Analysis of Mycobacterium ulcerans-specific T-cell cytokines for diagnosis of Buruli ulcer disease and as potential indicator for disease progression

Abstract

BackgroundBuruli ulcer disease (BUD), caused by Mycobacterium (M.) ulcerans, is the third most common mycobacterial disease after tuberculosis and leprosy. BUD causes necrotic skin lesions and is a significant problem for health care in the affected countries. As for other mycobacterial infections, T cell mediated immune responses are important for protection and recovery during treatment, but detailed studies investigating these immune responses in BUD patients are scarce. In this study, we aimed to characterise M. ulcerans-specific CD4+ T cell responses in BUD patients and to analyse specific cytokine-producing T cells in the context of disease severity and progression.Methodology/Principal findingsFor this case-control study, whole blood samples of BUD patients (N = 36, 1.5–17 years of age) and healthy contacts (N = 22, 3–15 years of age) were stimulated with antigen prepared from M. ulcerans and CD4+ T cells were analysed for the expression of TNFα, IFNγ and CD40L by flow cytometry. The proportions and profile of cytokine producing CD4+ T cells was compared between the two study groups and correlated with disease progression and severity. Proportions of cytokine double-positive IFNγ+TNFα+, TNFα+CD40L+, IFNγ+CD40L+ (p = 0.014, p = 0.010, p = 0.002, respectively) and triple positive IFNγ+TNFα+CD40L+ (p = 0.010) producing CD4+ T cell subsets were increased in BUD patients. In addition, TNFα+CD40L-IFNγ- CD4+ T cells differed between patients and controls (p = 0.034). TNFα+CD40L-IFNγ- CD4+ T cells were correlated with lesion size (p = 0.010) and proportion were higher in ‘slow’ healers compared to ‘fast healers’ (p = 0.030).ConclusionsWe were able to identify M. ulcerans-specific CD4+ T cell subsets with specific cytokine profiles. In particular a CD4+ T cell subset, producing TNFα but not IFNγ and CD40L, showed association with lesion size and healing progress. Further studies are required to investigate, if the identified CD4+ T cell subset has the potential to be used as biomarker for diagnosis, severity and/or progression of disease.

Cell-mediated and serology-based tests for Mycobacterium ulcerans disease: A systematic review and meta-analysis

“…List of abbreviations not in text: Acyl carrier protein 2& 3(ACP2, ACP3); Acyltransferase with acetate specificity type 1 &2 (ATac1, ATac2); Acyltransferase with propionate specificity (ATp); Enoylreductase (ER); Ketoreductase type A and B (KRA, KR B); Ketosynthase type C (KS C); Ketosynthase domain (Ksalt), dehydratase (DH), and Phorbol 1-myristate 1-acetate (PMA) [34]. Cluster of differentiation 4 (CD4); CD 40 Ligand (CD40L); Tumor necrosis factor alpha (TNFα) [32].…”

“…A summary of these 12 studies are presented in Table 1. Nine out of the 12 CMI and/or serological studies identified were conducted in Africa, specifically Benin [14], Cameroon [31], Cote d'Ivoire [29], Zaire (now Democratic Republic of Congo, DRC) [27], Ghana [15,19,[30][31][32]34] and Uganda [27]. The remaining three studies, were conducted in Australia [8,26,28].…”

“…All 12 studies started with the production of diverse antigens of the infective organism M. ulcerans. The antigens used for the detection assays were either pathogen-derived proteins (PDP) [26][27][28][29][30]32], recombinant proteins (RCP) [8,14,15,19,26,31] or both [26]. The pathogen-derived preparations were produced by heat-inactivation of M. ulcerans cells [8,25,26] or by mechanical disruption (sonication, agitation or homogenization) [26-28, 32, 36].…”

“…This was measured by induration on the skin. Other immunodetection methods used were ELISA [8,14,19,26,30,32,34], Western blot for M. ulcerans antibodies and cytokine specific screening [8,19,26,29,31].…”

“…All the studies that used BU patient samples for their assay also included healthy controls [8,14,15,19,26,27,29,31,32]. It was only Stanford et al [27], Dobos et al [29] and Okenu et al [30] that sought to address the cross-reactivity challenge by including patients other than BU with TB and leprosy, in their assays.…”