Phenotype, Function, and Mobilization of 6-Sulfo LacNAc-Expressing Monocytes in Atopic Dermatitis

Abstract

Mononuclear phagocytes (MPs) are important immune regulatory cells in atopic dermatitis (AD). We previously identified 6-sulfo LacNAc-expressing monocytes (slanMo) as TNF-α- and IL-23-producing cells in psoriatic skin lesions and as inducers of IFN-γ-, IL-17-, and IL-22-producing T cells. These cytokines are also upregulated in AD and normalize with treatment, as recently shown for dupilumab-treated patients. We here asked for the role of slanMo in AD. Increased numbers of slanMo were found in AD skin lesions. In difference to other MPs in AD, slanMo lacked expression of FcɛRI, CD1a, CD14, and CD163. slanMo from blood of patients with AD expressed increased levels of CD86 and produced IL-12 and TNF-α at higher amounts than CD14⁺ monocytes and myeloid dendritic cells. While CD14⁺ monocytes from patients with AD revealed a reduced IL-12 production, we observed no difference in the cytokine production comparing slanMo in AD and healthy controls. Interestingly, experimentally induced mental stress, a common trigger of flares in patients with AD, rapidly mobilized slanMo which retained their high TNF-α-producing capacity. This study identifies slanMo as a distinct population of inflammatory cells in skin lesions and as proinflammatory blood cells in patients with AD. slanMo may, therefore, represent a potent future target for treatment of AD.

Keywords: 6-sulfo LacNAc-expressing monocytes; CD86; IL-12; atopic dermatitis; dendritic cells; immunology; inflammation.

Figure 1

slan⁺ cells are a distinct population of dermal monocytes in atopic dermatitis. Immunohistochemical staining of 6-sulfo LacNAc-expressing monocytes (slanMo) in normal human skin (A) and skin samples from an atopic dermatitis patient (B). The results of multiple samples are shown in (C): healthy skin n = 10 and skin from atopic dermatitis patients n = 9. (D) Immunofluorescent stainings of slan (red) in combination with either HLA-DR, CD1a, CD14, or CD163 (green) was performed. Cell nuclei were stained with DAPI (blue). (A,B,D) Representative results of 10 donors that were analyzed. Statistical significance (C) was calculated using an unpaired t-test.

Figure 2

Expression of FcεRI by 6-sulfo LacNAc-expressing monocytes (slanMo) in skin and blood. (A) Immunofluorescent staining of slan (red) and FcεRI (green) in normal human skin (left) or skin from atopic dermatitis patients (right). Cell nuclei were stained with DAPI (blue). Representative results of 10 donors analyzed are shown. (B) Blood slanMo and CD14⁺ monocytes from patients with atopic dermatitis were analyzed for the expression of FcεRI by flow cytometry. A Representative result of 14 donors is shown.

Figure 3

Expression of surface maturation markers by blood 6-sulfo LacNAc-expressing monocytes (slanMo) from healthy donors and atopic dermatitis patients. slanMo were stained within freshly isolated or 24-h cultured peripheral blood mononuclear cells and analyzed for the expression of HLA-DR, CD83, and CD86 by flow cytometry (Ctrl n = 10, AD n = 14). Statistical significance was calculated using a one-way ANOVA followed by Tukey’s posttest.

Figure 4

IL-12 and TNF-α production in different subtypes of dendritic cells (DCs) and monocyte subsets from healthy donors and atopic dermatitis patients. (A) Gating strategy for the different subsets analyzed in (B,C) is demonstrated. (B) Representative histograms for IL-12 and TNF-α production are shown. Results of multiple donors (Ctrl n = 11, AD n = 15) are depicted in (C). Data are shown as mean + SEM. Statistical significance between Ctrl and AD within the different DC subsets was calculated using an unpaired t-test and a one-way ANOVA followed by Dunnett’s posttest was used to analyze differences in cytokine production between 6-sulfo LacNAc-expressing monocytes (slanMo) and the other subsets in AD.

Figure 5

6-Sulfo LacNAc-expressing monocytes (slanMo) are mobilized by mental stress and retain their capacity to produce TNF-α. (A) Blood cortisol levels were measured before and after induction of mental stress [Tier Social Stress Test (TSST)] in atopic dermatitis patients or after a resting period (no TSST) (n = 12). Frequency of slanMo in peripheral blood mononuclear cells (B) and their TNF-α production (C) were determined by flow cytometry (n = 7). Data are shown as mean + SEM and statistical significance was calculated using a one-way ANOVA followed by Tukey’s posttest.