Clinical Immunology (2011) 138, 50–59
available at www.sciencedirect.com
Clinical Immunology
www.elsevier.com/locate/yclim
Specific cytokine patterns of pulmonary tuberculosis
in Central Africa
Johannes Nemeth b,c,d , Heide-Maria Winkler a,b , Lucas Boeck f ,
Ayola A. Adegnika b,c , Ella Clement e , Toung M. Mve e ,
Peter G. Kremsner b,c , Stefan Winkler a,b,⁎
a
Department of Internal Medicine I, Division of Infectious Diseases and Tropical Medicine,
Medical University of Vienna, Austria
b
Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon
c
Institute for Tropical Medicine, Department of Parasitology, University of Tübingen, Germany
d
Department of Medicine, University Hospital Basel, Basel, Switzerland
e
Hôpital de Nkembo, Libreville, Gabon
f
Clinic of Pneumology, University Hospital Basel, Basel, Switzerland
Received 19 September 2009; accepted with revision 14 September 2010
Available online 15 October 2010
KEYWORDS
T cell;
Tuberculosis;
Cytokines;
FoxP3
Abstract Different cytokines have been suggested to be involved in the pathogenesis of
pulmonary tuberculosis (TB). The frequencies of Mycobacterium tuberculosis (MTB) specific CD4+
and CD8+ T cells, CD4+CD25+ Forkhead Box Protein (FoxP)3+ T cells, interleukin (IL)-6, interferon
(IFN)-γ, Tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β and IL-10 were
assessed in HIV-negative, pulmonary tuberculosis (TB) patients (n = 30) and in healthy controls
(n = 23) in Gabon. Peripheral blood mononuclear cells (PBMC) were stimulated with purified
protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6). In patients, a
pronounced pro-inflammatory cytokine response with highly significant increased levels of IL-6
and TNF-α accompanied by increased TGF-β was detectable. Differences in IFN-γ responses
between patients and healthy individuals were less pronounced than expected. FoxP3 expression
did not differ between groups. A distinct cytokine pattern is associated with active pulmonary TB
in patients from Central Africa.
© 2010 Elsevier Inc. All rights reserved.
Introduction
⁎ Corresponding author. Department of Internal Medicine I,
Division of Infectious Diseases and Tropical Medicine, Medical
University of Vienna, Waehringerguertel 18-20, A-1090 Vienna,
Austria. Fax: +43 1404004418.
E-mail address: stefan.winkler@meduniwien.ac.at (S. Winkler).
Tuberculosis (TB) is one of the most important infectious
diseases with nearly 10 million new cases of active TB
occurring every year and about two billion people infected
with Mycobacterium tuberculosis (MTB) globally [1]. The
1521-6616/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.clim.2010.09.005
Specific cytokine patterns of PT in Central Africa
majority of infected individuals never develop active disease
since invading pathogens are controlled by immune system.
Patients undergoing depletion of CD4+ T cells in the course
of human immunodeficiency virus infection or functional
neutralization of tumor necrosis factor (TNF)-α due to antiTNF-α-antibodies are at markedly increased risk to develop
active TB [2,3]. Similarly, disorders of IFN-γ receptors
predispose for severe courses of mycobacterial infections
[4]. Type 1 biased CD4+ T cells play a central role in protection against TB by promoting activation of macrophages
with the production of IFN-γ and TNF-α [5]. Thus, it has been
suggested that a defective type 1 activation is a possible
mechanism to explain the progression of latent MTB infection
to active disease.
However, studies assessing IFN-γ production capacity in
active TB yielded contradictory results. Decreased IFN-γ
production after stimulation with mycobacterial antigens
was reported in TB patients, underlining the assumption
that active disease may be associated with defective or
suppressed type 1 responses [6–8]. In contrast, increased
IFN-γ production in peripheral blood as well as at the site of
infection was shown to be a hallmark of active TB, a fact
which can even be used for diagnostic purposes [9–13].
A possible simultaneous up-regulation of inhibitory
cytokines and regulatory T cells could explain this discrepancy. The regulatory capability of T cells has been linked to
the expression of the transcription factor Forkhead Box
Protein (FoxP)-3 [14–16]. Previous studies suggested that
T cell derived over-regulation could promote suppression of
cytokine responses in active TB, allowing thereby progression of disease [17–24].
In the recent study, we assumed that stimulation of T cells
with purified protein derivative (PPD) reflects global cellular
responsiveness in this population from Central Africa with
high prevalence of latent TB, high BCG coverage and endemic non-tuberculous mycobacteria [1]. In contrast, early
secretory antigenic target-6 (ESAT-6) induced cytokine
responses were assumed to be highly specific for MTB.
Thus, this experimental setting enabled us to compare global
cellular responsiveness with highly MTB-specific cytokine
production in patients with active TB and healthy individuals. Moreover, measuring multiple pro- and anti-inflammatory mediators should reflect as closely as possible
the immune network underlying the development of active,
pulmonary TB.
Patients and methods
Patients
This cross sectional study was conducted at the Albert
Schweitzer Hospital in Lambaréné and at the Hospital N´
Kembo, Libreville, Gabon, from January to November 2006.
TB rates in Gabon are expected to be between 100 and
299 per 100,000, according to current WHO estimation [1].
Besides MTB, other mycobacterial infections such as Mycobacterium ulcerans are endemic. BCG vaccination is free of
charge and recommended for all neonates. Vaccine coverage
is high in our study area.
Adult patients with active pulmonary TB seeking medical
care at the Albert Schweizer Hospital in Lambarènè and the
51
Hospital N´Kembo as well as healthy volunteers were invited
to participate within the study.
The diagnostic laboratories of the Albert Schweizer
Hospital and the Hospital N´Kembo were screened on a
daily basis. If a newly detected, positive smear was reported,
an investigator approached the respective patient to ask
whether he or she was willing to participate in the study.
Active pulmonary TB was defined by sputum-positive
smears by Ziehl–Neelsen staining, chest-X rays consistent
with TB, and clinical history, including at least three of the
following symptoms: night sweat, cough lasting for more
then 3 weeks, unintended weight loss, malaise, fever, lassitude, known exposure to open tuberculosis. If the patient
was not clinically ill according to the stated criteria or had
no radiographic changes consistent with TB, the patient was
not included. Smear negative patients with the clinical
diagnosis of active TB were not included.
Exclusion criteria of both, patients and healthy subjects,
were age younger than 16 years (1), HIV antibodies detectable in sera (2), pregnancy (3) and any other acute illness (4)
as assessed by clinical examination. Noteworthy, healthy
volunteers were also excluded if they had a history of active
TB or if somebody with open TB was living in the same
household.
At inclusion participants were screened for malaria and
HIV (Determine HIV-1/2® test, Abbott Laboratories®, Tokyo,
Japan). Demographic data and clinical history were recorded
at inclusion and 45 ml blood was drawn from a cubital vein.
No further follow up was conducted in the course of this
study. Written informed consent was obtained from all
participants. The study protocol was approved by the ethics
committee of the International Foundation for the Albert
Schweitzer Hospital in Lambaréné.
Methods
Flow cytometry for intracellular staining of cytokines
Peripheral blood mononuclear cells (PBMC) were isolated
from heparinized blood by ficoll-diatrizoate centrifugation
and plated out in 24-well plates (2 × 106 cells per well). Cells
were cultured in 1 ml ultra culture medium (UCM) (Bio
Whittaker, Walkersville, MD) supplemented with L-glutamine
(2 mM/l; Sigma, St. Louis, MI), gentamicin (170 mg/l; Sigma)
and 2-mercaptoethanol (3.5 μl/l; Merck, Darmstadt,
Germany) for 18 h at 37 °C in 5% CO2 and stimulated with
purified protein derivate PPD (Statens Serum Institute,
Copenhagen, Denmark) at a final concentration of 10 μg/
ml or with ESAT6 (Statens Serum Institute, Copenhagen,
Denmark) with a final concentration of 5 μg/ml. In order to
amplify TCR signalling and to facilitate the initial phase of Tcell activation, the co-stimulatory MAb CD28 (Pharmingen
San Diego, CA) was added at a final concentration of 5 μg/ml.
Brefeldin A (10 μg/ml final concentration, Sigma) was added
after 6 h to block protein secretion. After 18 h, cells were
harvested on ice, washed twice in phosphate-buffered saline
(PBS), and fixed with 2% formaldehyde (1 ml per 2 × 106 cells)
for 20 min. After two additional washes in PBS, the cells were
re-suspended in Hank's balanced salt solution (supplemented
with 0.3% bovine serum albumin and 0.1% sodium-azide). The
cells were washed twice with PBS and made permeable with
saponin (0.1%; Sigma), re-suspended with 50 μl of saponin
52
buffer diluted antibodies and incubated for 25 min in the
dark. The following monoclonal antibodies were used: antiCD4, PerCP and APC labelled, anti-CD8, PerCP labelled
(Becton and Dickinson, Mountain View, CA); anti-CD25, APC
labelled (Becton and Dickinson); anti-FoxP3, phytoerythrin
(PE) labelled (eBioscience, San Diego, CA); anti-TGF-β, PE
labelled (IQProducts, Groningen, The Netherlands); MAb IFNγ (clone: B 27), fluorescein-isothiocyanate (FITC) labelled;
MAb IL–2 (MQ1-17H12), PE labelled; IL-10 (JES3-9D7), PE
labelled; TNF-α (MAB-11), PE labelled (all Becton and
Dickinson).
Four-color staining was performed, and at least 105
cells were analyzed on a FACS-Calibur (Becton Dickinson)
equipped with a two-laser system (488- and 630-nm
wavelength, respectively). The data were analyzed with
CELL-Quest Pro software (Becton and Dickinson) and results
were expressed as the percentage of cytokine-producing
cells in each population.
Detection of cytokines in plasma and supernatants
Supernatants were collected from wells stimulated for 18 h
as described above but without addition of Brefeldin A and
frozen at −20° until assayed. In both plasma and supernatants IL-6, IL-10, TNF-α, IFN-γ and TGF-β were quantified
using pre-validated commercially available ELISAs (R&D
Systems, Minneapolis, MN). All tests were performed in
duplicate, following the instructions of the manufacturer.
Isolation of CD4+CD25+ and CD4+CD25− cells
PBMC were prepared as described above. CD25high cells
(1–2% of PBMC) were isolated by positive selection from
PBMC with anti-CD25 magnetic microbeads (2 μl per 107
PBMC) (Miltenyi Biotec, Bergisch-Gladbach, Germany), and
separated over an LS column (Miltenyi Biotec, BergischGladbach, Germany). For highest purity the CD25high cells
were applied to a second, freshly prepared LS column and
washed with PBS (including 0.5% BSA, 2 mM EDTA). FACS
analysis proved purity greater than 96%.
FoxP3 gene expression by qPCR
Total RNA was isolated from purified CD4+CD25high and PBMC
cells using TRI REAGENT (Molecular Research Center, Inc.,
Ohio, USA) following the recommendations of the supplier.
500 ng of total RNA was reverse transcribed into first-strand
cDNA using Transcriptor-RT (Roche Diagnostics, Basel,
Switzerland), dNTPs (10 μM each) and random hexamer
primers.
Quantitative RT-PCR was performed by LightCycler
technology using the Fast Start SYBR Green I Kit (Roche
Diagnostics) for amplification and detection. RT-PCR conditions for FoxP3 expression were a 10 min 95 °C denaturation
step, amplification with 60 cycles of 5 min at 95 °C, 5 min at
68 °C and 15 min at 72 °C and finally melting point analysis
in 0.2 °C steps using 10 μM of the following primers: FoxP3_
forward (F), 5'-TCAAGCACTGCCAGGCG-3'; FoxP3_reverse
(R), 5'-CAGGAGCCCTTGTCGGAT-3'. The primer pair span
intron/exon boundaries to minimize amplification of genomic DNA.
In all assays each LightCycler capillary was loaded with
2 μl DNA Master Mix, 2.4 μl MgCl2 (25 mM), 12.6 μl H2O, 0.5 μl
of each primer (10 μM) and 2 μl first-strand cDNA of the
sample to a total volume of 20 μl. All samples were run
J. Nemeth et al.
in duplicate. Target gene expression was normalized to
either β2-microglobulin (β2M) or 5-aminolevulinic acid
synthase (ALAS). Primer sequences of ALAS: F, 5'-CCACTGGAAGAGCTGTGTGA-3'; R, 5'-ACCCTCCAACACAACCAAAG-3'.
RT-PCR efficiency was determined for each primer pair
using cDNA dilutions. Primer specificity was tested with
melting point analysis, gel electrophoresis and subsequently
DNA sequencing.
Statistics
Statistical analysis was performed using a standard statistical package (SPSS 15.0 for Windows; SPSS Inc., Chicago, IL).
The Mann–Whitney U-test was applied for group differences. A
Bonferroni–Holm procedure was used to correct for multiple
comparisons between groups.
Results
Participants
30 patients and 23 healthy individuals were included in
this study. Both groups were comparable regarding age
and gender. Body temperature was significantly higher and
hemoglobin concentrations were significantly lower in TB
patients when compared to healthy controls (p b 0.001 and
p = 0.033, respectively; Table 1).
Levels of circulating cytokines in peripheral plasma
Plasma levels of circulating cytokines except for IL-6 were
similar in patients and healthy subjects. IL-6 plasma levels
(p = 0.012) were significantly increased (corrected for multiple comparisons) in TB patients (Fig. 1: plasma).
Levels of cytokines released in the supernatant
Following cytokines were increased in the supernatant of TB
patients when compared to healthy individuals (corrected
for multiple comparisons): TNF-α, spontaneously secreted in
the control well (p = 0.002) and after ESAT-6 stimulation
(p = 0.002; Fig. 1A). The same pattern was observed for IL-6
(control well: p = 0.012; ESAT-6: p = 0.002; Fig. 1E). TGF-β
secretion was increased among TB patients, both in the
control well as after stimulation (control well: p = b 0.001;
Table 1 Demographic characteristics and results of
laboratory testing for study participants.
Study participants
TB (n = 30)
Sex, number of male/female
Age, median years (range)
Admission temperature
median °C (range)
WBC count 10x9 cells per l
18/12
29 (16–79)
38 (36.5–39.0)*
Hemoglobin concentration,
g/dl
Control
(n = 23)
15/8
28 (20–31)
36.5
(36.1–37.0)
6.6 (3–8.8)
5.0
( 2.8–7.9)
10.2 (7.3–15.3)* 13.3
(8.9–16.9)
Specific cytokine patterns of PT in Central Africa
53
Figure 1 (plasma): Plasma levels of different cytokines in TB patients (n = 30) and controls (n = 21) from Central Africa. (A, B, C, D, E)
Tumour necrosis factor (TNF)-α (A), interleukin (IL)-10 (B), transforming growth factor (TGF)-β (C), interferon (IFN)-γ (D) and IL-6
(E) in supernatants following stimulation with purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6),
respectively. White boxes indicate tuberculosis (TB) patients, grey boxes indicate healthy individuals. Comparability of groups was
analyzed by Mann–Whitney U-test. A Bonferroni–Holm procedure was used to correct for multiple comparisons between groups.
Significant differences are marked by *.
PPD: p = b 0.001; ESAT-6: p = 0.002). Remarkably, TGF-β
secretion appeared not to be influenced by short time
stimulation.
+
+
Frequency of PPD specific CD4 and CD8 T cells
expressing IFN-γ, TNF-α, IL-10, IL-2 and TGF-β
Differences between healthy individuals and patients after
PPD stimulation were minor. TNF-α producing CD4+ T cells
(p b 0.001) and IL-10 producing CD8+ T cells (p = 0.003) were
significantly increased in patients (Figs. 2, 3).
Frequency of ESAT-6 specific CD4+ and CD8+ T cells
expressing IFN-γ, TNF-α, IL-10, IL-2 and TGF-β
After stimulation with the MTB specific antigen ESAT-6
significant differences between patients and healthy individuals were restricted to CD4+ T cells. TNF-α producing CD4+ T
54
J. Nemeth et al.
100
100
101
102
TGFb PE
101
102
TNFa PE
103
103
104
104
100
100
101
101
102
102
TN
NFa PE
103
104
103
104
100
101
102
TGFb PE
103
104
100
101
102
TNFa PE
103
104
Figure 2 Representative two-parameter dot plots indicating the frequency of PPD and ESAT-6-specific CD4+ T cells expressing IFN-γ
and/or TNF-α or TGF-β, respectively. PBMC were incubated with medium alone (A), with PPD (B) and ESAT-6 (C).
cells (p = b 0.001) and CD4+ T cells (p = 0.003) co-producing IL2 and IFN-γ were significantly increased in patients when
compared with healthy individuals. CD4+ T cells co-producing
TNF-α and IFN-γ appeared to be increased in TB patients.
Differences, however, were not significant after correction
for multiple analysis (Figs. 2, 4).
Levels of FoxP3 in PBMCs, CD25high T cells and after
stimulation with mycobacterial antigens
FoxP3 mRNA from PBMCs was assessed in 19 participating
individuals. Target gene expression was normalized to 5aminolevulinic acid synthase (ALAS). No differences between
patients and controls were detected (Fig. 4; Table 2).
Furthermore, CD25high T cells were sorted with magnetic
banding and analyzed for FoxP3mRNA expression. Similarly,
no difference between patients and healthy individuals was
found. FoxP3mRNA appeared to be inducible by stimulation.
However, after correction differences did not reach statistical significance. Expression of FoxP3+ in CD4+CD25+ T cells
was analyzed on a single cell level by flow cytometry after
stimulation with mycobacterial antigens. Again, no significant differences were found between patients and healthy
individuals (Fig. 5B, Table 2).
Discussion
In the present work, we report a distinct cytokine profile
consistent of simultaneously increased TNF-α, IL-6 and TGFβ in a TB patient cohort from Central Africa.
Spontaneous TNF-α secretion as well as MTB specific TNFα production in CD4+T cells was increased in patients with
active TB. TNF-α blocking therapy is a risk factor to reactivate latent TB infection [2], highlighting the role of TNFα in granuloma maintenance during latent TB. The role of
TNF-α during active TB is less clear and might not only be
beneficial. Overproduction of TNF-α could be responsible for
immunopathology and contributive to the progression of
disease [25]. In the recent study-cohort, MTB specific TNF-α
appeared to be highly specific for active TB, a fact which
might be useful for diagnostic purposes in the future.
In contrast, IFN-γ, which is now extensively used for
immune-based diagnostics [12,13], was not significantly
increased in patients when compared to healthy individuals.
After ESAT-6 stimulation only CD4+T cells co-producing IL-2
and IFN-γ were significantly increased in patients. This is in
line with a recent study, which reported increased IL-2 and
IFN-γ co-producing T cells in TB patients [26]. Moreover,
measurement of IL-2 together with IFN-γ has been shown to
increase the discriminatory power of immune based
Specific cytokine patterns of PT in Central Africa
55
Figure 3 Frequency of cytokine expressing CD4+ T and CD8+ T cells obtained after stimulation of peripheral blood mononuclear cells
(PBMC) with purified protein derivative (PPD) in TB patients ( n = 30) and in healthy individuals (n = 23) from Central Africa. White boxes
indicate tuberculosis (TB) patients, grey boxes indicate healthy individuals. Each figure indicates the frequency of the T cell subclass
producing either one cytokine or co-producing the respective cytokine with IFN-γ. Significant differences are marked by *. The Mann–
Whitney U-test was used to test for significance. A Bonferroni–Holm procedure was used to correct for multiple comparisons between
groups.
diagnostics [27]. T cells capable of producing more than one
cytokine were described to be advantageous during human
HIV infection [28,29]. Our data suggest that T cells capable
to produce two cytokines are a marker of active TB, a
finding which is in line with the studies mentioned above
[26,27].
56
J. Nemeth et al.
Figure 4 Frequency of cytokine expressing CD4+ T and CD8+ T cells obtained after stimulation of peripheral blood mononuclear cells
(PBMC) with early secretory antigenic target-6 (ESAT-6) in TB patients ( n = 30) and in healthy individuals (n = 23) from Central Africa.
Boxes (line indicates the median percentage) and whiskers (displaying the range). White boxes indicate tuberculosis (TB) patients,
grey boxes indicate healthy individuals. Each figure indicates the frequency of the T cell subclass producing either one cytokine or coproducing the respective cytokine with IFN-γ. Significant differences are marked by *. The Mann–Whitney U-test was used to test for
significance, a Bonferroni–Holm procedure was used to correct for multiple comparisons between groups.
TGF-β was secreted at higher levels in patients compared
with healthy controls and was poorly influenced by specific
stimulation. The T cell independent TGF-β secretion in TB
patients was not influenced by antigenic stimulation.
Increased levels of TGF-β in the supernatants without an
increased expression of TGF-β within the T cell subsets
suggest that the cytokine is mainly monocyte-derived. PBMCs
contain between 10% and 15% of monocytes, which have
Specific cytokine patterns of PT in Central Africa
Table 2 Induction of FoxP3 after stimulation with MTB
specific antigens on mRNA level.
Induction of FoxP3 mRNA after stimulation
medium
PPD
ESAT-6
patients (n = 8)
controls (n = 10)
1 (0)
1.9 (0.38)
1.4 (0.18)
1 (0)
1.5 (0.14)
0.8 (0.24)
been shown to produce spontaneously TGF-β in peripheral
blood of patients suffering from TB [30]. TGF-β is known as
inhibitor of most effector functions of T cells, limiting the
acquisition of CTL function of CD8+ T cells [31] and impairing
the development of naïve CD4+ T cells to type 1 or type 2
cells [32]. In active TB, inhibition of TGF-β was associated
with increased MTB specific T cell responses [33].
57
IL-6, in contrast, showed an increased spontaneous
production in control wells and was inducible by ESAT-6
stimulation. This expression-pattern was comparable to
the pattern of TNF-α. IL-6 plays a central role during
inflammation in general and, more specifically, during MTB
infection [34]. MTB itself has been shown to induce IL-6 [35],
which, in turn, has been described to impair macrophage
responses to IFN-γ [36].
We report an excess of IL-6, TNF-α and TGF-β spontaneously
secreted in the supernatant, suggesting that these cytokines are
in part or–in case of IL-6–completely produced by monocytes.
This observation suggests that during active, pulmonary TB a
rather unspecific inflammation takes place which is absent in
latent infection. Hence, the combination of MTB specific
cytokines with markers of inflammation could lead to immune
based diagnostics from peripheral blood which is able to
discriminate between latent infection and active disease. In
line, IL-6 together with TNF-α and IFN-γ have recently been
used as markers to monitor TB treatment success [37].
Figure 5 (A) Frequency of PPD and ESAT-6-specific CD4+ T cells expressing CD25 and FoxP3 obtained after stimulation of peripheral
blood mononuclear cells (PBMC) with early secretory antigenic target-6 (ESAT-6) boxes (line indicates the median percentage) and
whiskers (displaying the range). White boxes indicate tuberculosis (TB) patients, grey boxes indicate healthy individuals. The Mann–
Whitney U-test was used to test for significance, a Bonferroni–Holm procedure was used to correct for multiple comparisons between
groups. (B) Representative two-parameter dot plots indicating the frequency of PPD and ESAT-6-specific CD4+ T cells expressing CD25
and FoxP3. PBMC were incubated with medium alone (A) with PPD (B) and ESAT-6 (C). For measurement purposes, CD4+CD25+ T cells
were divided into FoxP3 high and FoxP3 low positive T cells. Upper numbers indicate the percentage of CD4+CD25+ T cells classified as
expressing high FoxP3. The lower number indicates the percentage of CD4+CD25+ T cells with low FoxP3 expression. However, all
analyses were done on total FoxP3, i.e. in this example the following numbers were used for calculation: 0.08%, 0.26%, and 0.21%,
respectively. Table 2: left column displays the mean (range) induction of FoxP3 mRNA after stimulation as measured by PCR. FoxP3
expression was normalized to FoxP3 mRNA levels in the control well. Differences are statistically not different.
58
After PPD stimulation, CD8+T cells producing IL-10 were
significantly increased. Given the very small absolute
amount of CD8+ T cells producing IL-10 the biological
relevance of this finding remains unclear. A link between
T cell mediated anergy through IL-10 during active TB has
been published earlier [38].
We were unable to detect differences of FoxP3 mRNA
expression in peripheral blood in patients with active TB
when compared to healthy individuals. This was shown not
only for PBMCs but also for CD25high T cells. These findings
differ from previous studies, describing increased levels of
FoxP3 in peripheral blood of patients with active TB [17–23].
In line with our findings, a recent study failed to detect
regulatory T cells solely based on their FoxP3 expression [39].
Additionally and in contrast to some of the other studies on
FoxP3 in human TB, we included only patients with open
pulmonary TB [22,23]. Lower FoxP3 levels in open pulmonary
TB compared with non-pulmonary TB have been suggested
earlier [23]. Different clinical manifestations of active TB
appear to have different levels of FoxP3, as a comprehensive
study on miliary TB suggests [40]. The lack of systemic immune suppression and elevated FoxP3 levels could be related
to the inclusion of subjects with non-tuberculous mycobacterial pneumonitis. Mycobacterium avium and Mycobacterium kansasii can cause pulmonary disease indistinguishable
from MTB infection without mycobacterial culture [41]. This
also could explain differences to other studies showing
elevated FoxP3 levels in pulmonary TB.
Summarized, we identified a highly specific cytokine
pattern indicative of active TB in patients Gabon. Surprisingly, FoxP3 was not increased, a finding which does not
support a central role of regulatory T cells in the pathogenesis of active, pulmonary TB. Differences of MTB specific IFNγ levels comparing TB patients and healthy individuals were
less pronounced than expected. However, IFN-γ production
was not suppressed in patients, a fact arguing against a
defective type 1 response during active TB. A significant
amount of IL-6, TNF-α and TGF-β levels was spontaneously
produced by cells from TB patients, reflecting most probably
a systemic, unspecific inflammation. This observation could
provide the rationale for novel immunological approaches to
detect active TB.
Conflict of interest statement
The authors declare no conflict of interest.
Acknowledgments
The investigators want to thank the study participants. Peter
Pongratz is acknowledged for the timely delivery of samples.
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