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

Abstract

Background: Buruli 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 findings: For 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).

Conclusions: We 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.

Fig 1. T_H1 cytokine producing CD4+ T cells in Buruli ulcer disease patients and healthy contacts.

Whole blood was cultured for 17.5 hrs with either M. ulcerans antigen or Staphylococcal enterotoxin b. Cells were analysed by flow cytometry for TNFα, IFNγ and CD40L producing CD4+ T cells. Proportions of double and triple cytokine producing T cells (IFNγ+TNFα+, TNFα+CD40L+, IFNγ+CD40L+ and IFNγ+TNFα+CD40L+) are indicated in (A). Combinations of cytokine producing CD4+ T cells are shown in (B). Medians are indicated in grey and groups are compared by non-parametric Mann-Whitney U test and p values indicated.

Fig 2. TNFα+CD40L-IFNγ- CD4+ T cells are correlated with size of lesions in Buruli ulcer disease.

Cytokine producing CD4+ T cells were determined as for Fig 1 and analysed in regards to type of lesion (A, B) or surface area of lesions (C, D) or widest diameter of the lesions (E). Multiple cytokine producing are shown in (A, C); TNFα+CD40L-IFNγ- and CD40L+IFNγ- are indicated in (B, D). P—values of non-parametric Mann-Whitney U analyses (A, B) and Spearman correlations (C–E) are presented.

Fig 3. TNFα+CD40L-IFNγ- CD4+ T cells differ between fast and slow healers in Buruli ulcer disease.

(A) BUD patients were divided into fast healers (≤ 111 days) and slow healers (> 111 days) based on the time required for complete healing and proportions of cytokine producing CD4+ T cells (B, C) or lesion sizes (D) are compared by non-parametric Mann Whitney U test. The healing rate within the first four weeks (in change if mm/week) was correlated with TNFα+CD40L-IFNγ- CD4+ T cells (E) by Spearman correlation or compared based on time until healing process starts after initiating antibiotic treatment (F).