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. 2019 Aug 23;4(38):eaax8704.
doi: 10.1126/sciimmunol.aax8704.

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

Ivana R Ferrer  1 , Heather C West  1 , Stephen Henderson  2 , Dmitry S Ushakov  3 , Pedro Santos E Sousa  1 , Jessica Strid  4 , Ronjon Chakraverty  1 , Andrew J Yates  5 , Clare L Bennett  6
Affiliations

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

Ivana R Ferrer et al. Sci Immunol. .

Abstract

A dense population of embryo-derived Langerhans cells (eLCs) is maintained within the sealed epidermis without contribution from circulating cells. When this network is perturbed by transient exposure to ultraviolet light, short-term LCs are temporarily reconstituted from an initial wave of monocytes but thought to be superseded by more permanent repopulation with undefined LC precursors. However, the extent to which this process is relevant to immunopathological processes that damage LC population integrity is not known. Using a model of allogeneic hematopoietic stem cell transplantation, where alloreactive T cells directly target eLCs, we have asked whether and how the original LC network is ultimately restored. We find that donor monocytes, but not dendritic cells, are the precursors of long-term LCs in this context. Destruction of eLCs leads to recruitment of a wave of monocytes that engraft in the epidermis and undergo a sequential pathway of differentiation via transcriptionally distinct EpCAM+ precursors. Monocyte-derived LCs acquire the capacity of self-renewal, and proliferation in the epidermis matched that of steady-state eLCs. However, we identified a bottleneck in the differentiation and survival of epidermal monocytes, which, together with the slow rate of renewal of mature LCs, limits repair of the network. Furthermore, replenishment of the LC network leads to constitutive entry of cells into the epidermal compartment. Thus, immune injury triggers functional adaptation of mechanisms used to maintain tissue-resident macrophages at other sites, but this process is highly inefficient in the skin.

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Figures

Figure 1.
Figure 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 (Matahari) 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) and donor (CD45.1)-derived cells. B. Graph showing the frequency ± SD of donor LC in mice receiving BMT with (circles) or without (triangles) T cells. Significance was determined with a 2-way ANOVA, ***P<0.001. Data are pooled from 2 independent experiments for each time point (n=5–10). C. Graph shows the number ± SD of Vβ8.3+ Matahari (Mh) T cells in the epidermis over time, per 0.1g total ear tissue (n=7–8). D. Top - representative histogram overlays show the expression of LC-associated proteins on donor-derived LC (from mice that received BMT + T cells) or host eLC (BMT alone), 10 weeks post-transplant. Bottom - summary data showing the median fluorescent intensity (MFI) for each sample. H = host, D = donor, each symbol is one mouse. Data are pooled from 2 independent experiments (n=8), and representative of >3 different experiments.
Figure 2.
Figure 2.. DC lineage cells do not become long-term replacement LC.
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. 10 weeks later splenocytes and epidermal LC 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+ LC 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 2 independent experiments, and analyzed using a 2-way ANOVA, ***P<0.001 (n = 5–6).
Figure 3.
Figure 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= 6–7). B. Graph shows the number ± SD per 0.1g total ear weight of donor CD11b+ cells (n=5–10). C. Representative contour plots at 3 weeks showing 3 distinct sub-populations 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 LC (EpCAM+Langerin+) (n=7–8). Data are pooled from 2 independent experiments and analyzed by 2-way ANOVA for frequency; significance for numbers was calculated with a 2-way ANOVA with Tukeys multiple comparisons test. E. Top - representative histogram overlays show surface expression levels of LC-defining proteins in the gated donor populations 3 weeks post-transplant. Bottom - graphs show summary data for the median fluorescent intensity (MFI). Symbols represent individual samples, analyzed using a repeated measurements 1-way ANOVA. Data are pooled from 2 independent experiments per time point (n=6). *P<0.05, **P<0.01, ***P<0.001.
Figure 4.
Figure 4.. EpCAM+ monocyte-derived cells are distinct from donor LC.
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 knock-out 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+ LC 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 2 independent experiments (n=6). Percent of Ccr2+/+ cells versus Ccr2−/− in each population ***P<0.001, 1-way ANOVA. F. Heat maps showing relative gene expression of defined genes grouped into panels according to distinct functional processes. Blood monocytes (grey) n = 2, EpCAM+ cells (cyan) n = 3, donor LC (magenta) n = 3, dermal monocytes (grey) n = 3.
Figure 5.
Figure 5.. Proliferation of monocytes and LC 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 0.1g total ear tissue (right) of Ki67+ cells within gated epidermal populations. Circles CD11bhigh; squares EpCAM+; triangles donor LC. Data are pooled from 2 independent experiments per time point (n = 7–8) and significance calculated using a 2-way ANOVA, ***P<0.001. C. Data from the experiments shown in B. were described with mathematical models. Upper panels - Fitted, empirical descriptions of the timecourses of Ki67+ and Ki67- CD11bhigh cells. Middle panels - fits to the total numbers and Ki67+ fraction of EpCAM+ cells, using the empirical descriptions of the CD11bhigh cell kinetics as a source. Lower panels - fits to timecourses 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 LC to influx (with 95 percent confidence interval) over time. F. Graph showing the estimated mean interdivision time (with 95 percent confidence interval) of donor LC at different times post-BMT with T cells. Parameter estimates are displayed in full in Table 1. G. Mice received EdU 3 weeks after BMT with T cells. 4 hours later, the skin and blood were harvested and cells 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 2 independent experiments. Mo. = blood monocytes, dLC = donor LC.
Figure 6.
Figure 6.. Long-term LC are homologous to eLC and up-regulate Id2.
A. Correlation matrix comparing differentially expressed genes between and blood monocytes, mLC 10 weeks post-BMT + T cells, and eLC from age-matched controls. B. Graphs show the relative FPKM count normalized to the maximum value for different transcription factors from the RNAseq data. Significance was calculated with a 1-way ANOVA, blood Ly6C+ monocytes n = 2, epidermal EpCAM+ cells n= 3, donor LC n = 3, age-matched eLC n = 3. C. BM cells were cultured for 6 days with GM-CSF, TFGβ and different combinations of BMP7, CSF1 and IL-34. Box and whiskers graph shows mean ± min. to max. numbers of DEC205+EpCAM+ cells in the cultures. Significance was calculated with a 1-way ANOVA for non-parametric samples with Dunn’s multiple comparisons test. Each symbol is data from one culture, n = 5 independent BM donors, in 3 independent experiments. D. The bar graph shows the mean expression ± SD of Runx3 or Id2 relative to GAPDH in sorted DEC205+EpCAM+ cells. Symbols are cells from 4 independent BM donors, in 3 independent experiments. Id2 expression in LC 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 where pulsed with EdU for 24 hours on day 2 or day 5. Symbols are cells from independent cultures (n = 2–4). Data was analyzed using a 1-way ANOVA. F. Line graph shows the frequency of viable DEC205+EpCAM+ LC 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.
Figure 7.
Figure 7.. Immune damage and loss of eLC opens the epidermal compartment.
Male mice received BMT with or without T cells. A. Graph shows the number ± SD of total CD11b+Langerin+ LC in mice receiving BMT alone (triangles) or BMT with T cells (circles). Data are pooled from 2 independent experiments (n=5–13). The white square and dotted line shows LC numbers ± SD in untreated controls (n=5). B-C. Epidermal sheets were stained with anti-Langerin and anti-CD45.2, and confocal images processed and quantified using the Definiens Developer software: eLC (cyan) are Langerin+CD45.2+; DETC (red) LangerinnegCD45.2+; and mLC (yellow) are Langerin+CD45.2neg. B. shows example images from mice receiving BMT + T cells, and the graph in C. is the volume of mLC compared to eLC from BMT controls. Data are from 1 transplant experiment with 3 BMT (20 fields of view analyzed) and 2 BMT+T cell recipients (14 fields of view analyzed) (n= 162 cells from BMT mice and 356 LC from BMT+ T cell recipients). D-F. Topical FITC was painted on the ear skin of control un-transplanted mice (No Tx), or BMT and BMT + T cell recipients 10 weeks post-transplant. 3 days later uptake of FITC was analyzed within MHCIIhighEpCAM+Langerin+ LC in draining LN. D. Bar graph showing the frequency ± SD of FITC+ cells within LC. E. Representative contour plots show FITC uptake in gated LC. F. Bar graph showing the FITC median fluorescent intensity ± SD within FITC+ LC. Data are pooled from 2 independent experiments (n=4–7), significance was analyzed using a 1-way ANOVA, ***P<0.001. G. BMT + T cell recipients received a second round of irradiation and BMT alone 8 weeks later. The schematic illustrate the experimental set-up. H. Flow plots show the outcome in the epidermis of independent mice, who have received the 1st transplant only (Tx 1), the 2nd transplant only (Tx 2) or both transplants (Tx 1 and 2). Contour plots are pre-gated on EpCAM+Langerin(PE-labelled)+ LC.
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