Figure 1.
Flowchart of enrolment, inclusion/exclusion criteria, diagnosis and classification of dengue patients.
Figure 2.
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.
Table 1.
Demographic and clinical characteristics of 30 individuals with diagnosis of dengue and 20 healthy controls.
Figure 3.
Increased expression of TLRs in mDCs and pDCs of dengue-infected patients.
Expression profile of TLR2, 3, 4 and 9 in mDCs (A) and pDCs (B) of dengue patients isolated at different times after the onset of disease symptoms (day 3, 5 and 15) (n = 30) and of healthy controls (n = 20). The evaluation of TLR expression was performed by flow cytometry based on mean fluorescence intensity (MFI) analysis. Data are presented as the median and 25–75 interquartile ranges Statistical analysis was performed by using the Kruskal-Wallis test followed by the Dunn's Multiple comparisons Test. *p<0.05, ** p<0.01 and ***p<0.001.
Figure 4.
Increased TLR2 expression in pDCs of patients with severe disease.
Analysis of TLR2 expression by flow cytometry of PB mDCs (A) and pDCs (B) in DF patients (n = 19) and DHF patients (n = 11) at different times after the onset of symptoms (days 3, 5 and 15). Data are presented as the median and 25–75 interquartile ranges. Statistical analysis was performed by the Mann Withney test.*p<0.05, ** p<0.01 and ***p<0.001.
Figure 5.
TLR3 and TLR9 in mDCs and TLR9 in pDCs have different expression levels depending on disease severity.
Expression profile of TLR2 and TLR9 in mDCs (A and B) and pDCs (C) of dengue patients isolated at different times after the onset of disease symptoms (day 3, 5 and 15) (n = 30) and of healthy controls (n = 20). The evaluation of TLR expression was performed by flow cytometry based on mean fluorescence intensity (MFI) analysis. Data are presented as the median and 25–75 interquartile ranges Statistical analysis was performed by using the Kruskal-Wallis test followed by the Dunn's Multiple comparisons Test. *p<0.05, ** p<0.01 and ***p<0.001.
Figure 6.
Expression of the co-stimulatory molecules CD80 and CD86 is affected in mDCs of DHF patients.
Expression of CD80 (A) and CD86 (B) was evaluated by flow cytometry based on the FMI analysis in mDCs and pDCs (C) of DF and DHF patients on day 3 of illness and compared to that of healthy controls (HC). Data are presented as the median and the statistical analysis of the expression of CD80 and CD86 was performed by the Mann Withney test.*p<0.05, ** p<0.01 and ***p<0.001.
Figure 7.
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.