Wound healing is a complex biological process that is impaired under diabetes conditions. Chronic non-healing wounds in diabetes are some of the most expensive healthcare expenditures worldwide. Early diagnosis and efficacious treatment strategies are needed. microRNAs (miRNAs), a class of 18–25 nucleotide long RNAs, are important regulatory molecules involved in gene expression regulation and in the repression of translation, controlling protein expression in health and disease. Recently, miRNAs have emerged as critical players in impaired wound healing and could be targets for potential therapies for non-healing wounds. Here, we review and discuss the mechanistic background of miRNA actions in chronic wounds that can shed the light on their utilization as specific wound healing biomarkers.

Cutaneous wound healing is a vital biological process that aids skin regeneration upon injury. Wound healing failure results from persistent inflammatory conditions observed in diabetes, or autoimmune diseases like psoriasis. Chronic wounds are incurable due to factors like poor oxygenation, aberrant function of peripheral sensory nervature, inadequate nutrients and blood tissue supply. The most significant hallmark of chronic wounds is heavily aberrant immune skin function. The immune response in humans relies on a large network of signalling molecules and their interactions. Research studies have reported on the dual role of host defence peptides (HDPs), which are also often called antimicrobial peptides (AMPs). Their duality reflects their potential for acting as antibacterial peptides, and as immunodulators that assist in modulating several biological signalling pathways related to processes such as wound healing, autoimmune disease, and others. HDPs may differentially control gene regulation and alter the behaviour of epithelial and immune cells, resulting in modulation of immune responses. In this review, we shed light on the understanding and most recent advances related to molecular mechanisms and immune modulatory features of host defence peptides in human skin wound healing. Understanding their functional role in skin immunity may further inspire topical treatments for chronic wounds.

Effects of peptidoglycan linked with zinc (PGM–Zn) were investigated on plaque–forming cell (PFC) generation to sheep red blood cell (SRBC) and SRBC–unrelated antibody production in primary and secondary immune response in mice depleted in vivo of CD4+ and/or CD8+ T lymphocytes. PGM–Zn in nondepleted mice stimulated the PFC generation and IgM or IgG and IgG1 production in primary and secondary reaction. Single depletion of CD4 or CD8+ T cells did not change this ability. The effects of PGM–Zn after CD8+ depletion were even greater than those in nondepleted mice. Depletion of both T cell subsets, however, completely abrogated immunostimulatory effects of PGM on PFC generation (primary and secondary response), as well as on primary SRBC–unrelated antibody production, leaving only the increase of IgG in secondary response unchanged. Immunostimulatory effects and isotype switching to IgG1 and IgG2a correlated with the changes in splenic CD4+, CD8+, CD5+ cells, pointing to the regulatory role of these cells and/or their cytokines in PGM–Zn–induced immunostimulation. Altogether the data suggest that PGM–Zn may potentiate the costimulatory signals coming from activated T cells and act on B cells without the T cell help.

Aims/hypothesis: Upregulation of miR-146a-5p and miR-29-3p is observed in chronic non-healing wounds in diabetes. Their single or combined inhibition's molecular and cellular effects were assessed in human keratinocytes (HaCaT cells) and in vivo using a mouse model of type 1 diabetes. Methods: As primary outcomes, we screened for proteome changes in HaCaT cells by LC-MS/MS after transfection with miR-146a-5p or miR-29a-3p inhibitors individually or in combination and following stimulation with TNF-α. Moreover, as a secondary outcome, we collected the data and cryopreserved and paraffin-embedded skin biopsies to estimate the tissue response to miRNA inhibition using immunofluorescence and histological analysis. Cryopreserved biopsies were also used for the LC-MS/MS proteome profiling to identify targets and cellular pathways involved in observed tissue changes. Results: We identified a panel of extracellular matrix proteins, mainly laminins, whose levels changed after transfection with miR-146a-5p or miR-29a-3p inhibitors in HaCaT cells, counteracting TNF-α effects. There was a difference in wound closure rate in vivo between the dual inhibition of miR-146a-5p and miR-29a-3p and scramble controls on day 8 ( p <0.01) and day 9 ( p <0.05), although not at day 10. Histological analysis at day 10 shows a loose papillary layer in the scramble inhibition group, indicating incomplete wound closure compared with dual miRNA inhibition. Moreover, the dual action of the inhibitors decreased inflammation at day 3 and day 10 (both p <0.001) and reactive oxygen species formation ( p <0.01) 3 days post wounding, while increasing the angiogenesis on day 3 ( p <0.01) and day 10 ( p <0.001). This was consistent with cytoskeletal rearrangements and collagen alterations observed in proteome profiling. Conclusions/interpretation: These findings demonstrate that dual inhibition of miR-146a-5p and miR-29a-3p in vitro synergises in a bidirectional manner, resulting either in intermediate effects or in cancelling each other’s activity for the levels of specific proteins of basal lamina that impair proliferation and cell motility, compared with the individual inhibitors. Topical supplementation of miR-146a-5p and miR-29a-3p inhibitors to diabetic mouse wounds resulted in a reduction in wound size on days 8 and 9, which correspond to the later stages of healing, but did not lead to complete healing by day 10. However, dual inhibition demonstrates favourable effects on high oxidative stress, elevated inflammation and poor angiogenesis. These effects are superior to single miRNA inhibition, suggesting that combined miRNA inhibition could be a promising therapeutic strategy for diabetic wound healing. Nevertheless, further studies in humans are warranted. Graphical