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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 D, García S, Fournet A, Aguirre A, Pino K, Ferres M, Kalergis AM, Lopez-Lastra M, Veas F - Virol. J. (2011)

Bottom Line: Currently, neither specific therapy nor vaccines are available against this pathogen.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: UMR-MD3-University Montpellier 1, Comparative Molecular Immuno-Physiopathology Lab, Faculté de Pharmacie, 34093 Montpellier, France.

ABSTRACT

Background: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

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Impact of ANDES virus infection on iDC phenotype and functions. (A) The impact of ANDV infection on DCs viability was detected by using the Annexin V-propidium iodide (PI) method. In these assays, DCs treated with camptothecin A (4 μM for 18 h) were used as a positive apoptosis control. (B) DCs surface markers; CD80, CD86, CD83 and HLA-DR were analyzed in ANDV-infected iDCs by flow cytometry four days post-ANDV infection, while LPS-pulsed DCs and uninfected iDCs (mock) were used as controls. Bar graphs represent the fold-increase expression of these surface markers as compared their expression in mock control. Data are means of three independent experiments: *, p< 0.05; **, p < 0.01. (C) The endocytic capacity of ANDES infected iDCs, LPS-matured DCs (mDC), uninfected iDCs incubated at 37°C (iDC 37), and uninfected iDCs incubated at 4°C (iDC 4) was assessed using a FITC-conjugated Dextran (30 μg). Endocytosis was analyzed by flow cytometry after 2 h of incubation. Mean fluorescence intensity values within the gate for the different endocytically active stages were plotted. Bar graphs show the fold-increases of the mean fluorescence intensities (MFI), relative to the mock control (iDCs 37). For each experiment, 10 000 gated cells were evaluated. Data are means of five independent experiments. *, p< 0.05; **, p < 0.01.
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Figure 2: Impact of ANDES virus infection on iDC phenotype and functions. (A) The impact of ANDV infection on DCs viability was detected by using the Annexin V-propidium iodide (PI) method. In these assays, DCs treated with camptothecin A (4 μM for 18 h) were used as a positive apoptosis control. (B) DCs surface markers; CD80, CD86, CD83 and HLA-DR were analyzed in ANDV-infected iDCs by flow cytometry four days post-ANDV infection, while LPS-pulsed DCs and uninfected iDCs (mock) were used as controls. Bar graphs represent the fold-increase expression of these surface markers as compared their expression in mock control. Data are means of three independent experiments: *, p< 0.05; **, p < 0.01. (C) The endocytic capacity of ANDES infected iDCs, LPS-matured DCs (mDC), uninfected iDCs incubated at 37°C (iDC 37), and uninfected iDCs incubated at 4°C (iDC 4) was assessed using a FITC-conjugated Dextran (30 μg). Endocytosis was analyzed by flow cytometry after 2 h of incubation. Mean fluorescence intensity values within the gate for the different endocytically active stages were plotted. Bar graphs show the fold-increases of the mean fluorescence intensities (MFI), relative to the mock control (iDCs 37). For each experiment, 10 000 gated cells were evaluated. Data are means of five independent experiments. *, p< 0.05; **, p < 0.01.

Mentions: The impact of DC infection by ADNV on cell viability, maturation and endocytosis was assessed. The apoptotic level, evaluated by flow cytometry using the Annexin V assay [17], showed that infection of iDCs with ANDV does not affect cell viability (Figure 2A), observations in line with what has been reported for other hantaviruses [6,22]. In addition, ANDV infection did not induce any detectable cytopathic effect (data not shown). DCs play a pivotal role as antigen-presenting cells in the antiviral immune response. It is known that infection of iDCs by diverse viruses stimulates cell homing to inflammatory sites as well as their maturation into antigen-presenting cells (APC), a process essential for the initiation and modulation of T cell-mediated immune responses. Hence, we next evaluated whether ANDV infection of iDCs had a direct effect on the expression of key cell surface proteins CD80, CD83, CD86 and HLA-DR, known to be associated with a mature DCs (mDCs) phenotype. Immature DCs were infected with ANDV as described above. ANDV infection of iDCs induced a significant increase of HLA-DR, a marker protein that plays a pivotal role in guiding the development and activation of CD4+ T helper cells. Markers CD83 and CD86 also increased, albeit to a much lower extent than in LPS-pulsed iDCs conditions. As expected, expression of all surface markers increased in LPS-pulsed iDCs as compared to mock-DCs (Figure 2B). These data suggest that ANDV infection of human DCs induced cell maturation, and that ANDV infection alters the expression of HLA-DR on DCs. Constitutive expression of HLA-DR molecules is associated to professional antigens presenting cells (APCs) such as DCs, this basal expression can be enhanced in an environment enriched with proinflammatory cytokines [7,23].


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 D, García S, Fournet A, Aguirre A, Pino K, Ferres M, Kalergis AM, Lopez-Lastra M, Veas F - Virol. J. (2011)

Impact of ANDES virus infection on iDC phenotype and functions. (A) The impact of ANDV infection on DCs viability was detected by using the Annexin V-propidium iodide (PI) method. In these assays, DCs treated with camptothecin A (4 μM for 18 h) were used as a positive apoptosis control. (B) DCs surface markers; CD80, CD86, CD83 and HLA-DR were analyzed in ANDV-infected iDCs by flow cytometry four days post-ANDV infection, while LPS-pulsed DCs and uninfected iDCs (mock) were used as controls. Bar graphs represent the fold-increase expression of these surface markers as compared their expression in mock control. Data are means of three independent experiments: *, p< 0.05; **, p < 0.01. (C) The endocytic capacity of ANDES infected iDCs, LPS-matured DCs (mDC), uninfected iDCs incubated at 37°C (iDC 37), and uninfected iDCs incubated at 4°C (iDC 4) was assessed using a FITC-conjugated Dextran (30 μg). Endocytosis was analyzed by flow cytometry after 2 h of incubation. Mean fluorescence intensity values within the gate for the different endocytically active stages were plotted. Bar graphs show the fold-increases of the mean fluorescence intensities (MFI), relative to the mock control (iDCs 37). For each experiment, 10 000 gated cells were evaluated. Data are means of five independent experiments. *, p< 0.05; **, p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Impact of ANDES virus infection on iDC phenotype and functions. (A) The impact of ANDV infection on DCs viability was detected by using the Annexin V-propidium iodide (PI) method. In these assays, DCs treated with camptothecin A (4 μM for 18 h) were used as a positive apoptosis control. (B) DCs surface markers; CD80, CD86, CD83 and HLA-DR were analyzed in ANDV-infected iDCs by flow cytometry four days post-ANDV infection, while LPS-pulsed DCs and uninfected iDCs (mock) were used as controls. Bar graphs represent the fold-increase expression of these surface markers as compared their expression in mock control. Data are means of three independent experiments: *, p< 0.05; **, p < 0.01. (C) The endocytic capacity of ANDES infected iDCs, LPS-matured DCs (mDC), uninfected iDCs incubated at 37°C (iDC 37), and uninfected iDCs incubated at 4°C (iDC 4) was assessed using a FITC-conjugated Dextran (30 μg). Endocytosis was analyzed by flow cytometry after 2 h of incubation. Mean fluorescence intensity values within the gate for the different endocytically active stages were plotted. Bar graphs show the fold-increases of the mean fluorescence intensities (MFI), relative to the mock control (iDCs 37). For each experiment, 10 000 gated cells were evaluated. Data are means of five independent experiments. *, p< 0.05; **, p < 0.01.
Mentions: The impact of DC infection by ADNV on cell viability, maturation and endocytosis was assessed. The apoptotic level, evaluated by flow cytometry using the Annexin V assay [17], showed that infection of iDCs with ANDV does not affect cell viability (Figure 2A), observations in line with what has been reported for other hantaviruses [6,22]. In addition, ANDV infection did not induce any detectable cytopathic effect (data not shown). DCs play a pivotal role as antigen-presenting cells in the antiviral immune response. It is known that infection of iDCs by diverse viruses stimulates cell homing to inflammatory sites as well as their maturation into antigen-presenting cells (APC), a process essential for the initiation and modulation of T cell-mediated immune responses. Hence, we next evaluated whether ANDV infection of iDCs had a direct effect on the expression of key cell surface proteins CD80, CD83, CD86 and HLA-DR, known to be associated with a mature DCs (mDCs) phenotype. Immature DCs were infected with ANDV as described above. ANDV infection of iDCs induced a significant increase of HLA-DR, a marker protein that plays a pivotal role in guiding the development and activation of CD4+ T helper cells. Markers CD83 and CD86 also increased, albeit to a much lower extent than in LPS-pulsed iDCs conditions. As expected, expression of all surface markers increased in LPS-pulsed iDCs as compared to mock-DCs (Figure 2B). These data suggest that ANDV infection of human DCs induced cell maturation, and that ANDV infection alters the expression of HLA-DR on DCs. Constitutive expression of HLA-DR molecules is associated to professional antigens presenting cells (APCs) such as DCs, this basal expression can be enhanced in an environment enriched with proinflammatory cytokines [7,23].

Bottom Line: Currently, neither specific therapy nor vaccines are available against this pathogen.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: UMR-MD3-University Montpellier 1, Comparative Molecular Immuno-Physiopathology Lab, Faculté de Pharmacie, 34093 Montpellier, France.

ABSTRACT

Background: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus