Neutrophil Activation and Early Features of NET Formation Are Associated With Dengue Virus Infection in Human

The involvement of the immune system in the protection and pathology of natural dengue virus (DENV) has been extensively studied. However, despite studies that have referred to activation of neutrophils in DENV infections, the exact roles of neutrophils remain elusive. Here, we explored the phenotypic and functional responses of neutrophils in a cohort of adult dengue patients. Results indicated that during an acute DENV infection, neutrophils up-regulate CD66b expression, and produce a more robust respiratory response as compared with that in convalescent or healthy individuals; this confirmed in vivo neutrophil activation during DENV infection. Spontaneous decondensation of nuclei, an early event of neutrophil extracellular trap (NET) formation, was also markedly increased in cells isolated from DENV-infected patients during the acute phase of the infection. In vitro incubation of NETs with DENV-2 virus significantly decreased DENV infectivity. Interestingly, increased levels of NET components were found in the serum of patients with more severe disease form—dengue hemorrhagic fever (DHF), but not uncomplicated dengue fever, during the acute phase of the infection. Levels of pro-inflammatory cytokines IL-8 and TNFα were also increased in DHF patients as compared with those in healthy and DF subjects. This suggested that NETs may play dual roles during DENV infection. The increased ability for NET formation during acute DENV infection appeared to be independent of PAD4-mediated histone H3 hyper-citrullination. Our study suggests that neutrophils are involved in immunological responses to DENV infection

Comparison of antibody responses before and after booster doses with the Pfizer-BioNTech or Oxford–AstraZeneca vaccines in healthcare workers in Thailand

The severe acute respiratory syndrome 2 (SARS-CoV-2) has spread rapidly worldwide, not only causing significant morbidity and mortality but also dramatically increasing health care spending. To manage this in Thailand, healthcare workers first received two doses of the CoronaVac vaccine followed by a booster vaccine with either BNT162b2 vaccine (Pfizer-BioNTech; PZ) or ChAdOx1 nCoV-19 vaccine (Oxford–AstraZeneca; AZ). Given that the difference in anti-SARS-CoV-2 levels following vaccination may vary depending on the vaccine and on demographic characteristics, we measured the antibody response after the second CoronaVac dose and after the booster with either the PZ or AZ vaccine. Our results in 473 healthcare workers show that the variation in antibody response to the full CoronaVac dose depends on demographic characteristics such as age, gender, body mass index, and underlying disease. After receiving a booster dose, anti-SARS-CoV-2 levels were significantly higher in participants who received the PZ vaccine than in people who received the AZ vaccine. Overall, however, receiving a booster dose of either the PZ or AZ vaccine promoted strong antibody responses, even in the old and those with obesity or diabetes mellitus. In conclusion, our results support the use of a booster vaccination program after full vaccination with the CoronaVac vaccine. This approach effectively enhances immunity against SARS-CoV-2, especially in clinically vulnerable groups and healthcare workers

Neutrophil Activation and Early Features of NET Formation Are Associated With Dengue Virus Infection in Human

International audienceThe involvement of the immune system in the protection and pathology of natural dengue virus (DENV) has been extensively studied. However, despite studies that have referred to activation of neutrophils in DENV infections, the exact roles of neutrophils remain elusive. Here, we explored the phenotypic and functional responses of neutrophils in a cohort of adult dengue patients. Results indicated that during an acute DENV infection, neutrophils up-regulate CD66b expression, and produce a more robust respiratory response as compared with that in convalescent or healthy individuals; this confirmed in vivo neutrophil activation during DENV infection. Spontaneous decondensation of nuclei, an early event of neutrophil extracellular trap (NET) formation, was also markedly increased in cells isolated from DENV-infected patients during the acute phase of the infection. In vitro incubation of NETs with DENV-2 virus significantly decreased DENV infectivity. Interestingly, increased levels of NET components were found in the serum of patients with more severe disease form-dengue hemorrhagic fever (DHF), but not uncomplicated dengue fever, during the acute phase of the infection. Levels of pro-inflammatory cytokines IL-8 and TNFα were also increased in DHF patients as compared with those in healthy and DF subjects. This suggested that NETs may play dual roles during DENV infection. The increased ability for NET formation during acute DENV infection appeared to be independent of PAD4-mediated histone H3 hyper-citrullination. Our study suggests that neutrophils are involved in immunological responses to DENV infection

Activation of conventional T cells during acute phase of dengue viral infection.

<p>CD8⁺ and CD4⁺ conventional T cells were identified by the expression of CD8 or CD4 together with CD3 within lymphocytes population. The expression of CD69 on CD8⁺ or CD4⁺ conventional was gated in comparison to isotype control. Each dot represents percentage of CD69⁺CD8⁺ (a–e) and CD69⁺CD4⁺ (f–j) conventional T cells of each patients in DF (a, f) and DHF (b, g) group at 3 different time points, the line indicate median of each group. e, h) Dot plot summarized percentage of CD69⁺ CD8⁺ (c) and CD69⁺ CD4⁺ (h) conventional T cells during day -1 of each group of patients in comparison to OFI and healthy controls. Percentage of CD69⁺ CD8⁺ (d, e) and CD69⁺ CD4⁺ (i, j) conventional T cells during the course of dengue infection in DF (d, i) and DHF (e, j), each line connects data from each patient at various time points. Mann-Whitney test (a–c, f–h), and Wilcoxon signed rank test (d–e, i–j) were used for statistical comparison, *p<0.05, **p<0.01, ***p<0.001, p<0.05 was considered as statistically significant difference.</p

Cytokines production by iNKT cells from dengue infected patients with and without stimulation with α-GalCer.

<p>Each dot represents percentage of interferon gamma (IFN-γ)⁺ (a–b) and IL-4⁺ (e–f) iNKT cells from DF (a,e) and DHF (b,f) at day 0 and month 6, with (stimulated) and without (unstimulated) α-GalCer stimulation. At day 0 (c, g, i) and 6 months (d, h, j), percentage of IFN-γ⁺ iNKT cells (c,d), IL-4⁺ iNKT cells (g, h) and IFN-γ/IL-4 ratio (i, j) upon α-GalCer stimulation, comparing cells from healthy, OFI, DF and DHF groups. Mann-Whitney test, were used for statistical comparison, p<0.05 was considered as statistically significant difference (*p<0.05, **p<0.01, ***p<0.001).</p

Upregulation of CD1d expression on monocytes during acute DV infection.

<p>a) Monocytes were gated on their typical FSC/SSC appearance and on the presence of CD14. b) Representative histogram showed the level of CD1d expression on monocytes at different time points, day -1 (black line), day 0 (gray line), 2 week (dotted line) in comparison to isotype control (shaded gray) in DF and DHF groups and in healthy control. The level of CD1d expression was expressed as the difference in mean fluorescence intensity (dMFI) between CD1d staining and isotype control of each sample. c) Each dot represents CD1d dMFI on monocytes of each patient in DF and DHF groups at 3 different time points, the line represents median of each group. d) Monocytes CD1d dMFI during febrile phase (day -1) (left) and 2 weeks (right) of healthy, OFI, DF and DHF. e) Monocytes CD1d dMFI during the course of dengue infection. Each line connects data from each patient at different time points. Mann- Whitney test (c–d), and Wilcoxon signed rank test (e) were used for statistical comparison, p<0.05 was considered as statistically significant difference (*p<0.05, **p<0.01).</p

The frequency of peripheral blood iNKT cells during dengue infection with different disease severity.

<p>a) Representative dot plots show the percentage of iNKT cells within lymphocytes in healthy, other febrile illness (OFI), dengue fever (DF), and dengue hemorrhagic fever (DHF) at day -1, day 0 and day 2 weeks. b, c) Each dot represents percentage of iNKT cells of each patient in DF (b) and DHF (c) groups at 3 different time points, the line represents median of each group. d) The percentage of iNKT cells during febrile phase (day -1) of healthy, OFI, DF and DHF. Mann-Whitney test (b–d) was used for statistical comparison, p<0.05 was considered as statistically significant difference. No significant difference was found.</p

iNKT cells were activated during acute phase of dengue infection.

<p>a) Representative dot plot and histogram demonstrate gating strategy for iNKT cells and CD69 expression on iNKT cells. iNKT cells were gated, within lymphocytes population, on CD3⁺, PBS57-loaded CD1d tetramer⁺ (in comparison with unloaded CD1d tetramer control). CD69⁺ iNKT cells were gated in comparison to isotype control. b) Flow cytometry analysis of the expression of CD69 on iNKT cells in Healthy, OFI, DF, and DHF at day -1, day 0 and 2 weeks. Representative histograms show the expression of CD69 (black line) in comparison to isotype control (gray shade). c, d) Each dot represents percentage of CD69 positive iNKT cells of each patient in DF (c) and DHF (d) groups at 3 different time points, the line indicate median of each group. e) Dot plot summarized percentage of CD69 positive iNKT cells during febrile phase (day -1) of each group of patients in comparison to controls. f, g) Percentage of CD69 positive iNKT cells during the course of dengue infection, each line connects data from each patient at various time points. Mann-Whitney test (c–e), and Wilcoxon signed rank test (f–g) were used for statistical comparison, p<0.05 was considered as statistically significant difference (*p<0.05, **p<0.01, ***p<0.001).</p