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. 2023 Jan 10;56(1):78-92.e6.
doi: 10.1016/j.immuni.2022.12.004.

Role of MR1-driven signals and amphiregulin on the recruitment and repair function of MAIT cells during skin wound healing

Anastasia du Halgouet  1 , Aurélie Darbois  1 , Mansour Alkobtawi  2 , Martin Mestdagh  1 , Aurélia Alphonse  1 , Virginie Premel  1 , Thomas Yvorra  3 , Ludovic Colombeau  3 , Raphaël Rodriguez  3 , Dietmar Zaiss  4 , Yara El Morr  1 , Hélène Bugaut  1 , François Legoux  1 , Laetitia Perrin  1 , Selim Aractingi  2 , Rachel Golub  5 , Olivier Lantz  6 , Marion Salou  7
Affiliations

Role of MR1-driven signals and amphiregulin on the recruitment and repair function of MAIT cells during skin wound healing

Anastasia du Halgouet et al. Immunity. .

Abstract

Tissue repair processes maintain proper organ function following mechanical or infection-related damage. In addition to antibacterial properties, mucosal associated invariant T (MAIT) cells express a tissue repair transcriptomic program and promote skin wound healing when expanded. Herein, we use a human-like mouse model of full-thickness skin excision to assess the underlying mechanisms of MAIT cell tissue repair function. Single-cell RNA sequencing analysis suggested that skin MAIT cells already express a repair program at steady state. Following skin excision, MAIT cells promoted keratinocyte proliferation, thereby accelerating healing. Using skin grafts, parabiosis, and adoptive transfer experiments, we show that MAIT cells migrated into the wound in a T cell receptor (TCR)-independent but CXCR6 chemokine receptor-dependent manner. Amphiregulin secreted by MAIT cells following excision promoted wound healing. Expression of the repair function was probably independent of sustained TCR stimulation. Overall, our study provides mechanistic insights into MAIT cell wound healing function in the skin.

Keywords: CXCR6 chemokine receptor; MAIT cells; TCR signaling; amphiregulin; scRNA-seq; skin excision; tissue repair.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
MAIT cells accelerate wound closure Full-thickness wounds were performed on B6-MAITCAST mice and splinted with a silicone ring to prevent epithelial contraction. (A) Longitudinal follow-up of wound surface (ratio wound over ring areas) for Mr1+/+ (black circle) or Mr1−/− (gray square) littermates. Blind experiment. t test. (B) Wound surface at days 4 and 7. Pooled data from four independent experiments (n4 = 13; n7 = 9). Mann-Whitney test. (C) Hematoxylin and eosin-saffron staining of Mr1+/+ and Mr1−/− wounds 4 days after excision and longitudinal follow-up of wound gap (distance between the epithelial tongues). Pooled data from two independent experiments analyzed blindly (n1 = 2/3; n2 = 3/4; n4 = 5). Mann-Whitney test.
Figure 2
Figure 2
Skin MAIT cells accumulate in the wound and constitute a homogeneous type 17 T cell population with a tissue repair program (A) Flow cytometry staining (left), frequency (middle), and number (left, ratio wound over control numbers) of skin MAIT cells from wound and control sites at various time points. Pooled data from seven (n = 22) and four (nD4 = 10, nD6 = 7, and nD35 = 4) independent experiments for frequencies and numbers, respectively. Wilcoxon test. (B) MAIT cells sorted from thymus, wound (D4), and steady-state skin were analyzed by scRNA-seq and integrated. UMAP (Uniform Manifold Approximation and Projection, top) and features plot for Zbtb16 and Rorc expression (bottom) are displayed. (C) Cluster were defined by signature enrichment (Table S1; STAR Methods). (D) UMAP from (B) split according to dataset origin. (E) Rorc-GFP reporter expression by MAIT cells from wound and control sites. Pooled data from three independent experiments (n = 8). Please also see Figure S2B. (F) Average gene expression from MAIT cells in wound site and steady-state skin. (G) Differentially expressed genes in non-cycling MAIT17 cells from skin (wound and steady state) as compared to thymus. The average expression was calculated on scaled data after subsetting MAIT17 clusters from (B). (H) Nr4a1-GFP reporter expression by skin MAIT cells from wound (red) and control (orange) skin sites, by steady-state non-MAIT TCRβ+ (dark gray) and by TCRβ− (light gray) cells. Data are representative of two independent experiments (n = 5). (I) Tissue repair signature score on non-cycling MAIT17 cells. Tukey’s multiple comparison test. Please also see Figure S2E. (J) Average expression of tissue repair and MAIT17 signatures on clusters from (B). Please also see Figure S2F.
Figure 3
Figure 3
MAIT cells are recruited into the inflamed skin (A) Ki67 expression by MAIT cells. Data are from two (n = 5) independent experiments. Wilcoxon test. (B) Parabiosis protocol (left) and CD45.2/2 and CD45.1/2 staining (middle). Percentage of partner-derived MAIT, ɣδ T, and mainstream T cells in the skin (right) at steady-state and in the wound and control sites 4 days after excision. Data are from three independent experiments (nsteady state+control = 9; nexcision = 5). Sídák multiple comparison test. Please also see Figure S3A. (C) Tissue residency and circulating signature scores on non-cycling MAIT17 cells. Tukey’s multiple comparison test. (D) CD69 and CD103 expression by MAIT cells. Pooled data from six independent experiments (nCD69 = 23; nCD103 = 27). Wilcoxon test. (E) Graft protocol and CD45.2 and CD45.1 staining (left). CD45.2 donor cell frequency in MAIT and ɣδ T cells from the donor skin (D0) and after 6 days in the graft (middle). Absolute number of recipient CD45.1+ MAIT cells in grafts from D0, D6, and D12 (right, grafts from same donor are linked). Pooled data from two independent experiments (nD0 = 5; nD6 = 3/6; nD12 = 6). Mann-Whitney and Wilcoxon tests as appropriate. (F) Example of Kaede green and red expression (left) and frequency of photoconverted cell (right) in skin MAIT and ɣδ T cells. Pooled data from two independent experiments (nD0 = 4, nD2 = 5). Paired t test. (G) The number of MAIT cells in the inguinal and brachial LNs draining the wound or the control sites. Pooled data from two independent experiments (n = 6). Paired t test. Please also see Figures S3B and S3C. (H) Numbers of MAIT cells (ratio wound over control sites) 4 days after excision in FTY720- or PBS-treated mice. Pooled data from two independent experiments (nPBS = 5; nFTY720 = 6). Mann-Whitney test. Please also see Figure S3D.
Figure 4
Figure 4
MAIT cell recruitment and skin healing are independent of MR1 (A) Grafts were performed on Mr1+/+ animals. MAIT cell staining (left) and numbers (right) in Mr1−/− and Mr1+/+ grafts before transplant (D0) and longitudinally after grafting. Pooled data from 2 independent experiments (Mr1+/+: nD0 = 3, nD6,12 = 4; Mr1−/−: nD0 = 3, nD6 = 4, nD12 = 6). Unpaired t test. (B) Mr1−/− and Mr1+/+ mice were parabiosed for 5 weeks. MAIT cell staining in the skin of the Mr1−/− parabiont skin (left) and numbers at wound and control sites (right) for Mr1−/− and Mr1+/+ parabionts and Mr1−/− control mice. Pooled data from two independent experiments (nMr1−control = 3; nMr1−parabiont = 9; nMr1+parabiont = 9). Tukey multiple comparison test. (C) Percent of wound closure in Mr1−/− and Mr1+/+ parabionts and control Mr1−/− mice. Pooled data from two independent experiments (nMr1−/−alone = 3; nMr1−/−paired = 8; nMr1+/+paired = 8). Mann-Whitney and Wilcoxon tests as appropriate. (D) Mean fluorescence intensity of GFP expression on MAIT cells at wound and control skin sites in Nr4a1-GFP animals. Pooled data from two independent experiments (n = 6). Paired t test. (E) MAIT cell numbers at wound and control sites 4 days after transfer into Cd3e−/−Mr1+/+ and Mr1−/− mice. Pooled data from two independent experiments (nMr1+/+ = 4; nMr1−/− = 4). Mann-Whitney test. Please also see Figures S4A–S4C. (F) Longitudinal follow-up of wound surface of transferred Cd3e−/−Mr1+/+ and Mr1−/− mice and non-transferred Cd3e−/−Mr1+/+ control mice. Pooled data from two independent experiments with one blinded (nWithTransfer = 4/4; nWithoutTransfer =8). Please also see Figure S4D.
Figure 5
Figure 5
CXCR6 is necessary for MAIT cell recruitment into the skin (A) MAIT and ɣδ T cell staining (left) and numbers (wound over control sites) (right) in skin control and wound (D4) sites following Ptx injection. Pooled data from two independent experiments (n = 6). Mann-Whitney test. Please also see Figures S5A and S5B. (B) Chemokine receptor gene expression by non-cycling MAIT17 cells from integrated single-cell datasets as in Figure 1B. (C) CXCR6 expression by MAIT cells. Data are representative of 10 independent experiments. (D) CXCL16 protein quantity in total skin lysate from wound (D4) or steady-state skin of Mr1+/+ and Mr1−/− mice. Pooled data from two independent experiments (n = 4). Mann-Whitney test. (E) MAIT cell staining (left) and numbers (right) in control and wound (D4) skin sites following ɑ-CXCL16 or isotype control i.p. injection. Pooled data from two independent experiments (n = 7/10). Mann-Whitney test. (F) Expanded MAIT cells were deleted for Cxcr6 by CRISPR-Cas9 modification. Congenic marker and CXCR6 expression on the injected pool (top left) or recovered cells (bottom left). Quantitation of recovered Cxcr6−/− cells (ratio of Cxcr6−/− over Cxcr6+/+) at different sites (right). Pooled data from two independent experiments (ninjection = 2; nother = 6). Tukey’s multiple comparison test.
Figure 6
Figure 6
MAIT cell-derived Areg exerts a tissue repair function (A) Representative immunofluorescence images of wounds from Mr1+/+ and Mr1−/− animals (DAPI in blue, K14 in green) (left). Scale bar represents 100 μm. The epidermal tongues are underlined with white dashed lines and their length is quantified (right, D2/D4, 2 tongues per slide). Pooled data from one (D2: n = 3) and two independent experiments (D4: n = 5/4) analyzed blindly. Mann-Whitney test. (B) Representative immunofluorescence images of wounds from Mr1+/+ and Mr1−/− animals (DAPI in blue, Ki67 in red). The white dashed line separates the epidermal tongue and the underlying dermis (left). Proliferation in the epidermis is quantified by the Ki67/DAPI ratio and normalized to the average expression in Mr1−/− animals for each experiment (right). Data are from two independent experiments (n = 5/6) analyzed blindly. Unpaired t test. Please also see Figure S6B. (C) Dot plot showing RNA expression of repair molecules by non-cycling MAIT17 cells from integrated single-cell datasets as in Figure 1B. (D) Feature plot of Areg expression projected on the UMAP of the integrated datasets. (E) Ex vivo Areg staining on skin MAIT cells (blue: control skin; red: wound skin; gray: full staining except the biotinylated anti-Areg antibody). Pooled data from two independent experiments (n = 6). Wilcoxon test. (F) Areg expression by thymic enriched MAIT cells following 36 h of in vitro activation by 5-OP-RU or IL-18. One experiment (n = 8) representative of 2. Dunn’s multiple comparison test. (G) Wound surfaces at days 4 and 6 after excision on Zbtb16-cre−Aregfl/fl (black) and Zbtb16-cre+Aregfl/fl (gray). Pooled data from two independent experiments with one blind (full symbols) (ncre− = 8; ncre+ = 10). Mann-Whitney tests. (H) Wound surfaces after excision (D5 and D7) of Cd3e−/− animals transferred with thymic MAIT cells expanded from Zbtb16-cre−Aregfl/fl (black) and Zbtb16-cre+Aregfl/fl (gray) littermate mice. Pooled data from two independent experiments with one blindly analyzed (full symbols) (ncre− = 9; ncre+ = 8). Mann-Whitney tests.
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