Immunity to Aspergillus fumigatus: the basis for

Medical Mycology Supplement 1 2005, 43, S181 /S188

Immunity to Aspergillus fumigatus: the basis for immunotherapy and vaccination S. BELLOCCHIO*, S. BOZZA*, C. MONTAGNOLI*, K. PERRUCCIO$, R. GAZIANO*, L. PITZURRA* & L. ROMANI* *Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy and $Division of Hematology, Clinical Immunology, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy

Efficient responses to fungi require different mechanisms of immunity. Dendritic cells (DCs) are uniquely able to decode the fungus-associated information and translate it into qualitatively different T helper (Th) immune responses. Murine and human DCs phagocytose conidia and hyphae of Aspergillus fumigatus through distinct recognition receptors. The engagement of distinct receptors translates into disparate downstream signaling events, ultimately affecting cytokine production and co-stimulation. Adoptive transfer of different types of DCs activates protective and non-protective Th cells as well as regulatory T cells, ultimately affecting the outcome of the infection in mice with invasive aspergillosis. The infusion of fungus-pulsed or RNA-transfected DCs also accelerates recovery of functional antifungal Th l responses in mice with allogeneic hematopoietic stem cell transplantation. Patients receiving T cell-depleted allogeneic hematopoietic stem cell transplantation are unable to develop antigen-specific T cell responses soon after transplant due to defective DC functions. Our results suggest that the adoptive transfer of DCs may restore immunocompetence in hematopoietic stem cell transplantation by contributing to the educational program of T cells. Thus, the remarkable furictional plasticity of DCs can be exploited for the deliberate targeting of cells and pathways of cell-mediated immunity in response to the fungus. Keywords

Aspergillus, dendritic cells, immunotherapy, vaccination

Introduction Aspergilli are respiratory pathogens, and pulmonary infections are usually acquired through the inhalation of conidia [1,2]. With a diameter of about 2.5 /3.5 mm, the conidia are able to reach small airways and the alveolar space. A. fumigatus accounts for only 0.3 of aerial saprophytic fungal spores in the environment, yet this species is responsible for more than 90% of all human pulmonary fungal infections. This fact alone suggests that A. fumigatus possesses some unique

Correspondence: Luigina Romani, Department of Experimental Medicine and Biochemical Sciences-Microbiology Section, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. Tel/ Fax: /39 075 585 7411; E-mail: [email protected]

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features that favors its pathogenicity in human tissues. Invasive aspergillosis (IA) is the most common manifestation of A. fumigatus infection in immunocompromised patients [3]. Several million immunocompromised patients are at risk of IA each year. Examples include IA attack rates of 10% /15% in allogenic bone marrow recipients, 7% in acute leukemia, 40% in inherited chronic granulomatous disease and an increasing rate in medical ICU patients [4]. Although distinct clinicopathologic forms of IA have been described [5], IA is a highly lethal disease with approximately 90% mortality in bone marrow and liver transplant recipients, and a 40% /75% mortality in those with AIDS, leukaemia or lymphoma, heart and lung transplant recipients, and patients on long term corticosteroids [4]. DOI: 10.1080/14789940500051417

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Innate defense mechanisms to Aspergillus In the past decade, a dramatic shift has occurred in our understanding of the mechanisms of innate immunity. The appreciation that activation of the innate immune system initiates, amplifies and drives antigen-specific immune responses, together with the identification of discrete cell types, specific receptors and the signaling pathways involved in the activation of innate immunity, has provided a multitude of new targets for exploitation by the developments of adjuvants for vaccines. It has became apparent that understanding how immune responses are activated will enable the construction of better vaccines and immunomodulatory strategies that are effective at eliciting acquired protective immunity to fungi [6]. The model has brought dendritic cells (DCs) to prominence as promising targets for intervention for immunotherapy and vaccine development and has shifted the emphasis from the ‘antigen’ towards the ‘adjuvant’ [7]. The host defense of the airways against fungi includes mucous membrane, mucociliary clearance and local secretion of inflammatory mediators by innate immunity cells [8]. The airway mucus constitutes a physical, chemical and biological barrier of secretory products from the mucus membrane that facilitates the elimination of inhaled particles, including fungal spores. This fluid contains glycoproteins, proteoglycans, lipids, lysozyme and other substances like surfactant. Pulmonary surfactant is a complex mixture of lipids and proteins that lowers the surface tension at the air-liquid interface of the lung. Surfactant proteins (SPs) are members of the human contingent of the collectin (collagen-lectin) family, known to act as soluble pattern recognition receptors (PRRs) activating the innate immune system for microbe opsonization, activation and recruitment of phagocytic cells [9]. Through recognition of carbohydrate structures, SPs can bind a variety of fungi [9] and fungal pathogenassociated molecular patterns (PAMPs), such as b(1-6)glucan [10], and may also have a direct inhibitory activity on fungal yeasts [11]. SPs have long been known to beneficially dampen the persistent inflammatory response evoked by inhaled antigens and, indeed, SP-A down-regulated the production of proinflammatory cytokines by alveolar macrophages in response to fungi [9]. A recent study provided evidence that this occurs through modulation of toll-like receptor (TLR) signaling [12]. Interestingly, SPs share sequence homology with the mannose-binding lectin (MBL) a serum lectin that binds different fungi and promotes complement deposition [13]. Recognition of diverse fungal and microbial species by MBL occurs

through the selective binding to carbohydrates such as mannose and fucose, which are prevalent on glycolipids and glycoprotein that decorate the surface of fungi, but not to sialic acid and other carbohydrates that commonly occur at terminal sugars on mammalian cell surface molecules. It is not surprising therefore that patients with defective MBL [14] or MBL gene polymorphisms [15] have increased susceptibility to fungal infections. Based on their structural similarities, SPs and MBL have been grouped into the collectin family. This group also includes pentraxin 3 which has a nonredundant role in resistance to A. fumigatus by promoting conidial recognition and phagocytosis as well as activation of effector phagocytes and DCs [16]. Although epithelial and endothelial cells may internalize conidia [1,2], effector mechanisms of the innate immune system have long been recognized as major host defenses against IA [17]. Resident alveolar macrophages ingest and kill resting conidia, while polymorphonuclear neutrophils (PMNs) / through oxygen and non oxygen-dependent mechanisms / attack hyphae germinating from conidia that escape macrophage surveillance [2,18]. Little is understood about mechanisms of recognition and entry of A. fumigatus conidia in pulmonary alveolar macrophages, which are known to represent the first line of defense against Aspergillus conidia [1,17]. Murine macrophages have been shown to recognize and internalize A. fumigatus conidia through lectin-like attachment sites in the presence of collectins. Recently, the involvement of the TLRs has also been reported [6,19,20] (Fig. 1). It is of interest that Aspergillus hyphae, at variance with conidia, appear to be sensed by human monocytes through TLR4 and CD14 [20,21], a finding that indicates the ability of TLRs to discriminate among fungal morphotypes. Similarly, conidia appear to signal through TLR2, TLR4 and TLR9 and hyphae through TLR4 and TLR9 on murine PMNs [22]. The finding that distinct TLRs activate specialized effector functions on effector cells and govern both antifungal activity and inflammatory pathology suggest that TLR manipulation in vivo through the use of TLR agonists may induce an optimal microbicidal activity in absence of a detrimental inflammatory response. Alveolar macrophages ingest inhaled conidia very rapidly, destroy them intracellularly through oxidative mechanisms [23], and prevent germination to hyphae, the invasive form of the fungus. Resting conidia are to some extent more resistant than swollen conidia to both oxygen-dependent and oxygen-independent metabolites. In terminal airways, complement and antibodies cannot be readily available and therefore alveolar macrophages are able to recognize and bind conidia – 2005 ISHAM, Medical Mycology, 43, S181 /S188

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Fig. 1 Pattern recognition receptors as activators of innate and adaptive immunity to Aspergillus fumigatus. Recognition of A. fumigatus and fungal pathogen-associated molecular patterns by toll-like receptors (TLRs), Mannose receptor (MR), complement receptor (CR3) and receptors for Fc (FcgR) leads to activation of specialized antifungal effector functions in neutrophils, such as respiratory burst and degranulation, and production of IL-12 p70 by dendritic cells (see text). As dendritic cells are equipped with pattern recognition receptors and TLRs, they are uniquely able to decode the fungus-associated information and translate it into qualitatively different adaptive Th immune responses. The engagement of distinct receptors by conidia and hyphae translates into downstream signaling events, ultimately regulating co-stimulation, cytokine production and development of Th and regulatory T cells. The functional plasticity of DCs at the pathogen/immune system interface may offer new interpretative clues to fungal virulence.

even in the absence of opsonins. In addition, conidia poorly activate the complement system by the classical pathway and, even opsonized, they will trigger only a modest oxidative burst. Various enzymes, such as elastases and proteases produced by the fungus, might play an important role in the ability of conidia to evade phagocytosis by alveolar macrophages; to resist hydrolysis by endogenous peptides; and in the ability of germinating conidia to cross anatomical barriers [2]. As the second line of defence, circulating polynuclear and mononuclear cells will then be recruited at the site of infection [17]. If the above lines of host cellular defences are suppressed (e.g., by corticosteroids) or absent (e.g., as a result of neutropenia), A. fumigatus can germinate into hyphae and invade the lung parenchyma and blood vessels, producing tissue infarction, hemorrhagic necrosis, and death. In some, but not all, patients who remain persistently and profoundly immunocompromised, A. fumigatus can disseminate to distal sites including the brain, kidney, liver, and skin. The pathogenic determinants responsible for distal seeding of A. fumigatus to target organs are unknown. DCs have a primary role in surveillance for pathogens at the mucosal surfaces and are recognized as the initiators of immune responses to them [24,25]. DCs are strategically located at the interface of potential pathogen entry sites and take up antigen, move into secondary lymphoid tissues and activate both helper and cytotoxic T cells. Pathogen-mediated activation – 2005 ISHAM, Medical Mycology, 43, S181 /S188

induces DCs to undergo maturation consisting of antigen acquisition downregulation, increased expression of the major histocompatibility antigen complex (MHC) and co-stimulatory molecules, interleukin (IL)12 production, and altered expression of chemokine receptors [26]. As they mature, DCs migrate to the T cell areas of lymphoid organs, where they translate the tissue-derived information into the language of T helper (Th) cells, providing them with an antigenspecific ‘signal 1’, a co-stimulatory ‘signal 2’ and a ‘signal 3’ which determines the polarization of naive Th cells into Th1 or Th2 cells. In addition to DCs initiating immunity, certain subpopulations of DCs are able to downregulate immune responses [27]. The ability of DCs to influence the pattern of cytokine secretion by T cells represents a critical function, which can profoundly influence the final outcome of the immune response to pathogens. Several factors appear to influence the ability of DCs to polarize T cell cytokine responses, including the DC subsets, the nature of the maturation stimuli and the host microenvironment [28]. At the end, DCs represent the critical link between innate and adaptive immunity upon which appropriate concerted action is required for a successful host defence against an invading pathogen. A dense network of DCs has been described in the respiratory tracts [29,30]. In the resting state, respiratory tract DCs are specialized for uptake/processing but not for presentation of antigen, the latter requiring cytokine maturation signals that presumably occur

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after migration to regional lymph nodes [31,32]. The evidence that pulmonary DCs, through production of IL-10, mediate unresponsiveness to respiratory antigens [33,34] indicates that local production of immunoregulatory cytokines may affect the ability of DCs to instruct the appropriate T cell responses to the invading pathogens. Efficient responses to the different forms of fungi require different mechanisms of immunity [6]. DCs are uniquely able to decode the fungus-associated information and translate it in qualitatively different Th immune responses, in vitro and in vivo [35]. In infections, they are central in the balancing act between immunopathology and protective immunity generated by host-fungus interactions. In the case of Aspergillus, by using distinct pattern recognition receptors, including TLRs, human and murine DCs were found to be able to finely discriminate between conidia and hyphae of Aspergillus in terms of induction of adaptive Th responses [22,35 /37]. It is believed that microbial detection by DCs through TLRs is responsible for pathogen discrimination and the initiation of the appropriate effector response (Fig. 2). It is intriguing that the TLR9 agonist CpG-oligodeoxynucleotide (ODN) could convert an Aspergillus allergen to a potential protective antigen, suggesting the potential for TLR agonists to act upon the degree of flexibility of the immune recognition pathways to antigens and allergens [38]. These results suggest that the proper manipulation of DC functioning in vivo may translate into beneficial effects in infections.

Adaptive immunity to Aspergillus Studies on the epidemiology of IA in bone marrow transplantation recipients indicated a reduced neutropenia-related infection and an increased ‘late-onset’ infection, concomitant with the occurrence of graftversus-host disease (GVHD) [4,39]. These findings, together with the occurrence in non-neutropenic patients [40], attest to the importance of specific defects in both the innate and adaptive immune effector mechanisms in the pathogenesis of the disease [41,42]. The recent evidence that, in healthy individuals and in patients surviving IA, a significant antigen-specific proliferation of IFN-g-producing T cells occurred [42], confirms the crucial role of a Th1 reactivity in the control of infection [43 /45]. Adaptive immune responses are especially critical for control of the infection in the most common, sub-acute forms of IA. Two general patterns of Th activation characterize adaptive immune responses in IA. The Th1 response is associated with increased production of

Fig. 2 The immune response to A. fumigatus : the cross-talk between the innate and adaptive immune systems. Cells of the innate immune system discriminate between different forms of the fungus and produce sets of chemokines, cytokines and co-stimulatory molecules through which signals are sent to the adaptive T helper (Th) immune system. Protective and nonprotective Th cells release a distinct panel of cytokines, capable of delivering activating and inhibitory feedback signals to effector phagocytes. Together, the innate and adaptive immune systems contribute to the inflammatory response.

inflammatory cytokines interferon (IFN)-g, IL-2 and IL-12 and stimulation of antifungal effector cells (macrophages and PMNs). Alternatively Th2-type responses are associated with suppression of antifungal effector cell activity, decreased production of IFN-g and increased concentrations of IL-4 and IL-10, which promote humoral responses (IgE) to Aspergillus and allergy [6,8]. Hosts exhibiting Th2-predominant responses will experience a progressive infection. An association has been reported between serum IL-10 levels and the progression of IA in non-neutropenic patients [46]. Favorable outcome or stabilization of disease was seen in patients with low or decreasing serum IL-10 levels, whereas failure correlated with increasing serum IL-10 levels until patient death despite aggressive antifungal therapy. This study also shows that the IL-10 correlation with outcome of IA was similar, irrespective of the underlying immunosuppressive disease state, thus implying that IL-10 could be a surrogate marker for identifying patients at risk for IA. Further evidence indicate that the administration of – 2005 ISHAM, Medical Mycology, 43, S181 /S188


Th1 type cytokines, such as IFN-g and tumor necrosis factor (TNF)-a, protected mice from a lethal challenge of Aspergillus, whereas neutralization of Th2 type cytokines (such as IL-4) augmented resistance to the fungus. Conversely, administration of Th2 type cytokines (IL-4, IL-10) increased susceptibility to the infection and reduced survival [47]. The importance of Th1/Th2 dysregulation in the outcome of IA in humans was recently supported by work analyzing lymphocyte responses in patients with active infection. T cell responses to A. fumigatus antigen were compared in healthy patients vs. patients with hematological malignancies who were receiving treatment for probable or proven IA. On exposure to Aspergillus antigen, lymphoproliferative responses in healthy individuate exhibited a pattern of increased IFN-g production. Patients with clinical evidence of IA who were responding to antifungal therapy similarly demonstrated strong Th1 lymphoproliferative responses, with IFN-g/lL-10 ratios greater than 1.0. Patients with stable or progressive IA on antifungal therapy, however, exhibited poor lymphocyte stimulation indexes and low IFN-g/IL-10 ratios consistent with a Th2-predominant response [42]. Together, clinical and experimental observations suggest that a Th1/ Th2 dysregulation with suppression of host Th1 CD4 lymphocyte response and a switch to a Th2-type immune response may contribute to the development of an unfavorable outcome of IA [42,45].

Immunomodulation in aspergillosis Fungal-mediated suppression of the protective host Th1 response may play a key role in the progression of IA. Antigens and allergens of A. fumigatus have been described as being capable of inducing distinct patterns of Th cytokine production [2,48]. Clinical and experimental evidence indicates that the production of cytokines from Th2 [49,50] or Th1 [51,52] cells or both [53,54], in response to different Aspergillus proteins, contributes to the pathogenesis of allergic and autoimmune diseases upon exposure to the fungus. Although several mechanisms have been reported to contribute to the evasion of host immune responses [55], active secretion of immunosuppressive metabolites or mycotoxins during invasive growth may be a key factor by which Aspergillus suppresses host Th1 immunity in subacute disease [2]. Mycotoxins are also the most abundantly secreted substances during the culture of clinical A. fumigatus strains. In addition to their immunologic activity, many mycotoxins have also potent anti-angioneogenic effects which may contribute to pathologic features seen during angioinvasive growth – 2005 ISHAM, Medical Mycology, 43, S181 /S188

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of Aspergillus (coagulative necrosis). Gliotoxin, an epipolythiodioxo-piperazine metabolite, is the most abundant mycotoxin produced by A. fumigatus. Gliotoxin has been reported to have a broad range of immunosuppressive properties including potent inhibition of nuclear factor (NF)-kB activation and macrophage/neutrophil oxidative killing [56]. As gliotoxin has been detected in the tissues of infected animals at concentrations at least as high as that required to see immunological effects in vitro, the release of gliotoxin could aid the evasion of the fungus from antifungal effector cells.

Vaccination against Aspergillus: when, where and how Given that Aspergillus is a ubiquitous airborne, mainly indoor, organism, exposure cannot be avoided and therefore protection of all patients at risk would represent a breakthrough in healthcare. Adequate protection with antifungal prophylaxis is not always possible or effective [4]. Therefore, the development of a vaccine to protect these patients would be desirable. A vaccine that is not fully protective, but improves the chances of survival with antifungal therapy, would still be extremely useful. Patients awaiting transplant (when their immune systems are still fully functional), HIV infected patients and long-term immunosuppressed patients would be the primary recipients. Prior work on an Aspergillus vaccine is limited. A wide spectrum of vaccination strategies against aspergillosis, involving combination of a variety of proteins, glycoproteins, live or inactivated conidia used as immunogens or the adoptive transfer of immunity cells have been attempted since 1939. An ‘endotoxin’ isolated from A. fumigatus gave limited protection to hyperimmunized rabbits subsequently inoculated with spores. No protective effect could later be demonstrated in rabbits immunized with ‘endotoxin’, killed conidia or prior sublethal infections [57,58]. Some protection was seen in ducks and mice [59] using live conidia injected subcutaneously. Studies in turkey poults used various growth phase extracts and different adjuvants and the ‘best’ combination of germlings and adjuvant reduced mortality by 38% /57% [60,61]. A set of in vivo experiments with peptides derived from the ribotoxin of A. fumigatus (Asp f1) showed modulation of the immune response in mice consistent with Th1 driven response and protection [62]. In several recent experiments, immunization against A. fumigatus in murine models of IA has been studied by different groups. It has been clearly shown that vaccination with A. fumigatus extracts can confer protection against

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subsequent challenges with A. fumigatus conidia in immunosuppressed mice [45,63]. Protection was also observed with additional strategies of vaccination, including the use of adjuvants such as CpG ODN and DCs pulsed with different fungal preparations [38]. Protected mice develop strong Th1 responses characterized by high levels of IFN-g and IL-2 and low levels of IL-4 [38].

What type of vaccine to prevent IA? Although humoral immunity has not been thought to be important in natural defense against aspergillosis, it is understood that antibodies could contribute to the efforts of host effector cells by addition of, or improvement of, opsonization [64]. An earlier intriguing report indicated that passive transfer of immune serum could prolong survival after challenge with Aspergillus [65]. However, research studies on host defenses against aspergillosis suggest likely targets for immune-boosting from vaccination would be the manipulation of innate and adaptive immune responses. The immune system can be stimulated specifically by vaccination or nonspecifically in an antigen-independent manner by modulators of innate immune functions. With regard to manipulation of adaptive immunity, a rationale for a vaccine comes from prior work showing that the resolution of a prior Aspergillus infection leads to resistance against subsequent challenge [45]. A crude extract filtrate antigen protected neutropenic mice from aspergillosis through the induction of specific CD4/ Th1 cells. These cells were also able to confer protection upon adoptive transfer into naive recipient mice. This and more recent studies [66] have provided the rationale for investigating immunotherapy with antigen-specific T cells to restore protective antimicrobial responses in immunocompromised patients.

A dendritic cell-based vaccine in hematopoietic transplantation Recent experimental evidence suggest thats vaccination against Aspergillus through the use of fungus-pulsed DCs is a feasible option [67]. It also has also been shown that fungal RNA acts as potent DC activator [67 /69]. Although extracellular mRNA induced DC activation by signalling through a nucleotide receptor [70], fungal RNA also activated TLR expression on DCs. The expression of several TLRs was upregulated upon exposure to fungal RNA from Aspergillus conidia [69]. As DCs efficiently took up extracellular

fungal RNA [69], this indicates that DCs are allowed to orchestrate the immune response against both intracellular and extracellular fungi. Bozza et al . [67,69] found that the infusion of fungus-pulsed or RNA-transfected DCs induced antifungal resistance through the induction of Th1 cells producing IFN-g (Table 1). DCs also accelerated the recovery of both myeloid and lymphoid cells in mice with allogenic hematopoietic stem cell transplantation (HSCT), an experimental model in which autologous reconstitution of host stem cells is greatly reduced to the benefit of a long-term, donor type chimerism in more than 95% of the mice and low incidence of GVHD [71]. Patients receiving T cell-depleted HSCT are unable to develop antigen-specific T cell responses soon after transplant [72]. However, functional recovery of the T cell system after T cell-depleted allogeneic HSCT has been demonstrated [73], and both donor and recipient DCs may participate to the reconstitution of the T cell repertoire in transplantation through distinct pathways of antigen presentation [74]. The accelerated recovery of functional Th1 cells producing IFN-g by the infusion of fungus-pulsed or RNA-transfected DCs suggests that DCs may contribute to the educational program of T cells in HSCT during reconstitution, as already proposed [74]. Recent studies point to the use of selected TLR ligands or TLR activating agents as candidate adjuvants capable of activating DCs for Th1 priming to Aspergillus [37,38]. For instance, thymosin a1 promoted the production of IL-12p70 and IFN-a in human DCs through a TLR-dependent pathway. Thus, the ability to modulate DC functioning qualifies thymosin a1 as a candidate adjuvant capable of manipulating both the innate and adaptive immune responses to the fungus.

Conclusions and perspectives It has become apparent that improving our understanding on how immune responses are activated will enable the construction of better vaccines and vaccine strategies that are effective at eliciting acquired protective immunity to fungi. The model has brought DCs to Table 1 Dendritic cell vaccination against Aspergillus fumigatus [67,69]. DC pulsed with:

Th priming

Resistance to infection

Aspergillus conidia Conidial RNA Aspergillus hyphae Hyphal RNA

Th1 Th1 Th2 Th2

Increased Increased Decreased Decreased

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prominence as promising targets for intervention for immunotherapy and vaccine development [75] and has shifted the emphasis from the ‘antigen’ towards the ‘adjuvant’ [7]. The promise of a fungal vaccine will also demand an adjuvant capable of both stimulating the appropriate type of response best tailored to combating the infection and being effective in conditions of immunosuppression [76,77].

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