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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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Detection of ANDV infection of primary human dendritic cells. (A) Vero E6 epithelial cells and human immature DCs infected with ADNV (strain CHI-7913). ANDV-nucleocapsid (N) protein detected by IFA by incubating ANDV-infected with a mouse anti-ANDV N MAb revealed by an FITC-conjugated anti-mouse IgG Ab (Green, right column) while the corresponding cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue, left column). Negative control (mock) cells were incubated with supernatant from uninfected Vero-E6 cells. (B) Total RNA was extracted from uninfected LPS-Pulsed DCs (lane 1), ANDV-infected LPS-pulsed DCs (lane 2), uninfected iDCs (lane 3), ANDV-infected iDCs (lane 4), ANDV-infected Vero-E6 cells (lane 5), and used as template in a RT-PCR reaction designed to specifically amplify the viral S RNA. This assay also included a negative RT-PCR control (lane 6). MW is a molecular weight marker (1 Kb, Fermentas, Burlington, Canada). (C) DCs generated from primary monocytes, recovered from four healthy donors, were incubated with ANDV (lanes 1, 4, 7, 10), UV-irradiated ANDV (lanes 2, 5, 8, 11) or pulsed with LPS (lanes 3, 6, 9, 12). Total RNA was extracted from cell supernatants and used as a template in a RT-PCR reaction designed to specifically amplify the viral S RNA. MW is a molecular weight marker (100 pb, Fermentas).
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Figure 1: Detection of ANDV infection of primary human dendritic cells. (A) Vero E6 epithelial cells and human immature DCs infected with ADNV (strain CHI-7913). ANDV-nucleocapsid (N) protein detected by IFA by incubating ANDV-infected with a mouse anti-ANDV N MAb revealed by an FITC-conjugated anti-mouse IgG Ab (Green, right column) while the corresponding cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue, left column). Negative control (mock) cells were incubated with supernatant from uninfected Vero-E6 cells. (B) Total RNA was extracted from uninfected LPS-Pulsed DCs (lane 1), ANDV-infected LPS-pulsed DCs (lane 2), uninfected iDCs (lane 3), ANDV-infected iDCs (lane 4), ANDV-infected Vero-E6 cells (lane 5), and used as template in a RT-PCR reaction designed to specifically amplify the viral S RNA. This assay also included a negative RT-PCR control (lane 6). MW is a molecular weight marker (1 Kb, Fermentas, Burlington, Canada). (C) DCs generated from primary monocytes, recovered from four healthy donors, were incubated with ANDV (lanes 1, 4, 7, 10), UV-irradiated ANDV (lanes 2, 5, 8, 11) or pulsed with LPS (lanes 3, 6, 9, 12). Total RNA was extracted from cell supernatants and used as a template in a RT-PCR reaction designed to specifically amplify the viral S RNA. MW is a molecular weight marker (100 pb, Fermentas).

Mentions: Primary isolate, ANDV strain CHI-7913, was shown to efficiently propagated in the epithelial Vero-E6 cell line (Figure 1A), as previously reported [20], and that their titrated supernatants efficiently targeted and infected human iDCs (Figure 1A). At 4 days post-infection ANDV N-protein was detected by IFA using an anti-ANDV N MAb. Consistent with the cell immunofluorescence data, the presence of ANDV RNA in extensively washed infected DCs was confirmed by an ANDV-specific RT-PCR [21] (Figure 1B). The presence of both, viral antigens and RNA, in DCs does not necessarily imply viral replication, as the observations can be explained by the uptake of exogenous viral particles by cells without productive infection. To check for this option, infected DCs were extensively washed and fresh medium was added. Cell supernatants were recovered, total RNA isolated as described above and subjected to an ANDV-specific RT-PCR. The presence of viral RNA in DC supernatants was confirmed (Figure 1C), suggesting shedding of de novo generated viral particles. To assess the viral infection capacity, supernatants from ANDV-positive DCs were used to infect Vero-E6 cells. Five days post-infection, the presence of ANDV in Vero-E6 cells was confirmed by both IFA and RT-PCR as described above (data not shown). Together these observations strongly suggest that, consistent with other members of the Hantavirus genus of the Bunyaviridae, ANDV is able to infect and replicate in human DCs. Consequently, we explored the possible effects of this viral infection on both DC phenotypes and functionalities.


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)

Detection of ANDV infection of primary human dendritic cells. (A) Vero E6 epithelial cells and human immature DCs infected with ADNV (strain CHI-7913). ANDV-nucleocapsid (N) protein detected by IFA by incubating ANDV-infected with a mouse anti-ANDV N MAb revealed by an FITC-conjugated anti-mouse IgG Ab (Green, right column) while the corresponding cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue, left column). Negative control (mock) cells were incubated with supernatant from uninfected Vero-E6 cells. (B) Total RNA was extracted from uninfected LPS-Pulsed DCs (lane 1), ANDV-infected LPS-pulsed DCs (lane 2), uninfected iDCs (lane 3), ANDV-infected iDCs (lane 4), ANDV-infected Vero-E6 cells (lane 5), and used as template in a RT-PCR reaction designed to specifically amplify the viral S RNA. This assay also included a negative RT-PCR control (lane 6). MW is a molecular weight marker (1 Kb, Fermentas, Burlington, Canada). (C) DCs generated from primary monocytes, recovered from four healthy donors, were incubated with ANDV (lanes 1, 4, 7, 10), UV-irradiated ANDV (lanes 2, 5, 8, 11) or pulsed with LPS (lanes 3, 6, 9, 12). Total RNA was extracted from cell supernatants and used as a template in a RT-PCR reaction designed to specifically amplify the viral S RNA. MW is a molecular weight marker (100 pb, Fermentas).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3104372&req=5

Figure 1: Detection of ANDV infection of primary human dendritic cells. (A) Vero E6 epithelial cells and human immature DCs infected with ADNV (strain CHI-7913). ANDV-nucleocapsid (N) protein detected by IFA by incubating ANDV-infected with a mouse anti-ANDV N MAb revealed by an FITC-conjugated anti-mouse IgG Ab (Green, right column) while the corresponding cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue, left column). Negative control (mock) cells were incubated with supernatant from uninfected Vero-E6 cells. (B) Total RNA was extracted from uninfected LPS-Pulsed DCs (lane 1), ANDV-infected LPS-pulsed DCs (lane 2), uninfected iDCs (lane 3), ANDV-infected iDCs (lane 4), ANDV-infected Vero-E6 cells (lane 5), and used as template in a RT-PCR reaction designed to specifically amplify the viral S RNA. This assay also included a negative RT-PCR control (lane 6). MW is a molecular weight marker (1 Kb, Fermentas, Burlington, Canada). (C) DCs generated from primary monocytes, recovered from four healthy donors, were incubated with ANDV (lanes 1, 4, 7, 10), UV-irradiated ANDV (lanes 2, 5, 8, 11) or pulsed with LPS (lanes 3, 6, 9, 12). Total RNA was extracted from cell supernatants and used as a template in a RT-PCR reaction designed to specifically amplify the viral S RNA. MW is a molecular weight marker (100 pb, Fermentas).
Mentions: Primary isolate, ANDV strain CHI-7913, was shown to efficiently propagated in the epithelial Vero-E6 cell line (Figure 1A), as previously reported [20], and that their titrated supernatants efficiently targeted and infected human iDCs (Figure 1A). At 4 days post-infection ANDV N-protein was detected by IFA using an anti-ANDV N MAb. Consistent with the cell immunofluorescence data, the presence of ANDV RNA in extensively washed infected DCs was confirmed by an ANDV-specific RT-PCR [21] (Figure 1B). The presence of both, viral antigens and RNA, in DCs does not necessarily imply viral replication, as the observations can be explained by the uptake of exogenous viral particles by cells without productive infection. To check for this option, infected DCs were extensively washed and fresh medium was added. Cell supernatants were recovered, total RNA isolated as described above and subjected to an ANDV-specific RT-PCR. The presence of viral RNA in DC supernatants was confirmed (Figure 1C), suggesting shedding of de novo generated viral particles. To assess the viral infection capacity, supernatants from ANDV-positive DCs were used to infect Vero-E6 cells. Five days post-infection, the presence of ANDV in Vero-E6 cells was confirmed by both IFA and RT-PCR as described above (data not shown). Together these observations strongly suggest that, consistent with other members of the Hantavirus genus of the Bunyaviridae, ANDV is able to infect and replicate in human DCs. Consequently, we explored the possible effects of this viral infection on both DC phenotypes and functionalities.

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