Figure 1.

The characteristic distribution of neutrophils and macrophages in human DP. (A) Cluster analysis using the Uniform Manifold Approximation and Projection (UMAP) technique of single-cell sequencing from healthy non-DM and Diabetic foot ulceration non-healers skin samples revealed 19 distinct cell clusters. (B) GO biological process enrichment of all distinct cells in healthy and DFU-Non-healer. (C) UMAP of macrophage clusters, annotated and colored by the sample type and clustering. (D) GO biological process enrichment of macrophages in healthy and DFU-Non-healer. (E) Violin plots showing expression levels of key upstream regulators in phagocytic, inflammation, and NAD metabolism-related pathways in macrophage clusters. (F) Schematic diagram of clinical sample collection. (G) Neutrophils were identified by CD15(red) in gingivae. (H) Representative images of TUNEL staining in each group and observed under a fluorescence microscope (the nucleus was blue, and the apoptotic cell nucleus appeared green), and the percentages of apoptotic cells were quantitatively analyzed. (I) NETs were identified by Cit-H3 (red), MPO (green), and DAPI (blue) in the gingivae of periodontitis and those with diabetic periodontitis. (J-K) Immunofluorescence staining of gingival tissues, in which CD68 positive represents macrophage, CD86 positive represents M1 phenotype macrophage, and CD206 positive represents M2 phenotype macrophage. (L-R) Quantification of neutrophils, TUNEL positive apoptotic cells, NET, macrophage infiltration, and M1 and M2 polarization. The results were presented as means ± S.D. *p < 0.05; **p < 0.01; #p > 0.05 by 2-tailed, unpaired Student's t test. CP: chronic periodontitis, DP: diabetic periodontitis. The white dotted line indicates the gingival epithelial basement membrane.