Murine Chronic Granulomatous Disease Resistance and Restrains

Exogenous Pentraxin 3 Restores Antifungal Resistance and Restrains Inflammation in Murine Chronic Granulomatous Disease

This information is current as Carmen D'Angelo, Antonella De Luca, Teresa Zelante, of September 28, 2021. Pierluigi Bonifazi, Silvia Moretti, Gloria Giovannini, Rossana Giulietta Iannitti, Silvia Zagarella, Silvia Bozza, Silvia Campo, Giovanni Salvatori and Luigina Romani J Immunol 2009; 183:4609-4618; Prepublished online 4

September 2009; Downloaded from doi: 10.4049/jimmunol.0900345 http://www.jimmunol.org/content/183/7/4609

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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Exogenous Pentraxin 3 Restores Antifungal Resistance and Restrains Inﬂammation in Murine Chronic Granulomatous Disease1

Carmen D’Angelo,* Antonella De Luca,* Teresa Zelante,* Pierluigi Bonifazi,* Silvia Moretti,* Gloria Giovannini,* Rossana Giulietta Iannitti,* Silvia Zagarella,* Silvia Bozza,* Silvia Campo,† Giovanni Salvatori,† and Luigina Romani2*

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by life-threatening bacterial and fungal infections and hyperinflammation. The susceptibility to aspergillosis in experimental CGD (p47phox؊/؊ mice) is associated with the failure to control the inherent inflammatory response to the fungus and to restrict the activation of inflammatory Th17 cells. We assessed whether pentraxin (PTX)3, a member of a family of multimeric pattern-recognition proteins with potent anti-Aspergillus activity, could limit pathogenic inflammation in p47phox؊/؊ mice by curbing the IL–23/Th17 inflammatory axis in response to the fungus. We found that the production of PTX3 was delayed in CGD mice in infection but exogenous administration of PTX3 early in infection restored antifungal resistance Downloaded from and restrained the inflammatory response to the fungus. This occurred through down-regulation of IL-23 production by dendritic cells and epithelial cells which resulted in limited expansion of IL-23R؉ ␥␦؉ T cells producing IL-17A and the emergence of Th1/Treg responses with minimum pathology. Thus, PTX3 could be therapeutically used for the exploitation of NADPH-independent mechanism(s) of antifungal immune protection with limited immunopathology in CGD. The Journal of Immunology, 2009, 183: 4609–4618.

hronic granulomatous disease (CGD)3 is an immunode- Possible mechanisms involved in CGD hyperinflammation in- http://www.jimmunol.org/ ficiency caused by the lack of the superoxide-producing clude decreased degradation of phagocytosed material, redox-de- C phagocyte NADPH oxidase (1). It is often accompanied pendent termination of proinflammatory mediators and/or signal- by an increased susceptibility to infection with microorganisms (2) ing, as well as redox-dependent cross-talk between phagocytes and including Aspergillus (3). However, CGD patients not only suffer lymphocytes (4). It has been proposed that different inflammatory from recurrent infections, but also present with inflammatory, non- responses are triggered as a function of the level of reactive oxy- infectious conditions, such as granulomata and obstruction of the gen species and have specific characteristics in terms of physiol- gastrointestinal and genitourinary tracts and inflammatory bowel ogy and pathophysiology (4). Defective tryptophan catabolism has disease (4). In addition to the more classical inflammatory lesions, recently been shown to contribute to the excessive inflammation in by guest on September 28, 2021 CGD patients and mice have increased frequency of autoimmune CGD mice with aspergillosis (9). Tryptophan metabolism regu- diseases (5, 6), a finding consistent with the observation that gen- lates both antimicrobial resistance and protective tolerance to in- eration of neo-antigenic determinants by reactive oxygen and ni- flammatory microbial stimuli (10, 11). We have shown that the trogen species may contribute to epitope spreading in autoimmu- enzyme IDO and the other kynurenine pathway enzymes represent nity (7). Although inflammation in CGD may confer some forms not only effector host defense pathways, but also a means of bal- of protection against aspergillosis, excessive inflammation may be ancing the generation of Th17 and regulatory T cells (Tregs) (9, deleterious. It has reported that even low virulent mutant strains of 12). As superoxide is a cofactor of IDO, the rate-limiting enzyme Aspergillus nidulans caused mortality in experimental pulmonary aspergillosis in CGD (p47phoxϪ/Ϫ) mice as a result of an excessive in tryptophan degradation along the kynurenine pathway (13), su- inflammatory response (8). This finding emphasizes the unique peroxide-induced activation of IDO is a central mechanism by pathophysiologic features of aspergillosis in CGD. which the optimal balance of antifungal host defense and immune tolerance occurs. Tregs and IL-17-producing T cells mediate opposing responses in aspergillosis, as clearly shown in CGD mice where the augmen- *Department of Experimental Medicine and Biochemical Sciences, University of Pe- tation of IL-17-driven inflammation occurred concomitantly with rugia, Perugia, Italy and †Sigma-Tau, Pomezia, Rome, Italy the reduction of anti-inflammatory Treg cell responses, resulting in Received for publication January 30, 2009. Accepted for publication July 29, 2009. excessive inflammation (9). Neutralization of IL-17A resulted in The costs of publication of this article were defrayed in part by the payment of page beneficial effects in mice with aspergillosis (9, 14), a finding sug- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. gesting that limiting Th17 cell activation could be therapeutically 1 This work was supported by the Chronic Granulomatous Disorder Research Trust exploited to control infection and inflammation in aspergillosis (Grant Number J4G/06/05). (15). These data suggest that in addition to attempt to reverse the 2 Address correspondence and reprint requests to Dr. Luigina Romani, Microbiology primary defect of the NADPH oxidase, limiting and resolving the Section, Department of Experimental Medicine and Biochemical Sciences, University inflammatory response is key to a successful control of the infec- of Perugia, Via del Giochetto, Perugia, Italy. E-mail address: [email protected] tion and the associated pathology in the condition of NADH 3 Abbreviations used in this paper: CGD, chronic granulomatous disease; Treg, regulatory T cell; PTX3, pentraxin 3; WT, wild type; i.n., intranasal; i.t., intratracheal; deficiency. BAL, bronchoalveolar lavage; DC, dendritic cell; TLN, thoracic lymph node; ROI, Current prophylaxis with trimethoprim-sulfamethoxazole, itra- reactive oxygen intermediate; PMN, polymorphonuclear neutrophil. conazole and, in selected cases, additional IFN-␥ is efficient in Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 CGD patients, but imperfect (16). Inflammatory complications are www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900345 4610 PROTECTION BY PTX3 IN CGD MICE

typically treated with systemic corticosteroids, which further sup- determined by pan-cytokeratin Ab staining. DCs and epithelial cells were press immunity and have substantial side effects (16). Gene-re- exposed to unopsonized Aspergillus conidia at the ratio of 1:1 at 37°C for ␮ placement therapy for patients lacking a suitable stem cell donor is 18 h in the presence of 5 g/ml PTX3 before the assessment of mRNA expression. still experimental and faces major obstacles and risks (17, 18). Additional novel therapeutic options are necessary for this rare Flow cytometry disorder (19). Staining for cell Ag expression was done as described (12). Cells were In the present study, we evaluated whether pentraxin (PTX)3, a analyzed with a FACScan flow cytofluorometer (BD Biosciences) member of a family of multimeric pattern-recognition proteins equipped with CellQuest software. Before labeling, FcR blocking was per- with potent anti-Aspergillus activity (20), could have beneficial formed. Control staining of cells with irrelevant Ab was used to obtain background fluorescence values. Data are expressed as a percentage of effects in CGD mice with aspergillosis. Considering that PTX3 positive cells over total cells analyzed. acts not only as a nonredundant component of the humoral arm of innate immunity but is also involved in tuning inflammation (21) Antifungal effector activity and ROI production and autoimmunity (22), the interesting hypothesis was that PTX3 For fungicidal activity, total lung cells or purified neutrophils were incu- could limit pathogenic inflammation in CGD. We found that the bated with unopsonized conidia at 1:1 ratio at 37°C. The effect of PTX3 on production of PTX3 was delayed in CGD mice in infection, such antifungal effector activity of neutrophils was assessed either by pre-exposing the cells to 100 or 10 ng/ml PTX3 for 2 h before the addition of that early administration of PTX3 increased antifungal resistance conidia or by exposing cells to conidia pretreated with 5 ␮g/ml PTX3 and and restrained the inflammatory response to the fungus. extensively washed thereafter. For the conidiocidal activity, the percentage of colony forming unit inhibition (mean Ϯ SE), referred to as conidiocidal

Materials and Methods activity, was determined as described (27). PMN production of ROI was Downloaded from done by quantifying the release of superoxide anion (O Ϫ) in the culture Animals 2 supernatants through the measure of the superoxide dismutase-inhibitable Eight- to 10-wk-old C57BL6 wild-type (WT) mice were purchased from reduction of cytochrome c (27). The A550 was measured in a Microplate Charles River Laboratories or The Jackson Laboratory. Breeding pairs of Reader BioRad 550 (Bio-Rad) and the results were expressed as nanomoles Ϫ phoxϪ/Ϫ 6 homozygous p47 mice, raised on C57BL6 background (23), were O2 /10 cells. purchased from Harlan Laboratories and bred under specific-pathogen free conditions at the breeding facilities of the University of Perugia, Perugia, Cytokine assays phoxϪ/Ϫ http://www.jimmunol.org/ Italy. p47 mice recapitulate several of the features of the human Lung homogenates and serum were assessed for human (for exogenous disease and are highly susceptible to Aspergillus infection (23–25). Exper- PTX3) or murine PTX3 by using the DuoSet ELISA kit DY1826 or iments were performed according to the Italian Approved Animal Welfare DY2166 (R&D Systems), respectively. The levels of cytokines in pooled Assurance A-3143-01. lung homogenates and culture supernatants were determined by specific ELISAs (BD Pharmingen and R&D Systems). A. fumigatus, culture conditions, infection, and treatments RT-PCR and real-time PCR The A. fumigatus was obtained from a fatal case of pulmonary aspergillosis at the Infectious Diseases Institute of the University of Perugia (9, 12). For Total RNA extraction (TRIzol; Invitrogen Life Technologies) and synthe- infection, mice were anesthetized by i.p. injection of 2.5% avertin (Sigma- sis and PCR of cDNA were done as described (12, 14, 29). RT-PCR for Aldrich) before intranasal (i.n.) instillation of a suspension of 2 ϫ 107 PTX3 was done with the 5Ј-CCT GCT TTG TGC TCT CTG GT-3Ј and by guest on September 28, 2021 resting conidia/20 ␮l saline for three consecutive days or intratracheal (i.t.) 5Ј-TCT CCA GCA TGA TGA ACA GC-3Ј primers, corresponding to po- injection of 5 ϫ 106 resting conidia. Quantification of fungal growth in the sitions 117–292 of murine PTX3 mRNA (accession number X83601 http:// lungs was done by the colony forming unit (mean Ϯ SE) and chitin assay www.ncbi.nlm.nih.gov/nuccore/1181243?report ϭ GenBank); for mBD1 (micrograms of glucosamine per pair of lungs) (12). For reinfection, mice with the 5Ј-GGC ATT CTC ACA AGT CTT GGA CGA AG-3Ј and 5Ј- surviving the primary i.n. infection were similarly reinfected 40 days later AGC TCT TAC AAC AGT TGG GCT TAT CTG G-3Ј primers, and for and assessed for chitin content 2 wk later. For histology, paraffin-embedded cathelicidin with the 5Ј-GGCGGTCACTATCACTGCTGCTG-3Ј and 5Ј- sections were stained with Periodic acid-Schiff. Bronchoalveolar lavage CACTCGGAACCTCACAGACTTGG-3Ј primers. Real-time PCR was (BAL) was performed by cannulating the trachea and washing the airways performed using the Stratagene Mx3000P QPCR System and SYBR green with PBS to collect the BAL fluid. Total and differential cell counts were chemistry (Stratagene). The thermal profile for SYBR green real-time PCR done by staining BAL smears with May-Gru¨nwald Giemsa reagents was at 95°C for 10 min, followed by 40 cycles of denaturation for 30 s at (Sigma-Aldrich) before analysis. Different doses of PTX3 (obtained under 95°C and an annealing/extension step of 30 s at 72°C. Each data point was endotoxin-free conditions by immunoaffinity of culture supernatants of examined for integrity by analysis of the amplification plot. CHO cells transfected with PTX3 (26), Sigma-Tau) and Voriconazole (VFend, Pfizer), alone or in combination, were administered i.p. or i.n. Statistical analysis (PTX3) or intragastrically (Voriconazole) for 5 days starting the day of the Data were analyzed by GraphPad Prism 4.03 program (GraphPad Soft- first Aspergillus intranasal injection. Controls receive sterile saline. ware). The log-rank test was used for paired data analysis of the Kaplan- Cell purification and cell cultures Meier survival curves. Student’s t test or ANOVA and Bonferroni’s test were used to determine the statistical significance of differences in organ Purified peritoneal CD11bϩGr-1ϩ polymorphonuclear neutrophils (PMN) clearance and in vitro assays. Significance was defined as p Ͻ 0.05. In vivo (Ͼ98% pure on FACS analysis) were obtained as described (27). Lung groups consisted of 4–6 animals. The data reported were pooled from three CD11cϩ dendritic cells (DCs) (between 5 to 7% positive for CD8␣ and to five experiments, unless otherwise specified. between 30 to 35% positive for Gr-1) were isolated by magnetic cell sorting with MicroBeads (Miltenyi Biotec) (12). CD4ϩ were separated by Results magnetic cell sorting from thoracic lymph nodes (TLN), as described (12). PTX3 mRNA expression and production in mice with Lung epithelial cells were isolated as described (6, 28). In brief, the trachea were aseptically cut open lengthwise, washed, resuspended in prewarmed aspergillosis ϩ ϩ ␮ HBSS (Ca2 /Mg2 -free; 44 mM NaHCO3; 0.25 M sodium pyruvate; 50 Given the nonredundant role of PTX3 in anti-Aspergillus innate ␮ g gentamicin; 0.1 mg/ml DNase I; and 1.4 mg/ml pronase E were added immune response in vivo (20), we assessed whether PTX3 expres- shortly before use) and incubated at 37°C for 1 h for enzymatic digestion. phoxϩ/ϩ After blocking enzymatic activity with the addition of 10% FBS, the tra- sion and protein production would be different in p47 or phoxϪ/Ϫ cheal residues were transferred to 10 ml RPMI 1640 medium supplemented p47 mice during infection. PTX3 mRNA was not observed with 5% FBS and gentamicin. After centrifugation, cells were seeded in at the basal level in either type of mice, but promptly increased tissue culture plates for 3–4 h at 37°C to separate unattached epithelial after 6 h postinfection in p47phoxϩ/ϩ mice, reaching a peak level cells from contaminating cells and adherent fibroblasts. The average yield ϫ 5 Ϯ ϫ 5 expression at 18–24 h, to decline thereafter. In contrast, a delayed of tracheal cells was 1.7 10 cells/trachea ( 0.58 10 (SD)). Cell phoxϪ/Ϫ viability determined by trypan blue exclusion was Ͼ90%. Cytocentrifuge expression of PTX3 occurred in p47 mice, being PTX3 preparations of these cells revealed that ϳ99% expressed cytokeratin, as mRNA detected at 48 and 72 h postinfection (Fig. 1A). Consistent The Journal of Immunology 4611

FIGURE 1. Protective activity of PTX3 in p47phoxϪ/Ϫ mice with aspergillosis. Mice were injected intranasally (i.n.) with live Aspergil- lus conidia and assessed for PTX3 mRNA expression by RT-PCR (A) and production by ELISA (B)inthe lung at different time points after the infection. mPTX3, murine PTX3. The data are representative of six mice/group. For positive controls, PTX3-expressing lung DCs were p Ͻ 0.05, p47phoxϪ/Ϫ vs ,ء .used p47phoxϩ/ϩ mice. C, Mice were in- Downloaded from fected i.n. as above and treated daily, for 5 consecutive days, with different doses of PTX3 given i.p. D, Mice were i.n. infected and treated daily with 1 mg/kg PTX3 given i.p. or i.n. Chitin assay (␮g/lungs) were per-

formed in groups of mice sacriﬁced a http://www.jimmunol.org/ day after the last PTX3 treatment. E, Serum levels (by ELISA) of exog- enously administered PTX3 (hPTX3), given i.n. or i.p, daily for 5 days to p Ͻ 0.05, PTX3 i.n ,ء .infected mice vs i.p. F, Chitin content of PTX3- treated (1 mg/kg/i.p.) p47phoxϪ/Ϫ mice surviving the primary i.n. infection and similarly reinfected 40 days later. Assay was done 3 days after re- by guest on September 28, 2021 infection. G, Mice were infected in- tratracheally (i.n.) and PTX3-treated as in C. Shown are the results pooled from four experiments (six animals/ group) in the i.n. model and three experiments in the i.t. model (four ani- p Ͻ 0.05, PTX3- ,ء .(mals/group treated vs untreated mice. Bars indicate the SEs.

with the mRNA expression, PTX3 protein production in the lung Efficacy of PTX3 in p47phoxϪ/Ϫ mice with aspergillosis occurred earlier in p47phoxϩ/ϩ (between 6 to 24 h) than p47phoxϪ/Ϫ To assess whether early supply of exogenous PTX3 would impact mice (between 48 to 72 h), the high levels observed in the latter on antifungal resistance of p47phoxϪ/Ϫ mice, we administered likely due to the abundance of recruited PMNs, known to store PTX3 in the specific granules (30) (Fig. 1B). Both mRNA expres- PTX3 daily for 5 days, starting the day of the infection, to phoxϩ/ϩ phoxϪ/Ϫ sion and protein production returned to basal level at 168 h post p47 or p47 mice according to different treatment infection in both types of mice (data not shown). These results schedules that include: 1) different types of infection (i.n. vs i.t.), indicate that the production of PTX3 is somehow delayed in 2) different dosages, and 3) different routes of administration. Mice p47phoxϪ/Ϫ mice, in which the elevated PTX3 levels seen late in were monitored for survival, local fungal growth and dissemina- infection likely reflects the state of hyperinflammation, as sug- tion, inflammatory cell recruitment, and lung histopathology. gested (31, 32). Given the crucial role of PTX3 in promoting PTX3 significantly increased survival and reduced the fungal conidia phagocytosis (20), the defective PTX3 production ob- growth in the lungs of either types of mice infected i.n. (Fig. 1C) served in p47phoxϪ/Ϫ mice may prevent the optimal handling of and reduced fungal dissemination into the brain (data not shown). conidia early in infection. The beneficial effect of PTX3 was: 1) dose-dependent, being the 4612 PROTECTION BY PTX3 IN CGD MICE

FIGURE 2. PTX3 targets PMNs and epithelial cells. A, Conidiocidal activity, i.e., percentage of colony forming unit inhibition (mean Ϯ SE) of total lung cells (harvested a day after the last PTX3 treatment) from mice infected and treated with 1 mg/kg PTX3 given i.n. or i.t. B, Con- idiocidal activity and C, ROI production of puriﬁed PMNs from naive mice exposed to different concentra- tions of PTX3 in vitro a, or to PTX3- opsonized conidia b, as detailed in ,p Ͻ 0.05 ,ء .Materials and Methods PTX3-treated vs untreated mice or untreated (Ϫ) cells. D, mBD1 and Downloaded from cathelicidin mRNA expression (by RT-PCR) on total lung cells from WT mice infected with live Aspergillus conidia i.n. or i.t., at different time ,p Ͻ 0.05 ,ء .points after the infection infected vs uninfected (0) mice. E, mBD1 mRNA expression (by RT- http://www.jimmunol.org/ PCR) in total lung cells from i.n. infected mice, either untreated or ,ء .treated with PTX3 as indicated p Ͻ 0.05, PTX3-treated vs untreated (none) mice. Shown are the results pooled from two experiments (four animals/group). F, mBD1 mRNA expression (by RT-PCR) on lung epithelial cells puriﬁed from naive mice and by guest on September 28, 2021 exposed for 18 h to live unopsonized conidia and/or 5 ␮g/ml PTX3 before the assessment of mRNA expression. p Ͻ 0.05, exposed vs unexposed ,ء (Ϫ) cells. Shown are the results pooled from three experiments.

maximum effect observed at the dosage of 1 mg/kg (Fig. 1C) and PTX3 targets PMNs and epithelial cells 2) partially dependent on route of administration, being less effi- To define possible cellular targets underlying the PTX3 activity in cacious upon local (i.n.) than systemic (i.p.) administration (Fig. infection, we measured the conidiocidal activity in PTX3-treated mice 1D). The levels of PTX3 were much higher in the serum (Fig. 1E) and found that PTX3 treatment increased the antifungal activity of but not in the lung (data not shown) of mice treated with PTX3 i.p. effector lung cells of p47phoxϪ/Ϫ, more than WT, mice (Fig. 2A). We than i.n., a finding suggesting that PTX3-elicited systemic effects have shown that PMNs, more than alveolar macrophages, have de- are important for its antifungal activity in the lung. Interestingly, phoxϪ/Ϫ mice surviving the primary i.n. infection upon treatment with fective conidiocidal activity in p47 mice with aspergillosis (9). PTX3 also showed increased resistance to re-infection 40 days PTX3 was able to increase the conidiocidal activity of PMNs from phoxϪ/Ϫ later (Fig. 1F). PTX3 was not effective when given prophylacti- both WT and p47 mice (Fig. 2B), although unable to activate cally, (a week before the infection, data not shown), which indi- the respiratory burst of WT PMNs in response to conidia (Fig. 2C). cates that PTX3 alone did not modify antifungal immune reactivity Considering that PMN recruitment was decreased in the BAL and and that pathogen opsonization is important for its antimicrobial lungs (Fig. 3, A and B) after PTX3 treatment, this indicates that PTX3 activity. exerts a fine control of quality over PMN function through NADPH On assessing the efficacy of PTX3 in mice infected i.t., we found oxidase-independent mechanisms. that the beneficial effect was partially reduced, particularly in In an attempt to unravel possible mechanisms of NADPH oxi- p47phoxϪ/Ϫ mice (Fig. 1G), a finding indicating that PTX3 may dase-independent activity of PTX3, we assessed whether ␤-de- target cells of the upper airways. fensins and cathelicidin, known for their role in host defense and The Journal of Immunology 4613

FIGURE 3. PTX3 ameliorates lung inflammatory pathology. A, Periodic acid-Schiff-stained medial sections of lungs from i.n. infected p47phoxϩ/ϩ or p47phoxϪ/Ϫ mice either untreated (Ϫ) or treated with 1 mg/kg PTX3 either i.n. or i.p. and assessed 1 day after the end of treatment. Note the presence of few infiltrates of inflammatory mononuclear cells scattered in an otherwise intact lung parenchyma in p47phoxϩ/ϩ mice as opposed to the massive infil- tration of neutrophils, associated with Downloaded from the presence of hyphae (arrows) and signs of diffuse interstitial pneumonia in p47phoxϪ/Ϫ mice and its amelioration by PTX3 treatment. Bars indicate magnification. B, Percentages of neutrophils (PMN) and mononuclear cells (MNC) recruited in the bron- http://www.jimmunol.org/ choalveolar lavage fluid (BAL) of mice sacrificed as above. Photographs were taken using a high-resolution Microscopy Olympus DP71. by guest on September 28, 2021

inflammation in the lung (33, 34), were induced by PTX3 in vivo findings are in line with the observation that lung inflammation as well as in vitro on epithelial cells, known to be central partic- following Aspergillus infection involves transient inflammatory ipants in innate and adaptive immune response as well as mucosal cell influx into tissues and alveolar spaces, perivascular edema, inflammation (35). Interestingly, we found first that the expression and partial consolidation. Nevertheless, following fungal clear- of mBD1 and cathelicidin occurred differently in the i.n. vs i.t. ance, inflammation rapidly resolves, leaving little structural or infection, being the expression of both higher in the i.n. than the i.t. morphological evidence to indicate that the response occurred. To- infection (Fig. 2D). Second, PTX3 increased the mBD1 mRNA gether, these data indicate that, by promoting conidial phagocyto- expression both in vivo (Fig. 2E) and on epithelial cells in vitro sis, PTX3 may limit cell recruitment and tissue inflammation, a (Fig. 2F), a finding consistent with the ability of PTX3 to signif- desirable activity in condition of NADPH deficiency. icantly promote conidia phagocytosis by these cells (data not shown). The cathelicidin mRNA was also up-regulated by PTX3 in PTX3 affects cytokine production and Th cell activation in CGD vivo but this effect was not observed on epithelial cells in vitro mice with aspergillosis (data not shown). Although the relative role of mBD1 and cathe- As the high susceptibility of p47phoxϪ/Ϫ mice to infection and licidin in aspergillosis has yet to be defined, the ability of PTX3 to inflammation was associated with failure to activate protective Th1 induce, directly or indirectly, their expression suggests that PTX3 and Treg cell responses and the occurrence of inflammatory Th17 may exploit NADPH-independent mechanisms of antifungal resis- cells (9, 14), we evaluated parameters of inflammatory/anti- tance in aspergillosis. inflammatory cytokine production and adaptive Th immunity in WT and p47phoxϪ/Ϫ infected mice upon treatment with PTX3. To PTX3 ameliorates inflammatory pathology this purpose, we evaluated the mRNA expression and actual cy- The protective effects of PTX3 in infection included amelioration tokine production of inflammatory (IL-12p70, IL-23, IL-17A and of lung pathology, as indicated by the extensive reduction of neu- TNF-␣) and anti-inflammatory (IL-10) cytokines in the lungs as trophilic and inflammatory cell infiltrates in the lung parenchyma well as the presence of specific transcription factors for Th1 (Tbet), (Fig. 3A) and BAL (Fig. 3B) of infected WT and p47phoxϪ/Ϫ mice Th2 (Gata3), Th17 (Rorc), and Treg (Foxp3) in CD4ϩ T cells treated with PTX3 i.n. and, even more, i.p. The numbers and extent purified from TLN. In infection, the levels of IL-12p70, IL-23, of granulomas formations were also greatly reduced by PTX3 IL-17A, and TNF-␣ (both in terms of mRNA expression and actual treatment, as observed by gross pathology (data not shown). These cytokine production) were elevated in the lungs of p47phoxϪ/Ϫ 4614 PROTECTION BY PTX3 IN CGD MICE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Effect of PTX3 on inflammatory/anti-inflammatory cytokine production and Th cell activation. Mice were intranasally infected with Aspergillus conidia, treated with 1 mg/kg PTX3 as in legend to Fig. 1 and assessed, a day after the last treatment, for inflammatory/anti-inflammatory cytokine production by real time PCR on total lung cells or by ELISA on lung homogenates (A) and for activation of Th cell-specific transcription factors on CD4ϩ T cells purified from p Ͻ 0.05, PTX3-treated vs untreated mice. Bars are SE. Shown are the results of pooled ,ءء .p Ͻ 0.05, infected vs naive mice ,ء .(thoracic lymph nodes (TLN) (B homogenates, each assessed in triplicates, from four experiments (six animals/group) in the i.n. model and three experiments in the i.t. model (four animals/group). mice but were greatly reduced by treatment with PTX3, particu- PTX3 restricts the expansion of ␥␦ϩCD3ϩ T lymphocytes in the larly at the highest dose given i.p. (Fig. 4A). In the case of WT lungs in CGD mice with aspergillosis mice, treatment with PTX3 actually increased p35 mRNA expres- ϩ ϩ It has recently been shown that a population of ␥␦TCR2 T lym- sion as well as IL-12p70 and TNF-␣ productions. These results are phocytes producing IL-17A is rapidly expanded in the lungs of not only in line with previous data (20, 26) and the data of Fig. 5 Ϫ Ϫ p47phox / mice after A. fumigatus infection and contributes to the (see below), but also suggest that the opposite effects on p35 mRNA Ϫ Ϫ generation of the local inflammatory state and failure to restrict fungal expression seen in p47phox / and WT mice are likely dependent on growth (9). To evaluate whether treatment with PTX3 would affect mechanistic differences rather than the stage of infection. Concomi- 2ϩ Ϫ Ϫ the expansion of ␥␦TCR cells, largely responsible for the elevated tantly, the levels of IL-10 were greatly reduced in p47phox / mice IL-17A production early in infection (9), we performed phenotypic upon infection but restored by PTX3 treatment (Fig. 4A). phoxϪ/Ϫ In terms of adaptive Th immunity, in line with previous findings analysis of total lungs cells in WT and p47 mice after treat- ␥␦ϩ ϩ (9), Tbet/Foxp3 mRNA expressions (indicative of Th1/Treg activa- ment with PTX3. In contrast to WT mice, a population of CD3 phoxϪ/Ϫ tion) were increased in TLN CD4ϩ T cells from WT mice, while T cells was expanded (from 3 to 9%) in the lungs of p47 mice Rorc/Gata3 expressions (indicative of Th17/Th2 activation) were in- upon infection, but was reduced (to 2 and 4%) upon treatment with creased in p47phoxϪ/Ϫ mice. However, administration of PTX3 in- PTX3 at 1 mg/kg/i.p or i.n., respectively (Fig. 5A). Consistent with the creased the expression of Tbet/Foxp3 and decreased that of Rorc/ histological findings, treatment with PTX3, at the dose of 1 mg/kg/ ϩ Ϫ Gata3 in p47phoxϪ/Ϫ mice, leaving largely unaffected mRNA i.p., also concomitantly restricted the expansion of Gr-1 F4–80 expression in CD4ϩT cells from WT mice (Fig. 4B). Together, these neutrophils, a population of which was 6-fold expanded in the lungs Ϫ Ϫ data suggest that PTX3 affects the balance of inflammatory/anti- of p47phox / mice upon infection. As IL-17A is a potent chemoat- inflammatory cytokine production in the lungs of p47phoxϪ/Ϫ mice tractant of neutrophils in the lungs (36), it is likely that the restriction ϩ with aspergillosis, inhibits the activation of disease-promoting Th17 of ␥␦TCR2 cell expansion may have resulted in the reduced neu- cells and promotes the development of protective Th1/Treg cells trophil recruitment. Together, these findings suggest that the protec- likely accounting for resistance to infection and reinfection. tive effect of PTX3 in p47phoxϪ/Ϫ mice relies on the concomitant The Journal of Immunology 4615 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Effect of PTX3 on ␥␦ϩ T cells cell recruitment and IL-23 and IL-23R expression in the lungs of infected mice. A, Phenotypic analysis of total lung cells from i.n. infected mice, treated with 1 mg/kg PTX3 i.p or i.n. Numbers referred to percentages of ␥␦ϩCD3ϩ T cells or Gr-1ϩ F4–80Ϫ neutrophils over total cells analyzed, a day after the last PTX3 administration. B and C, Cytokine gene expression (real time PCR) on dendritic or epithelial cells purified from lungs of uninfected mice and stimulated in vitro with conidia in the presence of 5 ␮g/ml PTX3. n.d., not detectable, i.e., below the p Ͻ 0.05, PTX3-treated vs untreated cells. Bars are SE. D and E, Cytokine ,ءء .p Ͻ 0.05, stimulated vs unstimulated (Ϫ) cells ,ء .detection limit of the assay receptor mRNA expression in total lung cells or purified ␥␦ϩCD3ϩ cells from mice i.n. infected and treated with 1 mg/kg PTX3 i.p. The results shown represent one representative experiment of three. restriction of ␥␦TCR2ϩ T cell expansion and Gr-1ϩ neutrophil p35 mRNA expression was increased in WT DCs, a finding con- recruitment. sistent with the in vivo results (Fig. 4). Moreover, PTX3 increased the levels of il10 mRNA expression in response to the fungus in phoxϪ/Ϫ PTX3 down-regulates IL-23/IL-23R expression in p47 mice either type of cells from both types of mice, even though in WT Ϫ Ϫ The inhibition of ␥␦TCRϩ cells expansion in the lungs may occur more than p47phox / mice (Fig. 5, B and C). These data indicate through different mechanisms (9). Because IL-17A expression in that PTX3, by altering the balance between inflammatory (IL-23/ the lung is dependent on IL-23 (37), we have evaluated whether IL-12) and anti-inflammatory cytokines (IL-10) cytokines in DCs down-regulation of IL-23 production by DCs and epithelial cells and epithelial cells could have an impact on both the innate and would be a mechanism by which PTX3 restrict ␥␦TCRϩ cells adaptive immunity to the fungus. expansion in p47phoxϪ/Ϫ mice. Purified cells from the lungs of To assess whether levels of IL-23/IL-12 production also corre- uninfected mice were exposed in vitro to conidia in the presence of late with levels of IL-23R or IL-12␤2R expression in the lungs of PTX3 for 18 h, before the assessment of cytokine mRNA expres- mice, we measured cytokine receptors expression in total lung sion by real time PCR. For comparison, we also assessed levels of cells (Fig. 5D) or purified ␥␦TCRϩ (Fig. 5E) cells from PTX3- p35 and il10 mRNA expression as a measure of IL-12p70 or IL-10 treated or -untreated mice. Paralleling cytokine production, the ex- production. The results (Fig. 5, B and C) showed that p19 mRNA pression of IL-23R was higher in p47phoxϪ/Ϫ than WT mice after expression was higher in either type of cells from p47phoxϪ/Ϫ than the infection, but was significantly reduced upon treatment with WT mice but was significantly reduced in p47phoxϪ/Ϫ mice in the PTX3 on both total lung cells and purified ␥␦TCRϩ cells. In con- presence of PTX3. Although not expressed in epithelial cells, the trast, the expression of IL-12␤2R was increased in total lung cells, 4616 PROTECTION BY PTX3 IN CGD MICE

FIGURE 6. Effect of PTX3 on the therapeutic efﬁcacy of voriconazole. p47phoxϪ/Ϫ mice were i.n. infected with Aspergillus conidia and treated with PTX3 and/or Voriconazole (Vor) at the doses indicated for 5 days after the infection. Fungal growth in the lungs and brain (A), cytokine mRNA expression in the lungs and Downloaded from Th transcription factors in CD4ϩ T cells from TLN (B) were done a day ,p Ͻ 0.05 ,ء .after the last treatment PTX3Ϫ or Vor-treated vs PTX3ϩ Vor-treated mice. Bars are SE. Shown are the results pooled from three experiments (in vivo data, six animals/ http://www.jimmunol.org/ group) or results of one representative experiment (real-time PCR). C, Naive mice. by guest on September 28, 2021

but not on purified ␥␦TCRϩ cells, from WT mice and in mg/kg for voriconazole). Mice were monitored for fungal p47phoxϪ/Ϫ mice upon PTX3 treatment. Purified ␥␦TCRϩ cells growth in the lungs and brain and activation of protective Th1/ expressed high levels of IL-17A that were unmodified by PTX3 Treg cell responses. Each single agent given alone, at the op- treatment (Fig. 5E), a finding suggesting that ␥␦TCRϩ cells are not timal dose, significantly reduced the fungal growth in the lungs directly targeted by PTX3. As PTX3 apparently also failed to mod- and brain and concomitantly decreased TNF-␣ production and ulate IL-23R expression on purified ␥␦TCRϩ cells in vitro (data increased IL-10 production (data not shown). At the suboptimal not shown), these results indicate that cytokine production and dose, each single treatment reduced to some extent the fungal cytokine receptors expression are coordinately regulated in infec- growth in the lungs and, to a lesser extent, in the brain. How- tion by PTX3. ever, combination therapy with PTX3 and Voriconazole at suboptimal doses resulted in virtually no detectable fungal growth PTX3 increases the therapeutic efficacy of voriconazole in the lungs and brain and the occurrence of protective antifun- Current prophylaxis with itraconazole and, in selected cases, ad- gal responses, as judged by the high levels of IFN-␥/Tbet and ditional IFN-␥ is efficient, but imperfect in CGD patients (38). A il10/Foxp3 mRNA expression associated with inhibition of significant recent progress toward new antifungal (e.g., voricon- il17a/Rorc (Fig. 6). Therefore, PTX3 appears to work synergis- azole and posaconazole) therapy has allowed survival of most pa- tically with voriconazole to restrict the fungal growth, decrease tients into adulthood (16). All the above findings prompted us to the lung inflammatory response, and activate protective Th1/ evaluate whether the immunomodulatory activity of PTX3 could Treg cell responses. be exploited to increase the therapeutic efficacy of voriconazole in p47phoxϪ/Ϫ mice with aspergillosis. To this purpose, mice received Discussion PTX3 or voriconazole alone at the optimal doses (i.e., at 1 mg/kg/ The results of our study shows that restraining pathogenic inflamma- i.p. for PTX3 and 1 mg/kg/intragastrically for voriconazole) or tion could be a successful therapeutic strategy in CGD. Excessive together at the doses at which none of the agents achieved the inflammation in CGD is not solely the result of unresolved infection, maximum therapeutic effect (i.e., at 0.2 mg/kg for PTX3 and 0.5 but results from an intrinsic defect in the control of inflammation. One The Journal of Immunology 4617 important corollary of these new findings is whether therapeutic strat- PTX3 administered early in infection. Finally, the higher levels of egies in CGD should target infection or inflammation. The recent defensins induced upon PTX3 administered i.p. than i.n, indicate discovery that IL-17A-producing T cells crucially contribute to acute that PTX3 could set the lung for antifungal defense by remote and persistent inflammation in CGD mice with aspergillosis (9) offers control, the nature of which is presently under investigation. novel therapeutic targets for this rare disorder. We show in this study The action of PTX3 in DCs is also of interest. In fact, PTX3 that PTX3 could successfully limit Th17-driven inflammation in CGD differentially conditioned the activity of lung DCs from WT or to the benefit of the occurrence of antifungal protective Th1 responses CGD mice, potentiating the production of IL-12p70 in WT DCs and anti-inflammatory Tregs. but curbing that of IL-23 in DCs from CGD mice. This not only Much evidence suggests that ␥␦ϩ T cells function in an innate confirms previous data on the ability of PTX3 to activate DCs for manner, although they are arguably the most complex and ad- antifungal Th1 responses (20), but also suggests that NADPH ox- vanced cellular representatives of the innate immune system (39). idase may condition the DCs response to PTX3. That NADPH Indeed ␥␦ϩ T cells are activated in response to stress to the sur- oxidase is a regulator of DC activity has already been reported (6, rounding tissue and perform a number of functions depending 54–56) as well as the molecular mechanisms leading to the dif- upon the location and type of stress that has occurred (40). ␥␦ϩ T ferential production of IL-12/IL-23 in DCs (57, 58). How PTX3 cells are involved in resolving inflammation in the lung (41, 42) differentially condition DCs from WT and CGD mice is not but are also an important source of IL-17A (9, 37), which can known. In this regard, it is of interest that phagocytosis of apo- exacerbate autoimmune disease (43). It has been suggested that ptotic cells curbs IL-23 release by DCs (59) and that PTX3 regu- some ␥␦ϩ T cells are poised to produce IL-17A in a rapid manner lates the clearance of apoptotic cells by DCs (60). Irrespective of ϩ (43). Expansion of IL-17A-producing ␥␦ T cells occurred in My- the mechanism, the ability of PTX3 to condition DC activity from Downloaded from cobacterium bovis bacille Calmette-Gue´rin-infected lung and was CGD mice further suggests that PTX3 may have NADPH oxidase- dependent on IL-23 (37). Indeed, naive ␥␦ϩ T cells have been independent activity. As a matter of fact, PTX3 did not activate the shown to produce IL-17A in response to IL-23 alone, a cytokine respiratory burst of neutrophils, while potentiating their antifungal known to expand pathogenic Th17 cells (44, 45). A similar mech- effector activity. The same reasoning would apply for epithelial anism is at work in pulmonary aspergillosis as we have recently cells, whose immune regulatory activity beyond the antimicrobial

found that Th17 activation is defective in mice with genetic defi- function has recently been proposed (6, 35). http://www.jimmunol.org/ ciency or therapeutic ablation of IL-23 (14) and that ablation of One interesting finding of the present study was that PTX3 ␥␦ϩ T cells early in infection, improves resistance to the infection worked synergistically with voriconazole to restrict the fungal (9). Despite the fact that IL-23 may serve a protective role in condi- growth and decrease the lung inflammatory pathology. Despite the tion of Th1 deficiency (14, 46) and IL-17A is important for an optimal fact that the routine use of IFN-␥ and itraconazole have reduced Th1 response (37), it appears that a tight control of the IL-23/IL-23R the incidence of infections in CGD (3), fungal infections have axis is needed to control innate and adaptive antifungal response in remained a persistent problem in CGD patients. It is desirable that CGD mice. In this regard, it is of interest that PTX3 exert such a PTX3 will be used in combination antifungal therapy in these control over the IL-23 and IL-23R expression in the lungs of CGD- patients. infected mice. IL-23 expression was indeed profoundly down-regu- Collectively, this study suggests that PTX3 could be success- by guest on September 28, 2021 lated in both DCs and epithelial cells, the latter cells known to be fully exploited as a novel therapeutic agent with anti-inflammatory crucially involved in the cross-talk with ␥␦ϩ T cells (35, 47). Con- properties in CGD and points to the existence of NADPH-inde- comitantly, IL-23R expression was decreased in lung ␥␦TCRϩ cells pendent mechanism(s) of antifungal immune protection that are upon PTX3 treatment, a finding pointing to the harmonious regulation amenable to manipulation to restore the appropriate control of in- of the IL-23/IL-23R axis by PTX3. fection and inflammation in CGD. One interesting observation of the present study is that PTX3 was directly able to target epithelial cells for innate antifungal Acknowledgments resistance. Not only was the phagocytic ability of purified murine We thank Dr. Cristina Massi Benedetti for editorial support. and human epithelial cells increased by PTX3, but the expression of epithelial defensins was also promoted by PTX3. In response to Disclosures infection, defensins are produced within minutes to hours by neu- The authors have no financial conflict of interest. trophils and specialized epithelial cells (48). Some defensins act as References chemoattractant for immature DCs (49), thus acting as a potent 1. Segal, B. H., T. L. Leto, J. I. Gallin, H. L. Malech, and S. M. Holland. 2000. immunological adjuvant that provides a link between innate and Genetic, biochemical, and clinical features of chronic granulomatous disease. adaptive immunity. Interestingly, the antimicrobial activity of de- Medicine 79: 170–200. 2. 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