Immunohistochemical Monitoring of Wound Healing in Antibiotic Treated Buruli Ulcer Patients

Author Summary Coagulative tissue necrosis and local immunosuppression caused by the M. ulcerans macrolide toxin mycolactone are typical features of Buruli ulcer disease (BU). In particular in BU endemic remote rural areas of West Africa, patients often report with large ulcerated lesions. Despite the availability of an effective dual antimycobacterial antibiotic therapy, some ulcerative lesions may take long time to healing and represent a major burden for the patients as well as for the health system. Proper wound healing is a well-orchestrated process involving numerous cellular and acellular components. Here we have performed immunohistochemical studies with tissue from BU lesions collected before, during and after antibiotic treatment. We identified a set of markers which are appropriate to evaluate formation of granulation tissue (alpha-smooth muscle positive fibroblasts), matrix deposition (pro-collagen 1, fibronectin and tenascin C), cell activation (phosphorylated S6), hyper proliferation of the epidermis (cytokeratin 16) and apoptosis (cleaved caspase 3) during wound healing. These markers may become suitable for assessing progression of tissue repair and for investigating the functional basis of impaired wound healing. Background While traditionally surgery has dominated the clinical management of Buruli ulcer (BU), the introduction of the combination chemotherapy with oral rifampicin and intramuscular streptomycin greatly improved treatment and reduced recurrence rates. However management of the often extensive lesions after successful specific therapy has remained a challenge, in particular in rural areas of the African countries which carry the highest burden of disease. For reasons not fully understood, wound healing is delayed in a proportion of antibiotic treated BU patients. Therefore, we have performed immunohistochemical investigations to identify markers which may be suitable to monitor wound healing progression. Methodology/Principal findings Tissue specimens from eight BU patients with plaque lesions collected before, during and after chemotherapy were analyzed by immunohistochemistry for the presence of a set of markers associated with connective tissue neo-formation, tissue remodeling and epidermal activation. Several target proteins turned out to be suitable to monitor wound healing. While alpha-smooth muscle actin positive myofibroblasts were not found in untreated lesions, they emerged during the healing process. These cells produced abundant extracellular matrix proteins, such as pro-collagen 1 and tenascin and were found in fibronectin rich areas. After antibiotic treatment many cells, including myofibroblasts, revealed an activated phenotype as they showed ribosomal protein S6 phosphorylation, a marker for translation initiation. In addition, healing wounds revealed dermal tissue remodeling by apoptosis, and showed increased cytokeratin 16 expression in the epidermis. Conclusion/Significance We have identified a set of markers that allow monitoring wound healing in antibiotic treated BU lesions by immunohistochemistry. Studies with this marker panel may help to better understand disturbances responsible for wound healing delays observed in some BU patients.