Background: Poorly healing wounds are one of the major complications in patients suffering from recessive dystrophic epidermolysis bullosa (RDEB). At present, there are no effective means to analyze changes in cellular and molecular networks occurring during RDEB wound progression to predict wound outcome and design betted wound management approaches. Objectives: To better define mechanisms influencing RDEB wound progression by evaluating changes in molecular and cellular networks. Methods: We developed a non-invasive approach for sampling and analysis of wound-associated constituents using wound-covering bandages. Cellular and molecular components from seventy-six samples collected from early, established and chronic RDEB wounds were evaluated by FACS-based immuno-phenotyping and ELISA. Results: Our cross-sectional analysis determined that progression of RDEB wounds to chronic state is associated with the accumulation (up to 90 %) of CD16 + CD66b + mature neutrophils, loss of CD11b + CD68 + macrophages, and a significant increase (up to 50 %) in a number of CD11c + CD80 + CD86 + activated professional antigen presenting cells (APC). It was also marked by changes in activated T cells populations including a reduction of CD45RO + peripheral memory T cells from 80 % to 30 % and an increase (up to 70 %) in CD45RA + effector T cells. Significantly higher levels of MMP9, VEGF-A and cathepsin G were also associated with advancing of wounds to poorly healing state. Conclusions: Our data demonstrated that wound-covering bandages are useful for a non-invasive sampling and analysis of wound-associated constituents and that transition to poorly healing wounds in RDEB patients as associated with distinct changes in leukocytic infiltrates, matrix-remodeling enzymes and pro-angiogenic factors at wound sites.

Recessive dystrophic epidermolysis bullosa (RDEB) patients develop poorly healing skin wounds that are frequently colonized with microbiota. Because T cells play an important role in clearing such pathogens, we aimed to define the status of adaptive T cell-mediated immunity in RDEB wounds. Using a non-invasive approach for sampling of wound-associated constituents, we evaluated microbial contaminants in cellular fraction and exudates obtained from RDED wounds. Infectivity and intracellular trafficking of inactivated Staphylococcus aureus was accessed in RDEB keratinocytes. S. aureus and microbial antigen-specific activation of RDEB wound-derived T cells were investigated by fluorescence-activated cell sorting-based immune-phenotyping and T-cell functional assays. We found that RDEB wounds and epithelial cells are most frequently infected with Staphylococcus sp. and Pseudomonas sp. and that S. aureus essentially infects more RDEB keratinocytes and RDEB-derived squamous cell carcinoma cells than keratinocytes from healthy donors. The RDEB wound-associated T cells contain populations of CD4 + and CD8 + peripheral memory T cells that respond to soluble microbial antigens by proliferating and secreting interferon gamma (IFNγ). Moreover, CD8 + cytotoxic T lymphocytes recognize S. aureus-infected RDEB keratinocytes and respond by producing interleukin-2 (IL-2) and IFNγ and degranulating and cytotoxically killing infected cells. Prolonged exposure of RDEB-derived T cells to microbial antigens in vitro does not trigger PD-1-mediated T-cell exhaustion but induces differentiation of the CD4 high population into CD4 high CD25 + FoxP3 + regulatory T cells. Our data demonstrated that adaptive T cell-mediated immunity could clear infected cells from wound sites, but these effects might be inhibited by PD-1/ Treg-mediated immuno-suppression in RDEB.