Fig 2: Chronic wound fluid (CWF) induces pro‐inflammatory responses and impairs wound healing in a 3D in vitro wound model. (a) Wound closure analysis on day 4 shows significantly delayed healing in skin constructs treated with 4% CWF compared to controls (each group and time point n ≥ 11). (b) Flow cytometry analysis indicates a significantly increased expression of the pro‐inflammatory macrophage marker Msr‐1 and a trend towards reduced CD163 expression in CWF‐treated models (n = 5). (c) qPCR analysis demonstrates elevated expression of the pro‐inflammatory chemokine CCL2 in CWF‐treated models (n = 6). (d) Cytometric bead array (CBA) reveals higher concentrations of the pro‐inflammatory cytokines IL‐6 and IL‐1β in supernatants of CWF‐treated models (corrected by subtracting the amount added by CWF, as shown in Table 3) (each group and time point n ≥ 11). (e) ELISA analysis shows a significant reduction in the concentration of the anti‐inflammatory growth factor TGF‐β in CWF‐treated models (n = 7). (f) Volcano plot of RNA sequencing analyses of PBS‐ versus CWF‐treated wound models on day 4 (each n = 2). Dots depicted on the right‐hand side of the log2Foldchange value 0 represent significantly upregulated gene transcripts (blue), while the dots on the left‐hand side represent significantly downregulated transcripts (red). Statistical analyses: Data are expressed as mean ± SEM. (a), (e) Unpaired t‐test; (b), (c) Mann–Whitney U test; (d) Ordinary one‐way‐ANOVA. Asterisks mark statistically significant difference (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 3: sCD83 attenuates CWF‐induced inflammatory responses and promotes wound healing in a 3D in vitro wound model. (a) Wound closure analyses on day 4, show significantly accelerated healing in CWF‐exposed models, when treated with sCD83 compared to PBS‐controls (each group and time point n ≥ 14). (b) Flow cytometry reveals increased expression of the pro‐regenerative macrophage marker CD163 and decreased expression of the pro‐inflammatory marker Msr‐1, in sCD83‐treated models (n = 10). (c) Analyses of supernatants demonstrate elevated levels of pro‐healing growth factors such as VEGF (n = 11) and TGF‐β (n = 7) following sCD83 treatment. (d) qPCR analyses show significant upregulation of cytokeratins KRT6a and KRT14, markers of epithelial stem cell activation and motility, and a reduction in CCL2 expression. Additional pro‐inflammatory gene transcripts (CXCL2, IL1β, TNFα) showed a trend towards reduction (n = 6). (e) Volcano plot of RNA sequencing analyses of PBS versus sCD83‐treated CWF‐exposed wound models, on day 4 (each n = 2). Dots depicted on the right‐hand side of the log2Foldchange value 0 represent significantly upregulated gene transcripts (blue), while the dots on the left‐hand side represent significantly downregulated transcripts (red). (f), (g) Representative heat maps of differently expressed transcripts in CWF‐exposed wound models of PBS‐ and sCD83‐treated samples on day 4. (f) Represents gene transcripts associated with inflammation and (g) genes for either pro‐inflammatory or pro‐regenerative macrophages. Statistical analyses: Data are expressed as mean ± SEM. (a), (b), (c) Unpaired t‐test; (d) Mann–Whitney U test. Asterisks mark statistically significant difference (*p < 0.05, **p < 0.01).