Aberrant stromal tissue factor and mycolactone-driven vascular permeability, exacerbated by IL-1β, orchestrate pathogenic fibrin formation in Buruli ulcer lesions
Authors: Hsieh Louise Tzung-Harn , Dos Santos Scott J , Ogbechi Joy , Loglo Aloysius D. , Salguero Francisco J. , Ruf Marie-Thérèse , Pluschke Gerd , Simmonds Rachel E. 
Published: August 2021

AbstractThe neglected tropical disease Buruli ulcer, caused byMycobacterium ulceransinfection, displays coagulative necrosis in affected skin tissues. We previously demonstrated that exposure to theM. ulceransexotoxin 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 vitroassays 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 SummaryTo 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.