Altered Ratio of IFN-c/IL-10 in Patients with Drug
Resistant Mycobacterium tuberculosis and HIVTuberculosis Immune Reconstitution Inflammatory
Syndrome
Keira H. Skolimowska1,3, Molebogeng X. Rangaka1, Graeme Meintjes1,3,4, Dominique J. Pepper4,
Ronnett Seldon1, Kerryn Matthews1, Robert J. Wilkinson1,2,3,4, Katalin A. Wilkinson1,2*
1 Clinical Infectious Diseases Research Initiative, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa, 2 MRC
National Institute for Medical Research, London, United Kingdom, 3 Division of Medicine, Imperial College London, London, United Kingdom, 4 Infectious Diseases Unit,
GF Jooste Hospital, Cape Town, South Africa
Abstract
We have described a clinical relationship between HIV-Tuberculosis Immune Reconstitution Inflammatory Syndrome (TBIRIS) and anti-tubercular drug resistance. Here we studied the immune response of TB-IRIS patients from whom a drugresistant (n = 11) or drug-susceptible (n = 25) Mycobacterium tuberculosis (MTB) strain was isolated after presenting with TBIRIS. ELISpot analysis and multiplex cytokine analysis of the supernatant collected from peripheral blood mononuclear cells
stimulated overnight with the heat-killed H37Rv MTB laboratory strain was used. Although there was no statistical
difference in IFN-gamma ELISpot responses between the two groups, the results point towards higher bacterial load in the
drug-resistant patients, possibly due to failed therapy. The ratio between secreted IFN-gamma/IL-10 and IL-2/IL-10 was
significantly lower in TB-IRIS patients in whom the cause of TB was a drug-resistant strain compared to those with a fully
sensitive strain (p = 0.02). Since host immune responses are dependent on the bacterial load, we hypothesise that the
impaired cytokine balance is likely to be caused by the poorly controlled bacterial growth in these patients.
Citation: Skolimowska KH, Rangaka MX, Meintjes G, Pepper DJ, Seldon R, et al. (2012) Altered Ratio of IFN-c/IL-10 in Patients with Drug Resistant Mycobacterium
tuberculosis and HIV- Tuberculosis Immune Reconstitution Inflammatory Syndrome. PLoS ONE 7(10): e46481. doi:10.1371/journal.pone.0046481
Editor: Yoshihiko Hoshino, National Institute of Infectious Diseases, Japan
Received June 11, 2012; Accepted August 31, 2012; Published October 10, 2012
Copyright: ß 2012 Skolimowska et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by the Wellcome Trust (081667, 084323, 088316, 084670) and European-Developing Countries Clinical Trial Partnership (EDCTP)
(060613). KAW is supported by the Medical Research Council, UK. DJP was supported by funding from the United States Agency for International Development
and President’s Emergency Plan for AIDS Relief (PEPFAR) via the Perinatal HIV Research Unit. GM and DJP were also funded in part through a Fogarty International
Center South African TB/AIDS Training Award (NIH/FIC 1U2RTW007373-01A1, U2RTW007370 ICORTA). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: katalin.wilkinson@uct.ac.za
isoniazid, rifampicin, any fluoroquinolone, and at least one of
three injectable second-line drugs) TB is an increasing concern
globally, and the importance in high HIV-1 prevalence settings
has been highlighted [10,11]. Of the nearly 500000 new cases of
multidrug-resistant tuberculosis yearly, only 30000 are detected
and reported; misdiagnosis causes death, nosocomial and
community transmission, and amplification of drug resistance
[12,13]. In South Africa, there are an estimated 13000 cases of
MDR-TB emerging each year, however, nationwide estimates
mask regional variability. Thus, in Khayelitsha, a densely
populated peri-urban township outside Cape Town, where close
to 6000 tuberculosis cases were notified in 2008 (giving an
estimated case notification rate of 1158/100000/year), the burden
of MDR-TB cases was estimated to 51/100000/year, or 4.4% of
the cases notified in that year [14].
We have recently described a relationship between TB-IRIS
and antitubercular drug resistance in a cohort of 100 patients who
were considered likely cases of paradoxical TB-IRIS [9]. The
prevalence of unsuspected drug-resistant TB amongst TB-IRIS
patients was 10.1%, after exclusion of known rifampicin resistance
and alternative opportunistic diseases. The symptoms and signs of
Introduction
Antiretroviral therapy (ART) reduces the risk of tuberculosis
(TB) in HIV-1 infected persons by as much as 80% [1,2]. The
rollout of ART in South Africa has been associated with reduced
TB case notification rates in some communities [3]. However, TB
remains the most significant clinical challenge to the successful
rollout of ART, with a high prevalence of diagnosed and
undiagnosed TB in HIV-1 infected persons starting ART [4],
that can be further complicated by the immune reconstitution
inflammatory syndrome (TB-IRIS) [5]. Two forms of TB-IRIS are
recognized: (1) paradoxical, in patients established on TB
treatment before ART, who manifest with recurrent or new
symptoms and clinical features of TB after initiation of ART; and
(2) unmasking TB-IRIS, defined in patients not on treatment for
TB when ART is started, but who present with active TB within 3
months of starting ART, with heightened intensity of clinical
manifestations and marked inflammatory component to the
presentation [6–9].
Multidrug resistant (tuberculosis resistant to at least isoniazid
and rifampicin), and extensively drug resistant (resistant to
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Immunology of MDR TB-IRIS
(CXCL10), IL-12p40, IL-2; Chemokines MIP-1alpha (CCL3),
MIP-1beta (CCL4), RANTES (CCL5), IL-8 (CXCL8); Th-2 type
IL-4, IL-5, IL-13; and growth factors GM-CSF and IL-15, was
performed in 96-well filter plates, on the Bio-Plex platform (BioRad Laboratories, Hercules, USA), using customized MilliplexTM
kits (MPXHCYTO-60K, Millipore, St Charles, Missouri, USA),
following the manufacturer’s instructions.
TB-IRIS in the context of drug-resistant and drug-sensitive disease
were clinically indistinguishable. It has been reported that patients
with MDR-TB display impaired Th1 type responses and
enhanced regulatory T cell responses compared to patients with
drug-susceptible TB [15]. We found that TB-IRIS is associated
with hypercytokinemia [16], and therefore hypothesised that there
might be a difference in the immune response detectable between
TB-IRIS patients with drug-resistant compared to drug-sensitive
TB. The association between TB-IRIS and heightened type 1
helper T cell responses to a number of Mycobacterium tuberculosis
(MTB) antigens, as well as elevated secretion of various cytokines
has been described [16] [17]. Here we studied the IFN-gamma
ELISpot response to a range of MTB antigens, and 17 cytokines
secreted into the supernatant of peripheral blood mononuclear
cells stimulated overnight with the heat killed laboratory strain
H37Rv of MTB. We describe an altered balance between the Th1
and regulatory responses with decreased Th1 (IFN-gamma and
IL-2) and increased IL-10 cytokine secretion in TB-IRIS patients
in whom the cause of TB was a drug resistant strain compared to
those with a sensitive strain.
Data were analysed using Graphpad Prism 5 Software
(Graphpad Software, Inc.). The normality of samples was assessed
using D’Agostino and Pearson omnibus normality test. Normally
distributed paired samples were compared by using a student’s
paired t-test, while non-parametric paired samples were analysed
using the Wilcoxon-matched pairs test. Unpaired, non-parametric
samples were compared using the Mann-Whitney U test and
unpaired, normally distributed samples were compared using the
unpaired student’s t-test. A p-value,0.05 was considered statistically significant. Data are quoted as median (with interquartile
range, IQR) unless otherwise stated.
Study Population and Methods
Results
Patients
The baseline characteristics of the patients are summarised in
Table 1. Patients were recruited at the time of paradoxical TBIRIS diagnosis, and were all on first line therapy for tuberculosis at
the time of sampling. There was no statistically significant
difference in age, gender, and nadir CD4 count between the
patients who had TB-IRIS caused by drug resistant (MDR or RM)
or fully drug sensitive (FS) MTB isolates. There was a trend
towards higher proportions of patients with previous TB, as well as
longer duration of IRIS symptoms in the group with drug resistant
isolates, although neither of these differences were statistically
significant. C-reactive protein (CRP) was elevated in both groups:
median 151 mg/L (IQR 65-208) in the MDR+RM group, and
124 mg/L (IQR 80-180) in the FS group (p = 0.65).
The IFN-gamma ELISpot response to various MTB antigens
was not statistically different between the patient groups (Figure 1).
The median response to ESAT-6 tended to be higher in the FS
group, with 931 (IQR 168-1887) IFN-gamma SFC/106 PBMC,
compared to 300 (34-1808) in the MDR+RM group, although the
difference was not statistically significant (p = 0.53). The response
to the 38 kDa antigen also tended to be higher in the FS group,
510 (47-1078) SFC/106 PBMC compared to 255 (30-1523) in the
MDR+RM group (p = 0.9). A similar trend towards higher
responses in the FS group to the alpha-crystallins Acr1 (median
727 IQR 141-1340, compared to 322 IQR 137-2076 in the
MDR+RM group) and Acr2 (median 516 IQR 33-1018,
compared to 247 IQR 34-1111 in the MDR+RM group) was
observed (both p.0.5). Responses to PPD and hkH37Rv were not
different between the groups: median 582 (IQR 248-958) vs 785
(IQR 256-1633) to PPD, and 214 (IQR 37-678) vs 133 (IQR 331146) to hkH37Rv in the MDR+RM and FS groups respectively
(p = 0.5 in both cases).
The concentrations of 17 cytokines and chemokines in the
supernatant of PBMC cultures with and without stimulation from
TB-IRIS patients with drug-sensitive (FS, n = 10) and drugresistant (MDR, n = 10) disease are shown in Table 2, with
comparative p values corrected for multiple comparisons using
Bonferroni correction. Immune activation was evident in both
groups of patients, as shown by the significant increase in
inflammatory cytokine concentrations of TNF, IL-1beta, and IL6 (FS group only), upon stimulation of PBMC with hkH37Rv.
Similarly, a significant increase in the concentration of IL-10 was
Statistical analysis
The study site and study population has previously been
described [9]. This prospective observational study was conducted
at GF Jooste Hospital (Cape Town, South Africa), with the
catchment area including Khayelitsha. The study was approved by
the University of Cape Town Faculty of Health Sciences Human
Research Ethics Committee (FHS HREC 337/2004). The study
involved 250 consecutively enrolled patients, of whom 100 were
described previously [9]. All participants provided written
informed consent. Samples for immunological analysis were
available from 8 TB-IRIS patients with multi-drug (MDR)
resistant and 3 TB-IRIS patients with rifampicin mono-resistant
(RM) TB. Cases with fully sensitive (FS) TB, who had samples
available for immunological analysis (n = 25), were included as
controls.
ELISpot analysis
Peripheral blood mononuclear cells (PBMC) were separated
and set up in the IFN-gamma ELISpot assay as described [17].
Antigenic stimuli were endotoxin free and included the Region of
Difference-1 (RD1) encoded ESAT-6 (Rv3875) at a final
concentration of 10 mg/ml. Additional antigens included alphacrystallins Acr1 (encoded by Rv2031c) and Acr2 (encoded by
Rv0251c), as well as the MTB cell wall associated 38 kDa protein
(encoded by Rv0934), all at 10 mg/ml. Purified Protein Derivative
(PPD, SSI Denmark) was used at 5 mg/ml. The heat killed (hk)
laboratory strain of MTB H37Rv was used to stimulate cells at a
multiplicity of infection of 1 bacillus: 1 cell (MOI = 1:1, 200000/
well). No antigenic stimulus was used as negative control, while the
positive control was phytohemagglutinin (PHA, Sigma-Aldrich,
10 mg/ml). Results are expressed as IFN-gamma spot forming cells
per million (SFC/106) PBMC.
Multiplex cytokine analysis
Additional PBMC cultures were set up with MTB hkH37Rv
(MOI = 1:1) as described for 24 hours, and the supernatants from
stimulated and unstimulated cultures were collected and cryopreserved for future batched cytokine analysis [16]. These supernatants were available from 7 MDR, 3 RM and 10 FS cases.
Measurement of the pro-inflammatory cytokines TNF, IL-1beta,
IL-6; anti-inflammatory IL-10; Th1-type IFN-gamma, IP-10
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Immunology of MDR TB-IRIS
Table 1. Baseline characteristics of the patients.
TB-IRIS with drug resistant
MTB isolate (MDR1 or RM2)
TB-IRIS with fully
sensitive (FS) MTB
isolate
p value
Number of cases
11
25
N/A
Median age (years, IQR)
28 (26–34)
30 (24–36)
0.93
Gender (female/male)
7F, 4M
14F, 11M
0.73*
Nadir CD4 count (cells/ml, IQR)
50 (17–94)
55 (33–93)
0.69
Previous TB (%, n)
45.5% (5)
32% (8)
0.47*
Duration of TB treatment before ART (median days, IQR)
54 (33–84)
49 (24–84)
0.74
Median days to IRIS (IQR)
14 (8–44)
10 (6–16)
0.11
Duration of IRIS (median days, IQR)
170 (65–229)
101 (48–155)
0.31
C Reactive Protein (CRP, mg/L, median and IQR)
151 (65–208)
124 (80–180)
0.65
1
- multi-drug resistant;
- rifampicin mono-resistant;
*- Fisher’s Exact test.
doi:10.1371/journal.pone.0046481.t001
2
Figure 1. IFN-gamma ELISpot response of peripheral blood mononuclear cells (PBMC) stimulated with a range of Mycobacterium
tuberculosis antigens, expressed as IFN-gamma spot forming cells per million (SFC/106) PBMC. TB-IRIS patients with multi-drug resistant
(MDR) TB are shown in black full circles, rifampicin mono-resistant (RM) cases are shown as a cross, and fully sensitive (FS) TB cases are shown as open
circles.
doi:10.1371/journal.pone.0046481.g001
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Immunology of MDR TB-IRIS
Table 2. Multiplex cytokine analysis (corrected for multiple comparisons).
Median pg/ml IQR
FS (n = 10)
unstimulated
FS (n = 10) stimulated
p(1)
TNF
202
40597
0.013
190-237
12326-80459
77
9422
77-152
1349-24296
IL-1beta
IL-6
545
114700
229-7752
47775-246122
IL-10
12
163
8-19
101-1037
IFN-gamma
2
759
1-2
63-6906
IP-10 (CXCL10)
2958
151311
1067-6454
7948-726412
0
33
0-3
14-350
1
132
0-1
66-1234
1428
279656
1-1887
115527-397732
822
90146
638-4299
58661-167796
23194
82344
6574-85501
20247-102345
3200
82256
233-20728
61533-93089
GMCSF
0
3901
0-0
1227-5655
IL-15
3
3
2-3
2-4
1
2
0-1
1-4
0
1
0-0
0-1
5
8
3-5
5-53
IL-12p40
IL-2
MIP-1alpha (CCL3)
MIP-1beta (CCL4)
RANTES (CCL5)
IL-8 (CXCL8)
IL-4
IL-5
IL-13
0.006
0.026
MDR (n = 10)
unstimulated
MDR (n = 10)
stimulated
p(2)
p(3)
213
18177
0.001
.1
182-250
3212-76079
0.026
.1
0.26
.1
0.013
.1
0.018
.1
.1
.1
0.86
.1
0.018
0.89
0.013
.1
0.013
.1
.1
.1
0.01
.1
0.004
.1
N/A
N/A
N/A
N/A
N/A
N/A
.1
.1
112
7285
93-212
191-37595
2200
107578
114-6020
10829-282391
0.001
10
310
6-44
126-1090
0.001
1
103
1-1
15-841
0.26
0.052
0.003
0.003
0.008
.1
0.003
0.001
N/A
N/A
N/A
0.08
1262
13159
606-3303
1199-289846
0
33
0-2
0-130
0
34
0-0
9-123
1252
40981
278-2069
15793-331725
168
47396
76-1119
19502-199932
18091
55152
4604-52786
20975-183281
7422
78256
0-16700
53533-93923
96
1676
65-106
510-7852
0
0
0-0
0-0
0
0
0-0
0-0
0
0
0-0
0-1
4
12
4-9
5-25
(1)
p value comparing FS unstimulated with FS stimulated.
p value comparing MDR unstimulated with MDR stimulated.
p value comparing FS stimulated with MDR stimulated.
N/A: comparison not applicable due to very low values.
doi:10.1371/journal.pone.0046481.t002
(2)
(3)
significantly increased upon stimulation in both groups. Similarly,
the neutrophil chemoattractant IL-8 (CXCL8) was significantly
increased in both groups, as well as GM-CSF. The Th2 cytokines
IL-4, IL-5 and IL-13 were essentially undetectable in these
samples. Of note, the baseline characteristics and ELISpot
responses of the subset of 10 FS patients included in the cytokine
determinations did not differ from the full set of FS patients (data
not shown).
Because of the opposing trends of the Th1 cytokines and the
regulatory IL-10 concentrations observed between the MDR and
FS groups, we next evaluated the ratio of IFN-gamma/IL-10 in
PBMC stimulated with hkH37Rv, and found it to be significantly
detected upon stimulation in both groups, however, this tended to
be greater in the MDR group (median 310 pg/ml compared to
median 163 pg/ml in the FS group, p.0.5). Of the Th-1 type
cytokines, IFN-gamma and IL-2 significantly increased upon
stimulation in both groups (to median 759 and 132 pg/ml
respectively), but to a lesser extent in the MDR group (to median
103 and 34 pg/ml respectively). Changes in IP-10 (CXCL10)
concentrations were not significant in either group following
correction for multiple comparisons, however the increase upon
stimulation in the FS group was 11.5 fold greater compared to the
MDR group. Of the chemokines known to attract Th1 cells via
CCR5: MIP-1alpha (CCL3) and MIP-1beta (CCL4) were
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Immunology of MDR TB-IRIS
immunity using two different systems. Since this correlation was
only significant in the FS group (the corresponding Spearman
r = 0.16, p = 0.65 in the MDR group), it indirectly points towards
impaired Th1 immunity in the MDR group that may be related to
higher bacterial load. These data overall point towards an altered
balance between the Th1 and regulatory cytokine responses in
TB-IRIS patients in whom the cause of TB is an MDR strain
compared to those with a fully sensitive strain.
Discussion
We studied the immune response of TB-IRIS patients in whom
the cause of TB was a drug resistant strain compared to those with
a fully sensitive strain, with the hypothesis that there would be a
difference between the groups. We found an altered balance
between the Th1 and regulatory responses with decreased Th1
(IFN-gamma and IL-2) and increased IL-10 cytokine secretion in
the drug resistant group.
We recently described in a cross-sectional study that TB-IRIS is
associated with hypercytokinemia [16]. Here we used the same
panel of cytokines to explore the hypothesis that there is a
difference between the immune response in TB-IRIS patients
whose TB was caused by fully sensitive compared to those with
drug-resistant MTB. Our hypothesis was suggested by data
indicating that patients with MDR-TB have enhanced regulatory
T cells and impaired Th1 responses [15,18]. We wished to
determine whether this finding would stand in the context of TBIRIS induced hypercytokinemia.
The IFN-gamma ELISpot responses to a range of MTB
antigens showed no statistically significant differences between the
two groups of patients, although a trend towards higher responses
to the RD-1 encoded secreted antigen ESAT-6 in the FS group
was noted. Frequencies of circulating ESAT-6 specific IFNgamma secreting T cells were previously described to be higher in
patients with minimal disease and low bacterial burdens compared
to patients with culture positive active disease [19]. Since all
patients were on first line therapy at the time of diagnosis with
paradoxical TB-IRIS and sampling, it is possible that the bacterial
load of the patients in the MDR+RM group was higher, due to
failed therapy, compared to those with fully sensitive TB. Indeed,
the response to the 38 kDa cell wall associated protein and the
intracellular Acr1 and Acr2 tended to be higher in the FS group,
possibly indicating that treatment in this group results in more
bacterial destruction, thus these antigens becoming accessible to
circulating, antigen specific T cells in the blood. Having previously
shown large expansions of IFN-gamma secreting cells in HIV-TB
co-infected patients who develop TB-IRIS as well as in similar
controls who do not develop IRIS [17] it was perhaps not
surprising that our present results showed no difference between
the two groups of TB-IRIS patients.
We also evaluated cytokines secreted into the supernatant of
peripheral blood mononuclear cells stimulated overnight with heat
killed whole bacilli. We found that stimulation resulted in
increased cytokine/chemokine concentrations in both groups with
a few exceptions, however without a significant difference between
the two groups. A reduced ratio of IFN-gamma/IL-10 has been
found to relate to increased disease severity in pulmonary and
extrapulmonary TB [20]. Disease progression in MDR-TB
patients has also been associated with the detection of IL-10 in
whole blood [21]. IL-10 is an anti-inflammatory cytokine secreted
by many hematopoietic cells and has a central role in infection by
limiting the immune response to pathogens in order to prevent
damage to the host [22]. However, by suppressing macrophage
and dendritic cell functions required for the capture, control, and
Figure 2. The ratio of secreted cytokines measured by luminex
analysis in the supernatants of peripheral blood mononuclear
cells (PBMC) stimulated with heat killed H37Rv (MOI = 1:1) for
24 hours. Results from TB-IRIS patients with multi-drug resistant (MDR)
TB are shown in black full circles (n = 7), rifampicin mono-resistant (RM)
cases are shown as a cross (n = 3), and fully sensitive (FS) TB cases are
shown as open circles (n = 10).
doi:10.1371/journal.pone.0046481.g002
higher in the FS group (median 3.8 IQR 0.2-19.7) compared to
the MDR group (median 0.38 IQR 0.03-1.08), p = 0.02 (Figure 2).
A similar trend was observed for the IFN-gamma induced protein
CXCL10 (IP-10), with the ratio of IP-10/IL-10 being 305 (IQR
53-1666) in the FS group, compared to 86.6 (IQR 5.3-421) in the
MDR group (p = 0.2, data not shown). The ratio of IL-2/IL-10
was also significantly higher in the FS group (median 1.13 IQR
0.1-2.4) compared to the MDR group (median 0.16, IQR 0.0-0.4,
p = 0.02, Figure 2). The individual patient’s data on hkH37Rv
stimulated release of IFN-gamma, IL-2, IL-10, and the respective
ratios of IFN-gamma/IL-10 and IL-2/IL-10 are summarised in
Table 3. While there was variability between patients in the two
groups (Figure 2), the IFN-gamma/IL-10 ratio of 7/10 patients in
the FS group was higher than the median ratio seen in the MDR
group. In the same time, 10/10 patients in the MDR group had an
IFN-gamma/IL-10 ratio lower than the median seen in the FS
group. The same was observed for the IFN-gamma/IL-2 ratio as
well. In order to further relate cytokine release to the ELISpot
responses, we also included the response to ESAT-6 and the heat
killed whole bacilli (hkH37Rv) on an individual patient basis in
Table 3. We found a significant correlation between the ESAT-6
response and IFN-gamma release into the supernatant of PBMC
stimulated with hkH37Rv in the FS group (Spearman r = 0.73,
p = 0.02), indicating agreement between the readout of Th1
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Immunology of MDR TB-IRIS
Table 3. Individual patient’s data on hkH37Rv stimulated release of IFN-c, IL-2, IL-10, ratios of IFN-gamma/IL-10, IL-2/IL-10 and
ELISpot responses to ESAT-6 and hkH37Rv.
Patient
group
Patient
identifier
IFN-gamma
pg/ml
IL-2 pg/ml
IL-10 pg/ml
IFN-gamma/IL-10
IL-2/IL-10
ESAT-6
SFC/106 PBMC
hkH37Rv SFC/106
PBMC
FS
43
296
99
5149
0.06
0.02
1510
585
62
10000
2402
1864
5.36
1.29
2610
1725
72
14571
1096
761
19.15
1.44
2073
360
77
1203
152
68
17.66
2.24
1866
126
83
2270
287
106
21.47
2.71
2114
834
110
39
12
173
0.23
0.07
342
1146
113
5874
1649
269
21.86
6.14
101
1561
118
314
112
138
2.27
0.81
931
3571
215
71
82
84
0.84
0.97
74
0
257
37
18
152
0.24
0.12
34
67
759
132
163
3.82
1.13
1221
710
19
43
12
3763
0.01
0.00
1808
50
921
122
349
2.64
0.35
3240
52
814
125
752
1.08
0.17
2740
725
79
1
0
20
0.03
0.00
300
0
105
3007
544
1124
2.67
0.48
34
630
127
19
24
53
0.35
0.45
1742
3277
141
120
63
150
0.80
0.42
0
80
196
86
33
204
0.42
0.16
174
214
238
1
0
270
0.00
0.00
27
74
247
233
34
1079
0.22
0.03
40
0
103
34
310
0.38
0.16
237
214
Median
MDR
Median
465
doi:10.1371/journal.pone.0046481.t003
initiation of immune responses to MTB, it might also be linked to
the mechanisms by which MTB evades the host immune response,
mediating long-term infection in the lung [23–25]. While we found
no significant difference between the two groups of patients in the
concentrations of IL-10 secreted by MTB stimulated PBMC, we
did find a significant difference in the ratio of Th1 cytokines (IFNgamma and IL-2) to IL-10, which were significantly lower in the
MDR group. Thus, our data is in line with suggestions that
protective Th1 responses are impaired in some drug-resistant TB
patients. However, host immune responses are dependent on the
bacterial load, and as discussed earlier, the bacterial load of the
patients in the MDR group may be higher due to failed therapy,
we hypothesise that the impaired cytokine balance is likely to be
caused by the poorly controlled bacterial growth in these patients.
Limitations of our work include (i) the small sample size, (ii) the
lack of mycobacterial strain typing and (iii) the lack of cellular
analysis. In a previous study however, we showed that TB-IRIS is
not associated with increased proportions of regulatory
CD4+FoxP3+ T cells [17]. Moreover, Guyot-Revol et al have
shown that while FoxP3 expression is increased in patients with
TB, IL-10 and TGF-b1 mRNA did not correlate with regulatory
T-cell markers [26], suggesting that the source of IL-10 in these
TB patients could be other cell types. Indeed, it has been shown
that the frequency of MTB antigen specific CD8+ T cells secreting
IL-10 is elevated in MDR-TB patients [27].
MTB is a successful pathogen because it has the ability to persist
in an immune competent host via a number of immune evasion
strategies, such as altered antigen presentation to prevent the
recognition of infected macrophages by T cells, and evading
macrophage killing mechanisms [28]. While some of these
mechanisms are more prominent in heavily infected cells, the
resulting immune response will be dependent on the bacterial
load. Determining the tuberculous bacterial load and its link to
pathogenesis remains one of the greatest challenges in our
understanding of human tuberculosis.
Acknowledgments
We thank Valerie Mizrahi for helpful comments on the manuscript.
Author Contributions
Conceived and designed the experiments: KAW RJW GM. Performed the
experiments: KHS RS KM KAW. Analyzed the data: KAW MXR.
Contributed reagents/materials/analysis tools: RJW GM MXR DJP.
Wrote the paper: KAW RJW GM KHS. Patient recruitment: DJP MXR
GM.
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