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. 2005 Aug 18:5:64.
doi: 10.1186/1471-2334-5-64.

Inducible nitric oxide synthase (iNOS) expression in monocytes during acute Dengue Fever in patients and during in vitro infection

Patrícia C F Neves-Souza  1 , Elzinandes L Azeredo, Sonia M O Zagne, Rogério Valls-de-Souza, Sonia R N I Reis, Denise I S Cerqueira, Rita M R Nogueira, Claire F Kubelka
Affiliations

Inducible nitric oxide synthase (iNOS) expression in monocytes during acute Dengue Fever in patients and during in vitro infection

Patrícia C F Neves-Souza et al. BMC Infect Dis. .

Abstract

Mononuclear phagocytes are considered to be main targets for Dengue Virus (DENV) replication. These cells are activated after infection, producing proinflammatory mediators, including tumour-necrosis factor-alpha, which has also been detected in vivo. Nitric oxide (NO), usually produced by activated mononuclear phagocytes, has antimicrobial and antiviral activities.

Methods: The expression of DENV antigens and inducible nitric oxide synthase (iNOS) in human blood isolated monocytes were analysed by flow cytometry using cells either from patients with acute Dengue Fever or after DENV-1 in vitro infection. DENV-1 susceptibility to iNOS inhibition and NO production was investigated using NG-methyl L-Arginine (NGMLA) as an iNOS inhibitor, which was added to DENV-1 infected human monocytes, and sodium nitroprussiate (SNP), a NO donor, added to infected C6/36 mosquito cell clone. Viral antigens after treatments were detected by flow cytometry analysis.

Results: INOS expression in activated monocytes was observed in 10 out of 21 patients with Dengue Fever and was absent in cells from ten healthy individuals. DENV antigens detected in 25 out of 35 patients, were observed early during in vitro infection (3 days), significantly diminished with time, indicating that virus replicated, however monocytes controlled the infection. On the other hand, the iNOS expression was detected at increasing frequency in in vitro infected monocytes from three to six days, exhibiting an inverse relationship to DENV antigen expression. We demonstrated that the detection of the DENV-1 antigen was enhanced during monocyte treatment with NGMLA. In the mosquito cell line C6/36, virus detection was significantly reduced in the presence of SNP, when compared to that of untreated cells.

Conclusion: This study is the first to reveal the activation of DENV infected monocytes based on induction of iNOS both in vivo and in vitro, as well as the susceptibility of DENV-1 to a NO production.

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Figures

Figure 1
Figure 1
Dot and contour plots showing DENV-Ag and iNOS monocyte and lymphocyte expression in dengue patients. Human PBMLs a from healthy individual (Figures 1A,1D,1G) were used as control. Cells from a 4-day dengue infected patient (Figures 1B,1C,1E,1F,1H,1I) were labelled with anti-CD14-PE and CD14+ gated cells – R1 (Figure 1B) were considered as logical gate or monocyte gate and R2 is as the lymphocyte gate in the FCS vs. SSC dot plot (Figure 1C). R1 and R2 were used for further analysis during this work. Alternatively, human PBMLs were labelled either with an antibody to DENV Ags and anti-mouse IgG-PE (Figures 1D-1F) or with anti-iNOS-FITC (Figures 1G-1I) and analysed by FACS in either the R1, monocyte gate (Figures 1D-1F) or in the R2, lymphocyte gate (Figures 1F-1I). x-axis represent mean of cell population size (FCS) and y-axis represent cell granularity (SSC) or fluorescence intensity.
Figure 2
Figure 2
Frequencies of DENVAg+ and iNOS+ monocytes in dengue patients. Human PBMLs from dengue individuals were labelled with an antibody to DENV-Ag and an anti-mouse IgG-PE or with an antibody to iNOS FITC-labelled and data obtained by FACS in the monocyte gate. Percentages of DENV-Ag+ infected cells were determined in 11 healthy individuals and 35 with dengue (Figure 2A; 2 patients were measured in duplicate samples at different time points). Percentages of iNOS+ infected cells were determined in 10 healthy individuals and 22 with dengue (Figure 2B; 14 patients were measured in duplicate samples at different time points). Horizontal lines represent percentage averages of labelled cells from the population of individuals in each of the discrete categories.
Figure 3
Figure 3
Confocal microscopy of DENV-Ag expression after in vitro infection of monocyte-rich cultures. Adherent human PBMLs were incubated for three days either with cell culture medium (Figure 3A, 189.5 μm/field), or with infectious DENV (Figure 3B, 125 μm/field). Cells were labelled with antibody to DENV-Ag and anti-mouse IgG-FITC.
Figure 4
Figure 4
DENV-Ag and iNOS expression after in vitro Dengue infection of monocytes. Adherent human PBMLs were incubated for three days with infected DENV (Figures 4A, 4B and 4C). Cells were labelled either with isotype matched antibodies (Figures 6A and 6B) or antibodies to CD14-PE (y-axis) and DENV-Complex-Alexa-654 (x-axis) and were analysed by FACS (Figure 4C, ungated). Cells incubated for three to six days with cell culture medium, inactivated DENV or infectious DENV were labelled with antibody to DENV-Ag and anti-mouse IgG-PE and/or anti-iNOS-FITC and analysed by FACS. Isotype matched antibodies were used for both labelings (Figure 4D, 3 days for DENV-Ag and 6 days for iNOS labelling). x-axis represent mean of fluorescence intensity and y-axis represent percentage of maximum cell number counts. Figure 4E shows a kinetic curve comparing DENV-Ag and iNOS expression on cells from the monocyte gate. Each point represent average ± SEM percentages from samples set in triplicates. * P < 0.05 and in Student's T-test showing difference in DENV-Ag and iNOS expression compared to uninfected controls (CT). Data are obtained from one representative experiment out of three performed with different cell donors.
Figure 5
Figure 5
Effect of an iNOS inhibitor and of a NO donor on DENV-Ag expression after cell infection. Figures 5A and 5B – Effect of iNOS inhibitor – NGMLA on monocyte infection. Adherent PBMLs were incubated with either Mock C6/36 cell supernatant or DENV inoculum for 4 days in presence or absence of 400 μM NGMLA. Figure 4A shows histogram with profile of infected monocytes after NGMLA treatment and labelling controls. Figure 4B shows percentages of DENV-Ag+ in gated monocytes obtained by flow cytometry. Figures 5C and 5D -. Effect of NO donor – SNP, on C6/36 cell infection. C6/36 cells were incubated with DENV for 2 days in absence or presence of 10 or 100 μM SNP. Figure 4C shows a histogram with profiles of infected C6/36 cells obtained by FACS from a culture treated with 10 μM SNP or controls. The histogram gates have been made where ≥99% than cells in the cell control are excluded. Figure 4Dshows percentages of DENV-Ag+ cells calculated from ungated cells after labelling with antibody to DENV-Complex and anti-mouse IgG-PE. In histograms x-axis represent mean of fluorescence intensity and y-axis represent percentage of maximum cell number counts. Cells were labelled with antibody to DENV-Complex followed by anti-mouse IgG-PE. Average ± SEM are represented from samples set in triplicates. * P < 0.05 in Student's T-test showing difference in DENV infection after treatment. Data are obtained from one representative experiment out of two for each cell type using two different monocyte donors.
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