Immuno-inhibitory MCs and MC-CD8+ T-cell interactions appear to be down-regulated in AA.

<p>Immunohistochemical identification of c-Kit+/IL-10+ MCs and CD8+ T-cells in human scalp skin of controls (A) and AA patients (B), higher magnification of a single MCs in inserted panels. Higher magnification of IL-10/CD8/c-Kit triple IHC which shows c-Kit+ MCs (blue arrows), c-Kit+/IL-10+ MCs (green arrows), IL-10+ cells (red arrows) and CD8+ T-cells (brown arrows) (C). Quantitative analysis of IL-10+ MCs (detected by c-Kit) in AA patients compared to controls (D). Analysis derived from 20 areas of 11 HFs of 7 healthy controls and 14 areas of 5 HFs of 3 AA patients for non-lesional skin and 20 areas of 16 HFs of 7 AA patients for lesional skin, ***p≤0.001, ±SEM, Mann-Whitney-U-Test. Representative pictures of IL-10- (E,F) and IL-10+ (G,H) MCs (detected by c-Kit) interacting with CD8+ T-cells. Representative pictures showing low (I) and high (J) PD-L1 IR of human healthy skin MCs <i>in situ</i>. Immunohistochemical identification of PD-L1+/c-Kit+ MCs and CD8+ T-cells in human healthy skin (I-L) and AA patients (M). Brown arrows indicate CD8+ cells, blue arrows indicate c-Kit+ MCs, red arrows indicate PD-L1+ cells, green arrows indicate PD-L1+/c-Kit+ MCs. This staining was performed on one section/subject of 8 healthy individuals of non-lesional skin from 4 AA patients, and of lesional skin from 12 AA patients. Size bars: 200 µm (A–C), 50 µm (L,M), 20 µm (I,J) and 10 µm (E–H). Connective tissue sheath (CTS), hair follicle (HF), hair matrix (HM), outer root sheath (ORS), perifollicular dermis (PFD), sebaceous glands (SG).</p

Immunostainings.

<p>List of all immunostainings which were performed and relevant details (For list of primary antibodies, see Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094260#pone.0094260.s001" target="_blank">File S1</a>).</p><p>ABC-AP: Avidin-biotin complex, alkaline phosphatase; ABC-HRP: Avidin-biotin complex, horseradish peroxidase; AEC: 3-amino-9-ethylcarbazole; DAB: 3,3′-diaminobenzidine, DAPI: diamidino-2-phenylindole, Envision-HRP: Envision- horseradish peroxidise; FITC: Fluorescein isothiocyanate; IF: immunofluorescence, SIGMAFAST™: Fast Red TR/Naphthol AS-MX tablets, Rho: Rhodamine.</p

Schematic summary: MC immuno-phenotype and MC-CD8+ T-cell interactions in healthy compared to lesional AA skin.

<p>In human healthy skin, MCs are mostly non-degranulated and they express the SCF receptor, c-Kit, and MHCI molecules. Most of them express IL-10, TGFβ1 while only some express OX40L and PD-L1 and very few express CD30L and ICAM-1 (A). In lesional AA skin, the degranulation of MCs is increased (release of tryptase, heparin and histamine) and the expression of tryptase is increased, while the contents of TGFbeta1, IL-10 are decreased. Moreover, the numbers of OX40L+, CD30L+, 4-1BBL+ and ICAM-1+ MCs are up-regulated, while MCs positive for IL-10 and PD-L1 are down-regulated (B). In human healthy skin, rare MCs are found in close contact with CD8+ T-cells and most of them express OX40L. Therefore, we hypothesized that OX40/OX40L might mediate this interaction. Rarely, we found IL-10+ MCs interacting with CD8+ T-cells (C). In lesional AA skin, many MCs interact with CD8+ T-cells. During this cross-talk, most MCs express OX40L but instead, in some rare cases, 4-1BBL and ICAM-1 were expressed. These ligands might stimulate the activation and proliferation of CD8+ T-cells. Since MCs during the interaction are also degranulating, we hypothesize an activation of PAR-2 (tryptase receptor) on CD8+ T-cells. Finally, we suggest that MCs may operate as autoantigen-presenting cells (D). This schematic drawing was prepared using the Biomedical-PPT-Toolkit-Suite of Motifolio Inc., USA.</p

Human AA lesions show increased density, proliferation and degranulation of perifollicular MCs.

<p>The immunohistochemical identification and evaluation of MCs by c-Kit (A,D), TB (B,E) or Ki-67/tryptase (C,F) revealed a strong increase of MC numbers in AA (D–F) compared to control healthy (A–C) skin. Red arrows indicate MCs. C-Kit/tryptase double-IF shows immature c-Kit+ MCs (stained in green) and mature c-Kit+/tryptase+ MCs in AA skin (stained in green and red) (G). See inserted panels in the bottom left of each Figure for higher magnification views of the area highlighted in the small boxes. Reference area for the quantitative analysis using (immuno-)histomorphometry for cell counting in the connective tissue sheath (CTS) and perifollicular dermis (PFD). CTS+PFD represents the total area including the space demarcated up to 200 µm from the HF basement membrane (C,F). Fold change of MC density detected by c-Kit, TB and tryptase stainings (H). Black line indicates the control. Analysis derived from 69–81areas (HFs) of 11–17 AA patients and from 50–69 areas (HFs) of 5–7 healthy controls, ±SEM, *p≤0.05, **p≤0.01, ***p≤0.001, Mann-Whitney-U-Test or Student t-test (for c-Kit, TB and tryptase compared to respective controls and for comparing bars between CTS and PFD), Kruskal-Wallis test (p<0.0001) followed by Dunn's test (for comparing c-Kit, TB and tryptase within CTS and PFD). Identification of MCs by Ki-67/tryptase IHC (I,J,M), Ki-67/tryptase IF (K), Ki-67/c-Kit IHC (L) and TB (N) showing non-degranulating, non proliferating MCs (blue arrows), degranulating, non-proliferating MCs (green arrows), non-degranulating MCs undergoing proliferation (red arrows) and proliferating degranulating MCs (orange arrows). Quantitative analysis of MC proliferation by Ki-67/tryptase IHC (O). Analysis derived from 81 areas (HFs) of 17 AA patients and 50 areas (HFs) of 7 healthy controls, ±SEM, Mann-Whitney-U-Test (ns). Quantitative analysis of MC degranulation by TB histochemistry and Ki-67/tryptase IHC (P). Black line indicates the control. Analysis derived from 69–81 areas (HFs) of 11–17 AA patients and 50–69 areas (HFs) of 5–7 healthy controls, ±SEM, *p≤0.05, **p≤0.05, ***p≤0.001 Mann-Whitney-U-Test (compare to control), Mann-Whitney test (TB compare to tryptase) (ns). Scale bars: 100 µm (A–G) and 20 µm (I–N) Connective tissue sheath (CTS), hair shaft (HS), inner root sheath (IRS), outer root sheath (ORS), perifollicular dermis (PFD), toluidine blue (TB), sebaceous gland (SG).</p

MC numbers and interactions with CD8+ T-cells are increased in the humanized-mouse model of AA.

<p>Representative pictures of tryptase/CD8 double-staining in control (A) and AA-like (B) mice. Tryptase+ MCs are labelled in pink while CD8+ cells are labelled in brown. Quantitative analysis of tryptase+ MCs (C), c-Kit+ MCs (D), CD8+ cells (E) and tryptase+ MC-CD8+ cell interactions (F) in AA-like mice compared to control mice. Analysis deriving from 1–6 areas (HFs)/mouse of 3 mice/group from 2 experiments, ±SEM, Student's t-test or Mann-Whitney-U-Test (ns). Scale bars: 50 µm.</p

The number of perifollicular CD8+ T-cells and MC-CD8+ T-cell interactions are increased in AA.

<p>Immunohistochemical identification of tryptase+ MCs and CD8+ T-cells in human scalp skin of controls (A) and AA patients (B). Quantitative analysis of CD8+ T-cells (C) and of their interactions with tryptase+ MCs (D). Analysis derived from 56 areas (HFs) from 13 AA patients and 44 areas (HFs) of 7 healthy controls, ***p≤0.001, ±SEM, p value was calculated by Mann-Whitney-U-Test. Non-degranulating MCs (E,G,H) and degranulating MCs (F,I,J) close to CD8+ T-cells Scale bars: 50 µm (A–B) and 10 µm (E–J). Connective tissue sheath (CTS), dermal papilla (DP), outer root sheath (ORS), perifollicular dermis (PFD).</p

MCs in lesional AA skin up-regulate co-stimulatory molecules for CD8+ T-cells.

<p>Immunohistochemical identification of OX40L+/tryptase+ MCs, detected using OX40L/CD8/tryptase staining (A–E) and CD30L+/c-Kit+ MCs, detected using CD30L/c-Kit (M), showing the expression pattern of OX40L (A–E) and CD30L (M) within MCs, in human scalp skin of controls (A,B,D,M) and AA patients (C,E). Higher magnification of OX40L+/tryptase+ (F) and CD30L+/c-Kit+ (N) MCs. Representative pictures of OX40L+ (G) and OX40L− (H) (detected by tryptase) and CD30L− (O) and CD30L+ (P) (detected by c-Kit) MCs interacting with CD8+ T-cells. Immunohistochemical identification of OX40L+/tryptase+ (I,J) and CD30L+/c-Kit+ (Q,R) MCs and CD8+ T-cells in human HFs from lesional (I,Q), non-lesional (J) AA and healthy (R) skin. Brown cells are OX40L+ (A–J) or CD8+ (M–R) cells (brown arrows), blue cells are CD8+ (A–J) or c-Kit+ (M–R) cells (blue arrows), pink cells are tryptase+ cells (A–J) (pink arrows), red cells are CD30L+ cells (M–R) (red arrows), pink-brown cells are OX40L+/tryptase+ cells (A-J) and blue-red cells are CD30L+/c-Kit+ cells (M–R) (green arrows). Quantitative analysis of the % of OX40L+/tryptase+ MCs among all MCs (K), OX40L+/tryptase+ MCs interacting with CD8+ T-cells (L) and the % of CD30L+/c-Kit+ MCs among all MCs (S) in AA patients compared to controls. Analysis derived from 17–21 areas of 6–14 HFs of 6–7 healthy control and of 11–21 areas of 4–12 HFs of 3 AA patients for non-lesional skin and of 17–21 areas of 16–17 HFs of 7 AA patients for lesional skin. ±SEM ***p≤0.001, **p≤0.01, *p≤0.05 ±SEM, One-Way ANOVA or Kruskal-Wallis test followed respectively by Bonferroni's or Dunn's multiple comparison tests. Immunohistochemical identification of 4–1BBL+/c-Kit+ and 4-1BBL-/c-Kit+ MCs, detected using 4–1BBL/c-Kit/CD8 staining (T–W) and of ICAM-1+/tryptase+ and ICAM-1-/tryptase+ MCs, detected using ICAM-1/CD8/Tryptase staining (X-AA), around the HF bulb of AA patient (T,X) and control (W,Z). Representative pictures of 4–1BBL+/c-Kit+ (U), 4–1BBL-/c-Kit+ MCs (V), ICAM-1-/tryptase+ (Y) and ICAM-1+/tryptase+ (AA) MCs interacting with CD8+ T-cells. Brown cells are CD8+ (T–W) or ICAM-1+ (X-AA) cells (brown arrows), blue cells are c-Kit+ MCs (T–W) or CD8+ cells (X-AA) (blue arrows), red cells are 4–1BBL+ cells (T–W) (red arrows), pink cells are tryptase+ cells (X-AA) (pink arrows), blue-red cells are 4–1BBL+/c-Kit+ MCs (T–W) and pink-brown cells are ICAM-1+/tryptase+ MCs (X-AA) (green arrows). These stainings were observed in one section/subject of 6–8 healthy individuals and non-lesional skin from 4 AA patients and lesional skin from 11 AA patients. Scale bars: 20 µm (A–C, N–P,AA), 10 µm (D–E,U,V,Y), 5 µm (F–H), 50 µm (I–J,M,Q,R,T,W,X,Z). Connective tissue sheath (CTS), dermal papilla (DP), hair follicle (HF), hair matrix (HM), perifollicular dermis (PFD).</p

AA MCs contain less TGFβ1 and more tryptase compared to control MCs.

<p>Quantitative analysis of TGFβ1 IR in perifollicular MCs in AA patients compared to controls (A). Analysis derived from 272 MCs around 29 HFs of 10 AA patients and 175 MCs around 19 HFs of 2 healthy controls, ±SEM, Mann-Whitney-U-Test (ns). Representative pictures of TGFβ1+ MCs in human scalp skin of controls (B) and AA patients (C) stained by TGFβ1(green)/c-Kit(red) double-staining. Representative picture of TGFβ1(red)/c-Kit(green) double-staining (D). Quantitative analysis of tryptase IR in perifollicular MCs in AA patients compared to controls (E). Analysis derived from 272 MCs around 41 HFs of 14 AA patients and 182 MCs around 19 HFs of 2 healthy controls, ***p≤0.001, ±SEM, Mann-Whitney-U-Test. Representative pictures of tryptase+ MCs in human scalp skin of control (F,H) and AA patients (G,I) stained by Ki-67/tryptase double-staining. Scale bars: 20 µm (B–D) and 50 µm (F–I). Connective tissue sheath (CTS), hair follicle (HF), outer root sheath (ORS).</p

Experimental design and specific questions addressed.

<p>Experimental design and specific questions addressed.</p

MC numbers, degranulation and interactions with CD8+ T-cells are increased in the C3H/HeJ-mouse-model of AA.

<p>Representative pictures of c-Kit/CD8 double-staining in normal (NM) (A), sham-grafted (mSH) (B), failed-grafted (fAA) (C) and AA (mAA) (D) mice. C-Kit+ MCs are labelled in pink while CD8+ T-cells are labelled in brown. Quantitative analysis of c-Kit+ (E), CD8+ T-cells (F) and c-Kit+ MC/CD8+ T-cell interactions (G) in mAA compared to NM, mSH and fAA control mice. Representative pictures of c-Kit+ MC-CD8+ T-cell interaction (H). Representative pictures of mMCP6/CD8 double-staining in normal (NM) (l), sham-grafted (mSH) (J), failed-grafted (fAA) (K) and AA (mAA) (L) mice. mMCP6+ MCs are labelled in red while CD8+ T-cells are labelled in brown. Quantitative analysis of mMCP6+ cells (M), % of degranulation (N) and mMCP6+ MC-CD8+ T-cell interactions (O) in mAA compared to NM, mSH, fAA control mice. Immunohistochemical identification of non-degranulating (black arrows) and degranulating (red arrow) of mMCP6+ MCs (P) and mMCP6+MC-CD8+ T-cell interaction (Q). Analysis derived from 6 HFs/mouse of 2–4 mice/group ±SEM, One-Way ANOVA or Kruskal-Wallis test followed by Bonferroni's test or Dunn's test (*p≤0.05, **p≤0.01, ***p≤0.001). Scale bars: 50 µm (A–D, M–L) and 10 µm (G,P,Q). AA mice (mAA), failed-grafted AA mice (fAA), normal mice (NM), sham-grafted mice (mSH).</p