PTX3 Binds MD-2 and Promotes TRIF-Dependent Immune Protection in Aspergillosis

This information is current as Silvia Bozza, Silvia Campo, Brunilde Arseni, Antonio of September 25, 2021. Inforzato, Lindstedt Ragnar, Barbara Bottazzi, Alberto Mantovani, Silvia Moretti, Vasileios Oikonomous, Rita De Santis, Agostinho Carvalho, Giovanni Salvatori and Luigina Romani

J Immunol 2014; 193:2340-2348; Prepublished online 21 Downloaded from July 2014; doi: 10.4049/jimmunol.1400814 http://www.jimmunol.org/content/193/5/2340 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2014/07/18/jimmunol.140081 Material 4.DCSupplemental References This article cites 41 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/193/5/2340.full#ref-list-1

Why The JI? Submit online. by guest on September 25, 2021

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

PTX3 Binds MD-2 and Promotes TRIF-Dependent Immune Protection in Aspergillosis

Silvia Bozza,* Silvia Campo,† Brunilde Arseni,† Antonio Inforzato,† Lindstedt Ragnar,† Barbara Bottazzi,‡ Alberto Mantovani,‡ Silvia Moretti,* Vasileios Oikonomous,* Rita De Santis,† Agostinho Carvalho,* Giovanni Salvatori,† and Luigina Romani*

The long pentraxin 3 (PTX3) modulates different effector pathways involved in innate resistance to , including complement activation or promotion of phagocytosis by interacting with FcgRs. However, whether and how TLRs modulate PTX3 mediates antifungal resistance is not known. In this study, we demonstrate that PTX3 binds myeloid differentiation 2 (MD-2) in vitro and exerts its protective antifungal activity in vivo through TLR4/MD-2–mediated signaling. Similar to Tlr42/2 mice, Md22/2 mice displayed high susceptibility to pulmonary aspergillosis, a phenotype associated with a proinflammatory cytokine profile and impaired antifungal activity of polymorphonuclear neutrophils. Treating Md22/2 mice with PTX3 failed to Downloaded from confer immune protection against the fungus, whereas adoptive transfer of MD-2–competent polymorphonuclear neutrophils restored it. Mechanistically, engagement of MD-2 by PTX3-opsonized Aspergillus conidia activated the TLR4/Toll/IL-1R domain-containing adapter inducing IFN-b–dependent signaling pathway converging on IL-10. Thus, we have identified a novel receptor mechanism, involving the TLR4/MD-2/Toll/IL-1R domain-containing adapter inducing IFN-b–mediated signaling, whereby PTX3 elicits antifun- gal resistance with limited immunopathology in A. fumigatus infection. The Journal of Immunology, 2014, 193: 2340–2348. http://www.jimmunol.org/

nvasive aspergillosis is an infection caused by fungi of the pulmonary aspergillosis in immunocompetent individuals (8). genus Aspergillus that typically affects severely immuno- TLR4 signaling in response to its prototype ligand LPS relies on I compromised hematologic patients undergoing intensive che- the action of two accessory molecules, myeloid differentiation motherapy and bone marrow or solid organ transplantation (1–3). protein 2 (MD-2) (9) and CD14 (10), which are essential for the Despite the availability of new azole and echinochandin drugs that stabilization of TLR4 expression on the cell surface following have significantly improved the management of this severe infection, activation by LPS (11, 12). As a matter of fact, TLR4 is unable to the prevention, diagnosis, and therapy of aspergillosis remain ex- induce a complete response to LPS in the absence of MD-2 (9), tremely difficult, rendering it a leading cause of death among im- a glycoprotein that belongs to the MD-2–related lipid recognition by guest on September 25, 2021 munocompromised patients, with a 1-y mortality reaching 75% (1). family (13), that is a critical compound in host response to Gram- The immune response to Aspergillus fumigatus is critically de- negative bacteria and plays a central role in physiologic adaptation pendent on the recognition of fungal motifs, mostly cell wall com- to various insults (14). ponents, by pattern recognition receptors (4). TLR4 has been Of interest, naturally occurring alternatively spliced isoforms of demonstrated as one major pattern recognition receptor required for human MD-2 acted as a negative regulator of LPS-mediated TLR4 resistance to A. fumigatus infection (5). Consistently, a missense activation (15), a finding indicating that targeting MD-2 could mutation affecting the extracellular domain of human TLR4 was inhibit TLR4 inflammatory signaling, as recently suggested (16). found to be associated with increased susceptibility to Aspergillus Due to the conserved nature of the TLR4/MD-2/CD14 complex, pneumonia (6) and colonization (7) in recipients of allogeneic he- several reports have suggested that it is biologically relevant and matopoietic stem cell transplants, as well as to chronic cavitary responsive to several microorganisms, including bacteria (17, 18) and viruses (19). However, little is known about whether and how the TLR4/MD-2/CD14 complex mediates immune responses to *Department of Experimental Medicine, University of Perugia, Perugia 06132, Italy; †Department of Immunology, Sigma-Tau S.p.A., Pomezia 00040, Italy; and A. fumigatus. ‡Istituto Clinico Humanitas, Rozzano, Milan 20089, Italy Fungal sensing is assisted by the action of several molecules, Received for publication March 31, 2014. Accepted for publication June 19, 2014. including collectins, ficolins, pentraxins, and complement compo- This work was supported by Italian Projects PRIN 2009HL28E8_002 (to S.B.), nents that act as opsonins and facilitate the interaction of phagocytes Research Grant RS_R_280100 from Sigma-Tau (to L.R.), and European Commu- with fungi (20). Among these, the long pentraxin 3 (PTX3) has been nity Grant ERC-2011-AdG-293714 (to L.R.). found to have a nonredundant protective role in the immune re- Address correspondence and reprint requests to Prof. Luigina Romani, Department of Experimental Medicine, University of Perugia, Piazzale Gambuli, Polo Unico sponse to A. fumigatus (21). PTX3 modulates different effector Sant’Andrea delle Fratte, Perugia 06132, Italy. E-mail address: luigina. pathways involved in innate resistance to A. fumigatus, including [email protected] complement activation, promotion of phagocytosis, and regulation The online version of this article contains supplemental material. of inflammation (22–26). The molecular mechanisms underlying Abbreviations used in this article: BAL, bronchoalveolar lavage; DC, ; the opsonic activity of PTX3 and increased phagocytosis of conidia HSCT, hematopoietic stem cell transplantation; IKK, IkB kinase; i.n., intranasal; IRF3, IFN regulatory factor 3; MD-2, myeloid differentiation protein 2; PAS, peri- by neutrophils involved FcgRII-, CD11b-, and complement- odic acid-Schiff; PMN, polymorphonuclear neutrophil; PTX3, pentraxin 3; sMD-2, dependent mechanisms (26). However, the PTX3 protective ac- soluble MD-2; TLN, thoracic lymph node; Treg, regulatory T; TRIF, Toll/IL-1R tivity against CMV was impaired in TLR4-deficient mice (27), domain-containing adapter inducing IFN-b; WT, wild-type. a finding suggesting some level of cooperation between TLR4 and Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 PTX3 in host antimicrobial immunity. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400814 The Journal of Immunology 2341

In the current study, we sought to investigate the contribution of Collection of bronchoalveolar lavage fluid the TLR4/MD-2/CD14 complex to the PTX3-dependent activity Lungs were filled thoroughly with 1-ml aliquots of pyrogen-free saline through against A. fumigatus. Our findings demonstrate that PTX3 activates a 22-gauge bead-tipped feeding needle introduced into the trachea. Bron- the TLR4/Toll/IL-1R domain-containing adapter inducing IFN-b choalveolar lavage (BAL) fluid was collected in a plastic tube on ice and (TRIF)–dependent signaling pathway converging on IDO and IL-10 centrifuged at 400 3 g at 4˚C for 5 min. For differential BAL fluid cell counts, € upon binding MD-2. cytospin preparations were made and stained with May-Grunwald Giemsa reagents (Sigma-Aldrich). At least 200 cells/field/10 fields were counted, and the absolute number of each cell type was calculated (25). Photographs were taken using a using a BX51 microscope (Olympus, Milan, Italy), and images Materials and Methods were captured using a high-resolution DP71 camera (Olympus). Binding assays Treatment HEK293TCM, HEK293CM, and HEK293T cells expressing human TLR4A/ MD-2/CD14 or TLR4A and CD14/MD-2 (Invivogen), respectively, were Human PTX3 was obtained under endotoxin-free conditions by immu- used. The control line was HEK293Null. Binding of PTX3 to HEK293 noaffinity of culture supernatants of HEK293 cells expressing the protein cells was evaluated by incubating histidine- (R&D Systems) or biotin- and was administered i.p. at the dose of 1 mg/kg for 4 d starting the day of the Aspergillus i.n. injection. Controls received the diluent alone. A total of tagged PTX3 (prepared according to a Pierce protocol) followed by PE- 6 conjugated anti–polyhistidine Ab (R&D Systems) or FITC-streptavidin 10 purified peritoneal polymorphonuclear neutrophils (PMNs) was ad- (BD Pharmingen) and FACS analysis. To immobilize PTX3-coupled to ministered i.v. to recipient mice, 3 h postinfection. Soluble MD-2 (R&D MD-2, 20 mg biotinylated PTX3 was added to 0.5 mg streptavidin-coated Systems) was administered i.p. at 250 mg/kg for 4 d starting the day of the magnetic beads (binding capacity: 20 mg biotinylated IgG/mg beads) infection. (Dynabeads M-280; Life Technologies). After washing to remove unbound Hematopoietic stem cell transplantation Downloaded from PTX3, 1.5 mg MD-2 (R&D Systems) was then added to PTX3-coated beads. According to the product data sheet, the protein was purified Bone marrow cells from C57BL/6, BALB/c or Md22/2 donor mice were form Escherichia coli and therefore is not glycosylated. It has already been resuspended in HBSS (Life Technologies) containing 10% FBS (Life reported (28) that MD-2 spontaneously form disulfide bridges giving rise Technologies) at a concentration of 100 3 106 cells/ml. T cell depletion to dimer or higher-order oligomers. Magnetic beads were then collected, was obtained upon incubation of cells with MicroBeads (Miltenyi Biotec, washed in PBS and 0.8% Nonidet P-40, suspended in 50 mg sample buffer Bergisch Gladbach, Germany) conjugated to rat anti-mouse CD5 mAb under reducing condition, boiled at 100˚C, and run in SDS-PAGE (15 mg/ (Ly-1; clone 53-7.3) for 15 min at 4˚C followed by magnetic sorting in line) reducing gel and stained by Coomassie. The binding of full-length a magnetic cell separator (CliniMACS cell separation system; Miltenyi http://www.jimmunol.org/ (R&D Systems), N terminus, and C terminus PTX3 to immobilized MD-2 Biotec). Unbound cells were carefully aspirated, washed, and resuspended and vice versa were evaluated by adding specific recombinant at a concentration of 100 3 106 cells/ml. C57BL/6, BALB/c, and Md22/2 followed by detection Abs (R&D Systems), streptavidin-conjugated HRP recipient mice were exposed to a lethal dose of 9 Gy, infused i.v. with (Amersham), and TMB substrate. In the competition assay, MNB4 or T cell–depleted donor cells ($10 3 106), and infected with Aspergillus MNB1 were used (Alexis). The reaction was stopped by HCl 2N and the conidia a week later (29). absorbance was quantified at 450 and 540 nm in a microplate reader. To characterize the specific binding, biotinylated PTX3 (0.2–110 nM con- Cells sidering a molecular mass of 45 kDa for the PTX3 monomer) was added to Dendritic cells (DC), , and epithelial cells were purified from wells coated with 26 pmol MD-2, and the amount of bound PTX3 was + + converted to nanomolar concentration using a standard curve of bio- lungs as described (25, 29). Murine CD11b Gr-1 PMN were positively selected with magnetic beads (Miltenyi Biotec) from the peritoneal cavity by guest on September 25, 2021 tinylated PTX3. Kd was calculated by nonlinear fitting of the saturation curves. Binding of MD-2 and PTX3 to heat-inactivated conidia of of uninfected WT 8 h after the i.p. injection of 1 ml endotoxin-free 10% A. fumigatus was assessed using 96-well MultiScreen-HTS-BV filtration thioglycollate solution (5). Endotoxin was depleted from all solutions with Detoxi-gel (Pierce, Rockford, IL). On FACS analysis, Gr-1+ PMNs were plates (Millipore, Billerica, MA) that allow removal of liquids by vacuum. . Briefly, conidia were added to wells previously blocked with 1% (w/v) 98% pure and stained positive for the CD11b myeloid marker. nonfat dry milk in HBS-T (20 mM HEPES, 150 mM NaCl, 2 mM CaCl2,2 Phagocytosis and antifungal effector activity mM MgCl2, and 0.1% Tween 20 [pH 7]) and incubated with either MD-2 or PTX3. Following extensive washing with HBS-T, bound proteins were For conidia internalization and killing, cells were pre-exposed to either PTX3 revealed by either a sheep anti-MD-2 polyclonal Ab (R&D Systems) and ligand for 1 h before stimulation with live Aspergillus conidia or to PTX3- an HRP-conjugated donkey anti-sheep polyclonal Ab or a rabbit anti- opsonized conidia at 37˚C. The percentage of cells with internalized conidia PTX3 polyclonal Ab and an HRP-conjugated donkey anti-rabbit second- was calculated on Giemsa-stained preparation (5). After staining, aliquots of ary Ab. In some experiments, conidia were preincubated with saturating cells were spun down on slides on a cytocentrifuge and examined for conidia concentrations of MD-2 prior to incubation with PTX3. HRP activity was internalization by light microscopy. For each experiment, at least five fields measured by the addition of TMB. Substrate solutions were collected in in each slide were counted, and at least 200 cells were analyzed in each well. a 96-well Maxisorp microtiter plate (Nunc, Roskilde, Denmark), HCl 2N Cytospin preparations were observed using a BX51 microscope (Olympus), was added, and the absorbance was quantified at 450 nm in a microplate reader, as described above. Values from conidia-free wells were subtracted to account for unspecific binding of proteins to wells. Table I. Real-time murine PCR primers used in this study

Mice Primer Sequence Female 8–10-wk-old C57BL/6 mice (wild-type [WT]) were purchased Ifng Forward,59-ACTGGCAAAAGGATGGTGAC-39 from Charles River Laboratories (Calco, Italy). Breeding pairs of homo- 2/2 2/2 Reverse,59-TGAGCTCATTGAATGCTTGG-39 zygous Tlr4 and Trif mice on the C57BL/6 background were bred Il4 Forward,59-CGGCATTTTGAACGAGGTCACAGG-39 under specific-pathogen free conditions at the breeding facilities of the 2/2 Reverse,59-CCCTTTGCTATGGTGTCCTT-39 University of Perugia (Perugia, Italy). Md2 mice were provided by Il17a Forward,59-GGACTCAGATGACTTGCCACTC-39 Shin-Ichiroh Saitoh (Division of Infectious Genetics, Institute of Medical Reverse,59-CTGCCGCTGCTCCTCCTC-39 Science, University of Tokyo, Tokyo, Japan). Experiments were performed Il10 Forward,59-CCCTTTGCTATGGTGTCCTT-39 according to the Italian Approved Animal Welfare Assurance 245-2011-B. Reverse,59-TGGTTTCTCTTCCCAAGACC-39 Tbet Forward,59-GGACGATCATCTGGGTCACATTGT-39 Microorganism, culture conditions, and infection Reverse,59-GCCAGGGAACCGCTTATATG-39 Gata3 Forward,59-CTACCGGGTTCGGATGTAAGTCG-39 Resting conidia from A. fumigatus 293 were obtained as described (25). Reverse,5 -GTTCACCACACTCCCTGCCTTCT-3 For infection, mice were anesthetized in a small plastic cage by inhalation 9 9 Rorc Forward,59-ACAACAGCAGCAAGTGATGG-39 of 3% isoflurane in oxygen, before intranasal (i.n.) instillation of a sus- Reverse,5 -CCTGGATTTATCCCTGCTGA-3 pension of 6 3 107 conidia/20 ml saline. Mice were monitored for survival 9 9 Foxp3 Forward,59-CCCAGGAAAGACAGCAACCTTTT-39 (%), fungal growth (log10 CFU/organ, mean), histopathology (periodic acid- Reverse,5 -TTCTCACAACCAGGCCACTTG-3 Schiff [PAS] staining of lung tissue sections), and cytokine expression. 9 9 2342 PTX3/MD-2 INTERACTION IN ASPERGILLOSIS and images were captured using a high-resolution DP71 camera (Olympus). etry image analysis using Image Lab 3.1.1 software (Bio-Rad). The pixel The percentage of CFU inhibition (mean 6 SE), referred to as conidiocidal density of bands was normalized against total proteins. activity, was determined as described (25). Western blotting Quantification of cytokines and transcription factors by real-time RT-PCR Protein phosphorylation was assessed on murine PMN lysates incubated with rabbit polyclonal Abs, recognizing the unphosphorylated form of p38, CD4+ T cells were separated by magnetic cell sorting with MicroBeads and ERK, and IFN regulatory factor 3 (IRF3) or p-p38 (Thr180/Tyr182), p-p44/ MidiMacs (Miltenyi Biotec) from thoracic lymph nodes (TLNs). Real-time p42 ERK (Thr202/Tyr294), and p-IRF3 (Ser396) followed by HRP–anti- RT-PCR was performed using the Stratagene Mx3000P QPCR System and rabbit IgG, as per the manufacturer’s instruction (Cell Signaling Tech- SYBR Green chemistry (Stratagene, Cedar Creek, TX). Lung cells and CD4+ nology). Anti–p-IkB kinase (IKK)b (Ser181) rabbit Abs (Cell Signaling T cells were lysed, and total RNA was reverse transcribed with cDNA Syn- Technology) was used for Western blotting of p-IKKb. For IDO, cells were thesis Kit (Bio-Rad), according to the manufacturer’s instructions. The PCR incubated with rabbit polyclonal Ab (Millipore), followed by HRP–anti- primers were as listed in Table I. Amplification efficiencies were validated rabbit IgG, as per the manufacturer’s instruction (Cell Signaling Tech- and normalized against Gapdh. The thermal profile for SYBR Green real- nology). Chemiluminescence detection was performed with LiteAblot Plus time PCR was as follows: 95˚C for 10 min, followed by 40 cycles of dena- chemiluminescence substrate (Euro Clone) using the ChemiDoc XRS + turation for 30 s at 95˚C and an annealing/extension step of 30 s at 72˚C. Each imaging system (Bio-Rad), and quantification was obtained by densitom- data point was examined for integrity by analysis of the amplification plot. Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. PTX3 binds to HEK293 cells expressing TLR4 and MD-2. (A) HEK293 cells expressing TLR4/MD-2/CD14, TLR4A, CD14/MD-2, or the empty vector (Null) were incubated with a histidine-tagged PTX3 (25 mg/2 3 105 cells). FACS analysis was performed using an anti-histidine Ab. (B) FACS analysis of HEK293 cells expressing TLR4 alone were incubated with PBS (thick line) or rMD-2 (10 mg/106 cells) (thin line) and sub- sequently with recombinant biotinylated PTX3 (2 mg). PTX3 was detected by FITC–streptavidin. Cells incubated with FITC-streptavidin alone (dotted line). (C) SDS-PAGE analysis of streptavidin beads incubated with biotinylated PTX3, MD-2, and PTX3 with MD-2. The relative positions of the proteins on the gel are indicated by the arrows: MD-2 (MD-2 recombinant protein), biotinylated PTX3 (PTX3), MD-2 plus streptavidin beads (MD-2+beads), PTX3, MD-2, and streptavidin beads (PTX3+MD-2+beads), and PTX3 biotinylated plus streptavidin-beads (PTX3+beads). (D) Immobilized MD-2 was incubated with a 0.4–58 nM range of biotinylated PTX3. Data were analyzed with nonlinear fitting methods. Inset: Scatchard plot of binding data. (E) PTX3-coated plates were incubated with a 2–130 nM range of MD-2 followed by anti–MD-2 Ab, anti-mouse biotinylated Ab, and HRP-STR. (F) MD-2–coated plates (5 mg/ml) were incubated with full-length, N terminus, or C terminus PTX3 followed by polyclonal bio- tinylated anti-PTX3 and HRP–STR. (G) MD-2–coated plates were incubated with biotinylated PTX3 preincubated with MNB4 or MNB1 at the indicated Abs: protein molar ratio followed by HRP–STR. (H) Heat-inactivated conidia of A. fumigatus (5 M/well) were incubated with MD-2 at the indicated concentrations. Bound MD-2 was revealed with an anti–MD-2 polyclonal Ab followed by an HRP-conjugated secondary Ab (n =106 SE). (I) Heat-inactivated conidia were preincubated with MD-2 (at 1 mg/ml),andthenPTX3wasappliedatthereportedtiters.BoundPTX3wasassessed with an anti-PTX3 polyclonal Ab and an HRP-conjugated secondary Ab (n =106 SE). The Journal of Immunology 2343

Cytokine assays Results The cytokine levels in the culture supernatants was determined by ELISA PTX3 binds efficiently to MD-2 , Kit (BioLegend). The detection limits of the assays were 1.9 pg/ml for We first analyzed whether PTX3 could physically interact with the IFN-b and ,2.7 pg/ml for IL-10. TLR4/MD-2/CD14 complex. PTX3 was found to bind HEK293 transfectant cells expressing the TLR4/MD-2/CD14 complex and Statistical analysis only marginally to HEK293 transfectant cells transfected with We calculated statistical significance by one- or two-way ANOVA or TLR4 alone (Fig. 1A). No binding of PTX3 to CD14/MD-2 or null paired t test with Prism software (GraphPad, San Diego, CA). Survival was transfectants was observed. The interaction of PTX3 with the calculated using Kaplan–Meier calculations. A p value of ,0.05 was con- sidered statistically significant. The data shown in each figure represent the TLR4/MD-2/CD14 complex was dependent on MD-2. Indeed, mean of three or more independent experiments. The in vivo groups con- FACS analysis of HEK293 cells expressing TLR4 and pre- sisted of six to eight mice per group. incubated with MD-2 showed enhancement of the mean fluores- Downloaded from http://www.jimmunol.org/

FIGURE 2. MD-2 is essentially required for protection against A. fumigatus. Mice were infected i.n. with 6 3 107 resting conidia of A. fumigatus, by guest on September 25, 2021 and survival and fungal growth (A) (expressed as log10 CFU/lungs), BAL morphometry (percentage of PMNs) (B), and lung histology (PAS staining) were evaluated. Note the sustained inflammatory cell recruitment in the lungs and BAL of Md22/2 mice as well as the presence of peribronchiolar lymphocyte infiltrates. (C) Relative mRNA ex- pression of cytokines in the lung and Th-associated transcription factors in CD4+ T cells purified from the TLNs assessed at 5 d postinfection. Data are pooled from three independent experiments (six to eight animals per group) at 5 d postinfection. ***p , 0.001. Naive, uninfected mice. 2344 PTX3/MD-2 INTERACTION IN ASPERGILLOSIS cence intensity for PTX3 as compared with the mean fluorescence a condition in which no binding was observed against an irrelevant intensity of the same cells unexposed to MD-2 (Fig. 1B). Ac- proteins (data not shown). Accordingly, MD-2 binds the immo- cordingly, MD-2 interacts with a biotinylated PTX3 previously bilized PTX3 in a concentration-dependent fashion and a saturat- anchored to streptavidin-conjugated beads (Fig. 1C). Of note, ing dose of ∼30 nM (Fig. 1E). Interestingly, only the N terminus biotinylated PTX3 is less efficiently eluted from the beads than (amino acids 18–172), but not the glycosylated C terminus of MD-2 bound through PTX3, likely because of the strength of PTX3 (aa 173–381), bound to MD-2 (Fig. 1F). As a matter of fact, biotin–streptavidin interaction. Binding of biotinylated PTX3 to the N terminus Ab MNB4 inhibited PTX3 binding to MD-2 at MD-2 was dose dependent, with a Kd of 3.4 nM (Fig. 1D), in either 1:1 (30% inhibition) or 10:1 (45% inhibition) molar ratios, Downloaded from http://www.jimmunol.org/ FIGURE 3. Neutrophils reconstitution in Md22/2 mice confers resistance to A. fumigatus infection. HSCT mice were infected i.n. with Aspergillus live conidia and a week later the infusion of 10 3 106 viable T-depleted bone marrow cells. Fungal growth (A) and lung histology (B) (PAS-stained sections) were assessed 3 d after the infection. Data are pooled from two independent experiments (six to eight animals per group). Scale bars, 200 mm. (C) Phagocytosis and conidiocidal activity of WT by guest on September 25, 2021 and Md22/2 PMNs. For conidia internalization and killing assays, cells were pre-exposed to either PTX3 ligand (PTX3 L) for 1 h before stimulation with live Aspergillus conidia or to PTX3-opsonized conidia (PTX3 O). After a Diff Quik staining, aliquots of cells were spun down on slides and examined for conidia internalization by light microscopy. Data rep- resent mean 6 SD for at least three independent experiments. None, unstimulated cells. (D) Fungal growth in mice receiving 106 PMNs administered i.v., 3 h postinfection. (E) Lung histology (PAS-stained sections) of infected mice receiving an adoptive transfer of PMNs. Data are pooled from three inde- pendent experiments (six to eight animals per group). Scale bars, 200 mm. *p , 0.05, **p , 0.01, ***p , 0.001. None, mice not receiving PMNs. The Journal of Immunology 2345 whereas the C terminus Ab MNB1 only slightly reduced binding (Supplemental Fig. 1). Thus, MD-2 on PMNs is crucially required at a molar ratio of 10:1 (18% reduction) (Fig. 1G). This finding for PTX3-mediated protection against A. fumigatus infection and indicates that PTX3 binds to MD-2 through its N-terminal do- inflammatory pathology. main. Of great interest, consistent with the opsonic activity of Given that MD-2 can be found on the cell surface in association MD-2 (30), not only was MD-2 able to bind Aspergillus conidia with the ectodomain of TLR4 or as a secreted protein capable of (Fig. 1H), but it also strongly enhanced the binding of conidia to binding with high affinity to TLR4 (28) and that both PTX3 and PTX3 (Fig. 1I), a finding pointing to an important role of MD-2 in conidia can bind soluble MD-2 (sMD-2) (A. Inforzato, personal conidia recognition by PTX3. communication), we investigated the possible contribution of sMD-2 to PTX3-mediated protection in infection by treating WT MD-2 is required for antifungal resistance with limited and Md22/2 mice with sMD-2. The treatment did not affect the immunopathology in pulmonary aspergillosis local fungal growth in either type of mice (Fig. 4A), a finding We next evaluated the role of MD-2 in the response to A. fumigatus suggesting that sMD-2 may not sequester conidia. However, sMD-2 2/2 infection by infecting and treating with PTX3 Md2 mice. treatment greatly increased the local inflammatory pathology par- 2/2 Md2 mice were highly susceptible to pulmonary aspergillosis ticularly in WT mice (Fig. 4B), even in condition of concurrent compared with WT animals, as demonstrated by poorer survival administration of PTX3 (data not shown), a finding suggesting that and higher fungal load in the lungs (Fig. 2A), a marked inflam- sMD-2 may sequester PTX3, thereby preventing its immunoregu- matory response characterized by PMNs in the BAL fluid and lung latory activity during infection. infiltrates (Fig. 2B) and imbalanced Th1/Th17/regulatory T (Treg) PTX3 activates the anti-inflammatory TLR4/TRIF signaling cell responses (Fig. 2C). Treatment with PTX3 decreased the Downloaded from fungal burden (Fig. 2A), ameliorated the inflammatory pathology pathway via MD-2 (Fig. 2B), and restored protective Th1/Treg cell responses Regulation of inflammation is an essential component of host (Fig. 2C) in WT but not in Md22/2 mice. protective response in Aspergillus pneumonia (31) and is achieved To characterize the cell type required for MD-2 activity, we through a p38MAPK/TRIF-dependent pathway leading to IDO resorted to bone marrow chimeric mice in which either donor or and IL-10 (29, 32). PTX3 is a fine tuner of inflammation (25, 33,

recipient cells were obtained from MD-2–deficient mice. We 34). We assessed whether PTX3 would activate the p38MAPK/ http://www.jimmunol.org/ infected chimeric mice with A. fumigatus and evaluated the fungal TRIF pathway in response to the fungus in PMN in vitro and its growth and inflammatory pathology in the lung. We found that dependency on MD-2. We found that the exposure to Aspergillus mice reconstituted with Md22/2 myeloid cells were more sus- conidia greatly increased phosphorylation of p38, IRF3, and IDO ceptible to the infection, as indicated by the high fungal burden in WT but not Md22/2 PMNs, a finding indicating that MD-2 is (Fig. 3A) and the presence of numerous abscesses and inflam- required to activate the TRIF pathway in response to the fungus matory cell recruitment in the lung (Fig. 3B). Susceptibility to the (Fig. 5A). PTX3, albeit slightly, increased phosphorylation of p38 infection was instead apparently decreased in recipient Md22/2 and IRF3 in WT but not Md22/2 PMNs (Fig. 5A), which indicates mice (Fig. 3B), a finding suggesting that MD-2 may have different that the TRIF pathway mediates the activity of PTX3. Along the effects depending on cell types. Consistent with the prominent same reasoning, although PTX3 was unable to further stimulate by guest on September 25, 2021 antifungal role of MD-2 on myeloid cells, we found that phago- IDO protein expression in WT PMNs (Fig. 5A), the finding that cytosis and killing of conidia was severely impaired in Md22/2 IDO was poorly expressed and not induced upon stimulation in the PMNs compared with WT counterparts and was insensitive to absence of MD-2 (Fig. 5A) strongly argues for a critical role of PTX3 (Fig. 3C). Accordingly, WT but not Md22/2 PMNs de- MD-2 in IDO activation in response to the fungus. PTX3 slightly creased fungal colonization (Fig. 3D) and reduced neutrophilic decreased ERK phosphorylation in response to conidia, an effect influx and lung inflammation upon adoptive transfer into Md22/2 observed in WT but not Md22/2 PMNs (Fig. 5A) and had no mice (Fig. 3E). No antifungal effector impairment was observed effects on activation of NF-kB transcription factors (IKKb) in macrophages, DC, and epithelial cells from Md22/2 mice (Fig. 5A). Consistent with these results, the production of IFN-b

FIGURE 4. sMD-2 sequesters PTX3 and prevents its immunoregulatory activity during infection. Fungal growth (A) and lung histology (B) (PAS-stained sections) in mice treated with sMD-2 administered i.p. at 250 mg/kg for 4 d starting the day of the infection. Data are pooled from three independent experiments (six to eight animals per group). Scale bars, 200 mm. 2346 PTX3/MD-2 INTERACTION IN ASPERGILLOSIS Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. PTX3 activates the anti-inflammatory TLR4/TRIF signaling pathway via MD-2. (A) Levels of p38, ERK, IRF3, IKKb, and IDO in PMNs exposed to Aspergillus conidia, either unopsonized or opsonized with PTX3. Shown are immunoblots of cell lysates with phosphorylation-specific Abs and fold increases (pixel density) in the phosphorylated to total protein ratios. Data are representative of three independent experiments. (B) Cytokine pro- duction in culture supernatants of PMNs treated as above. None, unstimulated cells. Tlr42/2 or Trif2/2 mice were infected i.n. with 6 3 107resting conidia of A. fumigatus and evaluated for fungal growth (expressed as log10 CFU/lungs) (C) and lung histology (D) (PAS-stained sections). Scale bars, 200 mm. (E) Relative mRNA expression of Il10 in the lungs was assessed at 5 d postinfection. Data are pooled from three independent experiments (six to eight animals per group). **p , 0.01, ***p , 0.001. Naive, uninfected mice. and IL-10 was observed in WT PMNs but not in Md22/2 PMNs that PTX3 activates the TLR4/TRIF-dependent signaling pathway (Fig. 5B). converging on IDO and IL-10 upon binding MD-2. To corroborate these in vitro findings, we assessed the activity of By screening signaling pathways in DC in response to the PTX3 in vivo in Trif2/2 or Tlr42/2 mice infected with A. fumi- fungus, we have demonstrated a TRIF-dependent pathway leading gatus. PTX3 was unable to restrict the fungal growth (Fig. 5C), to IL-10 and IDO that was associated with the induction of pro- inflammatory pathology (Fig. 5D), and IL-10 expression (Fig. 5E) tective tolerance in infection in vivo (32). Resistance and tolerance in either type of mice. As PTX3 fully restored immune competence mechanisms of immunity are essential components of the host– in Myd882/2 mice (data not shown), this finding points to a requi- fungus interaction in the vertebrate host (4). An imbalance between site role for MD-2 in the activation of the TLR4/TRIF-mediated pro- and anti-inflammatory signals may prevent successful host/ signaling pathway responsible for the anti-inflammatory activity of fungal interaction, thus leading to infection and disease. Indeed, PTX3 in aspergillosis. despite the occurrence of severe aspergillosis in immunocompro- mised patients, clinical evidence indicates that aspergillosis also Discussion occurs in the setting of a heightened inflammatory response, such as The findings of this study define a previously undescribed mo- in nonneutropenic patients after allogeneic hematopoietic stem cell lecular mechanism of PTX3 interaction with the host innate im- transplantation (HSCT) (35), in chronic granulomatous disease (36), mune system that could be of relevance in aspergillosis. We found and in cystic fibrosis (37). Therefore, paradoxically, increased in- The Journal of Immunology 2347

flammatory innate response may predispose to either fungal infec- Disclosures tions or dysregulated immune responses to the fungus. The authors have no financial conflicts of interest. The finding that PTX3, in addition to its opsonic capacity (21) and complement activation ability (26), activates the tolerance pathway to the fungus expands upon the role PTX3 may have in antifungal References 1. Kontoyiannis, D. P., K. A. Marr, B. J. Park, B. D. Alexander, E. J. Anaissie, immunity to include a fine regulation of inflammation, as already T. J. Walsh, J. Ito, D. R. Andes, J. W. Baddley, J. M. Brown, et al. 2010. Pro- suggested (25, 33, 34). As a matter of fact, a recent multicenter spective surveillance for invasive fungal infections in hematopoietic stem cell study involving patients undergoing HSCT and patients with inva- transplant recipients, 2001-2006: overview of the Transplant-Associated Infec- tion Surveillance Network (TRANSNET) Database. Clin. Infect. Dis. 50: 1091– sive aspergillosis further confirmed the nonredundant role of PTX3 1100. in aspergillosis by showing that genetic deficiency of PTX3 could 2. Pagano, L., M. Caira, A. Nosari, M. T. Van Lint, A. Candoni, M. Offidani, affect the antifungal capacity of neutrophils and contribute to the T. Aloisi, G. Irrera, A. Bonini, M. Picardi, et al. 2007. Fungal infections in recipients of hematopoietic stem cell transplants: results of the SEIFEM B-2004 risk of invasive aspergillosis in high-risk patients (38). study—Sorveglianza Epidemiologica Infezioni Fungine Nelle Emopatie On the local level, PTX3 was found to bind MD-2, an interaction Maligne. Clin. Infect. Dis. 45: 1161–1170. 3. Pappas, P. G., B. D. Alexander, D. R. Andes, S. Hadley, C. A. Kauffman, that strongly enhanced the binding of PTX3 to conidia, thus favoring A. Freifeld, E. J. Anaissie, L. M. Brumble, L. Herwaldt, J. Ito, et al. 2010. In- fungal internalization. However, consistent with the ability of sMD-2 vasive fungal infections among organ transplant recipients: results of the to bind PTX3 also (A. Inforzato, personal communication), sMD-2 Transplant-Associated Infection Surveillance Network (TRANSNET). Clin. In- fect. Dis. 50: 1101–1111. was found to sequester PTX3, thereby preventing its immunoreg- 4. Romani, L. 2011. Immunity to fungal infections. Nat. Rev. Immunol. 11: 275– ulatory activity during infection. These findings may offer a plausible 288. explanation for the complex biological activities of PTX3 in vivo, 5. Bellocchio, S., C. Montagnoli, S. Bozza, R. Gaziano, G. Rossi, S. S. Mambula, Downloaded from A. Vecchi, A. Mantovani, S. M. Levitz, and L. Romani. 2004. The contribution which may also include MD-2–independent effects. Indeed, PTX3 of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to not only failed to increase, but also actually apparently decreased fungal pathogens in vivo. J. Immunol. 172: 3059–3069. 2/2 6. Bochud, P. Y., J. W. Chien, K. A. Marr, W. M. Leisenring, A. Upton, M. Janer, phosphorylation of p38 and IRF3 in Md2 PMNs. S. D. Rodrigues, S. Li, J. A. Hansen, L. P. Zhao, et al. 2008. Toll-like receptor 4 Irrespective of the plethora of possible pathways activated by polymorphisms and aspergillosis in stem-cell transplantation. N. Engl. J. Med. PTX3, the finding that PTX3 binds MD-2 is novel. PTX3 is known 359: 1766–1777. 7. Carvalho, A., C. Cunha, A. Carotti, T. Aloisi, O. Guarrera, M. Di Ianni, to interact with members of the complement and lectin pathway F. Falzetti, F. Bistoni, F. Aversa, L. Pitzurra, et al. 2009. Polymorphisms in Toll- http://www.jimmunol.org/ (39). However, due to its capacity to bind pathogen-derived like receptor and susceptibility to infections in allogeneic stem cell ligands, it was also found to activate a TLR2-dependent proin- transplantation. Exp. Hematol. 37: 1022–1029. 8. Carvalho, A., A. C. Pasqualotto, L. Pitzurra, L. Romani, D. W. Denning, and flammatory response (40). We have already shown in CMV in- F. Rodrigues. 2008. Polymorphisms in toll-like receptor genes and susceptibility fection (27) and confirmed in this study that PTX3’s protective to pulmonary aspergillosis. J. Infect. Dis. 197: 618–621. 9. Shimazu, R., S. Akashi, H. Ogata, Y. Nagai, K. Fukudome, K. Miyake, and activity is impaired in TLR4-deficient mice. The observation that M. Kimoto. 1999. MD-2, a molecule that confers lipopolysaccharide respon- PTX3 binds MD-2 may not only explain this finding but also siveness on Toll-like receptor 4. J. Exp. Med. 189: 1777–1782. points to a previously undefined role of MD-2 in antifungal im- 10. Wright, S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, and J. C. Mathison. 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS munity. Indeed, not only were MD-2–deficient mice insensitive to binding protein. Science 249: 1431–1433. the protective antifungal effect of PTX3, but they were also highly 11. Akashi, S., S. Saitoh, Y. Wakabayashi, T. Kikuchi, N. Takamura, Y. Nagai, by guest on September 25, 2021 susceptible to the infection, showing a defective ability to control Y. Kusumoto, K. Fukase, S. Kusumoto, Y. Adachi, et al. 2003. Lipopolysac- charide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity both the local fungal growth and inflammatory pathology and than that with MD-2 or CD14. J. Exp. Med. 198: 1035–1042. exhibiting imbalanced Th1/Th17/Treg cell responses. Considering 12. Gray, P., J. Dagvadorj, K. S. Michelsen, C. Brikos, A. Rentsendorj, T. Town, T. R. Crother, and M. Arditi. 2011. Myeloid differentiation factor-2 interacts that MD-2–deficient mice were also highly susceptible to vaginal with Lyn kinase and is tyrosine phosphorylated following lipopolysaccharide- candidiasis (data not shown), altogether these results point to induced activation of the TLR4 signaling pathway. J. Immunol. 187: 4331–4337. a previously unappreciated role for MD-2 in antifungal immunity. 13. Inohara, N., and G. Nun˜ez. 2002. ML — a conserved domain involved in innate immunity and lipid metabolism. Trends Biochem. Sci. 27: 219–221. Phosphorylation of MD-2 on specific tyrosines is required for 14. Tissie`res, P., and J. Pugin. 2009. The role of MD-2 in the opsonophagocytosis of mounting a response to LPS and considered to be a regulatory step Gram-negative bacteria. Curr. Opin. Infect. Dis. 22: 286–291. employed to curtail an overexuberant host immune response (12). 15. Gray, P., K. S. Michelsen, C. M. Sirois, E. Lowe, K. Shimada, T. R. Crother, S. Chen, C. Brikos, Y. Bulut, E. Latz, et al. 2010. Identification of a novel human Naturally occurring alternatively spliced isoforms of human MD-2 MD-2 splice variant that negatively regulates Lipopolysaccharide-induced TLR4 acted as a negative regulator of LPS-mediated TLR4 activation signaling. J. Immunol. 184: 6359–6366. 16. Wang, X., P. M. Grace, M. N. Pham, K. Cheng, K. A. Strand, C. Smith, J. Li, (15), a finding indicating that targeting MD-2 could inhibit TLR4 L. R. Watkins, and H. Yin. 2013. Rifampin inhibits Toll-like receptor 4 signaling inflammatory signaling, as recently suggested (16). However, by targeting myeloid differentiation protein 2 and attenuates neuropathic pain. unlike LPS, metal-induced TLR4 dimerization did not require FASEB J. 27: 2713–2722. 17. Cai, S., R. L. Zemans, S. K. Young, G. S. Worthen, and S. Jeyaseelan. 2009. MD-2 (41), a finding suggesting that MD-2 could be indispensable Myeloid differentiation protein-2-dependent and -independent neutrophil accu- for TLR4-induced . This may explain why MD-2 mulation during Escherichia coli pneumonia. Am. J. Respir. Cell Mol. Biol. 40: activity was apparently different among different types of innate 701–709. 18. Jain, A. V., Y. Zhang, W. B. Fields, D. A. McNamara, M. Y. Choe, G. H. Chen, immune cells, although the mechanism underlying this phenom- J. Erb-Downward, J. J. Osterholzer, G. B. Toews, G. B. Huffnagle, and enon is presently unknown. M. A. Olszewski. 2009. Th2 but not Th1 immune bias results in altered lung functions in a murine model of pulmonary Cryptococcus neoformans infection. In conclusion, this study shows that MD-2 is a critical compound Infect. Immun. 77: 5389–5399. in host response to Aspergillus and that its interaction with PTX3 19. Kurt-Jones, E. A., L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, plays a central role in physiologic regulation of inflammation E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg. 2000. Pattern recognition receptors TLR4 and CD14 mediate during infection. This finding may also explain how PTX3, response to respiratory syncytial virus. Nat. Immunol. 1: 398–401. a multitasking molecule, is capable of performing different bio- 20. Bottazzi, B., A. Doni, C. Garlanda, and A. Mantovani. 2010. An integrated view of logical activities in vivo and likely predict its effectiveness in vivo humoral innate immunity: pentraxins as a paradigm. Annu. Rev. Immunol. 28: 157–183. 21. Garlanda, C., E. Hirsch, S. Bozza, A. Salustri, M. De Acetis, R. Nota, in the different clinical settings. A. Maccagno, F. Riva, B. Bottazzi, G. Peri, et al. 2002. Non-redundant role of the long pentraxin PTX3 in anti-fungal innate immune response. Nature 420: 182–186. 22. Nauta, A. J., B. Bottazzi, A. Mantovani, G. Salvatori, U. Kishore, W. J. Schwaeble, A. R. Gingras, S. Tzima, F. Vivanco, J. Egido, et al. 2003. Acknowledgments Biochemical and functional characterization of the interaction between pentraxin We thank Cristina Massi-Benedetti for editorial assistance. 3 and C1q. Eur. J. Immunol. 33: 465–473. 2348 PTX3/MD-2 INTERACTION IN ASPERGILLOSIS

23. Deban, L., H. Jarva, M. J. Lehtinen, B. Bottazzi, A. Bastone, A. Doni, delivered siRNA targeting PI3K/Akt/mTOR inflammatory pathways protects T. S. Jokiranta, A. Mantovani, and S. Meri. 2008. Binding of the long pentraxin from aspergillosis. Mucosal Immunol. 3: 193–205. PTX3 to factor H: interacting domains and function in the regulation of com- 33. Inforzato, A., P. C. Reading, E. Barbati, B. Bottazzi, C. Garlanda, and plement activation. J. Immunol. 181: 8433–8440. A. Mantovani. 2012. The “sweet” side of a long pentraxin: how 24. Ma, Y. J., A. Doni, T. Hummelshøj, C. Honore´,A.Bastone,A.Mantovani, affects PTX3 functions in innate immunity and inflammation. Front. Immunol. 3: N. M. Thielens, and P. Garred. 2009. Synergy between ficolin-2 and pentraxin 3 407. boosts innate immune recognition and complement deposition. J. Biol. Chem. 284: 34. Deban, L., R. C. Russo, M. Sironi, F. Moalli, M. Scanziani, V. Zambelli, 28263–28275. I. Cuccovillo, A. Bastone, M. Gobbi, S. Valentino, et al. 2010. Regulation of 25. D’Angelo, C., A. De Luca, T. Zelante, P. Bonifazi, S. Moretti, G. Giovannini, leukocyte recruitment by the long pentraxin PTX3. Nat. Immunol. 11: 328–334. R. G. Iannitti, S. Zagarella, S. Bozza, S. Campo, et al. 2009. Exogenous pen- 35. Ortega, M., M. Rovira, X. Filella, M. Almela, J. Puig de la Bellacasa, traxin 3 restores antifungal resistance and restrains inflammation in murine E. Carreras, and J. Mensa. 2004. Prospective evaluation of procalcitonin in adults chronic granulomatous disease. J. Immunol. 183: 4609–4618. with febrile neutropenia after haematopoietic stem cell transplantation. Br. J. 26. Moalli, F., A. Doni, L. Deban, T. Zelante, S. Zagarella, B. Bottazzi, L. Romani, Haematol. 126: 372–376. A. Mantovani, and C. Garlanda. 2010. Role of complement and Fcgamma 36. Romani, L., F. Fallarino, A. De Luca, C. Montagnoli, C. D’Angelo, T. Zelante, receptors in the protective activity of the long pentraxin PTX3 against Asper- C. Vacca, F. Bistoni, M. C. Fioretti, U. Grohmann, et al. 2008. Defective tryp- gillus fumigatus. Blood 116: 5170–5180. tophan catabolism underlies inflammation in mouse chronic granulomatous 27. Bozza, S., F. Bistoni, R. Gaziano, L. Pitzurra, T. Zelante, P. Bonifazi, disease. Nature 451: 211–215. K. Perruccio, S. Bellocchio, M. Neri, A. M. Iorio, et al. 2006. Pentraxin 3 37. Iannitti, R. G., A. Carvalho, C. Cunha, A. De Luca, G. Giovannini, protects from MCMV infection and reactivation through TLR sensing pathways A. Casagrande, T. Zelante, C. Vacca, F. Fallarino, P. Puccetti, et al. 2013. Th17/ leading to IRF3 activation. Blood 108: 3387–3396. Treg imbalance in murine cystic fibrosis is linked to indoleamine 2,3-dioxyge- 28. Visintin, A., A. Mazzoni, J. A. Spitzer, and D. M. Segal. 2001. Secreted MD-2 is nase deficiency but corrected by kynurenines. Am. J. Respir. Crit. Care Med. a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to 187: 609–620. Toll-like receptor 4. Proc. Natl. Acad. Sci. USA 98: 12156–12161. 38. Cunha, C., F. Aversa, J. F. Lacerda, A. Busca, O. Kurzai, M. Grube, J. Lo¨ffler, 29. de Luca, A., S. Bozza, T. Zelante, S. Zagarella, C. D’Angelo, K. Perruccio, J. A. Maertens, A. S. Bell, A. Inforzato, et al. 2014. Genetic PTX3 deficiency C. Vacca, A. Carvalho, C. Cunha, F. Aversa, and L. Romani. 2010. Non- and aspergillosis in stem-cell transplantation. N. Engl. J. Med. 370: 421–432. hematopoietic cells contribute to protective tolerance to Aspergillus fumigatus 39. Inforzato, A., A. Doni, I. Barajon, R. Leone, C. Garlanda, B. Bottazzi, and Downloaded from via a TRIF pathway converging on IDO. Cell. Mol. Immunol. 7: 459–470. A. Mantovani. 2013. PTX3 as a paradigm for the interaction of pentraxins with 30. Tissie`res, P., I. Dunn-Siegrist, M. Scha¨ppi, G. Elson, R. Comte, V. Nobre, and the complement system. Semin. Immunol. 25: 79–85. J. Pugin. 2008. Soluble MD-2 is an acute-phase protein and an opsonin for 40. Jeannin, P., B. Bottazzi, M. Sironi, A. Doni, M. Rusnati, M. Presta, V. Maina, Gram-negative bacteria. Blood 111: 2122–2131. G. Magistrelli, J. F. Haeuw, G. Hoeffel, et al. 2005. Complexity and comple- 31. Carvalho, A., C. Cunha, R. G. Iannitti, A. De Luca, G. Giovannini, F. Bistoni, mentarity of outer membrane protein A recognition by cellular and humoral and L. Romani. 2012. Inflammation in aspergillosis: the good, the bad, and the innate immunity receptors. Immunity 22: 551–560. therapeutic. Ann. N. Y. Acad. Sci. 1273: 52–59. 41. Raghavan, B., S. F. Martin, P. R. Esser, M. Goebeler, and M. Schmidt. 2012.

32. Bonifazi, P., C. D’Angelo, S. Zagarella, T. Zelante, S. Bozza, A. De Luca, Metal allergens nickel and cobalt facilitate TLR4 homodimerization indepen- http://www.jimmunol.org/ G. Giovannini, S. Moretti, R. G. Iannitti, F. Fallarino, et al. 2010. Intranasally dently of MD2. EMBO Rep. 13: 1109–1115. by guest on September 25, 2021