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Differences in type I interferon signaling antagonism by dengue viruses in human and non-human primate cell lines.

Medina FA, Torres-Malavé G, Chase AJ, Santiago GA, Medina JF, Santiago LM, Muñoz-Jordán JL - PLoS Negl Trop Dis (2015)

Bottom Line: The ability of DENVs to inhibit IFN-α/β signaling is conserved.Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes.DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Dengue Branch, San Juan, Puerto Rico, United States of America.

ABSTRACT

Background/objectives: In vitro studies have shown that dengue virus (DENV) can thwart the actions of interferon (IFN)-α/β and prevent the development of an antiviral state in infected cells. Clinical studies looking at gene expression in patients with severe dengue show a reduced expression of interferon stimulated genes compared to patients with dengue fever. Interestingly, there are conflicting reports as to the ability of DENV or other flaviviruses to inhibit IFN-α/β signaling.

Methodology/principal findings: In order to determine the relative inhibition of IFN-α/β signaling by DENVs, a method combining flow cytometry and a four-parameter logistic regression model was established. A representative isolate from DENV-1, -3 and -4 and seventeen representative isolates encompassing all DENV-2 genotypes were evaluated. All of the DENVs evaluated in this study were capable of inhibiting IFN-α/β signaling. Most of the strains were able to inhibit IFN-α/β to a degree similar to DENV strain 16681; however, DENV-2 sylvatic strains demonstrated an increased inhibition of phosphorylated signal transducer and activator of transcription (pSTAT1). Surprisingly, we were unable to observe inhibition of pSTAT1 by DENV-2 sylvatic strains or the Asian strain 16681 in non-human primate (NHP) cell lines. Analysis in primary Rhesus macaque dendritic cells suggests that DENVs are capable of inhibiting IFN signaling in these cells. However, contrary to human dendritic cells, production of IFN-α was detected in the supernatant of DENV-infected Rhesus macaque dendritic cells.

Conclusions: The ability of DENVs to inhibit IFN-α/β signaling is conserved. Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes. DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

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DENV2 16681 blocks STAT1 phosphorylation but can’t inhibit production of IFN-α in primary myeloid dendritic cells.Dendritic cells from representative were mock-infected or infected with DENV-1 16681 at an MOI of 5 for twenty-four hours. (A) Cells were stimulated for 30 min. with IFN-β (500 U/ml) and co-stained with anti-pSTAT1 Alexa 647- and anti-dengue prM Alexa 488-conjugated antibodies. Cell fluorescence was measured on a BD FACS Calibur and data analysis was conducted using FlowJo software. (B) The concentrations of IFN-α in the supernatants were quantified by ELISA. Results shown for flow cytometry are representative of two human and two Rhesus macaque samples. Results shown for IFN-α production were done in triplicate and represent the values obtained from two Rhesus macaques.
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pntd.0003468.g007: DENV2 16681 blocks STAT1 phosphorylation but can’t inhibit production of IFN-α in primary myeloid dendritic cells.Dendritic cells from representative were mock-infected or infected with DENV-1 16681 at an MOI of 5 for twenty-four hours. (A) Cells were stimulated for 30 min. with IFN-β (500 U/ml) and co-stained with anti-pSTAT1 Alexa 647- and anti-dengue prM Alexa 488-conjugated antibodies. Cell fluorescence was measured on a BD FACS Calibur and data analysis was conducted using FlowJo software. (B) The concentrations of IFN-α in the supernatants were quantified by ELISA. Results shown for flow cytometry are representative of two human and two Rhesus macaque samples. Results shown for IFN-α production were done in triplicate and represent the values obtained from two Rhesus macaques.

Mentions: In order to determine if the observed reduction in pSTAT1 is also observed in primary cells we differentiated CD14+ monocytes into myeloid dendritic cells. Analysis of human dendritic cells infected with or without dengue and stimulated with IFN revealed results comparable to those obtained with human cell lines (A549, Huh7). Phosphorylation of STAT1 was observed in uninfected dendritic cells after stimulation with IFN. These levels of pSTAT1 did not increase in DENV infected human and Rhesus macaque dendritic cells after stimulation with IFN (Fig. 7A). An increase in pSTAT1 was observed in human and Rhesus macaque dendritic cells that were infected with DENV, but not stimulated with IFN, suggesting that IFN was being produced. Testing of Rhesus macaque dendritic cell supernatants twenty-four hours post-infection revealed the presence of IFN-α. No IFN-α was detected in human dendritic cell supernatants (Fig. 7B).


Differences in type I interferon signaling antagonism by dengue viruses in human and non-human primate cell lines.

Medina FA, Torres-Malavé G, Chase AJ, Santiago GA, Medina JF, Santiago LM, Muñoz-Jordán JL - PLoS Negl Trop Dis (2015)

DENV2 16681 blocks STAT1 phosphorylation but can’t inhibit production of IFN-α in primary myeloid dendritic cells.Dendritic cells from representative were mock-infected or infected with DENV-1 16681 at an MOI of 5 for twenty-four hours. (A) Cells were stimulated for 30 min. with IFN-β (500 U/ml) and co-stained with anti-pSTAT1 Alexa 647- and anti-dengue prM Alexa 488-conjugated antibodies. Cell fluorescence was measured on a BD FACS Calibur and data analysis was conducted using FlowJo software. (B) The concentrations of IFN-α in the supernatants were quantified by ELISA. Results shown for flow cytometry are representative of two human and two Rhesus macaque samples. Results shown for IFN-α production were done in triplicate and represent the values obtained from two Rhesus macaques.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359095&req=5

pntd.0003468.g007: DENV2 16681 blocks STAT1 phosphorylation but can’t inhibit production of IFN-α in primary myeloid dendritic cells.Dendritic cells from representative were mock-infected or infected with DENV-1 16681 at an MOI of 5 for twenty-four hours. (A) Cells were stimulated for 30 min. with IFN-β (500 U/ml) and co-stained with anti-pSTAT1 Alexa 647- and anti-dengue prM Alexa 488-conjugated antibodies. Cell fluorescence was measured on a BD FACS Calibur and data analysis was conducted using FlowJo software. (B) The concentrations of IFN-α in the supernatants were quantified by ELISA. Results shown for flow cytometry are representative of two human and two Rhesus macaque samples. Results shown for IFN-α production were done in triplicate and represent the values obtained from two Rhesus macaques.
Mentions: In order to determine if the observed reduction in pSTAT1 is also observed in primary cells we differentiated CD14+ monocytes into myeloid dendritic cells. Analysis of human dendritic cells infected with or without dengue and stimulated with IFN revealed results comparable to those obtained with human cell lines (A549, Huh7). Phosphorylation of STAT1 was observed in uninfected dendritic cells after stimulation with IFN. These levels of pSTAT1 did not increase in DENV infected human and Rhesus macaque dendritic cells after stimulation with IFN (Fig. 7A). An increase in pSTAT1 was observed in human and Rhesus macaque dendritic cells that were infected with DENV, but not stimulated with IFN, suggesting that IFN was being produced. Testing of Rhesus macaque dendritic cell supernatants twenty-four hours post-infection revealed the presence of IFN-α. No IFN-α was detected in human dendritic cell supernatants (Fig. 7B).

Bottom Line: The ability of DENVs to inhibit IFN-α/β signaling is conserved.Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes.DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Dengue Branch, San Juan, Puerto Rico, United States of America.

ABSTRACT

Background/objectives: In vitro studies have shown that dengue virus (DENV) can thwart the actions of interferon (IFN)-α/β and prevent the development of an antiviral state in infected cells. Clinical studies looking at gene expression in patients with severe dengue show a reduced expression of interferon stimulated genes compared to patients with dengue fever. Interestingly, there are conflicting reports as to the ability of DENV or other flaviviruses to inhibit IFN-α/β signaling.

Methodology/principal findings: In order to determine the relative inhibition of IFN-α/β signaling by DENVs, a method combining flow cytometry and a four-parameter logistic regression model was established. A representative isolate from DENV-1, -3 and -4 and seventeen representative isolates encompassing all DENV-2 genotypes were evaluated. All of the DENVs evaluated in this study were capable of inhibiting IFN-α/β signaling. Most of the strains were able to inhibit IFN-α/β to a degree similar to DENV strain 16681; however, DENV-2 sylvatic strains demonstrated an increased inhibition of phosphorylated signal transducer and activator of transcription (pSTAT1). Surprisingly, we were unable to observe inhibition of pSTAT1 by DENV-2 sylvatic strains or the Asian strain 16681 in non-human primate (NHP) cell lines. Analysis in primary Rhesus macaque dendritic cells suggests that DENVs are capable of inhibiting IFN signaling in these cells. However, contrary to human dendritic cells, production of IFN-α was detected in the supernatant of DENV-infected Rhesus macaque dendritic cells.

Conclusions: The ability of DENVs to inhibit IFN-α/β signaling is conserved. Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes. DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

Show MeSH
Related in: MedlinePlus