis explaining increased endothelial permeability or vascular leakage in DSS.

The Ubiquitin−Proteasome Pathway Is Important for Dengue Virus Infection in Primary Human Endothelial Cells

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are the most severe forms of dengue virus infection with hemorrhage and plasma leakage. However, pathogenic mechanisms of DHF and DSS remain poorly understood. We therefore investigated host responses as determined by changes in the cellular proteome of primary human endothelial cells upon infection with dengue virus serotype 2 (DEN-2) at a multiplicity of infection (MOI) of 10 for 24 h. Two-dimensional PAGE and quantitative intensity analysis revealed 38 significantly altered protein spots (16 upregulated and 22 downregulated) in DEN-2-infected cells compared to mock controls. These altered proteins were successfully identified by mass spectrometry, including those involved in oxidative stress response, transcription and translation, cytoskeleton assembly, protein degradation, cell growth regulation, apoptosis, cellular metabolism, and antiviral response. The proteomic data were validated by Western blot analyses [upregulated ubiquitin-activating enzyme E1 (UBE1) and downregulated annexin A2] and an immunofluorescence study (upregulated MxA). Interestingly, we found that MxA was colocalized with DEN-2 viral capsid protein, strengthening its role as an antiviral protein. Moreover, we also identified upregulation of a proteasome subunit. Our functional study revealed the significant role of ubiquitination in dengue infection and UBE1 inhibition by its specific inhibitor (UBEI-41) caused a significant reduction in the level of viral protein synthesis and its infectivity. Our findings suggest that various biological processes were triggered in response to dengue infection, particularly antiviral IFN and ubiquitin-proteasome pathways.

Vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein 1 and is important for viral replication and release

Our previous study using expression proteomics demonstrated that many proteins, particularly five forms of heterogeneous nuclear ribonucleoproteins (hnRNPs), were up-regulated in human endothelial cells upon dengue virus infection. To address functional significance of these proteins in response to dengue virus infection, we performed a functional proteomics study to identify hnRNPs-interacting proteins in the infected EA.hy926 cells. Immunoprecipitation followed by 2-D PAGE and mass spectrometric analyses revealed 18 and 13 interacting partners of hnRNP C1/C2 and hnRNP K, respectively. Interestingly, vimentin was a common partner for both hnRNP C1/C2 and K. The interaction between vimentin and these hnRNPs was confirmed by reciprocal immunoprecipitation followed by Western blot analysis and also by double immunofluorescence staining. Disruption of vimentin intermediate filament by acrylamide not only dissociated these complexes but also reduced nuclear hnRNPs expression, whereas cytosolic hnRNPs expression was unchanged. We also demonstrated that vimentin was strongly associated with dengue non-structural protein 1 (NS1). Disruption of vimentin intermediate filament not only dissociated this complex but also reduced dengue NS1 expression, as well as viral replication and release. Our data report for the first time that vimentin interacts with hnRNPs and dengue NS1, and plays a crucial role in replication and release of dengue virus.

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.

Protective effect of epigallocatechin-3-gallate (EGCG) via Nrf2 pathway against oxalate-induced epithelial mesenchymal transition (EMT) of renal tubular cells

This study evaluated effect of oxalate on epithelial mesenchymal transition (EMT) and potential anti-fibrotic property of epigallocatechin-3-gallate (EGCG). MDCK renal tubular cells were incubated with 0.5 mM sodium oxalate for 24-h with/without 1-h pretreatment with 25 μM EGCG. Microscopic examination, immunoblotting and immunofluorescence staining revealed that oxalate-treated cells gained mesenchymal phenotypes by fibroblast-like morphological change and increasing expression of vimentin and fibronectin, while levels of epithelial markers (E-cadherin, occludin, cytokeratin and ZO-1) were decreased. EGCG pretreatment could prevent all these changes and molecular mechanisms underlying the prevention by EGCG were most likely due to reduced production of intracellular ROS through activation of Nrf2 signaling and increased catalase anti-oxidant enzyme. Knockdown of Nrf2 by small interfering RNA (siRNA) abrogated all the effects of EGCG, confirming that the EGCG protection against oxalate-induced EMT was mediated via Nrf2. Taken together, our data indicate that oxalate turned on EMT of renal tubular cells that could be prevented by EGCG via Nrf2 pathway. These findings also shed light onto development of novel therapeutics or preventive strategies of renal fibrosis in the future.

Association of Alix with late endosomal lysobisphosphatidic acid is important for dengue virus infection in human endothelial cells.

The most severe form of dengue virus (DENV) infection is dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is accompanied by increased vascular permeability indicating that endothelial cells are the targets of DENV infection. However, molecular mechanisms underlying DENV replication in endothelial cells remained poorly understood. We therefore examined changes in subcellular proteomes of different cellular compartments (including cytosolic, membrane/organelle, nucleus, and cytoskeleton) of human endothelial (EA.hy926) cells upon DENV2 infection using a 2-DE-based proteomics approach followed by Q-TOF MS and MS/MS. A total of 35 altered proteins were identified in these subcellular locales, including an increase in the level of Alix (apoptosis-linked gene-2-interacting protein X) in the cytosolic fraction of DENV2-infected cells compared to mock control cells. Double immunofluorescence staining revealed colocalization of Alix with late endosomal lysobisphosphatidic acid (LBPA). This complex has been proposed to be involved in the export of DENV proteins from late endosomes to the cytoplasm. Subsequent functional study revealed that pretreatment with an anti-LBPA antibody prior to DENV challenge significantly reduced the level of viral envelope protein synthesis and DENV replication. Our data indicate that Alix plays a pivotal role in the early phase of DENV replication, particularly when it arrives at the late endosome stage. Blocking this step may lead to a novel therapeutic approach to reducing the level of DENV replication in vivo.

Cellular adaptive response of distal renal tubular cells to high-oxalate environment highlights surface alpha-enolase as the enhancer of calcium oxalate monohydrate crystal adhesion.

Hyperoxaluria is one of etiologic factors of calcium oxalate kidney stone disease. However, response of renal tubular cells to high-oxalate environment remained largely unknown. We applied a gel-based proteomics approach to characterize changes in cellular proteome of MDCK cells induced by 10mM sodium oxalate. A total of 14 proteins were detected as differentially expressed proteins. The oxalate-induced up-regulation of alpha-enolase in whole cell lysate was confirmed by 2-D Western blot analysis. Interaction network analysis revealed that cellular adaptive response under high-oxalate condition involved stress response, energy production, metabolism and transcriptional regulation. Down-regulation of RhoA, which was predicted to be associated with the identified proteins, was confirmed by immunoblotting. In addition, the up-regulation of alpha-enolase on apical surface of renal tubular epithelial cells was also confirmed by immunoblotting of the isolated apical membranes and immunofluorescence study. Interestingly, blockage of alpha-enolase expressed on the cell surface by antibody neutralization significantly reduced the number of calcium oxalate monohydrate (COM) crystals adhered on the cells. These results strongly suggest that surface alpha-enolase plays an important role as the enhancer of COM crystal binding. The increase of alpha-enolase expressed on the cell surface may aggravate kidney stone formation in patients with hyperoxaluria.

Secreted products of macrophages exposed to calcium oxalate crystals induce epithelial mesenchymal transition of renal tubular cells via RhoA-dependent TGF-β1 pathway.

Kidney stone disease is associated with renal fibrosis by the unclear mechanisms. We hypothesized that calcium oxalate (CaOx), a major crystalline component of kidney stones, could induce secretion of fibrotic factors from macrophages leading to “epithelial mesenchymal transition/transdifferentiation” (EMT) of renal tubular cells. Western blot analysis revealed an increased level of vimentin (mesenchymal marker) but decreased levels of E-cadherin and cytokeratin (epithelial markers) in MDCK cells treated with “secreted products from CaOx-exposed macrophages” (CaOx-M-Sup). Immunofluorescence study confirmed the increased level of vimentin and decreased level of cytokeratin, and also revealed the increased level of fibronectin (another mesenchymal marker). The data also showed decreased levels and disorganization of F-actin (cytoskeletal marker) and zonula occludens-1 (ZO-1) (tight junction marker) induced by CaOx-M-Sup. ELISA demonstrated the increased level of transforming growth factor-β1 (TGF-β1), the well-defined EMT inducer, in CaOx-M-Sup. Downstream signaling of TGF-β1 was involved as demonstrated by the decreased level of RhoA. Interestingly, pretreatment with a proteasome inhibitor (MG132) could restore RhoA to its basal level, most likely through ubiquitin-proteasome pathway (UPP). Moreover, MG132 successfully sustained cytoskeletal assembly and tight junction, and could prevent the cells from EMT. Altogether, these data demonstrate for the first time that CaOx-M-Sup could induce EMT in renal tubular cells by TGF-β1 signaling cascade via RhoA and UPP. This may be, at least in part, the underlying mechanism for renal fibrosis in kidney stone disease.

Macropinocytosis is the Major Mechanism for Endocytosis of Calcium Oxalate Crystals into Renal Tubular Cells

During an initial phase of kidney stone formation, the internalization of calcium oxalate (CaOx) crystals by renal tubular cells has been thought to occur via endocytosis. However, the precise mechanism of CaOx crystal endocytosis remained unclear. In the present study, MDCK renal tubular cells were pretreated with inhibitors specific to individual endocytic pathways, including nystatin (lipid raft/caveolae-mediated), cytochalasin D (actin-dependent or macropinocytosis), and chlorpromazine (CPZ; clathrin-mediated) before exposure to plain (non-labeled), or fluorescence-labeled CaOx monohydrate (COM) crystals. Quantitative analysis by flow cytometry revealed that pretreatment with nystatin and CPZ slightly decreased the crystal internalization, whereas the cytochalasin D pretreatment caused a marked decrease in crystal uptake. Immunofluorescence study and laser-scanning confocal microscopic examination confirmed that the cytochalasin D-pretreated cells had dramatic decrease of the internalized crystals, whereas the total number of crystals interacted with the cells was unchanged (crystals could adhere but were not internalized). These data have demonstrated for the first time that renal tubular cells endocytose COM crystals mainly via macropinocytosis. These novel findings will be useful for further tracking the endocytosed crystals inside the cells during the course of kidney stone formation.