Fig 2: Neutrophils persist in wound bed after the acute inflammatory phase, producing extracellular traps. (A) Neutrophils are present in the wound beds of C57BL/6J mice at early time points, as visible in representative haematoxylin and eosin (H&E) staining. Arrows indicate regions of interest, and dashed line demarcates boundaries. Black scale bar = 50 µm. (B) Neutrophils predominate throughout the wound beds of C57BL/6J mice on wound day (WD) 1 and WD3, visible in prominent myeloperoxidase (MPO) immunofluorescence (IF) staining (green). Few macrophages are present (Red, F4/80). White scale bar = 200 µm. (C) Per cent neutrophil (Ly6G+ cells from total CD45+ cells) levels are consistent in the blood throughout the wound time course but drop in the wound bed at WD11, as measured by FACS. ****p < 0.0001, as calculated by two‐way ANOVA. N = 2 vs. 4. Results are representative of at least two independent experiments. (D) Macrophage (F4/80) levels are largely absent from the blood and low in the wound bed during the early phase of healing but increase dramatically at WD11, as measured by FACS. ***p < 0.004, as calculated by two‐way ANOVA. n.s., not significant. N = 2 vs. 4. Results are representative of at least two independent experiments. (E) Citrullinated histone H3 (H3Cit, red) co‐localized with Ly6G+ neutrophils (green), beginning at WD3 in the wound beds of IF‐stained C57BL/6J mice, indicating the formation of extracellular traps. (F) Neutrophil extracellular trap‐positive cells (MPO+, SYTOX green +) are present at late wound time points, but are absent in the wound beds of PAD4−/− mice, as measured by FACs. ****p < 0.0001, as calculated by two‐way ANOVA. N = 7 vs. 4. Results are representative of at least two independent experiments. (G) Cytoplasmic U1 snRNA is present in the wound bed of C57BL/6J mice, while it localized exclusively in the nuclei of unwounded controls, as visualized by representative FISH. The solid white line delineates a hair follicle. White scale bar = 80 µm

Fig 3: DCs uptake preference of intradermal injected DiO-loaded PLGA nanoparticles in IMQ-induced psoriasis-like mice. (A) Representative immunofluorescence staining of psoriasis-like mice skin section (a) that intradermally injected with DiO-loaded PLGA nanoparticles (after injection for 4 h), enlarged in (b). (B) Representative immunofluorescence staining of SDLN from psoriasis-like mice that intradermally injected with DiO-loaded PLGA nanoparticles (after injection for 1 h), enlarged in (b). Scale bar: 250 µm (enlargement: 50 µm). Blue: nucleus, green: DiO, red: anti-CD11c. (C) Flow cytometry gating strategy to identify and analyze DCs populations in different lymphoid organs. Gating is shown in one representative data of mice at 1 h exposure after treated with S-NPs. After the initial live gating in a forward scatter (FSC) and side scatter (SSC) plot, particle-positive cells were first gated. Among them, DCs from SDLN and spleens were gated in a CD45 versus CD11c plot. (D) PBS controls and different sizes of DiO-loaded PLGA nanoparticles were intradermally administrated in IMQ-induced psoriasis-like mice, and the single-cell suspensions obtained from lymphoid organs were analyzed at 1, 4, 8, 24, and 48 h after particle exposure. Among particle-positive subset in SDLN (D) and spleens (E), comparison between S-NPs and L-NPs detected in these tissues were made. Data are presented as mean ± SEM (n = 6), **P < 0.01, *P < 0.05.

Fig 4: Single-Cell RNA Sequencing of Colonic Macrophages, Related to Figure 3(A, left) tSNE computed on the top 22 PCs obtained on the 1000 most variable genes (vst method). Cells are colored by the sample. (A, right) tSNE computed on the top 22 PCs obtained on the 1000 most variable genes (vst method). Cells are colored by the cluster (number of neighbors = 30, resolution = 0.3).(B) Distribution of the log-normalized expression levels of genes used for sorting terminally-differentiated ?? across clusters (gated on alive, CD45+, CD3-, CD19-, CD103-, CD11b+, F4/80+, CD64+, Ly6C-, MHCII+).(C, top panel) Distribution of the log-normalized expression levels of fibroblast/myofibroblasts markers across clusters. (C, bottom panel). Distribution of the log-normalized expression level of epithelial cell markers across clusters.(D) Clusters 4 and 6 from Kang et al., consist of terminally differentiated (mature) M?. Venn diagram of the set of cluster 4, 6 and 7 markers (top 50 upregulated genes, gene AF251705 is excluded because it is not associated to any official gene name, Kang et al.) and the terminally differentiated macrophage signature, defined as the genes showing log2(FC) 3 3 from P1 (monocytes) to P4 (mature ??) by Schridde et al. (65 genes).

Fig 5: M?s Are Required for Epithelial Cell Survival in the Distal Colon and Form “Balloon-like” Protrusions Inserted in between Epithelial Cells(A) Scheme of ?? depletion. CD64WT or CD64DTR littermates received two injections of diphtheria toxin (DT) 24 h apart.(B) Maximum z-projection (30 µm) of proximal and distal colon transversal sections 44 h after the first DT injection. Apoptotic cells were revealed with cleaved caspase 3 staining (red), F-actin (green). Scale bar: 50 µm.(C) Number of apoptotic epithelial cells per crypt in the distal or proximal colon. Pooled data from three independent experiments; dots represent average number per individual mouse. Mean ± SEM, multiple comparison Kruskal-Wallis test, *p < 0.05.(D) Serum fluorescence intensities 5–10 min after intra-rectal administration of hypotonic solution of hydrazide-AlexaFluor633. All mice were injected with DT. Pooled data from two independent experiments; dots represent average number per individual mouse. Mean ± SEM, Mann-Whitney test, *p < 0.05.(E) Morphological differences of ??s in the proximal and distal colon. Whole-mount staining of the distal and proximal colon of CD11c: Cre/R26mTmG mice. mGFP (green), CD11b (blue), CD103 (red), membrane tdTomato (gray). BLPs are indicated with arrows, the border between epithelium and the stroma is indicated with the dashed line. Z-projections of 20–40 µm; scale bars: 50 µm.(F) Single M? forming BLPs (left) or thin extensions (right). Yellow star: cell bodies; green arrows: BLPs; green arrowheads: extensions. Maximum z-projection of 10–15 µm; scale bar: 2 µm.(G) Number of BLPs, normalized per crypt (left) or per M? (right). Dots represent average number per individual mouse; left: pooled data from seven independent experiments; right: pooled data from another two independent experiments.(H) Number of M?s in the proximal and distal colon, analyzed by imaging (F4/80+MHCII+CD103- cells per crypt; each dot represents average number per individual mouse; data pooled from three independent experiments) and by flow cytometry (presented as percentage of CD45+ cells; dots represent individual mouse; data pooled from four independent experiments).(I) Number of extensions, normalized per M?. Dots represent average number per individual mouse; four independent experiments.In (G–I), mean ± SEM, Mann-Whitney test, *p < 0.05, **p < 0.01, ****p < 0.0001. See also Figure S1 and S2, Video S1.