THE IMMUNE RESPONSE TO A MOLD
Aspergillus species are ubiquitous moulds with worldwide
distribution. Exposure most commonly occurs when airborne
spores are inhaled into the lungs or sinuses. Once inhaled,
spores reach distal areas of the lung by virtue of their small
size. The most common infecting species is A. fumigatus,
followed by A. flavus and A. niger (Marr et al, 2002; Husain
et al, 2003). In normal hosts, isolation of Aspergillus generally
reflects colonization and not infection (Uffredi et al, 2003).
The clinical manifestations of aspergillosis vary depending
upon the nature of the host. In atopic individuals with an
allergic or hypersensitivity response, the fungus triggers
immune phenomena including allergic rhinitis, asthma, hypersensitivity
pneumonitis and allergic bronchoplumonary aspergillosis
(ABPA) (Horner et al, 1995). In patients with cavitary
pulmonary lesions, saprophytic colonization by Aspergillus
leads to aspergillomas. Finally, immunocompromised patients
may develop invasive aspergillosis (IA). This article will focus
on the latter group of patients. The degree of fungal invasion,
response to therapy and clinical outcome of IA depends upon
the type and depth of immune suppression. In susceptible
hosts, Aspergillus conidia germinate to form hyphae, the
Table I. Defects in host defences that predispose patients to infections
with specific fungi.
Fungal pathogen Host factor
Candida (mucosal) Impaired cell mediated immunity
Candida (disseminated) Impaired mucosa or integument,
neutropenia
Aspergillus Neutropenia, high-dose corticosteroids
Cryptococcus Impaired cell mediated immunity,
corticosteroids
Zygomycetes Neutropenia, deferoxamine treatment,
corticosteroids, diabetic ketoacidosis
Fusarium Neutropenia, impaired integument,
corticosteroids
Scedosporium Neutropenia
Trichosporon Neutropenia, impaired integument
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570 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 569–582
invasive form of the organism. An essential aspect of the
immune response to Aspergillus is recognition and killing of
conidia and activation of appropriate host defences to confront
fungi that have escaped killing and transitioned to the hyphal
form. In highly compromised patients, early detection of IA,
institution of antifungal therapy and improvement in host
immune status are crucial determinants in the outcome of
infection (von Eiff et al, 1995).
Qualitative and quantitative disorders of phagocyte function
are the most important host factors predisposing patients to
IA. In patients with neoplasms, virtually all cases are due to the
therapy used to treat the malignancy, rather than the
underlying disease itself. The major risk factor for IA is
chemotherapy-induced neutropenia, with the risk being
directly proportional to both the severity and the duration of
the neutropenia (Gerson et al, 1984). Treatment with high
doses of corticosteroids, which depresses neutrophil and
macrophage function, also predisposes patients to IA. Other
risk factors include chronic granulomatous disease, advanced
AIDS and chronic graft-versus-host disease. The incidence of
invasive fungal infections occurring after resolution of neutropenia
is also increasing with the widespread use of highly
immunosuppressive regimens, such as CAMPATH1-H or
fludarabine for low intensity transplantation. The most
frequent sites of involvement are the lungs followed by the
sinuses. Infection may disseminate from the respiratory tract
to other sites including the eyes, brain, liver, spleen, kidney,
skin and bone (Stevens et al, 2000). Occasionally, direct
inoculation of the fungus during invasive procedures or
injection drug use can lead to soft tissue and even disseminated
infection. IA in neutropenic hosts is characterized by extensive
hyphal infiltration, angioinvasion, coagulative necrosis, intraalveolar
haemorrhage, extra-pulmonary dissemination and
high mortality (Berenguer et al, 1995). In hosts that are less
severely compromised, such as transplant recipients receiving
corticosteroids and calcineurin inhibitors, IA tends to be more
indolent and is associated with a relatively higher survival rate.
Inhaled Aspergillus conidia that are not repelled by respiratory
tract mucociliary defences are mostly phagocytosed by
macrophages and dendritic cells. These cells constitute the
initial line of defence and have a dual role as antifungal
effectors and as activators of the immune response (Kan &
Bennett, 1988). Resident and monocyte-derived macrophages
ingest and kill conidia, thus preventing transition into the
invasive hyphal form (Schaffner et al, 1982, 1983; Waldorf
et al, 1984a; Levitz et al, 1986; Philippe et al, 2003). After
recognition and binding of conidia, actin-dependent pseudopodia
capture and internalize the fungal particles. Swelling of
the conidia inside the macrophage appears to be a prerequisite
for fungal killing (Philippe et al, 2003). Aspergillus containing
phagosomes mature by fusion with endocytic compartments.
Conidial killing proceeds with acidification of the phagolysosome
(Ibrahim-Granet et al, 2003). Production of reactive
oxidant intermediates within alveolar macrophages is important
for conidial killing, although non-oxidative killing mechanisms
also play a role. In mice, impairment of NADPH
oxidase inhibits killing without impairing phagocytosis. Corticosteroids
inhibit production of reactive oxidant intermediates
and killing of phagocytosed fungi, which may help to
explain the elevated rates of IA in steroid recipients (Philippe
et al, 2003). Cyclosporin A exerts only a modest effect on
phagocytic defences and in the absence of corticosteroids does
not increase the progression of IA (Roilides et al, 1994;
Berenguer et al, 1995). Experimental models, as well as reports
of IA in patients receiving anti-TNF-a antibodies, suggest
cytokine networks are essential for the activation of leucocyte
antifungal activity (Warris et al, 2001).
Humoral factors participate in the host response to Aspergillus.
Resting conidia, germinating conidia and hyphae are
potent activators of the complement cascade and induce
deposition of complement components upon the fungal
surface. Resting conidia activate the alternative pathway and
induce neutrophil chemotaxis. As the fungus matures into
swollen conidia and then hyphae there is progressive dependence
on the classical pathway (Kozel et al, 1989). In alveolar
fluid, surfactant proteins A (SP-A) and D (SP-D) enhance
chemotaxis, binding, phagocytosis and oxidative killing (Madan
et al, 1997). These C-type lectins also agglutinate Aspergillus
conidia, thereby immobilizing the pathogen. However,
despite the importance of these humoral factors in experimental
systems, the predisposing factor for the vast majority of
patients with IA is phagocytic dysfunction and not defects in
the humoral immunity.
Macrophages and dendritic cells activate host defences in
response to Aspergillus. At the surface of these cells are TLRs
that identify microbial products (Akira et al, 2001). Signalling
pathways associated with each TLR vary and activation of
different receptors may result in dissimilar biological responses.
For example, TLR 2 and TLR 4 differentially mediate
the release of specific cytokines in response to the fungus
(Braedel et al, 2004). When stimulated by Aspergillus conidia,
macrophages produce proinflammatory cytokines, including
TNF-a, IL-1a and IL-1b through TLR 4-dependent mechanisms.
Aspergillus hyphae, on the other hand, stimulate
production of the anti-inflammatory cytokine IL-10 through
TLR 2-dependent mechanisms. Thus, activation of specific cell
surface receptors during germination may allow Aspergillus to
counteract host defences (Netea et al, 2003). TNF-a release,
initially by resident macrophages and later by recruited
immune cells, is associated with chemokine production
[including Macrophage Inflammatory Protein (MIP)-1a and
MIP-2] and influx of neutrophils and monocytes into infected
tissues (Mehrad et al, 1999). Mice lacking functional CC
chemokine receptor 1(CCR1) are much less efficient at
blocking tissue invasion from the blood and have increased
susceptibility to infection (Gao et al, 1997). Aspergillus conidia
and hyphae induce nuclear factor (NF)-jB translocation, and
release of TNF-a and MIP-2 in a TLR 2- and TLR 4-dependent
manner. Neutrophil recruitment is severely impaired in mice
lacking both functional TLR 2 and TLR 4, but is less impaired
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2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 569–582 571
in single TLR 2 or TLR4 deficient mice, suggesting that both
receptors are required for an optimal immune response to
Aspergillus (Meier et al, 2003). In humans, optimal TNF-a
signalling in response to the various morphotypes of Aspergillus
requires TLR 2, CD14 and MyD88 (Mambula et al, 2002;
Levitz, 2004). Administration of corticosteroids suppresses
macrophage production of IL-1a, TNF-a, and MIP-1a, all of
which are protective against aspergillosis (Brummer et al,
2003).
Dendritic cells modulate the antifungal host response.
Aspergillus antigens induce activation and maturation of these
cells. Ingestion of Aspergillus conidia and hyphae proceeds
through distinct phagocytic mechanisms and elicited responses
differ depending upon the morphotype encountered. Following
exposure to Aspergillus, dendritic cells migrate to the spleen
and draining lymph nodes and induce local and peripheral Th
cell reactivity to the fungus (Bozza et al, 2002). The development
of specific Th responses is an essential determinant of the
host’s susceptibility or resistance to IA. As with other fungi,
production of Th1 cytokines appears to be protective, whereas
Th2 responses are not (Cenci et al, 1998; Cenci et al, 1999,
2001; Del Sero et al, 1999). In that regard, proinflammatory
signals, including granulocyte-macrophage colony-stimulating
factor (GM-CSF), TNF-a, IFN-c, IL-1, IL-6, and IL-12, IL-18
as well as the chemokines MIP-1, monocyte chemoattractant
protein (MCP)-1, and MIP-2, are associated with protection,
and IL-4 and IL-10 with invasion. Cell surface and secreted
pattern recognition receptors mediate the development of Th
responses. TLR-associated MyD88-dependent signalling is
crucial for priming antifungal Th1 responses (Bellocchio et al,
2004). The secreted pattern recognition receptor PTX3 binds
to select microbial products, including Aspergillus conidia, and
has a non-redundant role in resistance to the fungus. PTX3-
deficient mice are susceptible to invasive pulmonary aspergillosis
with impaired recognition of the fungus by alveolar
macrophages and dendritic cells and inappropriate induction
of a Th2 response (Garlanda et al, 2002).
When bronchoalveolar macrophages fail to control the
fungus, conidia germinate into hyphae, pierce through the cell
and grow extracellularly. Neutrophils and monocytes recruited
from the circulation phagocytose and damage fungi that have
escaped killing and are transitioning (or have transitioned)
into hyphae. As described above, proinflammatory signals,
including TNF-a, complement, chemokines, and surfactant
proteins, recruit neutrophils into sites of infection. Since
hyphae are too large to be completely phagocytosed, hyphal
damage is achieved via extracellular means. Non-oxidative
mechanisms include release of lysozyme and neutrophil
cationic peptides. Oxidative killing is mediated by myeloperoxidase
(MPO)-dependent and MPO-independent oxidative
systems (Washburn et al, 1987). The importance of oxidative
killing is demonstrated in chronic granulomatous disease. This
hereditary disease is characterized by impaired production of
oxidative intermediates and elevated rates of invasive infections
due to several catalase positive pathogens, including
Aspergillus (Cohen et al, 1981; Diamond & Clark, 1982).
Phagocytosis of Aspergillus is enhanced by opsonization and
proinflammatory molecules (Marr et al, 2001). TNF-a augments
the capacity of neutrophils to damage hyphae, possibly
through enhanced oxidative mechanisms, and increases anticonidial
phagocytic activity of resident macrophages (Roilides
et al, 1998a). Granulocyte colony-stimulating factor (G-CSF),
GM-CSF and especially IFN-c enhance monocyte and neutrophil
activity against hyphae (Gaviria et al, 1999). IL-15
enhances hyphal damage and IL-8 release by neutrophils
challenged with Aspergillus (Winn et al, 2003a). IL-8 recruits
neutrophils to sites of inflammation and mediates release of
antimicrobial peptides. By contrast, production of IL-4 by
CD4+ T lymphocytes impairs neutrophil antifungal activity
(Cenci et al, 1997). IL-10 suppresses oxidative burst and
antifungal activity of mononuclear cells against hyphae, while
increasing their phagocytic activity (Roilides et al, 1997).
Corticosteroids reduce oxidative burst and superoxide anion
release by neutrophils, thereby inhibiting hyphal killing
(Roilides et al, 1993; Brummer et al, 2003). Treatment with
G-CSF and IFN-c may prevent this impairment.
A variety of factors that may augment virulence by immune
evasion have been described in Aspergillus, but their contribution
in vivo is difficult to gauge. Gliotoxin inhibits ciliary,
macrophage, neutrophil, and lymphocyte function and induces
apoptosis in immune cells (Mullbacher & Eichner, 1984; Pahl
et al, 1996; Tsunawaki et al, 2004). Other Aspergillus-derived
factors inhibit oxidative killing, interfere with ciliary function,
inactivate complement, disrupt production of proinflammatory
cytokines and promote adhesion to endothelium (Washburn
et al, 1986; Jahn et al, 2001
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