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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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Secretion of active gMMP-9 and endothelial permeability enhancement are triggered by ANDV infection of iDCs. (A) The gMMP-9 expression in ANDV-infected iDCs was assessed by Western blotting. Uninfected iDCs (Mock), ANDV-infected iDCs (ANDV) and LPS-treated iDCs (LPS) were collected 3 h post-infection. Forty micrograms of total proteins from cell lysates were separated in an 8% SDS-PAGE gel. Following an electrical protein transfer onto a nitrocellulose membrane, an anti-gMMP-9 antibody was used to probe the presence of gMMP-9 that was revealed using an HRP-conjugated anti-mouse IgG Ab. (B) Supernatants from ANDV-infected DCs or Mock cells collected 3 h post-infection. Gelatinase activity was assayed by zymography. Gelatinolytic activity was quantified by gel densitometry using the Image J Software. Data are presented as the fold-increase of gMMP-9 activity in supernatants. Statistical significance (**, p < 0.01) was determined from five independent experiments. (C) Enhancement of the endothelial cells permeability induced by supernatant from ANDV-infected iDCs. HUVEC confluent monolayers plated onto collagen-coated transwell inserts were incubated with either ANDV supernatant, mock control (uninfected DCs supernatant) or with TNF-α (50 ng/ml) as positive control. Following 18 h at 37°C in CO2 5% after addition, within the top chamber, of 500 μg/ml FITC-conjugated Dextran, paracellular permeability was measured by reading in the bottom chamber containing the infiltrated FITC-dextran at an excitation wavelength of 485 nm and an emission of 530 nm. Data represent means of five independent experiments. **, p < 0.01.
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Figure 4: Secretion of active gMMP-9 and endothelial permeability enhancement are triggered by ANDV infection of iDCs. (A) The gMMP-9 expression in ANDV-infected iDCs was assessed by Western blotting. Uninfected iDCs (Mock), ANDV-infected iDCs (ANDV) and LPS-treated iDCs (LPS) were collected 3 h post-infection. Forty micrograms of total proteins from cell lysates were separated in an 8% SDS-PAGE gel. Following an electrical protein transfer onto a nitrocellulose membrane, an anti-gMMP-9 antibody was used to probe the presence of gMMP-9 that was revealed using an HRP-conjugated anti-mouse IgG Ab. (B) Supernatants from ANDV-infected DCs or Mock cells collected 3 h post-infection. Gelatinase activity was assayed by zymography. Gelatinolytic activity was quantified by gel densitometry using the Image J Software. Data are presented as the fold-increase of gMMP-9 activity in supernatants. Statistical significance (**, p < 0.01) was determined from five independent experiments. (C) Enhancement of the endothelial cells permeability induced by supernatant from ANDV-infected iDCs. HUVEC confluent monolayers plated onto collagen-coated transwell inserts were incubated with either ANDV supernatant, mock control (uninfected DCs supernatant) or with TNF-α (50 ng/ml) as positive control. Following 18 h at 37°C in CO2 5% after addition, within the top chamber, of 500 μg/ml FITC-conjugated Dextran, paracellular permeability was measured by reading in the bottom chamber containing the infiltrated FITC-dextran at an excitation wavelength of 485 nm and an emission of 530 nm. Data represent means of five independent experiments. **, p < 0.01.

Mentions: Upon antigen capture, DCs undergo a process of maturation and a lymph nodes homing, where they activate the adaptive immune system. The process of DC trafficking is complex. This phenomenon requires the enhancement of the vascular permeability, which could involve the expression of several components, such as cell to cell junction proteins including VE-cadherin, PECAM-1, occludin, claudins, as well as soluble factors, including, IL-1β, TNF-α, VEGF, and Kinins [32-34]. The gelatinolytic matrix metalloproteases (gMMP)-2 and 9 are reported as one of the major actors of this crucial phenomenon during fetal development and wound healing [35]. Interestingly, factors such as the inflammatory cytokines IL-8, TNF-α and IL-1β as well as VEGF [36] are in the upstream of the gMMP expression pathway in human monocytes [37]. Additionally, even if iDC produces gMMP, DC increase the MMP-9 production along DC maturation progression [38]. Interestingly, certain viruses can play opposite roles in DC maturation, for example the human Cytomegalovirus (hCMV) inhibits cell maturation to escape the immune system (4), whereas some others, such as HIV-1 [18], Dengue virus [11], and West Nile virus [39], enhance DC maturation, the secretion of active gMMP-9, as well as plasma vascular leakage. Thus, to extend our previous observation on the fact that DCs have a proinflammatory profile that can be compatible with the secretion of gMMP-9, we analyzed the expression and activity of gMMP-9 in cells and its presence in DC supernatants (SN). Selection seemed adequate as the levels of IL-10, which has been reported to inhibit MMP-9 induction, is weakly altered by ADNV infection (Figure 3B) and DC maturation is known to increase the level of active MMP-9 secretion. Intra-cellular expression of gMMP-9 in ANDV-infected DCs (3 h-post infection) was characterized by Western blotting (Figure 4A), using a mouse an anti-gMMP-9 MAb. Data show that gMMP-9 expression is indeed elevated in ANDV-infected DCs as compared with mock iDCs (Figure 4A). Gelatinolytic MMP-9 is secreted as a proenzyme, which remains inactive unless it is activated by the removal of the propeptide domain by proteolytic enzymes. The gelatinolytic activity of gMMP-9 was therefore assessed in cell supernatants (SN) by gelatin zymography [18]. Strikingly, ANDV-infected DC supernatants exhibited an elevated gMMP-9 activity as compared with SN-mock (Figure 4B). Furthermore, gMMP-9 activity was similar to that obtained by the LPS-induction of DC maturation (SN-LPS; Figure 4B).


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)

Secretion of active gMMP-9 and endothelial permeability enhancement are triggered by ANDV infection of iDCs. (A) The gMMP-9 expression in ANDV-infected iDCs was assessed by Western blotting. Uninfected iDCs (Mock), ANDV-infected iDCs (ANDV) and LPS-treated iDCs (LPS) were collected 3 h post-infection. Forty micrograms of total proteins from cell lysates were separated in an 8% SDS-PAGE gel. Following an electrical protein transfer onto a nitrocellulose membrane, an anti-gMMP-9 antibody was used to probe the presence of gMMP-9 that was revealed using an HRP-conjugated anti-mouse IgG Ab. (B) Supernatants from ANDV-infected DCs or Mock cells collected 3 h post-infection. Gelatinase activity was assayed by zymography. Gelatinolytic activity was quantified by gel densitometry using the Image J Software. Data are presented as the fold-increase of gMMP-9 activity in supernatants. Statistical significance (**, p < 0.01) was determined from five independent experiments. (C) Enhancement of the endothelial cells permeability induced by supernatant from ANDV-infected iDCs. HUVEC confluent monolayers plated onto collagen-coated transwell inserts were incubated with either ANDV supernatant, mock control (uninfected DCs supernatant) or with TNF-α (50 ng/ml) as positive control. Following 18 h at 37°C in CO2 5% after addition, within the top chamber, of 500 μg/ml FITC-conjugated Dextran, paracellular permeability was measured by reading in the bottom chamber containing the infiltrated FITC-dextran at an excitation wavelength of 485 nm and an emission of 530 nm. Data represent means of five independent experiments. **, p < 0.01.
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Figure 4: Secretion of active gMMP-9 and endothelial permeability enhancement are triggered by ANDV infection of iDCs. (A) The gMMP-9 expression in ANDV-infected iDCs was assessed by Western blotting. Uninfected iDCs (Mock), ANDV-infected iDCs (ANDV) and LPS-treated iDCs (LPS) were collected 3 h post-infection. Forty micrograms of total proteins from cell lysates were separated in an 8% SDS-PAGE gel. Following an electrical protein transfer onto a nitrocellulose membrane, an anti-gMMP-9 antibody was used to probe the presence of gMMP-9 that was revealed using an HRP-conjugated anti-mouse IgG Ab. (B) Supernatants from ANDV-infected DCs or Mock cells collected 3 h post-infection. Gelatinase activity was assayed by zymography. Gelatinolytic activity was quantified by gel densitometry using the Image J Software. Data are presented as the fold-increase of gMMP-9 activity in supernatants. Statistical significance (**, p < 0.01) was determined from five independent experiments. (C) Enhancement of the endothelial cells permeability induced by supernatant from ANDV-infected iDCs. HUVEC confluent monolayers plated onto collagen-coated transwell inserts were incubated with either ANDV supernatant, mock control (uninfected DCs supernatant) or with TNF-α (50 ng/ml) as positive control. Following 18 h at 37°C in CO2 5% after addition, within the top chamber, of 500 μg/ml FITC-conjugated Dextran, paracellular permeability was measured by reading in the bottom chamber containing the infiltrated FITC-dextran at an excitation wavelength of 485 nm and an emission of 530 nm. Data represent means of five independent experiments. **, p < 0.01.
Mentions: Upon antigen capture, DCs undergo a process of maturation and a lymph nodes homing, where they activate the adaptive immune system. The process of DC trafficking is complex. This phenomenon requires the enhancement of the vascular permeability, which could involve the expression of several components, such as cell to cell junction proteins including VE-cadherin, PECAM-1, occludin, claudins, as well as soluble factors, including, IL-1β, TNF-α, VEGF, and Kinins [32-34]. The gelatinolytic matrix metalloproteases (gMMP)-2 and 9 are reported as one of the major actors of this crucial phenomenon during fetal development and wound healing [35]. Interestingly, factors such as the inflammatory cytokines IL-8, TNF-α and IL-1β as well as VEGF [36] are in the upstream of the gMMP expression pathway in human monocytes [37]. Additionally, even if iDC produces gMMP, DC increase the MMP-9 production along DC maturation progression [38]. Interestingly, certain viruses can play opposite roles in DC maturation, for example the human Cytomegalovirus (hCMV) inhibits cell maturation to escape the immune system (4), whereas some others, such as HIV-1 [18], Dengue virus [11], and West Nile virus [39], enhance DC maturation, the secretion of active gMMP-9, as well as plasma vascular leakage. Thus, to extend our previous observation on the fact that DCs have a proinflammatory profile that can be compatible with the secretion of gMMP-9, we analyzed the expression and activity of gMMP-9 in cells and its presence in DC supernatants (SN). Selection seemed adequate as the levels of IL-10, which has been reported to inhibit MMP-9 induction, is weakly altered by ADNV infection (Figure 3B) and DC maturation is known to increase the level of active MMP-9 secretion. Intra-cellular expression of gMMP-9 in ANDV-infected DCs (3 h-post infection) was characterized by Western blotting (Figure 4A), using a mouse an anti-gMMP-9 MAb. Data show that gMMP-9 expression is indeed elevated in ANDV-infected DCs as compared with mock iDCs (Figure 4A). Gelatinolytic MMP-9 is secreted as a proenzyme, which remains inactive unless it is activated by the removal of the propeptide domain by proteolytic enzymes. The gelatinolytic activity of gMMP-9 was therefore assessed in cell supernatants (SN) by gelatin zymography [18]. Strikingly, ANDV-infected DC supernatants exhibited an elevated gMMP-9 activity as compared with SN-mock (Figure 4B). Furthermore, gMMP-9 activity was similar to that obtained by the LPS-induction of DC maturation (SN-LPS; Figure 4B).

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