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Production Through TGF-
-Dependent and -Independent Mechanisms1
,
,
Departments of
* Pediatrics,
Internal Medicine, and
Medical Microbiology and Immunology, and
Comprehensive Cancer Center, University of Wisconsin Medical School, University of Wisconsin Hospital and Clinics, Madison, WI 53792
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production in response
to the fungus; isogenic, attenuated BAD1 knockout yeast did not have
this effect. Phagocytes released 4- to 5-fold more TGF-
in vitro in
response to wild-type yeast vs BAD1 knockout yeast. Treatment of
inhibitory, conditioned supernatant with anti-TGF- mAb
neutralized detectable TGF- and restored phagocyte TNF-
production. Similarly, addition of anti-TGF- mAb into cultures
of phagocytes and wild-type yeast reversed BAD1 inhibition of TNF-
production. Conversely, TGF- treatment of phagocytes cultured with
knockout yeast suppressed TNF- production. Hence, TGF- mediates
BAD1 suppression of TNF- by wild-type B. dermatitidis
cultured in vitro with phagocytes. In contrast to these findings,
neutralization of elevated TGF- levels during experimental pulmonary
blastomycosis did not restore BAD1-suppressed TNF- levels in the
lung or ameliorate disease. Soluble BAD1 was found to accumulate in the
alveoli of infected mice at levels that suppressed TNF- production
by phagocytes. However, in contrast to yeast cell surface BAD1, which
induced TGF-, soluble BAD1 failed to do so and TNF- suppression
mediated by soluble BAD1 was unaffected by neutralization of TGF-.
Thus, BAD1 of B. dermatitidis induces suppression of
TNF- and progressive infection by both TGF--dependent and
-independent mechanisms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the factors that account for virulence of B. dermatitidis are incompletely understood, a bona fide virulence determinant of the fungus has recently been identified (3). BAD1 (formerly termed WI-1), a 120-kDa protein, is a major Ag and adhesion-promoting protein on B. dermatitidis. Tandem repeats of BAD1 display immunodominant B cell epitopes (4, 5) and also mediate attachment to CD18 and CD14 receptors on human macrophages (4). BAD1null strains of B. dermatitidis created by gene targeting and mutation of the BAD1 locus exhibit greatly reduced pathogenicity (3). In contrast to wild-type yeast, BAD1 knockout strains are nonpathogenic in a murine model, even at high inocula of 105 organisms. These observations underscore the prominent role of BAD1 in pathogenicity of B. dermatitidis.
Mechanisms that underlie the virulence-promoting effect of BAD1 have been partially elucidated. Adherence is one of them. Yeast that lack BAD1 bind poorly to the lung ex vivo and to macrophages in vitro (3). Such findings imply that knockout yeast are unable to establish infection in the lower respiratory tract because they bind poorly to structures in the airway or alveoli. Poorly adherent yeast might be more easily dislodged from the lung or, when lacking the capacity to enter resting lung macrophages, more easily recognized and killed by neutrophils.
Recent results point to additional defects in B.
dermatitidis yeast mutated at the BAD1 locus.
Many and diverse microbes escape host elimination by modulation of host
immunity (5). In a recent study (6), we found
that BAD1 modulates host immunity early in the course of infection and
thereby facilitates establishment of B. dermatitidis in the
lung. BAD1 interferes with host immunity by blocking production of the
proinflammatory cytokine, TNF-, by both macrophages and neutrophils.
Restoration of TNF- production by gene therapy ameliorated the
progression of infection with a wild-type virulent isolate. These
findings provided an additional mechanism by which BAD1 confers
virulence on B. dermatitidis, but did not address how BAD1
suppresses TNF- production. In that study, we found that BAD1
neither binds TNF- nor interferes with binding of TNF- to its
receptor. However, we observed that wild-type B.
dermatitidis yeast induce release of a soluble factor from
phagocytes, or themselves produce a soluble factor, which acts in a
paracrine and autocrine fashion to suppress TNF- production from
phagocytes.
In this study, we investigated BAD1-dependent soluble factor(s) that
suppress TNF- production. We show that BAD1 displayed on the surface
of B. dermatitidis yeast induces phagocyte TGF-
production, which suppresses TNF- production. In contrast, soluble
BAD1 released from yeast in lung alveoli in vivo or added to cell
culture in vitro also suppresses phagocyte TNF- production, but in a
manner independent of TGF-. These findings clarify how BAD1 subverts
host immunity and allows B. dermatitidis to establish
infection and disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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American Type Culture Collection strain ATCC 26199 of B. dermatitidis, a wild-type, virulent isolate originally obtained from a human patient, was used in this study, together with an isogenic nonpathogenic BAD1 knockout strain 55 recently described (3). Isolates of B. dermatitidis were maintained in the yeast form on Middlebrook 7H10 agar slants with oleic acid-albumin complex, grown at 39°C. Liquid cultures of yeast were grown in Histoplasma macrophage medium.
Mice
Inbred BALB/c strains of mice were obtained from the National Cancer Institute (Frederick, MD). Male mice 6–7 wk of age at the time of purchase were housed and cared for throughout these experiments according to guidelines of the University of Wisconsin Animal Care Committee, which approved all aspects of this work.
Reagents
Complete tissue culture medium consisted of RPMI 1640
supplemented with 10% heat-inactivated FBS (HyClone Laboratories,
Logan, UT), 25 mM HEPES buffer, L-glutamine, sodium
pyruvate, penicillin, and streptomycin (BioWhittaker, Walkersville,
MD). Experiments were performed under conditions designed to minimize
endotoxin contamination. Medium and serum contained <0.005 U/ml
endotoxin. Plasticware was obtained prepackaged and endotoxin free.
Casein, LPS derived from Escherichia coli, and Histopaque
1077 and 1119 were purchased from Sigma-Aldrich (St. Louis, MO).
Recombinant human TGF-1, biotinylated anti-human TGF-1 Ab,
and monoclonal anti-human TGF-1 Ab were purchased from R&D
Systems (Minneapolis, MN).
Recombinant, replication-deficient adenoviruses expressing either
murine TGF-1 or -galactosidase as a control were prepared as
described (7) and purchased from the vector core facility
at University of Michigan (Ann Arbor, MI).
Hybridoma 1D11.16 reactive with TGF-1 (8) was obtained
from American Type Culture Collection and used to prepare ascites in
athymic nude BALB/c mice. mAb was precipitated with ammonium sulfate or
used as ascites. The concentration and activity of mAb was determined
by testing it in parallel with commercial anti-TGF-1 from R&D
Systems.
Antigens
Secreted BAD1 was purified as described (9). Briefly, yeast were grown in liquid Histoplasma macrophage medium in a gyratory shaker at 37°C for 2 wk. BAD1 was purified from supernatants in two steps using anion exchange chromatography followed by hydrophobic interaction chromatography. Homogeneity of purified BAD1 was analyzed by SDS-PAGE and silver stain.
Attachment of BAD1 to B. dermatitidis
Reattachment of purified BAD1 to B. dermatitidis knockout strain 55 was described previously (10). Briefly, 20 µg purified BAD1 was added to 107 strain 55 yeast. Cells were incubated for 1 h at 37°C, washed three times with PBS, and analyzed by fluorescence microscopy and FACS analysis for staining with anti-BAD1 mAb DD5-CB4. Strain 55 cells became uniformly coated with BAD1 and the quantity of BAD1 displayed resembled parent strain 26199.
Isolation and assay of peritoneal macrophages and PMNs
Peritoneal exudate cells (PEC)3 were isolated as follows. At 16 and 3 h before PEC isolation, mice were injected i.p. with 3 ml of 10% casein in PBS. Cells were harvested in 3 ml of cold PBS supplemented with 0.05% EDTA. After two washes with HBSS (Life Technologies, Rockville, MD), PEC were enriched for polymorphonuclear leukocytes (PMNs) and macrophages on a two-step Histopaque gradient (Sigma-Aldrich) according to the method of Hilger and Danley (11). To establish that PMNs or macrophages were enriched, each cell fraction was stained with Diff-Quick (Dade Behring, Newark, DE), and cell composition was analyzed by light microscope. Purity of each fraction was >95%.
To assay TGF- production, PMNs were adjusted to 2 x
106/ml in RPMI 1640 and 0.5 ml of the cell
suspension was added to each well of a 24-well tissue culture plate
(Costar, Corning, NY). Preliminary experiments demonstrated that a
ratio of PMN:yeast of 1:1 and 24 h of incubation were optimal for
the production of TGF-, and these conditions were used throughout
the study. Macrophages were placed into 24-well plates at a
concentration of 2 x 106/ml (volume, 0.5
ml/well), incubated for 1 h at 37°C, and washed to remove
nonadherent cells. A ratio of macrophages:yeast of 4:1 and an
incubation time of 48 h were shown to be optimal for production of
TGF-, and these conditions were used to measure TGF- content in
wells.
To assay phagocyte killing of B. dermatitidis in vitro, PMNs
were adjusted to 1 x 106 cells/ml and
placed into 1.5-ml polypropylene microtubes (ISC Bioexpress,
Kaysville, UT). Wild-type yeast was added to achieve E:T ratios of
1:1–100:1. Cultures were exposed to varied amounts of TGF- or
TNF- (R&D Systems) or medium as a control. After incubation at
37°C in 5% CO2 for 3 h in a Nutator (BD
Biosciences, Sparks, MD), cultures were harvested with 0.01%
Triton X-100, which was found in preliminary assays not to effect
viability of B. dermatitidis. Lysates were
diluted in PBS and cultured on BHI agar to determine the number of CFU
of viable yeast. Percentage of killing was calculated by the following
formula: 1 - (coculture CFU/control CFU) x 100. In
macrophage killing assays, the cells were cultured at 1 x
106 cells/ml in 24-well plates and exposed to
TNF-, IFN-
, or medium control for 18 h. Supernatant was
removed and yeast was added to wells to yield an E:T ratio of
1:1–1:10. After incubation for 24 h, cultures were harvested and
the effect on viability of yeast was determined as for PMNs.
Harvest of bronchoalveolar lavage fluid and lung tissue
After mice were anesthetized, alveolar fluid was harvested
through a 20-guage catheter placed intratracheally. A volume of 1 ml
PBS containing 0.5% EDTA was instilled and reaspirated once, yielding
a total volume of 
0.8 ml lavage fluid per mouse. Individual fluid
samples were centrifuged at 2500 rpm for 10 min. Supernatants were
collected, frozen at -20°C, and thawed once for testing.
Whole lungs were harvested from sacrificed mice. Before removal of lungs, the pulmonary vasculature was perfused through the left ventricle with PBS containing 0.05% EDTA. After removal, whole lungs were homogenized in 1.5 ml of lysis buffer containing complete protease inhibitors (Boehringer Mannheim, Indianapolis, IN) using a tissue grinder (Fisher Scientific, Pittsburgh, PA). Homogenates were incubated on ice for 30 min and centrifuged at 2500 rpm for 10 min. Supernatants were passed through a 0.45-µm pore size filter (Gelman Sciences, Ann Arbor, MI) and stored at -20°C.
Measurement of cytokines and other cellular products
Levels of TGF- were measured after acidification to activate
latent TGF-, using a commercial ELISA (R&D Systems); limit of
detection was >10 pg/ml. In neutralization experiments, levels of
total and active TGF- were measured by bioassay using Mv-1-Lu mink
cells (American Type Culture Collection, Manassas, VA) as
described (12). Briefly, cells were cultured on 96-well
tissue culture plates (Costar) at a concentration of 7 x
104 cells per milliliter of medium. Plates were
incubated for 5 h at 37°C in a humidified 5%
CO2 atmosphere. A standard curve was prepared
with 0–50 ng/ml rTGF- (R&D Systems). A volume of 50 µl of
standard or sample per well was added to mink cells. The plates were
incubated an additional 72 h before 20 µl of MTT (5 mg/ml in
PBS) was added. The cells were further incubated for 1–4 h at 37°C
until formazan crystals formed. Addition of 100 µl of 0.04 M
HCl in isopropanol dissolved the crystals, and OD was measured
at 570 nm with an automatic ELISA plate reader (Tecan Spectra; SLT
Instruments, Salzburg, Austria). The limit of detection in this
assay is >3 pg/ml.
A commercial ELISA kit was used to measure TNF- in supernatants (R&D
Systems). ELISAs were developed with streptavidin HRP and substrate
tetramethylbenzidine (Sigma-Aldrich). OD450 of wells was
measured with an automatic plate reader as above. The limit of
detection in this assay is >16 pg/ml.
IL-10 in supernatant was also measured by ELISA (R&D Systems). The limit of detection with the assay is >15 pg/ml.
Nitrite in supernatant of phagocytes was measured using Griess’ reagent (R&D Systems) as described (13). The limit of detection in the assay is >3 µmol/L.
BAD1 protein content in bronchoalveolar lavage fluid was measured by ELISA. Briefly, rabbit anti-BAD1 Ab (concentration, 2 µg/ml) was adhered to microtiter wells of a 96-well plate (Immunoplates; Nunc, Roskilde, Denmark). After wells were blocked with 2% BSA, 100 µl alveolar fluid was added to each well and plates were incubated for 2 h at 37°C. Plates were washed twice with PBS before anti-BAD1 mAb DD5-CB4 was added (concentration, 100 ng/ml). Following incubation for an additional 1 h and washing of wells, an anti-mouse HRP conjugate was added, developed, and read on a plate reader as above. Each assay was done with a standard of known quantities of purified BAD1. The limit of detection in the assay is 15 pg/ml.
Adsorption of TGF- from conditioned supernatant
TGF- was removed from supernatant on an affinity column made
with anti-TGF- mAb 1D11.16 and an AminoLink Plus Immobilization
kit (Pierce, Rockford, IL). Briefly, 650 µg mAb was linked to 1.5 ml
of resin. Coupling efficiency was >95%. One milliliter of supernatant
(1000 pg/ml TGF-) was incubated with the resin for 1 h. The
column was washed with 14 ml of PBS and 1-ml wash fractions were
collected and measured by OD280. The first four
fractions, which contained >95% of the total protein of the wash,
were pooled, concentrated to the original 1-ml volume, and designated
TGF--adsorbed supernatant. Bound TGF- was then eluted from the
column with Tris-glycine buffer (pH 2–3). Eluted fractions were
pooled, dialyzed against PBS, and concentrated to the original 1-ml
volume. TGF- content in the adsorbed sample and the eluate was
quantified by ELISA and bioassay.
Experimental infection
Mice were infected intratracheally with B. dermatitidis. Before infection, mice were anesthetized by i.p. injection of etomidate (30 mg/kg; Bedford Laboratories, Bedford, OH). Skin over the trachea was incised and underlying tissue was separated. A 30-gauge needle (BD Biosciences, Rutherford, NJ) was bent and attached to a tuberculin syringe (BD Biosciences) containing B. dermatitidis yeast. The needle was inserted into the trachea and 30 µl of inoculum dispensed using a stepper device (Tridak, Brookfield, CT). Incised skin was closed with cyanoacrylate adhesive (Nexaband; Veterinary Products Laboratories, Phoenix, AZ). Mice recovered under a heating lamp. At selected time points after infection, burden of lung infection was measured by plating homogenized lung on brain heart infusion (Difco, Detroit, MI) agar and enumeration of yeast CFU. The detection limit was 10 organisms.
To neutralize TGF- during infection, mice were injected i.v. with
0.25–1.5 mg of mAb (1D11.16) 4–6 h before infection, and then with
the same dose of mAb i.v. every 2–3 days afterward. Control mice
received the same dose of rat IgG (Sigma-Aldrich) by a similar
schedule.
Statistical analysis
Differences between wild-type yeast (strain 26199) and BAD1
knockout yeast (strain 55) in stimulation of TGF- or suppression of
TNF- production were analyzed using methods for standard analysis of
variance (14). Differences in the number of CFU in tissue
between groups of infected mice were analyzed statistically using the
Wilcoxon rank test for nonparametric data (14, 15).
Differences between groups were considered statistically significant if
the two-sided p value was <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recently observed that BAD1 on wild-type B.
dermatitidis suppresses TNF- production by both neutrophils and
macrophages (6). In mixing experiments, we found that
wild-type yeast was able to suppress phagocyte stimulation by
BAD1null yeast, even when as few as
103 wild-type yeast were added to wells
containing 106 BAD1null
yeast and 106 phagocytes. Because wild-type yeast
were likely to interact with only a small proportion of phagocytes in
such wells in this short-term assay, the mixing data suggested that
BAD1 on wild-type yeast evokes release of a soluble factor(s) that acts
in an autocrine and paracrine manner to antagonize production of
TNF- by phagocytes.
To explore the idea of soluble factors in this study, we investigated
the inhibitory activity of conditioned supernatants that were made from
phagocytes cocultured in vitro with either wild-type yeast or
BAD1null yeast. These supernatants were added at
the initiation of fresh in vitro coculture of phagocytes with the
BAD1null yeast, a strain that has previously been
shown to be a potent stimulus of TNF- production (6).
Conditioned supernatant from cells that had been cultured with
wild-type yeast abolished TNF- production by PMNs and macrophages in
response to BAD1null yeast (Fig. 1
). TNF- levels were reduced to the
range of values seen when cells were cultured in medium alone.
Conditioned supernatant from cells that had been cultured with the
BAD1null yeast or with no yeast did not inhibit
TNF- production; TNF- levels were comparable to those seen in
response to the BAD1null strain alone.
Conditioned supernatant inhibited TNF- in a dose-dependent manner
(Fig. 1B); 100 µl suppressed levels maximally and was used
for subsequent assays. Hence, phagocytes release a soluble factor into
medium in response to BAD1 on B. dermatitidis, which in turn
suppresses TNF- production.
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Selected soluble factors have been reported to inhibit TNF-
produced by phagocytes or to have immunosuppressive properties,
including TGF-1, IL-10, and NO (16, 17, 18). We analyzed
conditioned supernatants for differential production of these factors
to explain inhibition of TNF- production. Supernatants from both
PMNs and macrophages selectively accumulated TGF-1 in response to
wild-type yeast, as compared with BAD1null yeast
(Fig. 2). PMNs released 5-fold more
TGF-1 in response to wild-type yeast as compared with
BAD1null yeast. Macrophages released nearly
4-fold more TGF-1 in response to the wild-type yeast. Differential
TGF-1 responses to the isogenic strains peaked at 24 h for PMNs
and 48 h for macrophages, but a time course of TGF-1 released
in response to the strains showed similar trends throughout an
incubation period of 6–48 h for PMNs and 18–96 h for macrophages
(data not shown).
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production in
response to BAD1 on B. dermatitidis. Stimulation of NO in
phagocytes was comparable for the isogenic strains (data not shown).
Hence, of factors analyzed, only TGF-1 accumulated selectively in
response to BAD1 on the wild-type strain, in a pattern consistent with
regulation of TNF- production by phagocytes.
Neutralization and adsorption of TGF-1 in conditioned
supernatant reverses inhibition of TNF- production
Our findings led us to postulate that TGF-1 in response to BAD1
on B. dermatitidis suppresses TNF- and accounts for the
inhibitory effect of conditioned supernatant shown in Fig. 1
. We
investigated this possibility by addition of anti-TGF-1 mAb
1D11.16 to conditioned supernatant to neutralize TGF-1, while
measuring the effect on TNF- production in response to B.
dermatitidis. In these experiments, depicted in Fig. 3A,
BAD1null yeast as a control stimulated robust
TNF- and little TGF-1 production by phagocytes, but addition of
conditioned supernatant (from cells cocultured with wild-type yeast and
enriched in TGF-1) suppressed the phagocyte TNF- response to the
BAD1null strain. Remarkably, addition of
anti-TGF-1 mAb to this supernatant neutralized TGF-1 activity
measured by bioassay and extinguished the supernatant’s inhibitory
effect, restoring TNF- production in response to the
BAD1null strain. Rat IgG control Ab added to the
conditioned supernatant had no effect on TGF-1 activity or TNF-
production. In a second approach, to exclude the effects of Ag-Ab
complexes, we used an Ab affinity column to adsorb TGF-1 from the
inhibitory conditioned supernatant and found that the adsorbed
supernatant no longer suppressed TNF- production. By contrast,
eluate from the affinity column containing TGF-1 suppressed TNF-
production in a concentration-dependent manner (Fig. 3B).
These data indicate that TGF-1 in conditioned supernatant is
responsible for suppressing phagocyte TNF- in response to the
BAD1null strain and imply that BAD1 on wild-type
B. dermatitidis suppresses TNF- by inducing phagocytes to
release TGF-1.
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1 on phagocyte
TNF- production in response to B. dermatitidis
We showed above that the suppressive effect of conditioned
supernatant on TNF- production is due to TGF-1. We next sought to
extend this finding by investigating whether addition or removal of
TGF-1 from the inception of in vitro coculture of phagocytes with
yeast could respectively down-regulate or up-regulate TNF- in
response to B. dermatitidis. For both PMNs and macrophages,
addition of rTGF-1 into the coculture suppressed production of
TNF- in response to the BAD1null strain of
B. dermatitidis and exerted a concentration-dependent effect
(Fig. 4A). Addition of as
little as 100 pg of TGF-1 to wells suppressed TNF- production,
and addition of 2 ng TGF-1 (amount achieved in conditioned
supernatant) suppressed TNF- production down to levels detected
during coculture of phagocytes and wild-type B. dermatitidis
yeast.
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1 mAb was added to
wells from the inception of coculture of wild-type yeast and phagocytes
to attempt to reverse suppression of TNF-. Anti-TGF-1 restored
production of TNF- in response to the wild-type yeast, doing so in a
concentration-dependent manner (Fig. 4B). Production of
TNF- nearly doubled following the addition of 1 µg mAb, and, in
the presence of 10 µg, phagocytes produced nearly 90% of the level
that was observed in response to the BAD1null
strain of B. dermatitidis. These findings show that
TGF-1 is both necessary and sufficient to modulate TNF-
production in response to B. dermatitidis yeast during
short-term in vitro coculture.
In vivo production of TGF- in lungs of mice infected with
isogenic strains of B. dermatitidis
We sought to correlate the in vitro findings with in vivo levels
of TGF-1 produced in lung in response to the isogenic strains. To
determine whether wild-type yeast selectively induced production of
TGF-1 at this primary site of infection, we quantified TGF-1
protein in the lung. After intratracheal infection with B.
dermatitidis yeast, levels of TGF-1 were 2- to 3-fold higher
for the wild-type strain compared with the
BAD1null strain between 24 and 96 h
postinfection, time points during which the extent of infection was
comparable for the two strains (Fig. 5).
This trend of increased TGF-1 in the lung in response to wild-type
yeast was also seen at 6 days postinfection, when infection with
wild-type yeast progressed, and at subsequent time points (data not
shown). These elevated TGF-1 levels were accompanied by reciprocal
alterations in TNF-, similar to what was seen in vitro. Results were
similar in lung homogenates and alveolar fluid lavage samples (data not
shown). Hence, induction of TGF-1 selectively in response to BAD1 on
B. dermatitidis is evident in vivo in the lung compartment
of infected mice, in addition to being observed during in vitro
coculture experiments.
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1
inhibited neutrophil killing of yeast by nearly 100% when added to
cocultures at concentrations that were detected in lung during
infection (Fig. 6). Addition of TNF-
to cocultures also had a marked functional effect, significantly
augmenting killing of the fungus by both neutrophils and macrophages.
Thus, excesses of TGF-1 in the lung could impair host defense
directly by suppressing phagocyte killing of yeast and indirectly by
suppressing production of TNF-, which would further reduce phagocyte
killing of yeast and have other potential downstream effects on host
defense.
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1 on experimental
infection
We sought to investigate whether TGF-1 alone is sufficient to
explain down-regulation of TNF- and progression of wild-type
B. dermatitidis in vivo in an experimental model of
blastomycosis. To test this concept, we administered anti-TGF-1
mAb 1D11.16 to mice just before and during the course of infection with
wild-type B. dermatitidis. Administration of 0.25 or 0.5 mg
per dose of anti-TGF-1 had little effect on TGF-1 (or
TNF-) levels in the lungs of treated mice. In contrast, the level of
TGF-1 was sharply reduced in mice that received 1.5 mg
anti-TGF-1 compared with rat IgG control (Fig. 7A). Nevertheless, the TNF-
level was unaffected by the drop in TGF-1 level and remained 2-
to 3-fold less than the lung TNF- level in mice that were infected
with the BAD1null yeast strain of B.
dermatitidis. In conjunction with these cytokine measurements, we
analyzed the influence of TGF-1 neutralization on the course of
experimental infection. Neutralization of TGF-1 did not ameliorate
the progression of wild-type B. dermatitidis infection, as
measured by lung CFU (Fig. 7B). Similarly, treatment of mice
with rTGF-1 via adenovirus did not exacerbate B.
dermatitidis infection (data not shown). Thus, elevated levels of
TGF-1 alone are not sufficient to explain TNF- suppression or
progression of wild-type B. dermatitidis infection. There
may be TGF-1-dependent and -independent mechanisms by which BAD1
enhances virulence of the fungus.
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production
through TGF-1-dependent and -independent mechanisms
BAD1 is released from wild-type B. dermatitidis yeast
in copious amounts during liquid culture. Nearly 25 mg of BAD1 per
liter of culture supernatant has been reported to accumulate after 5
days of growth in vitro for wild-type B. dermatitidis strain
ATCC 26199 (19), the strain used for the present study.
Importantly, soluble BAD1 can sharply suppress the production of
TNF- by phagocytes in response to a positive stimulus, either the
BAD1null strain of B. dermatitidis or
Saccharomyces cerevisiae (6). Hence, we
explored whether the inhibition of TNF- by surface-bound BAD1 and
soluble BAD1 were mediated by differential mechanisms, i.e., one
dependent on TGF-1 and the other one independent of TGF-1. In
these experiments, depicted in Fig. 8, yeast cell surface BAD1 and soluble BAD1 were tested for an influence
on suppression of TNF- and concomitant induction of TGF-1.
Furthermore, anti-TGF-1 mAb was tested for its ability to
reverse suppression of TNF- mediated by BAD1 in each of these forms.
BAD1 displayed on the yeast surface (wild-type yeast or BAD1-coated
knockout yeast) or added in soluble form during coculture markedly
suppressed production of TNF- in response to
BAD1null yeast (Fig. 8, upper panel).
In response to surface BAD1 on wild-type yeast or BAD1-coated knockout
yeast, the TGF-1 levels were concurrently elevated and ranged from 4
to 5 ng/ml. By contrast, the TGF-1 level was 10-fold lower when
soluble BAD1 was used to suppress TNF- production; the TGF-1
level was in fact similar to that in response to the
BAD1null yeast alone. Addition of
anti-TGF-1 mAb to the cultures neutralized TGF-1 activity
measured by bioassay and restored TNF- production in response to
wild-type yeast and BAD1-coated knockout yeast, yielding TNF- levels
similar to those in response to the BAD1null
strain (Fig. 8, lower panel). By contrast, addition of
anti-TGF-1 into wells where soluble BAD1 suppressed TNF- did
not restore TNF- production. These data suggest that soluble BAD1
suppresses TNF- by a mechanism that is independent of TGF-1. The
results might explain why in vivo neutralization of TGF-1 in mice
infected with wild-type yeast failed to restore lung TNF- levels and
ameliorate progression of experimental pulmonary infection. Such
conclusions are based on the premise that wild-type B.
dermatitidis yeast release soluble BAD1 in vivo at the site of
infection.
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A). We added these amounts of
BAD1 in vitro into wells with phagocytes and stimulatory
BAD1null yeast. As with microgram amounts of
soluble BAD1 tested in prior experiments (6), nanogram
amounts of soluble BAD1 sharply suppressed TNF- production by
phagocytes (Fig. 9B). TNF- levels were 3- to 5-fold lower
than those measured in the absence of BAD1, and TGF-1 levels were
not augmented. Thus, BAD1 is released by B. dermatitidis
yeast into alveolar fluid, and the levels detected are sufficient to
suppress TNF- production by phagocytes in response to the fungus.
Moreover, mechanism(s) by which soluble BAD1 suppresses TNF- appear
to be independent of TGF-1-mediated suppression of TNF-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(6). Production of TNF- is essential in
promoting host defense against B. dermatitidis, other
pathogenic fungi such as Cryptococcus neoformans and
Histoplasma capsulatum, and the intracellular bacterial
pathogen Mycobacterium tuberculosis (20, 21, 22, 23).
In an experimental model of B. dermatitidis infection,
restoration of suppressed TNF- by gene therapy stemmed the
progression of blastomycosis (6), emphasizing the
biological relevance of BAD1 modulation of host immunity in a model
that mimics human infection. Because TNF- suppression is important
in understanding the pathogenesis of this and other infectious
diseases, we explored the mechanism(s) responsible for BAD1 inhibition
of TNF- production. We postulated that BAD1 could act directly on
the phagocyte by inducing toxicity, apoptosis, or a signaling pathway
that shuts off TNF- production, or alternatively, act indirectly by
inducing release of inhibitory product(s) that act in an autocrine and
paracrine fashion. Such mechanisms are not mutually exclusive.
In this work, we demonstrate a mechanism that entails BAD1 induction of
TGF-, which in turn sharply suppresses TNF- production by
phagocytes. We initially suspected a suppressive mechanism involving a
soluble inhibitor from results of a mixing experiment in our recent
study (6). Stimulatory BAD1 knockout yeast
(106 cells) were mixed with wild-type yeast
(103 cells) and added to wells containing
106 phagocytes (E:T ratio of 1000:1 with respect
to wild-type yeast). Hence, in theory, wild-type yeast in wells
interacted with only 1 in 1000 phagocytes. Despite this, wild-type
yeast eliminated TNF- production in the wells. This result is
consistent with release of a factor(s) into medium that modulates
activity of all cells in the well, even though wild-type yeast interact
with only a small proportion. Our data indicate that phagocytes respond
to BAD1 on wild-type yeast by releasing a soluble factor(s) that
down-regulate TNF-, and at least one such factor is TGF-. First,
conditioned supernatant prepared from wild-type yeast inhibited
production of TNF- in response to BAD1 knockout yeast, whereas
control supernatants had little effect. Second, the amount of TGF-
in these supernatants correlated with their ability to inhibit TNF-
production. Third, removal of TGF- activity in the supernatant
reversed its inhibitory effects. Fourth, addition of neutralizing
anti-TGF mAb to wells with inhibitory wild-type yeast and
phagocytes reversed suppression of TNF- production. Last, addition
of rTGF- to wells with stimulatory BAD1 knockout yeast suppressed
production of TNF- in response to this strain. These results
together demonstrate that wild-type yeast, and presumably surface BAD1,
down-regulates TNF- production in response to B.
dermatitidis by inducing TGF- release from macrophages and
neutrophils. Although we did not explore the precise mechanism(s) by
which TGF- suppresses TNF- production, Bogdan et al.
(17) previously analyzed this interaction. TGF- acts at
the posttranscriptional level, suppressing translation of TNF-. This
mechanism contrasts with that of IL-10, another inhibitor of TNF-,
which promotes degradation of TNF- mRNA.
Parasites and bacteria can also elicit production of TGF-
(24, 25, 26, 27, 28, 29, 30, 31), which promotes evasion of immunity and sometimes
disease progression. Leishmania amazonensis and
Leishmania braziliensis elicit TGF-, which promotes
virulence and parasite escape from killing by host macrophages and is
important for determining disease susceptibility to experimental
leishmanial infection through influence on the Th phenotype (24, 25). Similarly, Leishmania chagasi, a causative agent
of visceral leishmaniasis, induces production of TGF- within liver
granulomas, and this locally secreted product inhibits IFN-
production and a Th1-associated cure of visceral leishmaniasis in a
murine model of disease (26, 27). TGF- serves as the
soluble mediator, accounting for the observation that engagement of
CTLA-4 on T cells leads to suppression of cellular immunity in a murine
model of kalaazar (27). Thus, increased TGF- secretion
is directly responsible for CTLA-4-mediated arrest of antiparasite
defense in the infected host. In Trypanosoma cruzi
infection, TGF- blocks the ability of IFN- to induce macrophage
killing of the parasite, and treatment of resistant mice with TGF-
greatly enhances disease progression in a murine model, illustrating
that TGF- also regulates T. cruzi infection in vitro and
in vivo (28, 29). In M. tuberculosis infection,
TGF- down-regulates in vitro proliferation of human PBMCs from
patients in response to mycobacterial Ags (30, 31).
Natural inhibitors of TGF- such as decorin and latency-associated
peptide corrected depressed T cell proliferation and led to significant
reductions in bacterial growth in vitro in mononuclear cells infected
with M. tuberculosis. These findings with regard to
parasites and bacteria suggest that induction of host TGF-
represents a general theme of immune evasion and virulence, and
underscores the fundamental relevance of observations reported in this
study with B. dermatitidis. Whereas microbial component(s)
responsible for TGF--mediated immune suppression have so far
remained obscure in parasites and bacteria, our data provide strong
evidence that BAD1 on the yeast is responsible for induction of TGF-
and concurrent TNF- suppression.
Because of precedents in the literature for the role of TGF- in
disease progression, we investigated this possibility in an
experimental model of blastomycosis. We observed that levels of TGF-
in alveolar fluid collected during the first 3 wk of infection were
severalfold higher in response to wild-type yeast compared with BAD1
knockout yeast. However, mAb neutralization of TGF- did not
influence the course of blastomycosis in mice that were infected with
wild-type yeast. Neutralization did reduce TGF- levels in alveolar
lavage fluid; however, several reasons could account for the lack of
biological effect. First, timing of neutralization may be important, as
TGF- can have pleiotropic effects on the host depending upon when
cells are exposed to the cytokine. For example, naive T cells may be
activated by TGF-, whereas activated T cells may conversely be
inhibited (16). We elected to neutralize TGF- in our
studies at time 0, i.e., right at the time of initial infection. It is
possible that the timing thus interfered with T cell activation. A
second explanation is more plausible and relates to the effects of
neutralization on restoration of TNF- production. We previously
showed that TNF- is critical in host defense against B.
dermatitidis (6), and in this work we showed that
TGF- suppresses the production of TNF- in vitro. Hence, it was of
great interest to monitor whether TGF- neutralization restored
TNF- levels to those observed in the setting of disease containment
due to infection with BAD1 knockout yeast. These studies pointed to
dissociation between neutralization of TGF- in vivo and restoration
of TNF- levels in lung alveolar fluid. TNF- levels appeared to be
uninfluenced despite a substantial decline in TGF- in mice that
received neutralizing Ab, and, in fact, the TNF- levels remained far
below those detected in the lungs of mice infected with the BAD1
knockout. These findings suggested to us that TGF- may not be solely
responsible for suppressed TNF- levels in vivo, and that there may
be a more complex regulatory mechanism between BAD1 and TNF-, which
encompasses factors in addition to TGF-.
Our in vitro data demonstrate that, although cell surface-bound BAD1
suppresses TNF- in a TGF--dependent manner, soluble BAD1
conversely suppresses TNF- in a manner that is independent of
TGF-. We showed that soluble BAD1 sharply suppressed the production
of TNF- by neutrophils and macrophages in response to stimulatory
knockout yeast, but TGF- levels were not elevated, nor did TGF-
neutralization reverse the suppression (as it did when cell surface
BAD1 was responsible for suppression). Hence, soluble BAD1 suppression
of TNF- could explain why TGF- neutralization in vivo neither
restored TNF- levels nor ameliorated disease progression. In support
of this concept, we found soluble BAD1 in lung alveolar lavage fluids
during the course of infection. The BAD1 concentrations detected in
vivo ranged from 10 to 50 ng/ml. Technical difficulties involved in
adequately sampling sites of heavy infection could underestimate BAD1
concentrations in the lung alveoli. Nevertheless, we demonstrated that
even these low levels of soluble BAD1 in lung could fully suppress
TNF- production in vitro independent of TGF- and thus potentially
account for in vivo observations. The polysaccharide constituent
glucuronoxylomannan of the capsule of C. neoformans is shed
in the lung and other infected body sites and also suppresses host
immune functions, including phagocyte production of TNF-
(32, 33, 34, 35, 36).
In summary, we show in this work that yeast cell surface BAD1
suppresses TNF- in a manner dependent on TGF-, whereas soluble
BAD1 also suppresses TNF- but does so independently of TGF-.
Further understanding of these regulatory mechanisms will clarify how
BAD1 and other microbe virulence factors enhance pathogenicity and
promote disease progression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bruce S. Klein, University of Wisconsin, 600 Highland Avenue, K4/434, Madison, WI 53792. E-mail address: bsklein{at}facstaff.wisc.edu
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; PEC, peritoneal exudate cell.
Received for publication November 5, 2001. Accepted for publication March 19, 2002.
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exacerbates primary and secondary pulmonary histoplasmosis by differential mechanisms. J. Immunol. 160:6072. plays a critical role in maintaining secondary immunity in the absence of IFN-. J. Immunol. 160:1359. in leishmanial infection: a parasite escape mechanism. Science 257:545. as a virulence mechanism for Leishmania braziliensis. Proc. Natl. Acad. Sci. USA 90:3442. in murine visceral leishmaniasis. J. Immunol. 161:6148. mediates CTLA-4 suppression of cellular immunity in murine kalaazar. J. Immunol. 164:2001. (TGF-). J. Exp. Med. 174:539. in human tuberculosis: suppression of antigen-drive blastogenesis and interferon production. Proc. Natl. Acad. Sci. USA 93:3193.. Proc. Natl. Acad. Sci. USA 94:3926.This article has been cited by other articles:
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  M. L. Bohse and J. P. Woods RNA Interference-Mediated Silencing of the YPS3 Gene of Histoplasma capsulatum Reveals Virulence Defects Infect. Immun., June 1, 2007; 75(6): 2811 - 2817. [Abstract] [Full Text] [PDF]
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 M. L. Bohse and J. P. Woods Expression and Interstrain Variability of the YPS3 Gene of Histoplasma capsulatum Eukaryot. Cell, April 1, 2007; 6(4): 609 - 615. [Abstract] [Full Text] [PDF]
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 T. T. Brandhorst, G. M. Gauthier, R. A. Stein, and B. S. Klein Calcium Binding by the Essential Virulence Factor BAD-1 of Blastomyces dermatitidis J. Biol. Chem., December 23, 2005; 280(51): 42156 - 42163. [Abstract] [Full Text] [PDF]
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M. L. Bohse and J. P. Woods Surface Localization of the Yps3p Protein of Histoplasma capsulatum Eukaryot. Cell, April 1, 2005; 4(4): 685 - 693. [Abstract] [Full Text] [PDF]
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 T. T. Brandhorst, M. Wuthrich, B. Finkel-Jimenez, T. Warner, and B. S. Klein Exploiting Type 3 Complement Receptor for TNF-{alpha} Suppression, Immune Evasion, and Progressive Pulmonary Fungal Infection J. Immunol., December 15, 2004; 173(12): 7444 - 7453. [Abstract] [Full Text] [PDF]
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 D. J. Weiss, O. A. Evanson, M. Deng, and M. S. Abrahamsen Gene Expression and Antimicrobial Activity of Bovine Macrophages in Response to Mycobacterium avium subsp. paratuberculosis Vet. Pathol., July 1, 2004; 41(4): 326 - 337. [Abstract] [Full Text] [PDF]
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J. N. Steenbergen, J. D. Nosanchuk, S. D. Malliaris, and A. Casadevall Interaction of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum with Acanthamoeba castellanii Infect. Immun., June 1, 2004; 72(6): 3478 - 3488. [Abstract] [Full Text] [PDF]
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P. J. Rooney and B. S. Klein Sequence Elements Necessary for Transcriptional Activation of BAD1 in the Yeast Phase of Blastomyces dermatitidis Eukaryot. Cell, June 1, 2004; 3(3): 785 - 794. [Abstract] [Full Text] [PDF]
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