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* Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523; and
Trudeau Institute, Saranac Lake, NY 12983
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, has been documented in individuals diagnosed with active
tuberculosis. In addition, IL-10 production is increased within the
lungs of mice that have chronic mycobacterial infection. Therefore, we
hypothesized that the down-regulatory properties of IL-10 might
contribute to the reactivation of chronic Mycobacterium
tuberculosis infection in mice. To determine the influence of
IL-10 on the course of infection, transgenic mice producing increased
amounts of IL-10 under the control of the IL-2 promotor were infected
with M. tuberculosis via the respiratory route. Mice
that overexpressed IL-10 showed no increase in susceptibility during
the early stages of infection, but during the chronic phase of the
infection showed evidence of reactivation tuberculosis with a highly
significant increase in bacterial numbers within the lungs.
Reactivation was associated with the formation of macrophage-dominated
lesions, decreased mRNA production for TNF and IL-12p40, and a decrease
in Ag-specific IFN-
secretion. These data support the hypothesis
that IL-10 plays a pivotal role during the chronic/latent stage of
pulmonary tuberculosis, with increased production playing a potentially
central role in promoting reactivation
tuberculosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3
million individuals go on to develop active disease each year
(1). The factors that contribute to an individual’s
susceptibility to reactivate an apparently latent M.
tuberculosis infection are currently unknown, although
malnutrition and immunosupression are among some of the suggested
reasons (2, 3). A current area of active research is that
of determining genetic linkage for susceptibility to develop
reactivation tuberculosis. In man, several genes such as
Nramp1 (4, 5), 15q, and
Xq (6) have been linked to tuberculosis
suggesting that susceptibility cannot be conferred by a single gene
alone but is in fact multifactoral in nature. This theory is supported
by studies in the mouse model that have identified associations with
loci on chromosomes 3 and 9 (7), the H-2 locus
(8), and more recently the sst1 gene
(9), suggesting that susceptibility is very unlikely to be
mapped to a single gene. Therefore, the identification of immune
correlates of protection may be a more feasible approach with which to
identify the very large number of latently infected individuals who are
at risk of developing reactivation disease. Using mouse models of reactivation tuberculosis, we have previously demonstrated that mouse strains such as the CBA/J and DBA/2 fail to up-regulate the cell surface adhesion molecules CD11a and CD54 on their circulating lymphocytes during an infection with M. tuberculosis (10). The failure of circulating T cells to increase their expression of adhesion molecules correlated with the absence of lymphocyte foci within the lung granulomas. We hypothesized that this absence of lymphocytes contributed to the eventual breakdown of the lesion and reactivation of tuberculosis, and that the measurement of CD11a and CD54 expression on circulating lymphocytes might be a useful correlate of protection. It is likely that the failure to up-regulate the expression of these molecules is the consequence of an upstream event, although the mechanisms contributing to this event in the reactivation susceptible mouse strains are currently unknown.
In this regard, IL-10 is a cytokine that has been shown to have
immunosuppressive activity that may contribute to mycobacterial
disease. In the presence of IL-10, it has been shown that both T cell
proliferation and IFN- production is inhibited (11, 12, 13, 14)
and the action of IL-10 has been linked to its down-regulation of
macrophage activation. IL-10 can inhibit TNF and NO secretion
(15, 16), down-regulate the expression of costimulatory
molecules (11), and MHC class II (16, 17);
therefore, it compromises both macrophage microbicidal mechanisms and
Ag presentation. The ability of IL-10 to down-regulate immune responses
and the fact that IL-10 can be detected in tuberculosis patients
(18, 19) have led researchers to investigate whether IL-10
plays a role in susceptibility to tuberculosis. More specifically, it
has been suggested that the removal of IL-10 will enhance protective
immunity. Despite the known immunosuppressive properties of IL-10,
infection of IL-10 gene-disrupted mice with Mycobacterium
bovis bacillus Calmette-Guérin resulted in only a
small and transient increase in resistance to infection, and the
outcome of chronic disease was unaltered (20, 21). More
significantly, infection of IL-10 gene-disrupted mice with M.
tuberculosis resulted in either a minor increase in resistance
(22) or no increase at all (23). The studies
described used gene-disrupted mice that were bred onto a naturally
resistant mouse strain, capable of generating a vigorous immune
response during M. tuberculosis infection and showing no
signs of reactivation disease until well into old age (24, 25). In this regard, we hypothesized that if C57BL/6 IL-10
gene-disrupted mice were slightly more resistant to tuberculosis, the
overexpression of IL-10 may result in an increased susceptibility of
this mouse strain to infection perhaps more akin to the susceptible
mouse strains that we have previously characterized.
To determine whether increased IL-10 production can influence the
outcome of disease, we chose to study M. tuberculosis
infection in transgenic C57BL/6 mice that express IL-10 under the
control of the IL-2 promoter (IL-10 transgenic mice) (26).
These mice thus generate excess IL-10 when the IL-2 gene is induced,
such as occurs during the vigorous response seen in M.
tuberculosis-infected C57BL/6 mice. Our data demonstrate that the
increased production of IL-10 during an infection with M.
tuberculosis conferred a reactivation susceptible phenotype on the
naturally resistant C57BL/6 mouse strain. Lesions within the lungs of
IL-10 transgenic mice were dominated by macrophages, mRNA for TNF and
IL-12p40 was reduced in the lung, and the capacity of lung cells to
secrete Ag-specific IFN- was compromised. This phenotype closely
resembles that seen in the reactivation susceptible mouse strains. The
demonstration that lesions within the lungs of CBA/J mice infected with
M. tuberculosis contain substantial levels of IL-10 supports
the hypothesis that this cytokine plays a major role in the development
of reactivation tuberculosis in man.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These studies were performed using specific pathogen-free C57BL/6 and CBA/J mice (The Jackson Laboratory, Bar Harbor, ME) at 6–8 wk of age. The IL-10 transgenic mice were a kind gift from Drs. H. Cheroute and M. Kronenberg (La Jolla Institute for Allergy and Infectious Disease, La Jolla, CA) (26). Mice were kept in ABL-3 biohazard conditions throughout the study and maintained on sterile chow and water ad libitum. The specific pathogen-free nature of the mouse colonies was demonstrated by testing sentinel animals. These were shown to be negative for 12 known mouse pathogens. All experimental protocols were approved by the Colorado State University Animal Care and Users Committee.
Bacterial infections
M. tuberculosis strain Erdman was grown from low
passage seed lots in Proskauer-Beck liquid media containing 0.02%
Tween 80 to mid-log phase, then aliquoted and frozen at -70°C until
use. Mice were infected via the aerosol route with a low dose
(102) of bacteria. Briefly, the nebulizer
compartment of a Middlebrook airborne infection apparatus (Glas-col,
Terre Haute, IN) was filled with 5 ml of distilled water containing a
suspension of bacteria known to deliver 100 bacteria/lung. The
numbers of viable bacteria in the lungs were followed against time by
plating serial dilutions of individual partial organ homogenates onto
nutrient Middlebrook 7H11 agar and counting bacterial colony formation
after 21 days incubation at 37°C. The data were expressed as the
log10 value of the mean number of bacteria
recovered from four individual animals.
Histology
The right caudal lung lobe from each mouse (n = 4/group) was infused with 10% neutral-buffered formalin. Tissues were sectioned for light microscopy with lobe orientation designed to allow for the maximum surface area of each lobe to be seen. Sections were stained with H&E. Sections were examined by a veterinary pathologist without prior knowledge of the experimental groups, and evaluated at least twice to verify the reproducibility of the observations.
Immunohistochemistry
Formalin-fixed lung tissue was embedded in paraffin, and serial sections of tissue were cut 7-µm thick. Paraffin was removed using EZ-DeWax solution (BioGenex Laboratories, San Ramon, CA), and the Ag retrieval procedure was performed using the Ag Retrieval Citra solution according to the manufacturer’s protocol (BioGenex Laboratories). Tissue endogenous peroxidase was inactivated using peroxidase block reagent (Innogenex, San Ramon, CA) and nonspecific binding blocked by incubating the sections for 30 min with 5% mouse serum (Sigma-Aldrich, St. Louis, MO). The sections were incubated overnight at 4°C with goat polyclonal Ab specific for murine IL-10, or an irrelevant IgG goat Ab (Santa Cruz Biotechnology, Santa Cruz, CA). The sections were washed with PBS containing 0.5% Tween 20 followed by incubation with the secondary polyclonal donkey anti-goat Ab conjugated to HRP (Serotec, Raleigh, NC) for 40 min at room temperature. Samples were developed using AEC substrate (BioGenex Laboratories). Sections were counterstained using hematoxylin (BioGenex Laboratories) and mounted using crystal mount.
Isolation of cells from infected lungs and spleen
Mice were euthanized and the pulmonary cavity opened. The lung was cleared of blood by perfusing through the pulmonary artery with 10 ml of saline containing 50 U/ml of heparin (Sigma-Aldrich). Lungs were removed from the pulmonary cavity and placed in cold DMEM (Life Technologies, Grand Island, NY). After removal of the connective tissue and trachea, the lungs were disrupted using sterile razor blades and incubated for 30 min at 37°C in a final volume of 2 ml DMEM containing collagenase XI (0.7 mg/ml; Sigma-Aldrich) and type IV bovine pancreatic DNase (30 µg/ml; Sigma-Aldrich). Ten milliliters of DMEM containing supplements (10% heat-inactivated FCS; Life Technologies), 1% 1 M HEPES buffer (1 M; Sigma-Aldrich), 1% L-glutamine (200 nM; Sigma-Aldrich), and 2% MEM-nonessential amino acids (100x; Sigma-Aldrich) was added to stop the action of the enzyme. Digested lungs were then gently dispersed through a nylon screen, and centrifuged at 300 x g. Remaining RBCs were lysed using ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3). Cells were resuspended in DMEM plus supplements. Spleens were harvested from individual mice and the cells dispersed through a nylon screen. RBCs were lysed using ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3) and spleen cells were resuspended in DMEM plus supplements.
Flow cytometry
Cells from the lung or spleen were obtained from each individual
mouse and incubated with specific Ab (25 µg/ml) for 30 min at 4°C
and in the dark followed by two washes in D-RPMI lacking biotin and
phenol red (Irvine Scientific, Santa Ana, CA). Cells were analyzed
using a BD Biosciences FACSCalibur and data analyzed using CellQuest
(BD Biosciences, San Diego, CA). Lymphocytes were gated by forward and
side scatter, and CD4+ and
CD8+ T cells identified by the presence of
specific fluorescent-labeled Ab. Cell surface markers were analyzed
using FITC-labeled anti-CD11a (clone 2D7) and PE anti-CD54
(clone 3E2), PerCP-labeled anti-CD4 (clone RM4-5), and
allophycocyanin-labeled anti-CD8 (clone 53-6.7). Appropriate
isotype control Abs were included in each analysis. All Abs were
purchased from BD PharMingen (San Diego, CA). Measurement of
intracellular IFN- was conducted by preincubating lung cells with
0.1 µg/ml anti-CD3
(clone 145-2C11) and 1 µg/ml
anti-CD28 (clone 37.51) in the presence of 3 µM monensin for
4 h at 37°C, 5% CO2. Cells were stained
with Abs for cell surface molecules as described above before a
permeabilization step conducted according to the manufacturer’s
instructions (Fix/Perm kit; BD PharMingen). FITC anti-IFN-
(clone XMG1.2) or IgG1 isotype control Ab was incubated with the cells
for a further 30 min, washed twice, and resuspended in D-RPMI before
analysis.
Lung cell culture
Lung cells were resuspended at 5 x 106 cells/ml in DMEM plus supplements. Cells were cultured with OVA (10 µg/ml; Sigma-Aldrich), or culture filtrate proteins (CFPs)3 from M. tuberculosis (10 µg/ml; received from National Institutes of Health contract AI-75320). After 5 days of culture at 37°C and 5% CO2, the plates were frozen at -70°C until further analysis.
Cytokine ELISA
Supernatants were harvested from lung cell cultures and assayed
for the presence of IFN- and IL-10 by ELISA. Briefly, the primary Ab
(IFN- clone R4-6A2, IL-10 clone JES5.2A5; BD PharMingen) was
incubated overnight in 96-well round bottom Immulon 2 plates in
carbonated coating buffer. Excess Ab was washed away using PBS-Tween
20. The wells were blocked with 3% BSA in PBS-Tween 20. The samples
were dispensed in duplicate into the wells. A standard curve was
prepared using IFN- (Genzyme, Cambridge, MA) or IL-10 (BD
PharMingen) for each individual plate. Cytokine production was detected
by the addition of a secondary biotinylated Ab (IFN- clone XMG1.2,
IL-10 clone SXC-1; BD PharMingen) and followed by avidin-peroxidase
(Zymed Laboratories, San Francisco, CA) and
3,3',5,5'-tetramethylbenzidine substrate (DAKO,
Carpinteria, CA).
PCR analysis of mRNA
A portion of lung tissue was suspended in Ultraspec (Cinna/Biotecx, Friendswood, TX), homogenized, and frozen rapidly for storage at -70°C. Total cellular RNA was extracted and reverse transcribed using murine Moloney leukemia virus reverse transcriptase (Life Technologies). PCR was performed with specific primers for IL-2 or IL-10. The PCR product was Southern blotted and probed with specific labeled oligonucleotides, and the blots were developed using the ECL kit (Amersham, Arlington Heights, IL). The hypoxanthine phosphoribosyltransferase housekeeping gene was also amplified for each sample and used to confirm that equivalent amounts of readable RNA were present in all the samples. Alternatively, the detection of IL-10, TNF, or IL-12p40 mRNA was conducted using a TaqMan 7700 real-time PCR machine (Applied Biosystems, Foster City, CA). For quantification purposes, Ribosomal DNA for each sample was also assayed as an endogenous normalizer. Quantification of message was conducted using the Delta Delta Ct method.
Statistical analysis
Statistical significance was determined with the Student’s t test and was found to be significant when p < 0.05, or highly significant when p < 0.005.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether the production of IL-10 could contribute to
the susceptibility of CBA/J mice to reactivate M.
tuberculosis infection, we stained sections of lung tissue from
CBA/J and C57BL/6 mice for the expression of IL-10. As early as 50 days
postinfection with M. tuberculosis, high levels of IL-10
could be detected within the lesions of CBA/J mice (Fig. 1
, A and B). IL-10
was predominantly found within the macrophage populations at the center
of the lesions, and could also be detected within bronchoepithelial
cells lining the airways. After 150 days of infection with M.
tuberculosis, abundant IL-10 could be detected in macrophages
within the lung lesions from CBA/J mice (Fig. 1, E and
F). In contrast, the lung lesions of C57BL/6 mice contained
only occasional macrophages and endothelial cells that stained weakly
positive for IL-10 after 50 (Fig. 1, C and D) or
150 days (Fig. 1, G and H) postinfection.
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To determine whether naturally reactivation-resistant C57BL/6
could reactivate M. tuberculosis infection if they produced
increased IL-10, we infected mice that were capable of overexpressing
IL-10 (IL-10 transgenic mice) and monitored the bacterial growth over
time. Whereas C57BL/6 mice were able to contain the bacterial load
within the lungs for the duration of the experiment, the lung bacterial
burden of IL-10 transgenic mice increased significantly during the
latter part of the study to 2 log more than the controls (Fig. 2). The increase in bacterial numbers
within the lungs of IL-10 transgenic mice occurred at a similar rate to
that seen in the reactivation-susceptible CBA/J mouse strain.
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To determine whether IL-10 was being produced in response to
mycobacterial Ag, lung cells were harvested from individual mice and
incubated with CFPs from M. tuberculosis. Lung cells from
IL-10 transgenic mice produced significantly more IL-10 than wild-type
C57BL/6 mice both 21 and 50 days postinfection when cultured with CFP
(Fig. 3). Ag-specific IL-10 production
could not be detected in lung cell cultures from CBA/J mice. Comparable
levels of IL-2 mRNA could be detected within the lungs of C57BL/6 and
IL-10 transgenic mice during the infection, and IL-10 transgenic mice
produced up to 4-fold more IL-10 mRNA within the lungs compared with
wild-type mice, as determined by RT-PCR (data not shown).
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We have previously demonstrated that circulating lymphocytes
isolated from reactivation-prone mice failed to express the adhesion
molecules CD11a and CD54 as brightly on their surface as similar cells
from C57BL/6 mice (10). Indeed, IL-10 exposure has also
been shown to decrease the expression of CD11a on the cell surface of
lymphocytes (27). CD4 T cells isolated from the spleen of
CBA/J mice expressed less CD11a on their surface (Fig. 4), as we have previously demonstrated
(10). The failure to increase CD11a expression was evident
up to 120 days postinfection, demonstrating that this was not a
transient event in the CBA/J mouse strain. CD4 T cells from the spleens
of IL-10 transgenic mice expressed a moderate expression of CD11a on
their surface, equivalent to CD4 T cells from the spleens of C57BL/6
mice (Fig. 4).
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, A and B). In
addition, lung digests revealed that CBA/J and IL-10 transgenic
mice had fewer CD4 T cells within the lungs in comparison to the
C57BL/6 mouse strain (Fig. 5C).
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IL-10 production can down-regulate macrophage responses, which
subsequently influence the capacity of the host to generate a T
cell-mediated response. To determine whether IL-10 overproduction could
influence the immune response within the lungs of IL-10 transgenic or
CBA/J mice, we quantified the amount of TNF and IL-12p40 mRNA during
the course of infection. The overexpression of IL-10 in the IL-10
transgenic mice correlated with delayed expression of TNF mRNA in the
lungs of these mice when compared with the expression in C57BL/6 mice
(Fig. 6A). The expression of
TNF mRNA was more substantially delayed in CBA/J mice, and this also
correlated with the high levels of IL-10 seen in the lung lesions of
these mice (see Fig. 1
). The pattern of IL-12p40 expression in both the
IL-10 transgenic and CBA/J mice was markedly different from that seen
for the C57BL/6 mice in that both mice expressed low levels of IL-12p40
mRNA in the lungs throughout infection (Fig. 6B).
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is reduced in IL-10 transgenic
mice
By its immunoregulatory action on the macrophage, IL-10 can also
influence Ag-specific IFN- production (11, 12);
therefore, we determined whether mycobacterial-specific IFN-
production was also reduced in the lungs of IL-10 transgenic mice. Lung
cells were isolated from mice that had been infected with M.
tuberculosis and cultured with CFP Ags from M.
tuberculosis. Ag-specific IFN- production was reduced in the
lung cell cultures of CBA/J and IL-10 transgenic mice when compared
with similar cultures from C57BL/6 mice (Fig. 7).
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A major characteristic of M. tuberculosis infection in
the reactivation prone mouse strains is the predominance of macrophage
type lesions within the lung. To determine whether the increased
expression of IL-10 and associated decreased expression of CD11a on
lymphocytes was associated with the formation of macrophage-dominated
lung lesions, lung tissue was collected, sectioned, and stained with
H&E. C57BL/6 mice developed characteristic lesions (25)
consisting of epithelioid macrophages associated with prominent
intralesional scattered or aggregated areas
of lymphocytes that persisted until at
least 200 days (Table I and Fig. 8, A and B). Initially, the lesions within the lungs
of IL-10 transgenic mice resembled those seen in the C57BL/6 strain
with prominent lymphocytic perivascular/peribronchiolar accumulations
and abundant macrophage clusters (Fig. 8C, day 50). However,
of particular interest was the observation that after 200 days of
infection with M. tuberculosis, only a few loose lymphocyte
aggregates were observed. The lesions had progressed to consist
predominantly of foamy macrophages (Fig. 8D, day 200) with
cellular necrosis, and cholesterol deposition similar to that observed
in the lungs of reactivation-prone mouse strains.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a mouse model of progressive primary tuberculosis, IL-10 was not detected until at least 6 mo (180 days) following an infection with M. tuberculosis (29), and we demonstrate in this study that the C57BL/6 mouse strain produced very little IL-10 during chronic M. tuberculosis infection. These results suggest that the production of IL-10 is not associated with the initial control of infection, but that this cytokine may be more important during the chronic or latent phase of tuberculosis in this model. Therefore, this may explain why only a moderate and transient enhanced early resistance can be found when C57BL/6 IL-10 gene-disrupted mice are infected with mycobacteria (20, 21, 23, 30). This increased resistance to infection in the absence of IL-10 is documented to be associated with an increased expression of inflammatory mediators such as TNF (21), inducible NO synthase (21), and IL-12 (23). Although IL-10 appears to reduce the production of these and other inflammatory molecules (16, 31), the transient and minimal reduction in bacterial numbers early in the course of infection of IL-10-deficient mice suggests that this molecules does not act to limit early protective responses in vivo.
Although early protective mechanisms appear unaffected by IL-10, we clearly show in this study that the expression of IL-10 during chronic infection is detrimental to both the protective antibacterial response and to the development of stable mononuclear granulomas. The overexpression of IL-10 in the IL-10 transgenic mice clearly correlates with the reduced expression of the cytokines TNF and IL-12p40, which are not only antibacterial, but also important in the generation of stable granulomas (32, 33, 34). That this reduced cytokine expression is also seen in the CBA/J mice, which also expressed high levels of IL-10 in the lung (albeit from macrophages rather than T cells), suggests that IL-10 is an active down-regulator of protective responses in the chronically infected murine lung. Why the early protective response is unaffected by IL-10 expression when the response within the chronically infected lung is altered poses an interesting question for further study. It is likely that the potent TH1-inducing nature of M. tuberculosis (33, 35) overrides any early inhibitory IL-10 effects, but that as disease develops, the levels of IL-10 increase (artificially in the IL-10 transgenic and naturally in the CBA/J mice) and the cytokine is able to limit the expression of protection.
Enhanced resistance to infection with other pathogens has also been demonstrated in IL-10 gene-disrupted mice (36, 37). However, it is also apparent that despite reducing bacterial numbers, the failure to down-regulate an acute immune response can also be fatal to the host due to the generation of an overwhelming inflammatory response (38, 39, 40). That IL-10 does not appear to be essential for the control of an infection with M. tuberculosis, or that its absence does not result in progressive inflammatory responses within the lung suggests that the down-regulation of the immune response to M. tuberculosis could be mediated by an independent mechanism. Alternatively, we demonstrate in this study that the overexpression of IL-10 results in the exacerbation of disease; therefore, it seems possible that a continued immune response during chronic M. tuberculosis infection may in fact be critical for the containment of a long-term infection. Studies that have sought to determine the immune parameters that are required for the maintenance of a chronic infection with M. tuberculosis have identified numerous molecules that include T cell subsets (41, 42), TNF (43, 44), and NO (45). It is also of interest to note that these same molecules are required for the initial control of infection (46, 47, 48), adding further support to the hypothesis that a continued immune response is necessary to prevent the reactivation of infection in the mouse model.
As we have demonstrated, the overexpression of IL-10 in our model was
associated with a reduced capacity of lung cells to produce IFN- in
vitro in response to CFP Ags from M. tuberculosis, thus
demonstrating that the Ag-specific response could be compromised. The
presence of IL-10 within the lungs of infected mice also inhibited the
further recruitment of lymphocytes into this organ, demonstrated by the
finding that in comparison to wild-type mice, overexpression of IL-10
resulted in fewer CD4 T cells within the lungs during the infection.
The production of IFN- per cell was not dramatically altered
(C57BL/6 1.4 x 10-3; CBA/J 1.6 x
10-3; IL-10 trangenic 0.98 x
10-3 pg/ml); however, the significantly reduced
number of T cells within the lungs of IL-10 transgenic and CBA/J mice
would also result in a net reduction of IFN- within the lungs.
Perhaps of more importance was the finding that those CD4 T cells present within the lungs of IL-10 transgenic mice also failed to up-regulate the expression of CD11a on their surface. IL-10 has been shown to influence the expression of CD11a on the surface of T cells (27), and may have a local influence on lymphocytes as they enter the lungs, down-regulating the expression of CD11a and influencing the capacity of lymphocytes to migrate within the lesions. This failure to up-regulate the adhesion molecule CD11a resembles the phenotype of two mouse strains, CBA/J and DBA/2, that we have previously described as reactivation-susceptible (10). The reduced expression of CD11a on T cells from CBA/J mice is a systemic effect as shown in this study and previously (10). That the IL-10 trangenic mice did not exhibit reduced CD11a expression in the spleen (where the level of IL-2 induction, and thus IL-10, is reduced due to low levels of infection and thus, immune stimulation) supports the hypothesis that it is IL-10 expressed at the site of infection that is limiting the expression of CD11a on the T cells within the lungs of the IL-10 transgenic mice. Therefore, these studies identify the overproduction of IL-10 as a potential correlate of reactivation tuberculosis in the mouse model that results in the characteristic phenotype within the lungs that is seen in the naturally reactivation-susceptible mouse strains.
The loss of lymphocyte foci within the lungs of IL-10 transgenic mice
appears to have a significant impact on the integrity of the lung
lesions as the course of infection ensued. The absence of localized T
cells and the loss of Ag-specific IFN- production appeared to be
directly associated with the gradual increase in bacterial load within
the lungs. As this occurred, lung lesions in the IL-10 transgenic mice
were dominated by macrophages and with progressive degeneration,
characterized by necrosis and neutrophil influx. As such, these lesions
closely resembled the macrophage-dominated lesions that we have
previously described in two reactivation-prone mouse strains
(10). The overexpression of IL-10 within lesions of the
lung, whether via transgene expression or as a result of
IL-10-producing macrophages within the lesions, thus clearly
predisposes these mice to enter a stage of reactivation disease.
The identification of immune correlates of protection/susceptibility to tuberculosis in the mouse model takes on particular importance if these findings can be extrapolated to man. The identification of individuals that have the potential to reactivate a latent M. tuberculosis infection, perhaps by a significant increase in their capacity to produce IL-10, may help identify individuals who will go on to develop reactivation disease later in life and hence be a source of infection. Therapeutic treatment of these individuals with drugs or perhaps with anti-IL-10R Ab (49) could potentially prevent reactivation tuberculosis and thereby reduce the index cases for infectious tuberculosis within the community. In support of this concept, elevated levels of serum IL-10 have been reported in individuals with active tuberculosis (19) and in the pleural fluid of tuberculosis pleurisy patients (18). In addition, elevated IL-10 levels appear to be greater in individuals that are coinfected with M. tuberculosis and HIV (50, 51, 52), which reflects the ability of both M. tuberculosis and HIV to stimulate IL-10 production during infection (53). Therefore, observations that the production of IL-10 is associated with the development of active (or reactivation) disease in man are in keeping with the findings of the present study, which demonstrate that increased susceptibility to reactivation tuberculosis in the mouse model is strongly influenced by the expression of IL-10 during the chronic or latent phase of the infection.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Joanne Turner, Mycobacteria Research Laboratories, Department of Microbiology Immunology and Pathology, Colorado State University, Fort Collins, CO 80523. E-mail address: joanne.turner{at}colostate.edu
3 Abbreviations used in this paper: CFP, culture filtrate protein.
Received for publication March 26, 2002. Accepted for publication October 2, 2002.
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References |
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T-cell-receptor-positive (TCR+) and 
TCR+ T-cell responses to Mycobacterium tuberculosis by IL-10 and transforming growth factor . Infect. Immun. 67:6461. stimulated macrophages: regulation by endogenous tumor necrosis factor- and by IL-10. Int. Immunol. 6:693., and TNF-. J. Immunol. 157:798.[Abstract]
and nitric oxide synthase 2. J. Exp. Med. 192:347. on host immune response in chronic persistent tuberculosis: possible role for limiting pathology. Infect. Immun. 69:1847. is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2:561.[Medline]
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 B. Samten, J. C. Townsend, Z. Sever-Chroneos, V. Pasquinelli, P. F. Barnes, and Z. C. Chroneos An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis J. Leukoc. Biol., July 1, 2008; 84(1): 115 - 123. [Abstract] [Full Text] [PDF]
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 P Fanourgiakis, E Mylona, I I Androulakis, C Eftychiou, E Vryonis, A Georgala, A Skoutelis, and M Aoun Non-Hodgkin's lymphoma and tuberculosis coexistence in the same organs: a report of two cases Postgrad. Med. J., May 1, 2008; 84(991): 276 - 277. [Abstract] [Full Text] [PDF]
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S. D. Chakravarty, G. Zhu, M. C. Tsai, V. P. Mohan, S. Marino, D. E. Kirschner, L. Huang, J. Flynn, and J. Chan Tumor Necrosis Factor Blockade in Chronic Murine Tuberculosis Enhances Granulomatous Inflammation and Disorganizes Granulomas in the Lungs Infect. Immun., March 1, 2008; 76(3): 916 - 926. [Abstract] [Full Text] [PDF]
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J. J. Lazarus, M. A. Kay, A. L. McCarter, and R. M. Wooten Viable Borrelia burgdorferi Enhances Interleukin-10 Production and Suppresses Activation of Murine Macrophages Infect. Immun., March 1, 2008; 76(3): 1153 - 1162. [Abstract] [Full Text] [PDF]
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 G. L. Beamer, D. K. Flaherty, B. Vesosky, and J. Turner Peripheral Blood Gamma Interferon Release Assays Predict Lung Responses and Mycobacterium tuberculosis Disease Outcome in Mice Clin. Vaccine Immunol., March 1, 2008; 15(3): 474 - 483. [Abstract] [Full Text] [PDF]
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P. J. Maglione, J. Xu, A. Casadevall, and J. Chan Fc{gamma} Receptors Regulate Immune Activation and Susceptibility during Mycobacterium tuberculosis Infection J. Immunol., March 1, 2008; 180(5): 3329 - 3338. [Abstract] [Full Text] [PDF]
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R. Hussain, N. Talat, F. Shahid, and G. Dawood Longitudinal Tracking of Cytokines after Acute Exposure to Tuberculosis: Association of Distinct Cytokine Patterns with Protection and Disease Development Clin. Vaccine Immunol., December 1, 2007; 14(12): 1578 - 1586. [Abstract] [Full Text] [PDF]
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D. Ordway, M. Henao-Tamayo, M. Harton, G. Palanisamy, J. Troudt, C. Shanley, R. J. Basaraba, and I. M. Orme The Hypervirulent Mycobacterium tuberculosis Strain HN878 Induces a Potent TH1 Response followed by Rapid Down-Regulation J. Immunol., July 1, 2007; 179(1): 522 - 531. [Abstract] [Full Text] [PDF]
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S. Roque, C. Nobrega, R. Appelberg, and M. Correia-Neves IL-10 Underlies Distinct Susceptibility of BALB/c and C57BL/6 Mice to Mycobacterium avium Infection and Influences Efficacy of Antibiotic Therapy J. Immunol., June 15, 2007; 178(12): 8028 - 8035. [Abstract] [Full Text] [PDF]
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P. J. Maglione, J. Xu, and J. Chan B Cells Moderate Inflammatory Progression and Enhance Bacterial Containment upon Pulmonary Challenge with Mycobacterium tuberculosis J. Immunol., June 1, 2007; 178(11): 7222 - 7234. [Abstract] [Full Text] [PDF]
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 S. M. Newton, R. J. Smith, K. A. Wilkinson, M. P. Nicol, N. J. Garton, K. J. Staples, G. R. Stewart, J. R. Wain, A. R. Martineau, S. Fandrich, et al. A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion PNAS, October 17, 2006; 103(42): 15594 - 15598. [Abstract] [Full Text] [PDF]
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E. Giacomini, A. Sotolongo, E. Iona, M. Severa, M. E. Remoli, V. Gafa, R. Lande, L. Fattorini, I. Smith, R. Manganelli, et al. Infection of Human Dendritic Cells with a Mycobacterium tuberculosis sigE Mutant Stimulates Production of High Levels of Interleukin-10 but Low Levels of CXCL10: Impact on the T-Cell Response. Infect. Immun., June 1, 2006; 74(6): 3296 - 3304. [Abstract] [Full Text] [PDF]
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M. d. P. Jimenez, L. Walls, and J. Fierer High Levels of Interleukin-10 Impair Resistance to Pulmonary Coccidioidomycosis in Mice in Part through Control of Nitric Oxide Synthase 2 Expression. Infect. Immun., June 1, 2006; 74(6): 3387 - 3395. [Abstract] [Full Text] [PDF]
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D. Ordway, M. Harton, M. Henao-Tamayo, R. Montoya, I. M. Orme, and M. Gonzalez-Juarrero Enhanced Macrophage Activity in Granulomatous Lesions of Immune Mice Challenged with Mycobacterium tuberculosis. J. Immunol., April 15, 2006; 176(8): 4931 - 4939. [Abstract] [Full Text] [PDF]
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N. Ismail, H. L. Stevenson, and D. H. Walker Role of Tumor Necrosis Factor Alpha (TNF-{alpha}) and Interleukin-10 in the Pathogenesis of Severe Murine Monocytotropic Ehrlichiosis: Increased Resistance of TNF Receptor p55- and p75-Deficient Mice to Fatal Ehrlichial Infection Infect. Immun., March 1, 2006; 74(3): 1846 - 1856. [Abstract] [Full Text] [PDF]
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M. D. Woolard, D. Hudig, L. Tabor, J. A. Ivey, and J. W. Simecka NK Cells in Gamma-Interferon-Deficient Mice Suppress Lung Innate Immunity against Mycoplasma spp. Infect. Immun., October 1, 2005; 73(10): 6742 - 6751. [Abstract] [Full Text] [PDF]
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B. M. Sullivan, O. Jobe, V. Lazarevic, K. Vasquez, R. Bronson, L. H. Glimcher, and I. Kramnik Increased Susceptibility of Mice Lacking T-bet to Infection with Mycobacterium tuberculosis Correlates with Increased IL-10 and Decreased IFN-{gamma} Production J. Immunol., October 1, 2005; 175(7): 4593 - 4602. [Abstract] [Full Text] [PDF]
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A. Blumenthal, J. Lauber, R. Hoffmann, M. Ernst, C. Keller, J. Buer, S. Ehlers, and N. Reiling Common and Unique Gene Expression Signatures of Human Macrophages in Response to Four Strains of Mycobacterium avium That Differ in Their Growth and Persistence Characteristics Infect. Immun., June 1, 2005; 73(6): 3330 - 3341. [Abstract] [Full Text] [PDF]
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S. S. Kang and P. M. Allen Priming in the Presence of IL-10 Results in Direct Enhancement of CD8+ T Cell Primary Responses and Inhibition of Secondary Responses J. Immunol., May 1, 2005; 174(9): 5382 - 5389. [Abstract] [Full Text] [PDF]
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C. G. Feng, D. Jankovic, M. Kullberg, A. Cheever, C. A. Scanga, S. Hieny, P. Caspar, G. S. Yap, and A. Sher Maintenance of Pulmonary Th1 Effector Function in Chronic Tuberculosis Requires Persistent IL-12 Production J. Immunol., April 1, 2005; 174(7): 4185 - 4192. [Abstract] [Full Text] [PDF]
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C. Holscher, A. Holscher, D. Ruckerl, T. Yoshimoto, H. Yoshida, T. Mak, C. Saris, and S. Ehlers The IL-27 Receptor Chain WSX-1 Differentially Regulates Antibacterial Immunity and Survival during Experimental Tuberculosis J. Immunol., March 15, 2005; 174(6): 3534 - 3544. [Abstract] [Full Text] [PDF]
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 Y. Shen, L. Shen, P. Sehgal, D. Huang, L. Qiu, G. Du, N. L. Letvin, and Z. W. Chen Clinical Latency and Reactivation of AIDS-Related Mycobacterial Infections J. Virol., December 15, 2004; 78(24): 14023 - 14032. [Abstract] [Full Text] [PDF]
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S. Jang, S. Uematsu, S. Akira, and P. Salgame IL-6 and IL-10 Induction from Dendritic Cells in Response to Mycobacterium tuberculosis Is Predominantly Dependent on TLR2-Mediated Recognition J. Immunol., September 1, 2004; 173(5): 3392 - 3397. [Abstract] [Full Text] [PDF]
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J. Turner, K. M. Dobos, M. A Keen, A. A. Frank, S. Ehlers, I. M. Orme, J. T. Belisle, and A. M. Cooper A Limited Antigen-Specific Cellular Response Is Sufficient for the Early Control of Mycobacterium tuberculosis in the Lung but Is Insufficient for Long-Term Survival Infect. Immun., July 1, 2004; 72(7): 3759 - 3768. [Abstract] [Full Text] [PDF]
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J. M. Cliff, I. N. J. Andrade, R. Mistry, C. L. Clayton, M. G. Lennon, A. P. Lewis, K. Duncan, P. T. Lukey, and H. M. Dockrell Differential Gene Expression Identifies Novel Markers of CD4+ and CD8+ T Cell Activation Following Stimulation by Mycobacterium tuberculosis J. Immunol., July 1, 2004; 173(1): 485 - 493. [Abstract] [Full Text] [PDF]
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M. G. Bonecini-Almeida, J. L. Ho, N. Boechat, R. C. Huard, S. Chitale, H. Doo, J. Geng, L. Rego, L. C. O. Lazzarini, A. L. Kritski, et al. Down-Modulation of Lung Immune Responses by Interleukin-10 and Transforming Growth Factor {beta} (TGF-{beta}) and Analysis of TGF-{beta} Receptors I and II in Active Tuberculosis Infect. Immun., May 1, 2004; 72(5): 2628 - 2634. [Abstract] [Full Text] [PDF]
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G. Prevot, E. Bourreau, H. Pascalis, R. Pradinaud, A. Tanghe, K. Huygen, and P. Launois Differential Production of Systemic and Intralesional Gamma Interferon and Interleukin-10 in Nodular and Ulcerative Forms of Buruli Disease Infect. Immun., February 1, 2004; 72(2): 958 - 965. [Abstract] [Full Text] [PDF]
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R. C. Huard, S. Chitale, M. Leung, L. C. O. Lazzarini, H. Zhu, E. Shashkina, S. Laal, M. B. Conde, A. L. Kritski, J. T. Belisle, et al. The Mycobacterium tuberculosis Complex-Restricted Gene cfp32 Encodes an Expressed Protein That Is Detectable in Tuberculosis Patients and Is Positively Correlated with Pulmonary Interleukin-10 Infect. Immun., December 1, 2003; 71(12): 6871 - 6883. [Abstract] [Full Text] [PDF]
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M. Gonzalez-Juarrero, T. S. Shim, A. Kipnis, A. P. Junqueira-Kipnis, and I. M. Orme Dynamics of Macrophage Cell Populations During Murine Pulmonary Tuberculosis J. Immunol., September 15, 2003; 171(6): 3128 - 3135. [Abstract] [Full Text] [PDF]
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) and IL-10 transgenic mice (
) were infected
with 
), CBA/J (
), or IL-10 transgenic mice (
