Infection of human monocyte-derived dendritic cells by ANDES Hantavirus enhances pro-inflammatory state, the secretion of active MMP-9 and indirectly enhances endothelial permeability
© Marsac et al; licensee BioMed Central Ltd. 2011
Received: 1 April 2011
Accepted: 13 May 2011
Published: 13 May 2011
Andes virus (ANDV), a rodent-borne Hantavirus, is the major etiological agent of Hantavirus cardiopulmonary syndrome (HCPS) in South America, which is mainly characterized by a vascular leakage with high rate of fatal outcomes for infected patients. Currently, neither specific therapy nor vaccines are available against this pathogen. ANDV infects both dendritic and epithelial cells, but in despite that the severity of the disease directly correlates with the viral RNA load, considerable evidence suggests that immune mechanisms rather than direct viral cytopathology are responsible for plasma leakage in HCPS. Here, we assessed the possible effect of soluble factors, induced in viral-activated DCs, on endothelial permeability. Activated immune cells, including DC, secrete gelatinolytic matrix metalloproteases (gMMP-2 and -9) that modulate the vascular permeability for their trafficking.
A clinical ANDES isolate was used to infect DC derived from primary PBMC. Maturation and pro-inflammatory phenotypes of ANDES-infected DC were assessed by studying the expression of receptors, cytokines and active gMMP-9, as well as some of their functional status. The ANDES-infected DC supernatants were assessed for their capacity to enhance a monolayer endothelial permeability using primary human vascular endothelial cells (HUVEC).
Here, we show that in vitro primary DCs infected by a clinical isolate of ANDV shed virus RNA and proteins, suggesting a competent viral replication in these cells. Moreover, this infection induces an enhanced expression of soluble pro-inflammatory factors, including TNF-α and the active gMMP-9, as well as a decreased expression of anti-inflammatory cytokines, such as IL-10 and TGF-β. These viral activated cells are less sensitive to apoptosis. Moreover, supernatants from ANDV-infected DCs were able to indirectly enhance the permeability of a monolayer of primary HUVEC.
Primary human DCs, that are primarily targeted by hantaviruses can productively be infected by ANDV and subsequently induce direct effects favoring a proinflammatory phenotype of infected DCs. Finally, based on our observations, we hypothesize that soluble factors secreted in ANDV-infected DC supernatants, importantly contribute to the endothelial permeability enhancement that characterize the HCPS.
Hantaviruses are rodent-born enveloped RNA-viruses belonging to Bunyaviridae family. Two major severe pathologies associated to Hantaviruses have been reported: hemorrhagic fever with renal syndrome (HFRS) in the Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. HCPS is more frequently associated (40%) to fatal outcomes than HFRS (<1%) . Andes Hantavirus (ANDV) is the major etiological agent of the HCPS in South America, syndrome characterized by the presence of high amounts of pulmonary fluids leading to an edema evolving to a cardiogenic shock that synergistically acts with hypovolemia due to capillary leakage resulting in an abrupt cardiopulmonary collapse . Although disease severity directly correlates with the viral RNA load , considerable evidence exists suggesting that immune mechanisms rather than direct viral cytopathology are indeed responsible for the massive vascular dysfunction and plasma leakage of HFRS and HCPS [4, 5].
The hemorrhagic viruses, including the members of the Bunyaviridae as well as dengue viruses, target endothelial cells and immune cells, mainly monocyte-derived cells such as the professional antigen-presenting cells, Dendritic cells (DCs) [6–8]. DCs activation triggers their maturation and trans-endothelial migration occurring during wound healing or inflammation. These processes require extracellular matrix remodeling and involve changes in endothelial permeability regulated by the production of matrix metalloproteases (gMMPs) or vascular endothelial growth factor (VEGF). However, in excess, these soluble factors can have deleterious effects on endothelial cell integrity. Data from different reports show that endothelial cells infected by dengue virus trigger secretion of soluble factors such as VEGF and the decrease of VEGF-R2 receptor [9, 10]. We have recently reported in vitro and in vivo showing that soluble factors secreted from DV-infected DCs enhance endothelial permeability and down-regulate expression of endothelial junction proteins, Pecam-1 and VE-cadherin in a gMMP-9-dependent manner . More recently, complementary and convergent studies, to our own previous data on dengue, have reported that Hantavirus-infected endothelial cells enhances the permeability via the reduction of VE-cadherin expression due to its dissociation with VEGF-receptor2 (VEGF-R2) which, in turn, become associated with VEGF [12, 13]. An accurate understanding of Hantavirus pathogenesis is pivotal to design de novo therapeutic or vaccine approaches that are still lacking against this hemorrhagic viral infection. In this study, we show that ANDV-infected DC are quickly activated and rapidly progress to an intermediate maturation and pro-inflammatory state that contributes to the increase of soluble factors in their supernatant able to trigger the enhancement of endothelial permeability.
Virus and cells
The primary isolate, ANDV strain CHI-7913 was propagated in the epithelial Vero-E6 cell line (ATCC CRL 1586). Titrated supernatants of these cells were used to infect, at a MOI of 1 for 2 h, human iDCs derived from peripheral blood monocytes (PBMC), as previously described . In these experiments, UV (λ: 250 nm; 15 min)-irradiated ANDV was used as the negative control. Four days post-DC infection, ANDV N-protein was detected by indirect immunofluorescence (IFA) using a well characterized anti-ANDV N monoclonal antibody (MAb) . Total RNA was extracted using the High Pure viral nucleic acid kit (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacture's protocol and 1 μl of total RNA was amplified in a one step RT-PCR (SuperScript III One-Step RT-PCR with Platinum Taq, Invitrogen) using primers that recognize the nucleocapsid coding region (forward primer: 5' ACA CGA ACA ACA GCT CGT GAC '3 and reverse primer: 5' AGG CTC AAG CCC TGT TGG ATC '3). To assess the viral infectivity, from ANDV-positive DCs, their supernatants were used to infect Vero-E6 cells.
Phenotype profiling of ANDES-infected DC
The influence of ANDV (3 h post-viral) infection of iDCs (as described above) was assessed through the expression of key cell surface proteins CD80, CD83, CD86 and HLA-DR, known to be associated with the mature DCs (mDCs) phenotype. Pro-inflammatory profile of ANDV-infected iDCs was assessed through the expression level of some pro and anti-inflammatory cytokines as well as gMMP. As infection of iDCs by other Hantaviruses induces the pro-inflammatory cytokines expression . Lipopolysaccharide (LPS)-pulsed iDCs were used as a positive control for iDCs maturation, while iDCs incubated with fresh culture medium (mock) were used as a negative control. Supernatants of Mock-DCs, ANDV-infected DCs, and LPS-pulsed DCs were assessed for the production of the pro-inflammatory cytokine, TNF-α, (ELISA kit OptEIA™ Human TNF ELISA Kit II; Pharmingen, Franklin Lakes, NJ).
Functional assessment of ANDV-infected DC
The effect of ANDV infection on the endocytic capacity of DCs was assessed by measuring the FITC-conjugated Dextran up-take at 37°C . In this assay, 18 h LPS-pulsed iDCs were used as a control for cell maturation assessment, while iDCs, incubated at 4°C, were used as a control for the immature stage with lowest capacities of endocytosis. The apoptotic level of ANDV-infected DCs was assessed by flow cytometry using the Annexin V assay  using the same previous controls.
Gelatinolytic MMP-9 secretion
Intra-cellular expression of gMMP-9 in ANDV-infected DCs (3 h-post infection) was characterized by Western blotting, using a mouse anti-gMMP-9 MAb (MAB13416; Chemicon International, Temecula, CA). The gelatinolytic activity of gMMP-9 DC supernatants (SN) was assessed by gelatin zymography . In these experiments fresh medium (SN-Mock) was used as a negative control for maturation, while medium containing LPS (1 μg/ml) (SN-LPS) was used as a positive control.
Endothelial monolayer permeability assessment
The capacity to modify the vascular permeability of ANDV-infected DC supernatants was measured in vitro, using a monolayer of primary human umbilical vascular endothelial cells (HUVEC)  Briefly, HUVEC obtained from pooled donors were plated onto collagen-coated transwell inserts (0.4 μm-pore, 6.5 mm-diameter Transwell-COL; Costar) and once in confluence cells, were exposed for 18 h to mock-SN, virus- free SN from ANDV infected DCs, or to TNF-α (50 nM). Monolayer endothelial permeability was assayed with the standard cell permeability assay according to the Chemicon ECM-640 procedure (Millipore), by adding FITC-conjugated dextran to the upper chamber of the transwell inserts and evaluating the migration of FITC-conjugated Dextran to the lower chamber by using a TECAN fluorometer at 490/530 nm. In this transwell system, endothelial monolayer permeability is directly proportional to the flux of 70-kDa dextran passing through the HUVEC monolayer.
Data sets were compared using Kruskal & Wallis analysis of variance was performed with the GraphPad Prism 4.03 software. Two-sided p-values were considered.
Results and Discussion
Immature DCs efficiently capture and process antigen, a characteristic cell function that decreases with maturation [7, 8, 24–27]. Therefore, we next sought to establish whether the partial DC maturation phenotype (Figure 2B) was correlated with its decreased ability to capture antigens. To this purpose, we studied the effect of ANDV infection on the endocytic capacity of DCs by measuring the up-take of FITC-conjugated Dextran at 37°C . Results indicate that ANDV-infected DC exhibited a reduced endocytic capacity as compared to uninfected iDCs (Figure 2C). However, in our conditions, infected DCs exhibited a higher endocytic activity than the used dose of LPS to pulse iDCs (positive control). Together, these observations suggest that ANDV infection does indeed reduce antigen capture by DCs, supporting a role of ANDV infection in cell maturation.
ANDV belongs to the group of hemorrhagic fever viruses known for inducing different forms of vascular leakage in different organs. For some of these hemorrhagic fever or encephalitic viruses the mechanism associated to their capacity to enhance endothelial permeability in vitro and in vivo has been reported [11, 18, 39]. Upon viral infection, the vascular permeability phenomenon is principally due to viral-dependent dysfunctions of endothelial cells junctions that can be directly induced by the action of factors from viral activated endothelial cells, such as VEGF or indirectly induced by factors from immune viral-activated cells, via the MAP-kinase pathway [11, 18]. For these reasons, we wonder if ANDV infection induced the production of soluble factors that could indirectly enhance endothelial permeability. To evaluate this possibility, the capacity of ANDV infected DCs-supernatants to modify the permeability was measured through the amounts of FITC-conjugated dextran that passed thru a monolayer of primary human umbilical vascular endothelial cells (HUVEC) seeded onto transwell plates that considered as vascular model . Following the exposure of confluent HUVEC to SN from ANDV infected DCs, or to controls, the amounts of FITC-conjugated dextran detected in the lower chamber of the transwell system (Figure 4C) suggest that cell supernatant from ANDV-infected DCs contains soluble and active factors favoring the increase of the HUVEC monolayer permeability.
Altogether, the in vitro data presented herein show that ANDV infects and replicates in DCs, inducing a partial maturation and pro-inflammatory phenotype, while increasing the expression and secretion of active gMMP-9 as well as that supernatants from ANDV-infected DCs were able to enhance the permeability of a monolayer of primary human vascular endothelial cells.
The herein presented data and data recently published in the literature are not only convergent but also complementary, they allow to speculate that both indirect effects of ANDV on DCs and direct effects of pathogenic hantaviruses, such as ANDV, on endothelial cells generate conjugated inflammatory consequences that result in the increased endothelial permeability associated to this virus infection. A better understanding of major mechanisms involved in vascular pathogenesis observed in HCPS is pivotal to progress in the design of therapies that today remain unavailable for this serious life-threatening hemorrhagic viral disease.
DM was a FONDECYT post-doctorate fellowship; SG is PhD student and KP is a technician at the MLL lab; AF is from the technical staff of the FV lab.
We thank Dr. M. Rau (Oxford, UK) for critical reading and editing of the manuscript. We are grateful to Dr. Nicole Tischler, Fundación Ciencias para la Vida, Santiago-Chile, for kindly providing the anti-N MAb used in this study. We thank colleagues from the Pontificia Universidad Católica de Chile: Prof. Roberto Ebenspenger and Lorena Rubio for kindly providing HUVECs; Connie Martinez for titrated viruses. The present study was supported by CONICYT through grant FONDECYT-POSTDOCTORADO-2008-3085029 to DM, FONDECYT N° 1100756, and PHS grant 2U01AI045452-11 to MLL, Proyecto Instituto Milenio P-07-088-F to AK and MLL, Chile. We also thank IRD and University Montpellier 1, France, for supporting a part of this study.
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