Novel cutaneous mediators of chemical allergy

Figures & data

Figure 1. Ear ILC2 Phenotyping following TDI sensitization in mice. Flow cytometric analysis of ILC2 isolated from the ear at 2 days post dermal treatment (25 µl/ear) of female Balb/c mice with acetone, 0.5% TDI, or 4% TDI (as indicated on the X-axis). These concentrations were previously determined to be sensitizing following a single dermal application. While exposure at 4% induced irritation, 0.5% induced sensitization in the absence of irritation. Cells were isolated from the ear via enzymatic digestion (Liberase-TL) and then passed through cell strainer to obtain single cell suspensions. Cells were counted, stained, fixed, and data acquired on an LSRII flow cytometer (BD Biosciences). Following doublet-exclusion gating, ILC2s were identified as Lineage^-CD45⁺CD90⁺CD25⁺CD127⁺ cells. The lineage gate was composed of antibodies detecting the following antigens: Ter119, CD19, CD3e, CD11b, Ly-6G, CD2, and CD11c. ILC2s were then further characterized by analyzing median fluorescent intensities (MFI) for activation markers (A) ICOS, (B) KLRG1, and (C) CD25. Bars represent mean MFI ± SEM. n = 5 mice/treatment group. p Values are represented by asterisks (p < .01).

Figure 2. T_reg Phenotyping in mouse skin following TDI sensitization. Flow cytometric analysis of dermal T_reg cells following treatment (25 µl/ear) of female Balb/c mice with acetone, 0.5% TDI, or 4% TDI (as indicated on X-axis). These concentrations were previously determined to be sensitizing following a single dermal application. While exposure at 4% induced irritation, 0.5% induced sensitization in the absence of irritation. Cells were isolated from the ear via enzymatic digestion (Liberase-TL) and then passed through a cell strainer to obtain single cell suspensions. Cells were counted, stained, fixed, and data was acquired on an LSRII flow cytometer (BD Biosciences). Following doublet exclusion gating, T_reg cells were first gated on their expression of CD45, CD3, and CD4 then were further identified by CD25 and Foxp3 expression (defined as T_reg cells) at indicated timepoints. T_reg cell frequency and number were determined based on flow cytometry analysis and extrapolation of this data with total ear cellularity. Graph symbols represent mean (±SE) of 5 mice/group. p Values are presented as ^0.5% TDI and *4% TDI (p < .05). Dermal treatment groups are indicated by the following symbols: circle = acetone, square = 0.5% TDI, and triangle =4% TDI.

Figure 3. MiRNA expression in mouse skin following TDI sensitization. RT-PCR analysis of miRNA expression in the ears following a single dermal application (25 µl/ear) to female Balb/c mice of 4% TDI or acetone vehicle. This concentration was previously determined to be sensitizing following a single dermal application. Expression of (A) miR-210, (B) miR-22, and (C) miR-31 determined 7 days post-TDI exposure. Bars represent mean relative fold-change (±SE) of 4–5 mice/group. p Values are represented by asterisks (p < .05).