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Complement induction and complement evasion in patients with cerebral aspergillosis
Microbes and Infection 10 (2008) 1567e1576 www.elsevier.com/locate/micinf
Original article
Complement induction and complement evasion in patients with cerebral aspergillosis Gu¨nter Rambach a, Hans Maier b, Gianluca Vago c, Iradj Mohsenipour d, Cornelia Lass-Flo¨rl a, Alexandra Defant a, Reinhard Wu¨rzner a, Manfred P. Dierich a, Cornelia Speth a,* a
Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Fritz-Pregl-Str. 3, 6020 Innsbruck, Austria b Institute of Pathology, Innsbruck Medical University, Mu¨llerstr. 44, 6020 Innsbruck, Austria c Department of Pathology, Luigi Sacco Hospital, University of Milan, Milan, Italy d Department of Neurosurgery, Innsbruck Medical University, Anichstr. 35, 6020 Innsbruck, Austria Received 14 March 2008; accepted 25 September 2008 Available online 14 October 2008
Abstract Cerebral aspergillosis is a mostly lethal infection of the central nervous system. Former results identified low cerebral complement levels as one cause for insufficient immune reaction. Therefore we studied cerebral complement expression after fungal invasion and investigated putative mechanisms of Aspergillus spp to cope with the complement-induced selection pressure. Brain tissue derived from patients with cerebral aspergillosis or non-infected individuals was analyzed immunohistochemically for complement synthesis. Correlations between expression levels were determined statistically. Increased complement synthesis, a prerequisite for strengthened antifungal potency, was visible in resident astrocytes, neurons, oligodendrocytes as well as in infiltrating macrophages after fungal infection. Surprisingly, microglia, although regarded as major immune cells, only marginally participated in synthesis of most complement proteins. Several evasion mechanisms were found that help the fungus to establish a cerebral infection even in the presence of complement: Fungal hyphae limit the surface deposition of C3 and thus interfere with complement-dependent phagocytosis. Furthermore, the ‘‘sealing off’’ in brain abscesses not only inhibits fungal spreading but also forms protection shields against complement attack. Complement indeed seems to represent an important selection pressure and evokes the development of fungal evasion mechanisms. Counteractions for these evasion processes might represent interesting therapeutic approaches. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Aspergillus; Complement; Cerebral aspergillosis; Immune evasion
1. Introduction Invasive aspergillosis is an opportunistic infection that mainly affects immunocompromised individuals, such as AIDS patients, transplant recipients, patients with haematological diseases, or patients with autoimmune diseases treated with immunosuppressive regimens [1e3]. Central nervous system (CNS) aspergillosis is one of the most common extrapulmonary * Corresponding author. Tel.: þ43 512 9003 70705; fax: þ43 512 9003 73700. E-mail address: [email protected] (C. Speth). 1286-4579/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2008.09.011
sites of the disseminated infection; penetration into the CNS occurs in 20e60% of all cases of invasive aspergillosis and its lethality risk exceeds 90% [4]. Characteristic neuropathologic manifestations are cerebrovascular aspergillosis, single or multiple cerebral abscess formation, cerebral granuloma, mycotic cerebral aneurysm, and meningitis [5,6]. Clinical features include acute stroke-like illness with focal neurological deficits, fever, sensory impairment, altered mental and/or conscious status, headache, hemiplegia and seizures [2,5,6]. Antifungal therapy is often of poor efficacy, although presentday drugs like voriconazole have improved response and survival rates of the patients [7].
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As the CNS is separated from the periphery by the bloodbrain-barrier, the local innate immunity turns out to be the main antifungal defense system. One immune weapon whose components are produced locally in the CNS is the complement system, a cascade representing a central part of innate immunity [8,9]. All types of resident brain cells are able to synthesize complement factors, all together forming a functional and locally acting system [9]. Complement can start its attack immediately after contact with the pathogen and features a broad spectrum of action against microorganisms including fungi. Aspergillus conidia and hyphae are potent complement activators with subsequent opsonization of the fungal surface by complement fragments to support phagocytosis and killing by monocytes/macrophages [10]. Furthermore, soluble cleavage products such as C3a and C5a harbor a variety of pro-inflammatory biological functions in the brain such as cell stimulation and induction of chemotaxis [9]. The available concentration of complement proteins is a critical parameter for the antifungal immune defense, which is illustrated by the fact that C deficiency is correlated with enhanced susceptibility to a disseminated infection by A. fumigatus [11]. The spontaneous complement production within the CNS is low; the constitutively present complement levels are sufficient to achieve a weak opsonization, but insufficient to support phagocytosis and killing [10]. Our former results demonstrate elevated complement concentrations in the cerebrospinal fluid (CSF) derived from a patient with cerebral aspergillosis. The source of the increased complement levels either locally produced or diffused through a leaky bloodebrain-barrier could not be clarified at that time. This inflammatory CSF induces a better complement deposition on the Aspergillus surface, an increased phagocytosis and killing by infiltrating granulocytes, and the accumulation of complement activation products compared to non-inflammatory CSF. However, complement protein concentrations and the corresponding antifungal immune capacity of the serum are not achieved [10]. Since complement represents such an effective and fast weapon of immunity, many pathogens have developed strategies to overcome its attack, either by avoiding recognition or by inhibiting complement-mediated eradication. The most successful evasion principle is probably that of disguise by preferred usage of non- or poorly activating surface moieties or by acquisition of complement regulatory molecules from the host. Once detected by complement, fungi still have a potent arsenal to avoid their eradication by removal, consumption or destruction of complement [12e14]. For Aspergillus the relevance of complement, its protein levels and effective fungal evasion strategies is enlightened by the fact that the degree of complement deposition on the surface of the fungus inversely correlates with its pathogenicity [15]. The surface of the pathogen is optimized to limit complement activation. The fungal genes arp1 and alb1 mask complement-activating surface antigens and their disruption results in a more efficient surface binding of C3 and subsequently in an increased phagocytosis by neutrophils [16,17]. Furthermore, A. fumigatus secretes a soluble factor which
selectively inhibits activation of the alternative complement pathway and interferes with opsonization of the fungus and with C3b-dependent phagocytosis and killing [18,19]. Studies by Sturtevant revealed the synthesis of a membrane-bound proteolytic enzyme that was able to degrade complement factor C3 (reviewed in Refs. [21,20]). This is confirmed by recent own experiments showing complement-degrading proteolysis in the supernatant of Aspergillus when grown in CSF (unpublished). Our results presented here show that resident brain cells upregulate their complement expression after fungal invasion into the CNS; infiltrating macrophages also contribute to increased complement concentrations. However, the high lethality of cerebral aspergillosis in spite of the increased complement levels indicates the efficacy of fungal complement evasion strategies. We could demonstrate the limited surface deposition of C3 even in the presence of complement, probably due to secretion of a fungal complement inhibitor. In addition, the formation of brain abscesses, a host mechanism to inhibit further spreading of invading pathogens, results in protection against complement attack. 2. Materials and methods 2.1. Patients and brain material Brain tissue specimens derived from sixteen cases of cerebral aspergillosis were analyzed. The patients suffered from various immunocompromising underlying diseases such as HIV infection (10 cases), tumor therapy (2 cases), acute myeloid leukemia (2 cases), aplastic anemia (1 case) and organ transplantation (1 case). Two patients were female, 14 male with an average age of 49y (þ/18.1y). Fifteen samples were taken from cerebrum, either with or without stem ganglia, one sample was from pons. Pathologically either fresh or encapsulated Aspergillus abscesses were seen, except in two cases where Aspergillus hyphae were distributed in the brain tissue without indication of abscess formation. Further typical pathological findings were reactive gliosis and inflammatory infiltrates; in most brain tissue samples widespread necroses were visible, often together with hemorrhage and edema. Brain tissue samples derived from seven individuals without aspergillosis and without neurological involvement were used as control. Those individuals had died from cardiac diseases (5 cases), pneumonia (1 case) or pancreas carcinoma (1 case). Three out of the seven patients were male, 4 female, with an average age of 68y (þ/9.25y). The CNS tissue sections were prepared from cerebrum, either with or without stem ganglia, and controlled microscopically for the absence of any pathological finding such as infiltrates, reactive astrocytosis, edema, or cell death. 2.2. Immunohistochemistry Brain sections from the patients with cerebral aspergillosis as well as from the uninfected individuals were analyzed for the expression of complement factors. Paraffin-embedded
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tissue sections were stained by the ABC method [22]. For the present study polyclonal rabbit anti-human antibodies recognizing C1q, C4, C3 or C5 (Dako Cytomation, Denmark) were used. Briefly, the sections were deparaffinized in xylene and rehydrated through graded concentrations of ethanol to TBS (0.05 M TriseHCl, 0.15 M NaCl, pH7.6). Endogenous peroxidase activity was blocked with H2O2 (3% in TBS, 5 min) followed by three washing steps in TBS. Unspecific blocking was counteracted by incubation in a blocking solution (Dako). The antigens were unmasked by proteinase K (Dako). Sections were incubated over night with the primary antibody, diluted to a concentration of 10 mg/ml in a diluent solution with background reduction (Dako). Control sections were incubated with an unspecific antibody of the same isotype and in the same concentration. After extensive washing in TBS the biotinylated secondary antibody was diluted in PFA (1% FCS, 0.1% sodium azide, 1% normal macaque serum 5% human AB serum in PBS) and incubated for 30 min. After washing the bound antibody was detected using DAB as chromogen (Dako) with signal amplification by the corresponding ABC kits (Vector, USA). The sections were counterstained by hematoxylin (Sigma, USA) and the slides were mounted in Permount medium (Fisher Scientific). 2.3. Analysis of immunohistochemistry The complete immunohistochemical slides were manually scored for the percentage of immunolabelled cells (<10%, <50%, >50%) within a respective cell type, including astrocytes, microglia, neurons and perivascular macrophages, which were identified based on location and morphology by a very experienced pathologist. Furthermore, the staining intensity of fungal hyphae was scored based on the following scale: negative [], weak [þ], moderate [þþ], strong [þþþ]. The correlation between two series (X, Y ) of n measurements was calculated using the Pearson product-moment correlation coefficient (¼sample correlation coefficient) according to the formula: rsy ¼
X ðxi xÞðyi yÞ ðn 1Þsx sy
;
where x and y are the sample means of X and Y, sx and sy are the sample standard deviations of X and Y and the sum is from i ¼ 1 to n.
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different brain cell types nor a significant extracellular staining of the surrounding tissue (Fig. 1AeD). In contrast, a high C1q expression and secretion was detected in the brain tissue of most patients with cerebral aspergillosis (Fig. 1EeM). A detailed analysis showed C1q-expressing reactive astrocytes (Fig. 1E, I), neurons (Fig. 1F, K) and oligodendrocytes (Fig. 1G, L). Positive cells were not limited to areas with direct presence of Aspergillus hyphae, but distributed throughout the tissue, suggesting that complement expression is induced by (a) soluble inflammatory factor(s) rather than by immediate contact between cells and hyphae. Although microglia are regarded as the main immune cell type in the brain, C1q-positive microglia were interestingly found in only one tissue sample. Instead of that infiltrating macrophages, not present in the control tissue, strongly expressed C1q (Fig. 1H, M). Stained tissue sections were assessed semi-quantitatively for the percentage of C1q-positive cells and the capacity of complement production was compared between different cell types. This analysis could help to identify the predominant complement-producing cell type in the brain and thus the putative target for stimulation of complement synthesis to improve antifungal innate immunity. Whereas no expression was seen in the astrocytes of uninfected individuals, this cell type was the most potent local C1q-producer in patients with cerebral aspergillosis (Fig. 2A, Table 1). Beside astrocytes, neurons and oligodendrocytes were the main local producers of C1q. While there was no immunohistochemical staining in uninfected brain tissue samples, the tissue sections of 7 out of 16 patients revealed staining for C1q (Fig. 2B, C). However, those brains with neurons expressing C1q were only partly the same like those with positive oligodendrocytes (Table 1). Statistical analysis revealed a correlation of only 45% between expression of C1q in neurons and that in oligodendrocytes. Similarly, the correlation between neuronal and astrocytic C1q synthesis was weak (Table 1), indicating that the induction of this complement factor underlies different mechanisms for the various cell types. Whereas microglia staining positive for C1q were found only in 1/16 tissue samples of patients with cerebral aspergillosis, infiltrating macrophages with strong synthesis of C1q could be detected in 16/16 brain samples (Fig. 2D, E). In 15/16 cases even more than 50% of macrophages produced C1q. Again, there was no correlation between expression of C1q in macrophages/microglia and the production of C1q in other cell types (Table 1).
3. Results 3.1. Expression of complement factor C1q in patients with cerebral aspergillosis To evaluate the cerebral expression of C1q, immunohistochemistry was performed in tissue sections of patients with cerebral aspergillosis and of non-infected individuals. Some examples of immunohistochemical staining of brain tissue samples are shown in Fig. 1. Generally, uninfected control brains revealed neither visible expression of C1q in the
3.2. Cerebral expression of complement factors C4, C3 and C5 in patients with cerebral aspergillosis Similar to C1q, the constitutively low production of complement factors C4 and C3 in the brain was highly increased in presence of the fungus. Astrocytes (Figs. 3A and 4A; Table 1) and infiltrating macrophages (Figs. 3E and 4E) were the main producers of C4 and C3. Neurons (Figs. 3B and 4B) and oligodendrocytes (Figs. 3C and 4C) contributed to cerebral C4 and C3 levels to a lesser extent, whereas microglia
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Fig. 1. Expression of complement factor C1q in brain tissue of uninfected individuals (AeD) and patients with cerebral aspergillosis (EeM). Immunohistochemistry was performed in paraffin-embedded sections with a specific C1q-antibody. Astrocytes (A, E, I), neurons (B, F, K), oligodendrocytes (C, G, L), and infiltrating macrophages (H, M) are shown; no infiltrating macrophages were present in the brain of uninfected individuals (D). Magnification: 400.
did not play a significant role as complement producers (Figs. 3D and 4D). Astrocytes and macrophages were also main producers of complement factor C5 with 86% and 75% of the tissue samples harboring C5-positive cells, respectively (Table 1). The role of microglia was more pronounced in the synthesis of C5 than in the synthesis of C1q, C4 and C3, and stained positive for C5 in 90% of the tissue samples. In contrast neurons only marginally contributed to the production of C5 (Table 1). In general there was no clear correlation between the expression levels of different complement factors and between the expression in different cell types, indicating distinctive mechanisms of regulation (Table 1). 3.3. Incomplete complement deposition on fungal hyphae in different locations Since an Aspergillus-produced complement inhibitor is described in literature, the deposition of complement on the fungal surface was also evaluated (Fig. 5, Table 1). Fungal hyphae were present perivascularly, within the brain parenchyma and/or in the meninges. Independent from the location,
an intense deposition was visible for C1q and C4 (Fig. 5A, B). However, in 7 out of 16 brain samples the fungal hyphae did not stain at all for C3, and staining was only weak in further 8 tissue samples (Fig. 5A, B), indicating an incomplete complement deposition and thus an inadequate complementdependent immune attack. Similarly fungal hyphae in 5 out of 15 brain tissue samples did not stain for complement factor C5 (Table 1). 3.4. Abscess: sealing off against fungal spreading but also against complement attack In one patient the focus of cerebral infection was organized as a mature abscess with a central necrotic area surrounded by a capsule of granulomatous fibrous tissue. Immunohistochemical staining revealed the effect of this encapsulation on the distribution of complement factors C1q, C4 and C3 (Fig. 6A, D, G). Whereas the fibrous collagenized tissue of the abscess was intensely stained for complement proteins, especially for C1q (Fig. 6A, B) and C4 (Fig. 6D, E), the intensity decreased towards the center of the abscess. The central necrotic area contained no or only minor amounts of the different complement proteins and
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0% <10% <50% >50%
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 + Asp
D numberof brains
number of brains
C 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
control
17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
+ Asp
17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
control
E number of brains
control
B number of brains
number of brains
A 17
control
1571
+ Asp
17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
+ Asp
control
+ Asp
Fig. 2. Expression of complement factor C1q in astrocytes (A), neurons (B), oligodendrocytes (C), microglia (D) and infiltrating macrophages (E) of uninfected individuals (control) and patients with cerebral aspergillosis (þAsp). Paraffin-embedded sections were stained immunohistochemically to visualize C1q. The slides were analyzed microscopically to quantify the percentage of positive cells. Not all cell types were present in all slides.
Table 1 Percentage positive staining and correlations between positive staining of different cell types. Fungal infection
C1q staining
C4 staining
C3 staining
C5 staining
Hyphae
þ þ þ þ þ þ þ
0% 73% 0% 44% 0% 6% 0% 44% e 100% 42% 100% 93%
0% 71% 0% 36% 0% 0% 0% 57% e 85% 0% 86% 100%
29% 81% 0% 73% 14% 25% 14% 75% e 93% 42% 100% 56%
0% 86% 0% 7% 0% 90% 0% 60% e 75% 0% 80% 63%
Correlation coefficient Astrocytes e neurons Astrocytes e oligodendrocytes Astrocytes e microglia Astrocytes e macrophages Neurons e oligodendrocytes Neurons e microglia Neurons e macrophages Oligodendrocytes e microglia Oligodendrocytes e macrophages Microglia e macrophages
þ þ þ þ þ þ þ þ þ þ
0.12 0.49 0.16 0 0.02 0.23 0 0.23 0 0
0.08 0.09 0.28 0.54 0.45 0.02 0.16 0.33 0.13 0.16
0.52 0.09 0 0.28 0.04 0 0.34 0 0.10 0
0.11 0.12 0.41 0.52 0.33 0.13 0.52 0.27 0.19 0.25
Percentage positive Astrocytes Neurons Microglia Oligodendrocytes Macrophages Tissue staining
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number of brains
A
17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
0% <10% <50% >50%
control
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
+ Asp
control
D 17 16
control
+ Asp
E
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
number of brains
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
number of brains
number of brains
C 17 16
B number of brains
1572
control
+ Asp
+ Asp
17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 control
+ Asp
Fig. 3. Expression of complement factor C4 in astrocytes (A), neurons (B), oligodendrocytes (C), microglia (D) and infiltrating macrophages (E) of uninfected individuals (control) and patients with cerebral aspergillosis (þAsp). Paraffin-embedded sections were stained immunohistochemically to visualize C4. The slides were analyzed microscopically to quantify the percentage of positive cells. Not all cell types were present in all slides.
no deposition on the fungal surface was visible (Fig. 6 C, F, I), implying that the abscess capsule may protect the fungus within the abscess from any efficient complement attack. 4. Discussion To increase the efficiency of antifungal complement attack in the CNS represents an interesting therapeutic concept, but requires a deeper understanding of the underlying mechanisms. Former in vitro results have shown that the limited constitutive complement levels in the CSF restrict the antifungal attack by this part of the immune system [10]. In this study we could show by immunohistochemical staining that the low production of several complement proteins is upregulated in the course of cerebral fungal infection. The increased expression of C3 is of special importance since this central factor participates in all three-complement activation pathways. The immunohistochemical analyses presented here confirm former results showing enhanced complement concentrations in the CSF from a patient with cerebral aspergillosis [10]. This increase of complement expression enables a better opsonization of the fungal hyphae as shown by former in vitro experiments. Higher concentrations of complement in the CSF are associated with higher phagocytic activity of microglia, granulocytes and monocytes/macrophages, a stimulated oxidative burst and a more efficient killing [10]. Further consequences of increased complement
concentrations might include the chemotactic recruitment of inflammatory cells by C3a, a fragment of C3, and the stimulation of microglia and astrocytes via induction of proinflammatory cytokines [9]. The experiments identified resident brain cells as main producers, and also infiltrated monocytes/macrophages participate in complement production. Thus, a leaky bloode brain-barrier may contribute to enhance local complement levels, but is not the predominant factor. Particularly astrocytes upregulate the synthesis of complement after an infectious stimulus, followed to a lesser extent by neurons and oligodendrocytes. It appears surprising that microglia, generally considered to be the preeminent immune cells of the CNS, are only minor producer of most complement factors by C5 being the only exception. There seems to be a separation between microglia as immune effector cells which respond to contact with complement factors by activation and phagocytosis [23], and other cell types in the CNS which synthesize the complement factors. This separation might prevent autoactivation and excess stimulation of microglia. Interestingly, the precise signaling pathway for complement induction seems to vary for the different cell types, since there is no correlation between their percentages of producing cells within one tissue sample. A brain section with low percentage of C1q-positive astrocytes might harbor a high percentage of neurons expressing C1q. This individuality of complement induction might reflect the cell
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0% <10% <50% >50%
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
+ Asp
control
D
E
number of brains
number of brains
C
control
+ Asp
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
number of brains
control
B 17 16 number of brains
number of brains
A 17 16
control
1573
+ Asp
+ Asp
17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 control
+ Asp
Fig. 4. Expression of complement factor C3 in astrocytes (A), neurons (B), oligodendrocytes (C), microglia (D) and infiltrating macrophages (E) of uninfected individuals (control) and patients with cerebral aspergillosis (þAsp). Paraffin-embedded sections were stained immunohistochemically to visualize C3. The slides were analyzed microscopically to quantify the percentage of positive cells. Not all cell types were present in all slides.
type-specific distribution of the various signaling molecule receptors, since the inflammatory milieu composed of cytokines, chemokines and other extracellular effector molecules is supposed to be responsible for complement upregulation. This hypothesis is underlined by the fact that pro-inflammatory cytokines like IFN-g, TNF-a, IL-1b and IL-6 have a prominent effect on complement synthesis in glial and neuronal cells [9,24,25]. The indirect induction by proinflammatory cytokines is also implied by the presence of complement-producing cells in rather distance from fungal hyphae. A direct contact between brain cells and hyphae is not sufficient to stimulate complement production as demonstrated in vitro by experiments with incubation of brain cells with fungal hyphae (data not shown). Furthermore there is no correlation between the syntheses of different complement factors; brain tissue samples with high C1q expression are not the same like those with high C4 or C3 synthesis. This is surprising, since pro-inflammatory cytokines were described to induce a broad spectrum of acute phase proteins [26] complement proteins; e.g. IFN-g and TNF-a stimulate the production of all three factors C1q, C4 and C3 [27]. A possible explanation might be that the induction of a complement protein does not result from the action of a single cytokine but from a complex mixture of pro- and antiinflammatory cytokines which stimulate different promoter elements in different complement genes [27].
Despite the complement induction with all its putative positive effects on the antifungal defense cerebral aspergillosis is associated with a high lethality. Two explanations are possible: the stimulation of complement synthesis may be too late in the course of infection or not prominent enough to enable an efficient antifungal reaction; alternatively Aspergillus may be protected by the establishment of efficient complement evasion mechanisms. We could detect two relevant processes by which the fungus escapes from cerebral complement attack. First, our immunohistochemical studies showed significant deposition of the early complement factors C1q and C4 on the hyphal surface in the brain, but only minor opsonization by C3. Whereas C1q and C4 are proteins involved in early steps of the complement cascade, C3 and its fragments are part of more terminal steps of the complement cascade and mediate most antifungal mechanisms. The finding that C3-derived fragments are absent from the fungal hyphae is a hint for the presence of an Aspergillus-derived complement inhibitor which has been described by Washburn et al. [18,19]. In vitro, this soluble inhibitor CI rather selectively interfered with complement activation, decreased binding of the C3-fragment C3b to the fungal surface and inhibited C3b-dependent phagocytosis and killing [18,19]. This fungus-derived inhibitor was described to represent an important complement evasion strategy [12]. The production of CI during fungal growth under the physiological conditions of the brain had not been investigated, but seems plausible now according to our results.
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A
perivascular
parenchymal
meningeal
C1q
10µm
10µm
10µm
10µm
10µm
10µm
10µm
10µm
10µm
C4
C3
B
16
number of brains
14 12 10 8
negative
+ ++ +++
6 4 2 0
C1q
C4
C3
Fig. 5. Deposition of the complement factors C1q, C4 and C3 on hyphae of Aspergillus spp. in brain sections of patients with cerebral aspergillosis. (A) Immunohistochemical analysis of paraffin-embedded sections with specific antibodies reveals an intense deposition of C1q and C4 (brown color), but almost no C3 (blue color) on the fungal hyphae (magnification 1000). A semiquantitative analysis of hyphal staining intensity for C1q and C4 in comparison to C3 in the brain specimens is shown in (B).
A second complement escape mechanism is the formation of mature abscesses. Being a host defense strategy to prevent fungal spread, it also constitutes the manifestation of persisting microbial infection. Our experiments revealed that despite high complement levels in the surrounding fibrous layer the central necrotic area containing the fungal hyphae harbors only
marginal amounts of complement proteins. These amounts do not appear to be sufficient to enable a deposition on the hyphae as they did not stain in immunohistochemistry. Thus a useful immune mechanism meant to protect the host is at the same time a competent evasion mechanism for the fungus. However, most patients died before a mature abscess was formed and only early
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Fig. 6. Protection of Aspergillus spp. against complement attack by formation of a mature abscess, as shown by immunohistochemistry of paraffin-embedded sections. While the complement factors C1q (AeC), C4 (DeF), and C3 (GeI) are present in the surrounding fibrous layer (brown color), their amount in the central necrotic area of the abscess is low. Hyphae in the center reveal a weak or absent surface deposition with these complement proteins (C, F, I). Magnification: 100 (A, D, G), 400 (B, C, E, F, H, I).
stages of abscess formation were visible in these cases. This is a clear difference to murine or rat models of cerebral aspergillosis where all animals showed focal abscesses [28,29]. This discrepancy between humans and the animal model can be explained by the fact that the mice were infected intracerebrally by intracisternal injection. This situation is different in humans where cerebral aspergillosis mostly disseminates from a pulmonary infection. Thus the patients are already severely ill before the fungus reaches the CNS and therefore are more likely to die before maturation of an abscess. A further complement evasion mechanism, namely the production of secretory proteases by Aspergillus species, is currently studied by our group. Preliminary results show evidence for the secretion of complement-degrading proteases.
Increasing knowledge about fungal immune escape mechanisms may hopefully lead to new targets for novel supportive therapies against CNS-penetrating fungal pathogens. The development of those complement-based therapies should, however, avoid the pitfall of excessive complement activation, since presence of complement and complement activation products was associated with neurodegenerative diseases such as Alzheimer disease, Parkinson or multiple sclerosis [30]. Acknowledgements ¨ sterreichische NatioThis study was supported by the O nalbank (Project No. 11944). The authors thank T. Erbeznik for support in statistical analysis.
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References [1] F.P. Silveira, S. Husain, Fungal infections in solid organ transplantation, Med. Mycol. 45 (2007) 305e320. [2] M. Ruhnke, G. Kofla, K. Otto, S. Schwartz, CNS aspergillosis: recognition, diagnosis and management, CNS Drugs 21 (2007) 659e676. [3] K.A. Marr, R.A. Carter, M. Boeckh, P. Martin, L. Corey, Invasive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors, Blood 100 (2002) 4358e4366. [4] E. Jantunen, L. Volin, O. Salonen, A. Piilonen, T. Parkkali, V.J. Anttila, A. Paetau, T. Ruutu, Central nervous system aspergillosis in allogeneic stem cell transplant recipients, Bone Marrow Transplant. 31 (2003) 191e196. [5] W.W. Hope, T.J. Walsh, D.W. Denning, The invasive and saprophytic syndromes due to Aspergillus spp. Med. Mycol. 43 (Suppl. 1) (2005) 207e238. [6] T. Nadkarni, A. Goel, Aspergilloma of the brain: an overview, J. Postgrad. Med. 51 (2005) 37e41. [7] S. Schwartz, M. Ruhnke, P. Ribaud, E. Reed, P. Troke, E. Thiel, Poor efficacy of amphotericin B-based therapy in CNS aspergillosis, Mycoses 50 (2007) 196e200. [8] K. Francis, J. van Beek, C. Canova, J.W. Neal, P. Gasque, Innate immunity and brain inflammation: the key role of complement, Expert Rev. Mol. Med. 5 (2003) 1e19. [9] C. Speth, M.P. Dierich, P. Gasque, Neuroinvasion by pathogens: a key role of the complement system, Mol. Immunol. 38 (2002) 669e679. [10] G. Rambach, M. Hagleitner, I. Mohsenipour, C. Lass-Flo¨rl, H. Maier, R. Wu¨rzner, M.P. Dierich, C. Speth, Antifungal activity of the local complement system in cerebral aspergillosis, Microbes Infect. 7 (2005) 1285e1295. [11] R.F. Hector, E. Yee, M.S. Collins, Use of DBA/2 N mice in models of systemic candidiasis and pulmonary and systemic aspergillosis, Infect. Immun. 58 (1990) 1476e1478. [12] J. Behnsen, A. Hartmann, J. Schmaler, A. Gehrke, A.A. Brakhage, P.F. Zipfel, The opportunistic human pathogenic fungus Aspergillus fumigatus evades the host complement system, Infect. Immun. 76 (2008) 820e827. [13] G. Vogl, I. Lesiak, D.B. Jensen, S. Perkhofer, R. Eck, C. Speth, C. Lass-Flo¨rl, P.F. Zipfel, A.M. Blom, M.P. Dierich, R. Wu¨rzner, Immune evasion by acquisition of complement inhibitors: the mould Aspergillus binds both factor H and C4b binding protein, Mol. Immunol. 45 (2008) 1485e1493. [14] P.F. Zipfel, R. Wu¨rzner, C. Skerka, Complement evasion of pathogens: common strategies are shared by diverse organisms, Mol. Immunol. 44 (2007) 3850e3857.
[15] S. Henwick, S.V. Hetherington, C.C. Patrick, Complement binding to Aspergillus conidia correlates with pathogenicity, J. Lab. Clin. Med. 122 (1993) 27e35. [16] H.F. Tsai, Y.C. Chang, R.G. Washburn, M.H. Wheeler, K.J. KwonChung, The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence, J. Bacteriol. 180 (1998) 3031e3038. [17] H.F. Tsai, R.G. Washburn, Y.C. Chang, K.J. Kwon-Chung, Aspergillus fumigatus arp1 modulates conidial pigmentation and complement deposition, Mol. Microbiol. 26 (1997) 175e183. [18] R.G. Washburn, D.J. DeHart, D.E. Agwu, B.J. Bryant-Varela, N.C. Julian, Aspergillus fumigatus complement inhibitor: production, characterization and purification by hydrophobic interaction and thinlayer chromatography, Infect. Immun. 58 (1990) 3508e3515. [19] R.G. Washburn, C.H. Hammer, J.E. Bennett, Inhibition of complement by culture supernatants of Aspergillus fumigates, J. Infect. Dis. 154 (1986) 944e951. [20] J. Sturtevant, J.P. Latge, Interactions between conidia of Aspergillus fumigatus and human complement component C3, Infect. Immun. 60 (1992) 1913e1918. [21] J.F. Tomee, H.F. Kauffman, Putative virulence factors of Aspergillus fumigatus, Clin. Exp. Allergy 30 (2000) 476e484. [22] S.M. Hsu, L. Raine, Protein A, avidin, and biotin in immunohistochemistry, J .Histochem. Cytochem. 29 (1981) 1349e1353. [23] U.K. Hanisch, H. Kettenmann, Microglia: active sensor and versatile effector cells in the normal and pathologic brain, Nat. Neurosci. 10 (2007) 1387e1394. [24] A. Thomas, P. Gasque, D. Vaudry, B. Gonzalez, M. Fontaine, Expression of a complete and functional complement system by human neuronal cells in vitro, Int. Immunol. 12 (2000) 1015e1023. [25] S.R. Barnum, J.L. Jones, Differential regulation of C3 gene expression in human astroglioma cells by interferon-gamma and interleukin-1 beta, Neurosci. Lett. 197 (1995) 121e124. [26] J.R. Cardinaux, I. Allaman, P.J. Magistretti, Pro-inflammatory cytokines induce the transcription factors C/EBPbeta and C/EBPdelta in astrocytes, Glia 29 (2000) 91e97. [27] J.E. Volanakis, Transcriptional regulation of complement genes, Annu Rev. Immunol. 13 (1995) 277e305. [28] T.M. Chiller, J.C. Luque, Development of a murine model of cerebral aspergillosis, Int. Rev. Neurobiol. 79 (2007) 99e126. [29] S. Zimmerli, U. Knecht, S.L. Leib, A model of cerebral aspergillosis in non-immunosuppressed nursing rats, Acta Neuropathol. 114 (2007) 411e418. [30] S.D. Skaper, The brain as a target for inflammatory processes and neuroprotective strategies, Ann. N Y Acad. Sci. 1122 (2007) 23e34.
Original article
Complement induction and complement evasion in patients with cerebral aspergillosis Gu¨nter Rambach a, Hans Maier b, Gianluca Vago c, Iradj Mohsenipour d, Cornelia Lass-Flo¨rl a, Alexandra Defant a, Reinhard Wu¨rzner a, Manfred P. Dierich a, Cornelia Speth a,* a
Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Fritz-Pregl-Str. 3, 6020 Innsbruck, Austria b Institute of Pathology, Innsbruck Medical University, Mu¨llerstr. 44, 6020 Innsbruck, Austria c Department of Pathology, Luigi Sacco Hospital, University of Milan, Milan, Italy d Department of Neurosurgery, Innsbruck Medical University, Anichstr. 35, 6020 Innsbruck, Austria Received 14 March 2008; accepted 25 September 2008 Available online 14 October 2008
Abstract Cerebral aspergillosis is a mostly lethal infection of the central nervous system. Former results identified low cerebral complement levels as one cause for insufficient immune reaction. Therefore we studied cerebral complement expression after fungal invasion and investigated putative mechanisms of Aspergillus spp to cope with the complement-induced selection pressure. Brain tissue derived from patients with cerebral aspergillosis or non-infected individuals was analyzed immunohistochemically for complement synthesis. Correlations between expression levels were determined statistically. Increased complement synthesis, a prerequisite for strengthened antifungal potency, was visible in resident astrocytes, neurons, oligodendrocytes as well as in infiltrating macrophages after fungal infection. Surprisingly, microglia, although regarded as major immune cells, only marginally participated in synthesis of most complement proteins. Several evasion mechanisms were found that help the fungus to establish a cerebral infection even in the presence of complement: Fungal hyphae limit the surface deposition of C3 and thus interfere with complement-dependent phagocytosis. Furthermore, the ‘‘sealing off’’ in brain abscesses not only inhibits fungal spreading but also forms protection shields against complement attack. Complement indeed seems to represent an important selection pressure and evokes the development of fungal evasion mechanisms. Counteractions for these evasion processes might represent interesting therapeutic approaches. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Aspergillus; Complement; Cerebral aspergillosis; Immune evasion
1. Introduction Invasive aspergillosis is an opportunistic infection that mainly affects immunocompromised individuals, such as AIDS patients, transplant recipients, patients with haematological diseases, or patients with autoimmune diseases treated with immunosuppressive regimens [1e3]. Central nervous system (CNS) aspergillosis is one of the most common extrapulmonary * Corresponding author. Tel.: þ43 512 9003 70705; fax: þ43 512 9003 73700. E-mail address: [email protected] (C. Speth). 1286-4579/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2008.09.011
sites of the disseminated infection; penetration into the CNS occurs in 20e60% of all cases of invasive aspergillosis and its lethality risk exceeds 90% [4]. Characteristic neuropathologic manifestations are cerebrovascular aspergillosis, single or multiple cerebral abscess formation, cerebral granuloma, mycotic cerebral aneurysm, and meningitis [5,6]. Clinical features include acute stroke-like illness with focal neurological deficits, fever, sensory impairment, altered mental and/or conscious status, headache, hemiplegia and seizures [2,5,6]. Antifungal therapy is often of poor efficacy, although presentday drugs like voriconazole have improved response and survival rates of the patients [7].
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As the CNS is separated from the periphery by the bloodbrain-barrier, the local innate immunity turns out to be the main antifungal defense system. One immune weapon whose components are produced locally in the CNS is the complement system, a cascade representing a central part of innate immunity [8,9]. All types of resident brain cells are able to synthesize complement factors, all together forming a functional and locally acting system [9]. Complement can start its attack immediately after contact with the pathogen and features a broad spectrum of action against microorganisms including fungi. Aspergillus conidia and hyphae are potent complement activators with subsequent opsonization of the fungal surface by complement fragments to support phagocytosis and killing by monocytes/macrophages [10]. Furthermore, soluble cleavage products such as C3a and C5a harbor a variety of pro-inflammatory biological functions in the brain such as cell stimulation and induction of chemotaxis [9]. The available concentration of complement proteins is a critical parameter for the antifungal immune defense, which is illustrated by the fact that C deficiency is correlated with enhanced susceptibility to a disseminated infection by A. fumigatus [11]. The spontaneous complement production within the CNS is low; the constitutively present complement levels are sufficient to achieve a weak opsonization, but insufficient to support phagocytosis and killing [10]. Our former results demonstrate elevated complement concentrations in the cerebrospinal fluid (CSF) derived from a patient with cerebral aspergillosis. The source of the increased complement levels either locally produced or diffused through a leaky bloodebrain-barrier could not be clarified at that time. This inflammatory CSF induces a better complement deposition on the Aspergillus surface, an increased phagocytosis and killing by infiltrating granulocytes, and the accumulation of complement activation products compared to non-inflammatory CSF. However, complement protein concentrations and the corresponding antifungal immune capacity of the serum are not achieved [10]. Since complement represents such an effective and fast weapon of immunity, many pathogens have developed strategies to overcome its attack, either by avoiding recognition or by inhibiting complement-mediated eradication. The most successful evasion principle is probably that of disguise by preferred usage of non- or poorly activating surface moieties or by acquisition of complement regulatory molecules from the host. Once detected by complement, fungi still have a potent arsenal to avoid their eradication by removal, consumption or destruction of complement [12e14]. For Aspergillus the relevance of complement, its protein levels and effective fungal evasion strategies is enlightened by the fact that the degree of complement deposition on the surface of the fungus inversely correlates with its pathogenicity [15]. The surface of the pathogen is optimized to limit complement activation. The fungal genes arp1 and alb1 mask complement-activating surface antigens and their disruption results in a more efficient surface binding of C3 and subsequently in an increased phagocytosis by neutrophils [16,17]. Furthermore, A. fumigatus secretes a soluble factor which
selectively inhibits activation of the alternative complement pathway and interferes with opsonization of the fungus and with C3b-dependent phagocytosis and killing [18,19]. Studies by Sturtevant revealed the synthesis of a membrane-bound proteolytic enzyme that was able to degrade complement factor C3 (reviewed in Refs. [21,20]). This is confirmed by recent own experiments showing complement-degrading proteolysis in the supernatant of Aspergillus when grown in CSF (unpublished). Our results presented here show that resident brain cells upregulate their complement expression after fungal invasion into the CNS; infiltrating macrophages also contribute to increased complement concentrations. However, the high lethality of cerebral aspergillosis in spite of the increased complement levels indicates the efficacy of fungal complement evasion strategies. We could demonstrate the limited surface deposition of C3 even in the presence of complement, probably due to secretion of a fungal complement inhibitor. In addition, the formation of brain abscesses, a host mechanism to inhibit further spreading of invading pathogens, results in protection against complement attack. 2. Materials and methods 2.1. Patients and brain material Brain tissue specimens derived from sixteen cases of cerebral aspergillosis were analyzed. The patients suffered from various immunocompromising underlying diseases such as HIV infection (10 cases), tumor therapy (2 cases), acute myeloid leukemia (2 cases), aplastic anemia (1 case) and organ transplantation (1 case). Two patients were female, 14 male with an average age of 49y (þ/18.1y). Fifteen samples were taken from cerebrum, either with or without stem ganglia, one sample was from pons. Pathologically either fresh or encapsulated Aspergillus abscesses were seen, except in two cases where Aspergillus hyphae were distributed in the brain tissue without indication of abscess formation. Further typical pathological findings were reactive gliosis and inflammatory infiltrates; in most brain tissue samples widespread necroses were visible, often together with hemorrhage and edema. Brain tissue samples derived from seven individuals without aspergillosis and without neurological involvement were used as control. Those individuals had died from cardiac diseases (5 cases), pneumonia (1 case) or pancreas carcinoma (1 case). Three out of the seven patients were male, 4 female, with an average age of 68y (þ/9.25y). The CNS tissue sections were prepared from cerebrum, either with or without stem ganglia, and controlled microscopically for the absence of any pathological finding such as infiltrates, reactive astrocytosis, edema, or cell death. 2.2. Immunohistochemistry Brain sections from the patients with cerebral aspergillosis as well as from the uninfected individuals were analyzed for the expression of complement factors. Paraffin-embedded
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tissue sections were stained by the ABC method [22]. For the present study polyclonal rabbit anti-human antibodies recognizing C1q, C4, C3 or C5 (Dako Cytomation, Denmark) were used. Briefly, the sections were deparaffinized in xylene and rehydrated through graded concentrations of ethanol to TBS (0.05 M TriseHCl, 0.15 M NaCl, pH7.6). Endogenous peroxidase activity was blocked with H2O2 (3% in TBS, 5 min) followed by three washing steps in TBS. Unspecific blocking was counteracted by incubation in a blocking solution (Dako). The antigens were unmasked by proteinase K (Dako). Sections were incubated over night with the primary antibody, diluted to a concentration of 10 mg/ml in a diluent solution with background reduction (Dako). Control sections were incubated with an unspecific antibody of the same isotype and in the same concentration. After extensive washing in TBS the biotinylated secondary antibody was diluted in PFA (1% FCS, 0.1% sodium azide, 1% normal macaque serum 5% human AB serum in PBS) and incubated for 30 min. After washing the bound antibody was detected using DAB as chromogen (Dako) with signal amplification by the corresponding ABC kits (Vector, USA). The sections were counterstained by hematoxylin (Sigma, USA) and the slides were mounted in Permount medium (Fisher Scientific). 2.3. Analysis of immunohistochemistry The complete immunohistochemical slides were manually scored for the percentage of immunolabelled cells (<10%, <50%, >50%) within a respective cell type, including astrocytes, microglia, neurons and perivascular macrophages, which were identified based on location and morphology by a very experienced pathologist. Furthermore, the staining intensity of fungal hyphae was scored based on the following scale: negative [], weak [þ], moderate [þþ], strong [þþþ]. The correlation between two series (X, Y ) of n measurements was calculated using the Pearson product-moment correlation coefficient (¼sample correlation coefficient) according to the formula: rsy ¼
X ðxi xÞðyi yÞ ðn 1Þsx sy
;
where x and y are the sample means of X and Y, sx and sy are the sample standard deviations of X and Y and the sum is from i ¼ 1 to n.
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different brain cell types nor a significant extracellular staining of the surrounding tissue (Fig. 1AeD). In contrast, a high C1q expression and secretion was detected in the brain tissue of most patients with cerebral aspergillosis (Fig. 1EeM). A detailed analysis showed C1q-expressing reactive astrocytes (Fig. 1E, I), neurons (Fig. 1F, K) and oligodendrocytes (Fig. 1G, L). Positive cells were not limited to areas with direct presence of Aspergillus hyphae, but distributed throughout the tissue, suggesting that complement expression is induced by (a) soluble inflammatory factor(s) rather than by immediate contact between cells and hyphae. Although microglia are regarded as the main immune cell type in the brain, C1q-positive microglia were interestingly found in only one tissue sample. Instead of that infiltrating macrophages, not present in the control tissue, strongly expressed C1q (Fig. 1H, M). Stained tissue sections were assessed semi-quantitatively for the percentage of C1q-positive cells and the capacity of complement production was compared between different cell types. This analysis could help to identify the predominant complement-producing cell type in the brain and thus the putative target for stimulation of complement synthesis to improve antifungal innate immunity. Whereas no expression was seen in the astrocytes of uninfected individuals, this cell type was the most potent local C1q-producer in patients with cerebral aspergillosis (Fig. 2A, Table 1). Beside astrocytes, neurons and oligodendrocytes were the main local producers of C1q. While there was no immunohistochemical staining in uninfected brain tissue samples, the tissue sections of 7 out of 16 patients revealed staining for C1q (Fig. 2B, C). However, those brains with neurons expressing C1q were only partly the same like those with positive oligodendrocytes (Table 1). Statistical analysis revealed a correlation of only 45% between expression of C1q in neurons and that in oligodendrocytes. Similarly, the correlation between neuronal and astrocytic C1q synthesis was weak (Table 1), indicating that the induction of this complement factor underlies different mechanisms for the various cell types. Whereas microglia staining positive for C1q were found only in 1/16 tissue samples of patients with cerebral aspergillosis, infiltrating macrophages with strong synthesis of C1q could be detected in 16/16 brain samples (Fig. 2D, E). In 15/16 cases even more than 50% of macrophages produced C1q. Again, there was no correlation between expression of C1q in macrophages/microglia and the production of C1q in other cell types (Table 1).
3. Results 3.1. Expression of complement factor C1q in patients with cerebral aspergillosis To evaluate the cerebral expression of C1q, immunohistochemistry was performed in tissue sections of patients with cerebral aspergillosis and of non-infected individuals. Some examples of immunohistochemical staining of brain tissue samples are shown in Fig. 1. Generally, uninfected control brains revealed neither visible expression of C1q in the
3.2. Cerebral expression of complement factors C4, C3 and C5 in patients with cerebral aspergillosis Similar to C1q, the constitutively low production of complement factors C4 and C3 in the brain was highly increased in presence of the fungus. Astrocytes (Figs. 3A and 4A; Table 1) and infiltrating macrophages (Figs. 3E and 4E) were the main producers of C4 and C3. Neurons (Figs. 3B and 4B) and oligodendrocytes (Figs. 3C and 4C) contributed to cerebral C4 and C3 levels to a lesser extent, whereas microglia
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Fig. 1. Expression of complement factor C1q in brain tissue of uninfected individuals (AeD) and patients with cerebral aspergillosis (EeM). Immunohistochemistry was performed in paraffin-embedded sections with a specific C1q-antibody. Astrocytes (A, E, I), neurons (B, F, K), oligodendrocytes (C, G, L), and infiltrating macrophages (H, M) are shown; no infiltrating macrophages were present in the brain of uninfected individuals (D). Magnification: 400.
did not play a significant role as complement producers (Figs. 3D and 4D). Astrocytes and macrophages were also main producers of complement factor C5 with 86% and 75% of the tissue samples harboring C5-positive cells, respectively (Table 1). The role of microglia was more pronounced in the synthesis of C5 than in the synthesis of C1q, C4 and C3, and stained positive for C5 in 90% of the tissue samples. In contrast neurons only marginally contributed to the production of C5 (Table 1). In general there was no clear correlation between the expression levels of different complement factors and between the expression in different cell types, indicating distinctive mechanisms of regulation (Table 1). 3.3. Incomplete complement deposition on fungal hyphae in different locations Since an Aspergillus-produced complement inhibitor is described in literature, the deposition of complement on the fungal surface was also evaluated (Fig. 5, Table 1). Fungal hyphae were present perivascularly, within the brain parenchyma and/or in the meninges. Independent from the location,
an intense deposition was visible for C1q and C4 (Fig. 5A, B). However, in 7 out of 16 brain samples the fungal hyphae did not stain at all for C3, and staining was only weak in further 8 tissue samples (Fig. 5A, B), indicating an incomplete complement deposition and thus an inadequate complementdependent immune attack. Similarly fungal hyphae in 5 out of 15 brain tissue samples did not stain for complement factor C5 (Table 1). 3.4. Abscess: sealing off against fungal spreading but also against complement attack In one patient the focus of cerebral infection was organized as a mature abscess with a central necrotic area surrounded by a capsule of granulomatous fibrous tissue. Immunohistochemical staining revealed the effect of this encapsulation on the distribution of complement factors C1q, C4 and C3 (Fig. 6A, D, G). Whereas the fibrous collagenized tissue of the abscess was intensely stained for complement proteins, especially for C1q (Fig. 6A, B) and C4 (Fig. 6D, E), the intensity decreased towards the center of the abscess. The central necrotic area contained no or only minor amounts of the different complement proteins and
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0% <10% <50% >50%
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 + Asp
D numberof brains
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C 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
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17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
+ Asp
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E number of brains
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number of brains
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Fig. 2. Expression of complement factor C1q in astrocytes (A), neurons (B), oligodendrocytes (C), microglia (D) and infiltrating macrophages (E) of uninfected individuals (control) and patients with cerebral aspergillosis (þAsp). Paraffin-embedded sections were stained immunohistochemically to visualize C1q. The slides were analyzed microscopically to quantify the percentage of positive cells. Not all cell types were present in all slides.
Table 1 Percentage positive staining and correlations between positive staining of different cell types. Fungal infection
C1q staining
C4 staining
C3 staining
C5 staining
Hyphae
þ þ þ þ þ þ þ
0% 73% 0% 44% 0% 6% 0% 44% e 100% 42% 100% 93%
0% 71% 0% 36% 0% 0% 0% 57% e 85% 0% 86% 100%
29% 81% 0% 73% 14% 25% 14% 75% e 93% 42% 100% 56%
0% 86% 0% 7% 0% 90% 0% 60% e 75% 0% 80% 63%
Correlation coefficient Astrocytes e neurons Astrocytes e oligodendrocytes Astrocytes e microglia Astrocytes e macrophages Neurons e oligodendrocytes Neurons e microglia Neurons e macrophages Oligodendrocytes e microglia Oligodendrocytes e macrophages Microglia e macrophages
þ þ þ þ þ þ þ þ þ þ
0.12 0.49 0.16 0 0.02 0.23 0 0.23 0 0
0.08 0.09 0.28 0.54 0.45 0.02 0.16 0.33 0.13 0.16
0.52 0.09 0 0.28 0.04 0 0.34 0 0.10 0
0.11 0.12 0.41 0.52 0.33 0.13 0.52 0.27 0.19 0.25
Percentage positive Astrocytes Neurons Microglia Oligodendrocytes Macrophages Tissue staining
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D 17 16
control
+ Asp
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15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
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15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
number of brains
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C 17 16
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+ Asp
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Fig. 3. Expression of complement factor C4 in astrocytes (A), neurons (B), oligodendrocytes (C), microglia (D) and infiltrating macrophages (E) of uninfected individuals (control) and patients with cerebral aspergillosis (þAsp). Paraffin-embedded sections were stained immunohistochemically to visualize C4. The slides were analyzed microscopically to quantify the percentage of positive cells. Not all cell types were present in all slides.
no deposition on the fungal surface was visible (Fig. 6 C, F, I), implying that the abscess capsule may protect the fungus within the abscess from any efficient complement attack. 4. Discussion To increase the efficiency of antifungal complement attack in the CNS represents an interesting therapeutic concept, but requires a deeper understanding of the underlying mechanisms. Former in vitro results have shown that the limited constitutive complement levels in the CSF restrict the antifungal attack by this part of the immune system [10]. In this study we could show by immunohistochemical staining that the low production of several complement proteins is upregulated in the course of cerebral fungal infection. The increased expression of C3 is of special importance since this central factor participates in all three-complement activation pathways. The immunohistochemical analyses presented here confirm former results showing enhanced complement concentrations in the CSF from a patient with cerebral aspergillosis [10]. This increase of complement expression enables a better opsonization of the fungal hyphae as shown by former in vitro experiments. Higher concentrations of complement in the CSF are associated with higher phagocytic activity of microglia, granulocytes and monocytes/macrophages, a stimulated oxidative burst and a more efficient killing [10]. Further consequences of increased complement
concentrations might include the chemotactic recruitment of inflammatory cells by C3a, a fragment of C3, and the stimulation of microglia and astrocytes via induction of proinflammatory cytokines [9]. The experiments identified resident brain cells as main producers, and also infiltrated monocytes/macrophages participate in complement production. Thus, a leaky bloode brain-barrier may contribute to enhance local complement levels, but is not the predominant factor. Particularly astrocytes upregulate the synthesis of complement after an infectious stimulus, followed to a lesser extent by neurons and oligodendrocytes. It appears surprising that microglia, generally considered to be the preeminent immune cells of the CNS, are only minor producer of most complement factors by C5 being the only exception. There seems to be a separation between microglia as immune effector cells which respond to contact with complement factors by activation and phagocytosis [23], and other cell types in the CNS which synthesize the complement factors. This separation might prevent autoactivation and excess stimulation of microglia. Interestingly, the precise signaling pathway for complement induction seems to vary for the different cell types, since there is no correlation between their percentages of producing cells within one tissue sample. A brain section with low percentage of C1q-positive astrocytes might harbor a high percentage of neurons expressing C1q. This individuality of complement induction might reflect the cell
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Fig. 4. Expression of complement factor C3 in astrocytes (A), neurons (B), oligodendrocytes (C), microglia (D) and infiltrating macrophages (E) of uninfected individuals (control) and patients with cerebral aspergillosis (þAsp). Paraffin-embedded sections were stained immunohistochemically to visualize C3. The slides were analyzed microscopically to quantify the percentage of positive cells. Not all cell types were present in all slides.
type-specific distribution of the various signaling molecule receptors, since the inflammatory milieu composed of cytokines, chemokines and other extracellular effector molecules is supposed to be responsible for complement upregulation. This hypothesis is underlined by the fact that pro-inflammatory cytokines like IFN-g, TNF-a, IL-1b and IL-6 have a prominent effect on complement synthesis in glial and neuronal cells [9,24,25]. The indirect induction by proinflammatory cytokines is also implied by the presence of complement-producing cells in rather distance from fungal hyphae. A direct contact between brain cells and hyphae is not sufficient to stimulate complement production as demonstrated in vitro by experiments with incubation of brain cells with fungal hyphae (data not shown). Furthermore there is no correlation between the syntheses of different complement factors; brain tissue samples with high C1q expression are not the same like those with high C4 or C3 synthesis. This is surprising, since pro-inflammatory cytokines were described to induce a broad spectrum of acute phase proteins [26] complement proteins; e.g. IFN-g and TNF-a stimulate the production of all three factors C1q, C4 and C3 [27]. A possible explanation might be that the induction of a complement protein does not result from the action of a single cytokine but from a complex mixture of pro- and antiinflammatory cytokines which stimulate different promoter elements in different complement genes [27].
Despite the complement induction with all its putative positive effects on the antifungal defense cerebral aspergillosis is associated with a high lethality. Two explanations are possible: the stimulation of complement synthesis may be too late in the course of infection or not prominent enough to enable an efficient antifungal reaction; alternatively Aspergillus may be protected by the establishment of efficient complement evasion mechanisms. We could detect two relevant processes by which the fungus escapes from cerebral complement attack. First, our immunohistochemical studies showed significant deposition of the early complement factors C1q and C4 on the hyphal surface in the brain, but only minor opsonization by C3. Whereas C1q and C4 are proteins involved in early steps of the complement cascade, C3 and its fragments are part of more terminal steps of the complement cascade and mediate most antifungal mechanisms. The finding that C3-derived fragments are absent from the fungal hyphae is a hint for the presence of an Aspergillus-derived complement inhibitor which has been described by Washburn et al. [18,19]. In vitro, this soluble inhibitor CI rather selectively interfered with complement activation, decreased binding of the C3-fragment C3b to the fungal surface and inhibited C3b-dependent phagocytosis and killing [18,19]. This fungus-derived inhibitor was described to represent an important complement evasion strategy [12]. The production of CI during fungal growth under the physiological conditions of the brain had not been investigated, but seems plausible now according to our results.
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A
perivascular
parenchymal
meningeal
C1q
10µm
10µm
10µm
10µm
10µm
10µm
10µm
10µm
10µm
C4
C3
B
16
number of brains
14 12 10 8
negative
+ ++ +++
6 4 2 0
C1q
C4
C3
Fig. 5. Deposition of the complement factors C1q, C4 and C3 on hyphae of Aspergillus spp. in brain sections of patients with cerebral aspergillosis. (A) Immunohistochemical analysis of paraffin-embedded sections with specific antibodies reveals an intense deposition of C1q and C4 (brown color), but almost no C3 (blue color) on the fungal hyphae (magnification 1000). A semiquantitative analysis of hyphal staining intensity for C1q and C4 in comparison to C3 in the brain specimens is shown in (B).
A second complement escape mechanism is the formation of mature abscesses. Being a host defense strategy to prevent fungal spread, it also constitutes the manifestation of persisting microbial infection. Our experiments revealed that despite high complement levels in the surrounding fibrous layer the central necrotic area containing the fungal hyphae harbors only
marginal amounts of complement proteins. These amounts do not appear to be sufficient to enable a deposition on the hyphae as they did not stain in immunohistochemistry. Thus a useful immune mechanism meant to protect the host is at the same time a competent evasion mechanism for the fungus. However, most patients died before a mature abscess was formed and only early
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Fig. 6. Protection of Aspergillus spp. against complement attack by formation of a mature abscess, as shown by immunohistochemistry of paraffin-embedded sections. While the complement factors C1q (AeC), C4 (DeF), and C3 (GeI) are present in the surrounding fibrous layer (brown color), their amount in the central necrotic area of the abscess is low. Hyphae in the center reveal a weak or absent surface deposition with these complement proteins (C, F, I). Magnification: 100 (A, D, G), 400 (B, C, E, F, H, I).
stages of abscess formation were visible in these cases. This is a clear difference to murine or rat models of cerebral aspergillosis where all animals showed focal abscesses [28,29]. This discrepancy between humans and the animal model can be explained by the fact that the mice were infected intracerebrally by intracisternal injection. This situation is different in humans where cerebral aspergillosis mostly disseminates from a pulmonary infection. Thus the patients are already severely ill before the fungus reaches the CNS and therefore are more likely to die before maturation of an abscess. A further complement evasion mechanism, namely the production of secretory proteases by Aspergillus species, is currently studied by our group. Preliminary results show evidence for the secretion of complement-degrading proteases.
Increasing knowledge about fungal immune escape mechanisms may hopefully lead to new targets for novel supportive therapies against CNS-penetrating fungal pathogens. The development of those complement-based therapies should, however, avoid the pitfall of excessive complement activation, since presence of complement and complement activation products was associated with neurodegenerative diseases such as Alzheimer disease, Parkinson or multiple sclerosis [30]. Acknowledgements ¨ sterreichische NatioThis study was supported by the O nalbank (Project No. 11944). The authors thank T. Erbeznik for support in statistical analysis.
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