Research ArticleSKIN INFLAMMATION

A wave of monocytes is recruited to replenish the long-term Langerhans cell network after immune injury

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Science Immunology  23 Aug 2019:
Vol. 4, Issue 38, eaax8704
DOI: 10.1126/sciimmunol.aax8704
  • Fig. 1 Immune injury leads the gradual replenishment of the epidermis with LC-like cells.

    (A) Male recipients received female BM alone (BMT) or with CD4 and CD8 (Mh) T cells. Chimerism was measured within the mature CD11b+Langerin+ LC population at different time points. Representative flow plots show the relative frequency of host (CD45.2)–derived and donor (CD45.1)–derived cells. (B) Graph showing the frequency ± SD of donor LCs in mice receiving BMT with (circles) or without (triangles) T cells. Significance was determined with a two-way ANOVA, ***P < 0.001. Data are pooled from two independent experiments for each time point (n = 5 to 10). (C) Graph shows the number ± SD of Vβ8.3+ Mh T cells in the epidermis over time per 0.1 g of total ear tissue (n = 7 to 8). (D) Top: Representative histogram overlays show the expression of LC-associated proteins on donor-derived LCs (from mice that received BMT + T cells) or host eLCs (BMT alone) 10 weeks after transplant. Bottom: Summary data showing the median fluorescent intensity (MFI) for each sample. Each symbol is one mouse. Data are pooled from two independent experiments (n = 8) and are representative of more than three different experiments.

  • Fig. 2 DC lineage cells do not become long-term replacement LCs.

    (A) Schematic showing the experimental procedure. Male mice received female BMT with T cells. BM was composed of a 1:1 mixture of cells from syngeneic female Clec9aYFP and VavTom mice. Ten weeks later, splenocytes and epidermal LCs were assessed for the relative contribution of cells expressing Tomato (Tom) or YFP. (B) Representative contour plots showing gated CD11c+MHCII+ cells in the spleen or CD11b+EpCAM+Langerin+ LCs in the epidermis of mice that received BMT with or without T cells. (C) Summary bar graphs showing the frequency of red Tom+ or yellow YFP+ cells within splenic CD11c+MHCII+ (left) or epidermal EpCAM+Langerin+ (right) cells in mice receiving BMT alone (open bars) or BMT with T cells (filled bars). Bars show the mean and range of data points. Data are pooled from two independent experiments and analyzed using a two-way ANOVA, ***P < 0.001 (n = 5 to 6).

  • Fig. 3 LC repopulation is preceded by influx of CD11b+ cells.

    Mice received BMT with T cells, and the epidermis was analyzed at different time points. (A) Left: Dot plot shows the gating of single CD11bint to highCD45.1+ donor myeloid cells. Right: Summary graph showing the frequency ± SD donor CD11b+ cells in mice receiving BMT alone (triangles) or BMT + T cells (circles) (n = 6 to 7). (B) Graph shows the number ± SD per 0.1 g of total ear weight of donor CD11b+ cells (n = 5 to 10). (C) Representative contour plots at 3 weeks showing three distinct subpopulations within single CD11bint to highCD45.1+ cells. (D) Summary graphs showing the frequency ± SD (left) and number ± SD (right) of cells within each of the gated populations shown in (C): circles, CD11bhigh (EpCAMnegLangerinneg); squares, EpCAM+; triangles, donor LCs (EpCAM+Langerin+) (n = 7 to 8). Data are pooled from two independent experiments and analyzed by two-way ANOVA for frequency; significance for numbers was calculated with a two-way ANOVA with Tukey’s multiple comparisons test. (E) Top: Representative histogram overlays show surface expression levels of LC-defining proteins in the gated donor populations 3 weeks after transplant. Bottom: Graphs show summary data for the MFI. Symbols represent individual samples, analyzed using a repeated-measures one-way ANOVA. Data are pooled from two independent experiments per time point (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 EpCAM+ monocyte-derived cells are distinct from donor LCs.

    (A) Schematic showing the populations of cells and phenotypic markers used to isolate cells for sequencing. (B) Dendrogram showing clustering of samples. (C) Schematic illustrating competitive chimera experiments to test the requirement for monocyte-derived cells. Male mice received female BMT with T cells. BM was composed of a 1:1 mixture of cells from congenic wild-type (CD45.1+Ccr2+/+) or CCR2-deficient (CD45.2+Ccr2−/−) mice. Epidermal cells were analyzed 3 weeks later. (D) Representative contour plots showing the frequency of wild-type or knockout cells within gated donor epidermal myeloid cells (host cells were excluded at this time point by the use of Langerin.EGFP recipients and exclusion of GFP+ LCs from our analyses). (E) Summary data showing the frequency of Ccr2+/+ donor cells within each population. Bar graphs show the mean and range of data points; data are pooled from two independent experiments (n = 6). Percent of Ccr2+/+ cells versus Ccr2−/− in each population. ***P < 0.001, one-way ANOVA. (F) Heat maps showing relative gene expression of defined genes grouped into panels according to distinct functional processes. Blood monocytes (gray), n = 2; EpCAM+ cells (cyan), n = 3; donor LCs (magenta), n = 3; dermal monocytes (mustard), n = 3.

  • Fig. 5 Proliferation of monocytes and LCs in situ combine to replenish the LC network.

    Mice received BMT with T cells. Total numbers and Ki67 expression of epidermal cells were analyzed at different time points and described with mathematical models. (A) Representative histograms show gating of Ki67+ cells in the EpCAM+ and donor LC populations 3 weeks after BMT with T cells. (B) Graphs show the frequency ± SD (left) and number ± SD per 0.1 g of total ear tissue (right) of Ki67+ cells within gated epidermal populations. Circles, CD11bhigh; squares, EpCAM+; triangles, donor LCs. Data are pooled from two independent experiments per time point (n = 7 to 8), and significance was calculated using a two-way ANOVA (***P < 0.001). (C) Data from the experiments shown in (B) were described with mathematical models. Top: Fitted, empirical descriptions of the time courses of Ki67+ and Ki67 CD11bhigh cells. Middle: Fits to the total numbers and Ki67+ fraction of EpCAM+ cells, using the empirical descriptions of the CD11bhigh cell kinetics as a source. Bottom: Fits to time courses of mature LC numbers and the Ki67+ fraction using either CD11bhigh (P1) or EpCAM+ cells (P2) as a source. (D) The model of a linear development pathway had the strongest statistical support. Numbers indicate parameter estimates from the model. (E) Graph showing the relative contribution of proliferation in donor LCs to influx (with 95% confidence interval) over time. (F) Graph showing the estimated mean interdivision time (with 95% confidence interval) of donor LCs at different times after BMT with T cells. Parameter estimates are displayed in full in Table 1. (G) Mice received EdU 3 weeks after BMT with T cells. Four hours later, the skin and blood were harvested, and cells were analyzed for incorporation of EdU. Representative contour plots show overlaid gated CD11b+Langerinneg (yellow) or CD11b+Langerin+ (magenta) populations in the epidermis, or Ly6C+CD115+ monocytes in the blood. FMO is the fluorescent minus one stain without the EdU detection reagent. (H) Summary graph showing the mean ± SD frequency of EdU+ cells in the different groups. Circles are individual mice (n = 6). Data are pooled from two independent experiments. Mo., blood monocytes; dLC, donor LCs.

  • Fig. 6 Long-term LCs are homologous to eLCs and up-regulate Id2.

    (A) Correlation matrix comparing differentially expressed genes between blood monocytes, mLCs 10 weeks post-BMT + T cells, and eLCs from age-matched controls. (B) Graphs show the relative FPKM (fragments per kilobase of transcript per million reads) count normalized to the maximum value for different transcription factors from the RNA-seq data. Significance was calculated with one-way ANOVA: blood Ly6C+ monocytes, n = 2; epidermal EpCAM+ cells, n = 3; donor LCs, n = 3; age-matched eLCs, n = 3. (C) BM cells were cultured for 6 days with GM-CSF, TGFβ, and different combinations of BMP7, CSF-1, and IL-34. Box and whiskers graph shows mean ± minimum to maximum numbers of DEC205+EpCAM+ cells in the cultures. Significance was calculated using one-way ANOVA for nonparametric samples with Dunn’s multiple comparisons test. Each symbol is data from one culture, n = 5 independent BM donors, in three independent experiments. (D) The bar graph shows the mean expression ± SD of Runx3 or Id2 relative to glyceraldehyde phosphate dehydrogenase (GAPDH) in sorted DEC205+EpCAM+ cells. Symbols are cells from four independent BM donors in three independent experiments. Id2 expression in LCs generated in the absence versus the presence of IL-34 was analyzed by paired t test. (E) Bar graph shows the mean frequency ± SD of EdU+ cells on day 6 of culture after cells were pulsed with EdU for 24 hours on day 2 or 5. Symbols are cells from independent cultures (n = 2 to 4). Data were analyzed using one-way ANOVA. (F) Line graph shows the frequency of viable DEC205+EpCAM+ LCs in GM-CSF/TGFβ cultures with or without IL-34. Symbols represent paired individual BM cultures and were analyzed using a paired t test (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 7 Immune damage and loss of eLCs open the epidermal compartment.

    Male mice received BMT with or without T cells. (A) Graph shows the number ± SD of total CD11b+Langerin+ LCs in mice receiving BMT alone (triangles) or BMT with T cells (circles). Data are pooled from two independent experiments (n = 5 to 13). The white square and dotted line show LC numbers ± SD in untreated controls (n = 5). (B and C) Epidermal sheets were stained with anti-Langerin and anti-CD45.2, and confocal images were processed and quantified using the Definiens Developer software: eLCs (cyan) are Langerin+CD45.2+, DETCs (red) are LangerinnegCD45.2+, and mLCs (yellow) are Langerin+CD45.2neg. Panel (B) shows example images from mice receiving BMT + T cells, and the graph in (C) is the volume of mLCs compared with eLCs from BMT controls. Data are from one transplant experiment with three BMT (20 fields of view analyzed) and two BMT + T cell recipients (14 fields of view analyzed) (n = 162 cells from BMT mice and 356 LCs from BMT + T cell recipients). (D to F) Topical FITC was painted on the ear skin of control untransplanted mice (No Tx) or BMT and BMT + T cell recipients 10 weeks after transplant. Three days later, uptake of FITC was analyzed within MHCIIhighEpCAM+Langerin+ LCs in draining LNs. (D) Bar graph showing the frequency ± SD of FITC+ cells within LCs. (E) Representative contour plots show FITC uptake in gated LCs. (F) Bar graph showing the FITC median fluorescent intensity ± SD within FITC+ LCs. Data are pooled from two independent experiments (n = 4 to 7), and significance was analyzed using one-way ANOVA (***P < 0.001). (G) BMT + T cell recipients received a second round of irradiation and BMT alone 8 weeks later. The schematic illustrates the experimental setup. (H) Flow plots show the outcome in the epidermis of independent mice, which have received the first transplant only (Tx1), the second transplant only (Tx2), or both transplants (Tx1 and Tx2). Contour plots are pregated on EpCAM+Langerin(PE-labeled)+ LCs.

  • Table 1 Parameter estimates from the best-fitting model describing the linear flow from incoming monocytes to CD11bhigh cells, EpCAM+ cells, and mature donor LCs.

    Data show the estimated value with 95% confidence interval.

    ParameterEstimate95% CI
    Mean time spent in EpCAM+0.10 days0.0084–0.30
    Efficiency of maturation from EpCAM+ to donor LCs0.0420.0068–0.053
    Mean residence time of donor LCs73 days14–1100
    Mean interdivision time in donor LCs at week 15.8 days2.4–14
    Mean interdivision time in donor LCs at week 26.8 days4.9–16
    Mean interdivision time in donor LCs at week 318 days14–66
    Mean interdivision time in donor LCs at week 454 days32–250
    Mean interdivision time in donor LCs at week 1078 days44–370
    Relative contribution of proliferation in donor LCs to
    influx at week 1
    1.40.84–11
    Relative contribution of proliferation in donor LCs to
    influx at week 2
    0.220.15–0.86
    Relative contribution of proliferation in donor LCs to
    influx at week 3
    0.240.16–0.74
    Relative contribution of proliferation in donor LCs to
    influx at week 4
    0.310.18–1.06
    Relative contribution of proliferation in donor LCs
    to influx at week 10
    137–44

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/38/eaax8704/DC1

    Fig. S1. Immune injury leads the gradual replenishment of the epidermis with LC-like cells.

    Fig. S2. Sorting strategies used to isolate cells for RNA-seq.

    Fig. S3. EpCAM+ monocyte-derived cells are distinct from donor LCs.

    Fig. S4. Proliferation of monocytes and LCs in situ combine to replenish the LC network.

    Fig. S5. Long-term mLCs are homologous to eLCs and up-regulate Id2.

    Fig. S6. Gating of BM-derived LCs in vitro.

    Fig. S7. Immune damage and loss of eLCs open the epidermal compartment.

    Table S1. Raw data file.

    Table S2. Differentially expressed genes between eLCs and mLCs.

    Materials and Methods

    References (6471)

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Immune injury leads the gradual replenishment of the epidermis with LC-like cells.
    • Fig. S2. Sorting strategies used to isolate cells for RNAseq.
    • Fig. S3. EpCAM+ monocyte-derived cells are distinct from donor LCs.
    • Fig. S4. Proliferation of monocytes and LCs in situ combine to replenish the LC network.
    • Fig. S5. Long-term mLCs are homologous to eLCs and up-regulate Id2.
    • Fig. S6. Gating of BM-derived LCs in vitro.
    • Fig. S7. Immune damage and loss of eLCs opens the epidermal compartment.
    • Table S2. Differentially expressed genes between eLCs and mLCs.
    • Materials and Methods
    • References (6471)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data file.

    Files in this Data Supplement:

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