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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

Flowchart of enrolment, inclusion/exclusion criteria, diagnosis and classification of dengue patients.
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Related In: Results  -  Collection


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pntd-0002060-g001: Flowchart of enrolment, inclusion/exclusion criteria, diagnosis and classification of dengue patients.

Mentions: Thirty ml of peripheral blood (PB) collected in EDTA-containing tubes. Blood samples were collected three times, on the 3rd and 5th day after the beginning of symptoms (acute samples), and 15 days after admission to the hospital (convalescence samples). Infection with DENV was confirmed if one of the following tests was positive, (1) Platelia Dengue NS1 Antigen kit (Bio-Rad Laboratories, Marnes La Coquette, France), (2) DENV specific real-time RT-PCR [24], (3) DENV IgM detection by ELISA UMELISA (Centro de Inmunoensayo, Instituto Pedro Kourí, La Habana, Cuba) or (4) virus isolation and propagation in C6/36 mosquito cells [25]. To determine whether the patient had a primary or secondary infection, the presence of dengue-specific IgM/IgG antibodies was evaluated, using the PanBio Dengue Duo Cassette System (PanBio Ltd, Queensland, Australia). Anti-dengue IgG levels were determined on days 3, 5 and 15 after the beginning of symptoms, and if there was no rise in IgG titer over time it was considered as secondary infection. Dengue cases were classified as DF or DHF according to the 1997 guidelines of the World Health Organization (WHO) [3], we applied the old guidelines since the new WHO guidelines published in 2009 are more directly focused on clinical practice and are not widely accepted for use in research [26]. Clinical characterization of DHF included the following criteria: fever, thrombocytopenia (platelet counts <100×103/mm3), hemorrhagic manifestations, positive tourniquet test, and hemoconcentration (20% changes in hematocrit value) or ascites as evidenced by plasma leakage. A flow chart explaining the inclusion/exclusion criteria is depicted in Figure 1.


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)

Flowchart of enrolment, inclusion/exclusion criteria, diagnosis and classification of dengue patients.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585035&req=5

pntd-0002060-g001: Flowchart of enrolment, inclusion/exclusion criteria, diagnosis and classification of dengue patients.
Mentions: Thirty ml of peripheral blood (PB) collected in EDTA-containing tubes. Blood samples were collected three times, on the 3rd and 5th day after the beginning of symptoms (acute samples), and 15 days after admission to the hospital (convalescence samples). Infection with DENV was confirmed if one of the following tests was positive, (1) Platelia Dengue NS1 Antigen kit (Bio-Rad Laboratories, Marnes La Coquette, France), (2) DENV specific real-time RT-PCR [24], (3) DENV IgM detection by ELISA UMELISA (Centro de Inmunoensayo, Instituto Pedro Kourí, La Habana, Cuba) or (4) virus isolation and propagation in C6/36 mosquito cells [25]. To determine whether the patient had a primary or secondary infection, the presence of dengue-specific IgM/IgG antibodies was evaluated, using the PanBio Dengue Duo Cassette System (PanBio Ltd, Queensland, Australia). Anti-dengue IgG levels were determined on days 3, 5 and 15 after the beginning of symptoms, and if there was no rise in IgG titer over time it was considered as secondary infection. Dengue cases were classified as DF or DHF according to the 1997 guidelines of the World Health Organization (WHO) [3], we applied the old guidelines since the new WHO guidelines published in 2009 are more directly focused on clinical practice and are not widely accepted for use in research [26]. Clinical characterization of DHF included the following criteria: fever, thrombocytopenia (platelet counts <100×103/mm3), hemorrhagic manifestations, positive tourniquet test, and hemoconcentration (20% changes in hematocrit value) or ascites as evidenced by plasma leakage. A flow chart explaining the inclusion/exclusion criteria is depicted in Figure 1.

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