Bleomycin hydrolase downregulation in lesional skin of adult atopic dermatitis patients is independent of FLG gene mutations Laurence Pellerin, PhD,

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Presentation transcript:

Bleomycin hydrolase downregulation in lesional skin of adult atopic dermatitis patients is independent of FLG gene mutations Laurence Pellerin, PhD, Carle Paul, MD, PhD, Anne-Marie Schmitt, MD, PhD, Guy Serre, MD, PhD, Michel Simon, PhD Journal of Allergy and Clinical Immunology Volume 134, Issue 6, Pages 1459-1461.e7 (December 2014) DOI: 10.1016/j.jaci.2014.07.056 Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Reduced detection of BLMH in AD skin. Total epidermal proteins of 28 controls (Co) and 28 patients (AD) were immunodetected. Representative Ponceau red staining and blots (A). Immunodetected BLMH amounts were quantified and are indicated (AU/μg total proteins) using box plots (B) and individual values (C). Only statistically significant P values are shown. Journal of Allergy and Clinical Immunology 2014 134, 1459-1461.e7DOI: (10.1016/j.jaci.2014.07.056) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 In normal keratinocytes, BLMH is downregulated by IL4, IL13, and IL25. Keratinocytes from 2 healthy volunteers (V1 and V2) were cultured without (-) and with (100 ng/mL) the indicated cytokines. Levels of HRNR, FLG, and BLMH mRNA were evaluated by real-time RT-PCR (V1, left bars; V2, right bars) (A). Expression of BLMH and caspase-14 was analyzed by Western blotting (B). Journal of Allergy and Clinical Immunology 2014 134, 1459-1461.e7DOI: (10.1016/j.jaci.2014.07.056) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Profilaggrin/filaggrin ratio is increased in AD skin. Total epidermal proteins from NL and L skin of 28 healthy controls (C) and 28 atopic dermatitis patients (AD) were immunodetected with AHF3 antibody for the expression of filaggrin. One control and 7 patients carried an FLG mutation (black shaded circle; FLGmut); the others did not (non-shaded circle; FLGwt). Representative Western blots (A). Profilaggrin and processing intermediates (referred to as proFLG) are indicated by a bracket and filaggrin monomers (FLG) by an arrow. Molecular masses are indicated on the right in kDa. Immunodetected amounts of profilaggrin and filaggrin were quantified (B). Profilaggrin/filaggrin ratio and the median values are indicated. Calculated P values are shown only when statistically significant. Journal of Allergy and Clinical Immunology 2014 134, 1459-1461.e7DOI: (10.1016/j.jaci.2014.07.056) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Procaspase-14 is increased, and its activation is reduced in atopic skin. Total epidermal proteins of 28 controls (C) and 28 atopic dermatitis patients (AD) were separated by SDS-PAGE, transferred to membranes, stained with Ponceau red, and immunodetected with an antibody recognizing both the procaspase-14 (pro) and the large subunit of the active caspase-14 (cas). Representative results for 2 controls (C1 and C2) and 2 patients (AD1 and AD2) are shown (A). Both the NL and L skin were analyzed. The immunodetected procaspase-14 (B) and active caspase-14 (C) were quantified, normalized to the total amounts of proteins in the samples, and plotted as arbitrary units (AU) per μg of proteins for each of the extracts. Bars represent the median values. The subjects were stratified according to the absence (FLGwt) or the presence (FLGmut) of an FLG gene mutation. The proform/active form ratios were calculated and are indicated using box plots showing the median (midline), first and third quartiles, and minimal and maximal values (D). Calculated P values are shown when significant. Journal of Allergy and Clinical Immunology 2014 134, 1459-1461.e7DOI: (10.1016/j.jaci.2014.07.056) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Detection of calpain-1. Total epidermal proteins of 28 controls (C; one was a carrier of an FLG mutation) and 28 atopic dermatitis patients (AD, 7 were carriers of an FLG mutation) were separated by SDS-PAGE, transferred to membranes, stained with Ponceau red, and immunodetected with an antibody recognizing calpain-1 (CALP1). Representative results corresponding to the extracts of 3 controls (C1-C3) and 3 patients (AD1-AD3) are shown (A). Both the NL and L skin were analyzed. The amounts of immunodetected calpain were quantified (AU per μg) and are indicated using box plots showing the median (midline), first and third quartiles, and minimal and maximal values (B). Calculated P values are shown when significant. Journal of Allergy and Clinical Immunology 2014 134, 1459-1461.e7DOI: (10.1016/j.jaci.2014.07.056) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions