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Clinical Trial
, 2014, 231036

Circulating Conventional and Plasmacytoid Dendritic Cell Subsets Display Distinct Kinetics During in Vivo Repeated Allergen Skin Challenges in Atopic Subjects

Affiliations
Clinical Trial

Circulating Conventional and Plasmacytoid Dendritic Cell Subsets Display Distinct Kinetics During in Vivo Repeated Allergen Skin Challenges in Atopic Subjects

Stelios Vittorakis et al. Biomed Res Int.

Abstract

Upon allergen challenge, DC subsets are recruited to target sites under the influence of chemotactic agents; however, details pertinent to their trafficking remain largely unknown. We investigated the kinetic profiles of blood and skin-infiltrating DC subsets in twelve atopic subjects receiving six weekly intradermal allergen and diluent injections. The role of activin-A, a cytokine induced in allergic and tissue repair processes, on the chemotactic profiles of DC subsets was also examined. Plasmacytoid (pDCs) and conventional DCs (cDCs) were evaluated at various time-points in the blood and skin. In situ activin-A expression was assessed in the skin and its effects on chemokine receptor expression of isolated cDCs were investigated. Blood pDCs were reduced 1 h after challenge, while cDCs decreased gradually within 24 h. Skin cDCs increased significantly 24 h after the first challenge, inversely correlating with blood cDCs. Activin-A in the skin increased 24 h after the first allergen challenge and correlated with infiltrating cDCs. Activin-A increased the CCR10/CCR4 expression ratio in cultured human cDCs. DC subsets demonstrate distinct kinetic profiles in the blood and skin especially during acute allergic inflammation, pointing to disparate roles depending on each phase of the inflammatory response. The effects of activin-A on modulating the chemotactic profile of cDCs suggest it may be a plausible therapeutic target for allergic diseases.

Figures

Figure 1
Figure 1
Study design. Flow diagram showing the time-points when the skin challenges with allergen and diluent were performed and when the samples were taken. The dose of Dermatophagoides pteronyssinus administered was 30 BU at the allergen site with equal volume of diluent at the opposing site every week (solid arrows). A screening period of 2 weeks was introduced to clinically verify that subjects did not exhibit seasonal allergic symptoms or an upper/lower respiratory infection.
Figure 2
Figure 2
Early- and late-phase reactions. Mean diameter of skin reactions after each allergen challenge with Dermatophagoides pteronyssinus measured at 15 min and 1 h (a), as well as 6 h (b), is presented in mm. No statistical significant differences were observed between challenges or at different time-points. Values are expressed as mm ± SEM.
Figure 3
Figure 3
DC subsets exhibit different kinetic patterns in the peripheral blood following repeated allergen skin challenges in vivo. Gating strategies utilized to identify cDC and pDC subsets in the peripheral blood by flow cytometry. (a) For cDC identification, a 3-step analysis was performed. Initially, CD33pos cells were selected (Gate 1) to differentiate between mature lymphoid cells or lymphoid precursors (CD33neg) from other cells of myeloid origin that include cDCs. Next, all CD(14+16)dim⁡  to  neg/ILT3pos cells are selected in Gate 2 to exclude monocytes, macrophages, NK cells, and neutrophils. Gate 3 is drawn around CD33bright/ILT3pos cells, so cDCs are characterized as CD33bright/ILT3pos/CD(14+16)dim⁡ to neg. (b) Regarding pDCs, initially all CD123pos cells are selected (Gate 1) and then gated on the basis of CD(14+16)neg expression (Gate 2) to exclude monocytes, lymphocytes, and most granulocytes. Gate 3 is drawn around CD123bright/ILT3bright cells, strictly selecting pDCs and excluding basophils, so pDCs are characterized as CD123bright/ILT3bright/CD(14+16)neg. Representative FACS plots are shown. The percentages of pDCs (c) and cDCs (d) in the peripheral blood at baseline and following in vivo allergen challenges are shown. Data are expressed as median with interquartile range (first and third quartiles).
Figure 4
Figure 4
Plasmacytoid DC kinetics in the skin upon in vivo allergen challenge. Immunofluorescence staining was performed on skin biopsies and examined by confocal microscopy. Counterstaining was performed with Hoechst to visualize nuclear DNA (blue, column 1). PDCs were stained with a monoclonal antibody against BDCA-2 and envisioned with a secondary goat anti-mouse antibody conjugated with AF488 (green, column 2). Column 3 is the result of merging columns 1 and 2. Representative microphotographs (×100) of pDCs are shown 24 h after the first challenge at the diluent (a) and allergen sites (b) and after the sixth challenge at the allergen site (c). PDC numbers were not significantly altered between different time-points, although a trend for increased pDCs was observed after the sixth allergen challenge (d). Data are expressed as median with interquartile range (first and third quartiles). *P < 0.05, WBC: whole blood cells.
Figure 5
Figure 5
Conventional DCs are recruited early to the skin upon in vivo allergen challenge. Immunofluorescence staining and data analysis were performed as described in Figure 4. Counterstaining was performed with Hoechst (blue, column 1). CDCs were stained with a monoclonal antibody against BDCA-1 and envisioned with a secondary goat anti-mouse antibody conjugated with AF568 (red, column 2). Column 3 is the result of merging columns 1 and 2. Representative microphotographs (×100) of cDCs are shown 24 h after the first challenge at the diluent (a) and allergen sites (b) and after the sixth challenge at the allergen site (c). Tissue cDCs significantly increased 24 h after the first allergen challenge compared to the diluent, and their numbers remained high after the sixth allergen challenge, although the difference was not significant (d). A significant inverse correlation was found between blood and skin tissue-infiltrating cDCs 24 h after the first allergen challenge (P = 0.0202; r = −0.667) (e). Data are expressed as median with interquartile range (first and third quartiles). *P < 0.05, WBC: whole blood cells.
Figure 6
Figure 6
Activin-A is increased in the skin after allergen challenge and correlates with BDCA+ cDCs. Activin-A in the skin was analysed using the alkaline phosphatase-antialkaline phosphatase method (red colour). (a) Activin A in normal skin (diluent site) was minimal and mostly located in a scattered fashion in the basal cells of the epidermis (black arrows) with minimal expression in the dermis (red arrows) (×100). (b) After the first allergen challenge, activin-A was more prominent and intense in the epidermis (black arrows) and in infiltrating inflammatory cells (red arrows) in the dermis (×100), also shown in (c) at higher magnification (×200). (d) Quantification of activin-A+ cells showed significantly higher expression at the allergen site 24 h after the first challenge compared to the diluent site. (e) The numbers of activin-A+ cells correlated with the numbers of BDCA-1+ cDCs in the dermis 24 h after allergen challenge (P = 0.0219; r = 0.662). Data are expressed as median with interquartile range (first and third quartiles). *P < 0.001.
Figure 7
Figure 7
Activin-A modifies the chemokine receptor profile of CD1c+ cDCs towards a skin-homing phenotype. CD1c+ cDCs were isolated from the peripheral blood of individuals with atopy to Dermatophagoides pteronyssinus (Der p1), cultured for 24 h with 1 μg/mL Der p1 in the presence of 50 ng/mL recombinant activin-A or PBS (control). DCs were stained with fluorochrome-conjugated antibodies against human CCR4, CCR10, CCR6, CCR9, and CXCR3 and analysed by flow-cytometry. (a) Activin-A induced an increase in CCR10 levels, concomitant with a decrease in CCR4 on CD1c+ cDCs during stimulation with Der p1 in vitro. No differences were observed regarding the expression of CCR6, CCR9, and CXCR3 in Der p1-stimulated CD1c+ cDCs in the presence or absence of activin-A. (b) Activin-A significantly increased the ratio of CCR10/CCR4 expressing cDCs. Data are representative of two independent experiments. *P < 0.05.

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