Ragweed Subpollen Particles of Respirable Size Activate Human Dendritic Cells

Ragweed (Ambrosia artemisiifolia) pollen grains, which are generally considered too large to reach the lower respiratory tract, release subpollen particles (SPPs) of respirable size upon hydration. These SPPs contain allergenic proteins and functional NAD(P)H oxidases. In this study, we examined whether exposure to SPPs initiates the activation of human monocyte-derived dendritic cells (moDCs). We found that treatment with freshly isolated ragweed SPPs increased the intracellular levels of reactive oxygen species (ROS) in moDCs. Phagocytosis of SPPs by moDCs, as demonstrated by confocal laser-scanning microscopy, led to an up-regulation of the cell surface expression of CD40, CD80, CD86, and HLA-DQ and an increase in the production of IL-6, TNF-a, IL-8, and IL-10. Furthermore, SPP-treated moDCs had an increased capacity to stimulate the proliferation of naı¨ve T cells. Co-culture of SPP-treated moDCs with allogeneic CD3+ pan-T cells resulted in increased secretion of IFN-c and IL-17 by T cells of both allergic and non-allergic subjects, but induced the production of IL- 4 exclusively from the T cells of allergic individuals. Addition of exogenous NADPH further increased, while heat-inactivation or pre-treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, strongly diminished, the ability of SPPs to induce phenotypic and functional changes in moDCs, indicating that these processes were mediated, at least partly, by the intrinsic NAD(P)H oxidase activity of SPPs. Collectively, our data suggest that inhaled ragweed SPPs are fully capable of activating dendritic cells (DCs) in the airways and SPPs’ NAD(P)H oxidase activity is involved in initiation of adaptive immune responses against innocuous pollen proteins

Ragweed Subpollen Particles of Respirable Size Activate Human Dendritic Cells

<div><p>Ragweed (<em>Ambrosia artemisiifolia</em>) pollen grains, which are generally considered too large to reach the lower respiratory tract, release subpollen particles (SPPs) of respirable size upon hydration. These SPPs contain allergenic proteins and functional NAD(P)H oxidases. In this study, we examined whether exposure to SPPs initiates the activation of human monocyte-derived dendritic cells (moDCs). We found that treatment with freshly isolated ragweed SPPs increased the intracellular levels of reactive oxygen species (ROS) in moDCs. Phagocytosis of SPPs by moDCs, as demonstrated by confocal laser-scanning microscopy, led to an up-regulation of the cell surface expression of CD40, CD80, CD86, and HLA-DQ and an increase in the production of IL-6, TNF-α, IL-8, and IL-10. Furthermore, SPP-treated moDCs had an increased capacity to stimulate the proliferation of naïve T cells. Co-culture of SPP-treated moDCs with allogeneic CD3⁺ pan-T cells resulted in increased secretion of IFN-γ and IL-17 by T cells of both allergic and non-allergic subjects, but induced the production of IL-4 exclusively from the T cells of allergic individuals. Addition of exogenous NADPH further increased, while heat-inactivation or pre-treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, strongly diminished, the ability of SPPs to induce phenotypic and functional changes in moDCs, indicating that these processes were mediated, at least partly, by the intrinsic NAD(P)H oxidase activity of SPPs. Collectively, our data suggest that inhaled ragweed SPPs are fully capable of activating dendritic cells (DCs) in the airways and SPPs' NAD(P)H oxidase activity is involved in initiation of adaptive immune responses against innocuous pollen proteins.</p> </div

Cytokine production of CD3⁺ pan-T cells in response to co-culture with SPP-exposed human moDCs.

<p>Freshly isolated CD3⁺ pan-T cells obtained from three ragweed allergic individuals and three non-allergic ones were co-cultured with allogeneic moDCs pre-treated with SPPs for 4 days under various conditions as indicated on the figure. The numbers of IFN-γ-, IL-17-, and IL-4-producing T cells were detected by ELISPOT assays. Each assay was performed three times per donor, and data from one representative experiment are shown. Data are presented as means ± SEM. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 vs untreated DCs, ^#<i>p</i><0.05, ^##<i>p</i><0.01, ^###<i>p</i><0.001. (DPI: diphenyleneiodonium; N/D: not detectable; SPP: subpollen particle).</p

Cytokine and chemokine profile of human moDCs exposed to SPPs.

<p>ELISA was used to determine the release of cytokines (IL-6, TNF-α, IL-10) and chemokine (IL-8) from human moDCs in response to freshly isolated SPPs, DPI-treated SPPs, and heat-inactivated SPPs, in the presence and absence of NADPH, after 24 h incubation. Data are presented as means ± SEM of four individual experiments. * <i>p</i><0.05; ** <i>p</i><0.01 vs untreated DCs, ^#<i>p</i><0.05. (DPI: diphenyleneiodonium; SPP: subpollen particle; SPP^H: heat-inactivated SPP).</p

Exposure to SPPs increases the intracellular ROS levels in human moDCs.

<p>Cells were incubated with freshly isolated ragweed SPPs for 1 h both in the presence and absence of NADPH. Cells were then loaded with the redox-sensitive H₂DCF-DA and changes in DCF fluorescence intensities were assessed by flow cytometry. Control experiments were performed using heat-inactivated SPPs and SPPs treated with DPI, an inhibitor of NADPH oxidases. Data are presented as means ± SEM of three individual experiments. *** <i>p</i><0.001 vs untreated DCs, ^##<i>p</i><0.01. (AU, arbitrary units; DCF: dichlorofluorescein; DPI: diphenyleneiodonium; SPP: subpollen particle; SPP^H: heat-inactivated SPP).</p

Uptake of SPPs by human moDCs.

<p>(A) Investigation of the percentage of human moDCs having attached/internalized SPPs by means of flow cytometry. Cells were incubated for 4 h with CellVue® Jade-labeled SPPs at 37°C or 4°C. The fluorescence of untreated cells (thin line) was used as a threshold level above which cells were considered to have attached SPPs (thick line). Numbers indicate the percentage of SPP attached/internalized cells in a representative measurement from four independent experiments. (B) Visualization of SPPs in human moDCs by confocal laser scanning microscopy. Human moDCs were cultivated with CellVue® Jade-labeled SPPs (green) for 4 h, stained with PE-conjugated anti-hDC-SIGN (red), fixed, and visualized by confocal microscopy. Cells are shown at 40× magnification. Scale bar  = 5 µm.</p

ROS generating capacity of freshly isolated SPPs.

<p>To determine ROS production by SPPs, H₂DCF-DA, a redox-sensitive dye, was added to the SPP suspension and the changes in DCF fluorescence intensity were measured by fluorometry. To prove that ROS production was due to intrinsic NAD(P)H oxidases present in SPPs, controls containing heat-inactivated SPPs, the supernatant of the freshly isolated SPPs, NADPH, a substrate of NAD(P)H oxidases, and DPI, an NADPH oxidase inhibitor, were included. Data are presented as means ± SEM of three individual experiments. ** <i>p</i><0.01; *** <i>p</i><0.001 vs PBS, ^#<i>p</i><0.05. (DCF: dichlorofluorescein; DPI: diphenyleneiodonium; SPP: subpollen particle; SPP^H: heat-inactivated SPP; SPP^SUP: supernatant of SPPs).</p

Phenotypic characterization of SPP-exposed human moDCs.

<p>Cells were treated with freshly isolated SPPs, DPI-treated SPPs, and heat-inactivated SPPs, individually and in combination with NADPH for 24 h. Expression of HLA-DQ and co-stimulatory molecules was analyzed by means of flow cytometry. Data are presented as means ± SEM of four independent experiments. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001 vs untreated DCs, ^#<i>p</i><0.05; ^##<i>p</i><0.01. (DPI: diphenyleneiodonium; SPP: subpollen particle; SPP^H: heat-inactivated SPP; SPP^SUP: supernatant of SPPs).</p

T cell-priming capacity of human moDCs exposed to SPPs.

<p>CFSE-labeled naïve CD4⁺ T cells were co-cultured with allogeneic moDCs pre-treated with SPPs under various conditions as indicated on the figure. After 5 days of co-cultivation, cell division was measured by flow cytometry. Numbers indicate the proportion of viable dividing T cells. Results are representative of four independent experiments. (DPI: diphenyleneiodonium; SPP: subpollen particle; SPP^H: heat-inactivated SPP).</p