Reactivation of M. tuberculosis infection in trans-membrane tumour necrosis factor mice.

Abstract

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.

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Reactivation of M. tuberculosis Infection in Trans-
Membrane Tumour Necrosis Factor Mice
Ivy Dambuza1, Roanne Keeton1, Nasiema Allie1, Nai-Jen Hsu1, Philippa Randall1, Boipelo Sebesho1,
Lizette Fick1, Valerie J. F. Quesniaux2,3, Muazzam Jacobs1,4*
1Division of Immunology, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa, 2CNRS UMR6218, Orleans, France,
3Molecular Immunology and Embryology, University of Orleans, Orleans, France, 4National Health Laboratory Service, Sandringham, South Africa
Abstract
Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large
reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for
containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated
the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against
reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol
inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic
treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate
that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established
mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma
structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial
phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice
show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs
and reduced IL-12p70, IFNc and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication
relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune
pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.
Citation: Dambuza I, Keeton R, Allie N, Hsu N-J, Randall P, et al. (2011) Reactivation of M. tuberculosis Infection in Trans-Membrane Tumour Necrosis Factor
Mice. PLoS ONE 6(11): e25121. doi:10.1371/journal.pone.0025121
Editor: Bernhard Ryffel, French National Centre for Scientific Research, France
Received July 7, 2011; Accepted August 25, 2011; Published November 21, 2011
Copyright: ? 2011 Dambuza 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 National Research Foundation (South Africa), The Medical Research Council (South Africa), The National Health
Laboratory Services (South Africa), The University of Cape Town, the European Union (TB REACT Contract no 028190). 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: Muazzam.Jacobs@uct.ac.za
Introduction
Although a third of the global population has been exposed to
tuberculosis the majority harbours a latent form of infection [1].
This global reservoir potential poses significant challenges to
therapeutic intervention, made more difficult by poor understand-
ing of the immune mechanisms that exert pressure to maintain
bacilli in a state of latency. Real threats are associated with disease
reactivation, particularly in disease burden countries in which
immune-compromised individuals such as those with HIV/AIDS
form a significant part of the population. In low burden, first world
countries, reactivation of latent bacilli form the primary cause of
active disease as opposed to new infections in developing countries.
Host immune factors that allow for mycobacteria to remain in a
persistent state of latency have not been clearly defined although
considerable insight has been gained through the application of in
vitro models and animal studies that simulate M. tuberculosis
reactivation [2,3,4,5]. However, identifying factors responsible
for maintaining a latent infectious state and those that are
compromised to give rise to reactivation have proven to be
complex. Loss of function and neutralization studies has been key
to understand the effects of Tumour Necrosis Factor (TNF) in host
protection. We and others have shown that while TNF is critical to
control acute infection [6,7,8], it is similarly important to prevent
bacilli replication during chronic infection [9] or during drug
induced latent infection [10]. The reemergence of bacilli in the
absence of TNF correlated with a lack of proper granuloma
structures and the increase of pro-inflammatory cytokines. The
importance of TNF for maintaining latent infection was verified in
clinical studies in which anti-TNF therapy administered to patients
with chronic inflammatory diseases resulted in spontaneous
reactivation of tuberculosis [11,12,13,14]. The mechanisms
through which TNF mediates control of latent infection is unclear,
however studies have reported that administration of TNF
inhibitors interferes with TNF mediated phagosome maturation,
apoptosis, T cell activation and autophagy [15]. A study by Bruns
et al., 2009 showed that anti-TNF neutralizing antibodies reduced
the population of effector memory CD8 T cells resulting in
reduced antimicrobial activity against M. tuberculosis [16].
TNF is a multifunctional cytokine, initially synthesized as a
26 Kda non-glycosylated type II trans membrane protein (Tm-
TNF) which upon cleavage by the metalloprotease TNF-
converting enzyme (TACE) forms a soluble 17 KDa protein.
Both molecular forms of TNF are biologically functional as
homotrimeric proteins that bind and mediate signaling through
either TNFRp55 (TNFRSF1A, CD120a,TNFR1) or TNFRp75
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(TNFRS1B, CD120b, TNFR2) with Tm-TNF binding strongly
TNFRp75 [17]. We and others have previously reported that acute
M. tuberculosis infection could be controlled by Tm-TNF but that
soluble TNF was required to sustain host immune protection
[18,19,20,21]. Moreover, we have demonstrated that rapid and
lethal reactivation of M. tuberculosis was associated with lack of
proper bactericidal granuloma formation in latently infected
complete TNF2/2mice treated with isoniazid and rifampicin [10].
With the current development of new TNF inhibitor biologics
which specifically inhibit solTNF and spare Tm-TNF in the
treatment of chronic inflammatory disorders [22,23,24,25], we
investigated the role of Tm-TNF in controlling reactivation of
therapeutically induced latent infection. We show that Tm-TNF
mediates control of reactivating bacilli but that soluble TNF is
required to sustain long-term growth inhibition. We found that
susceptibility in reactivating Tm-TNF mice is associated with
unstructured granuloma formation and a defect of protective
cytokine synthesis.
Materials and Methods
Mice
C57Bl/6 wild type (WT) control mice, TNF2/2mice [26] and
Tm-TNF mice [27] were bred, maintained and housed in
individually ventilated cages under specific pathogen free condi-
tions in the animal facility of the University of Cape Town, South
Africa. For all the experiments, age matched mice on a C57Bl/6
background were used and genotypes were confirmed by PCR
analysis. All the experiments and protocols performed were in
accordance with the guidelines of the Research Ethics Committee
of the University of Cape Town, South Africa (Approval ID- REF
REC: 008/023).
Bacterial infection and chemotherapy
M. tuberculosis H37Rv was grown in Middlesbrook 7H9 broth
(Becton, Dickinson and Company, Le Pont de Claix, France)
supplemented with 10% Middlebrook OADC enrichment medi-
um (Life Technologies, Gaitherburg, MD), 0.5% glycerol and
0.05% Tween 80 at 37uC until log phase. Prior to usage,
mycobacterial aliquots were passed 306through a 29.5 G needle
to minimize bacterial clumping. Pulmonary infection with 100–
200 cfu live M. tuberculosis H37Rv bacteria was performed using a
Glas-Col Inhalation Exposure System, Model A4224. Inoculum
size was confirmed 24 h post-infection by determining the
bacterial burden in the lungs of infected mice.
For the M. tuberculosis reactivation model (Fig. 1), we used a
modified protocol as previously described [10]. Briefly, groups of
mice were infected by aerosol inhalation with 100–200 cfu viable
M. tuberculosis H37Rv bacilli. The infection was allowed to progress
for 21 days, termed the pre-immune phase (Fig. 1, line A), where
unrestricted bacilli replication occurred before commencement of
treatment with 25 mg/Kg INH-RIF (Sigma, St. Louis, USA) in
drinking water for 6 weeks. In this drug treatment phase (Fig. 1,
line D) bacilli numbers were reduced to at least less than
100 CFUs in the lungs after which treatment was withdrawn,
allowing reactivation of bacilli (Fig. 1, line E). Alternatively, in
control groups that received no therapy after 21 days, bacilli
burdens remained constant (steady state phase Fig. 1, line B).
Colony enumeration assay
Bacterial burdens in the lungs, livers and spleens of infected
mice were determined at specific time points after infection with
M. tuberculosis H37Rv. Organs were weighed and homogenized in
0.04% Tween 80 saline. Tenfold serial dilution of organ
homogenates were plated in duplicates on Middlesbrook 7H10
(Becton, Dickinson and Company) agar plates containing 10%
OADC (Life Technologies, Gaitherburg, MD) and incubated at
37uC for 19–21 days. Colonies on plates were enumerated and
bacterial burdens determined.
Pulmonary pathology
Mice were euthanized by carbon dioxide inhalation at specific
time points. Organs were weighed and fixed in 10% formalin and
paraffin-embedded. Two to 3 mm sections were stained with
haematoxylin and eosin (H&E) and a modified Ziehl-Nielson (ZN)
method as described [28]. For immunostaining, formalin-fixed
paraffin-embedded sections were deparaffinised and rehydrated
then stained with rabbit anti-mouse specific inducible nitric oxide
synthase (iNOS) [29], rat anti-mouse CD11b (clone M1/70) or rat
anti-mouse CD3 (clone 145-2c-11) antibodies. Sections were then
washed in PBS and incubated for 30 min at room temperature
with biotinylated secondary antibody then subsequently incubated
with avidin-biotin complexes (Vector Laboratories, CA, USA) for
30 min, washed and incubated with DAB substrate (Dako
Corporation, CA, USA).
Lung homogenate preparations
Whole lungs were removed from infected mice at specific time
points and were homogenized in 1 ml 0.04% Tween 80 saline
containing protease inhibitor (Sigma) and the supernatants were
collected after low-speed centrifugation, aliquoted and frozen at
280uC.
Cytokine ELISA
Supernatants from organ homogenates or from cultured cells
were harvested and assayed for cytokine concentration using
commercially available ELISA reagents for IFNc, IL-10, and IL-
12p70 (R&D Systems, Germany and BD PharMingen, San
Diego), according to the manufacturer’s instructions.
Figure 1. Graphic presentation of drug-based M. tuberculosis
reactivation mouse model. Line A: Preimmune phase, short bacterial
replication period post-infection with low dose M. tuberculosis; Line B:
Steady state phase: Control of M. tuberculosis growth through host
immunity and establishment of chronic infection. Line D: Drug
treatment phase: Reduction of bacilli replication through INH-RIF
chemotherapy. Line E: Reactivation phase: Reactivation of infection
upon cessation of antibiotics.
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Statistical Analysis
The data are expressed as the mean 6 SEM. Statistical analysis
was performed by ANOVA. For mortality studies, analysis was
performed using the log-rank test. For all tests, a p-value of ,0.05
was considered significant.
Results
Tm-TNF protects mice from severe tuberculosis
reactivation
In a study performed by McCune et al., 1966, it was observed
that immunizing mice with M. tuberculosis then re-infecting them
preceding treatment with anti-tuberculous drugs resulted in mice
reactivating with lower M. tuberculosis CFU numbers compared to
control groups [30]. This result was interpreted as the influence of
the host’s acquired immune resistance to mycobacteria. In this
study, we investigated the contribution of two molecular forms of
TNF, in particular Tm-TNF in host immunity in mice immunized
by prior infection with M. tuberculosis. The model used entailed
aerosol inhalation infection of WT mice, TNF2/2mice and Tm-
TNF mice with 100 viable M. tuberculosis H37Rv bacilli. The
infection was allowed to progress for 21 days before commence-
ment of treatment with 25 mg/Kg INH-RIF in drinking water for
6 weeks to reduce bacilli numbers to at least less than 100 CFUs in
the lungs after which treatment was withdrawn and tuberculosis
reactivation was monitored. Body weights were recorded through-
out the infection period and body weight loss was interpreted as
severe disease due to reappearing tuberculosis and correlated with
susceptibility to infection. Control groups of WT, Tm-TNF and
TNF2/2mice that did not receive chemotherapeutic treatment
were confirmed to have phenotypes as previously described [18].
We found that, of the therapeutically treated animals, WT mice
showed a steady increase in body weight over the duration of the
infection in contrast to TNF2/2mice which displayed significantly
lower body weights between 69 and 165 days post-infection
(Fig. 2A) coinciding with the period subsequent to the withdrawal
of treatment. TNF2/2mice also appeared sick with ruffled fur and
hunched backs and eventually became moribund and had to be
terminated on day 165. In contrast, Tm-TNF showed an increase
in body weight comparable to WT mice for the first 130 days post-
infection (Fig. 2A) but significant weight loss was recorded in Tm-
TNF mice between 237 days and 265 days post-infection while the
body weights in WT mice remained stable (Fig. 2A). For the
remainder of the experimental period, Tm-TNF mice maintained
slightly lower body weights relative to WT mice with no significant
differences observed (Fig. 2A) but exhibited no physical signs of
severe disease apparent in TNF2/2mice. These data indicate that
complete absence of TNF renders mice susceptible to severe
reactivating tuberculosis which is alleviated by the presence of
Tm-TNF.
Next, bacilli burdens in lungs and spleens of infected mice were
determined at specific time points to investigate the effect of the
different molecular forms of TNF on mycobacteria recrudescence
in the presence pre-existing immunity in WT mice, TNF2/2mice
and Tm-TNF mice. Bacilli burdens in WT mice were reduced by
3.5 log10in the lung (Fig. 2B) and by 2.5 log10in the spleen
(Fig. 2C) after exposure to 6 weeks INH-RIF treatment.
Withdrawal of antibiotic treatment resulted in spontaneous M.
tuberculosis reactivation with bacilli burdens reaching up to 4 log10
in the lung and 2.5 log10in the spleen (Fig. 2B & C, respectively).
In TNF2/2mice, INH-RIF treatment reduced the high bacilli
burden by more than 6 log10in lung (Fig. 2B) and 4 log10in spleen
(Fig. 2C) after 6 weeks exposure. The more efficacious reduction in
bacterial burden in the lung of TNF2/2relative to WT mice after
INH-RIF treatment is in agreement with the concept that reduced
immune pressure is intimately associated with improved antibiotic-
mediated mycobacterial clearance [31]. Within 133 days post-
treatment, mycobacteria reappeared in TNF2/2mice and bacilli
burden reached at least 6 log10in lung (Fig. 2B) and 4 log10in
spleen (Fig. 2C) of fully deficient TNF2/2mice. Tm-TNF mice
responded to INH-RIF treatment in a manner comparable to WT
mice and showed a similar slow kinetic in the rate of reactivation
comparable to that of WT mice. However, by 322 days post-
treatment, bacterial burdens in the lung of Tm-TNF mice
increased significantly by 2 log10compared to a 1 log10increase
observed in WT mice (Fig. 2B). Bacilli burdens in spleens of Tm-
TNF mice were comparable to WT mice at all time points
investigated (Fig. 2C). Therefore the data show that Tm-TNF is
required to promote early bacterial killing mechanisms even after
priming of the host by previous M. tuberculosis infection. However,
although Tm-TNF confers early protection against recrudescence
M. tuberculosis, Tm-TNF alone does not sustain long term control
of the infection, indicating that solTNF is needed for controlling
chronic infection.
Abnormal inflammatory response in the absence of TNF
during tuberculosis reactivation is delayed in Tm-TNF
mice
TNF has previously been shown to be at the apex of
inflammatory responses [32]. To determine whether Tm-TNF
was sufficient in mediating an inflammatory response during
tuberculosis reactivation, mouse lung weights were recorded at
specific time points during the infection period as a surrogate
marker of inflammation. Compared to lung weights determined at
day 21 post-infection, WT mice displayed no change in lung
weights after exposure to INH-RIF for 6 weeks. However, an
increase in lung weights was observed during later stages of disease
(322 days post-infection; Fig. 3A) which was consistent with the
increase in bacilli burdens at this time point (Fig. 2B). In sharp
contrast, by 21 days post-infection, TNF2/2mice already
displayed significantly higher lung weights compared to WT mice
(Fig. 3A). Paradoxically, 63 days post-infection at the end of the
therapy period, the lung weights had increased significantly
(p,0.001) compared to WT mice (Fig. 3A) but did not correlate
with the decreased number of bacilli at this time point (Fig. 2B).
This observation differed from a previous report where similar
lung weights were measured in TNF2/2mice in a model using a 3
fold lower infection dose and chemotherapy was initiated earlier
[10]. We postulate that here, a delay in onset of chemotherapy
until 21 days combined with the potential higher antigenic burden
of killed bacilli provided conditions for irreversible proinflamma-
tory stimulation and excessive inflammation in the absence of
TNF. Susceptibility of TNF2/2mice was confirmed with a further
significant increase (p,0.001) in lung weights noted at 113 days
post-infection, 50 days after cessation of therapy. In contrast, lung
inflammation in Tm-TNF mice was similar to WT mice during
early infection with similar lung weights at 21, 63 and 133 days
post-infection (Fig. 3A). However, control of inflammation was not
sustained as a significant increase (p,0.01) in lung weights was
evident in Tm-TNF mice at day 322 post-infection compared to
WT mice (Fig. 2.1B).
To further analyze airways inflammation in the absence of
TNF, or in the presence of Tm-TNF, the number of cells present
in the bronchoalveolar lavage (BAL) fluid was determined after
infection. The cellularity in the BAL fluid was significantly higher
in TNF2/2mice compared to WT mice, whereas Tm-TNF mice
had a number of cells comparable to WT mice at day 77 post-
infection (Fig. 3B) confirming that the control of inflammation
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during early infection was membrane TNF dependent. Consistent
with chronic lung weight data, Tm-TNF mice displayed
significantly increased number of cells in BAL fluid relative to
WT mice on day 378 post-infection (Fig. 3C). Together, the data
indicates that control of early inflammation is mediated primarily
by Tm-TNF but solTNF is required for regulation of inflamma-
tion during chronic infection. However, the lack of control of
inflammation appears to be strongly associated with the onset of
tuberculosis reactivation.
Tm-TNF is inadequate to maintain bactericidal
granulomas during reactivating tuberculosis
We next asked whether granuloma structures were formed in
the presence of Tm-TNF during tuberculosis reactivation. Studies
performed by Mohan et al., 2001, illustrated that TNF was
required for maintenance of granuloma structure during persistent
M. tuberculosis infection whereby upon treatment with TNF-
neutralizing antibody, mice displayed severe histopathology
marked with excessive inflammation and loss of structured
granulomas [33]. Lung sections were obtained from infected
WT mice, TNF2/2mice and Tm-TNF mice at the indicated time
points post-infection and pulmonary pathology analyzed. Untreat-
ed WT mice (Fig. 4A) and Tm-TNF mice (Fig. 4C) displayed small
compact lesions with tight lymphocytic wedges and a high degree
of clear airway spaces at day 33 post infection. In sharp contrast,
TNF2/2mice (Fig. 4B) showed enlarged unstructured lesions with
inflammation occupying larger areas of the lung and presenting
with evidence of necrosis. At the end of chemotherapeutic
treatment (day 63 p.i.), WT (Fig. 4D) and Tm-TNF (Fig. 4F)
mice had pulmonary pathology characterized by well-defined
granulomas and clear alveoli whereas TNF2/2mice displayed
higher inflammation characterized by peri-vascular and peri-
bronchiolar inflammation (Fig. 4E). Development of pathology
subsequent to withdrawal of chemotherapy (day 133 p.i.)
continued to show well-defined granulomas in WT (Fig. 4G) and
Tm-TNF (Fig. 4I) mice with clear alveoli, whereas TNF2/2mice
now presented with excessive inflammation with no defined
granulomas and limited alveolar space (Fig. 4H). The inability of
Tm-TNF mice (Fig. 4K) to control development of further
pathology was clearly evident at day 322 post-infection where, in
contrast to WT mice (Fig. 4J), Tm-TNF mice displayed enlarged
unstructured lesions with excess inflammatory responses and
interstitial pneumonia.
These observations demonstrate that during tuberculosis
reactivation, Tm-TNF does not sustain long term maintenance
of protective granuloma structure in the absence of soluble TNF
resulting in malformed lesions that associate with failure to inhibit
M. tuberculosis growth.
We therefore determined whether effector macrophage anti-
mycobacterial function was intact in Tm-TNF mice undergoing
M. tuberculosis reactivation. Studies have shown that cell mediated
mycobacterial killing function can be achieved through production
of toxic reactive intermediates (RNI) via the enzymatic action of
macrophage iNOS [34]. Previously, it was demonstrated that
inhibition of iNOS in mice chronically infected with M. tuberculosis
resulted in reactivation of tuberculosis disease with increased
organ bacillary burdens and extensive granulomatous response
[10,35]. In view of these findings we determined iNOS expression
immunohistochemically in lung tissue sections of reactivating
infected WT mice, TNF2/2mice and Tm-TNF mice after
completion of chemotherapy. iNOS expression patterns were
largely within the confinement of granuloma lesions in WT mice
(Fig. 5A) in contrast to TNF2/2mice where the pattern was
dispersed and associated with the diffused granuloma lesions
(Fig. 5B) analyzed 133 days post-infection. Tm-TNF mice
displayed similar iNOS expression to WT mice at day 133 days
post-infection (Fig. 5C). However, during reactivating chronic
infection at day 322, in contrast to WT mice (Fig. 5D), increased
iNOS expression was evident in Tm-TNF mice (Fig. 5E) with a
random distribution and evidence of lung tissue destruction. We
further characterized the effect of Tm-TNF signaling on
macrophage (CD11b+cells) and lymphocyte (CD3+cells) recruit-
ment to investigate whether a correlation existed between
macrophage recruitment and iNOS induction, and also to
determine Tm-TNF effects on the structural relationship between
macrophages and lymphocytes with respect to granuloma
formation during reactivation. It is clear that macrophage
distribution correlated strongly with iNOS induction where, in
WT mice it was localized on the periphery of established
granulomas (Fig. 6A) but randomly distributed in Tm-TNF mice
(Fig. 6B) at 322 days post-infection. Lymphocytes recruitment was
focused and predominantly occupied central areas of granulomas
in reactivating WT mice (Fig. 6C) but was unorganized in
reactivating Tm-TNF mice (Fig. 6D). Therefore, these observa-
tions indicate that, in hosts where tuberculosis reactivation occurs,
Tm-TNF on its own cannot sustain the structural integrity of
granulomas with respect to the cellular organization of macro-
phages and lymphocytes, and that iNOS induction by macro-
phages is insufficient for controlling mycobacterial growth if the
granuloma structure is not properly formed.
Defective protective cytokine induction in reactivating
Tm-TNF mice during late stages of the disease
Next we assessed the effect of Tm-TNF mediated immune
responses in infected mice subsequent to INH-RIF treatment with
particular reference to the quantification of IFNc and IL-12 levels
because of their reported functions in generating and maintaining
protective immunity against M. tuberculosis infection [36,37,38].
Studies by Feng et al., 2005, demonstrated that continuous IL-12
production is necessary for maintenance of pulmonary IFNc-
producing effector CD4+T cells and subsequent bacilli control
during chronic M. tuberculosis infection suggesting that interruption
of IL-12 signal transduction contribute to development of
reactivation of tuberculosis [39]. Therefore, we quantified
pulmonary IL-12 and IFNc production during tuberculosis
reactivation (Fig. 7). Comparable IL-12p70 and IFNc concentra-
tions were found in the lung of WT mice and Tm-TNF mice 133
days post-infection. However, there was a significant decrease in
IL-12p70 and IFNc pulmonary concentrations in Tm-TNF
compared to WT mice 322 days post-infection associated with
Figure 2. Effect of Tm-TNF on M. tuberculosis replication during reactivation. WT mice (black circles), TNF2/2mice (white diamonds) and
Tm-TNF mice (white squares) were treated for 6 weeks with 25 mg/Kg INH-RIF in drinking water subsequent to 3 weeks aerosol infection with 100–
200 CFU’s of M. tuberculosis H37Rv. (A) Body weights were recorded throughout the course of the infection period and bacterial burdens in lungs (B)
and spleens (C) were enumerated at time points indicated. Data are representative of two experiments and data points are expressed as the mean 6
SD of 5 mice/group (for CFUs). The body weight study consisted of between 6–16 mice/group where the data represents weights for remaining mice
in each group. The red ‘‘T’’ in the figure corresponds to the drug treatment phase. Significant differences (*p,0.05; **p,0.01) were determined by
Student’s t test for comparisons between two groups and ANOVA for comparisons between three groups.
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susceptibility of Tm-TNF mice to M. tuberculosis reactivation at this
time point. The concentration of anti-inflammatory IL-10 was
comparable 133 days post-infection but increased in WT mice by
day322post-infectionandremained significantlylowerinTm-TNF
mice (Fig. 7). These data suggest that Tm-TNF is not sufficient to
sustain protective cytokine induction in post-infection M. tuberculosis
chronic immunity and this phenotype is associated with lack of
control of bacilli burden in the lung and lethality of these mice.
Discussion
Estimates indicate that a third of the global population is
latently infected with the tuberculosis pathogen, M. tuberculosis [1].
The host mechanisms responsible for maintaining a latent
infection remain elusive. Several studies have established that
TNF is important in immune responses against mycobacterial
infections [6,28,40,41,42,43] but it is also a central mediator of
Figure 3. Induction of excessive inflammation in the absence of solTNF during reactivation of M. tuberculosis. WT (black circles), TNF2/2
(white diamonds) and Tm-TNF (white squares) mice were exposed by aerosol inhalation infection to 100–200 CFUs/mouse of M. tuberculosis H37Rv
for 3 weeks preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in drinking water. (A) Lung weights were measured at specific time points
and BAL derived cell numbers were determined 77 days (B) and 378 days (C) post-infection. The red ‘‘T’’ in the figure corresponds to the drug
treatment phase. Data are representative of 1 of 2 experiments performed and are expressed as mean 6 SD of 5 mice/group. Significant differences
(*p,0.05; **p,0.01; ***p,0.001) were determined by Student’s t test for comparisons between two groups and ANOVA for comparisons between
three groups.
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pathology in autoimmune diseases [44,45,46]. Patients on anti-
TNF treatment for chronic inflammatory diseases have an
increased incidence of tuberculosis [11,12,13,14] implicating
TNF in the preservation from latent tuberculosis. Although highly
efficacious, the currently used anti-TNF therapies i.e. etanercept,
infliximab, and adalimumab block both Tm-TNF and solTNF
[47,48,49,50]. Therefore, further research on TNF biology is
required for designing improved therapeutics that will alleviate
inflammatory diseases while maintaining protection against
mycobacteria.
We and others investigated the contribution made by the two
molecular forms of TNF, solTNF and Tm-TNF, in the induction
of protective immunity or immunopathology during M. tuberculosis
or M. bovis BCG infection [18,19,20,51]. A conclusion derived
Figure 4. Tm-TNF contributes to protective bactericidal granuloma formation during M. tuberculosis reactivation but is insufficient
to sustain structural integrity and bactericidal efficacy. WT mice (A,D,G,J), TNF2/2mice (B, E, H) and Tm-TNF mice (C,F,I,K) were infected by
aerosol inhalation with 100–200 CFUs/mouse M. tuberculosis H37Rv for 3 weeks preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in
drinking water. Lungs were removed at the indicated timepoints and tissue sections stained with haematoxylin and eosin to determine the
granulomatous response.
doi:10.1371/journal.pone.0025121.g004
Figure 5. Extra-granulomatous pulmonary expression of INOS is associated with susceptibility in Tm-TNF reactivating mice. WT
mice (A,D), TNF2/2mice (B) and Tm-TNF mice (C,E) were infected by aerosol inhalation with 100–200 CFUs/mouse M. tuberculosis H37Rv for 3 weeks
preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in drinking water. Lungs were removed at 133 and 322 days post infection and tissue
sections were stained with polyclonal rabbit anti-mouse antibody (see Materials and methods). Brown stain represents iNOS expression by activated
macrophages. Micrographs represent 4 animals/group and are shown at 632 magnification.
doi:10.1371/journal.pone.0025121.g005
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from these studies revealed that Tm-TNF expressing mice have an
intermediary phenotype i.e. they controlled acute infection where
gene deficient TNF mice were susceptible. However Tm-TNF
mice succumbed to chronic infection with increased bacillary
burdens and excessive granulomatous response which was not
observed in WT mice. Here, we report that Tm-TNF and solTNF
are both required for the maintenance of immune pressure during
reactivating tuberculosis. We found in our drug-induced tubercu-
losis reactivation model that TNF is an absolute requirement for
the containment of the re-emergence of tuberculosis reflected by
the rapid propagation of mycobacteria after cessation of antibiotic
treatment in TNF2/2mice. This observation confirms findings
previously reported by Botha and Ryffel, 2003 [10] and support
data showing that treatment of chronically infected mice [33,52]
or latently infected humans treated with TNF neutralizing anti-
bodies results in reappearanceof tuberculosis [11,12,13,14,16,53,54].
Furthermore, our results show that Tm-TNF contributes to the initial
containment of re-emerging tuberculosis but is not sufficient for long-
term mycobacteria containment resembling the outcome of chronic
infection. Together, these observations demonstrate that Tm-TNF
mediates early protection against reactivating M. tuberculosis infection
and that solTNF might be required at later stages for sustained
protection.
The formation of granulomas in response to mycobacterial
challenge leads to killing or alternatively, isolation and confine-
ment of bacilli to local sites of infection. They are dynamic
structures that promote cellular interaction to enhance bactericidal
efficacy, and the initial establishment and continued maintenance
of its structural integrity in murine models is critically dependent
on TNF. Nevertheless, the relationship between TNF and
granuloma structure as an indicator of protection mediated
against infectious mycobacteria remains controversial. More
recently, Lin et al, 2010 found that neutralization of TNF in
cynomolgus macaques resulted in dissemination of disease without
compromising the structural integrity of established granulomas
[55]. Previously, we proposed that Tm-TNF alone was sufficient to
develop distinct granulomas during respiratory M. tuberculosis
Figure 6. Macrophage (CD11b+cells) and lymphocyte (CD3+
cells) recruitment in WT and Tm-TNF mice. WT mice (A,C) and Tm-
TNF mice (B,D) were infected by aerosol inhalation with 100–200 CFUs/
mouse M. tuberculosis H37Rv for 3 weeks preceding chemotherapy with
25 mg/Kg INH-RIF for 6 weeks in drinking water. Lungs were removed
322 days post infection and tissue sections were stained with either
anti-CD11b anti-mouse (A,B) antibody or anti-CD3 anti-mouse antibody
(C,D) (see Materials and methods). Micrographs represent 4 animals/
group and are shown at 632 magnification.
doi:10.1371/journal.pone.0025121.g006
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infection, but referred to the diminished bactericidal capacity of
such granulomatous structures [18]. Moreover, these findings were
supported in clinical studies by Lliopoulos et al, 2006 who found
typical granulomas in biopsy specimens from patients on anti-TNF
therapy who developed tuberculosis [56]. Furthermore, although
presence of structurally defined granulomas are widely accepted as
the hallmark of protection, Johnson et al, 1998 reported adequate
protection in ICAM-1 deficient mice despite lacking structured
granulomas during M. tuberculosis infection [57]. These combined
observations therefore suggest that factors independent of TNF
may determine granuloma structural integrity and that such
structures alone cannot be used as a marker to define protection
against M. tuberculosis. Here, we show that during tuberculosis
reactivation, TNF2/2mice formed larger lesions with inflamma-
tion occupying larger areas of the lung with some evidence of
necrosis compared to WT mice, an observation that corroborates
previous findings by Mohan et al., 2001 and Botha and Ryffel,
2003 where a lack of proper defined granulomas in reactivating
TNF2/2mice were described [10,33]. Furthermore, our results
show that Tm-TNF mice were capable of granuloma structure
formation during initial tuberculosis reactivation comparable to
WT mice but that long term Tm-TNF dependent sustainability
was not enough to maintain protective granuloma structures and
susceptibility was reflected by formation of larger, more diffuse
lesions with excess inflammation and interstitial pneumonia in
reactivating mice. These results therefore illustrate a role for both
solTNF and Tm-TNF as a requirement for maintaining
granuloma structures during tuberculosis reactivation.
We further assessed iNOS induction as a parameter of
macrophage activation in situ within granulomas to understand
the lack of sustained Tm-TNF mediated protection during M.
tuberculosis reactivation. It is clear that Tm-TNF induced activation
of macrophages in a focused manner where iNOS expression was
localized to structured granulomas unlike in the complete absence
of TNF. The sustainability of this initial focused macrophage
response was however compromised during latter stages where
iNOS synthesis was notably randomized. The failure to sustain
focused bactericidal activity within granulomas was accompanied
by a reduction in overall cytokine profile that included IFNc, IL-
12p70 and IL-10. Clinically, similar findings were noted in
reactivating tuberculosis patients on anti-TNF therapy who
presented with a decreased T cell activation profile and reduction
in IFNc and IL-10 synthesis [58]. Lower IFNc levels, in particular,
critically impacts on macrophage responses by suppressing
phagosome maturation and promoting mycobacterial survival.
We therefore postulate that, in our studies, the dependence of IL-
12p70 synthesis on autologous TNF mediated signaling by antigen
presenting cells such as macrophages which are mostly located
within granulomas may eventually have been compromised in
Tm-TNF deficient mice. The inhibition of T cell activation and
lower IFNc production, as a consequence of diminished IL-12
levels, may have resulted in suppressed M. tuberculosis specific
phagosome maturation, thereby promoting bacterial survival and
replication during long term reactivation. The cytokine profile in
reactivating Tm-TNF mice reported here contrasts to that
observed in de novo infected Tm-TNF mice [18] with respect to
pulmonary IFNc AND IL-10 concentrations during late stage
infection despite similar observed susceptible phenotypes. We
postulate that cytokine induction under these very diverse
conditions may be driven by distinctive differences in immune
responses. Under conditions of de novo M. tuberculosis infection
innate cellular responses drive initial protective immunity whereas
under conditions of reactivation protective function may primarily
be driven by adaptive memory immunity established during
infection prior to pathogen resolution after chemotherapeutic
treatment.
In conclusion data presented here, illustrates that TNF
mediated immunity against M. tuberculosis infection requires both
Tm-TNF and solTNF during tuberculosis reactivation. Although
Tm-TNF protects mice against acute M. tuberculosis infection, long
term protection requires solTNF partly to down regulate
inflammatory responses in chronic infection and to sustain
immune pressure during recrudescence of M. tuberculosis infection.
Author Contributions
Conceived and designed the experiments: ID NA N-JH PR VJFQ MJ.
Performed the experiments: ID RK NA BS LF MJ. Analyzed the data: ID
RK NA N-JH PR BS VJFQ MJ. Contributed reagents/materials/analysis
tools: VJFQ MJ. Wrote the paper: ID RK NA N-JH PR BS VJFQ MJ.
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