Fig 2: DCIR mediates cockroach allergen binding and uptake.(A and B) Representative immunofluorescence images of dorsal skin sections and fluorescence analysis of DCIR staining in the skin tissues of patients with AD and controls (n = 8). Scale bar: 100 μm. (C) Quantification analysis of DCIR+tryptase+ cells in the lesion skin of patients with AD and controls. (D) Flow cytometry analysis of DCIR expression in human mast cell line cKit+FcεRI+ LAD2 cells. (E) Scheme of experimental protocol for the direct bindings of human recombinant DCIR (hrDCIR) to BSA, CRE, and Man-BSA. (F) Direct binding of different doses of hrDCIR (0–5.0 μg/mL) to BSA, CRE, or Man-BSA (n = 3). (G) Representative immunofluorescence images of FITC-CRE uptake by LAD2. Scale bar: 15 μm. (H and I) Flow cytometry analysis (H) and quantification (I) of FITC-CRE uptake at different doses (1–500 ng/mL) by LAD2 cells (n = 3). (J) Inhibition of FITC-CRE uptake in LAD2 cells pretreated with DCIR neutralizing antibody (α-DCIR) or IgG isotype (n = 4). Data represent mean ± SEM of 2 independent experiments. Data in B, C, and I were compared using a 2-tailed Student’s t test. Data in F and J were compared by 2-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: Lack of DCIR protects against cockroach allergen–induced skin allergic inflammation.(A) Representative skin images and EASI scores of PBS- and CRE-treated WT and DCIR–/– mice. (B) Representative H&E staining and epidermal thickness (μm) of skin tissues of PBS- and CRE-treated WT and DCIR–/– mice. (C) Representative Toluidine blue staining and quantification of cells with positive staining for Toluidine blue of skin tissue sections of PBS- and CRE-treated WT and DCIR–/– mice. Scale bar: 100 μm. Arrows represent mast cells. (D) Serum levels of specific IgE and IgG1 to CRE. (E) Quantitative PCR analysis of IL-4, IL-13, IL-33, and TNF-α expression in the skin tissues of PBS- and CRE-treated WT and DCIR–/– mice. Each circle represents 1 mouse. n = 8. Data represent mean ± SEM of 2 independent experiments. Data were compared by 2-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: DCIR regulates IgE-mediated mast cell activation and allergic responses.(A and B) BMMCs were confirmed and FcεRI expression was detected in BMMCs from WT and DCIR–/– mice by flow cytometry analysis. (C) Scheme of experimental protocol for IgE-mediated mast cell activation. (D) Flow cytometry analysis of CD107a expression in DNP-activated mast cells at different time points (n = 3). (E) ELISA analyses of β-hexosaminidase and histamine levels in supernatants of DNP-activated BMMCs from WT and DCIR–/– mice (n = 6). (F) Representative images of Evans blue–stained extravasation into ear skin of DNP-treated WT and DCIR–/– mice and quantification of the extravasation of Evans blue leakage into the skin (n = 6). (G) Representative Toluidine blue staining and quantification of cells with positive staining for Toluidine blue of skin tissue sections of DNP-treated WT and DCIR–/– mice (n = 6). Scale bar: 100 μm. (H) Heatmap of fold changes relative to the WT untreated group for the multiplex assays of cytokines and chemokines in supernatants of BMMCs from WT and DCIR–/– mice (n = 6). (I) ELISA analyses of cytokines and chemokines in supernatants of BMMCs from WT and DCIR–/– mice (n = 6). Data represent mean ± SEM of 2 independent experiments. Data were compared using a 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: DCIR regulates ROS generation and CaMKII oxidation.(A) Flow cytometry analysis of intracellular ROS production with CM-H2DCFDA. (B) Quantitative analysis of flow cytometry data (n = 3–5). (C) Schematic of the CaMKII kinase activity translocation reporter assay (CaMKII-KRT). (D and E) Representative immunofluorescence images of CaMKII-KTR transfection into WT or DCIR–/– BMMCs and then treated with or without DNP (white arrows indicate the location of the nucleus) (D) and quantification of cytosolic to nuclear KTR signal ratios (n = 12–20) (E). Scale bar: 10 μm. (F) Western blot analysis of ox-CaMKII expression in DNP-treated BMMCs from WT and DCIR–/– mice. (G and H) Representative immunofluorescence images of dorsal skin sections (G) and quantitative fluorescence analysis (H) of ox-CaMKII staining in the skin mast cells of CRE-treated WT and DCIR–/– mice (n = 16). Scale bar: 100 μm. Data represent mean ± SEM of 2 independent experiments. Data were compared by 2-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.