Limits...
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.

Show MeSH

Related in: MedlinePlus

IFN-α production by PBMCs in response to TLR9 ligand is impaired by DENV infection.PBMCs from healthy controls were challenge with wild type DENV or iDENV at a MOI of 5 and then stimulated with agonists for TLR3 (poly∶IC), TLR4 (LPS), TLR7 and TLR8 (R848) and TLR9 (CpG A). The production of IFN-α was measured by ELISA a 24 h post-stimulation. The antagonist of TLR9 (TTAGGG ODN) was used to neutralize the stimulatory effect of GpG A. The supernatant from C6/36 cells were used as mock and Influenza virus was used as a positive control for IFN-α production.. Data are presented as the mean of values of at least two independent experiments performed in triplicate; error bars indicate standard deviation. Statistical comparisons among groups were carried out using the Kruskal-Wallis test comparing between groups. *p<0.05 and** p<0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585035&req=5

pntd-0002060-g007: IFN-α production by PBMCs in response to TLR9 ligand is impaired by DENV infection.PBMCs from healthy controls were challenge with wild type DENV or iDENV at a MOI of 5 and then stimulated with agonists for TLR3 (poly∶IC), TLR4 (LPS), TLR7 and TLR8 (R848) and TLR9 (CpG A). The production of IFN-α was measured by ELISA a 24 h post-stimulation. The antagonist of TLR9 (TTAGGG ODN) was used to neutralize the stimulatory effect of GpG A. The supernatant from C6/36 cells were used as mock and Influenza virus was used as a positive control for IFN-α production.. Data are presented as the mean of values of at least two independent experiments performed in triplicate; error bars indicate standard deviation. Statistical comparisons among groups were carried out using the Kruskal-Wallis test comparing between groups. *p<0.05 and** p<0.01.

Mentions: It is known that DENV is a weak inducer of type I IFN production after infection of human DCs. Several possible mechanisms have already been proposed to explain this observation [37]–[39]. The differential expression profiles of TLR3 and TLR9 in DF and DHF patients presented here may also influence type I IFN secretion. However, whether recognition of viral components by TLRs triggers IFN production in DENV-infected cells, or, whether DENV infection affects the function of TLRs is largely unclear. To expand our knowledge on this subject, we tested the ability of PBMCs to respond to the TLR3, TLR4, TLR7/8 and TLR9 ligands, and to trigger the IFN-α pathway. To this end, cells were infected with wild-type DENV or inactivated DENV (iDENV) and 2 h post-addition of the virus, the specific TLR ligands were added to the cells. We decided to uses PBMCs for these studies to mimic natural infection thereby allowing cross talking between cell subsets. PBMCs treated with the TLR3, TLR7/8 and TLR9 agonists, and challenged with DENV, showed variable but enhanced IFN-α production when compared to of non-treated cells, except for the TLR4 agonist LPS, which produced similar levels of IFN-α as the control (Fig. 7). PBMCs infected with DENV in the presence of the TLR3, and 7/8 agonists further stimulated IFN-α production (p<0.05). The observation that iDENV produces a more robust IFN-α response than wild-type DENV is in line with prior published data indicating that the nonstructural proteins, NS2B, NS4B and NS5 are responsible for inhibition of type I IFN production [37]–[39]. Interestingly, DENV infection of PBMCs in the presence of the TLR9 agonist CpG led to a significant decrease of IFN-α expression (p<0.05), compared to PBMCs treated with CpG A only. To investigate the potential effect of TLR9 in more detail, we next infected PBMCs in the presence of CpG A and the TLR9 antagonist TTAGGG ODN, which neutralize the stimulatory effect of CpG A. The ligands were added 2 hours post-infection with DENV. The addition of antagonist indeed decreased IFN-α production when compared to cells treated with agonist only (p<0.05). Interestingly, a stronger reduction in IFN-α production was observed in the presence of the virus (Fig. 7; p<0.01) which may suggest a combined effect between DENV and the antagonist, in the IFN-α reduction. However, additional experiments are required to confirm this hypothesis.


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)

IFN-α production by PBMCs in response to TLR9 ligand is impaired by DENV infection.PBMCs from healthy controls were challenge with wild type DENV or iDENV at a MOI of 5 and then stimulated with agonists for TLR3 (poly∶IC), TLR4 (LPS), TLR7 and TLR8 (R848) and TLR9 (CpG A). The production of IFN-α was measured by ELISA a 24 h post-stimulation. The antagonist of TLR9 (TTAGGG ODN) was used to neutralize the stimulatory effect of GpG A. The supernatant from C6/36 cells were used as mock and Influenza virus was used as a positive control for IFN-α production.. Data are presented as the mean of values of at least two independent experiments performed in triplicate; error bars indicate standard deviation. Statistical comparisons among groups were carried out using the Kruskal-Wallis test comparing between groups. *p<0.05 and** p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002060-g007: IFN-α production by PBMCs in response to TLR9 ligand is impaired by DENV infection.PBMCs from healthy controls were challenge with wild type DENV or iDENV at a MOI of 5 and then stimulated with agonists for TLR3 (poly∶IC), TLR4 (LPS), TLR7 and TLR8 (R848) and TLR9 (CpG A). The production of IFN-α was measured by ELISA a 24 h post-stimulation. The antagonist of TLR9 (TTAGGG ODN) was used to neutralize the stimulatory effect of GpG A. The supernatant from C6/36 cells were used as mock and Influenza virus was used as a positive control for IFN-α production.. Data are presented as the mean of values of at least two independent experiments performed in triplicate; error bars indicate standard deviation. Statistical comparisons among groups were carried out using the Kruskal-Wallis test comparing between groups. *p<0.05 and** p<0.01.
Mentions: It is known that DENV is a weak inducer of type I IFN production after infection of human DCs. Several possible mechanisms have already been proposed to explain this observation [37]–[39]. The differential expression profiles of TLR3 and TLR9 in DF and DHF patients presented here may also influence type I IFN secretion. However, whether recognition of viral components by TLRs triggers IFN production in DENV-infected cells, or, whether DENV infection affects the function of TLRs is largely unclear. To expand our knowledge on this subject, we tested the ability of PBMCs to respond to the TLR3, TLR4, TLR7/8 and TLR9 ligands, and to trigger the IFN-α pathway. To this end, cells were infected with wild-type DENV or inactivated DENV (iDENV) and 2 h post-addition of the virus, the specific TLR ligands were added to the cells. We decided to uses PBMCs for these studies to mimic natural infection thereby allowing cross talking between cell subsets. PBMCs treated with the TLR3, TLR7/8 and TLR9 agonists, and challenged with DENV, showed variable but enhanced IFN-α production when compared to of non-treated cells, except for the TLR4 agonist LPS, which produced similar levels of IFN-α as the control (Fig. 7). PBMCs infected with DENV in the presence of the TLR3, and 7/8 agonists further stimulated IFN-α production (p<0.05). The observation that iDENV produces a more robust IFN-α response than wild-type DENV is in line with prior published data indicating that the nonstructural proteins, NS2B, NS4B and NS5 are responsible for inhibition of type I IFN production [37]–[39]. Interestingly, DENV infection of PBMCs in the presence of the TLR9 agonist CpG led to a significant decrease of IFN-α expression (p<0.05), compared to PBMCs treated with CpG A only. To investigate the potential effect of TLR9 in more detail, we next infected PBMCs in the presence of CpG A and the TLR9 antagonist TTAGGG ODN, which neutralize the stimulatory effect of CpG A. The ligands were added 2 hours post-infection with DENV. The addition of antagonist indeed decreased IFN-α production when compared to cells treated with agonist only (p<0.05). Interestingly, a stronger reduction in IFN-α production was observed in the presence of the virus (Fig. 7; p<0.01) which may suggest a combined effect between DENV and the antagonist, in the IFN-α reduction. However, additional experiments are required to confirm this hypothesis.

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