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*
Microbiology and
Anatomy Sections, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy; and
Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, Tor Vergata University, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aspergilli are respiratory pathogens and pulmonary infections are
usually acquired through the inhalation of conidia (3).
With a diameter of 
2.5–3.5 µm, the conidia are able to reach
small airways and the alveolar spaces, where germination of conidia and
subsequent tissue invasion occurs in immunologically impaired
individuals. Although epithelial and endothelial cells may internalize
conidia (3), effector mechanisms of the innate immune
system have long been recognized as major host defenses against IPA
(4). Resident alveolar macrophages ingest and kill resting
conidia, mainly through nonoxidative mechanisms, while neutrophils use
oxygen-dependent mechanisms to attack hyphae germinating from conidia
that escape macrophage surveillance (3, 4, 5). More recent
studies in mice (6, 7, 8) and humans (9, 10)
have shown that a Th1/Th2 dysregulation and a switch to a Th2 immune
response may contribute to the development of an unfavorable outcome
of IPA.
Dendritic cells (DC) have a primary role in surveillance for pathogens
at the mucosal surfaces and are recognized as the initiators of immune
responses to them (11, 12, 13). A dense network of DC has been
described in the respiratory tracts (14, 15, 16). In the
resting state, respiratory tract DC are specialized for uptake and
processing, but not for presentation of Ag, the latter requiring
cytokine maturation signals that presumably occur after migration to
regional lymph nodes (17, 18, 19). The evidence that pulmonary
DC, through production of IL-10, mediate unresponsiveness to
respiratory Ags (20, 21) indicates that local production
of immunoregulatory cytokines may affect the ability of DC to instruct
the appropriate T cell responses to the invading pathogens. This is
particularly relevant in the case of responses to fungi, as DC appeared
to be uniquely able to discriminate among the different forms of them
(22). Through the use of different pattern recognition
receptors (PRRs), receptors for a number of components of the
complement system (CR), and FcR (Fc
R), DC finely discriminated
between yeast and hyphae of Candida albicans and were
responsible for Th priming and education in C. albicans
saprophytism and infection (22). In the present study, we
assessed the functional activity of pulmonary DC in response to
A. fumigatus conidia and hyphae, both in vitro, and in
infection in vivo. We analyzed mechanisms and receptors for
phagocytosis of fungi by DC as well as DC migration, maturation, and Th
priming in vivo upon exposure to either form of the fungus. We found a
remarkable functional plasticity of DC in response to the different
forms of the fungus, as pulmonary DC were able to discriminate between
the different forms in terms of maturation, cytokine production, and
induction of local and peripheral Th cell reactivity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c (H-2d) mice, 8- to 10-wk old, were purchased from Charles River Breeding Laboratories, Calco, Italy. BALB/c mice were bred under specific pathogen-free conditions in the animal facility of the University of Perugia, Perugia, Italy. Procedures involving animals and their care were conducted in conformity with national and international laws and policies.
Microorganism, culture conditions, and infection
The strain of A. fumigatus was obtained from a fatal
case of pulmonary aspergillosis at the Infectious Diseases Institute of
the University of Perugia (6, 7, 8). The microorganism was
grown on Sabouraud dextrose agar (Difco, Detroit, MI) supplemented with
chloramphenicol for 4 days at room temperature. Abundant conidia were
elaborated under these conditions. Conidia were harvested by washing
the slant culture with 5 ml of 0.025% Tween 20 in saline and gently
scraping the conidia from the mycelium with a plastic pipette (or
shaking it vigorously). Cell debris was allowed to settle by gravity
and the suspension was decanted into 50-ml plastic conical tubes. After
extensive washing with saline, conidia were counted and diluted to the
desired concentrations. Swollen conidia were prepared by incubating the
resting conidia in Sabouraud’s broth until the spores swelled to
almost twice their resting diameter but had not germinated (4 h at
37°C). For generation of hyphae, resting or swollen conidia were
allowed to germinate (>98% germination) by further incubation in
Sabouraud’s broth (20 and 6 h, respectively). The viability of
resting and swollen conidia was >95%, as determined by serial
dilution and plating of the inoculum on Sabouraud dextrose agar. For
intratracheal (i.t.) injection, immunocompetent mice were anesthetized
by i.p. injection of 2.5% avertin (Sigma-Aldrich, St. Louis, MO); a
volume of 80-µl saline containing 2 x 108
conidia or 5 x 105 hyphae was injected
under direct visualization through the opening vocal cords using a
25-gauge metal catheter connected to the outlet of a micropipette. In
the case of infection with FITC-labeled fungal cells, control mice were
injected with 12 µg/20 µl FITC (3 mg/ml, F-4274; Sigma-Aldrich)
diluted in DMSO. Mice succumbing to fungal challenge were routinely
necropsied for histopathological confirmation of invasive
aspergillosis.
Labeling of conidia and hyphae with FITC
Live conidia and hyphae were suspended in 0.1 M of carbonate buffer (pH 9.3) at 2 x 108/ml and added to 200 µl of FITC in DMSO, as described (23). After incubation for 2 h at room temperature protected from light, the suspensions were diluted and washed twice in PBS (pH 7.2) to remove all detectable free-FITC as determined by fluorescence measurement of the supernatants compared with PBS alone. The conidia and hyphae pellets were then counted and diluted in PBS to the desired concentration. FITC labeling did not affect the viability of cells.
Propagation of fetal skin-derived DC (FSDC)
Immature FSDC were generated by retroviral immortalization, as described (24). Cells were cultured in IMDM (Life Technologies Italia, Milan, Italy) containing 5% filtered FCS, 50 mM of 2-ME, 2 mM of L-glutamine, and 50 µg/ml gentamicin (complete medium). For routine passaging, the cells were detached from tissue culture flasks (Falcon; BD Labware, Franklin Lakes, NJ) with 2 mM of EDTA in PBS.
Transmission electron microscopy (TEM)
For TEM, FSDC were incubated in suspension culture dishes with A. fumigatus conidia or hyphae, as in the phagocytic assay for 1 and 3 h. A total of 5 x 106 cells were pelleted at 8 x g for 5 min and washed twice with PBS. Cells and thin sections of the lungs were fixed in cold 2.5% glutaraldehyde in 0.1 M of sodium cacodylate/1% sucrose buffer for 4 h. The cells were postfixed in 1% osmium tetroxide (50 min), encapsulated in 1% agar, stained with uranyl acetate and phosphotungstic acid, and dehydrated in a series of graded ethanolic solutions finishing with propylene oxide before finally being embedded in Epon 812-Araldite mixture. Ultrathin sections (50 nm) were cut on a LKB ultramicrotome and placed under 200 mesh standard copper grids, contrasted with uranyl acetate and lead citrate, and examined with a Philips TEM 400 transmission electron microscope (Eindhoven, The Netherlands).
DC isolation and culture
DC were isolated from lungs as described by others
(25), with minor modifications. After perfusion of the
pulmonary vasculature with 5 ml of PBS containing 100 U/ml heparin, the
lungs were minced and incubated for 90 min at 37°C in digestion
buffer containing 0.7 µg/ml collagenase IV (C-5138, Sigma-Aldrich)
and 30 µg/ml type IV bovine pancreatic DNase I (D-4263,
Sigma-Aldrich). Large particulate matter was removed by passing the
cell suspension through a small loose nylon wool plug. After overnight
plastic adherence to remove macrophages, DC were reacted with 100 µl
of MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany)
conjugated to hamster anti-mouse CD11c mAbs (clone N-418), as
described (22). Positively selected DC (at 10–15%
yield recovery) routinely contained >90%
CD11chigh and were not expressing CD8
, in line
with previous findings (14, 18, 25). DC were purified from
thoracic (paratracheal and parathymic) lymph nodes and spleens
by magnetic cell sorting. After overnight plastic adherence to remove
macrophages, 108 collagenase D-treated
(Sigma-Aldrich) nonadherent cells were reacted with 100 µl of CD11c
MicroBeads before magnetic separation. Consistent with previous reports
(26, 27, 28), spleen and lymph node DC (at 1–2% yield
recovery) routinely express high levels of CD11c integrin and between
25 and 30% stained positive for CD8 (data not shown). For cytokine
determination, purified DC were pulsed with live conidia or hyphae, as
detailed in the phagocytic assay, for 2 h before the addition of
2.5 µg/ml amphotericin B (Sigma-Aldrich) to prevent fungal
overgrowth. Supernatants were harvested at 24 h. Control
experiments indicated that amphotericin B alone did not modify patterns
of cytokine production by DC.
Phagocytosis and inhibition studies
For phagocytosis, FSDC and purified pulmonary DC (2 x
105cells/200 µl) were incubated for different
times at 37°C with 106 conidia or 2 x
105 hyphae in 6-ml polypropylene tubes (N. 2063,
Falcon; BD Biosciences), in 200 µl of IMDM containing 5
µg/ml polymixin B (Sigma-Aldrich) and 50 µg/ml gentamicin but no
FCS to avoid nonspecific activation by serum components and endotoxin.
Phagocytic cells were separated from nonphagocytosed A.
fumigatus cells by centrifugation on a fetal serum gradient and a
0.1-ml sample of the harvested phagocytic cells was used for cytospin
preparation. After Diff-Quik staining, fungal cell internalization was
expressed according to the following formula: percentage of
internalization = number of cells containing one or more fungal
cells/100 cells counted. The phagocytic index (PI) indicates the number
of cells containing one (PI = 1) or more (PI > 1) fungal
elements per 100 cells. For inhibition studies, fungal internalization
at 2 h was assessed in the presence of the following reagents
(Sigma-Aldrich, unless otherwise specified): cytochalasin D, 20
µg/ml, added to the cultures, or pre-exposure of cells at 37°C,
before addition of fungi, to 20 µg/ml nocodazole for 15 min, 50 mM of
EDTA (Life Technologies) for 60 min, 10 µg/ml -mannan from
Saccharomyces cerevisiae or 250 µg/ml 
-glucan from
barley for 10 min, 50 µg/ml laminarin for 60 min, and 0.9 µg/ml
galactomannan polysaccharide from seeds of Ceratonia siliqua
for 60 min. For inhibition of the mannose receptors (MR), CR3 and
FcRII and III, by specific mAbs, cells were preincubated for 30 min
at 37°C with the saturating concentration of 20 µg/ml
F(ab')2 of the following purified mAbs: mouse
IgG1 (PAM-1) capable of inhibiting MR uptake of human
monocyte-derived DC (29), purified from ascites of the
corresponding hybridoma and rat IgG M1/70 and 2.4G2, which detect CD11b
and FcRII and III, respectively, from BD PharMingen (Palo Alto, CA).
Rat IgG2a mAb (NLDC-145, recognizing the multilectin receptor on
DC, DEC-205; Ref. 30) was obtained from BMA Biomedical AG
(Rheinstrosse, Switzerland). F(ab')2 of
Abs were generated by pepsin digestion with the ImmunoPure
F(ab')2 preparation KIT (Pierce, Rockford, IL),
following the manufacturer’s instructions. Isotype-matched
control Ab would not affect internalization (data not shown).
Phenotypic analysis
The cell surface phenotype of purified pulmonary, lymph node, and spleen DC was assessed by reacting cells with the following FITC-conjugated mAb reagents: rat IgG directed to I-Adb (3-5-34), CD80 (1G10), and CD86 (GL1), all from BD PharMingen. For DC labeling in vivo, lungs, lymph nodes, and spleens were reacted with PE-conjugated anti-CD11c (N418). Before all labeling experiments, FcR blocking was performed by incubating cells with 5% normal mouse serum. Unrelated isotype-matched mAbs were used as control. Analysis was performed on a FACScan (BD Biosciences). Data were evaluated both as the percentage of positive cells and as the median fluorescence intensity (MFI).
ELISPOT
IFN-- and IL-4-producing CD4+ T cells
were enumerated by ELISPOT assay, as described (22).
Purified CD4+ T splenocytes (>90% pure on FACS
analysis) were obtained by positive selection as described
(22). Cells were cultured (1 x
105 cell/well) in complete medium (RPMI 1640 with
10% FCS, 50 mM of 2-ME, and 50 µg/ml gentamicin sulfate) for 18
h in 96-well plates previously coated with rat anti-murine R4-6A-2
(for IFN-) or BVD4-1D11 (for IL-4) mAbs. Biotinylated AN-18.17.24
(for IFN-) or BVD6-24G2 (for IL-4) were used as the detecting
reagents; the enzyme used was avidin-alkaline-phosphatase conjugate
(Vector Laboratories, Burlingame, CA) and the substrate was
5-bromo-4-chloro-3-indolyl phosphate-p-toluidine salt (Life
Technologies). Results are expressed as the mean number of
cytokine-producing cells (±SE) per 104 cells,
calculated using replicates of serial twofold dilutions of cells.
Cytokine assays
The levels of TNF-, IL-4, IL-10, and IL-12p70 in culture
supernatants were determined by kit ELISA (R&D Systems, Milan, Italy).
The detection limits of the assays were <32 pg/ml for TNF-, <3
pg/ml for IL-4, <4 pg/ml for IL-10, and <16 pg/ml for IL-12p70.
Statistical analysis
The Student t test was used to determine the significance of values in experimental groups (significance was defined as p < 0.05). In vivo groups consisted of six to eight animals. The data reported were pooled from three to five experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Resident alveolar macrophages ingest and kill resting conidia,
while neutrophils attack extracellular hyphae germinating from conidia
that escape macrophage surveillance (3, 4). To determine
whether DC phagocytose conidia and hyphae of Aspergillus,
FSDC were exposed to either conidia or hyphae of the fungus and
internalization of fungal cells was evaluated at different times after
exposure, by light microscopy and TEM. As swelling of conidia is
a prerequisite step for hyphal development (3), the
internalization of swollen conidia was also assessed. FSDC ingested
conidia, swollen conidia, and hyphae of A. fumigatus in a
time-dependent manner, with optimum phagocytosis at 2 h (Fig. 1
, A–C). DC
internalized swollen conidia to an extent similar to that
of hyphae. In terms of the number of fungal elements internalized by
DC, swollen conidia and hyphae were only internalized as single
elements while conidia could be internalized as either single or
multiple elements (see also TEM). For conidia, the number of cells with
PI > 1 was a function both of time and multiplicity of infection
(Fig. 1, D and E). TEM revealed that the uptake
of fungal cells occurred through different forms of phagocytosis.
Internalization of conidia occurred predominantly by coiling
phagocytosis (Fig. 2A),
characterized by the presence of overlapping bilateral pseudopods, that
led to a pseudopodal stack before transforming into a phagosome wall.
In contrast, entry of hyphae occurred by a more conventional
zipper-type phagocytosis, characterized by the presence of symmetrical
pseudopods, which strictly followed the contour of the hyphae before
fusion (Fig. 2E). Three hours later, conidia (Fig. 2B) and hyphae (Fig. 2F) were present inside the
cells, but only the latter could be found in partially degraded forms
(Fig. 2G). In contrast, conidia were found to be still alive
inside the cells, and, interestingly, in close association with
mitochondria (Fig. 2, C and D). Studies in vivo
suggested that cells with DC-like morphology and topology
(15) had the ability to internalize Aspergillus
conidia and to migrate between epithelial cells. Soon after the
infection, conidia were found to be internalized by phagocytic cells
with the characteristics of DC morphology, as judged by numerous
cytoplasmatic extensions and an abundant cytoplasm, present in the
alveolar spaces (Figs. 3, A
and B). DC were also found to emit pseudopods which
engulf conidia and, eventually, make contact with epithelial cells
(Fig. 3B). At the site of contact, the epithelial cells
appear to invaginate (Fig. 3C), thus allowing DC as well
free conidia to translocate to the space below, within the alveolar
septal wall (Fig. 3D). Although macrophages are competing
for the ability to phagocytose conidia (3), and the close
proximity between macrophages and DC in the airways (15)
may impede a clear distinction between them, the extent to which
phagocytosis of conidia by bronchoalveolar macrophages is occurring in
vivo is not known at the moment. Our results suggest that both in vitro
and in vivo, DC appear to be endowed with the ability to phagocytose
both forms of the fungus, and, importantly, each fungal form appears to
be internalized by different phagocytic mechanisms and to have
different fates once inside the cells.
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As DC finely discriminated between the yeast and hyphae of
C. albicans by using distinct recognition receptors
(22)4, we
analyzed the phagocytosis of the different forms of A.
fumigatus by pulmonary DC in the presence of distinct inhibitors
or receptor ligands. Live unopsonized conidia or hyphae of the fungus
were both internalized mainly through a phagocytic process, as
internalization was inhibited in the presence of cytochalasin D and
nocodazole (Fig. 4). The internalization
of conidia, but not that of hyphae, was inhibited in the presence of
EDTA, a finding suggesting the involvement of a C-type lectin receptor
in the recognition of conidia. The inhibition of conidia
internalization observed in the presence of the NLDC-145 mAb, known to
bind to the lectin-like receptor DEC-205 (30), confirms
the involvement of a lectin-like receptor. In a previous study
(22), a >80% inhibition of phagocytosis of yeast
cells was observed in the presence of -mannan, a finding confirming
that MR is sufficient to mediate phagocytosis of fungal yeast
(31, 32). We performed uptake experiments in the presence
of concentrations of mannan known to inhibit the MR uptake
by immature DC (22), and of saturating concentrations of
the MR-reacting mAb, PAM-1. We found that internalization of conidia,
but not hyphae, was greatly inhibited by blocking MR with either PAM-1
or -mannan and, interestingly, galactomannan (Fig. 4). For hyphae,
the internalization was greatly inhibited by blocking CR3 with either
the M1/70 mAb or -glucan or laminarin. Interestingly, -glucan
partially inhibited conidia internalization, and this finding, together
with the significant, albeit very low, inhibition observed in the
presence of the M1/70 Ab, suggested that unopsonized conidia may also
exploit CR3 to enter DC. Internalization of hyphae was also inhibited
by blocking FcRII and III with the 2.4G2 mAb, thus implying the
involvement of these receptors in the recognition and entry of hyphae
in DC. As pulmonary DC are known to express functional MR (16, 33), DEC-205, CR3 (14, 18), and FcR
(34), all together these results indicated that different
recognition receptors mediate the entry of unopsonized conidia and
hyphae of A. fumigatus. MR and C-type lectins of
galactomannan specificity appear to mediate the entry of conidia. In
contrast, CR3 together with FcR is sufficient to mediate the entry
of unopsonized hyphae.
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To assess the pattern of cytokine production by DC upon
phagocytosis of conidia or hyphae of the fungus, purified DC from lungs
were exposed to either form of the fungus before evaluating cytokine
levels in supernatants by cytokine-specific ELISA at 24 h. Similar
to what was observed with C. albicans (22),
TNF- was produced in response to either type of fungal forms.
IL-12p70 was produced upon exposure to conidia, but not to hyphae,
while IL-4 was detected upon phagocytosis of hyphae, but not conidia
(Table I). Different from what was
observed with unopsonized Candida (22), the
internalization of Aspergillus hyphae also resulted in IL-10
production which could not be detected in response to conidia. On
measuring levels of cytokine production by FSDC upon exposure to
conidia or hyphae, a pattern similar to that observed with pulmonary DC
was found (data not shown). These results indicate that, upon exposure
to A. fumigatus conidia or hyphae, pulmonary DC
differentially produce IL-12 and IL-4/IL-10.
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As DC appear to phagocytose conidia and hyphae of
Aspergillus in vivo (Fig. 3
), we address the question of
whether pulmonary DC, after phagocytosis of conidia or hyphae of the
fungus, would migrate to the draining and peripheral lymphoid organs,
such as the thoracic lymph nodes and spleens. For this purpose,
FITC-labeled conidia and hyphae were i.t. injected at different times
after infection and the number of FITC-positive DC were enumerated in
lungs, lymph nodes, and spleens by FACS analysis. In accordance with
previous studies (18, 35), DC with low-level
autofluorescence are present in the lungs (Fig. 5). However, the number of
CD11c+FITC+ cells greatly
increased in the lungs of mice as soon as 3 h after the injection
of either FITC-labeled conidia (from 7 to 51%) or FITC-labeled hyphae
(from 7 to 44%). At 6 h after the infection,
CD11c+FITC+ cells also
appeared in the thoracic lymph nodes (from 7 to 22 and to 19%, for
conidia and hyphae, respectively) and in the spleens (from 4 to 12 and
to 15%, for conidia and hyphae, respectively). Because no similar
increase in the number of
CD11c+FITC+ cells was
observed upon injecting the mice with either FITC or DMSO alone (data
not shown), passive leakage of FITC from the airway mucosa to the
draining lymph nodes did not occur, as already reported
(18). Therefore, our results indicate that pulmonary DC
transport Aspergillus conidia or hyphae to the draining
lymph nodes and spleens. Interestingly, no fungal growth could be
observed upon assaying lymph nodes and spleens for the presence of
viable fungi, 3 days after challenge (data not shown), a finding
suggesting that conidia and hyphae may have undergone degradation for
efficient Ag presentation by DC, eventually.
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To assess whether airway DC from mice injected with
Aspergillus conidia or hyphae undergo functional maturation
during migration to the draining lymph nodes and spleens, the levels of
MHC class II Ags and CD80 and CD86 costimulatory molecules were
assessed in DC purified from lungs, lymph nodes, and spleens at
different times after infection. It was found that the exposure to
either form of the fungus did not increase the expression of the above
activation or costimulatory molecules in pulmonary DC at 3 h after
infection, except for the small increase in the MFI for CD80 upon
exposure to conidia. In contrast, the expressions of MHC class II Ags
and CD80 and CD86 molecules were all greatly increased in DC from
spleens, but particularly, lymph nodes of mice infected with either
type of the fungus, 6 h previously (Fig. 6). Therefore, pulmonary DC undergo
functional maturation upon migration from the airways to the local and
peripheral lymphoid organs in mice with aspergillosis.
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To correlate migration and maturation of pulmonary DC with their
ability to induce T cell priming in the lymph nodes and spleens, mice
were infected with conidia and hyphae of the fungus and were assessed 3
days later for Th1 or Th2 priming in the thoracic lymph nodes and
spleens. To this purpose, the number of IFN-- or IL-4-producing
CD4+ cells was enumerated by ELISPOT assay. We
found that the number of IFN-producing cells greatly increased in both
the lymph nodes and spleens of mice injected with
Aspergillus conidia, while IL-4-producing cells were
increased in mice exposed to hyphae (Fig. 7). A small, but significant,
lymphoproliferative response was also observed, at this time, in the
lymph nodes and spleens of infected mice (data not shown). Together,
these data indicate that Th1 and Th2 cell activation occurs in the
local and distant lymphoid organs of mice infected with A.
fumigatus.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Myeloid DC, unlike conventional phagocytic cells such as macrophages for which phagocytosis of conidia only had been reported (3, 4), phagocytosed both forms of the fungus through different phagocytic mechanisms. Similar to what was observed during uptake of C. albicans yeast and hyphae (22), FSDC engulfed conidia, via coiling phagocytosis, and hyphae through a more conventional zipper-type phagocytosis. Although the coiling phagocytosis is considered to reflect a disturbance of the more conventional zipper-type phagocytosis (36), it appears that DC use both forms of phagocytosis to internalize fungi. Once internalized, the fate of conidia and hyphae inside the cells was different. For hyphae, different stages of progressive degradation were seen. In contrast, conidia were apparently still alive 3 h after phagocytosis, a finding confirming the relative resistance of resting conidia to killing by effector phagocytes (5, 37). Interestingly, conidia were found to emit thick protrusions which promote close contact with mitochondria. The significance of this finding is presently under investigation.
Although killing of conidia would seem to be a necessary prerequisite to obtain efficient Ag presentation, it has been demonstrated that infected DC were still capable of stimulating lymphocyte proliferation (38). Therefore, either a small number of conidia are actually degraded by mature DC thus allowing their Ag processing and presentation, or, alternatively, Ags could be processed and regurgitated by other infected phagocytes and then transferred to DC for presentation.
Conidia of A. fumigatus are known to be ingested by professional and nonprofessional phagocytes, through a variety of mechanisms and opsonic requirements (3, 39). However, receptors for binding and phagocytosis on the different cell types had not been identified. For monocytes and macrophages, a mannosyl-fucosyl receptor (39) and receptors inhibited by glucan and chitooligosaccharides (40) have been described. Conidial binding to pneumocytes was instead glycosphingolipid-mediated (41).
Receptors that have been identified on immature DC include
lectins such as the MR and DEC-205 as well as Fc
RI and FcR
(42, 43, 44). Receptors for Ag capture on DC vary in their
ligand, specificity, and mode of delivery to Ag-processing compartments
(22, 45). In this study, we show that distinct receptors
on DC are involved in the internalization of unopsonized conidia or
hyphae of the fungus in vitro. Receptor-mediated entry of conidia was
greatly inhibited by blocking lectin-like receptors, including MR and
the DEC-205. A little but significant inhibition was also observed by
blocking the CR3, a finding in line with what was observed with
alveolar macrophages (39). Studies done in the presence of
different saccharides, known to inhibit the lectin-like attachment on
macrophages (39), revealed that pattern of saccharide
inhibition was different from that observed for the mannosyl-fucosyl
receptor on macrophages (data not shown). We found that conidia
internalization was strongly inhibited by galactomannan, whose
structure is similar to that reported for galactomannan of A.
fumigatus (46). Because galactomannans are abundantly
secreted during infection (3), it can be hypothesized that
secreted galactomannans may compete with conidia for binding to DC,
thus preventing DC activation. This will be in line with the
observation of high levels of galactomannans in invasive aspergillosis
(3, 47).
For hyphae, the internalization by DC was greatly inhibited by blocking
CR3 or FcR II and III with specific-blocking mAbs or specific
receptor ligands, such as -glucan and laminarin for CR3 (48, 49). However, -glucan and laminarin also bind to a recently
discovered PRR for fungal -1,3-D-glucans, the dectin-1
receptor (50), a finding implicating the possible
involvement of additional glucan receptors in host recognition of
Aspergillus. As the phenomenon was observed with hyphae,
irrespective of opsonization, this suggests that unopsonized hyphae are
endowed with the ability to activate opsonic receptors. Overall, these
results, similar to those obtained in candidiasis
(22),4 suggest that fungi have exploited
common pathways of entry into DC, which may include a lectin-like
pathway for unicellular forms and opsono-dependent pathways for
filamentous fungi.
Whether the engagement of these receptors also occurs in vivo is not known at the moment. However, a number of considerations makes it likely: 1) all of these receptors are known to be expressed on DC in the airways (14, 18, 33) and MR are principally involved in the uptake and transport of macromolecules by these cells (16, 18, 33); 2) activation of a complement occurs in response to A. fumigatus (51); and 3) the mannose-binding lectin gene polymorphisms are recognized as a susceptible factor for chronic necrotizing pulmonary aspergillosis (52).
One interesting observation of this study concerns the role of epithelial cells in assisting DC trafficking in the lungs. The activity of epithelial cells includes the formation of invaginations through which DC translocate to the space below. This mechanism is similar to that of caveolae plasma membrane invaginations, known to play an important role in normal transport in the lung (53). However, as chitin derivatives induce transient opening of tight junctions between epithelial cells (54), it is likely that the opening of the tight junctions between epithelial cells by DC may also occur, as recently reported in the gut (55). In addition, as bronchial epithelial cells constitutively produce IL-10 (56) and epithelial cells isolated from lungs produce IL-10 in response to conidia and hyphae of the fungus (data not shown), it is likely that the contribution of epithelial cells to the overall performance of DC in the lung may go beyond the assistance in cell migration to include an effect on cell maturation.
In normal circumstances, a state of tolerance to inhaled Ags is achieved through several mechanisms (57) including IL-10 production by local DC (21). It is known that DC of the respiratory tract are specialized for uptake and processing, but not for Ag presentation, because the latter requires cytokine maturation signals that are encountered after migration to regional lymph nodes (18, 58, 59). Although transfer of Ag between different types of APC in vivo cannot be excluded (60), direct migration of airway DC to the draining lymph nodes has been demonstrated (18, 59). Experiments using i.t. delivery of soluble proteins indicated the appearance of strong Ag-presenting activity in the DC of draining lymph nodes between 6 and 24 h after instillation (18, 58).
In this study, we show that pulmonary DC produce IL-12 in response to
conidia and IL-4 and IL-10 in response to hyphae. Different pathways
and receptors on DC regulate cytokine production in response to
different stimuli (44, 45). It is now recognized that
several intracellular pathogens exploit different pathways of IL-12
attenuation (61). IL-12 suppression by these pathways may
occur through different mechanisms. The yeast Histoplasma
capsulatum, for instance, by entry through CR3, down-modulates
IL-12 production on phagocytes in a manner dependent on its binding to
the 2 integrins (62). The FcR-mediated inhibition
appears to proceed by more than on mechanism, including IL-10
production upon ligation of FcRI (63). Therefore, the
different sets of cytokines produced by DC upon exposure to conidia or
hyphae may reflect the selective involvement of distinct recognition
receptors.
Pulmonary DC did not up-regulate the expression of MHC class II Ags and
costimulatory molecules upon phagocytosis of either form of the fungus,
a finding compatible with the features of airway DC. In contrast, the
appearance of CD11c+FITC+
cells in the thoracic lymph nodes and spleens as early as 3 h
after the i.t. injection of FITC-labeled conidia or hyphae is
consistent with the transport of fungi and migration of airway DC to
regional and peripheral lymphoid organs. This phenomenon was associated
with the up-regulated expression of activation and costimulatory
molecules, particularly for DC with conidia. Moreover, local activation
of cytokine-producing CD4+ T cells also occurred,
as IFN--producing cells were observed in response to the unicellular
form of the fungus and IL-4-producing cells were observed in response
to hyphae. Therefore, as with C. albicans
(64), DC discriminate between conidia and hyphae of
Aspergillus in terms of the type of Th cell responses
elicited.
From a conceptual point of view, it is intriguing that the host has evolved different types of responses toward unicellular (conidia or yeast) or filamentous forms of opportunistic fungi such as C. albicans and A. fumigatus. In particular, it would appear that the discriminative response toward Th2 is of weak teleological meaning. Although the evolutionary selection pressure on the immune system is such that virulent pathogens to which nonprotective Th2 responses still exist, in the case of fungi, one may argue that the filamentous, rather than the unicellular, fungal growth is of evolutionary advantage, at least under a selected condition of growth.
Much remains to be learned with regard to factors and mechanisms governing local immune reactivity upon exposure to Aspergillus conidia, such as the role and functional activity of the different PRRs, including collectins, pathogen-associated molecular patterns, and opsonins. In this regard, it is worth mentioning that metabolites of Aspergillus exist which have the capacity to negatively affect the activity of DC (65). Although circumstantial evidence indicates that a Th1/Th2 dysregulation and a switch to a Th2 immune response may contribute to the development of an unfavorable outcome for IPA (10, 66), little is known about the mechanisms of immunoregulation operating in conditions of continuous exposure to nonfatal low doses of Aspergillus conidia, as those which presumably occur in nature. Our finding that the mode of entry of conidia into DC was dependent on time and multiplicity of the infection may suggest that exposure to low or high doses of conidia may impact DC activation and the subsequent immune responses that are elicited differently.
All together, these results point to a unique role of DC in aspergillosis, as they behave as both sentinel for innate immune recognition and initiator of Th cell differentiation and functional commitment. In doing so, murine DC are exquisitely sensitive to the different forms of the fungus, a finding in line with the increasingly recognized importance of PRR in antifungal host defense (67, 68, 69). Considering that phagocytosis of inactivated Aspergillus conidia induced functional maturation of human DC derived from CD54+ progenitors and that ex vivo-generated DC could partially restore the antifungal effector T cell reactivity in vitro in hemopoietic transplantation (70), our findings provide important and novel insights into the key role of DC in the regulation of antifungal immune reactivity, which may go from immunity to autoimmunity and allergy (71).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Luigina Romani, Department of Experimental Medicine and Biochemical Sciences, Via del Giochetto, 06122 Perugia, Italy. E-mail address: lromani{at}unipg.it
3 Abbreviations used in this paper: IPA, invasive pulmonary aspergillosis; DC, dendritic cells; PRR, pattern recognition receptor; CR, complement receptor; i.t., intratracheal; FSDC, fetal skin-derived DC; TEM, transmission electron microscopy; PI, phagocytic index; MR, mannose receptor; MFI, median fluorescence intensity.
4 C. Montagnoli, A. Bacci, S. Bozza, A. Spreca, P. Allavena, F. Bistoni, P Puccetti, and L Romani. The interaction of dendritic cells with Candida albicans: different recognition receptors determine the state of immunity or tolerance to the fungus. Submitted for publication.
Received for publication October 15, 2001. Accepted for publication December 4, 2001.
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) and 1:5 (
). The PI indicates
the number of cells containing one (PI = 1) or more (PI > 1)
fungal elements per 100 cells. *, p < 0.05; 2 or
4 h vs 30 min internalization.
) of the fungus i.t. Three days later,
CD4+T cells were purified from thoracic lymph nodes and
spleens and the number of IFN-