Journal: Advanced Science

Article Title: Dual Lineages of Langerhans Cells Cooperate to Restore the Immune Barrier after Skin Injury

doi: 10.1002/advs.76816

Figure Lengend Snippet: Langerhans cells migrate toward the wound during re‐epithelization. (A) Revisit multi‐photon in vivo microscopy images of a 1 mm wound from the same mouse. Image shows x‐y view of epithelial cells (red nuclei) and LCs (green) in the epidermis. Dashed line indicates initial wound boundary. Left : Day of wound induction (Day 0). Middle : 5 days after wound induction. Right : zoomed view of the wound center at Day 5. Representative images from 3 mice. Scale bars, 100 µm. (B) Time‐lapse image of epithelial cells (red nuclei) and LCs (green) 2 days after wound induction. Dashed line, initial wound boundary. Solid line, basal membrane separating epidermis from dermis. Top : x‐y view. Bottom : x‐z view shows the epidermis (red) and dermis (collagen SHG, blue). Representative images from 3 mice. Scale bars, 100 µm. (C) Imaris track analysis of LCs (B) 2 days after wound induction. Colors project time (blue, 0 h; red, 6 h). Top : x‐y view. Bottom : x‐z view. Representative images from 3 mice. Scale bars, 100 µm. (D) Top : zoomed migration tracks from (C). The green frame is from the wound leading‐edge epithelial migration zone, and the teal frame is from the epithelial proliferation zone . Middle : time‐lapse frames show the movement of individually colored LCs across 6 h. Epithelial cell nuclei in gray. Other LCs in white. Bottom : vector arrows show the general movement direction of the matching color LC. Representative images from 3 mice. Scale bars, 100 µm. (E) Mean total displacement of individual LC tracks over 6 h plotted as a function of distance from the wound. n = 3 mice. (F) Mean track displacement in the x axis of individual LC tracks over 6 h plotted as a function of distance from the wound. Calculated by comparing the start and end values in the x axis of each track. Positive change indicates movement toward the wound. n = 3 mice. (E,F) Imaging performed 2 days after wound induction. Dashed line, initial wound boundary. The displacements of migrating cell tracks were averaged every 100 µm from the initial wound. Data are mean ± s.d. (G) Imaris cell count analysis of LCs (spots) at Day 0 ( left ) and 5 days after wound induction ( right ). Dashed lines separate LCs into 3 zones: wound (yellow spots), near (0–400 µm from the wound edge, green spots), and far (400‐700 µm from the wound edge, teal spots). Epithelial cell nuclei are shown in gray. Representative images from 3 mice. Scale bars, 100 µm. (H) Mean LC number comparing cell density between Day 0 and 5 days after wound induction according to the 3 zones established in (G). n = 3 mice. (I) Mean LC number comparing the cell density change from the addition of the wound and near zones between Day 0 and 5 days after wound induction ( left bars ). Total change in LC density across all 3 zones between Day 0 and 5 days after wound induction ( right bars ). n = 3 mice. (H,I) Data analyzed using paired two‐way ANOVA; data are mean ± s.d. with each dot representing individual mice. *** p < 0.001, **** p < 0.0001.

Article Snippet: Samples were then enriched for LCs by magnetic sorting following the standard protocol from the Epidermal Langerhans Cell MicroBead Kit (Miltenyi Biotec).

Techniques: In Vivo, Microscopy, Membrane, Migration, Plasmid Preparation, Imaging, Cell Characterization

Journal: Advanced Science

Article Title: Dual Lineages of Langerhans Cells Cooperate to Restore the Immune Barrier after Skin Injury

doi: 10.1002/advs.76816

Figure Lengend Snippet: Langerhans cell mobility is independent of epithelial cell migration. (A) Time‐lapse x‐y view of epithelial cells (red nuclei) and LCs (green) 2 days after wound induction. Top : control mouse. Bottom : Epi‐ Rac1 KO mouse. Dashed line indicates initial wound boundary. Representative images from 3 mice per group. Scale bars, 100 µm. (B) Imaris x‐y view track analysis of LCs (A) 2 days after wound induction. Colors project time (blue, 0 h; red, 6 h). Top : control mouse. Bottom : Epi‐ Rac1 KO mouse. Right : zoomed migration tracks from near the wound edge. Dashed line indicates initial wound boundary. Representative images from 3 mice per group. Scale bars, 100 µm. (C) Mean total displacement of individual epithelial cell tracks from control and Epi‐ Rac1 KO mice over 6 h plotted as a function of distance from the wound. n = 3 mice per group (D) Mean total displacement of individual LC tracks from control and Epi‐ Rac1 KO mice over 6 h plotted as a function of distance from the wound. n = 3 mice per group C,D, Imaging performed 2 days after wound induction. Dashed line, initial wound boundary. The displacements of migrating cell tracks were averaged every 100 µm from the initial wound. Data analyzed using unpaired two‐way ANOVA; data are mean ± s.d. * p < 0.05. (E) In‐vivo microscopy images shows x‐y view of epithelial cells (red nuclei) and LCs (green) in the epidermis 5 days after wound induction. Dashed line indicates initial wound boundary. Left : control mouse and LC‐ Rac1 KO mouse. Right : zoomed view of the wound center from 5 days after wound induction. Representative images from 4 mice per group. Scale bars, 200 µm. (F) Mean LC number inside the wound epidermis from control and LC‐ Rac1 KO mice. Imaging was performed 5 days after wound induction. LC density normalized to the individual mouse wound area quantified. Data analyzed using unpaired two‐tailed t ‐test; n = 4 mice per group; data are mean ± s.d. with each dot representing individual mice. **** p < 0.0001.

Article Snippet: Samples were then enriched for LCs by magnetic sorting following the standard protocol from the Epidermal Langerhans Cell MicroBead Kit (Miltenyi Biotec).

Techniques: Migration, Control, Imaging, In Vivo, Microscopy, Two Tailed Test

Journal: Advanced Science

Article Title: Dual Lineages of Langerhans Cells Cooperate to Restore the Immune Barrier after Skin Injury

doi: 10.1002/advs.76816

Figure Lengend Snippet: Langerhans cell density changes during the remodeling phase. (A) Revisit multi‐photon in vivo microscopy images of a 1 mm wound from the same mouse. Images show x‐y view of epithelial cells (red nuclei) and LCs (green) in the epidermis at 0, 5, 15 days, and 6 weeks after wound induction. Dashed line indicates initial wound boundary. Left : zoomed view of the wound center. Right : revisit images showing the classification of several zones according to their distance from the wound: wound, near, middle, and far. Representative images from 6 mice. Scale bars, 100 µm. (B) Mean LC number comparing cell density changes within designated zones established in (A) at 0, 5, 15 days, and 6 weeks after wound induction. n = 6 mice. (C) Timeline of LC number comparing cell density changes between the wound and near zones. n = 6 mice. (B,C) Data analyzed using unpaired two‐way ANOVA; data are mean ± s.d. * p < 0.05, ** p < 0.01. (D) Time‐lapse image in x‐y view of proliferative LCs (green) at 7 days after wound induction. Dermis SHG collagen is shown in gray. Red dashed circles indicate diving LCs. (E) Time‐lapse frames from yellow highlighted area in (D) show LC division across 5 h. White arrows indicate actively diving LC. (D,E) Representative images from 3 mice. White dashed circle/line indicates initial wound boundary. Scale bars, 50 µm. (F) Confocal immunofluorescent images of cell proliferation at the wound epidermis 7 days after wound induction. Images show x‐y view of LCs (green, MHC‐II), proliferation (red, Ki67), and cell nuclei (blue, DAPI). Left : composite image. Middle : MHC‐II and Ki67 positive cells. Right : zoomed example of proliferative LC. MHC‐II, major histocompatibility complex class II. White arrows show proliferative cells. White arrowheads show proliferative LCs. Representative images from 4 mice. The white dashed line indicates the initial wound boundary. Scale bars, 50 µm. (G) Timeline of mean proliferative (Ki67+) cell density at the wound during healing ( n = 3 mice) and in homeostasis ( n = 6 mice). (H) Timeline of percentage of proliferative LCs (MHC‐II+Ki67+) at the wound during healing ( n = 3 mice) and in homeostasis ( n = 6 mice). (I) Confocal immunofluorescent images of cell apoptosis at the wound epidermis 3 weeks after wound induction. Images show x‐y view of LCs (green, MHC‐II), apoptosis (red, CC3), and cell nuclei (blue, DAPI). Left : composite image. Middle : MHC‐II and CC3 positive cells. Right : zoomed example of apoptotic LC. CC3, cleaved caspase‐3. The white arrow shows an apoptotic cell. White arrowhead shows an apoptotic LC. Representative images from 4 mice. The white dashed line indicates the initial wound boundary. Scale bars, 50 µm. (J) Mean number of apoptotic cells (CC3+) and apoptotic LCs (CC3+MHC‐II+) at the wound 3 weeks after wound induction. n = 3 mice. (K) Percentage of apoptotic LCs (CC3+MHC‐II+) during homeostasis and at 3 weeks after wound induction. n = 3 mice. (J,K) Wound area quantified 0.49 mm 2 per mouse. Data analyzed using unpaired one‐way ANOVA; data are mean ± s.d. with each dot representing individual mice. * p < 0.05, **** p < 0.0001.

Article Snippet: Samples were then enriched for LCs by magnetic sorting following the standard protocol from the Epidermal Langerhans Cell MicroBead Kit (Miltenyi Biotec).

Techniques: In Vivo, Microscopy, Immunopeptidomics

Journal: Advanced Science

Article Title: Dual Lineages of Langerhans Cells Cooperate to Restore the Immune Barrier after Skin Injury

doi: 10.1002/advs.76816

Figure Lengend Snippet: Multiple sources of Langerhans cells repopulate the wound site. (A) Experimental design of dual‐labeled LC mouse line. Dual‐labeled LC mice received daily tamoxifen injections 5 days prior to wound induction. Embryonic LCs (eLCs) are labeled yellow and progenitor‐derived LCs are only green. (B) Revisit multi‐photon in vivo microscopy images of a 1 mm wound from the same mouse. Images show x‐y view of epithelial cells (red nuclei), eLCs (yellow), and progenitor‐derived LCs (green) in the epidermis at 5 and 15 days after wound induction. Dashed line indicates initial wound boundary. Left : zoomed view of the wound center. Representative images from 6 mice. Representative image from 3 mice. Scale bars, 100 µm. (C) Timeline of LC number comparing cell density changes among embryonic and progenitor‐derived LCs at the wound. n = 6 mice. (D) Timeline of changes in the percentage ratio between embryonic and progenitor‐derived LCs at the wound site. n = 6 mice. (C,D) Wound area quantified 0.16 mm 2 per mouse. Data analyzed using paired two‐way ANOVA; data are mean ± s.d. * p < 0.05, ** p < 0.01. (E) Multi‐photon in vivo microscopy of dual‐labeled LC mice 22 weeks after wound induction. Dashed line indicates initial wound boundary. Left : zoomed view of the wound center. Representative image from 3 mice. Scale bars, 100 µm.

Article Snippet: Samples were then enriched for LCs by magnetic sorting following the standard protocol from the Epidermal Langerhans Cell MicroBead Kit (Miltenyi Biotec).

Techniques: Labeling, Derivative Assay, In Vivo, Microscopy

Journal: Advanced Science

Article Title: Dual Lineages of Langerhans Cells Cooperate to Restore the Immune Barrier after Skin Injury

doi: 10.1002/advs.76816

Figure Lengend Snippet: Monocyte‐derived Langerhans cells integrate into the existing LC network after wound closure. (A) UMAP of monocyte and Langerhans cells re‐clustering. Clusters were defined as monocytes (Mono), pre‐monocyte‐derived LCs (pre‐mLC), monocyte‐derived LCs (mLC), and embryonic LCs (eLC). Embryonic LCs subclusters were merged as “eLC” for simplicity. (B) Left : Violin plots showing the expression of top markers for monocytes ( Cd14 ) and LCs ( Cd207 ). Right : Feature plots showing the expression of the gene mentioned in the same row. (C) RNA velocity pseudotime trajectories. The direction of the black arrows reflects the changes in the surrounding cellular states. Red arrows show the general trend. (D) Dot plot of selected marker genes for monocytes, LC differentiation, and embryonic LCs. (E) Experimental design for the in vivo identification of repopulating monocyte‐derived LCs. Tamoxifen was used to label existing embryonic LCs red prior to wound induction. Tamoxifen continued to be delivered up until the expected arrival of progenitor LCs (Day 10 after wound induction). Injections were delivered every 3 days after wound induction to avoid toxicity. Revisit imaging (camera icon) was performed on Day 5 and 13. (F) Revisit multi‐photon in vivo microscopy images of a 1 mm wound from the same mouse. Top : Images show x‐y view of epithelial cells (dim red nuclei), monocytes (green), and LCs (red) in the epidermis at 5 and 13 days after wound induction. Blue frame : zoomed example of a GFP high only cell. Teal frame : zoomed example of GFP low /tdTomato low double‐positive cells. Magenta frame : zoomed example of tdTomato low only cells. Orange frame : zoomed example of a tdTomato high only cell. White arrowheads highlight the same cell within each frame. Dashed line indicates initial wound boundary. Representative images from 3 mice. Scale bars: Top : 100 µm, Colored frames : 50 µm. (G) GO plot of selected biological process terms among the top 50 results enriched in each cluster using the top 200 markers. Highlighted color shades under GO terms match the corresponding cluster colors.

Article Snippet: Samples were then enriched for LCs by magnetic sorting following the standard protocol from the Epidermal Langerhans Cell MicroBead Kit (Miltenyi Biotec).

Techniques: Derivative Assay, Expressing, Marker, In Vivo, Imaging, Microscopy

Journal: Advanced Science

Article Title: Dual Lineages of Langerhans Cells Cooperate to Restore the Immune Barrier after Skin Injury

doi: 10.1002/advs.76816

Figure Lengend Snippet: Cxcr2 inhibition blocks eLC wound repopulation. (A) Heatmap of normalized log2 fold change of chemokine receptor gene expression from homeostatic epithelial (KTC) and Langerhans cells (LC). Cells isolated through FACS and sequenced through bulk RNA‐seq. Each column represents an independent sample, and each row is assigned to a specific gene. Red indicates maximum expression and blue indicates minimum expression. n = 4 mice (B) Experimental design for drug treatment. Starting on wound induction day, drug was injected once a day intradermally at the ear near the wound site. Control mice received vehicle (1% DMSO) injections. Wounds were imaged at wound closure (5 days after wound induction), and candidate drugs were further analyzed for migration dynamics through time‐lapse at 2 days after wound induction. Revisit imaging (camera icon) was performed on Day 0 and 5. Timelapse imaging (video icon) was performed on Day 2. (C) Mean LC number comparing cell density at the wound in response to drug treatment. Imaging was performed 5 days after wound induction. LC density normalized to the individual mouse wound area was quantified. Data analyzed using unpaired one‐way ANOVA; n = 6 control, n = 4 BX471, n = 4 INCB3344, n = 4 DAPTA, n = 3 Cenicriviroc, n = 5 Danirixin, n = 5 SB225002, and n = 4 CXCR3 antagonist‐treated mice.; data are mean ± s.d. with each dot representing individual mice. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (D) In vivo microscopy images show x‐y view of epithelial cells (red nuclei) and LCs (green) in the epidermis 5 days after wound induction. Top : control mouse (1% DMSO). Middle : CXCR2‐inhibited mouse (Danirixin). Bottom : CXCR2‐inhibited mouse (SB225002). Right : zoomed view of the wound center matching the image on the left. Dashed line indicates initial wound boundary. Representative images are shown. n = 6 control mice and n = 5 mice per drug‐treated group. Scale bars, 100 µm. (E) Confocal immunofluorescent images of CXCR2 expression at the epidermis during homeostasis and 2 days after wound induction. Images show x‐y view of LCs (green, MHC‐II), CXCR2 (red), and cell nuclei (blue, DAPI). Right : zoomed view in composite, green channel only, and red channel only. Dashed line indicates initial wound boundary. Representative images from 3 mice. Scale bars, 25 µm. (F) qRT‐PCR gene expression analysis of CXCR2 ligands in the skin during homeostasis (control) and 2 days after wound induction. Data analyzed using multiple unpaired two‐tailed t ‐test; n = 5 mice; data are mean ± s.d. with each dot representing individual mice. ** p < 0.01. (G) ELISA assay of CXCL1 present on wounds treated with CXCR2‐inhibitor (Danirixin) compared to control (DMSO) and homeostasis. (H) ELISA assay of CXCL2 present on wounds treated with CXCR2‐inhibitor (Danirixin) compared to control (DMSO) and homeostasis. (I) ELISA assay of CXCL3 present on wounds treated with CXCR2‐inhibitor (Danirixin) compared to control (DMSO) and homeostasis. (J) ELISA assay of CXCL5 present on wounds treated with CXCR2‐inhibitor (Danirixin) compared to control (DMSO) and homeostasis. (K) ELISA assay of CXCL7 present on wounds treated with CXCR2‐inhibitor (Danirixin) compared to control (DMSO) and homeostasis. (G–K) Data analyzed using unpaired one‐way ANOVA; n = 4 mice; data are mean ± s.d. with each dot representing individual mice. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Samples were then enriched for LCs by magnetic sorting following the standard protocol from the Epidermal Langerhans Cell MicroBead Kit (Miltenyi Biotec).

Techniques: Inhibition, Gene Expression, Isolation, RNA Sequencing, Expressing, Injection, Control, Migration, Imaging, In Vivo, Microscopy, Quantitative RT-PCR, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Dual Lineages of Langerhans Cells Cooperate to Restore the Immune Barrier after Skin Injury

doi: 10.1002/advs.76816

Figure Lengend Snippet: Monocyte‐derived Langerhans cells compensate for the loss of embryonic Langerhans cells at the wound. (A) Time‐lapse x‐y view of epithelial cells (red nuclei) and LCs (green) 2 days after wound induction. Top : control mouse 1% DMSO. Bottom : drug‐treated mouse CXCR2 (Danirixin) as shown in (Figure ). Dashed line indicates initial wound boundary. Representative images from 3 mice per group. Scale bars, 100 µm. (B) Imaris x‐y view track analysis of LCs (A) 2 days after wound induction. Colors project time (blue, 0 h; red, 6 h). Top : control mouse 1% DMSO. Bottom : drug‐treated mouse CXCR2 (Danirixin). Right : zoomed migration tracks from near the wound edge. Representative images from 3 mice per group. (C) Mean total displacement of individual epithelial cells tracks from control and CXCR2‐inhibited mice over 6 h plotted as a function of distance from the wound. n = 3 mice. (D) Mean total displacement of individual LC tracks from control and CXCR2‐inhibited mice over 6 h plotted as a function of distance from the wound. n = 3 mice. (E) Mean track displacement in the x axis of individual LC tracks from control and CXCR2‐inhibited mice over 6 h plotted as a function of distance from the wound. Calculated by comparing the start and end values on the x axis of each track. Positive change indicates movement toward the wound. n = 3 mice. (C–E) Imaging performed 2 days after wound induction. Dashed line, initial wound boundary. The displacements of migrating cell tracks were averaged every 100 µm from the initial wound. Data analyzed using unpaired two‐way ANOVA; data are mean ± s.d. * p < 0.05, **** p < 0.0001. (F) Revisit multi‐photon in vivo microscopy images of a 1 mm wound from the same mouse. Images show x‐y view of epithelial cells (dim red nuclei), embryonic LCs (orange/yellow), and progenitor‐derived LCs (green) in the epidermis at 5 ( Left ) and 17 ( Middle ) days after wound induction. Day 17 LCs at the wound quantified using Imaris spots analysis ( Right ). Top : control mouse 1% DMSO. Bottom : drug‐treated mouse CXCR2 (Danirixin). Dashed line indicates initial wound boundary. Representative images from 3 mice per group. Scale bar, 100 µm. (G) Mean LC number comparing cell density changes among embryonic LCs (eLC) and monocyte‐derived LCs (mLC) in response to CXCR2 inhibition. (H) Percentage ratio between eLCs and mLCs at the wound epidermis in response to CXCR2 inhibition. (G,H) Imaging performed 17 days after wound induction. LC density normalized to the individual mouse wound area was quantified. Data analyzed using unpaired two‐way ANOVA; n ≥3 mice; data are mean ± s.d. * p < 0.05.

Article Snippet: Samples were then enriched for LCs by magnetic sorting following the standard protocol from the Epidermal Langerhans Cell MicroBead Kit (Miltenyi Biotec).

Techniques: Derivative Assay, Control, Migration, Imaging, In Vivo, Microscopy, Inhibition