Aberrant stromal tissue factor and mycolactone-driven vascular permeability, exacerbated by IL-1β, orchestrate pathogenic fibrin formation in Buruli ulcer lesions

The neglected tropical disease Buruli ulcer, caused by Mycobacterium ulcerans infection, displays coagulative necrosis in affected skin tissues. We previously demonstrated that exposure to the M. ulcerans exotoxin mycolactone depletes the expression of thrombomodulin and impacts anticoagulation at the endothelial cell surface. Moreover, while widespread fibrin deposition is a common feature of BU lesions, the cause of this phenotype is not clear. Here, we performed sequential staining of serial tissue sections of BU patient biopsies and unbiased analysis of up to 908 individual non-necrotic vessels of eight BU lesions to investigate its origins. Most vessels showed evidence of endothelial dysfunction being thrombomodulin-negative, von Willebrand factor-negative and/or had endothelium that stained positively for tissue factor (TF). Primary haemostasis was rarely evident by platelet glycoprotein CD61 staining. Localisation of TF in these lesions was complex and aberrant, including diffuse staining of the stroma some distance from the basement membrane and TF-positive infiltrating cells (likely eosinophils). This pattern of abnormal TF staining was the only phenotype that was significantly associated with fibrin deposition, and its extent correlated significantly with the distance that fibrin deposition extended into the tissue. Hence, fibrin deposition in Buruli ulcer lesions is likely driven by the extrinsic pathway of coagulation. To understand how this could occur, we investigated whether clotting factors necessary for fibrin formation might gain access to the extravascular compartment due to loss of the vascular barrier. In vitro assays using primary vascular and lymphatic endothelial cells showed that mycolactone increased the permeability of monolayers to dextran within 24 hours. Moreover, co-incubation of cells with interleukin-1β exacerbated mycolactone’s effects, nearly doubling the permeability of the monolayer compared to each challenge alone. We propose that leaky vascular and lymphatic systems are important drivers of extravascular fibrin deposition, necrosis and oedema frequently seen in Buruli ulcer patients. Author Summary To date, the debilitating skin disease Buruli ulcer remains a public health concern and financial burden in low or middle-income countries, especially in tropical regions. Late diagnosis is frequent in remote areas, perhaps due to the painlessness of the disease. Hence patients often present with large, destructive opened ulcers leading to delayed wound closure or even lifelong disability. The infectious agent produces a toxin called mycolactone that drives the disease. We previously found evidence that the blood clotting system is disrupted by mycolactone in these lesions, and now we have further explored potential explanations for these findings by looking at the expression of coagulation regulators in BU. In detailed analysis of patient skin punch biopsies, we identified distinct expression patterns of certain proteins and found that tissue factor, which initiates the so-called extrinsic pathway of blood clotting, is particularly important. Mycolactone is able to disrupt the barrier function of the endothelium, further aggravating the diseased phenotype, which explains how clotting factors access the tissue. Altogether, such localised hypercoagulation in Buruli ulcer skin lesions may contribute to the development of the lesion. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Rachel Simmonds holds and Wellcome Trust Investigator Award in Science (202843/Z/16/Z) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval for analysing BU patient punch biopsies was obtained from the Ethikkommission beider Basel, Basel, Switzerland and the provisional national ethical review board of the Ministry of Health Benin (N IRB00006860) as well as from the Cameroon National Ethics Committee and the Ethics Committee of the Heidelberg University Hospital, Germany ([ISRCTN72102977][1]). A favourable ethical opinion for analysing normal human skin was given by Faculty of Health and Medical Science Ethics Committee of the University of Surrey (1174-FHMS-16). The normal human skin samples were collected by the Whiteley Clinic, Guildford, Surrey or were purchased from AMS Biotechnology. Written informed consent was obtained from adult patients or the guardians of child patients. The research related to human tissues complies with the ethical processes of University of Surrey. All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data are fully available without restriction. All relevant data are within the manuscript and its Supporting Information files [1]: /external-ref?link_type=ISRCTN&access_num=ISRCTN72102977