Aspergillosis Tlrs Govern Neutrophil Activity In

TLRs Govern Neutrophil Activity in Aspergillosis Silvia Bellocchio, Silvia Moretti, Katia Perruccio, Francesca Fallarino, Silvia Bozza, Claudia Montagnoli, Paolo Mosci, This information is current as Grayson B. Lipford, Lucia Pitzurra and Luigina Romani of September 28, 2021. J Immunol 2004; 173:7406-7415; ; doi: 10.4049/jimmunol.173.12.7406 http://www.jimmunol.org/content/173/12/7406 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TLRs Govern Neutrophil Activity in Aspergillosis1

Silvia Bellocchio,* Silvia Moretti,* Katia Perruccio,* Francesca Fallarino,* Silvia Bozza,* Claudia Montagnoli,* Paolo Mosci,* Grayson B. Lipford,† Lucia Pitzurra,* and Luigina Romani2*

Polymorphonuclear neutrophils (PMNs) are essential in initiation and execution of the acute inflammatory response and subsequent resolution of fungal infection. PMNs, however, may act as double-edged swords, as the excessive release of oxidants and proteases may be responsible for injury to organs and fungal sepsis. To identify regulatory mechanisms that may balance PMN- dependent protection and immunopathology in fungal infections, the involvement of different TLR-activation pathways was evaluated on human PMNs exposed to the fungus Aspergillus fumigatus. Recognition of Aspergillus and activation of PMNs occurred through the involvement of distinct members of the TLR family, each likely activating specialized antifungal effector functions. By affecting the balance between fungicidal oxidative and nonoxidative mechanisms, pro- and anti-inflammatory cy- Downloaded from tokine production, and apoptosis vs necrosis, the different TLRs ultimately impacted on the quality of microbicidal activity and inflammatory pathology. Signaling through TLR2 promoted the fungicidal activity of PMNs through oxidative pathways involving extracellular release of gelatinases and proinflammatory cytokines while TLR4 favored the oxidative pathways through the participation of azurophil, myeloperoxidase-positive, granules and IL-10. This translated in vivo in the occurrence of different patterns of fungal clearance and inflammatory pathology. Both pathways were variably affected by signaling through TLR3,

TLR5, TLR6, TLR7, TLR8, and TLR9. The ability of selected individual TLRs to restore antifungal functions in defective PMNs http://www.jimmunol.org/ suggests that the coordinated outputs of activation of multiple TLRs may contribute to PMN function in aspergillosis. The Journal of Immunology, 2004, 173: 7406–7415.

nvasive fungal infections have become the major cause of among the different granules of PMNs. Specific and secretory, my- infectious morbidity and mortality in patients with qualitative eloperoxidase (MPO)-negative granules are considered to play im- or quantitative defects of polymorphonuclear neutrophil portant roles in initiating the inflammatory response. Accordingly, I 3 (PMN) function, graft-vs-host disease, and receipts of steroids for the MPO-negative granules mainly mediate extracellular release of prevention and therapy (1). Consistent with their role, PMNs are their protein contents, such as gelatinases (7). In contrast, azuro- the predominant immune cells in the acute stage of most fungal phil MPO-positive granules are viewed as particularly active in the by guest on September 28, 2021 infections and play a crucial role in determining the type of pa- digestion of phagocytosed material (7). Although during phagocy- thology associated with fungal infections in different clinical set- tosis PMNs are known to release only small amounts of oxidants tings (2, 3). In this regard, PMNs may act as double-edged swords, in the surroundings (8), the findings that products of the oxidative as they are essential for pathogen eradication but an excessive stress may impede the phagocytic-dependent clearance of inflam- release of oxidants and proteases may be responsible for injury to matory products (9) and that host proteins rather than those of organs and fungal sepsis (4). This implicates that, for an efficient engulfed microbe are found to be the targets of oxidative reactions control of fungal pathogens, tight regulatory mechanisms are re- in PMNs (10, 11) suggest that reactive oxygen intermediates quired to balance protection and immunopathology. (ROIs) production may adversely affect the host ability to oppose Oxidant generation is not sufficient to mediate fungal killing by the inflammatory pathology. PMNs without complementary nonoxidative mechanisms medi- Consistent with their role in immune surveillance, human (12, 13) ated by granule constituents (5, 6). A clear distinction exists and murine (14) PMNs express TLRs, a family of conserved, mammalian cellular receptors that mediate cellular responses to structurally conserved pathogen-associated microbial products (15). All TLRs *Microbiology Section, Department of Experimental Medicine and Biochemical Sci- activate a core set of stereotyped responses, such as inflammation ences, University of Perugia, Perugia, Italy; and †Coley Pharmaceutical Group, Wellesley, MA 02481 (16), although the intricacies of how TLRs signal have provided the molecular basis for tailoring the immune responses to the provoking Received for publication June 3, 2004. Accepted for publication September 23, 2004. pathogen (16). Modulation of TLR signaling may be a two-edged The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance sword: TLRs are absolutely essential components of the innate im- with 18 U.S.C. Section 1734 solely to indicate this fact. mune response to pathogens and increased TLR expression and/or 1 This study was supported by the National Research Project on AIDS, Contract signaling may contribute to the pathophysiology of several important 50F.30 “Opportunistic Infections and Tuberculosis”, Italy. disease states (17). Understanding this dichotomy may ultimately lead 2 Address correspondence and reprint requests to Dr. Luigina Romani, Microbiology to innovative approaches to the treatment of diseases associated with Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. E-mail address: [email protected] altered innate immunity. As TLRs have been implicated in host defense against fungi (2, 3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; MPO, myeloperoxidase; ROI, reactive oxygen intermediate; BMT, bone marrow transplant; 18), the present study was undertaken to characterize the functional ZYM, zymosan; LTA, lipoteichoic acid; FLAG, flagellin; DCF, dichlorodihydrofluo- activity of TLRs in the PMNs response to Aspergillus fumigatus,a rescein diacetate; SOD, superoxide dismutase; MFI, mean fluorescence intensity; MMP, matrix metalloproteinase; AZI, azide; HEP, heparin; GLIO, gliotoxin; DEX, frequent cause of pneumonia and death in bone marrow-transplanted dexamethasone; HPRT, hypoxanthine phosphoribosyltransferase. (BMT) patients (3, 19). To this purpose, the involvement of different

TLR-activation pathways was evaluated in murine and human PMNs FACS analysis, Gr-1ϩ PMNs were Ͼ98% pure and stained positive for the exposed to the fungus in the presence of different TLR ligands. The CD11b myeloid marker. No differences in cell viability (between 95 and ability of each individual TLR to activate specialized antifungal 98% by the dye exclusion test) were observed on PMNs recovered from the different types of mice. effector functions on PMNs in response to conidia and hyphae of Aspergillus suggests a possible involvement of TLRs in governing the Phagocytosis, antifungal effector activity, and cultures function of PMNs in aspergillosis. For phagocytosis, PMNs were incubated at 37°C with unopsonized As- pergillus conidia for 60 min and the percentage of cells with internalized Materials and Methods conidia (% internalization) was calculated on Giemsa-stained preparations Animals (14). For fungicidal activity, PMNs were incubated with unopsonized fungal cells (at an effector to fungal cell ratio of 1:5) for 120 (conidia) or 60 Female, 8- to 10-wk-old C57BL/6 mice were from Charles River Labora- min (hyphae) at 37°C. For the conidiocidal activity, the percentage of CFU tories (Calco, Italy) or The Jackson Laboratory (Bar Harbor, ME). Breed- inhibition (mean Ϯ SE), referred to as conidiocidal activity, were deter- ing pairs of homozygous TLR2-, TLR4-, and TLR9-deficient mice, raised mined as described (20). To assess the damage to the hyphae, viability on a C57BL/6 background (14), were bred under specific pathogen-free staining with the fluorescence probe FUN-1 (Molecular Probes, Leiden, conditions at the breeding facilities of the University of Perugia (Perugia, The Netherlands) was examined in a Fluorescence Microplate Reader (Ti- Italy). Procedures involving animals and their care were conducted in con- tertek II; Flow Laboratories, Milan, Italy) at 485 nm excitation/620 nm formity with national and international laws and policies. emission (25). As controls, hyphae were incubated without cells and were treated with 96% ethanol. For staining of degranulated PMNs, the cytospin Microorganisms, culture conditions, and infections preparations were subjected to methylene blue (for azurophil granules) or The origin, characteristics, and culture conditions of the strains of A. fu- eosin (for nonazurophil granules) staining following standard procedures

migatus used in this study have already been described (14). Conidia were (14). The effects of TLR stimulation on PMN functions were assessed by Downloaded from preincubating PMNs for 120 min with the different TLR ligands (unopso- harvested by extensive washing of the slant culture with 5 ml of 0.025% ␮ ␮ Tween 20 in saline. For generation of hyphae, resting or swollen conidia nized ZYM (1, 10, and 100 g/ml), LTA (1, 10, 100 g/ml), poly(I:C) (10, Ͼ 50, 100 ␮g/ml), LPS (1, 10, 100 ␮g/ml), FLAG (0.1, 1, 10 ␮g/ml), R848 were allowed to germinate ( 98% germination) by incubation in Sab- ␮ ouraud broth (ϳ20 and 6 h, respectively) at 37°C. For Aspergillus infec- (100, 300, 900 nM), and CpG-ODN 2006 (0.01, 0.1, 1 M)) before the tion, conidia were given intranasally for 3 consecutive days (2 ϫ 107/20 ␮l addition of fungi, as detailed above. Photographs were taken using a high saline/each injection) to mice immunosuppressed with cyclophosphamide resolution Microscopy Color Camera AxioCam Color, using AxioVision (150 mg/kg i.p.) a day before. Mice were anesthetized by i.p. injection of Software Release 3.0 (Carl Zeiss, Milan, Italy). For cytokine determina- http://www.jimmunol.org/ 2.5% avertin (Sigma-Aldrich, St. Louis, MO). For the quantification of tion, PMNs were stimulated with the different TLR ligands in Iscove’s fungal growth in the lungs, the chitin assay was used (20). For histological medium with no serum but with polymixin B for 24 h. ␮ analysis, sections (3–4 m) of paraffin-embedded tissues were subjected to Detection of respiratory burst H&E staining (20). PMN production of ROIs was done by either measuring the oxidation of TLR ligands dichlorodihydrofluorescein diacetate (DCF; 5 ␮M), a cell-permeable com- Zymosan (ZYM, as a TLR2 stimulus) from Saccharomyces cerevisiae, pound which becomes fluorescent upon oxidation by hydrogen peroxide and other reactive oxygen species (26), by flow cytometry or quantifying lipoteichoic acid (LTA, as a stimulus for TLR2 and TLR6) from Staphy- . lococcus aureus, and polyinosine-polycytidylic acid (poly(I:C), as a TLR3 the release of superoxide anion (O2) in the culture supernatants through the stimulus) were purchased from Sigma-Aldrich. Ultra-pure LPS (as a TLR4 measure of the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (14). For flow cytometry, the results were expressed as median by guest on September 28, 2021 stimulus) from Salmonella minnesota Re 595 was from Labogen (Milan, . Italy). Flagellin (FLAG, as a TLR5 stimulus) was from Alexis (Vinci, fluorescence intensity (MFI). For O2,A550 was measured in a Microplate Italy). R848 (a stimulus for TLR7 and TLR8) and CpG oligonucleotides Reader Bio-Rad 550 (Bio-Rad, Rome, Italy) and the results were expressed . 6 2006 (CpG-ODN, as a TLR9 stimulus) of proven immunostimulatory se- as nanomoles O2/10 cells. quences were obtained as described (21, 22). Unopsonized ZYM was used to avoid complement receptor activity. To assess for the TLR specificity of Zymography selected TLR ligands, assays were done with the human embryonic kidney Gelatin zymography was performed as described (27). Gelatinase activity cell line HEK293 stably transfected with human TLR2 or TLR4 (23). Ul- was detected as white lysis bands against a blue background and quanti- Ϯ tra-pure LPS induced IL-8 production (161 12 pg/ml) in HEK293/TLR4 tative evaluation of the gelatinolytic activity was performed by scanning Ϯ but not in HEK293/TLR2; ZYM induced IL-8 production (222 19 pg/ the gel using a Bio-Rad Gel DOC 1000 imaging densitometer (Bio-Rad). ml) in HEK293/TLR2 but not in HEK293/TLR4 whereas LTA promoted Gelatinolytic activity of matrix metalloproteinase 9 (MMP9) was deter- Ϯ IL-8 production in HEK293/TLR2 only, although to a low extent (84 12 mined by scanning the lysis band in the 72-kD area. pg/ml). MPO assays Cell purification Western blotting was performed as described (28). Briefly, samples (106 Human PMNs were obtained from the heparinized whole blood of healthy cell equivalent) were probed with rabbit polyclonal anti-human MPO Ab donors or BMT patients, who gave their written informed consent, by lysis (Calbiochem, San Diego, CA) (1:2000) and visualized using ECL (Amer- with ammonium chloride and fractionation by Ficoll Hypaque (Pharmacia sham Pharmacia Biotech). MPO activity was determined by the oxidation Fine Chemicals, Uppsala, Sweden) centrifugation. The purity of PMNs Ј Ј Ͼ of 3,3 ,5,5 -tetramethylbenzinide by H2O2, and light absorption was mea- preparations was 97%, as determined by Giemsa morphology. BMT pa- sured at a 655 nm wavelength. Units of MPO activity (per 10 min) were tients received haploidentical, T cell-depleted peripheral blood cells, and calculated from a standard curve that used the peroxidase (Sigma-Aldrich) underwent conditioning with total-body irradiation followed by adminis- enzyme as the standard enzyme. MPO data were expressed as U/106 cells. tration of thiopepa, antithymocyte globulins, and fludarabine, as described (24). No postgrafting immune suppressive treatment was given for graft- Inhibition of PMN antifungal effector function vs-host disease prophylaxis. All postengraftment blood samples used in the present study showed 100% donor chimerism, assessed as described (24). The selective inhibitors of PMN function included azide (AZI; Sigma- Murine CD11bϩGr-1ϩPMNs were positively selected with magnetic beads Aldrich), a potent and specific inhibitor of MPO (29–31), heparin (HEP; (Miltenyi Biotec, Bologna, Italy) (14) from the peritoneal cavity of unin- Sigma-Aldrich), a compound that binds to ␣-defensins and inhibits their fected wild-type or TLR-deficient mice 8 h after the i.p. injection of 1 ml function (32) and gliotoxin (GLIO; Sigma-Aldrich), a fungal metabolite of endotoxin-free 10% thioglycolate solution. The PMNЈs recovery was that inhibits NADPH oxidase of human PMNs (33). PMNs were preincu- equivalent (between 15 and 18 ϫ 106 cells/mouse) between wild-type, bated for 15 min at 37°C with 0.1 mM AZI, a concentration known to TLR4-, and TLR9-deficient mice and slightly decreased (between 10 and inhibit MPO activity without altering other PMN functions (31) before 12 ϫ 106/mouse) in TLR2-deficient mice. RT-PCR analysis confirmed the being added to resting or germinated conidia. GLIO (1 ␮g/ml) or HEP (0.5 absence of the relevant TLR mRNA in TLR-deficient PMNs, either unex- U/ml) were added immediately before incubating PMNs with fungi. For posed or after the exposure to Aspergillus conidia and hyphae. Endotoxin degranulation, cells were treated for 18 h with 15 mM strontium chloride was depleted from all solutions with Detoxi-gel (Pierce, Rockford, IL) (Sigma-Aldrich), a compound devoid of cellular toxicity (34). The treat- and/or antagonized by 5 ␮g/ml polymixin B added to the cultures. On ment did not affect the cell viability or the ability to produce ROIs (data not 7408 TLR SIGNALING IN ASPERGILLOSIS shown). PMNs were exposed to 0.5 ␮M dexamethasone (DEX; Laborato- rio Farmacologico Milanese, Varese, Italy) and/or TLR ligands for 120 min before the addition of fungi. Assessment of neutrophil viability and apoptosis PMNs were exposed to different TLR ligands for 120 min before the addition of conidia and hyphae for a further 120 min. Apoptosis was quan- tified by staining with the human Annexin VFITC kit (Benden Med System Diagnostic, Vienna, Austria) and analyzed by flow cytometry. Four hours of stimulation was used based on preliminary experiments and according to previous studies (26). Cytokine assays The levels of cytokines in culture supernatants were determined by Kit ELISA (R&D Systems, Space Import-Export, Milan, Italy). The detection limits (picograms per milliliter) of the assays were Ͻ32 for TNF-␣ and Ͻ3 for IL-10. RT-PCR Total RNA was extracted (TRIzol; Invitrogen Life Technologies, Milano,

Italy) from peripheral PMNs after the exposure to unopsonized Aspergillus Downloaded from resting conidia or hyphae for 60 min, as suggested by initial experiments. Synthesis and PCR of cDNA were done as described (14). The forward and reverse PCR primers used for murine or human TLRs and hypoxanthine phosphoribosyltransferase (HPRT) were as described (14). The products were subjected to 32 cycles of PCR amplification at 94°C/30 s, 58°C/30 s, and 72°C/2 min. The synthesized PCR products were separated by elec- trophoresis on 2% agarose gel and visualized by ethidium bromide staining. http://www.jimmunol.org/ Analysis of p38 and NF-␬B activation FIGURE 1. Kinetics of TLR mRNA induction in PMNs upon exposure The activation of p38 and NF-␬B on murine PMNs exposed for 30 min at to A. fumigatus. A, Peripheral PMNs were exposed for 15, 60, and 120 min 37°C to Aspergillus conidia or hyphae was done as described (23). Blots of to unopsonized resting conidia and hyphae before the assessment of TLR cell lysates were incubated with rabbit polyclonal Abs recognizing either mRNA by RT-PCR. B, Peritoneal murine PMNs were exposed for 60 min. the unphosphorylated form of p38 MAPK, or the double-phosphorylated 180 182 DNA levels were normalized against the HPRT gene. None, Cells exposed (Thr /Tyr ) p38 MAPK (Cell Signaling Technology, Celbio, Milano, ␬ Italy), or Abs specific for the Rel A, 65 kDa DNA binding subunit of to the medium alone. C, Activation of NF- B and p38 on murine PMNs human NF-␬B (Zymed Laboratories, South San Francisco CA), followed exposed to Aspergillus conidia and hyphae. The assay was done by probing by HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology), as the cell lysates with specific anti-phospho-38 and anti-NF-␬B Abs. Shown by guest on September 28, 2021 per manufacturer’s instructions. Blots were developed with the Enhanced are the data from one representative experiment of three. Chemiluminescence detection kit (Amersham Pharmacia Biothech, Co- logno Monzese, Milano, Italy). Bands were visualized after exposure of the blots to Kodak RX film. To ensure similar protein loading in each lane, the phosphoblots were stripped and the membranes were reprobed with Abs of exposure, and that TLR mRNAs detected at 60 min were still against p38 and NF-␬B. present at 120 min (Fig. 1A). Although variability was observed Statistical analysis among different donors in the expression of TLR2, TLR4, TLR9, and TLR6 on unstimulated PMNs, the increased expression of The Student t test or ANOVA and Bonferroni’s test were used to determine TLR2, TLR4, and TLR9 upon the exposure to the fungus was a the statistical significance of differences in experimental groups was de- fined as p Ͻ 0.05. The data reported were pooled from five to eight ex- consistent finding. Flow cytometry with commercially available periments, unless otherwise specified. In vivo groups consisted of four to Abs to TLRs 2, 4, and 9 confirmed the increased expression of six animals. The in vivo data are from one representative experiment these TLRs in human PMNs (data not shown). As NF-␬B and p38 of five. MAPK activation are early events in triggering TLR-induced gene expression (15, 16) and also mediate the TLR-dependent responses Results to the fungus (14, 35), the nuclear translocation of NF-␬B as well TLR mRNA induction and activation in PMNs upon exposure to as p38 phosphorylation was also assessed in PMNs exposed to Aspergillus conidia or hyphae conidia or hyphae. NF-␬B activation and p38 phosphorylation To evaluate whether the exposure to A. fumigatus may affect the were observed in response to both fungal forms (Fig. 1C). There- levels of TLR mRNA and TLR-dependent signaling in PMNs, fore, TLR-dependent signaling is activated by the fungus in PMNs. freshly isolated PMNs were assessed for the presence of TLR mRNA by RT-PCR upon exposure to unopsonized conidia or hy- TLRs modulate PMN antifungal effector functions phae at different time points. No mRNAs for TLR1, TLR3, TLR5, To determine the effects of TLR stimulation on PMN functions, and TLR7 were observed on human and murine PMNs after the human cells were exposed to the different TLR ligands, at different exposure to conidia or hyphae at any time point (data not shown). concentrations, and assessed for phagocytosis, fungicidal activity, In contrast, after 60 min of exposure, the levels of TLR9 and TLR4 and quality of antifungal effector mechanisms, such as oxidant mRNA were increased upon the exposure to conidia or hyphae and production and degranulation. Although PMNs are considered to those of TLR2 mRNA upon exposure to conidia on both human exert maximal antifungal activity against extracellular hyphae (Fig. 1A) and murine (Fig. 1B) PMNs. The exposure to fungi did (36), they may also phagocytose and kill resting conidia (20, 37), not alter the levels of TLR6, TLR8, or TLR10 mRNAs (Fig. 1A). an activity that may be of benefit to the host in conditions of Time-course experiments reveal that the induction of some functional impairment of conidiocidal activity of alveolar macro- mRNAs (TLR8, TLR9, and TLR10) occurred earlier, after 15 min phages (38) or after swelling of conidia (39). We found that the The Journal of Immunology 7409

phagocytosis of conidia was impaired by LTA and poly(I:C) but unaffected by ZYM, LPS, FLAG, R848, and CpG-ODN (Fig. 2A). With respect to the fungicidal activity, only ZYM, LPS, or CpG- ODN either signiﬁcantly increased (ZYM) or left unaffected the conidiocidal and the hyphal damage activities, whereas LTA, poly(I:C), FLAG, and R848 all variably decreased both antifungal effector activities (Fig. 2A). Interestingly, a sustained and prolonged (still present after 20 h of pulsing) killing activity was observed upon exposure to LPS, partly to CpG-ODN but not to ZYM (data not shown). A dose dependency was observed for the above effects but, interestingly, opposite effects were also observed (CpG-ODN) upon increasing the concentrations (Fig. 2B). There- fore, these data suggest that the induction of TLR2, TLR4, and TLR9 expression upon exposure to fungi positively correlates with the induction of antifungal effector functions on PMNs. Interest- ingly, the pattern of TLR expression may not predict the respon- FIGURE 3. Effects of various inhibitors on PMN antifungal effector siveness to the relevant TLR ligands. functions. Peripheral blood PMNs were preincubated for 15 min at 37°C On assessing the relative contribution of oxidative and nonoxi- with 0.1 mM AZI before being added to resting or germinated conidia. dative mechanisms on the PMNs ability to kill Aspergillus conidia GLIO (1 ␮g/ml) or HEP (0.5 U/ml) were added immediately before incu- Downloaded from and hyphae, we found that the conidiocidal activity was markedly bating PMNs with fungi. For degranulation, cells were treated for 18 h with inhibited by GLIO, partially by AZI or by degranulation and un- 15 mM strontium chloride. Each treatment did not directly affect viability .p Ͻ 0.05, with or without (None) inhibitors ,ء .affected by HEP. In contrast, the ability to damage hyphae was of fungi greatly inhibited by AZI and degranulation, and unaffected by GLIO and HEP (Fig. 3). No direct adverse effects on fungal cells

were observed with these compounds (data not shown). Therefore, pathways while the hyphal damage is dependent on the MPO- http://www.jimmunol.org/ as in macrophages (29, 30), the conidiocidal activity of PMNs dependent system and granule constituents. mainly proceeds by parallel MPO-dependent and -independent To assess the effects of TLR stimulation on these pathways, we measured both the cell-associated (MFI) and the extracellular . (O2) ROI production and degranulation in response to conidia and hyphae in the presence of the different TLRs agonists. We found that ROI production was increased in response to conidia and hyphae and was modified by the different ligands. The intracellular ROI production against conidia was particularly increased by ZYM, inhibited by LTA and poly(I:C), and unaffected by the other by guest on September 28, 2021 TLR ligands. ZYM also increased the extracellular ROI release in response to hyphae along with poly(I:C) and LPS, while LTA decreased it (Fig. 4A). A similar pattern of production, although to a lesser degree, was observed upon stimulation with the TLR ligands alone (data not shown). With respect to degranulation, we found that, in line with previous studies (40), both conidia and hyphae induced PMN degranulation, although to a greater extent by hyphae for the azurophil degranulation. Degranulation of both types of granules were potentiated by poly(I:C), greatly decreased by LTA, and unaffected by FLAG, R848, and CpG-ODN. Interest- ingly, ZYM appears to mainly promote the nonazurophil degranulation while LPS mainly the azurophil degranulation (Fig. 4B). To further confirm the differential effects of TLR stimulation on the pattern of degranulation against the fungus, the levels of MMP9, a gelatinase found mainly in nonazurophil granules (7) and MPO, a marker of azurophil granules (7), were measured in culture supernatants of PMNs stimulated with conidia and hyphae and/or TLR ligands. According to the notion that peroxidase-negative granules mainly mediate extracellular release of their protein contents (7), we found that the active form of MMP9 was abundantly released in response to the fungus and to conidia more than hy- FIGURE 2. Antifungal activities of PMNs. A, Purified human PMNs phae. Quantification of zymograms by densitometric scanning re- from peripheral blood were pretreated with ZYM (10 ␮g/ml), LTA (10 vealed that MMP9-related gelatinolytic activity was significantly ␮ ␮ ␮ ␮ g/ml), poly(I:C) (50 g/ml), LPS (10 g/ml), FLAG (1 g/ml), R848 increased upon stimulation with ZYM or poly(I:C) only. In con- (300 nM), and unmethylated CpG oligonucleotide (CpG, 0.06 ␮M) or dif- trast, the release of MPO occurred more in response to hyphae than ferent concentrations of the above TLR ligands (B) for 120 min before the assessment of effector functions after a subsequent incubation with resting conidia and was increased by poly(I:C) and LPS. Both productions conidia (120 min) or hyphae (60 min). For phagocytosis and fungicidal were decreased by LTA, R848, and CpG-ODN (Fig. 4C). activity, see Materials and Methods. B, Values are the mean Ϯ SE of Together, these data indicated that the expression of the anti- -p Ͻ 0.05, with and fungal effector functions of PMNs may occur through the involve ,ء .samples taken from five independent experiments without (None) TLR ligands. ment of distinct members of the TLR family, each likely activating 7410 TLR SIGNALING IN ASPERGILLOSIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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Table I. Synopsis of the impact of the different TLR agonists on effector and immunomodulatory activities of PMNs in response to A. fumigatusa

Killing ROI Production Degranulation Cytokine

TLR Ligands Conidia Hyphae Intra Extra Nonazurophil Azurophil MMP9 MPO IL-10 Survival

ZYM 1 ϭ 11 1 ϭ 1 ϭϭ 2 LTA 22222 2 221 2 Poly(I:C) 22211 1 11ϭ 2 LPS ϭϭϭ1 ϭ 1211 ϭ R848 22ϭϭ ϭ ϭ 22 1 2 FLAG 2 ϭϭϭ ϭ ϭ ND ND ND ND CpG-ODN ϭϭϭϭϭ ϭ 22 ϭ 2

a 1, increased; ϭ, unchanged; 2, decreased. specialized antifungal effector functions (Table I). ZYM appears to proinflammatory and anti-inflammatory cytokine production is dif- promote the fungicidal activity of PMNs through oxidative path- ferently affected by the different stimuli. ways involving primarily nonazurophil granules while LPS appears to favor the oxidative pathways through the participation of TLRs govern clearance of the fungus and histopathology in vivo azurophil granules. However, ZYM, more than LPS, favor the ex- Because both TLR2 and TLR4 have been implicated in the re- Downloaded from tracellular degranulation. Both pathways were not significantly af- sponse of human PMNs to LPS (12) and ODN may also signal in fected by CpG-ODN, whereas, along with the phagocytosis, they a TLR9-independent manner on human PMNs (42), we resorted to were greatly inhibited by LTA. TLR2-, TLR4-, and TLR9-deficient mice to assess the fungicidal activity of PMNs together with parameters of inflammatory pa- TLRs modulate PMN apoptosis and necrosis thology in the lungs of infected mice. The conidiocidal activity

As PMNs become apoptotic once they have undergone phagocy- was greatly reduced in TLR4-deficient PMNs, partially reduced in http://www.jimmunol.org/ tosis and oxidase products are implicated in the process (41), we TLR2-deficient PMNs, and slightly increased in PMNs from measured PMN apoptosis and necrosis after exposure to conidia or TLR9-deficient mice, while the hyphal damage activity was im- hyphae in the presence of the different TLR ligands. Both apopto- paired in TLR4-deficient PMNs only and actually significantly in- sis and necrosis were observed upon exposure to the fungus and, creased in TLR9-deficient PMNs (Fig. 6A). Therefore, despite interestingly, apoptosis was significantly ( p Ͻ 0.05) increased by some redundancy in TLRs usage, TLR4 signaling is essential for exposure to hyphae and necrosis in response to conidia (from 17% the antifungal effector activity of PMNs against both conidia and of apoptotic cells in unstimulated PMNs to 25 and 15% in response hyphae of Aspergillus. to hyphae and conidia, respectively; from 4% of necrotic cells of The assessment of the fungal burden in the lungs of TLR-defi- by guest on September 28, 2021 unstimulated PMNs to 13 and 6% in response to conidia and hy- cient mice with aspergillosis revealed that the fungal burden was phae, respectively). Different effects were observed with the dif- higher in TLR2- or TLR4-deficient and lower in TLR9-deficient as ferent TLR ligands, being that the necrosis was markedly (Ͼ2- compared with wild-type mice (Fig. 6A). In contrast, histological fold) fostered by ZYM and poly(I:C), slightly (Ͻ2-fold) by R848 examination of the lungs revealed that the inflammatory pathology and CpG-ODN, and unaffected by LPS. The PMN death was un- was greatly reduced in TLR2-or TLR9-deficient mice but not in affected by LPS even after 20 h of incubation (data not shown). TLR4-deficient mice. The nonazurophil degranulation was virtu- Interestingly, apoptosis was greatly (Ͼ3-fold) increased by LTA, ally absent in TLR2-deficient PMNs, while the azurophil degran- despite the low ROI production observed, a finding that may dis- ulation was either decreased in TLR4-deficient PMNs or, interest- prove a general role for ROIs in the induction of apoptosis. These ingly, increased in TLR9-deficient PMNs (Fig. 6B). data suggest that an action on PMN survival and mechanism of Together, these data suggest that the control of the infection and death is a further mechanism through which TLRs may govern resolution of the inflammatory pathology are tightly regulated pro- immunopathology associated with the clearance of fungi. cesses upon which TLRs exert disparate activity. The inflammatory pathology associated with the clearance of the fungus ap- TLRs affect cytokine production by PMNs peared to be associated with the TLR2-dependent activation of the As PMNs release cytokines in response to fungi (2), we analyzed oxidant pathway and nonazurophil degranulation and largely in- the production of TNF-␣ and IL-10 following stimulation with the dependent of azurophil degranulation. different TLR ligands. The production of TNF-␣ was observed in response to each stimulus, although to a variable degree. However, TLR agonists restore the antifungal activity of defective PMNs the concomitant production of IL-10 was only observed in re- The elevated morbidity and mortality due to fungal sepsis in bone sponse to LTA and LPS (Fig. 5). Therefore, the balance between marrow transplantation or corticosteroid-treated patients (3)

FIGURE 4. Effects of TLR agonists on the antifungal functions of PMNs. Puriﬁed PMNs from peripheral blood were pretreated as in the Fig. 2 legend before the assessment of effector functions. A, Production of ROIs was done by either measuring the oxidation of DCF by ﬂow cytometry and expressing . 6 the results as MFI or by quantifying the release of superoxide anion (nanomoles O2/10 cells through the measure of the SOD-inhibitable reduction of p Ͻ 0.05, TLR ligands vs no TLR ligands ,ءء ;p Ͻ 0.05, fungus-stimulated vs unstimulated (Ϫ) cells ,ء .cytochrome c). Experiments were done in triplicate (None). B, PMN degranulation, as assessed by eosin (pink color) and methylene blue (blue color) staining. Photographs were taken using a high resolution Microscopy Color Camera AxioCam Color. C, Production of gelatinase and MPO by PMNs as assessed by gelatin zymography and Western blot analysis performed on culture supernatants of PMNs exposed to TLR agonists and fungi. The bands show the active 92-kDa MMP9 and the 60-kDa MPO (arrows). p Ͻ 0.05, TLR ligands ,ءء ;p Ͻ 0.05 fungus-stimulated vs unstimulated (Ϫ) cells ,ء .MPO activity was assessed as described in Materials and Methods vs no TLR ligands (None). 7412 TLR SIGNALING IN ASPERGILLOSIS

from either healthy donors upon treatment with DEX in vitro or from BMT patients at a different time posttransplantation. Consis- tent with the ﬁnding that ROI production by PMNs was unaffected by DEX (data not shown), the ability to damage hyphae more than the conidiocidal activity was impaired by the steroid. However, the defective activity could be rescued by signaling through TLR2, TLR4, and TLR9 (Fig. 7A). In BMT patients, the conidiocidal activity of PMNs was low in the early period posttransplant but was gradually recovering during the subsequent period (Fig. 7B), despite a similar degree of phagocytic ability (from 65% at 30 days to 80% at 180 days). Here again, the antifungal activity was fostered by signaling through TLR2, TLR4, and TLR9.

Discussion The present study shows that TLRs, by activating the antifungal state of PMNs through distinct mechanisms, play an important role FIGURE 5. TLR agonists affect cytokine production by PMNs. Cyto-

in governing the functions of PMNs in fungal infection and the Downloaded from kines were determined by speciﬁc ELISA in culture supernatants of human associated inﬂammatory pathology. Variable expression of TLRs ,p Ͻ 0.05 ,ء .PMNs exposed to the different TLR agonists for 24 h at 37°C TLR ligands vs no TLR ligands (none). has been reported on human PMNs (12, 13). Nevertheless, the ability of TLR agonists to activate PMN functions suggests that TLRs are important pattern recognition receptors for PMNs (13). prompted us to evaluate whether the impaired PMN function in- TLR2, TLR4, and TLR9 all have been implicated in host defense

duced by corticosteroid treatment or observed in bone marrow against A. fumigatus (2). We found here that these as well as other http://www.jimmunol.org/ transplantation could be rescued by selective TLR agonists. To this TLRs mediate the recognition and response to Aspergillus by purpose, the antifungal effector activity was assessed on PMNs PMNs in a morphotype-speciﬁc manner. The various fungal forms by guest on September 28, 2021

FIGURE 6. TLRs govern clearance of the fungus and histopathology in vivo. A, Purified peritoneal PMNs from uninfected wild-type or TLR2-, TLR4-, and TLR9-deficient mice were assessed for conidiocidal and hyphal damage activities as well as for pattern of degranulation (insets in B) as described in Materials and Meth- ods. The fungal growth (micrograms of glu- cosamine per lung) was assessed at 4 days p Ͻ 0.05, mutant vs wild-type ,ء .postinfection mice. B, Mice were treated with cyclophosphamide and injected with Aspergillus conidia intranasally. H&E-stained sections were prepared from lungs of infected mice at 4 days postinfection. Severe signs of bronchial wall damage and necrosis and abundant inflammatory cell recruitment are observed in the lungs of wild-type or TLR4-deficient mice, as opposed to that observed in TLR2- or TLR9- mice, whose lungs were characterized by scarce inflammatory cell recruitment with no evidence of bronchial wall damage and tissue destruction. Magnification ϫ200. The Journal of Immunology 7413

tors for ZYM and CD14 for LPS) cannot be excluded. In this regard, it is of interest that ROI formation is a common conse- quence of many pathologies and may represent a mechanism to enhance local inflammatory response (46). Similarly, dysregulation of MMP activity, despite the requirement of gelatinases for PMN migration (7), is implicated in tissue destruction under inflammatory conditions and MMP9 dysregulation is causally linked to lung inflammatory pathology (27). With active MPO, the emerging picture questions the conventional role of MPO as an essential component of the PMN antimicrobial machinery and calls for an immunomodulatory role resulting from the generation of auto-oxidants that inactivate certain granule contents (11) or protection of host enzymes from oxidative damage (6, 10). It is interesting therefore that MPO only is released upon TLR4 stimulation, a finding suggesting that signaling through TLR2 more that TLR4 is associated with inflammatory pathology. A number of findings point to LPS as a negative modulator of proinflammatory signals (47). The ability of LPS to signal through different intra-

cellular pathways (15), as well as the existence of intracellular Downloaded from pathways functioning as a counterregulatory pathway in endotoxin stimulation (16), may account for this effect. The ﬁndings with LTA and CpG-ODN are of interest. Stimula- tion with LTA abrogated the fungicidal activity of PMNs and this was associated with decreased ROI production and degranulation.

This finding, while confirming that oxidant production is required http://www.jimmunol.org/ FIGURE 7. TLR agonists restore antifungal activity of human PMNs for the concomitant activation of granule proteases (6), is in line treated with dexamethasone (DEX) or derived from BMT patients. A, PMNs were exposed to 0.5 ␮M DEX and/or TLR ligands (see legend to with the notion that modification of LTA renders bacteria suscep- Fig. 2 for details) for 120 min prior the addition of resting or germinated tible to PMN killing (48) and may explain the reduced capacity of -p Ͻ 0.05, TLR PMNs to kill fungi after the exposure to long-chain fatty acid ,ءء ;p Ͻ 0.05, with and without (None) DEX ,ء .conidia ligands vs no TLR ligands (DEX alone). B, PMNs, from at least two BMT containing lipid emulsions (49). As the LTA-dependent effects patients for each time point after transplant, were exposed to resting were present in TLR2-deficient mice (data not shown), it is likely conidia in the presence of the different TLR agonists. The results are the that signaling through TLR6 is responsible for the effects. With (p Ͻ 0.05, with CpG, given that TLR9-stimulation enhances innate effectors (50 ,ء .mean Ϯ SE of the different samples, each tested twice

Ͻ by guest on September 28, 2021 ءء and without (None) TLR ligands. , p 0.05, PMNs at 60 or 180 days and Th1 responses in murine aspergillosis (2), one would reason- vs PMNs at 30 days. ably expect a potentiation of antifungal effector activities. Actu- ally, CpG-ODN both stimulated and inhibited the antifungal effector activities of PMNs depending on the concentrations. A not only elicit a different response but also differ in their suscep- number of untoward effects, including tissue necrosis, were re- tibilities to cellular microbicidal mechanisms. Coordination of recently reported upon repeated CpG-ODN administration (51). It is sponses for fungicidal activity by PMNs is complex, requiring in- possible, therefore, that the slightly increased PMN necrosis ob- teractive effects of multiple PMN products (4). In line with the served with CpG-ODN is due to priming with doses of CpG-ODN finding that deficiency of both the oxidative and nonoxidative sys- that might foster enhanced, but perhaps less specifically, release of tems impairs the killing of the fungus (43, 44) and may predispose toxic PMN products, possibly having the potential to damage ef- to the infection (30), we found here that, similar to macrophages fector cells themselves. Therefore, a reduced inflammatory pathol- (43), the oxidative mechanisms mainly operate in the destruction ogy associated with the clearance of the fungus could explain the of conidia, while the oxidative burst in combination with nonoxi- resistance to aspergillosis of TLR9-deficient mice. Alternatively, dative mechanisms is responsible for hyphal damage by PMNs. It we are left with the possibility that ODN may activate PMNs in a is of interest that the fungal metabolite GLIO, a putative virulence TLR9-independent manner (42) or that TLR9 signaling may me- factor that specifically inhibits NF-␬B (45) and NADPH oxidase diate additional effects (52). (33), greatly impaired the conidiocidal activity of PMNs, leaving It is known that TLR signaling may affect PMN survival (26). unaffected the ability to damage hyphae. Theoretically, prolonging the life span of PMNs at inflammatory Similar to what was observed with murine PMNs (14), ZYM sites may enhance protection against pathogens, but, because ap- stimulated and LPS prolonged the fungicidal activity of PMNs. optosis is an important mechanism for PMN removal (therefore However, different mechanisms of antifungal activity appeared to containing the inflammatory response), decreased apoptosis may be at work. ZYM promoted the fungicidal activity of PMNs imply an exaggerated inflammatory response. Defective apoptosis through the elicitation of the oxidative pathway and the involve- has recently been suggested as a mechanism contributing to the ment of granules producing MMP9 while LPS favored the oxida- pathophysiology of chronic granulomatous disease (53). More- tive pathway through the involvement of azurophil granules re- over, the mechanisms by which PMNs and their histotoxic con- leasing mainly MPO. As the quantity and specificity of delivery of tents are cleared from inflamed sites greatly impacts on the reso- toxic PMN products ultimately will determine the relative effi- lution of the inflammatory response. While extravasated PMNs ciency of fungicidal activity vs inflammatory cytotoxicity to host disintegrate (undergo necrosis), during apoptosis PMNs retain cells (5), it follows that qualitative different responses originated their granule contents and lose the ability to secrete them in re- upon TLR2 or TLR4 signaling on PMNs, although the contribution sponse to secretagogues. Also, in contrast to necrotic PMNs, ap- of additional receptors (such as the ␤-glucan and mannose recep- optotic PMNs are eliminated through a phagocytic recognition 7414 TLR SIGNALING IN ASPERGILLOSIS mechanism that fails to provoke a proinflammatory response (54). 12. Kurt-Jones, E. A., L. Mandell, C. Whitney, A. Padgett, K. Gosselin, It is therefore very interesting that the anti-inflammatory state in- P. E. Newburger, and R. W. Finberg. 2002. 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