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Originally published online as doi:10.1189/jlb.1005581 on March 10, 2006

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(Journal of Leukocyte Biology. 2006;79:1150-1156.)
© 2006 by Society for Leukocyte Biology

Systemic suppression of interferon-{gamma} responses in Buruli ulcer patients resolves after surgical excision of the lesions caused by the extracellular pathogen Mycobacterium ulcerans

Dorothy Yeboah-Manu*,{dagger}, Elisabetta Peduzzi{dagger}, Ernestina Mensah-Quainoo{ddagger}, Adwoa Asante-Poku*, David Ofori-Adjei*, Gerd Pluschke{dagger} and Claudia A. Daubenberger{dagger},1

* Noguchi Memorial Institute for Medical Research, University of Ghana, Legon;
{dagger} Swiss Tropical Institute, Molecular Immunology, Basel, Switzerland; and
{ddagger} Ghana Health Service, Amasaman, Ga District, Ghana

1Correspondence: Swiss Tropical Institute, Molecular Immunology, Socinstrasse 57, CH 4002 Basel, Switzerland. E-mail: Claudia.Daubenberger{at}unibas.ch

ABSTRACT

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial infection in immunocompetent humans besides tuberculosis and leprosy. We have compared by ex vivo enzyme-linked immunospot analysis interferon-{gamma} (IFN-{gamma}) responses in peripheral blood mononuclear cells (PBMC) from BU patients, household contacts, and individuals living in an adjacent M. ulcerans nonendemic region. PBMC were stimulated with purified protein derivative (PPD) and nonmycobacterial antigens such as reconstituted influenza virus particles and isopentenyl-pyrophosphate. With all three antigens, the number of IFN-{gamma} spot-forming units was reduced significantly in BU patients compared with the controls from a nonendemic area. This demonstrates for the first time that M. ulcerans infection-associated systemic reduction in IFN-{gamma} responses is not confined to stimulation with live or dead mycobacteria and their products but extends to other antigens. Interleukin (IL)-12 secretion by PPD-stimulated PBMC was not reduced in BU patients, indicating that reduction in IFN-{gamma} responses was not caused by diminished IL-12 production. Several months after surgical excision of BU lesions, IFN-{gamma} responses of BU patients against all antigens used for stimulation recovered significantly, indicating that the measured systemic immunosuppression was not the consequence of a genetic defect in T cell function predisposing for BU but is rather related to the presence of M. ulcerans bacteria.

Key Words: ex vivo ELISpot analysis • immunosuppression • interleukin-12

INTRODUCTION

Buruli ulcer (BU) caused by Mycobacterium ulcerans is an infectious disease characterized by chronic, necrotizing ulceration of subcutaneous (s.c.) tissues and the overlying skin. The disease starts as a s.c. nodule, papule, or plaque, which eventually ulcerates and progresses over weeks to months until surgical excision or spontaneous healing occurs [1 ]. After tuberculosis and leprosy, BU is the third most common mycobacterial infection in immunocompetent humans [2 ]. The main burden of disease falls on children living in sub-Saharan Africa, but healthy people of all ages, races, and socioeconomic class are susceptible [3 ]. The effectiveness of antimycobacterial drug therapy has not been proven [3 ]. Consequently, surgery is presently the recommended treatment option [4 ]. In BU lesions, clumps of extracellular, acid-fast organisms surrounded by areas of necrosis are found, particularly in s.c. fat tissue [5 ]. M. ulcerans produces a family of macrolide toxin molecules, the mycolactones, which are associated with tissue destruction and local immunosuppression [6 ]. In cell culture experiments, mycolactones produce apoptosis and necrosis in many human cell types [7 , 8 ]. The toxin appears to play a role in inhibiting the recruitment of inflammatory cells to the site of infection, which explains at least in part why inflammatory responses are poor in BU lesions [5 ]. However, intralesional influx of leukocytes and granulomatous responses in the dermis and panniculus has been reported in late stages of the disease [9 , 10 ]. Spontaneous healing can occur and is often accompanied by a conversion of the Burulin (M. ulcerans sonicate) skin test from negative to positive. However, the immune mechanisms involved in protection against BU are largely unknown.

The importance of interferon-{gamma} (IFN-{gamma}) for immunity against mycobacterial infections in humans is demonstrated by the increased susceptibility of children carrying complete IFN-{gamma} receptor 1 (IFN-{gamma}R1) chain deficiency to environmental mycobacterial infection [11 ]. Apart from CD4+ T cells, {gamma}{delta} T cells, natural killer cells (NK), and CD8+ T cells are potent sources of IFN-{gamma}. CD4 T cells can be differentiated into T helper cell type 1 (Th1) and Th2, distinguished by their patterns of cytokine production after antigen activation. Apart from other cytokines, Th1 cells preferentially secrete IFN-{gamma}, and Th2 cells preferentially secrete interleukin (IL)-4 and IL-5. Th1 or Th2 development is determined by the cytokine environment during T cell activation in the primary response to antigen, and IL-12 and IFN-{gamma} are implicated in the decision to adopt a Th1 phenotype [12 ]. IFN-{gamma} binds to the IFN-{gamma}R1/IFN-{gamma}R2 complex and stimulates innate cell-mediated immunity through NK cells and activation of bactericidal mechanisms in macrophages. The central role of IFN-{gamma} in major histocompatibility complex class I- and class II-restricted antigen processing and presentation is well-documented [13 ]. At present, the contribution of IFN-{gamma} in immunity to extracellular M. ulcerans remains to be established.

Peripheral blood mononuclear cells (PBMC) of BU patients with active disease showed significantly reduced lymphoproliferation and IFN-{gamma} secretion in response to stimulation with live or killed preparations of Mycobacterium bovis, M. ulcerans, M. tuberculosis, and the recombinant protein Ag85 of M. tuberculosis [14 15 16 17 18 ]. Here, we have determined in a cross-sectional study the frequency of IFN-{gamma}-secreting cells in PBMC from BU patients, their household contacts, and controls from BU nonendemic areas by ex vivo enzyme-linked immunospot (ELISpot) analysis. The antigens used for stimulation included isopentenyl pyrophosphate (IPP), reconstituted influenza virus particles (virosomes), and tuberculin-purified protein derivative (PPD) of M. tuberculosis, which stimulate distinct T cell subsets, such as V{gamma}2V{delta}2 T cells [19 ], CD4 T cells [20 ], and CD4 and V{gamma}2V{delta}2 T cells [21 ], respectively. Results demonstrate that BU-associated reductions in IFN-{gamma} responses are not confined to stimulation with live or dead mycobacteria and mycobacterial antigens. Furthermore, it is shown for the first time that in individual BU patients, this suppression in IFN-{gamma} secretion improved in a time interval of 5–10 months.

MATERIALS AND METHODS

Study population
Thirteen BU patients, 19 clinically healthy household contacts who never had clinical BU, and 18 healthy persons living in a M. ulcerans nonendemic district in the Greater Accra region of Ghana were enrolled for the study (Table 1 ). All BU patients enrolled were residents of the BU-endemic Ga District and presented with preulcerative or ulcerative lesions at the Amasaman Health Centre. Informed consent was obtained from study participants or their parents or guardians before enrollment. For ethical reasons, the age range of the nonexposed controls, who were included into the analysis, was higher than that of the BU patients. Ethical approval for the study was obtained from the local ethical review board of the Noguchi Memorial Institute for Medical Research, University of Ghana (Legon). Clinical diagnosis of BU was reconfirmed as described [22 , 23 ] by one or more laboratory verification tests (Table 2 ), including culture of M. ulcerans, microscopic detection of acid-fast bacilli (AFB), or IS2404 polymerase chain reaction (PCR). The clinical pictures of the patients ranged from nodules or plaques to severe ulcerative forms (Table 2) . In the BU group, nine persons and in the other two groups, 10 persons each with a BCG scar were recruited (Table 1) . PBMC were isolated from venous peripheral blood using Ficoll-Hypaque gradient centrifugation following standard procedures and cryopreserved prior to the analysis.


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  		Table 1. Characteristics of BU Patients, Household Contacts, and Controls from a BU Nonendemic Area Enrolled in the Study

 

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  		Table 2. Clinical Data of Patients Presenting with Lesions As a Result of Confirmed M. ulcerans Infection

 
Antigens
After thawing, PBMC were directly stimulated with 50 µM IPP (Sigma Chemical Co., St. Louis, MO), 10 µg/ml tuberculin PPD derivative of M. tuberculosis (Statens Seruminstitut, Denmark), 10 µg/ml phytohemagglutinin (PHA; Sigma Chemical Co.), or 20 µg/ml immunopotentiating reconstituted influenza virosomes (IRIV; Berna Biotech, Switzerland). These influenza virosomes are spherical, unilamellar vesicles prepared by detergent removal from influenza surface glycoproteins and mixtures of natural and synthetic phospholipids containing H1N1 from influenza virus strain A/Singapore/6/86 [24 ]. Cell culture medium consisted of RPMI 1640, 10% heat-inactivated human AB serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco-BRL, Grand Island, NY).

Ex vivo IFN-{gamma} ELISpot analysis
PBMC were thawed, washed, and suspended at a concentration of 2 x 106 cells/ml in complete cell culture medium. The cells were then stimulated with the different antigens at the final concentrations indicated above and incubated for 24 h at 37°C, 5% CO2 humidified atmosphere. A 96-well nitrocellulose-bottomed plate (Millipore, Beford, MA) was coated overnight at 4°C with 10 µg/ml anti-human IFN-{gamma} primary antibody (Clone1-D1K; Mabtech, Sweden). The plates were then washed five times with phosphate-buffered saline (PBS) and blocked with cell culture medium for 1 h at room temperature. The medium was decanted, and preincubated cells (2x105 or 1x105) were added to each well in triplicate and incubated at 37°C for another 20 h. Assays were terminated by washing plates three times with PBS-Tween 80 (0.05%) followed by PBS another three times. Secondary antibody (biotin-labeled anti-human-IFN-{gamma}; Clone 7-B6-1) was added to each well at 1 µg/ml, and the plate was incubated for 2 h at room temperature (RT). Plates were washed again six times with PBS before application of streptavidine-alkaline phosphatase (1:1000 dilution in PBS, 0.5% fetal calf serum, 100 µl/well) for 1 h at RT. After washing wells seven times with PBS, distinct spots were developed by the incubation of plates at RT for 4–10 min following the addition of the developing buffer (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium, diluted 1:100, Bio-Rad, Hercules, CA). Spot development was stopped by washing the plates extensively with water and left to dry. Plates were later evaluated using the ELISpot Reader system (AID, Germany) to determine the number of spot-forming units (SFU).

Enzyme-linked immunosorbent assay (ELISA) quantification of IL-12 in cell culture supernatants
Levels of total IL-12 in culture supernatant of PBMC incubated with PHA (10 µg/ml) and PPD (10 µg/ml) for 96 h were determined by ELISA using a commercial kit (Mabtech). Samples were analyzed in triplicates, and results were expressed as the average of the three readings in an ELISA reader at 450 nm with reference to curves generated using serially diluted recombinant human IL-12. The sensitivity of the assay was 30 pg/ml for total IL-12.

Statistical analysis
Data were analyzed using the STATA program (Stata Corporation, College Station, TX). Comparisons among the paired samples were performed using the Wilcoxon signed-rank test, and the Wilcoxon rank-sum test was used to analyze the significance of observed difference in IL-12 secretion between patients and unexposed controls. For comparing the frequencies of antigen-specific IFN-{gamma}-secreting cells, the data were transformed to normality using Box-Cox transformation before being analyzed by linear regression. Data were considered statistically significant when P < 0.05.

RESULTS

Frequency reduction of IFN-{gamma}-secreting SFU in PBMC from BU patients
Thirteen patients with laboratory-reconfirmed M. ulcerans infection, 19 clinically healthy household contacts, and 18 individuals from a neighboring BU nonendemic area were enrolled for this study (Tables 1 and 2) . The frequency of immediate IFN-{gamma}-secreting cells in PBMC upon stimulation with IPP, IRIV, and PPD was analyzed using an ex vivo ELISpot assay. Figure 1 shows that the mean of SFU upon stimulation with PPD, IPP, and IRIV was significantly lower (P=0.0086, P=0.001, and P=0.0002, respectively) in BU patients compared with nonexposed controls. In household contacts, the mean of SFU after IPP and IRIV stimulation was significantly higher compared with BU patients (P=0.005 and P=0.001, respectively), and in PPD-stimulated cultures, no significant difference was observed (P=0.82).


Figure 1
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  		Figure 1. Quantification of ex vivo IFN-{gamma}-secreting cells by ELISpot analysis after stimulation of PBMC with IPP ({diamond}), IRIV ({circ}), and PPD ({Delta}). PBMC derived from BU patients, household contacts, and controls from a BU nonendemic area were thawed into cell culture medium and kept overnight in the presence or absence of the different stimuli. For the detection of IFN-{gamma}-producing cells, 2 x105 PBMC/well were plated in triplicate wells, and spots were developed after 24 h. Delta SFU = Mean of SFU of stimulated triplicate cultures – mean SFU of unstimulated triplicate cultures. Data from each individual analyzed are shown separately. Mean SFU/106 PBMC were 26 (6–102), 137 (57–327), and 262 (157–436) after stimulation with IPP in BU patients, household contacts, and nonendemic controls, respectively. In IRIV-stimulated cells, the mean SFU/106 cells were 22 (7–70), 135 (83–221), and 149 (56–394) in BU patients, household contacts, and nonendemic controls, respectively. After PPD stimulation, 88 (35–223), 39 (14–106), and 212 [100–445] SFU/106 PBMC were recorded in BU patients, household contacts, and nonendemic controls, respectively. The values given in brackets are the 95% confidence intervals of the geometric means of SFU. Data of Patients P03, P08, P09, and P12 are shown by shaded symbols.

 
Recovery from systemic immunosuppression after surgical treatment
All patients enrolled for the study were treated by wide surgical excision of the BU lesions. Blood samples were collected several months after the first sampling (Table 2) , and PBMC from both time-points were analyzed in parallel using the same IFN-{gamma} ELISpot assay as above. A 3.9-, 3.7-, and 3.6-fold median increase in cellular responses against IPP, IRIV, and PPD stimulation, respectively, was observed when PBMC, taken at the two time-points, were compared (Fig. 2A ). Statistical analysis confirmed this increase of responses as highly significant with P values of 0.021 and 0.003, respectively, after IPP and IRIV stimulation. PHA-stimulated control wells showed only a slight (1.7-fold) median increase between the two different time-points analyzed (P=0.17), and in PPD-stimulated cultures, the boost was not significant (P=0.09). One representative example of an ELISpot analysis is shown in Figure 2B , and the numbers of SFU detectable after IPP, IRIV, and PPD stimulation rose between the first and second sampling. In contrast, medium and PHA control wells remained at a comparable level between the two analyses.


Figure 2
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  		Figure 2. (A) Frequency increase of antigen-specific ex vivo IFN-{gamma}-secreting cells in BU patients. PBMC of 13 BU patients sampled at two time-points (Table 2) were thawed into cell culture medium and kept overnight in the presence or absence of IPP ({diamond}), IRIV ({square}), PPD ({Delta}), and PHA ({circ}). For the detection of IFN-{gamma}-producing cells, 2 x105 PBMC/well were plated in triplicate wells, and spots were developed after 24 h. Delta SFU were calculated as described in Figure 1 . Given is the ratio of SFU obtained after the second sampling/first sampling. Data of Patients P03, P08, P09, and P12 are shown by shaded symbols. (B) Representative IFN-{gamma} ELISpot patterns of BU Patient P21. PBMC of BU Patient P21 were obtained immediately before surgical excision of the BU lesion (first sampling) and 5 months later (second sampling). Parallel analysis of both samples was conducted in triplicates arranged horizontally with 2 x 105 cells/well plated. The different stimulators used are given on the right, and the numbers of SFU/well are shown in the lower-left corners.

 
IL-12 production in PPD-stimulated PBMC is not affected in BU patients
Next, we wanted to determine whether the systemic suppression of IFN-{gamma} responses in BU patients is related to a diminished capacity to secrete IL-12. PBMC of nine patients and 10 persons from BU nonendemic areas were stimulated with PHA or PPD, and the total IL-12 concentration in cell culture supernatants was measured by ELISA (Fig. 3 ). It is interesting that the IL-12 concentrations in PPD-stimulated PBMC of individuals living in M. ulcerans nonendemic regions were statistically lower compared with BU patients (P=0.011), and in PHA-stimulated cultures, no significant difference was observed (P=0.57; Fig. 3 ). The mean IL-12 concentrations in supernatants of PPD- and PHA-stimulated cultures of PBMC obtained before or after surgical treatment showed no difference (Fig. 3) .


Figure 3
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  		Figure 3. Total IL-12 concentrations in cell culture supernatants of PHA- and PPD-stimulated PBMC of nine BU patients obtained at the two different sampling time-points (Table 2) . Mean of total IL-12 concentration (given in pg/ml) in PPD-stimulated cultures was 543 and 750 after the first and second sampling, respectively, and in PHA-stimulated cultures, 348 and 472, respectively. PBMC of 10 individuals living in a neighboring M. ulcerans nonendemic region were treated similarly, and the mean total IL-12 concentrations after PPD and PHA stimulation were 276 and 402, respectively.

 
DISCUSSION

An ex vivo ELISpot assay for detection of IFN-{gamma} secretion permitting the direct detection of individual antigen-specific T cells at low frequencies was used in the current study [25 ]. The 48-h antigen challenge of PBMC suffices to engage cytokine production in the memory/effector T lymphocyte but not in naïve T cells and allows determination of frequencies and cytokine signatures of recirculating, antigen-specific T cells [25 ]. The frequencies of systemic IFN-{gamma}-producing SFU after stimulation with IPP, IRIV, or PPD were reduced significantly in BU patients compared with individuals living in a neighboring BU nonendemic area. This result is consistent with other reports demonstrating that PBMC from subjects with past or current M. ulcerans disease had reduced IFN-{gamma} production in response to PPD of M. tuberculosis and M. bovis or whole-killed M. bovis BCG or M. ulcerans [14 15 16 17 18 ]. However, our data strongly indicate that reduced IFN-{gamma} production is not confined to immune responses specific for mycobacterial antigens or whole mycobacteria but extends to CD4 T cell responses specific for influenza virus [20 ] and V{gamma}2V{delta}2 T cells [19 ].

Reduced IFN-{gamma} production in BU patients could be the consequence of a genetic abnormality in T cell function predisposing for mycobacterial infections [11 ]. Therefore, we analyzed PBMC of BU patients sampled at two consecutive time-points. When the paired PBMC samples were analyzed in parallel by ELISpot analysis, the numbers of IFN-{gamma}-secreting cells after IPP, IRIV, and PPD stimulation increased significantly after surgical treatment. Comparable numbers of SFU in PHA-stimulated PBMC in paired samples excluded a general suppression of cellular immune responses in BU and variations in quality of PBMC sample cryopreservation. To our knowledge, this is the first report demonstrating that antigen-specific IFN-{gamma} production in BU patients is coming back to normal levels after surgical treatment. Hence, confounding genetic defects do not seem to be responsible for the observed immunosuppression. The question of whether IFN-{gamma} production in patients undergoing spontaneous healing of BU will also improve over time is not addressed here.

IL-12 induces T and NK cells to produce several cytokines, including IFN-{gamma} [26 ]. Total IL-12 concentrations in culture supernatants of PPD-stimulated PBMC were statistically higher in BU patients (treated and untreated) than in the controls. This may reflect a compensatory mechanism of the immune system to restore IFN-{gamma} production and indicates that the observed reduction in systemic IFN-{gamma} responses is not caused by diminished IL-12 production. Different cytokine expression profiles in PBMC and skin lesions were described in patients suffering from the nodular and ulcerative forms of BU. Nodules were associated with higher IFN-{gamma} and lower IL-10 production and BU, with lower IFN-{gamma} and higher IL-10 production [17 ]. Within the limited number of BU patients enrolled in our study, a relationship between different stages of BU disease and reduction of IFN-{gamma} secretion was not observed.

V{gamma}2V{delta}2 T cells compose the majority of human {gamma}{delta} T cells in circulation and among their defined ligands, is IPP, a metabolite found in prokaryotic and eukaryotic cells including mycobacteria [27 ]. Studies in rhesus monkeys provided evidence that V{gamma}2V{delta}2 T cells contribute to adaptive immune responses in mycobacterial infections [28 ]. A correlation between the absence or loss of V{gamma}2V{delta}2 T cells and the extent of M. tuberculosis disease has been described [29 ]. An impaired ability of V{gamma}2V{delta}2 T cells to produce cytokines or proliferate in response to phosphorylated microbial metabolites was observed in active M. tuberculosis pulmonary disease [30 31 32 ]. It is interesting that in 10 out of 13 BU patients analyzed here, the ex vivo IFN-{gamma} secretion of IPP-reactive V{gamma}2V{delta}2 T cells increased significantly after surgical treatment.

The diffusible macrolide toxins produced by M. ulcerans are considered as virulence factors responsible for pathogenicity of M. ulcerans, and it has been hypothesized that lack of inflammatory responses in BU lesions are related to local immunosuppressive activities of the mycolactones [6 , 33 , 34 ]. The treatment-associated reversal of immune suppression may indicate that mycolactones exert, apart from local, systemic effects also. Alternatively, additional, other immune-suppressive mechanisms may be operative in chronic M. ulcerans disease. PBMC from healthy contacts of tuberculosis patients and tuberculosis patients with limited disease produce large quantities of IFN-{gamma} in response to whole mycobacteria, PPD of M. tuberculosis, early secretory antigenic target-6, and 16-kDa and 38-kDa proteins [35 36 37 38 39 ]. In contrast, PBMC of active tuberculosis patients with advanced disease produce low quantities of IFN-{gamma} after similar stimulation, and following effective drug treatment, the IFN-{gamma} secretion improved [39 , 40 ]. Possible candidate structures mediating immune suppression in M. tuberculosis isolates such as phenolic glycolipids have been described [41 , 42 ]. In leprosy induction of regulatory or suppressor T cells, mediating immune suppression in affected hosts has been proposed [43 , 44 ]. Our findings, within their limits as a result of the small number of BU patients analyzed and the timing of the blood samples drawn, may indicate that mycolactone-independent immunosuppressive mechanisms common to chronic mycobacterial infections contribute to the reduction of systemic IFN-{gamma} responses in BU patients.

ACKNOWLEDGEMENTS

This study was supported in part by the Stanley Thomas Johnson Foundation, the Ghana Government, and a stipend from the Amt für Ausbildungsbeiträge of the county Basel-Stadt for D. Y-M. We thank Samuel Owusu, Dr. Kwasi Addo, John Tetteh, and Charles Atiogbe from Noguchi Memorial Institute for Medical Research (Legon, Ghana); the BU team nurses at Amasaman Health Centre (Ghana) for technical and field support; and all the participants involved in the study for their time. We are grateful to Dr. Penelope Vounatsou for support in statistical analysis.

Received October 14, 2005; revised February 3, 2006; accepted February 6, 2006.

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