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Differential expression of Toll-like receptors in dendritic cells of patients with dengue during early and late acute phases of the disease.

Torres S, Hernández JC, Giraldo D, Arboleda M, Rojas M, Smit JM, Urcuqui-Inchima S - PLoS Negl Trop Dis (2013)

Bottom Line: In addition, we found a lower expression of TLR2 in patients with DF compared to DHF.This suggests that the virus can affect the interferon response through this signaling pathway.Changes in TLRs expression could play a central role in DC activation, thereby influencing the innate immune response.

View Article: PubMed Central - PubMed

Affiliation: Grupo Inmunovirología, Sede de Investigación Universitaria, Universidad de Antioquia, Medellín, Colombia.

ABSTRACT

Background: Dengue hemorrhagic fever (DHF) is observed in individuals that have pre-existing heterotypic dengue antibodies and is associated with increased viral load and high levels of pro-inflammatory cytokines early in infection. Interestingly, a recent study showed that dengue virus infection in the presence of antibodies resulted in poor stimulation of Toll-like receptors (TLRs), thereby facilitating virus particle production, and also suggesting that TLRs may contribute to disease pathogenesis.

Methodology/principal findings: We evaluated the expression levels of TLR2, 3, 4 and 9 and the co-stimulatory molecules CD80 and CD86 by flow cytometry. This was evaluated in monocytes, in myeloid and plasmacytoid dendritic cells (mDCs and pDCs) from 30 dengue patients with different clinical outcomes and in 20 healthy controls. Increased expression of TLR3 and TLR9 in DCs of patients with dengue fever (DF) early in infection was detected. In DCs from patients with severe manifestations, poor stimulation of TLR3 and TLR9 was observed. In addition, we found a lower expression of TLR2 in patients with DF compared to DHF. Expression levels of TLR4 were not affected. Furthermore, the expression of CD80 and CD86 was altered in mDCs and CD86 in pDCs of severe dengue cases. We show that interferon alpha production decreased in the presence of dengue virus after stimulation of PBMCs with the TLR9 agonist (CpG A). This suggests that the virus can affect the interferon response through this signaling pathway.

Conclusions/significance: These results show that during dengue disease progression, the expression profile of TLRs changes depending on the severity of the disease. Changes in TLRs expression could play a central role in DC activation, thereby influencing the innate immune response.

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Related in: MedlinePlus

Gating strategy for identification of mDCs, pDCs and monocytes from PB samples and TLR staining.(A) Non duplets population was fractioned in mDCs as mononuclear cells Lin− and CD11c High (P3); (pDCs) as CD123+ BDCA2+ (P2) and monocytes as CD14+ (P2) . (B) Representative examples of TLR2 expression in mDCs, pDCs, and monocytes. HC indicates healthy control and DF denotes for dengue fever.
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pntd-0002060-g002: Gating strategy for identification of mDCs, pDCs and monocytes from PB samples and TLR staining.(A) Non duplets population was fractioned in mDCs as mononuclear cells Lin− and CD11c High (P3); (pDCs) as CD123+ BDCA2+ (P2) and monocytes as CD14+ (P2) . (B) Representative examples of TLR2 expression in mDCs, pDCs, and monocytes. HC indicates healthy control and DF denotes for dengue fever.

Mentions: PB samples were used to determine the frequency of mDCs, pDCs and monocytes and their expression of TLRs. In all patients and healthy controls the proportion of mDCs, pDCs, and monocytes among blood mononuclear cells was 0.5% (0.4–1.6%) 0.3% (0.1–1%), and 6% (4–12%), respectively. PB was incubated with the appropriate antibodies at room temperature for 25 minutes. The whole blood was lysed with lysing Buffer (BD Biosciences, San Jose, CA) during 10 minutes at room temperature. Leucocytes were resuspended and washed in PBS containing 0.5% BSA and 0.1% sodium, fixed with 2% formaldehyde and stored at 4°C until analysis. For staining of intracellular receptors (TLR3 and TLR9), the cells were treated with fixation permeabilization buffer (eBiosciences, San Diego. CA) following manufacturer's recommendations. All samples were evaluated within 2–4 h of staining and 200,000 events were acquired per tube. Analyses were performed using the BD FACS Dive V6.1.1 (BD Biosciences), and the operating software on the FACS Canto II flow cytometer. TLR levels were reported as mean fluorescent intensity (MFI) compared to the isotype control. Logical gating was used to identify mDC, pDC and monocyte populations. The acquisition gate (P1) was common for all the populations and was established based on forward scatter (FSC) and side scatter (SSC) corresponding to the gate of mononuclear cells (approximately 130,000–170,000 events). For phenotyping of each sub-population a single tube analysis was performed. The following strategy was used: mDCs (Lin1−/CD11chigh) were gated as P3; Lin1 positive cells were excluded from the analysis (gate P2). The pDCs (BDCA-2+/CD123high) were gated as P2. Monocytes were identified as CD14+ versus side scatters and gated as P2 (Fig. 2A). Each specific sub-population was plotted as a histogram to show the expression of TLR2 (Fig. 2B), 3, 4 and 9. The data are presented as overall MFI for TLR on the TLR+ subset, after subtraction of isotype staining background. The following monoclonal antibodies were used: Lineage 1 FITC (anti-CD3, anti-CD14, anti-CD16, anti-CD19, anti-CD20, and anti-CD56 cocktail), anti-CD123 PE-Cy5, anti-CD11c PE-Cy5, anti-CD80 PE and anti-CD86 PE (BD Biosciences, San Jose, CA). Anti-TLR2, 3, 4 and 9 were PE conjugates (eBiosciences). Anti-BDCA-2 FITC was from Miltenyi Biotec (Auburn CA). Unstained cells and conjugated isotype antibodies were used as controls; all of them matched for concentration with the primary antibodies. All reagents were used according to manufacturer's instructions.


Differential expression of Toll-like receptors in dendritic cells of patients with dengue during early and late acute phases of the disease.

Torres S, Hernández JC, Giraldo D, Arboleda M, Rojas M, Smit JM, Urcuqui-Inchima S - PLoS Negl Trop Dis (2013)

Gating strategy for identification of mDCs, pDCs and monocytes from PB samples and TLR staining.(A) Non duplets population was fractioned in mDCs as mononuclear cells Lin− and CD11c High (P3); (pDCs) as CD123+ BDCA2+ (P2) and monocytes as CD14+ (P2) . (B) Representative examples of TLR2 expression in mDCs, pDCs, and monocytes. HC indicates healthy control and DF denotes for dengue fever.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585035&req=5

pntd-0002060-g002: Gating strategy for identification of mDCs, pDCs and monocytes from PB samples and TLR staining.(A) Non duplets population was fractioned in mDCs as mononuclear cells Lin− and CD11c High (P3); (pDCs) as CD123+ BDCA2+ (P2) and monocytes as CD14+ (P2) . (B) Representative examples of TLR2 expression in mDCs, pDCs, and monocytes. HC indicates healthy control and DF denotes for dengue fever.
Mentions: PB samples were used to determine the frequency of mDCs, pDCs and monocytes and their expression of TLRs. In all patients and healthy controls the proportion of mDCs, pDCs, and monocytes among blood mononuclear cells was 0.5% (0.4–1.6%) 0.3% (0.1–1%), and 6% (4–12%), respectively. PB was incubated with the appropriate antibodies at room temperature for 25 minutes. The whole blood was lysed with lysing Buffer (BD Biosciences, San Jose, CA) during 10 minutes at room temperature. Leucocytes were resuspended and washed in PBS containing 0.5% BSA and 0.1% sodium, fixed with 2% formaldehyde and stored at 4°C until analysis. For staining of intracellular receptors (TLR3 and TLR9), the cells were treated with fixation permeabilization buffer (eBiosciences, San Diego. CA) following manufacturer's recommendations. All samples were evaluated within 2–4 h of staining and 200,000 events were acquired per tube. Analyses were performed using the BD FACS Dive V6.1.1 (BD Biosciences), and the operating software on the FACS Canto II flow cytometer. TLR levels were reported as mean fluorescent intensity (MFI) compared to the isotype control. Logical gating was used to identify mDC, pDC and monocyte populations. The acquisition gate (P1) was common for all the populations and was established based on forward scatter (FSC) and side scatter (SSC) corresponding to the gate of mononuclear cells (approximately 130,000–170,000 events). For phenotyping of each sub-population a single tube analysis was performed. The following strategy was used: mDCs (Lin1−/CD11chigh) were gated as P3; Lin1 positive cells were excluded from the analysis (gate P2). The pDCs (BDCA-2+/CD123high) were gated as P2. Monocytes were identified as CD14+ versus side scatters and gated as P2 (Fig. 2A). Each specific sub-population was plotted as a histogram to show the expression of TLR2 (Fig. 2B), 3, 4 and 9. The data are presented as overall MFI for TLR on the TLR+ subset, after subtraction of isotype staining background. The following monoclonal antibodies were used: Lineage 1 FITC (anti-CD3, anti-CD14, anti-CD16, anti-CD19, anti-CD20, and anti-CD56 cocktail), anti-CD123 PE-Cy5, anti-CD11c PE-Cy5, anti-CD80 PE and anti-CD86 PE (BD Biosciences, San Jose, CA). Anti-TLR2, 3, 4 and 9 were PE conjugates (eBiosciences). Anti-BDCA-2 FITC was from Miltenyi Biotec (Auburn CA). Unstained cells and conjugated isotype antibodies were used as controls; all of them matched for concentration with the primary antibodies. All reagents were used according to manufacturer's instructions.

Bottom Line: In addition, we found a lower expression of TLR2 in patients with DF compared to DHF.This suggests that the virus can affect the interferon response through this signaling pathway.Changes in TLRs expression could play a central role in DC activation, thereby influencing the innate immune response.

View Article: PubMed Central - PubMed

Affiliation: Grupo Inmunovirología, Sede de Investigación Universitaria, Universidad de Antioquia, Medellín, Colombia.

ABSTRACT

Background: Dengue hemorrhagic fever (DHF) is observed in individuals that have pre-existing heterotypic dengue antibodies and is associated with increased viral load and high levels of pro-inflammatory cytokines early in infection. Interestingly, a recent study showed that dengue virus infection in the presence of antibodies resulted in poor stimulation of Toll-like receptors (TLRs), thereby facilitating virus particle production, and also suggesting that TLRs may contribute to disease pathogenesis.

Methodology/principal findings: We evaluated the expression levels of TLR2, 3, 4 and 9 and the co-stimulatory molecules CD80 and CD86 by flow cytometry. This was evaluated in monocytes, in myeloid and plasmacytoid dendritic cells (mDCs and pDCs) from 30 dengue patients with different clinical outcomes and in 20 healthy controls. Increased expression of TLR3 and TLR9 in DCs of patients with dengue fever (DF) early in infection was detected. In DCs from patients with severe manifestations, poor stimulation of TLR3 and TLR9 was observed. In addition, we found a lower expression of TLR2 in patients with DF compared to DHF. Expression levels of TLR4 were not affected. Furthermore, the expression of CD80 and CD86 was altered in mDCs and CD86 in pDCs of severe dengue cases. We show that interferon alpha production decreased in the presence of dengue virus after stimulation of PBMCs with the TLR9 agonist (CpG A). This suggests that the virus can affect the interferon response through this signaling pathway.

Conclusions/significance: These results show that during dengue disease progression, the expression profile of TLRs changes depending on the severity of the disease. Changes in TLRs expression could play a central role in DC activation, thereby influencing the innate immune response.

Show MeSH
Related in: MedlinePlus