Download Local Immune Responses in Human Tuberculosis: Learning From

SUPPLEMENT ARTICLE
Local Immune Responses in Human
Tuberculosis: Learning From the Site of Infection
Susanna Brighenti1 and Jan Andersson1,2
1Center for Infectious Medicine, and 2Division of Infectious Diseases, Department of Medicine, Karolinska Institutet, Karolinska University Hospital
Huddinge, Stockholm, Sweden
Host–pathogen interactions in tuberculosis should be studied at the disease site because Mycobacterium
tuberculosis is predominately contained in local tissue lesions. Although M. tuberculosis infection involves
different clinical forms of tuberculosis, such as pulmonary tuberculosis, pleural tuberculosis, and lymph node
tuberculosis, most studies of human tuberculosis are performed using cells from the peripheral blood, which
may not provide a proper reflection of the M. tuberculosis–specific immune responses induced at the local site of
infection. A very low proportion of M. tuberculosis–specific effector T cells are found in the blood compared
with the infected tissue, and thus there may be considerable differences in the cellular immune response and
regulatory mechanisms induced in these diverse compartments. In this review, we discuss differences in the
immune response at the local site of infection compared with the peripheral circulation. The cell types and
immune reactions involved in granuloma formation and maintenance as well as the in situ technologies used to
assess local tuberculosis pathogenesis are also described. We need to strengthen and improve the exploratory
strategies used to dissect immunopathogenesis in human tuberculosis with the aim to accelerate the
implementation of relevant research findings in clinical practice.
Decades of immunological studies on tuberculosis,
both in humans and animal models, have identified
a number of immune mechanisms potentially involved
in protection against Mycobacterium tuberculosis infection. Despite this progress, studies of immune responses in tuberculosis, including local production of
inflammatory mediators and induction of different
immune cells involved in disease progression, have
lagged behind, particularly in humans. Most studies
on human tuberculosis involve cells from the peripheral blood, which may not provide a representative
image of the specific immune responses present at the
site of the infected organ or in the microenvironment
of the granulomatous lesions. To explore pathogenesis
in human tuberculosis with the aim to develop new
strategies for prevention and treatment of disease, we
Correspondence: Susanna Brighenti, PhD, Department of Medicine, Center for
Infectious Medicine (CIM), Karolinska Institutet, Karolinska University Hospital
Huddinge, 141 86 Stockholm, Sweden ([email protected]).
The Journal of Infectious Diseases 2012;205:S316–24
Ó The Author 2012. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
[email protected]
DOI: 10.1093/infdis/jis043
S316
d
JID 2012:205 (Suppl 2)
d
Brighenti & Andersson
must intensify research to study the important host–
pathogen interactions at the local site of M. tuberculosis
infection. For this purpose, ex vivo and in situ studies on
relevant clinical materials obtained from M. tuberculosis–
infected patients should be given greater attention
and be properly compared with the vast amount of
data available from experimental animal models and
in vitro cell culture systems. In this review, we describe current knowledge regarding cellular immune
effector functions in human tuberculosis and discuss
the contribution and value of studies performed at
the local site of infection.
CELLULAR IMMUNE EFFECTOR
MECHANISMS IN HUMAN
TUBERCULOSIS
Cellular immunity and, in particular, T-cell–mediated
responses are central in the regulation of specific host–
M. tuberculosis interactions. Protective immunity in
tuberculosis has been shown to be dependent on Th1
CD41 T cells producing interferon c (IFN-c) and tumor
necrosis factor a (TNF-a) as well as cytolytic T cells
(CTLs) producing granule-associated cytolytic
effector molecules such as perforin, granzymes, and granulysin
[1]. Upon antigen-specific T-cell activation, effector cytokines are
produced that promote macrophage activation and control of M.
tuberculosis growth, mainly through the production of nitric
oxide (NO) and antimicrobial peptides including human
cathelicidin, LL37 [1]. These innate and adaptive antimicrobial effector functions are regulated by a complex network of
cells and immune mediators that may be negatively affected by
microbe-specific virulence factors. M. tuberculosis–infected
macrophages that fail to eradicate the bacteria recruit T cells to
the area of infection and promote the generation of chronic
inflammation and formation of granulomas in an attempt to
wall off and contain the infection [2].
Importantly, the onset of adaptive immunity in human
tuberculosis is delayed compared with other infections,
which allows the bacterial load in the lung to expand significantly at the early stages of infection [3]. Studies from the
site of tuberculosis infection in the murine lung have demonstrated defective trafficking of M. tuberculosis–infected
dendritic cells (DCs) to the lung-draining lymph nodes
where antigen-specific T cells are primed [4]. Furthermore,
virulent mycobacteria may delay T-cell responses by inhibiting
apoptosis of infected macrophages, which will reduce crosspresentation of M. tuberculosis antigens to bystander DCs
and subsequent priming of T cells [5]. There is also evidence
that early induction of regulatory T (Treg) cells delay local
effector T-cell responses in the lung [6], either by inhibition
of DC function or by direct suppression of T-cell effector
functions. Induction of suppressive immunoregulatory pathways, including excessive Th2 responses, could disturb the
balance of protective host immunity and result in progression of tuberculosis disease.
IMMUNE RESPONSES AT THE LOCAL SITE OF
M. TUBERCULOSIS INFECTION VERSUS THE
SYSTEMIC CIRCULATION
In chronic infections such as tuberculosis, it is of significant
relevance to study host–pathogen interactions in the infected
tissue because effector T cells are recruited to and accumulate
at the local site of bacterial replication [7–11]. Immune cells
use the bloodstream as a transportation system to traffic into
lymphoid and peripheral tissues in response to microbial
antigens, where their true activated morphology and function
are demonstrated. Blood is often sampled to study pathological conditions because it is easily accessible, although
functional analysis of peripheral blood mononuclear cells
(PBMCs) normally requires antigen-specific restimulation in
vitro. Importantly, circulating lymphocytes in the blood
represent only about 2% of the total lymphocyte pool,
whereas most lymphocytes are confined to lymphoid organs
but are also found in nonlymphoid tissues such as the lung
and bone marrow during steady-state conditions [12]. During
an infection, most microbe-specific T cells migrate to the local
tissue site in response to chemoattractant signals, where they
expand and exert their effector functions. Such compartmentalization of T-cell responses also seems to be the result of
preferential homing of activated cells back to their inductive
sites. These activated effector T-cell populations can be
studied without in vitro restimulation or other manipulation.
For the reasons summarized in Table 1, immune responses
detected in the peripheral circulation may be different
compared with those in the disease sites, which underlines
the importance of using complementary methods to assess
systemic and local immune responses. Accordingly, a number of important studies on human tuberculosis have reported significant differences in T-cell responses at the site of
M. tuberculosis infection compared with blood. Most of these
experiments have taken advantage of cells and fluids from
the pleura or bronchoalveolar lavage (BAL), whereas available data from M. tuberculosis–infected tissue, such as tissue
from the lung, pleura, and lymph nodes, are more limited.
Previous studies of tuberculosis patients demonstrate similar
T-lymphocyte subset profiles in lung tissue and BAL [13] as
well as in pleural biopsies and pleural effusions [8], indicating that BAL and pleural fluid samples accurately reflect
cell populations present in granulomatous lesions.
Accumulation of IFN-g–Producing CD41 T cells at the Site of
M. tuberculosis Infection
Compared to peripheral blood, a general increase in the
proportion of total CD31 T cells and particularly IFNc–producing CD41 T-cell subsets can be found in pleural
effusions from patients with a local tuberculosis pleuritis
[14–16]. Interestingly, the purified protein derivative response of peripheral blood T cells from pleural tuberculosis
patients was lower than from pleural fluid T cells but also
lower than from blood T cells from healthy individuals [17].
Furthermore, compartmentalization of ESAT-6–specific,
IFN-c–producing T cells has been shown to be highly enriched to about 15-fold in both lung [9, 18] and pleura [7]
compared with blood of patients with active tuberculosis.
These findings are consistent with other clinical observations
that M. tuberculosis–specific IFN-c–producing CD41 T cells
were markedly elevated at the site of tuberculosis disease
[8, 10, 11, 19, 20]. Similarly, CD41 T cells are collected at
M. tuberculosis–infected sites in the lungs of both macaques
[21] and mice [22]. These results confirm that potent effector T cells accumulate at the site of tissue inflammation in
vivo and are only present in low levels among peripherally
circulating lymphocytes. This selective accumulation is likely
a result of both active recruitment and local expansion of
T cells at sites of bacterial replication. Importantly, the antigen specificity of M. tuberculosis–reactive T cells has been
Immune Responses to M. tuberculosis in Humans
d
JID 2012:205 (Suppl 2)
d
S317
Table 1. Differences in Local and Systemic Immune Responses
Local Site of Infection
Systemic Circulation
Cells kept in a physiological milieu in the presence
of stromal cells and soluble factors
Lack of 3-dimensional tissue–organ structure
Close cellular interactions, paracrine signaling,
granuloma formation, necrosis, and caseation in the tissue
No granuloma formation or other organized cellular interactions
Presence of M. tuberculosis
bacilli and infected cells
Lack of M. tuberculosis bacilli and infected cells (except for miliary
tuberculosis patients)
Compartmentalization of different immune cell subsets
Migration of naive immune cells to local sites
Morphological modifications of immune cells including
epitheliod and giant cells
No epitheliod or giant cell formation
Tissue macrophages with high bactericidal activity or
regulatory function
Undifferentiated monocytes with low bactericidal activity
High frequencies of in vivo–activated M. tuberculosis–specific
T cells that express different effector functions
Low frequencies of M. tuberculosis–specific T cells that require in
vitro restimulation with antigen to become activated
Snapshot of a specific temporal window of tuberculosis disease
Easily accessible clinical samples, possible to perform
longitudinal analysis
shown to be significantly different in the lung versus blood of
pulmonary tuberculosis patients [23]. Additionally, differences in bactericidal activity of alveolar macrophages and
blood monocytes of these tuberculosis patients emphasize
that there is a fundamental compartmentalization of immune effector cells in the M. tuberculosis–infected lung [23].
Differences in Cytokine Profiles at the Site of M. tuberculosis
Infection Versus the Peripheral Blood
Mycobacterium tuberculosis–specific Th1 and Th17 cells activate antimicrobial effector functions in infected macrophages as well as in M. tuberculosis–specific CTLs to promote
innate and adaptive bacterial killing and aid containment of
tuberculosis infection. However, elevated IFN-c levels and
augmented apoptosis of cells in the pleural space compared
with peripheral blood may suggest that immune activation
and loss of M. tuberculosis–specific T cells occur concomitantly, thus favoring persistence of M. tuberculosis locally at
the site of infection [16]. This is consistent with the findings,
which demonstrate enhanced IFN-c and interleukin 2 (IL-2)
production but also elevated interleukin 10 (IL-10) levels in
pleural fluid versus serum [24]. Similarly, a significant rise in
IFN-c as well as IL-10 [25] or interleukin 4 (IL-4) [26] levels
was found in culture supernatants from M. tuberculosis–
stimulated BAL cells, but not in PBMCs, obtained from
patients with pulmonary tuberculosis. Patients with miliary
tuberculosis (extensive tuberculosis disease), instead, possessed significantly lower IFN-c levels but higher IL-4 levels
in BAL fluid cells compared with the peripheral blood [27].
Correspondingly, a predominant IFN-c response in BAL CD41
T cells was observed in patients with noncavitary tuberculosis
(mild tuberculosis disease), whereas IL-4 levels were relatively
higher in cavitary tuberculosis (moderate–advanced tuberculosis
disease) [28]. These results suggest that there may be a gradual
dysregulation of the local cytokine profile toward a Th2
S318
d
JID 2012:205 (Suppl 2)
d
Brighenti & Andersson
response, especially in patients with severe forms of tuberculosis disease [27].
Induction of Immunoregulatory Mechanisms at the Site of
M. tuberculosis Infection
Inflammatory responses, including IFN-c, interleukin 12
(IL-12), and Toll-like receptor 2 (TLR2), but low levels of
NO synthase in cells from sputum of active tuberculosis
patients were found to be associated with anti-inflammatory
mediators such as IL-10, Suppressors of cytokine signaling
(SOCS), and Transforming growth factor (TGF-bRII) at the
local site [29]. In this regard, both human and experimental
animal models provide evidence that Treg cells redistribute
from the blood to the lungs and draining lymph nodes upon
M. tuberculosis infection, where they are retained within
granulomas along with effector T cells [21, 22, 30]. A significantly higher proportion of CD41CD251FoxP31 Treg
cells in pleural fluid compared with peripheral blood of tuberculosis patients was also found to be inversely correlated
with local IFN-c production [31]. Hence, although Treg cells
migrate to sites of bacterial replication to modulate local
inflammation and prevent tissue pathology, inhibition of
IFN-c production could also prevent the induction of important antimicrobial effector functions.
Altogether, these results illustrate that in patients with active tuberculosis a cellular immune response is mounted and
organized primarily at the inflammatory site where a productive M. tuberculosis infection has been established. The
presence of anti-inflammatory cytokines or other negative
regulators may compete with and counteract innate as well as
Th1-mediated immunity at infected sites. Compartmentalization of mixed Th1 and immunosuppressive responses at the
site of disease could provide important clues to elucidate the
mechanisms responsible for impaired tuberculosis control
that would not be possible to detect only from studies of the
Figure 1. Schematic illustration of cells and effector molecules at the local site of Mycobacterium tuberculosis infection. (1) Upon infection, activated
monocytes and effector T cells as well as regulatory T (Treg) cells migrate from the blood and accumulate in the area of bacterial replication. The proportion
of M. tuberculosis–specific effector T cells producing cytokines and antimicrobial effector molecules are significantly higher at the disease sites compared
with the circulation. (2) M. tuberculosis infection induces both morphological and functional changes of immune cells present at the disease sites. M.
tuberculosis–infected macrophages can transform into epitheliod cells and also fuse to form multinucleated giant cells (MGCs). Classically activated
macrophages (CAMs) are more bactericidal and control M. tuberculosis replication better than do alternatively activated macrophages (AAMs). Secretion of
chemokines and cytokines result in expansion and activation of M. tuberculosis–specific T cells with different effector functions. Stromal cells (ie, epithelial
cells and fibroblasts) present in the tissue may also induce regulatory components in the local millieu at the site of infection. (3) An organized collection of
tightly clustered M. tuberculosis–infected macrophages forms the core of the tuberculosis granuloma. Chemokines and Th1 cytokines participate in the
recruitment of T cells, monocytes, and other immune cells that are contained in the mature granuloma. (4) Extensive inflammation in the granulomatous area
triggers cellular apoptosis that contribute to central necrosis (yellow mass ) and extracellular growth of M. tuberculosis (pink rods ). Excessive Th1-mediated
immune responses could also become potentially harmful and lead to uncontrolled immune activation. (5) The ratios of Th1/Th2 cells, CAMs/AAMs, and
effector T (Teffector)/Treg cells are of vital importance to maintain the immunological balance in the tissue and prevent the progression of tuberculosis
disease. A shift in the local immune response will tip the balance toward suboptimal immunity and impaired control of tuberculosis disease but can also
result in excessive cellular immunity and tissue destruction. Abbreviations: IL-2, interleukin 2; IL-4, interleukin 4; IL-10, interleukin 10; IL-13, interleukin 13;
iNOS, inducible nitric oxide synthase; LL-37, TGF-b, transforming growth factor-b; TNF-a, tumor necrosis factor a.
systemic circulation. Importantly, age-related differences in
the immune system seen in children and adults will affect
the susceptibility and outcome of tuberculosis infection [32].
Thus, assessment of local immune responses across the varying
spectrum of tuberculosis disease at different ages may unravel
valuable information about some of the host-specific factors
that contribute to immunopathogenesis in clinical tuberculosis.
LOCAL IMMUNE RESPONSES IN THE
GRANULOMA AT THE SITE OF
M. TUBERCULOSIS INFECTION
Chronic tuberculosis gives rise to a granulomatous inflammation involving morphological and functional changes of
immune cells, which creates a range of local microenvironments
in infected organs that must be considered upon analysis of
M. tuberculosis–specific immune responses (Figure 1) [2]. Formation of granulomas is a typical hallmark of human tuberculosis and is defined as an organized collection of immune cells
with the aim to contain M. tuberculosis infection. However,
pathogenic mycobacteria may also exploit early granuloma formation and promote spreading of bacteria to uninfected macrophages that are recruited to the infected area [33]. Hence, the
granuloma may have a beneficial function for the host as well as
the bacteria, depending on the stage of infection.
Macrophage Activation and Granuloma Formation in
M. tuberculosis–Infected Tissue
Initial granuloma formation is characterized by continuous
activation of M. tuberculosis–infected macrophages, which
Immune Responses to M. tuberculosis in Humans
d
JID 2012:205 (Suppl 2)
d
S319
Figure 2. Inducible nitric oxide synthase (iNOS) expression in human tuberculosis. Alveolar macrophages in the Mycobacterium tuberculosis–infected
lung and macrophages present in lymph node granulomas (solid line ) express CD68 and high levels of iNOS. In contrast, iNOS and nitric oxide (NO) are
very difficult to induce in human macrophages in vitro. Note the confluent granulomas in the cross-section of a lymph node (lower left image ).
Immunohistochemistry was used to show positive staining (brown; diaminobenzidine staining) and negative staining (blue; nuclear hematoxylin staining).
Magnification, 350 and 3125, respectively. Tissue samples from tuberculosis lung lesions were obtained from human immunodeficiency virus
(HIV)–negative adults with chronic, active noncavitary tuberculosis disease [35], whereas tuberculosis lymph nodes were collected from HIV-negative
children with a local tuberculosis lymphadenitis (neck region) [36]. Complete tables including clinical and bacteriological demographics of tuberculosis
patients have been published elsewhere [35, 36].
induces the cells to adhere closely together, assuming an
epitheliod shape and sometimes fusing to form multinucleated giant cells with a yet unknown functional role. Evidently, macrophages could enter different differentiation
programs, depending on the organ-specific location as well
as the microbial stimuli [34]. Here, classically activated
macrophages (CAMs) produce NO and are highly bactericidal, whereas alternatively activated macrophages (AAMs)
can produce immunosuppressive cytokines such as IL-10
and TGF-b and are poorly microbicidal. It is very difficult to
induce NO production in human blood–derived macrophages in vitro, even though NO expression can be detected
in macrophages in vivo in lung [35] and lymph nodes [36] of
tuberculosis patients (Figure 2). Whereas IFN-c promotes
classical macrophage activation and a hostile milieu in the
tuberculosis granuloma, Th2 cytokines, including IL-4 and
interleukin 13 (IL-13), could promote alternative macrophage activation characterized by arginase activation and
collagen deposition in the inflamed tissue [37], which are
typical traits of advanced tuberculosis disease. Importantly,
inducible NO synthase (iNOS) and arginase compete for the
same substrate in activated macrophages to produce NO or
collagen, respectively. Accordingly, it was recently described
that initial induction of iNOS-expressing CAMs was followed
by arginase-expressing AAMs in the lung, which would support a switch in macrophage polarization upon progression
of tuberculosis disease [38]. A shift in the CAM/AAM ratio in
S320
d
JID 2012:205 (Suppl 2)
d
Brighenti & Andersson
human tuberculosis granulomas may modulate T-cell effector functions and/or promote the expansion of Treg cells
that reduce the ability of the host to control M. tuberculosis
infection [2].
Granulomas are highly dynamic structures with multiple
appearances in infected organs during active tuberculosis
disease, including solid nonnecrotizing and caseous necrotic
granulomas [36, 39]. These heterogenous lesions are perceived
as palpable nodules in the infected organs, and the vigorous
inflammation induced at the site of infection could ultimately
destroy and displace the normal surrounding tissue. Upon
progressive disease, small, early cellular aggregates of nonnecrotic granulomas will advance to form large, mature necrotic granulomas with little cellular content [40]. Numerous
extracellular bacteria persist in the caseous necrotic fluid that
will drain into the infected tissue upon rupture of mature
granulomas [41]. Efficient spread of the infection is accomplished when viable bacteria reach the airways and are expelled from pulmonary tuberculosis patients as contagious
aerosols. Thus, extensive apoptosis in caseating granulomas
may also contribute to and enhance the immunopathogenesis
of tuberculosis [42]. Consequently, even though macrophage
and Th1-cell responses are important to induce inflammation
and immune protection in tuberculosis, uncontrolled activation of these cells results in massive necrosis and loss of
normal tissue architecture, which can lead to cavity formation
in pulmonary tuberculosis.
Figure 3. Impaired cytolytic T-cell (CTL) responses in human tuberculosis granuloma. Low expression of CD81 CTLs and the cytolytic and antimicrobial
effector molecules perforin and granulysin in serial cryosections of a human lymph node granuloma (solid line ). In situ immunohistochemical images show an
abundance of CD681 macrophages and CD41 T cells surrounding the necrotic core of the granuloma. A multinucleated giant cell (MGC) with nuclei in
a characteristic horseshoe shape is also depicted in the tissue. In contrast to the low expression of cytolytic effector molecules inside the granuloma,
granzyme A, perforin, and granulysin can be detected in the parafollicular areas of the lymph node. Note that there is no expression of perforin and granulysin
in the B-cell follicles (Bc foll), which do not contain any CD81 T cells. Positive staining (brown; diaminobenzidine staining) and negative staining (blue; nuclear
hematoxylin staining) are shown. Arrows depict some of the positive cells in the images with a low level of positive staining. Magnification, 3125.
Tuberculosis lymph node tissues were collected from human immunodeficiency virus (HIV)–negative children with a local tuberculosis lymphadenitis (neck
region) [36]. Complete tables including clinical and bacteriological demographics of tuberculosis patients have been published elsewhere [36].
Effector Cell Responses and Immunoregulation in the
Tuberculosis Granuloma
The specific host and bacterial factors that regulate granuloma
development and function are still poorly defined. Abundant
coexpression of IFN-c–inducible chemokines, as well as IFN-c
and TNF-a with M. tuberculosis 16S RNA in pulmonary granulomas, suggests that continuous cell recruitment and chronic
inflammation are involved in granuloma formation and maintenance [43]. Accordingly, Th17 cells promote infiltration of
IFN-c–producing CD41 T cells in the lung and support proper
granuloma formation at the site of infection [44]. Fenhalls et al
used in situ hybridization to demonstrate mixed expression of
IFN-c and IL-4 transcripts in granulomas, whereas high levels of
TNF-a were always associated with necrotic granulomas
[45]. T-cell–produced cytokines were absent in necrotic
granulomas, which supports the assumption that T-cell responses are downregulated upon progressive tuberculosis disease
[46]. A negative association between IL-4 and TLR2 expression
in pulomonary granulomas also implies that Th2 cytokines could
counteract important TLR-activating signals [47].
Using in situ image analysis, we have previously discovered
that the abundance of CD81 CTLs expressing the important
antituberculosis effector molecules perforin and granulysin is
very low in human granulomatous lesions both in lung [35] and
lymph nodes [36], indicating that CTL activation is impaired at
the site of bacterial persistence (Figure 3). Th1 cytokines,
including IFN-c, TNF-a, and interleukin 17, were not upregulated in M. tuberculosis–infected lymph nodes obtained from
Immune Responses to M. tuberculosis in Humans
d
JID 2012:205 (Suppl 2)
d
S321
patients with ongoing disease, whereas there was a significant
induction of TGF-b and IL-13. Several studies have shown that
the predominating lymphocyte population in the core of the
granuloma is memory CD41 T cells, whereas a lower number of
CD81 T cells are mainly located in the outer lymphocytic mantel
surrounding the tuberculosis granuloma [8, 41, 48]. One
hypothesis is that IFN-c–producing CD41 T cells could be involved in macrophage activation and macrophage-mediated
elimination of bacteria in the center of the granuloma.
However, we found that the levels of CD41FoxP31 Treg cells
and TGF-b were significantly elevated in the tuberculous granulomas, suggesting that a substantial proportion of the CD41 T
cells may be Treg cells and not activated effector T cells [36].
Such local compartmentalization of T-cell responses may prevent
the expansion and activation of CD81 CTLs and also contactdependent killing of M. tuberculosis–infected cells in the center of
the granuloma [39]. As described above, M. tuberculosis–specific,
IFN-c–secreting T cells but also FoxP31 Treg cells were
previously found to be particularly concentrated at the disease
sites compared with matched blood samples from patients with
different clinical forms of tuberculosis [30, 31]. Interestingly,
stromal cells in the tissue have been shown to direct local differentiation of regulatory DCs that can induce IL-10–producing
Treg cells and suppress imperative T-cell responses, especially in
the presence of an intracellular infection [49]. Thus, Treg-mediated suppression of effector responses may be significantly
enhanced in the microenvironment of the infected tissue. Similar
to a Th1/Th2 or CAM/AAM shift, a shift in the effector T-cell/
Treg cell ratio could promote immunosuppressive signals and
bacterial persistence in the tuberculosis lesions, providing a
protective survival niche for the bacteria (Figure 1).
QUANTITATIVE IN SITU IMAGE ANALYSIS TO
MEASURE IMMUNE RESPONSE AT THE LOCAL
SITE OF M. TUBERCULOSIS INFECTION
To increase the understanding of pathogenesis in human tuberculosis, it is necessary to develop new models and refine existing
technology to explore effector mechanisms at the site of infection
in general and in the tuberculosis granuloma in particular. In
contrast to peripheral blood, in situ image analysis of patient
tissue samples provides the opportunity to study the spatial
anatomical expression of different proteins and the organ-specific
cell–cell interactions in local compartments where the numbers of
pathogen responder cells are high. Immunocytochemical staining
and in situ quantitative computerized image analysis enable local
assessment of immune cells and effector molecules in cryopreserved cells and tissues. Complementary analysis of protein
and messenger RNA (mRNA) expression in tissue using in situ
image analysis, quantitative polymerase chain reaction, and in situ
hybridization can be combined with the corresponding analysis of
blood and fluids or homogenized tissue samples using multicolor
S322
d
JID 2012:205 (Suppl 2)
d
Brighenti & Andersson
flow cytometry, multiplex protein, and mRNA analyses. All these
methodologies require a minimal amount of cells or tissue, which
enables complex studies of very rare clinical materials.
The common view is that in situ techniques are descriptive in
nature, but basically it is possible to analyze the functional
expression and distribution of mRNA and protein in the context
of a physiologically relevant milieu that is difficult to reproduce
in cell-culture models. Protein expression can be quantified at
the single-cell level using microscopy and a highly sensitive
digital image analysis system with the ability to detect and
separate 16.7 million different colors (Figure 4) [35, 36]. This is
a well-established quantitative method that has been extensively
used primarily in humans and nonhuman primates for analysis
of a wide range of proteins, including cell surface and secreted
proteins and cytoplasmic, granule-associated, or nuclear
proteins, to describe the phenotype and function of different cell
types present in tissue. Multicolor staining can be used to study
coexpression of proteins in the immunological synapse. In
addition, identification of mycobacterial DNA or RNA transcripts
[43] as well as mycobacterial proteins [36] within human tuberculosis granulomas offers the advantage of gauging the spatial
interplay between mycobacteria and local host cells and relating
certain expression profiles to bacterial persistence and progression
of disease. Thus, this methodology provides important
information about the regulation of immune responses in
the microenvironment of the tuberculosis granuloma that
complements the knowledge gained from animal and in vitro
experiments. The development of novel humanized tissue culture
systems and the implementation of system biology approaches
involving assessments of genes and proteins affected by this
microbial invasion will further improve our opportunities
to dissect relevant signalling pathways at M. tuberculosis–
infected sites.
CLINICAL RELEVANCE OF STUDIES AT THE
LOCAL SITE OF M. TUBERCULOSIS INFECTION
A major challenge to the scientific community is to improve
diagnostic methods that can discriminate between active and
latent tuberculosis. Importantly, the accumulation of effector T cells at the site of infection can be used for an accurate
and rapid immunodiagnosis of active tuberculosis using
M. tuberculosis–specific IFN-c release assays (IGRAs). Here, it
has been demonstrated that cells from the site of M. tuberculosis
infectiondthat is, BAL [18, 50] or pleura fluid cells [11]dcan be
successfully used to increase the sensitivity of an M. tuberculosis–
specific enzyme-linked immunosorbent spot assay (ELISPOT)
to distinguish sputum smear–negative active tuberculosis
from latent tuberculosis cases. In contrast, low levels of effector memory T cells may persist in the blood of individuals
with active as well as latent tuberculosis, and, consequently,
IGRAs cannot differ between active and latent tuberculosis
detection of active tuberculosis in routine clinical practice in
countries with low tuberculosis incidence.
In vaccinology, evaluation of M. tuberculosis–specific immune
responses at the site of infection also provides unique
information about novel biomarkers or immune correlates that
can be used to monitor vaccine efficacy, as vaccination may
change the frequency, phenotype, and functional properties of
immune cells in local sites compared with blood. Many
studies also suggest that the type of immune response detected
at the site of infection strongly correlates with different
clinical symptoms and severity of disease or disease outcome.
Enhanced knowledge of the cellular responses involved in
protective immunity as well as disease progression in human
tuberculosis will generate superior insights into bacterial
pathogenesis and facilitate the implementation of relevant
findings in clinical practice.
Notes
Financial support. This work was supported by the Swedish Research
Council; the Heart and Lung Foundation; the Swedish International Development Cooperation Agency; the Swedish Society for Medical Research;
the Swedish Foundation for Strategic Research; and the von Kantzow
Foundation.
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
References
Figure 4. Illustration of quantitative in situ image analysis. A blank
immunohistochemical image showing CD31 T cells in a small nonnecrotic
lymph node granuloma is used to set the threshold for the positive staining
(diaminobenzidine, brown ). Next, the total cellular area (hematoxylin, blue )
is depicted, and thereafter it is possible for the computer software to
determine the percentage of positive area of the total cell area. A summary
of the field statistics is as follows: Field number 1; Total area measured
(lm2): 1.29e1005; cell area measured (lm2): 51116.54; percentage of cell
area in the total area: 45.56; stained area measured (lm2): 8062.68;
percentage of stained area in the cell area: 20.77; mean intensity of the
positive area: 131.50; total intensity of the positive area: 1.06. Normally,
average values from 10–50 microscopic fields of a tissue section will be
included in the analysis. Magnification, 3125.
when performed on PBMCs alone [11, 18, 50]. Local immunodiagnosis using ELISPOT is an important advancement for
1. Brighenti S, Andersson J. Induction and regulation of CD81 cytolytic
T cells in human tuberculosis and HIV infection. Biochem Biophys
Res Commun 2010; 396:50–7.
2. Flynn JL, Chan J, Lin PL. Macrophages and control of granulomatous
inflammation in tuberculosis. Mucosal Immunol 2011; 4:271–8.
3. Urdahl KB, Shafiani S, Ernst JD. Initiation and regulation of T-cell
responses in tuberculosis. Mucosal Immunol 2011; 4:288–93.
4. Wolf AJ, Desvignes L, Linas B, et al. Initiation of the adaptive immune
response to Mycobacterium tuberculosis depends on antigen production
in the local lymph node, not the lungs. J Exp Med 2008; 205:105–15.
5. Divangahi M, Desjardins D, Nunes-Alves C, Remold HG, Behar SM.
Eicosanoid pathways regulate adaptive immunity to Mycobacterium
tuberculosis. Nat Immunol 2010; 11:751–8.
6. Shafiani S, Tucker-Heard G, Kariyone A, Takatsu K, Urdahl KB.
Pathogen-specific regulatory T cells delay the arrival of effector T cells
in the lung during early tuberculosis. J Exp Med 2010; 207:1409–20.
7. Wilkinson KA, Wilkinson RJ, Pathan A, et al. Ex vivo characterization
of early secretory antigenic target 6–specific T cells at sites of active
disease in pleural tuberculosis. Clin Infect Dis 2005; 40:184–7.
8. Barnes PF, Mistry SD, Cooper CL, Pirmez C, Rea TH, Modlin RL.
Compartmentalization of a CD41 T lymphocyte subpopulation in
tuberculous pleuritis. J Immunol 1989; 142:1114–9.
9. Jafari C, Ernst M, Strassburg A, et al. Local immunodiagnosis of pulmonary tuberculosis by enzyme-linked immunospot. Eur Respir J
2008; 31:261–5.
10. Schwander SK, Torres M, Sada E, et al. Enhanced responses to Mycobacterium tuberculosis antigens by human alveolar lymphocytes during
active pulmonary tuberculosis. J Infect Dis 1998; 178:1434–45.
11. Nemeth J, Winkler HM, Zwick RH, et al. Recruitment of Mycobacterium tuberculosis specific CD41 T cells to the site of infection for diagnosis of active tuberculosis. J Intern Med 2009; 265:163–8.
Immune Responses to M. tuberculosis in Humans
d
JID 2012:205 (Suppl 2)
d
S323
12. Blum KS, Pabst R. Lymphocyte numbers and subsets in the human
blood. Do they mirror the situation in all organs? Immunol Lett 2007;
108:45–51.
13. Law KF, Jagirdar J, Weiden MD, Bodkin M, Rom WN. Tuberculosis in
HIV-positive patients: cellular response and immune activation in the
lung. Am J Respir Crit Care Med 1996; 153:1377–84.
14. Shiratsuchi H, Tsuyuguchi I. Analysis of T cell subsets by monoclonal
antibodies in patients with tuberculosis after in vitro stimulation with
purified protein derivative of tuberculin. Clin Exp Immunol 1984;
57:271–8.
15. Ribera E, Ocana I, Martinez-Vazquez JM, Rossell M, Espanol T, Ruibal A.
High level of interferon gamma in tuberculous pleural effusion. Chest
1988; 93:308–11.
16. Hirsch CS, Toossi Z, Johnson JL, et al. Augmentation of apoptosis
and interferon-gamma production at sites of active Mycobacterium
tuberculosis infection in human tuberculosis. J Infect Dis 2001; 183:
779–88.
17. Fujiwara H, Okuda Y, Fukukawa T, Tsuyuguchi I. In vitro tuberculin
reactivity of lymphocytes from patients with tuberculous pleurisy.
Infect Immun 1982; 35:402–9.
18. Jafari C, Thijsen S, Sotgiu G, et al. Bronchoalveolar lavage enzymelinked immunospot for a rapid diagnosis of tuberculosis: a Tuberculosis Network European Trials group study. Am J Respir Crit Care Med
2009; 180:666–73.
19. Place S, Verscheure V, de San N, et al. Heparin-binding, hemagglutininspecific IFN-gamma synthesis at the site of infection during active
tuberculosis in humans. Am J Respir Crit Care Med 2010; 182:848–54.
20. Walrath J, Zukowski L, Krywiak A, Silver RF. Resident Th1-like
effector memory cells in pulmonary recall responses to Mycobacterium tuberculosis. Am J Respir Cell Mol Biol 2005; 33:48–55.
21. Green AM, Mattila JT, Bigbee CL, Bongers KS, Lin PL, Flynn JL. CD4(1)
regulatory T cells in a cynomolgus macaque model of Mycobacterium
tuberculosis infection. J Infect Dis 2010; 202:533–41.
22. Scott-Browne JP, Shafiani S, Tucker-Heard G, et al. Expansion and
function of Foxp3-expressing T regulatory cells during tuberculosis.
J Exp Med 2007; 204:2159–69.
23. Sable SB, Goyal D, Verma I, Behera D, Khuller GK. Lung and blood
mononuclear cell responses of tuberculosis patients to mycobacterial
proteins. Eur Respir J 2007; 29:337–46.
24. Barnes PF, Lu S, Abrams JS, Wang E, Yamamura M, Modlin RL.
Cytokine production at the site of disease in human tuberculosis.
Infect Immun 1993; 61:3482–9.
25. Gerosa F, Nisii C, Righetti S, et al. CD4(1) T cell clones producing
both interferon-gamma and interleukin-10 predominate in bronchoalveolar lavages of active pulmonary tuberculosis patients. Clin
Immunol 1999; 92:224–34.
26. Herrera MT, Torres M, Nevels D, et al. Compartmentalized bronchoalveolar IFN-gamma and IL-12 response in human pulmonary
tuberculosis. Tuberculosis (Edinb) 2009; 89:38–47.
27. Sharma SK, Mitra DK, Balamurugan A, Pandey RM, Mehra NK.
Cytokine polarization in miliary and pleural tuberculosis. J Clin Immunol 2002; 22:345–52.
28. Mazzarella G, Bianco A, Perna F, et al. T lymphocyte phenotypic
profile in lung segments affected by cavitary and non-cavitary tuberculosis. Clin Exp Immunol 2003; 132:283–8.
29. Almeida AS, Lago PM, Boechat N, et al. Tuberculosis is associated with
a down-modulatory lung immune response that impairs Th1-type
immunity. J Immunol 2009; 183:718–31.
30. Guyot-Revol V, Innes JA, Hackforth S, Hinks T, Lalvani A. Regulatory
T cells are expanded in blood and disease sites in patients with
tuberculosis. Am J Respir Crit Care Med 2006; 173:803–10.
31. Chen X, Zhou B, Li M, et al. CD4(1)CD25(1)FoxP3(1) regulatory
T cells suppress Mycobacterium tuberculosis immunity in patients with
active disease. Clin Immunol 2007; 123:50–9.
S324
d
JID 2012:205 (Suppl 2)
d
Brighenti & Andersson
32. Donald PR, Marais BJ, Barry CE 3rd. Age and the epidemiology and
pathogenesis of tuberculosis. Lancet 2010; 375:1852–4.
33. Davis JM, Ramakrishnan L. The role of the granuloma in expansion
and dissemination of early tuberculous infection. Cell 2009; 136:37–49.
34. Day J, Friedman A, Schlesinger LS. Modeling the immune rheostat
of macrophages in the lung in response to infection. Proc Natl Acad
Sci U S A 2009; 106:11246–51.
35. Andersson J, Samarina A, Fink J, Rahman S, Grundstrom S. Impaired
expression of perforin and granulysin in CD81 T cells at the site of
infection in human chronic pulmonary tuberculosis. Infect Immun
2007; 75:5210–22.
36. Rahman S, Gudetta B, Fink J, et al. Compartmentalization of immune
responses in human tuberculosis: few CD81 effector T cells but
elevated levels of FoxP31 regulatory t cells in the granulomatous
lesions. Am J Pathol 2009; 174:2211–24.
37. Gordon S, Martinez FO. Alternative activation of macrophages:
mechanism and functions. Immunity 2010; 32:593–604.
38. Redente EF, Higgins DM, Dwyer-Nield LD, Orme IM, GonzalezJuarrero M, Malkinson AM. Differential polarization of alveolar
macrophages and bone marrow–derived monocytes following chemically and pathogen-induced chronic lung inflammation. J Leukoc Biol
2010; 88:159–68.
39. Kaplan G, Post FA, Moreira AL, et al. Mycobacterium tuberculosis
growth at the cavity surface: a microenvironment with failed immunity. Infect Immun 2003; 71:7099–108.
40. Fenhalls G, Stevens L, Moses L, et al. In situ detection of Mycobacterium tuberculosis transcripts in human lung granulomas reveals
differential gene expression in necrotic lesions. Infect Immun 2002;
70:6330–8.
41. Ulrichs T, Kosmiadi GA, Trusov V, et al. Human tuberculous granulomas induce peripheral lymphoid follicle-like structures to orchestrate
local host defence in the lung. J Pathol 2004; 204:217–28.
42. Keane J, Balcewicz-Sablinska MK, Remold HG, et al. Infection by
Mycobacterium tuberculosis promotes human alveolar macrophage
apoptosis. Infect Immun 1997; 65:298–304.
43. Fuller CL, Flynn JL, Reinhart TA. In situ study of abundant
expression of proinflammatory chemokines and cytokines in
pulmonary granulomas that develop in cynomolgus macaques
experimentally infected with Mycobacterium tuberculosis. Infect Immun 2003; 71:7023–34.
44. Umemura M, Yahagi A, Hamada S, et al. IL-17-mediated regulation of
innate and acquired immune response against pulmonary Mycobacterium
bovis bacille Calmette-Guerin infection. J Immunol 2007; 178:3786–96.
45. Fenhalls G, Stevens L, Bezuidenhout J, et al. Distribution of
IFN-gamma, IL-4 and TNF-alpha protein and CD8 T cells producing
IL-12p40 mRNA in human lung tuberculous granulomas. Immunology 2002; 105:325–35.
46. Fenhalls G, Wong A, Bezuidenhout J, van Helden P, Bardin P, Lukey PT.
In situ production of gamma interferon, interleukin-4, and tumor
necrosis factor alpha mRNA in human lung tuberculous granulomas.
Infect Immun 2000; 68:2827–36.
47. Fenhalls G, Squires GR, Stevens-Muller L, et al. Associations between Toll-like receptors and interleukin-4 in the lungs of patients
with tuberculosis. Am J Respir Cell Mol Biol 2003; 29:28–38.
48. Gonzalez-Juarrero M, Turner OC, Turner J, Marietta P, Brooks JV,
Orme IM. Temporal and spatial arrangement of lymphocytes within
lung granulomas induced by aerosol infection with Mycobacterium
tuberculosis. Infect Immun 2001; 69:1722–8.
49. Svensson M, Maroof A, Ato M, Kaye PM. Stromal cells direct local
differentiation of regulatory dendritic cells. Immunity 2004; 21:
805–16.
50. Jafari C, Ernst M, Kalsdorf B, et al. Rapid diagnosis of smear-negative
tuberculosis by bronchoalveolar lavage enzyme-linked immunospot.
Am J Respir Crit Care Med 2006; 174:1048–54.