Abstract
Multidrug-resistant tuberculosis (MDR-TB) is known as having a poor prognosis with a weak response to therapy and very high death rates. The aim of this work was to assess the immune response to the RD1-encoded antigen ESAT-6 of Mycobacterium tuberculosis in MDR-TB patients and compare to non-resistant (NR) TB patients and healthy controls (HC). Evaluation of interferon (IFN)-γ production showed that, although 55% of the MDR patients were responsive to ESAT-6, they produced lower IFN-γ levels (553 ± 11 pg/ml) when compared to NR-TB (1179 ± 163 pg/ml; P < 0·05) but not to controls (412 ± 65·7 pg/ml). Differences in the response to ESAT-6 and to its overlapping peptides mixture were also significant between MDR versus treated pulmonary NR-TB. Furthermore, a very low rate of response to PPD (23·5%) and to Ag85B (33·3%) was noted in MDR-TB patients as compared to the other groups. To determine the inflammatory response in patients’ groups, detection of tumour necrosis factor (TNF)-α was assessed in their sera before and during chemotherapy. Mean TNF-α levels in MDR-TB (43·8 ± 9 pg/ml) paralleled those found in treated pulmonary, and it was significantly different (P < 0·05) from the values found in untreated NR and HC. Interestingly, secretion of IFN-γ and TNF-α were predominant in MDR patients who presented with bilateral pulmonary lesions and lung cavitation. The present data indicate that the overall immune response to mycobacterial antigens is decreased in resistant TB and the major role inflammatory cytokines may play in perpetuating pulmonary tissue damage.
Keywords: ESAT-6, IFN-γ, MDR-TB, TNF-α, tuberculosis
Introduction
Resistance of Mycobacterium tuberculosis (Mtb) to anti-mycobacterial agents has recently received increased attention worldwide because morbidity and mortality from disease are extremely high, despite drug therapy [1,2]. The rise of multidrug-resistant tuberculosis (MDR-TB) cases represents a serious threat to TB control in high prevalence countries and it is the main reason for immediate concern. The World Health Organization estimates that 50 million people are infected with resistant Mtb. In 2000, 273 000 MDR were among the 8·7 million new diagnosed TB cases [3]; in Brazil, around 100 000 new TB cases are detected per year from which 0·5–1% are multidrug-resistant, and more than 60% are known to be concentrated in the states of Rio de Janeiro and São Paulo [4].
A Th1-type immune response, characterized by elevated production of interferon (IFN)-γ and interleukin (IL)-12, is essential to mount a protective immunity against Mtb [5,6]. In addition, tumour necrosis factor (TNF)-α, a proinflammatory cytokine, seems to play an important role in control of mycobacterial replication both in mice and humans [7,8].
The complete sequencing of the Mtb genome [9] has led to the screening and definition of immunodominant gene families, one of which is the esat-6 gene family that has been demonstrated to encode several immunodominant molecules. ESAT-6, the early secreted antigenic target, is essentially present in pathogenic mycobacteria and absent in all M. bovis BCG strains and most environmental mycobacteria [10]. The immune response to ESAT has been detected in vitro in TB patients but not in Bacille Calmette—Guérin (BCG)-vaccinated donors obtained from non-endemic areas [11,12]. Consequently, the use of ESAT-6 as a marker of M. tuberculosis infection has been proposed. Further analysis on the in vitro immune response to ESAT-6 in endemic countries has showed that despite the high rate of responders (varying from 30% to 80%) found among controls with non-TB contact history [13,14], levels of IFN-γ induced by ESAT-6, but not by antigen 85B (Ag85B) or PPD, were significantly lower in Brazilian community controls when compared to treated TB, pleural or cavitary TB [15]. MDR-TB is often accompanied by host immunosuppression [16,17] and recent studies have demonstrated decreased IFN-γ concomitant to enhanced IL-10 and IL-18 production in response to PPD [18]. Nevertheless, no information is available on the patients’ response to recombinant proteins of Mtb, in particular to rESAT-6.
In the present study, T cell responses to Mtb antigens were investigated in Brazilian MDR-TB patients and compared to the response of non-MDR-TB and healthy controls who are tuberculin skin test (TST) negative. Additionally, levels of the inflammatory cytokine TNF-α were also assessed in patients’ sera before and during chemotherapy. It is demonstrated that the overall immune response to Mtb antigens is decreased in MDR-TB versus NR-TB patients. Moreover, TNF-α was detected in the circulation in both groups of patients, mainly on those who presented with active disease.
Materials and methods
Studied population
Patients with MDR-TB and non-resistant pulmonary TB (NR-TB) were referred from the Ambulatory Service of the Health Unit, Reference Center for Tuberculosis (CRPHF), and from the District Hospital Raphael de Paula e Souza, Rio de Janeiro, Brazil. MDR patients showed culture positive isolates resistant to at least isoniazid (INH) and riphampin (RMP), and to any other specific anti-TB drug from the standardized regimens, as established according to the Lowenstein Jensen and BACTEC system, with proportional methods for sensitivity patterns. A total of 28 MDR patients (16 males and 12 females), mean age (± s.d.) of 35·3 ± 11·6 (ranging from 19 to 60 years), were enrolled into the study (Table 1). Management of MDR relied on treatment with at least three drugs to which an isolate is susceptible.
Table 1. Clinical feature of tuberculosis (TB) patients and controls.
| Treatment status | ||||
|---|---|---|---|---|
| Group (n) | Male/female | Treated | Untreated | Mean age ± s.d. (years) |
| MDR-TB (28) | 16/12 | 0 | 28† | 35·3 ± 11·6 |
| NR-TB (77) | 50/27 | 40* | 37* | 33 ± 12 |
| Controls | 13/10 | – | – | 30 ± 12 |
| TST(–) (23) | ||||
MDR-TB = multidrug-resistant TB patients; NR-TB = patients with non-resistant TB;
MDR-TB were all untreated patients when referring to the alternative TB therapy.
A total of 24 treated and 26 untreated NR-TB were evaluated for their in vitro
All MDR cases showed pulmonary disease and had failed to respond to current treatment for tuberculosis. All patients were assayed before initiation of alternative chemotherapy and nine individuals were recruited again while on treatment (after > 4 months of alternative treatment).
As for NR-TB, diagnosis of pulmonary TB (n = 77) was performed according to the criteria established by WHO and the Ministry of Health, Brazil. Patients (50 males and 27 females; mean age 33 ± 12 years, ranging from 14 to 60) with pulmonary TB diagnosis was defined by at least one sputum-positive smear by Ziehl—Neelsen staining or a sputum culture positive for M. tuberculosis, respiratory symptoms for ≥ 4 weeks, and suggestive lesions on the chest X-ray [15]. None of the treated TB patients in this category had so far presented any evidence of anti-TB chemotherapy failure.
Within the group of NR-TB (Table 1), patients were grouped according to their treatment stage, as untreated (< 7 days of chemotherapy) and treated patients (≥ 30 days of chemotherapy). In addition, seven of the NR-TB patients from the untreated group were assessed again while on treatment for TB (ranging from 2 to 6 months of therapy). All patients (MDR and NR) were tested negative for HIV [enzyme-linked immunosorbent assay (ELISA)] and had been treated with anti-tuberculosis chemotherapy regimens in accordance to the recommendations of the Ministry of Health. Individuals gave permission for blood sampling according to approval of the Ethical Committees of the Centers involved. None of the patients required hospital admission to receive treatment or clinical assistance. Pregnant or breast-feeding women and individuals with co-morbidity, such as diabetes and cancer, were not included.
The control group (n = 23; 13 males and 10 females) comprised healthy unrelated donors (mean age 30 ± 12, range 21–42) with negative TST (induration < 5 mm), who had no knowledge of prior contact or history of TB. In addition, six healthy individuals with positive TST reactions (≥ 10 mm) were included.
Mycobacterial antigens
M. tuberculosis recombinant ESAT-6, antigen 85B (Ag85B) and synthetic peptides, 20 amino acids (aa) long with a 10-aa overlap, were manufactured as described elsewhere [19]. Peptides were dissolved in phosphate-buffered saline (PBS)/1% dimethyl sulphoxide (DMSO; Sigma Chemical Co., St Louis, MO, USA) and, for stimulation of cultures, they were used as a mixture of nine overlapping peptides (PeptMix) corresponding to the full-length ESAT-6 protein in which each peptide was included at a concentration of 0·5 µg/ml. Proteins generated from the vector were used as negative control. PPD RT23 was purchased from the Statens Serum Institute (Copenhagen, Denmark). rESAT-6 and PPD were used in the in vitro assays at a final concentration of 5 µg/ml; Ag85B was used at 2·5 µg/ml and the mitogen phytohaemaglutinin (PHA; Gibco Laboratories, Gaithersburg, MD, USA) at 1%. All reagents and antigens were shown to contain < 0·1 U/ml endotoxin when assayed through the Limulus amebocyte lysate assay (Whittaker Bioproducts, Walkersville, MD, USA).
Cell culture and stimulation
After written and informed consent, heparinized venous blood was obtained from all individuals and peripheral blood mononuclear cells (PBMC) isolated by Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ, USA) density centrifugation. A total of 2 × 105 PBMC/well were cultured in 96-well round-bottomed plates (Costar Corporation, Cambridge, MA, USA) as described [15] when antigens or mitogen were added to the wells in duplicate. After 5 days, supernatants were recovered and kept frozen (−20°C) for further assay. Control wells comprised cells cultured in medium alone (with equivalent amounts of DMSO). For determination of cytokine levels in the circulation, blood was obtained from all patients and controls, and allowed to clot for 30 min before centrifugation. Serum samples were harvested and kept frozen until use.
Cytokine detection
Concentration of IFN-γ and TNF-α were determined by using commercial specific ELISAs, processed according to the manufacturer's specifications (R&D Systems Inc., Minneapolis, MN, USA). Cytokine levels were expressed as pg/ml of protein and IFN-γ values in the experimental wells were already subtracted from the values obtained in the control wells. Cut-off value for IFN-γ was 100 pg/ml, and for TNF-α 45 pg/ml, which corresponds to 3 s.d. above mean values found in the control group. The detection limit of the assay was 8 pg/ml and 4 pg/ml, respectively.
Statistical analysis
Results are reported as mean ± standard error of the mean (s.e.m.) for each group of experiments. For comparison of data among patients and controls, the non-parametric Kruskall—Wallis and Mann—Whitney tests were used whenever appropriate. Mann—Whitney and one-way anova with Dunnett's post-test was performed using GraphPad Prism version 4·0 for Windows. The statistical significance level adopted was P < 0·05.
Results
IFN-γ production in response to ESAT-6 and to its peptide mixture is decreased in patients with MDR-TB
Production of IFN-γ in response to rESAT-6 was evaluated in vitro in the PBMC from 20 patients with MDR-TB and compared to 50 non-resistant (NR) Brazilian TB patients and 23 healthy tuberculin negative controls (HC TST-). According to the previous published data [15], the rate of response to the recombinant antigen was found to be similar in all groups tested. For the MDR-TB patients, a total of 55% (11 of 20) responders was observed as compared to 60% (30/50) in the NR-TB and 56·5% (13/23) in the controls. Even though the rate of response was similar among the groups, mean positive IFN-γ values in response to ESAT-6 (Fig. 1a) were significantly lower (P < 0·05) in the MDR-TB group (IFN-γ± s.e.m. = 553 ± 111 pg/ml) when compared to the NR-TB (1179 ± 163 pg/ml), but similar to those in the HC (412 ± 66 pg/ml; P > 0·05). Significant differences (P < 0·03) were also detected when MDR-TB and tuberculin skin test-positive healthy donors (HC TST+) were compared (1553 ± 158 pg/ml).
Fig. 1.
Decreased in vitro response to ESAT-6 in multidrug-resistant tuberculosis (MDR-TB) patients. (a) Levels of interferon (IFN)-γ were assessed by enzyme-linked immunosorbent assay (ELISA) following ]in vitro stimulation of peripheral blood mononuclear cells (PBMC), obtained from multidrug-resistant (MDR; n = 20) and non-resistant (NR) TB (n = 50) patients, with ESAT-6, and compared to healthy controls (HC; n = 23). Values in the graphic represent mean positive IFN-γ levels ± s.e.m. *Statistical significance (P < 0·05) was noted between NR-TB versus MDR patients and **controls (P = 0·01). (b) The in vitro response to the PeptMix of ESAT-6 was assessed in NR (n = 49), MDR-TB (n = 17) and controls (n = 18). *Significant differences when compared to MDR and HC groups. Cytokine amounts in control unstimulated cultures were mainly undetectable unless indicated otherwise.
Within the group of NR-TB patients, patients were grouped according to their treatment stage, 26 being untreated and 24 treated. For MDR-TB, patients were considered as treated patients when related to the conventional TB therapy, but were all untreated when related to alternative therapy. A significant difference (P < 0·05) in mean IFN-γ was found between MDR-TB and treated NR pulmonary TB (1241 ± 187 pg/ml) but not between MDR-TB and untreated NR-TB patients (1124 ± 263 pg/ml).
Previous data have demonstrated that the response to ESAT-6 and to the mixture of all overlapping peptides is almost identical [19]. In order to determine how MDR-TB patients responded in vitro, PBMC from the different patients’ groups were also stimulated with a peptide mixture spanning the complete sequence of ESAT-6 (PeptMix) as a substitute for the whole protein. A total of 47% of the MDR-TB patients (eight of 17) showed a positive response to the PeptMix as compared to 79·6% in the NR-TB (39/49) and to 55·5% in the HC (10/18). As demonstrated in Fig. 1b, and similar to the response to ESAT-6, mean positive IFN-γ levels were noted to be significantly (P < 0·05) different between MDR-TB (384 ± 137 pg/ml) and NR-TB (1425 ± 256 pg/ml), but not to HC (481 ± 78 pg/ml), and between NR-TB versus HC (P = 0·04). As a positive control for the T cell response, it was observed that the rate of response to the mitogen PHA was not affected in the MDR-TB group when compared to the other groups (not shown).
Low rate of response to PPD and Ag85B was also detected in MDR-TB
When responses to PPD (n = 17) and Ag85B (n = 15) were evaluated, only 23·5% of the MDR-TB patients (four of 17) were found to respond to PPD compared to 95% responders in the NR-TB and 90% in the healthy control group (HC TST-). Accordingly, the rate of response to Ag85B was 33·3%, 88% and 90%, respectively (Fig. 2a). When the amount of IFN-γ in the cultures was compared (Fig. 2b), MDR-TB patients secreted lower IFN-γ values (864 ± 96 pg/ml) in response to PPD (P < 0·05) than the other groups (untreated NR = 1646 ± 281 pg/ml; treated NR = 1577 ± 150 pg/ml; or TST+ controls = 2192 ± 573 pg/ml), while they were not different when compared to HC TST- (1128 ± 249 pg/ml). The response to the Ag85B followed a similar trend but differences were not statistically significant (Fig. 2b).
Fig. 2.
Multidrug-resistant (MDR) patients show diminished response to PPD and Ag85B in vitro. (a) Interferon (IFN)-γ production in MDR-tuberculosis (TB) patients was assayed following stimulation of peripheral blood mononuclear cells (PBMC) with ESAT-6, PPD and Ag85B. Bars in the graphic represent the percentage of response to the recombinant proteins in each patients’ group. Except for ESAT-6, a lower rate of response to PPD and Ag85B is noted in the MDR group compared to non-resistant (NR)-TB and healthy controls (HC). (b) Values in the graphic indicate mean positive IFN-γ± s.e.m. in response to PPD and Ag85B in each group of individuals tested. *Indicates significant lower response (P < 0·05) when compared to the NR pulmonary TB.
Detection of enhanced TNF-α levels in the sera of TB patients
TNF-α is a proinflammatory cytokine which production is up-regulated by other cytokines, such as IFN-γ, both in vivo and in vitro[20]. We therefore measured TNF-α in the serum of patients with MDR-TB and compared to the other groups. A total of 28 MDR, 77 NR-TB (40 treated, 37 untreated) and 20 controls (HC TST-) were assayed. Evaluation of the inflammatory response in the MDR group showed enhanced TNF-α values (mean ± s.e.m. = 43·8 ± 9 pg/ml) in 35·7% of the patients (10 of 28, Fig. 3). In the control group, all individuals showed low to undetectable TNF-α (mean 11 ± 2·5 pg/ml, ranging from 0 to 34 pg/ml).
Fig. 3.
Tumour necrosis factor (TNF)-α levels are enhanced in the sera of tuberculosis (TB) patients. Serum TNF-α was assayed by specific enzyme-linked immunosorbent assay (ELISA). Each symbol in the graphic represents one individual. Multidrug-resistant (MDR) (n = 28), untreated (n = 37), treated (n = 40) non-responsive (NR)-TB and healthy controls (HC) (n = 20) were tested. Horizontal bars are mean TNF-α in each group. Significant differences were observed when compared to HC (P < 0·05, for treated NR-TB; P < 0·03, for MDR and untreated NR).
A total of 17·5% of the treated patients (seven of 40) were positive for TNF-α in the circulation vs. 45·4% detected in the untreated group. Mean TNF-α was 33·8 ± 6·1 and 82·8 ± 15 pg/ml, respectively. Because of the large interindividual variations, significant differences were observed only when patients’ groups were compared against the HC (Fig. 3). Moreover, when mean positive TNF-α values were considered, a significant difference (P = 0·043) was also noted between MDR-TB (87·9 ± 14 pg/ml) and the untreated NR group (160·4 ± 23 pg/ml).
Evaluation of TNF-α levels in the circulation was also performed in the same individuals at diagnosis and during treatment (> 2 months of chemotherapy) in 11 MDR-TB and seven NR-TB patients (Fig. 4a and inset). Lower serum TNF-α was detected in most individuals at the second visit, except in three patients (two MDR and one NR): in one individual (MDR), although TNF-α was slightly higher at the second evaluation, values were still below the cut-off; for the other two patients, TNF-α remained/turned to positive in the follow-up. In addition, increased in vitro IFN-γ response to ESAT-6 was noted in most of the five MDR-TB patients tested (Fig. 4b).
Fig. 4.
(a) Follow-up evaluation of serum tumour necrosis factor (TNF)-α was assessed in multidrug-resistant (MDR) (n = 11) and non-resistant tuberculosis (NR-TB) (n = 7; inset) patients, before and during chemotherapy. Lower TNF-α was observed in the circulation in all patients but in only two individuals (one MDR and one NR), TNF-α turned to remain positive during the follow-up. (b) Follow-up evaluation of in vitro IFN-γ response to ESAT-6 assayed in the peripheral blood mononuclear cells (PBMC) of MDR patients (n = 5), before and during chemotherapy. In most patients, high IFN-γ was present during the follow-up.
Relationship of cytokine response and clinical features of MDR-TB
Clinical features of MDR-TB group, as detailed in Table 2, showed the overall resistance pattern on 25% of the patients with resistance to three drugs, 32·2% to four drugs and 42·8% to five drugs. Chest radiographs were compatible with presence of lesions in both lungs in 89·3% (25 patients) and cavitation in 71·4% of the cases (20 patients). Positive cultures for Mtb were +28·6%, ++35·7%, and +++35·7% (Table 2). No correlation between cytokine levels and period of disease or resistance profile or culture growth was detected.
Table 2. Clinical profile of multidrug-resistant tuberculosis (MDR-TB) patients enrolled into the study.
| MDR-TB patients | Sex | Culture severity† | DIa | Pulmonary lesionb | Cavitation | Drug-resistancec |
|---|---|---|---|---|---|---|
| 1 | F | + + | 2 | 2 | Yes | 3 |
| 2 | F | + + + | 4 | 2 | No | 3 |
| 3 | M | + + + | 2 | 2 | No | 4 |
| 4 | M | + + | 1 | 1 | Yes | 3 |
| 5 | M | + + + | 4 | 2 | Yes | 4 |
| 6 | M | + + + | 3 | 1 | No | 5 |
| 7 | F | + + + | 2 | 2 | No | 3 |
| 8 | F | + + + | 1 | 2 | Yes | 4 |
| 9 | F | + + + | 2 | 1 | Yes | 4 |
| 10 | F | + + | 1 | 2 | Yes | 3 |
| 11 | M | + + | 1 | 2 | Yes | 5 |
| 12 | M | + | 4 | 2 | No | 5 |
| 13 | F | + | 3 | 2 | No | 5 |
| 14 | M | + | 1 | 2 | Yes | 5 |
| 15 | M | + + + | 1 | 2 | Yes | 5 |
| 16 | F | + + | 1 | 2 | Yes | 3 |
| 17 | F | + | 1 | 2 | Yes | 4 |
| 18 | M | + + | 2 | 2 | No | 4 |
| 19 | F | + | 2 | 2 | Yes | 5 |
| 20 | F | + + | 1 | 2 | Yes | 3 |
| 21 | M | + | 1 | 2 | No | 4 |
| 22 | M | + | 2 | 2 | Yes | 4 |
| 23 | M | + + | 2 | 2 | Yes | 4 |
| 24 | F | + + + | 5 | 2 | Yes | 5 |
| 25 | M | + | 2 | 2 | Yes | 5 |
| 26 | M | + + | 2 | 2 | Yes | 5 |
| 27 | M | + + + | 3 | 2 | Yes | 5 |
| 28 | M | + + | 3 | 2 | Yes | 5 |
Duration of infection (years); M = male; F = female.
Semi-quantitative scale for mycobacterial culture growth on agar plates: +, 20–100 colonies; ++, >100 colonies (isolated colonies); +++, colonies with confluence.
Pulmonary lesions: 1 = one lung; 2 = both lungs.
Drug-resistance profile: numbers indicate the total amount of drugs that patients are resistant to. Drugs are defined here as: Rifampin, Isoniazid, Pyrazinamide, Ethambutol and Streptomycin.
As related to the occurrence of cavitation, 64·3% of the patients who showed lung cavitation were positive for IFN-γ as opposed to 33·3% detected among patients without cavitation. When related to TNF-α, figures were 40% and 25%, respectively (Table 3). From all individuals who presented with elevated serum TNF-α levels (n = 10), 100% presented with pulmonary lesions in both lungs and 80% showed lung cavitation. Similar data were found among the IFN-γ positive individuals (91% and 82%). Moreover, concordance between the enhanced in vitro IFN-γ and serum TNF-α occurred in 50% of the patients. When NR-TB patients were evaluated, enhanced serum TNF-α was detected in 36·4% of the individuals who presented lung cavitation and in 20% of the patients without cavitation (Table 3). All NR-TB patients presented pulmonary lesion in one lung.
Table 3. Cytokine response and clinical features of tuberculosis.
| Cavitation | Pulmonary lesion | ||||
|---|---|---|---|---|---|
| Patients | Cytokine response | Yes | No | 1 | 2 |
| n = 20 | n = 8 | n = 3 | n = 25 | ||
| MDR-TB | TNF-α | 40%* | 25% | 0 | 40% |
| (n = 28) | IFN-γ | 63·3% | 33·3% | 50% | 55·5% |
| n = 18 | n = 32 | n = 50 | n = 0 | ||
| NR-TB | TNF-α | 36·4% | 20% | 45% | n.d. |
| (n = 50) | IFN-γ | 73·3% | 54·5% | 55% | n.d. |
Numbers in parentheses indicate total number of individuals in each group.
Represents percentage of individuals in each clinical situation who present positive cytokine values. Pulmonary lesions = when one or two lungs were affected. IFN-γ: interferon-γ; TNF-α: tumour necrosis factor-α; MDR-TB: multidrug-resistant tuberculosis; NR-TB: non-resistant tuberculosis
Discussion
The mechanism of immunosuppression at play in MDR-TB is not understood. Some hypotheses refer to modulation at the level of T cell and/or macrophage apoptosis during M. tuberculosis infection [22–24], or to an enhanced production of IL-10 in such patients [16,24]. Previously published data have already demonstrated poor PBMC proliferation and IFN-γ secretion in patients with active TB and advancing disease [25–27]. Accordingly, elevated IL-10 and IL-18 production together with lower IFN-γ were detected in vitro in response to PPD in MDR-TB [18].
This study confirms and extends previously published observations [16, 18, 21] on the in vitro low responsiveness of MDR-TB patients (evidenced by low IFN-γ production against PPD, M. tuberculosis, 30 kDa antigen) to the RD1 region encoded immunodominant antigen ESAT-6. In line with this, the response of MDR-TB patients to the overlapping peptides mixture of ESAT-6 was also lower when compared to NR-TB. Our own previous work [15] had demonstrated a differential IFN-γ response to ESAT-6 between TB patients and TST negative healthy donors (HC). Even so, and probably as a result of the depressed responses found in MDR-TB patients, the in vitro response to ESAT-6 did not discriminate between MDR-TB and HC in an endemic TB area.
It is our hypothesis that the specificity profile of responses to a panel of antigens might differentiate MDR-TB in an endemic TB setting. In conjunction with the lower response to ESAT-6, MDR-TB patients showed lower in vitro responses to PPD and Ag85B, whereas HC, although being low responders to ESAT, produced high IFN-γ when stimulated with PPD or Ag85B. Moreover, NR-TB patients were highly responsive to all three antigens. Further differences in the profile of in vitro responses to ESAT-6 have been demonstrated recently to identify contacts of tuberculosis patients who are at risk of developing disease [28], as opposed to healthy controls from the same endemic TB area. Such major differences seem to rely on the expression of regulatory cytokines, such as IL-4δ2, which was highly expressed among the ESAT-6 positive controls [29].
TNF-α is thought to exert a major role, both in inflammation as well as in protection against mycobacterial infection [7,30]. In humans, the participation of TNF-α was emphasized by the detection of TB reactivation in rheumatoid arthritis patients who were treated with anti-TNF antibodies [8,31]. Production of TNF-α was evaluated in the sera of TB patients and controls. The detection of IFN-γ and TNF-α, particularly in MDR-TB patients who presented with deteriorating clinical features, is in agreement with the description of Cardoso et al. [15], who reported enhanced IFN-γ release in response to ESAT-6 and the occurrence of cavitation in pulmonary (NR) TB. Despite the fact that all MDR-TB patients who had TNF-α in their sera showed lesions in both lungs and 80% presented with cavitation, mean positive TNF-α values were still lower than those found in the untreated pulmonary NR-TB. One possible explanation could be the patients’ ability in the latter group to produce higher amounts of IFN-γ. Enhanced secretion of TNF-α has been demonstrated to be up-regulated by IFN-γ, and also following neutralization of IL-10 [20,32]. On the other hand, it was demonstrated that concentration of serum cytokines did not necessarily parallel the in vitro response obtained from M. tuberculosis-stimulated cultures [33]. Indeed, our own data showed a 50% correlation between the two measurements in MDR cases.
When TB patients were assayed during treatment, decreasing amounts of TNF-α were observed in both groups (NR- and MDR-TB), in addition to high IFN-γ response, during the follow-up. These data are in accordance with previous reported work describing the recovery of immune response to various mycobacterial antigens, including ESAT-6 [34,35], and support the idea that monitoring antigen specific cellular responses in TB patients may provide an early indication of drug resistance [35]. On the other hand, persistent IFN-γ may account for the maintenance of tissue injury that follows bacterial persistence in the lungs.
To our knowledge this is the first time that a major relationship is described between such immunological parameters and clinical conditions in MDR-TB. The present data indicate that T cells from MDR patients respond to mycobacterial antigens in vitro at a lower extent when compared to NR-TB and that inflammatory response is also exacerbated. As a whole, patients’ clinical status may be the resultant of a predicted response of the host induced in vivo by the adapted mutated bacteria as one possible mechanism to subvert human immunological host defence or human interventions such as anti-TB treatment [36]. Altogether, the patients’ response suggests, as stated above, the major role that cytokines may play in perpetuating pulmonary tissue damage. Hence, cytokine response in resistant TB needs to be viewed clearer and a careful monitoring in patients’ clinical evolution and immunological parameters still needs to be performed in higher numbers of individuals, once disease-associated immunosuppression may prevent IFN-γ immunodiagnosis of more advanced TB.
Acknowledgments
The authors are grateful to E. B. Oliveira and D. S. Carvalho for technical assistance and P. Cabello for help with statistical evaluation. We also thank the nursing staff of the TB ward for helping with patient recruitment and sample collection. This work was supported by the EEC (INCO-DC), grant number ERBIC 18CT 980377, the Netherlands Leprosy Relief Foundation (NLR) and the Scientific Research for the Tropics (WOTRO) Fund of the Dutch Organization for Scientific Research (NWO). A. R. Fortes and K. C. Pereira are supported by a Student Fellowship programme (CAPES and CNPq, Brazil). M. M. Ribeiro-Carvalho is a Research Visitor at the Leprosy Laboratory, sponsored by an associated Research Program FIOCRUZ/FAPERJ.
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