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Infection and Immunity, October 2001, p. 6156-6164, Vol. 69, No. 10
Department of
Medicine1 and Department of
Pathology,2 University of California, San Diego,
La Jolla, California 92093, and Department of Biology,
Indiana University, Bloomington, Indiana 474053
Received 26 March 2001/Returned for modification 22 May
2001/Accepted 9 July 2001
Bacterial DNA and its synthetic immunostimulatory
oligodeoxynucleotide analogs (ISS-ODN) activate innate immunity and
promote Th1 and cytotoxic T-lymphocyte immune responses. Based on these activities, we investigated whether ISS-ODN could modify the course of
Mycobacterium avium infection. M. avium
growth in vitro was significantly inhibited by ISS-ODN treatment of
human and mouse macrophages, and M. avium growth in vivo
was similarly inhibited in C57BL/6 mice treated with ISS-ODN. This
protective effect of ISS-ODN was largely independent of tumor necrosis
factor alpha (TNF- Mycobacterium avium
infection is a common opportunistic infection in patients with AIDS
(22), while M. avium rarely causes disease in
immunocompetent individuals. In AIDS, disseminated M. avium
infection occurs only after severe depletion of
CD4+ T cells (10) and is a major
cause of morbidity and mortality. Although intensive antiretroviral
therapy prevents the onset of M. avium infection to some
extent (3), M. avium infection in AIDS is
extremely difficult to treat when encountered because it responds
poorly to available antimycobacterial therapies (33).
M. avium predominantly infects and multiplies within
macrophages (14). This organism is known to attach to and
enter macrophages with the help of complement, transferrin, and
integrin receptors expressed on the surface of macrophages (6,
42). Macrophages secrete several cytokines in response to
infection with this organism, including tumor necrosis factor alpha
(TNF- Immunostimulatory DNA sequences (ISS) were initially discovered in the
mycobacterial genome as short DNA sequences that selectively enhanced
NK cell activity (53). In addition, bacterial DNA or oligodeoxynucleotides containing ISS (ISS-ODN) activate
antigen-presenting cells, such as dendritic cells and macrophages.
ISS-ODN activation leads to the up-regulation of costimulatory
receptors (32), the release of IFN- Recent studies have also demonstrated the ability of ISS-ODN to
facilitate control of the intracellular pathogens Listeria monocytogenes, Leishmania major, and Francisella
tularensis (24, 27, 51). This effect is generally
attributed to the activation of innate immunity, which is crucial for
both the initial control of L. monocytogenes and L. major and the long-term control from subsequent induction of Th1
and CTL responses.
Here, we examine whether ISS-ODN can modify the course of M. avium infection. Our studies demonstrate that ISS-ODN is able to
restrict the growth of M. avium via the induction of
indoleamine 2,3-dioxygenase (IDO), revealing a novel facet of
ISS-ODN-induced activation of innate immunity. As a therapeutic
adjunct, ISS-ODN significantly enhances the ability of the conventional
antimycobacterial agent clarithromycin (CLA) to treat M. avium infection in mouse and human macrophages as well as in a
mouse model of M. avium infection.
Mice.
Female C57BL/6 mice (7 to 8 weeks old) and 129/SvEv
mice were purchased from The Jackson Laboratory (Bar Harbor, Maine) and Taconic Laboratories, Germantown, New York, respectively. The inducible
nitric oxide synthetase (iNOS Reagents and cytokines.
Endotoxin-free (<1 ng of DNA per
mg) phosphorothioate single-stranded oligodeoxynucleotides were
obtained from Trilink Biotechnologies, San Diego, Calif. The sequence
of the ISS-ODN was 5'-TGACTGTGAACGTTCGAGATGA-3'. The sequence of the mutated-ODN (M-ODN) was
5'-TGACTGTGAAGGTTAGAGATGA-3' (underlining
indicates immunostimulatory sequences [ISS]). We used 10 µg
of ISS-ODN or M-ODN per ml unless otherwise noted. L-Tryptophan (L-Try) was obtained from Gibco
BRL (Grand Island, N.Y.) and Dulbecco's modified Eagle's medium
(DMEM) was supplemented with L-Try for a final
concentration of 66 µg/ml. 1-Methyl-DL-tryptophan (M-Try)
was purchased from Aldrich Chemicals (Milwaukee, Wis.). Anti-CD3,
anti-CD28, anti-CD4, and anti-CD8 monoclonal antibodies as well as
monensin and murine recombinant IFN- Culture of M. avium and CFU assay.
M.
avium strain I13 isolated from an AIDS patient at University of
California, San Diego (34), was used in all experiments. Bacteria were cultured as described previously (21). The
numbers of CFU in a sample were determined by colony counting as
described previously (39).
Preparation of murine macrophages and isolation of human
monocytes.
Murine bone marrow-derived macrophages (mBMDMs) were
prepared from mouse bone marrow using L-cell conditioned medium, as
described previously (32). Human monocytes were isolated
from normal human buffy coats obtained from the San Diego Blood Bank by
Ficoll-Hypaque and Percoll gradient centrifugation (21)
and then cultured in Teflon beakers for 7 days to yield mature human
monocyte-derived macrophages (hMDMs).
M. avium infection of macrophages in vitro.
To study the protective effect of ISS-ODN treatment before infection,
5 × 104 mBMDMs or hMDMs were treated with
ISS-ODN for 3 days before infection. Macrophages treated with M-ODN or
with medium alone served as controls. After 3 days, the cells were
infected for 2 h with M. avium at a
macrophage/bacterium ratio of 2:1 for mBMDMs or 1:10 for hMDMs and
subsequently cultured in fresh medium without antibiotics. To examine
attachment and/or invasion of M. avium, the macrophages were
lysed immediately after washing, and the number of bacteria was
enumerated by the CFU assay. Intracellular growth of M. avium was determined on days 1, 3, and 7 after infection. To
account for all of the mycobacteria in each well, corresponding lysates were combined with the culture media, and then the number of CFU was
determined. To examine the efficacy of antimycobacterial treatments, CFU recovered from cells treated with medium alone was considered as
100% growth.
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.10.6156-6164.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Enhancement of Innate Immunity against
Mycobacterium avium Infection by Immunostimulatory DNA
Is Mediated by Indoleamine 2,3-Dioxygenase
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
), interleukin 12 (IL-12), nitric oxide, NADPH
oxidase, alpha/beta interferon (IFN-/
), and IFN-
. In contrast,
we found that the induction of indoleamine 2,3-dioxygenase (IDO) was
required for the antimycobacterial effect of ISS-ODN. To evaluate the
potential for synergism between ISS-ODN and other antimycobacterial
agents, treatment with a combination of ISS-ODN and clarithromycin
(CLA) was tested in vitro and in vivo. ISS-ODN significantly enhanced the therapeutic effect of CLA in both human and mouse macrophages and
in C57BL/6 mice. This study newly identifies IDO as being involved in
the antimicrobial activity of ISS-ODN and suggests the usefulness of
ISS-ODN when used in combination with conventional chemotherapy for
microbial infections.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
), interleukin 1 (IL-1), IL-6, granulocyte-macrophage
colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating
factor (G-CSF) (17, 38).
/, TNF-,
IL-12, and IL-18 (25, 28, 44, 47, 48, 55), and the priming
of Th1 responses and cytotoxic T-lymphocyte (CTL) responses to
exogenous antigens (9, 30). This immune profile has been
used to generate cellularly mediated immunity to coinjected antigens in
the development of novel vaccine strategies (9, 29, 44).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/) IL-12
p40/,
TNF-/, and NADPH oxidase (gp91
phox/) knockout mice are on the C57BL/6
background and were purchased from The Jackson Laboratory. IFN-/
receptor (IFN-/R/) and IFN- receptor
(IFN-R/) (129/EvSv background) knockout
mice were obtained from B & K Universal, Ltd. (East Yorkshire, United Kingdom).
were purchased from BD
Pharmingen (San Diego, Calif.). Abbott Laboratories (Abbott Park, Ill.)
kindly provided CLA.
M. avium infection of mice in vivo. To study the effect of treatment with ISS-ODN before infection, mice were injected intradermally (i.d.)with ISS-ODN (50 or 100 µg/mouse) 3 days before infection, while control mice received phosphate-buffered saline (PBS). In the preliminary experiments, the numbers of CFU in the spleen and lungs were similar in the M-ODN-treated mice and control (PBS treated) mice 4 weeks after infection, but were significantly higher than those in the ISS-ODN-treated mice (by 2 logs in spleen and 1 log in lungs, respectively). Therefore, treatment with M-ODN was excluded in this set of experiments. All mice were infected intravenously (i.v.) with M. avium (107/mouse). At 2, 4, and 6 weeks after infection, the mice were sacrificed, and the spleen, liver, and lungs from each mouse were collected and weighed. A section of each organ was homogenized with 0.25% sodium dodecyl sulfate (SDS) in PBS. The number of CFU in the tissue homogenates was determined by the CFU assay, and the results were expressed as CFU per organ.
To study the effect of ISS-ODN when combined with CLA in vivo, 25 mice were injected with M. avium (107/mouse). One week after infection, treatment with either ISS-ODN or M-ODN and CLA was initiated. Mice were divided into five treatment groups (n = 5 per group): group 1, no treatment; group 2, ISS-ODN alone; group 3, CLA alone; group 4, CLA and ISS-ODN; and group 5, CLA and M-ODN. CLA (200 mg/kg of body weight) was administered intraperitoneally three times a week for 4 weeks as described previously (13), and bacterial growth in the spleen, liver, and lungs was determined.Detection of intracellular IFN- by FACS and IFN- secretion
by ELISA.
Mice were injected i.d. with ISS-ODN (50 µg/mouse).
The control mice received M-ODN (50 µg/mouse) or PBS. All mice were
infected with 107 M. avium cells.
Three weeks after infection, the mice were sacrificed, and splenocytes
from mice receiving the same treatment were pooled. Intracellular
cytokine staining was performed with the Cytofix/Cytoperm kit (BD
Pharmingen) according to the manufacturer's instructions. Briefly, the
splenocytes were stimulated with anti-CD3 and anti-CD28 activating
antibodies in the presence of monensin to allow intracellular IFN-
to accumulate for 6 h. Next, the cells were stained for surface
CD4 and CD8 and fixed, and the plasma membranes were permeabilized, allowing for intracellular staining with anti-IFN-. The cells were
analyzed by fluorescence-activated cell sorting (FACS) with a
FACSCalibur flow cytometer (Becton Dickinson). To study IFN- secretion, splenocytes were incubated with anti-CD3 and anti-CD-28 antibodies or M. avium sonicates (20) as
antigens in vitro for 24 h, and then the supernatant was assayed
for IFN- secretion by enzyme-linked immunosorbent assay (ELISA) as
previously described (32).
RNA extraction, RT-PCR, and IDO activity assay. mBMDMs (2 × 106) were treated with ISS-ODN or M-ODN. After 3 days, the cells were infected with M. avium for 2 h. At 2, 24, and 48 h after infection, the M. avium-infected macrophages were lysed, and total RNA was isolated by using the Trizol reagent (Gibco BRL). The induction of IDO gene transcription was measured by semiquantitative reverse transcription-PCR (RT-PCR). First-strand cDNA preparation and PCR amplification were carried out with the SuperScript Pre-amplification System (Gibco BRL) and AdvanTaq Plus DNA polymerase (Clontech, San Francisco, Calif.), respectively. PCR products were visualized by electrophoresis on 2% agarose gels. The primer sequences used were as follows: IDO, 5'-TTATGCAGACTGTGTCCTGGCAAA-3' and 5'-TTTCCAGCCAGACAGATATATGCG-3'; and glucose-3-phosphate dehydrogenase (G3PDH), 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'.
Histological examination. Tissue sections of the spleen collected from the experimental and control mice were fixed overnight in 10% buffered formalin at room temperature and then embedded in paraffin. The paraffin-embedded tissue was further sectioned (5-µm thickness) and stained with hematoxylin-eosin. The sections were observed under an Olympus microscope. At least three sections of each organ from each of the experimental and control mice were evaluated.
Statistical analysis. Results are expressed as means ± standard deviations (SD), and statistical differences were determined with the nonpaired Student's t test (two-tailed distribution). A P value below 0.05 is considered to be statistically significant.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Treatment with ISS-ODN inhibits the growth of M. avium in vitro. M. avium is able to infect and replicate in macrophages (14). We performed the following studies to address whether ISS-ODN-induced activation of macrophages in vitro inhibits intracellular growth of M. avium.
(i) Treatment prior to infection.
To examine whether treatment
with ISS-ODN can stimulate macrophages to inhibit the growth of
M. avium, mBMDMs were first treated with ISS-ODN or M-ODN
(3, 10, and 30 µg/ml) for 72 h and then infected with M. avium. The number of CFU was counted on days 1, 3, and 7 postinfection (Fig. 1A). By day 7, treatment with ISS-ODN inhibited intracellular growth of M. avium in mBMDMs by 80% (P < 0.001). No
significant difference in M. avium growth was seen among the
different concentrations of ISS-ODN tested. Since viability of
macrophages can affect M. avium growth, we assessed the
viability of macrophages by trypan blue exclusion. At day 7 after
infection, mBMDMs treated with ISS-ODN, M-ODN, or medium alone were all
>90% viable. The experiments were terminated at day 7 after
infection, since the viability of mBMDMs began to decline from this
point on.
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(ii) Treatment after infection. To evaluate the therapeutic antimycobacterial effect of ISS-ODN, infected mBMDMs were treated with ISS-ODN for 7 days after infection, starting 2 h after the time of infection. Treatment with a single dose of ISS-ODN significantly decreased the intracellular growth of M. avium in mBMDMs by 68% (P < 0.05), compared to the number of CFU in infected cells treated with M-ODN or medium alone (Fig. 1B).
ISS-ODN transiently inhibits the growth of M. avium in vivo. To determine whether ISS-ODN can enhance resistance to M. avium infection in vivo, mice were treated with ISS-ODN and infected i.v. 3 days later with 107 organisms per mouse. In a preliminary experiment, bacterial growth in spleen and lung from mice treated with 50 µg of ISS-ODN was similar to bacterial growth in mice treated with 100 µg of ISS-ODN 4 weeks after infection (data not shown). Therefore, we used 50 µg of ISS-ODN per mouse for the in vivo experiment. At 2, 4, and 6 weeks after infection, the number of CFU was determined in the spleen, lungs, and liver. Based on the preliminary experiments, which demonstrated no differences in colony counts among the ISS-ODN, M-ODN, or control mice at 6 versus 8 weeks postinfection, all future experiments were terminated at 6 weeks postinfection.
At week 2, the lungs of M. avium-infected mice treated with ISS-ODN prior to infection contained a significantly lower number of viable bacteria compared to control PBS-treated mice (P < 0.05) (Fig. 2A). In addition, mice treated with ISS-ODN prior to M. avium infection had nearly 2 logs fewer bacteria in the spleen at 4 weeks (P < 0.05) compared to the PBS-treated mice (Fig. 2B). By 6 weeks, however, the numbers of splenic CFU were similar in control and ISS-ODN groups. There was no significant difference in the mycobacterial loads in the liver of mice treated with ISS-ODN prior to infection compared to those of PBS-treated mice at any of these time points (Fig. 2C). Thus, a single injection of ISS-ODN significantly reduced the mycobacterial growth in the spleen and lungs, but not in the liver of M. avium-infected mice. This protective effect was transient and was most apparent at 2 and 4 weeks after a single administration of ISS-ODN.
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ISS-ODN protection in vivo is not mediated through augmentation of
the T-cell response.
The observation that ISS-ODN protects
isolated macrophages in vitro (Fig. 1) and that the protective effect
observed in ISS-ODN-treated mice is transient (Fig. 2) suggests a
T-cell-independent mechanism of protection via innate immunity. To
further investigate the potential role of adaptive immunity in this
model of ISS-ODN-mediated protection against M. avium, mice
were treated with ISS-ODN or M-ODN (50 µg/mouse) and infected with
107 organisms/mouse, and then their T-cell
response was evaluated. The mice were sacrificed at 3 weeks
postinfection, and the spenocytes were restimulated with anti-CD3 and
anti-CD28 antibodies or M. avium sonicates to amplify the
response from preexisting memory and activated T cells. T cells were
then examined for their production of IFN-. FACS-based intracellular
cytokine staining was used to determine the frequencies of
IFN--producing CD4+ (Fig.
3A, B, and C) and
CD8+ (Fig. 3B) T cells, while the total quantity
of IFN- secreted by splenocytes was determined by ELISA (data not
shown). There was a dramatic increase in the IFN- response of the
CD4+ T cells in the infected versus uninfected
animals, demonstrating that the observed Th1 response is infection
specific. However, treatment of M. avium-infected mice with
ISS-ODN did not further increase the frequency of IFN--positive
CD4+ or CD8+ T cells. In
addition, treatment with ISS-ODN did not enhance the IFN- secretion
by splenocytes, which were restimulated by anti-CD3 and anti-CD28
antibodies or M. avium sonicates, compared to M-ODN
treatment (data not shown). These data, combined with the observation
that ISS-ODN protects isolated macrophages in vitro (Fig. 1) and that
the protective effect observed in ISS-ODN-treated mice is transient
(Fig. 2), suggest that it is unlikely T-cell-dependent immunity plays a
role in the antimycobacterial effect of ISS-ODN during early stages of
infection.
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ISS-ODN inhibition of M. avium growth in macrophages
is independent of iNOS, NAPDH oxidase, IL-12, TNF-, IFN-/, and
IFN-.
To further investigate the mechanisms of the
antimycobacterial effects of ISS-ODN, mice with targeted disruptions of
genes known to play roles in M. avium infection were used.
Oxygen radicals generated by NADPH oxidase and induction of nitric
oxide (NO) by iNOS result in antimicrobial activity against many
microorganisms (16, 35). IL-12, TNF-, and IFN- play
important roles in the clearance of M. avium (2, 13,
26). Furthermore, macrophages produce IL-12, TNF-, and
IFN-/ in response to ISS-ODN treatment (25, 44). To
study the role of these molecules in the antimycobacterial effect of
ISS-ODN, mBMDMs from NADPH oxidase/,
iNOS/, TNF-/,
IL-12 p40/,
IFN-/R/, and
IFN-R/ mice were treated with ISS-ODN for
3 days and then infected with M. avium. ISS-ODN inhibited
the intracellular growth of M. avium in mBMDMs from these
knockout mice by 60 to 85% (P < 0.05), similar to
wild-type mice (Table 1). These results
indicate that these gene products (e.g., nitrogen intermediates, oxygen
radicals, TNF-, etc.) are not central to the antimycobacterial
effect induced by ISS-ODN in vitro.
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Induction of IDO contributes to the antimycobacterial activity of ISS-ODN. Macrophages orchestrate the activation of innate immunity to directly attack an invading pathogen by secreting a battery of cytokines, expressing costimulatory molecules, and generating free radicals. However, macrophages also employ effector mechanisms to deny the pathogens required substrates for their growth. One such example is the depletion of tryptophan due to the induction of IDO. IDO is the rate-limiting enzyme in the catabolism of tryptophan. Induction of this enzyme limits the availability of this important amino acid to invading pathogens (11). To study the potential role of IDO in the antimycobacterial effect of ISS-ODN, we assessed (i) the induction of IDO activity as measured by semiquantitative RT-PCR in vivo and in vitro and (ii) the abrogation of the antimycobacterial effect of ISS-ODN by addition of excess L-Try or by using a competitive inhibitor for IDO, M-Try.
When mice were injected i.v. with 50 µg of ISS-ODN, IDO gene induction was observed in the lungs and spleen after 16 h, but not in the liver (Fig. 4A). Injection of M-ODN did not result in any detectable induction of IDO (data not shown). For in vitro studies, mBMDMs were treated with ISS-ODN for 3 days prior to M. avium infection. Cells were then lysed at 4, 8, and 24 h after infection, total RNA was extracted, and semiquantitative RT-PCR was performed. We found that optimal induction of IDO gene transcription required both treatments with ISS-ODN and M. avium infection (Fig. 4B).
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, injected
with saline; +, injected with ISS-ODN. (B) M. avium
infection with ISS-ODN treatment of mBMDMs induces IDO gene expression.
mBMDMs were treated with ISS-ODN for 3 days prior to infection with
M. avium. Total RNA was evaluated by semiquantitative
RT-PCR to assess the induction of IDO gene expression at 4, 8, and
24 h postinfection. Lanes: 1, medium alone; 2, ISS-ODN treatment
alone; 3, M. avium infection alone; 4, ISS-ODN treatment
and M. avium infection. (C) Addition of excess
L-Try or a competitive IDO inhibitor, M-Try, partially
inhibited the antimycobacterial effect of ISS-ODN. mBMDMs were treated
with ISS-ODN for 3 days in the presence of excess L-Try
(final concentration of 66 µg/ml) or M-Try (125 µM) and
subsequently infected with M. avium for 7 days in the
presence of L-Try or M-Try. Intracellular growth of
M. avium was assessed by the CFU assay. Each condition
was tested in triplicate, and the results are expressed as mean ± SD CFU per well. The results shown are representative of three
experiments. *, P < 0.01 compared to CFU
recovered from cells treated with ISS-ODN.
ISS-ODN is an adjunct to antimycobacterial therapy with CLA. Treatment of M. avium infection by conventional chemotherapy is difficult and requires prolonged administration (8, 33). In in vivo experiments, ISS-ODN did not affect the colony counts when administered in an established infection. Therefore, we hypothesized that coadministration of ISS-ODN along with a chemotherapeutic agent such as CLA may further enhance mycobacterial clearance.
mBMDMs were first infected with M. avium and then treated with CLA (0.1 µg/ml) in the presence or absence of ISS-ODN (10 µg/ml) or M-ODN (10 µg/ml), and M. avium growth in vitro was determined 7 days after infection. ISS-ODN and CLA (0.1 µg/ml), when used individually, reduced bacterial growth in mBMDMs by 68 and 84%, respectively (P < 0.01; Fig. 5A). When ISS-ODN was used together with CLA, bacterial counts were further reduced (95%, P < 0.01) compared to medium alone (Fig. 5A).
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ISS-ODN inhibits M. avium growth in human
macrophages in vitro.
To study the relevance of the
antimycobacterial effects of ISS-ODN in humans, hMDMs were treated with
ISS-ODN or M-ODN (3, 10, and 30 µg/ml) for 3 days and then infected
with M. avium. There was maximal inhibition of M. avium growth at 3 µg of ISS-ODN per ml (Fig.
6A) with no further increase in
inhibition at the higher concentrations (data not shown). At 7 days
postinfection, treatment with ISS-ODN inhibited intracellular growth of
M. avium by 91% (Fig. 6A, P < 0.001). No
changes in cell viability were observed in the various groups. To study
the therapeutic effects of ISS-ODN on established M. avium
infection, infected hMDMs were treated with ISS-ODN (10 µg/ml) for 7 days. Treatment with ISS-ODN significantly decreased the intracellular
growth of M. avium in hMDMs by 53% (P < 0.05) (Fig. 6B). When infected cells were treated with ISS-ODN together
with CLA (0.1 µg/ml), M. avium growth was further
inhibited up to 99% (P < 0.01), compared to medium
alone (Fig. 6B). These findings indicate that ISS-ODN can provide a significant antimycobacterial effect in human macrophages as well as in
the mouse model.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study demonstrates that ISS-ODN augments host resistance
against M. avium infection and that the induction of IDO
plays an important role in this effect. Furthermore, ISS-ODN synergizes with the chemotherapeutic drug CLA to further promote mycobacterial clearance. These data support recent reports showing that
administration of ISS-ODN provides protection against the intracellular
pathogens Listeria monocytogenes (27),
Leishmania major (51), and Francisella tularensis (15). Studies with L. monocytogenes showed that injection of mice with Escherichia
coli DNA or ISS-ODN resulted in a much lower bacterial burden in
the spleen and liver (27). Similarly, ISS-ODN conferred a
protective immunity against primary infection and resistance to
secondary infection with L. major (51). This antimicrobial activity was attributed mainly to ISS-ODN-induced release
of cytokines such as IL-12 and to the subsequent induction of a
protective Th1 and/or CTL response. Indeed, administration of exogenous
IL-12 or IFN-, which are induced by ISS-ODN, increases protection
against L. major (51).
Our study suggests that ISS-ODN-induced resistance to M. avium infection does not act via augmentation of the adaptive
T-cell response. First, the in vitro studies showed that treatment with ISS-ODN enhanced the ability of macrophages to resist M. avium growth despite the absence of T cells (Fig. 1). Second,
ISS-ODN-induced resistance to M. avium in vivo decayed by 6 weeks, at which point ISS-ODN-treated mice had the same mycobacterial
burden as control, PBS-treated mice (Fig. 2). The induction of a
protective antimycobacterial T-cell response would be expected to
provide a long-lasting antimycobacterial effect, rather than the
transient protective effect observed in our experiments. Third, our
data showed that T cells from ISS-treated, M. avium-infected
mice did not display any increased infection-specific IFN- response
over untreated mice upon restimulation in vitro (Fig. 3), indicating
that ISS-ODN treatment of M. avium-infected mice does not
enhance the anti-M. avium T-cell response in these animals.
This finding is supported by the observation by Doherty and Sher
(12) that the absence of cell-mediated immunity affects pathogen growth only in the chronic stage of M. avium
infection in T-cell-deficient mice (BL/scid).
To further investigate the possible factors for the antimycobacterial
effects of ISS-ODN, we used mice with targeted disruptions of genes
known to play protective roles against M. avium infection in
other systems (Table 1). ISS-ODN induces reactive oxygen species in
murine B cells and monocytes (54). However, oxygen
radicals and NO did not appear to play a role in the observed
ISS-ODN-mediated growth inhibition of M. avium in mBMDMs.
These findings are consistent with previous reports in which certain
strains of M. avium are resistant to the bactericidal effect
of NO or other oxygen radicals (1, 5), despite the fact
that reactive radicals show antimicrobial activity against many
microorganisms (16, 35). In addition, ISS-ODN-induced
cytokines such as TNF-, IL-12, and IFNs did not play significant
roles in the ISS-ODN-mediated antimycobacterial effect.
The reduced M. avium growth in the lung and spleen but the unaffected M. avium growth in the liver (Fig. 2) led us to search for an organ-specific inducible factor with antimicrobial effects. IDO is such a factor. IDO is induced by IFNs (18), and induction of this enzyme is required to prevent rejection of the allogeneic fetus by maternal T cells (37). IDO also inhibits the growth of a variety of intracellular organisms, such as Toxoplasma gondi (41), Plasmodium berghe (in a murine model of malaria [46]), Chlamydia psittaci (7), and Chlamydia trachomatis (4), by breaking down L-Try, which is required for their growth, to L-kynurenine. Furthermore, in vivo ISS-ODN administration induced organ-specific IDO gene expression. As shown in Fig. 4, the induction of IDO was observed in the spleen and lungs, but not in the liver of ISS-ODN-injected mice, which agrees with the observed pattern of M. avium inhibition in vivo (Fig. 2). Although mycobacteria are able to synthesize all amino acids required for their growth, including tryptophan (43), the inhibition of M. avium growth is likely due to the combined result of reduced tryptophan availability and increased levels of the toxic metabolites L-kynurenine, anthranilic acid, quinolinic acid, and picolinic acid (40). Indeed, as shown in Fig. 4, the depletion of tryptophan is not the sole mechanism responsible for the antimycobacterial effect of ISS-ODN, since repletion of L-Try or addition of an IDO inhibitor, M-Try, did not fully restore M. avium growth. Another mechanism by which ISS-ODN inhibits M. avium growth may be the enhancement of CD40 expression by ISS-ODN (32), which could result in enhanced CD40-CD40 ligand binding. We have previously shown that CD40-CD40 ligand signaling results in the inhibition of the intracellular growth of M. avium (21). Further study is necessary to determine whether other factors or mechanisms are involved in the antimycobacterial effect of ISS-ODN in vivo.
ISS-ODN was initially identified in mycobacterial genomes (50), and it is well known that mycobacterial DNA is immunostimulatory. This raises a question: why is it that the M. avium genomic DNA contained within infected cells such as macrophages does not facilitate the clearance or inhibit the growth of this intracellular pathogen? A potential explanation for this paradox may lie in the recent observation that agents such as chloroquine (31), which inhibits the acidification of lysosomes, neutralize the immunostimulatory effects of ISS-ODN. Interestingly, mycobacteria also inhibit the generation of low pH in the lysosomes (49). Therefore, neutralization of the acidic environment of the lysosome may protect M. avium both directly, by avoiding digestion, and indirectly, by blocking the detection of its own immunostimulatory genomic DNA by the host cell.
The antimycobacterial effect of ISS-ODN in vivo on established M. avium infection is less than its effect when used prior to
infection, while ISS-ODN significantly inhibits M. avium
growth in vitro (Fig. 1B). In vivo, M. avium delivered i.v.
can infect different macrophage populations, such as splenic
macrophages, alveolar macrophages, and peritoneal macrophages.
Peripheral macrophage populations have been shown to exhibit distinct
functional phenotypes, including cytokine production, response to
immunomodulatory stimuli, and clearance of pathogens (reviewed in
reference 45). Gangadharam and Pratt reported that
alveolar macrophages and peritoneal macrophages have the distinct
ability to ingest and control the multiplication of
Mycobacterium cells (19). Alveolar macrophages
are known to release more nitrate than peritoneal macrophages when
exposed to LPS and IFN- in C3H/HeJ mice (52). Indeed,
in our preliminary study, alveolar macrophages, splenic macrophages,
and BMDMs show unique individual patterns of cytokine secretion induced
by ISS-ODN (data not shown). It is likely that various macrophage
populations behave differently in response to ISS-ODN and M. avium infection.
Although treatment of macrophages with ISS-ODN inhibited M. avium growth in vitro, in vivo administration of ISS-ODN alone to mice with an established M. avium infection had no protective effect (Fig. 5B). However, when ISS-ODN was used together with CLA, there was a synergistic anti-M. avium therapeutic effect in mouse and human macrophages in vitro. This synergism might be due to more effective protein synthesis inhibition. CLA inhibits protein synthesis by binding to the 50S ribosome, while IDO induced by ISS-ODN interferes with protein synthesis by depletion of tryptophan. In in vivo experiments, ISS-ODN plus CLA showed a significant though modest synergistic therapeutic effect. Additional work is required with different doses and timing of ISS-ODN administration in order to optimize combined therapy as an alternate regimen in the treatment of established M. avium infection.
In summary, this study demonstrates that administration of ISS-ODN enhances resistance against M. avium infection through the induction of IDO. ISS-ODN itself provides limited protection against M. avium. However, this effect can be amplified upon codelivery with an antimycobacterial drug. The combined administration of ISS-ODN with other antibiotics or other antimicrobial agents provides a novel therapeutic strategy for microbial infections and warrants further investigation.
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ACKNOWLEDGMENTS |
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Funds from the National Institutes of Health (grants AI40682 and AI47078 to E.R., AI35258 and HL57911 to R.S.K., and AR44850 for D.A.C) and from Dynavax Technologies Corporation and Sequella Global TB Foundation grant VIP 012 supported this research.
We thank Sunil J. Sahdeo for excellent technical assistance. We also thank Lucinda Beck for helpful editorial assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0663. Phone: (858) 534-5444. Fax: (858) 534-5399. E-mail: eraz{at}ucsd.edu.
Editor: S. H. E. Kaufmann
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, October 2001, p. 6156-6164, Vol. 69, No. 10
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.10.6156-6164.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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