Antiviral Activity of the Long Chain Pentraxin PTX3 against Influenza Viruses Patrick C. Reading, Silvia Bozza, Brad Gilbertson, Michelle Tate, Silvia Moretti, Emma R. Job, Erika C. Crouch, Andrew This information is current as G. Brooks, Lorena E. Brown, Barbara Bottazzi, Luigina of September 26, 2021. Romani and Alberto Mantovani J Immunol 2008; 180:3391-3398; ; doi: 10.4049/jimmunol.180.5.3391

http://www.jimmunol.org/content/180/5/3391 Downloaded from

References This article cites 45 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/180/5/3391.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• 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 by guest on September 26, 2021

*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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Antiviral Activity of the Long Chain Pentraxin PTX3 against Influenza Viruses1

Patrick C. Reading,2* Silvia Bozza,† Brad Gilbertson,* Michelle Tate,* Silvia Moretti,† Emma R. Job,* Erika C. Crouch,¶ Andrew G. Brooks,* Lorena E. Brown,* Barbara Bottazzi,‡ Luigina Romani,3† and Alberto Mantovani3‡§

Proteins of the can act as natural inhibitors of influenza virus, limiting growth and spread of the virus in the early stages of infection before the induction of adaptive immune responses. In this study, we identify the long pentraxin PTX3 as a potent innate inhibitor of influenza viruses both in vitro and in vivo. Human and murine PTX3 bound to influenza virus and mediated a range of antiviral activities, including inhibition of hemagglutination, neutralization of virus infectivity and inhibition of viral neuraminidase. Antiviral activity was associated with binding of the viral hemagglutinin glycoprotein to sialylated ligands present on PTX3. Using a mouse model we found PTX3 to be rapidly induced following influenza infection and that PTX3؊/؊ mice Downloaded from were more susceptible than wild-type mice to infection by PTX3-sensitive virus strains. Therapeutic treatment of mice with human PTX3 promoted survival and reduced viral load in the lungs following infection with PTX3-sensitive, but not PTX3-resistant, influenza viruses. Together, these studies describe a novel antiviral role for PTX3 in early host defense against influenza infections both in vitro and in vivo and describe the therapeutic potential of PTX3 in ameliorating disease during influenza infection. The Journal of Immunology, 2008, 180: 3391–3398. http://www.jimmunol.org/

nfluenza is an important annual respiratory infection that re- The antiviral activities of collectins have been well described mains a leading cause of illness and death throughout the with strong evidence to suggest that surfactant -D (SP-D)4 I world. In the early stages of infection, innate immune mech- is of particular importance during influenza infections. Both sur- anisms represent the main line of host defense, acting to limit virus factant protein-A (SP-A) and SP-D mediate a range of activities spread in host tissues before the induction of the adaptive response. against influenza virus, including inhibition of hemagglutination, Innate mechanisms that may contribute to defense against influ- virus neutralization, virus aggregation, and opsonizaton of the vi- enza infection include the intrinsic resistance of , the rus for interaction with neutrophils (2–8), with SP-D acting as a induction of IFN and proinflammatory cytokines and activation of more potent inhibitor than SP-A in vitro (4). Levels of SP-D re- by guest on September 26, 2021 NK cells, macrophages, and neutrophils in the airways. In addi- covered from the airways by lavage increase significantly follow- ing infection of mice with influenza virus (3, 9–11), and SP-DϪ/Ϫ tion, serum and airway fluids contain a number of innate mice exhibit enhanced viral replication and illness (9, 11). capable of recognizing and inhibiting influenza viruses, including SP-AϪ/Ϫ mice also display enhanced susceptibility to influenza in- members of the collectin and pentraxin families, mucins and sal- fections in some studies (9, 10), but not others (12); however, the ivary scavenger receptor-rich glycoprotein 340 (1). Ϫ Ϫ effect is less pronounced than that observed in SP-D / mice. Of interest, the mechanisms by which SP-A and SP-D act against influenza virus are different. SP-D functions as a classic ␤ inhibitor, binding in a Ca2ϩ-dependent manner through its lectin *Department of Microbiology and Immunology, University of Melbourne, Parkville, domains to oligosaccharides on the viral hemagglutinin (HA) and † Victoria, Australia; Microbiology Section, Department of Experimental Medicine neuraminidase (NA) glycoproteins (6). As such, the degree or pat- and Biochemical Sciences, University of Perugia, Perugia and Fondazione “Istituto di Ricovero e Cura per le Biotecnologie Trapiantologiche,” Perugia; ‡Instituto Clinico tern of is a major factor in determining the sensitiv- Humanitas, Istituto Di Ricovero e Cura a Carattere Scientifico; §State University of ity of a particular virus strain to inhibition by SP-D (3). In contrast, ¶ Milan, Milan, Italy; and Department of Pathology and Immunology, Washington 2ϩ University School of Medicine, St. Louis, MO 63110 SP-A inhibits influenza viruses via Ca -independent binding of the viral HA to terminal sialic acid expressed on the SP-A mole- Received for publication August 7, 2007. Accepted for publication December 26, 2007. cule, thereby blocking the receptor binding site on the viral HA The costs of publication of this article were defrayed in part by the payment of page such that it can no longer access cellular receptors. SP-A is there- charges. This article must therefore be hereby marked advertisement in accordance fore classified as a ␥ inhibitor, acting in a similar manner to the with 18 U.S.C. Section 1734 solely to indicate this fact. serum inhibitor ␣2-macroglobulin (13, 14). 1 This work was supported by Project Grant 400226 and a Programme Grant from The The long chain pentraxin (PTX3) is a 45-kDa protein that as- National Health and Medical Research Council of Australia, and by Research Grant RIGP/05/CR90 from Tecnogen Societa`Per Azioni Italy (to L.R.). E.C.C. was sup- sembles to form high m.w. multimers linked by interchain disul- ported by Grants HL44015 and HL29594 from the National Institutes of Health. A.M. fide bonds (15). The C-terminal domain (203 aa) of PTX3 shares was supported by Cariplo Foundation (Project Next Generation Optical Network for Broadband European Leadership), Telethon, Project Fluinnate, and Mugen from the European Commission. P.C.R. is a National Health and Medical Research Council R. D. Wright Research Fellow. 4 Abbreviations used in this paper: SP-D, surfactant protein D; SP-A, surfactant pro- 2 tein A; HA, hemagglutinin; NA, neuraminidase; CHO, Chinese hamster ovary; HAU, Address correspondence and reprint requests to Dr. Patrick C. Reading, Department hemagglutinating unit; MDCK, Madin-Darby canine kidney; BAL, bronchoalveolar of Microbiology and Immunology, University of Melbourne, Parkville 3010, Victoria, lavage; HI, hemagglutination inhibition; SA, sialic acid. Australia. E-mail address: [email protected] 3 L.R. and A.M. contributed equally to this study. Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org 3392 ANTIVIRAL ACTIVITY OF PTX3 AGAINST INFLUENZA homology with the classic short pentraxins C-reactive protein and (both IgG1), were obtained by immunization of PTX3Ϫ/Ϫ mice (18) with SAP, whereas the N-terminal domain (178 aa) does not show any purified murine PTX3. C1q from human serum was purchased from Cal- significant homology with other known proteins. PTX3 plays a biochem. Recombinant human SP-D was expressed by CHO-K1 cells and purified by sequential maltosyl-agarose and gel filtration chromatography; complex nonredundant role in vivo, recognizing a diverse range of dodecamers were used in all experiments as verified by ultrastructural anal- pathogens, modulating complement activity by binding C1q and ysis. The rabbit anti-human SP-D Ab was prepared using purified, C-ter- facilitating pathogen recognition by macrophages and dendritic minal domains of natural SP-D as Ag (1). cells (reviewed in Ref. 16). Despite a well documented role in Detection of PTX3 or SP-D binding to influenza virus by ELISA innate host defense against certain bacteria and fungi (17–20), few studies have addressed the antiviral activities of PTX3. To this To compare the binding of PTX3 or SP-D to different influenza viruses, wells of a microtiter tray were coated with increasing concentrations of end, a recent study described the ability of human PTX3 to bind purified influenza virus in 50 ␮l of TBS (0.05 M Tris-HCl, 0.15 M NaCl and inhibit the infectivity of human and murine cytomegaloviruses (pH 7.2)), blocked for Ͼ1 h with 10 mg of BSA per ml, and washed with (21). Furthermore, the short pentraxin SAP has been shown to act TBS containing 0.05% Tween 20 (TBST). Wells were incubated for 2 h as a ␤ inhibitor against influenza viruses, binding in a Ca2ϩ-de- with 50 ␮l of biotin-labeled human PTX3 (bPTX3; 0.1 ␮g/ml) or rhSP-D (1.0 ␮g/ml) in TBS-T containing 5 mg of BSA per ml and either 20 mM pendent manner to mannose-rich glycans on the viral HA to inhibit 2ϩ CaCl2 (BSA5-TBST-Ca ) or 5 mM EDTA (BSA5-TBST-EDTA) and then both hemagglutination and viral infectivity (22, 23). Therefore, in washed. Binding of bPTX3 was detected by the addition of streptavidin- this study we have assessed the antiviral activity of PTX3 against conjugated HRP (BD Pharmingen). Binding of SP-D was detected by ad- a range of influenza viruses, both in vitro and in vivo. dition of rabbit antiserum against rhSP-D for 2 h, followed by the addition of HRP-conjugated swine anti-rabbit Ig (DakoCytomation). A similar pro- tocol was used to determine binding of bPTX3 (0.25 ␮g/ml) and asialo- Materials and Methods Downloaded from bPTX3 (0.25 ␮g/ml) to wells coated with increasing concentrations of Viruses either HKx31 or C1q. HKx31 (H3N2) influenza virus is a laboratory-derived high-yielding reas- HA and HA inhibition tests sortant of A/PR/8/34 (PR8, H1N1) with A/Aichi/2/68 (Aichi/68; H3N2). Other viruses used included H3N2 subtype viruses, i.e., A/Memphis/1/71 Tests were performed in round-bottom 96-well microtiter plates at room (Mem/71), A/Udorn/307/72 (Udorn/72), A/Port Chalmers/1/73 (PtChal/ temperature using 1% v/v chicken erythrocytes. Hemagglutination titers 73), A/Victoria/75 (Vic/75), A/Bangkok/1/79 (Bang/79), A/Beijing/353/89 were determined by titration of virus samples in TBS followed by addition (Beij/89), A/Beijing/32/92 (Beij/92), A/Guandong/25/93 (Guan/93); and of an equivalent volume of chicken erythrocytes. For hemagglutination http://www.jimmunol.org/ additional H3N2 reassortants A/Texas/1/77 x PR8 (Tex/77-X), A/Philip- inhibition tests, dilutions of SP-D or PTX3 were prepared in TBS alone (or pines/2/82 x PR8 (Phil/82-X); and H1N1 subtype viruses, i.e., PR8, in TBS supplemented with 10 mM CaCl2 or 5 mM EDTA) and 4 hemag- A/NWS/33 (NWS), A/Brazil/11/78 (Brazil/78), A/Victoria/57/83 (Vic/83) glutinating units (HAU) of virus was added. Following 30 min of incuba- and A/Victoria/36/88 (Vic/88). tion, chicken erythrocytes were added and the ability of PTX3 or SP-D to Additional virus strains tested for sensitivity to PTX3 and SP-D in- inhibit virus-induced hemagglutination was assessed. Results are expressed cluded H3N2 subtype viruses (1997–2005), i.e., A/Sydney/5/97, A/Mos- as the minimum concentration of PTX3 or SP-D required to fully inhibit cow/10/99, A/Panama/2007/99, A/Philippines/472/2002, A/Kumamoto/ the hemagglutinating activity of 4 HAU of virus and are expressed as 102/2002, A/Fujian/411/2002, A/Singapore/37/2004, A/Victoria/523/2004, ␮g/ml. A/Wellington/1/2004, A/New York/55/2004, A/California/7/2004, A/Bris- NA and NA inhibition assays bane/3/2005, A/Victoria/512/2005, A/Wisconsin/67/2005; additional by guest on September 26, 2021 H1N1 viruses, i.e., A/Beijing/262/95, A/New Caledonia/20/1999, A/Fu- Influenza virus NA activity and its inhibition by PTX3 were measured by jian/156/2000, A/England/51/2002, A/Singapore/14/2004, A/Malaysia/ an enzyme-linked microplate assay in which Arachis hypogaea (peanut) 1513/2004, A/New Caledonia/9/2004, A/Brisbane/193/2004, A/Shenzhen/ lectin was used to detect ␤-D-galactose-N-acetylglucosamine sequences ex- 141/2005; and type B influenza viruses, i.e., B/Shangdon/7/97, B/Brisbane/ posed after the removal of sialic acid from fetuin (26). Titrations of NA 32/2002, B/Shanghai/361/2002, B/Jiangsu/10/2003, B/Malaysia/2506/2004, activity and inhibition of NA activity by PTX3 or SP-D were performed as B/Florida/7/2004, B/Ohio/1/2005, B/Victoria/502/2005. described (3). Viruses were obtained from Alan Hampson and Ian Barr, World Health Organization Collaborating Centre for Reference and Research on Influ- Virus neutralization assay enza, Melbourne, Australia. Influenza viruses were propagated in the al- lantoic cavity of 10-day-old embryonated hens’ eggs and purified from Neutralization of virus infectivity was measured by fluorescent-focus re- allantoic fluid as described previously (8). duction in monolayers of Madin-Darby canine kidney (MDCK) cells cul- Reassortant influenza viruses used in this study were generated by eight- tured in 96-well plates as described (3). In brief, dilutions of PTX3 were plasmid reverse genetics as previously described (24). Viruses were 7:1 mixed with virus, incubated at 37°C for 30 min and inoculated onto MDCK reassortants consisting of either the PR8 (H1N1) backbone with the HA or monolayers. After adsorption of the virus for 45 min at 37°C, the inoculum NA from Udorn/72 (H3N2; PR8-Udorn/72 HA and PR8-Udorn/72 was removed, fresh medium was added and plates were incubated at 37°C NA, respectively) or the Udorn/72 backbone with the HA or NA gene from for an additional 6 to 7 h. Cells were fixed and stained with anti-NP mAb PR8 (Udorn/72-PR8 HA and Udorn/72 PR8 NA, respectively). Eight-plas- A-3, and the total number of fluorescent foci in four representative fields mid reverse genetics was also used to generate wild-type PR8 and was counted and expressed as a percentage of the number of foci in the Udorn/72 viruses. The rescued viruses were recovered after 3 days and corresponding area of duplicate control wells infected with virus alone. To amplified in the allantoic cavity of 10-day-old embryonated hens’ eggs. test for inhibition by sugars or by mannan, diluted PTX3 was incubated The identity of all viruses was confirmed by restriction digestion and full- with sugar or mannan for 20 min at room temperature before addition of length sequencing of RT-PCR products for the HA, NP, NA, M, and NS virus. The appropriate concentration of sugar or mannan was also included . in the virus control. Proteins, sera, and Abs Mice, infections, and PTX3 treatment Recombinant human (rh)PTX3 was purified from the supernatants of Chi- C57BL/6, PTX3ϩ/ϩ, and PTX3Ϫ/Ϫ mice in a mixed C57BL/7 ϫ 129S nese hamster ovary (CHO) cells stably expressing the protein and biotin- background were housed in specific pathogen-free conditions in the animal labeled (bPTX3) as described (15). Bio-PTX3 was desialylated under non- facility at the University of Perugia, Italy. Adult mice (6–10 wk) were used denaturing conditions for4hat37°C using 2 mU of Vibrio cholerae type in all experiments. Mice were lightly anesthetized with diethyl ether and III NA (Sigma-Aldrich) per microgram of protein. Naturally occurring hu- infected via the intranasal route with 2 ϫ 104 PFU of influenza virus in 20 man PTX3 was purified by immunoaffinity from human fibrosarcoma cells ␮l of saline. Recombinant human PTX3 (1 and 2 mg/kg, via i.p. route) was ␣ 8387 exposed to TNF- (20 ng/ml) for 24 h (PTX38387). Recombinant and administered on the day of the infection and daily thereafter for 4 consec- naturally occurring human PTX3 used in this study were glycosylated mul- utive days. Experiments were performed according to the Italian Approved timers consisting of 8 covalently bound multimers (15, 25). A cDNA en- Animal Welfare Assurance A-3143-01. To determine virus titers in lungs, coding murine PTX3 cDNA was subcloned into the pSG5 expression vec- mice were euthanized and lungs were removed, homogenized, and clarified tor, stably transfected in CHO cells, and recombinant protein was purified by centrifugation and supernatants frozen at Ϫ70°C. The samples were from the culture supernatant as described (15). Two mAbs, 2C3 and 6B11 assayed for infectious virus by measuring plaque formation in MDCK cells The Journal of Immunology 3393

from Klebsiella pneumoniae inaCa2ϩ-dependent manner (20, 27) whereas binding of PTX3 to C1q is Ca2ϩ independent (15); ac- cordingly, binding to influenza virus strain HKx31 (H3N2) was not inhibited by chelation of Ca2ϩ with 5 mM EDTA. Furthermore, binding of bPTX3 was not inhibited by inclusion of D-mannose or mannan, but was inhibited by preincubation of HKx31-coated plates with purified PTX3 that was not biotinylated, indicating the specific nature of the interaction between PTX3 and HKx31. In contrast, binding of human SP-D to HKx31 was Ca2ϩ-de- pendent and inhibited in the presence of either mannose or man- nan (Fig. 1B). We next tested binding of biotin-PTX3 to a range of purified influenza viruses known to differ in their degree of glycosylation of the HA glycoprotein, namely HKx31 (7 glycosylation sites on HA), Udorn/72 (H3N2; 7 sites), Beij/89-X (H3N2; 9 sites), Phil82-X (H3N2; 10 sites) and PR8 (H1N1; 5 sites); a particular feature of the PR8 strain is the lack of glycosylation sites on the FIGURE 1. Binding of human PTX3 and human SP-D to purified in- fluenza viruses. Biotin-labeled PTX3 (bPTX3, 1.0 ␮g/ml) (A) or recombi- exposed head of the HA molecule (28, 29). PTX3 bound specifi- ␮ cally to wells coated with purified HKx31 and Udorn/72, but not Downloaded from nant human SP-D (5 0 g/ml) (B)inBSA5-TBST containing 10 mM CaCl2 alone (F) or supplemented with 25 mM D-mannose (ƒ) or 5 mg/ml man- to wells coated with equivalent concentrations of Beij/89-X, Phil/ ‚ E nan ( ), or in BSA5-TBST containing 5 mM EDTA ( ) were applied to 82-X or PR8 (Fig. 1C). SP-D displayed a very different pattern of wells coated with increasing concentrations of purified HKx31, and bind- reactivity, binding with high avidity to heavily glycosylated Phil/ ing of PTX3 or SP-D was determined by ELISA. In the PTX3-binding 82-X and Beij/89-X strains, with intermediate avidity to HKx31 ELISA, additional wells coated with increasing concentrations of HKx31 and Udorn/72, and weakly to strain PR8 (Fig. 1D). Together, these were incubated with 220 nM recombinant PTX3 for 60 min at 37°C, before findings indicate that PTX3 binds only to particular strains of in- http://www.jimmunol.org/ ␮ the addition of bPTX3 (1.0 g/ml) for an additional 60 min at 37°C, and fluenza virus, and that binding is not determined by the presence of levels of bPTX3 bound were determined (Ⅺ). bPTX3 (C) or human SP-D Ca2ϩ, nor by the amount of mannose-containing oligosaccharides (D) were applied to wells coated with increasing concentrations of either HKx31 (FE), Mem/71 (fⅪ), Beij/89-X (Œ‚), Phil/82-X ((ƒ)) or PR8 on the virus. Œ᭛ ( ) and binding of PTX3 or SP-D was determined. Closed symbols Antiviral activity of PTX3 against influenza virus HKx31 indicate binding levels in BSA5-TBST containing 10 mM CaCl2. Speci- ficity of bPTX3 binding was determined by incubating virus-coated wells The potential of recombinant human PTX3 to mediate antiviral with the 220 nM of unlabeled PTX3 for 60 min at 37°C, before the addition activity against influenza virus strain HKx31 was compared with of bPTX3 (C, open symbols). Addition of 220 nM human C-reactive pro- that of recombinant human SP-D. First, PTX3 was shown to in- tein, a control protein that does not bind influenza viruses, had no effect on hibit HKx31-induced hemagglutination of chicken erythrocytes, by guest on September 26, 2021 binding of bPTX3 to any of the virus strains tested (data not shown). indicating that binding of PTX3 to virus was sufficient to block Specificity of SP-D binding was determined by adding SP-D in BSA - 5 access of the viral HA glycoprotein to erythrocyte cell surface TBST containing 5 mM EDTA (D, open symbols). Equivalent coating receptors (Fig. 2A). The ability of PTX3 to inhibit hemagglutina- levels of different purified virus preparations were confirmed using mAb 2ϩ 165, which binds to the host-derived carbohydrate Ag that is characteristic tion was Ca independent and unaffected by the inclusion of of egg-grow influenza viruses (data not shown). mannan. In contrast, hemagglutination inhibition of HKx31 by SP-D was inhibited in the presence of mannan, or by chelation of Ca2ϩ with EDTA. in the presence of trypsin. For collection of bronchoalveolar lavage (BAL) We next compared the ability of PTX3 and SP-D to inhibit the fluids, mice were euthanized and lungs were flushed 3 times with a 1-ml activity of the viral NA. Preincubation of HKx31 with PTX3 (Fig. volume of PBS through a blunted 23-gauge needle inserted into the trachea. 2B) or SP-D (Fig. 2C) inhibited the viral NA in a dose-dependent manner and inhibition by SP-D, but not PTX3, was abolished in Assay for PTX3 in BAL fluid the presence of mannan. Preincubation with PTX3 (Fig. 2D)or The presence of PTX3 in mouse BAL fluid was assayed by ELISA on wells SP-D (Fig. 2E) also inhibited the ability of HKx31 to infect sus- ␮ coated with mAb 2C3 (anti-murine PTX3) at 1 g/ml in 15 mM carbonate ceptible MDCK cells in a neutralization assay. The neutralizing buffer (pH 9.6). After washing, wells were blocked with 3% skim milk for 2 h at room temperature, and samples were titrated relative to a standard activity of SP-D, but not PTX3, was inhibited in the presence of curve of murine PTX3 (10 ng/ml–9.7 pg/ml). Bound PTX3 was detected mannan. The sensitivity of BJx109 (highly glycosylated), HKx31 with 0.25 ␮g/ml biotin-labeled mAb 6B11 (anti-murine PTX3) followed by (intermediate levels of glycosylation), and PR8 (poorly glycosy- streptavidin-conjugated HRP and substrate. lated) to inhibition by SP-D or PTX3 was also examined using NA inhibition ELISA (Fig. 2, B and C) and neutralization assay (Fig. Results 2, D and E). In both assays a clear hierarchy was observed in the Binding of PTX3 to purified influenza viruses sensitivity of virus strains to SP-D (BJx109 Ͼ HKx31 Ͼ PR8). In SP-D, a collectin present in respiratory fluids, and the short pen- contrast, only HKx31 was sensitive to inhibition by PTX3. To- traxin SAP bind to influenza viruses in a Ca2ϩ-dependent manner gether, these studies clearly demonstrate that SP-D and PTX3 me- (4, 6, 22, 23) and mediate antiviral activity by binding to mannose diate antiviral activity against influenza viruses via a distinct residues on the viral HA/NA glycoproteins. Therefore, studies mechanism. were undertaken to assess the ability of the long pentraxin PTX3 to bind influenza virus by ELISA. Wells coated with increasing Antiviral activity of PTX3 against different strains of influenza concentrations of purified viruses were probed with biotin-PTX3 virus in the presence or absence of Ca2ϩ (Fig. 1A). PTX3 binds to li- We next examined a range of influenza isolates for their sensitivity gands such as the extracellular matrix protein TSG-6 and Omp A to PTX3 (and for comparison to SP-D) using an hemagglutination 3394 ANTIVIRAL ACTIVITY OF PTX3 AGAINST INFLUENZA

FIGURE 3. Inhibition of HA activity of H3N2 and H1N1 subtype vi- Downloaded from ruses by PTX3 or SP-D. Results shown are the minimum concentration of PTX3 or SP-D required to fully inhibit the hemagglutinating activity of 4 HAU of each strain of influenza virus tested. Experiments were performed as described in Materials and Methods. HI titers were determined at least twice for each virus strain, with similar results, and the data from a single experiment are shown. The dashed line represents the highest concentration FIGURE 2. Antiviral activity of PTX3 or SP-D against HKx31. A, In- of inhibitor tested. If PTX3 or SP-D were unable to inhibit hemagglutina- http://www.jimmunol.org/ hibition of virus-induced hemagglutination. Dilutions of PTX3 or SP-D tion at the highest concentration tested titers were recorded as Ͼ20 ␮g/ml 2ϩ were prepared in TBS containing 10 mM Ca alone or supplemented with or Ͼ10 ␮g/ml, respectively, and bars are marked with a plus (ϩ) symbol 5 mg/ml mannan, or in TBS containing 5 mM EDTA. Results show con- to indicate the Ͼ titer. centrations of PTX3 or SP-D required to fully inhibit hemagglutination (small circles) by 4 HAU of HKx31. Large circles indicate virus-induced hemagglutination. B and C, Inhibition of influenza virus NA activity. HKx31 (F), PR8 (᭜) or BJx109 (Œ) were mixed with increasing concen- PTX3 binds to the HA glycoprotein of PTX3S strains of trations of PTX3 (B) or SP-D (C) and assayed for NA activity as described. influenza virus To examine the effect of mannan on PTX3 or SP-D, titrations of either by guest on September 26, 2021 protein were prepared in the presence of 5 mg/ml mannan before the ad- To determine which glycoproteins were bound by PTX3, we used dition of HKx31 (E). D and E, Neutralization of virus infectivity. HKx31 eight-plasmid reverse genetics to construct wild-type viruses S R (F), PR8 (᭜) or BJx109 (Œ) were mixed with increasing concentrations of Udorn/72 (PTX3 ) and PR8 (PTX3 ), as well as reassortant vi- PTX3 (D) or SP-D (E), incubated for 30 min at 37°C and the amount of ruses consisting of 1) PR8 backbone with the HA or NA gene of infectious virus remaining was determined by fluorescent focus assay. In- Udorn/72 (PR8-Udorn72 HA and PR8-Udorn72 NA viruses, re- hibition of neutralization by mannan was examined by adding mannan (to spectively); or 2) Udorn/72 backbone with the HA or NA gene of a final concentration of 5 mg/ml) to PTX3 or SP-D (E) dilutions 20 min PR8 (Udorn/72-PR8 HA and Udorn72-PR8 NA, respectively). E before the addition of HKx31 ( ). Each virus was tested for sensitivity to PTX3 in HI assay or in a virus neutralization assay. As expected, wild-type Udorn/72 was sensitive to HI (Fig. 4A) and neutralization (Fig. 4B) by PTX3 but inhibition (HI) assay. In previous studies, we have reported that wild-type PR8 was resistant. This sensitivity to both HI and neu- early and late H3N2 subtype viruses differ in their degree of gly- tralization by PTX3 was found to correlate with expression of the cosylation and therefore in their sensitivity to collectins, such that Udorn/72 HA by the reassortant viruses. Furthermore, cells in- strains isolated after 1977 were particularly sensitive to SP-D (3). fected with wild-type Udorn/72 or Udorn/72-PR8 NA bound Data presented in Fig. 3 show the minimum concentration of PTX3 bPTX3; however, cells infected to similar levels with wild-type or SP-D required to inhibit the hemagglutinating activity of a range PR8 or Udorn/72-PR8 HA did not (Fig. 4C). Together, these data of H3N2 and H1N1 subtype viruses. Consistent with previous re- indicate that PTX3 binds to the HA glycoprotein of sensitive virus ports, we found later H3N2 isolates (1977–1993) particularly sen- strains. sitive to HI by human SP-D. In contrast, PTX3 showed a very different pattern of reactivity against H3N2 subtype viruses, with Recognition of sialic acid on PTX3 by influenza virus HA early isolates (1968–1973) sensitive to inhibition by PTX3 but Collectins such as SP-D, MBL, and conglutinin are classified as ␤ later strains (1977–1993) resistant to the highest concentration inhibitors of influenza viruses and act via Ca2ϩ-dependent binding tested. Furthermore, whereas H1N1 subtype isolates were either to mannose-containing glycans on the viral HA/NA glycoproteins sensitive or resistant to SP-D, all isolates tested were resistant to (6–8). In contrast, ␣2-macroglobulin and collectin SP-A mediate inhibition by PTX3 (Fig. 3). We tested an additional range of hu- antiviral activity by providing sialic acid residues that are recog- man influenza viruses for sensitivity to PTX3, including recent nized by the HA glycoprotein of influenza viruses and are classi- H3N2 isolates (1997–2005; refer to Materials and Methods), fied as ␥ inhibitors (13, 14, 30). Oligosaccharide analysis of PTX3 H1N1 isolates (1995–2005; refer to Materials and Methods) and has indicated the presence of complex type sugars displaying vary- type B influenza virus isolates (1997–2004; refer to Materials and ing degrees of sialylation attached to the single N-linked glyco- Methods); all strains tested (1995–2005) were found to be resistant sylation site at Asn220 in the pentraxin domain of the PTX3 mol- to PTX3 (data not shown). ecule (25). To assess the contribution of oligosaccharide to The Journal of Immunology 3395

FIGURE 6. PTX3 protects from HKx31 infection in vivo. C57BL6 Downloaded from FIGURE 4. PTX3 binds to influenza virus HA glycoprotein. A, Inhibition mice were infected via the intranasal route with 2 ϫ 104 of HKx31 (circles) of virus-induced hemagglutination by PTX3. Dilutions of PTX3 were prepared or PR8 virus (squares). PTX3 was administered beginning the day of the 2ϩ in TBS containing 10 mM Ca and their ability to inhibit hemagglutination infection and daily for 4 consecutive days at a dose rate of 1 (EⅪ)or2 of wild-type Udorn/72 or PR8, or the reassortant viruses Udorn/72-PR8 HA, (Qµ) mg/kg by the i.p. route. Control mice received the diluent alone Udorn/72-PR8 NA, PR8-Udorn/72 HA or PR8-Udorn/71-NA was tested. Re- Ff ( ). Survival (%) (A and C) and viral load (log10/gram/organ PFU as sults show concentrations of PTX3 required to inhibit (small circles) 4 HAU determined by standard plaque assay of lung tissues on MDCK cells) (B of each virus. B, Neutralization of reassortant viruses by PTX3. Wild-type and D) are shown. Results are representative of three independent exper- http://www.jimmunol.org/ F Ⅺ .p Ͻ 0.001; PTX3-treated vs untreated mice ,ءء ,p Ͻ 0.05 ,ء .Udorn/72 ( )orPR8( ), or an equivalent infectious dose of the reassortant iments viruses Udorn/72-PR8 HA(ƒ), Udorn/72-PR8 NA(), PR8-Udorn/72 HA (Œ) or PR8-Udorn/72-NA (‚) was mixed with increasing concentrations of PTX3, incubated for 30 min at 37°C and the amount of infectious virus remaining was determined by fluorescent focus assay. C, Binding of bPTX3 to cells infected with reassortant viruses. MDCK cells were infected with wild-type Udorn/72 interactions with influenza viruses we compared the ability of en- (clear histogram, black line) or PR8 (black histogram, black line), or the re- zymatically desialylated bPTX3 (asialo-bPTX3) to bind to HKx31 assortant viruses Udorn/72-PR8 HA (clear histogram, dashed line) or Udorn/ by ELISA. Treatment with V. cholerae type I⌱⌱ sialidase com- 72-PR8 NA (grey histogram, black line), and binding of bPTX3 (1.0 ␮g/ml) pletely inhibited the ability of bPTX3 to bind HKx31 (Fig. 5A). to infected cells was assessed at 6 h postinfection. Hemadsorption assays con- Consistent with previous reports (25), desialylated bPTX3 bound by guest on September 26, 2021 firmed a similar percentage of cells (70–80%) expressed the HA glycoprotein complement component C1q more effectively than untreated at the cell surface in each sample at this time. bPTX3 (Fig. 5B), confirming that the desialylated protein retained binding activity for other known ligands of PTX3. Furthermore, asialo-bPTX3 failed to inhibit hemagglutination or neutralize in- fectivity of HKx31 (data not shown). Together, these data indicate that like ␣2-macroglobulin and SP-A, PTX3 provides sialic acid residues which are bound by the HA glycoprotein of certain virus strains, and therefore acts as a ␥ inhibitor against influenza viruses.

Administration of PTX3 to mice reduces morbidity following infection with HKx31 To explore the role of PTX3 in vivo, C57BL/6 mice were infected with either HKx31 (PTX3S) or PR8 (PTX3R) and treated with different doses of human PTX3 daily for 4 days, beginning the day of infection. Mice were monitored for survival over the next 15 days or killed at day 5 postinfection and viral load quantified in the lungs by standard plaque assay. Infection with HKx31 resulted in 40% survival of untreated mice; however, daily treatment with either 1 mg/kg or 2 mg/kg PTX3 promoted 100% survival of HKx31-infected animals (Fig. 6A). The antiviral effects of PTX3 treatment were reflected in lung viral titers at day 5 postinfection (Fig. 6B), with viral titers reduced 10- to 100-fold in mice treated with 1 mg/kg or 2 mg/kg, respectively, when compared with those of untreated animals. In contrast, PTX3 treatment had no effect on FIGURE 5. Recognition of sialic acid on PTX3 by influenza virus HA is survival following infection with PR8 virus (Fig. 6C) and 80% of required to mediate antiviral activity. Wells coated with increasing concentra- tions of either HKx31 (A) C1q (B) were probed with 2.0 ␮g/ml bPTX3 (fŒ) mice succumbed to disease in the absence or presence of thera- or asialo-bPTX3 (Ⅺ‚), and levels of bound pentraxin were determined by peutic PTX3 treatment (2 mg/kg). PTX3 treatment, did however, ELISA as described in Materials and Methods. Neither bPTX3 nor desialy- have some effect in reducing viral load in the lungs of PR8-in- lated bPTX3 showed significant binding to ELISA wells coated with equiva- fected mice treated with 1 mg/ml and 2 mg/kg of PTX3 suggesting lent concentrations of virus strains PR8 or BJx109 (data not shown). an opsonin-independent effect in antiviral resistance (Fig. 6D). 3396 ANTIVIRAL ACTIVITY OF PTX3 AGAINST INFLUENZA

lungs 5 days after infection. We found virus titers were consis- tently higher in the lungs of PTX3Ϫ/Ϫ infected with HKx31 com- pared with PTX3ϩ/ϩ mice (Fig. 7B) however virus titers were not significantly different in the lungs of PTX3Ϫ/Ϫ or PTX3ϩ/ϩ mice infected with PR8. These data indicate an important role for en- dogenous PTX3 in limiting virus replication following intranasal infection with the PTX3S strain HKx31 but not with the PTX3R PR8 strain.

Discussion Innate inhibitors of influenza A viruses, including the collectins SP-A and SP-D and the pentraxin SAP, are constitutively ex- pressed in fluids lining the respiratory tract. Previous reports have described the activity of SAP against influenza viruses in vitro (22, 23), however in vivo studies indicate a limited role in host defense against influenza (32). Herein we report that long pentraxin PTX3 mediates a range of antiviral activities against influenza viruses in

vitro but also makes an important contribution to early host de- Downloaded from fense in vivo. Together, these studies identify PTX3 as a novel inhibitor of influenza viruses and highlight its potential for thera- peutic use during influenza infection. In this study we describe marked differences in the mechanisms FIGURE 7. Murine PTX3 is induced during influenza infections and by which long pentraxin PTX3 acts against influenza viruses when plays a protective role in vivo against HKx31 virus. A, Levels of PTX3 in compared with the collectin SP-D, or to the short pentraxin SAP. http://www.jimmunol.org/ BAL fluids during influenza virus infection. C57BL/6 mice were infected PTX3 acts as a potent ␥ inhibitor, providing sialylated ligands that with 105 PFU of either HKx31 or PR8 and at days 3 and 5 postinfection, mimic the structure of the cellular receptors used by influenza mice were killed and levels of PTX3 in BAL fluids determined by ELISA. viruses thereby blocking the receptor-binding site of HA. In con- PTX3 levels were also determined from the BAL of uninfected (un) mice. trast, SAP (and SP-D) act as classic ␤ inhibitors, binding to man- Data shown represent the mean (ϮSD) titer of murine PTX3 in BAL (n ϭ nose-rich glycans on the viral HA to sterically block access of the p Ͻ 0.001, BAL PTX3 levels from day 5 PR8- ,ءء .(mice per group 5 Ϫ/Ϫ viral HA to cell surface receptors (22, 23). Despite in vitro activity infected mice compared with day 5 HKx31-infected mice. B, PTX3 against influenza viruses, SAPϪ/Ϫ mice did not display enhanced mice are more susceptible to infection with HKx31 virus. PTX3ϩ/ϩ and Ϫ/Ϫ ϫ 4 susceptibility to influenza infection (32). The sequence and regu-

PTX3 mice were infected with 2 10 PFU of either HKx31 or PR8 by guest on September 26, 2021 lation of SAP have diverged markedly from man to mouse such and virus titers, expressed as log10/gram/organ, were quantified on MDCK p Ͻ 0.001, that murine SAP binds influenza virus poorly when compared with ,ءء .cells at day 5 postinfection by standard plaque assay Ϫ/Ϫ PTX3ϩ/ϩ mice compared with PTX3Ϫ/Ϫ mice, n ϭ 5 per group. Results human SAP; even so, no protection was afforded to SAP mice are representative of two independent experiments. transgenic for human SAP following influenza infection (32). PTX3 is highly conserved in evolution with ortholog genes de- scribed in mammals as well as in birds and the most ancient ver- This reduction was not, however, sufficient to protect animals from tebrate Takifugu rubripes (33). Consistent with this notion, we the severe disease and subsequent lethality of the PR8 virus. found murine PTX3 to bind effectively to purified HKx31 and to mediate a range of antiviral activities in vitro at equivalent doses Role of endogenous PTX3 during infection of mice with PTX3S R to that of human PTX3 (data not shown). Furthermore, mice with or PTX3 virus strains a targeted deletion in the PTX3 gene were markedly more suscep- We found recombinant murine PTX3 to be active against HKx31 tible to HKx31 infection (Fig. 7B), and treatment of C57BL/6 mice and other “early” H3N2 virus strains, but not against PR8 virus, in with human PTX3 effectively reduced the viral load in the lungs of vitro. To assess the role of endogenous PTX3 in the murine model infected animals (Fig. 6B). Together, these data indicate that en- of influenza infection we first determined levels of PTX3 present dogenous murine PTX3 is an important antiviral host defense pro- in airway fluids from uninfected and virus-infected mice. After tein and can play a role in limiting influenza infection in vivo. infection with 105 PFU of HKx31, PTX3 levels in BAL were noted Naturally occurring ␥ inhibitors of influenza virus have been to rise during the course of infection (Fig. 7A). Of interest, infec- known to exist in serum of different species (including human) and tion of mice with an equivalent dose of the PTX3R PR8 virus in fluid secretions such as lung and pleural fluids for many years induced higher levels of PTX3 at day 5 postinfection ( p Ͻ 0.001 (reviewed in Ref. 34, 35). The high molecular-weight glycoprotein compared with HKx31-infected mice at day 5), likely reflecting the ␣2-macroglobulin is the major inhibitor in horse, guinea pig and severe disease associated with infection of mice by this virulent pig sera and equine ␣2-macroglobulin has been particularly well virus (31). Inoculation of mice with 105 PFU of HKx31 or PR8 characterized (14, 36, 37). Resistance to equine ␣2-macroglobulin that had been UV inactivated to destroy virus infectivity did not is associated with a single alteration at position 226 in induce levels of PTX3 above those observed in uninfected mice at the receptor binding pocket of HA and is associated with a change day 3 or day 5 postinfection (data not shown). The induction of in receptor specificity from sialic acid (SA) linked to galactose by murine PTX3 during influenza infections is consistent with a role ␣2,6 linkages (SA␣2,6Gal) to SA␣2,3Gal (38, 39). We found in host defense. wild-type HKx31 and a horse serum-resistant mutant of this virus To formally address the role of endogenous PTX3 during influ- to be equally sensitive to inhibition by murine or human PTX3 enza infection we infected PTX3ϩ/ϩ or PTX3Ϫ/Ϫ mice with an (data not shown), indicating definitive differences in mode of ac- equivalent dose of HKx31 or PR8 and assessed viral growth in the tion between equine ␣2-macroglobulin and PTX3. The molecular The Journal of Immunology 3397 mechanisms underlying the ability of human PTX3 to bind to and Treatment of C57BL/6 mice with human PTX3 clearly demon- inactivate influenza viruses will be the subject of future studies. strate its therapeutic potential in ameliorating disease and reducing Innate inhibitors such as PTX3 could influence the selection of morbidity associated with influenza infection, however as recent influenza virus receptor variants in natural hosts; however, direct H1N1, H3N2 and type B influenza viruses are resistant to PTX3 it experimental support for this hypothesis is lacking. The H3N2 is likely that the role of PTX3 during current human influenza subtype has circulated widely in the human population since its infections is limited. This does not, however, rule out the potential introduction from an avian source in 1968. Previous studies have for endogenous or therapeutic PTX3 to modulate disease should a demonstrated that early H3N2 subtype isolates recognize both new subtype emerge within the human population in the future. As SA␣2,3Gal and SA␣2,6Gal linkages whereas isolates from 1975 our data using H3N2 virus strains demonstrated, when a new sub- to 1994 recognize only SA␣2,6Gal (40). Thus, it appears that evo- type emerges it may be sensitive to inhibition by PTX3 and the lution of this subtype has selected for receptor variants focused on acquisition of resistance may be a feature associated with its con- recognition of SA␣2,6Gal receptors. Our findings that early H3N2 tinued survival in the human population. Given the devastating isolates (1968–1973) were sensitive to PTX3 while later isolates morbidity and mortality that may be associated with the appear- (1975–2005) were resistant correlate with the presence of ance of a new subtype of human influenza, the role of PTX3 as an SA␣2,3Gal residues on PTX3, such that later H3N2 isolates are innate antiviral protein may be particularly important in future in- unable to recognize this linkage. Indeed, biochemical analysis of fluenza outbreaks. human PTX3 confirmed the presence of SA␣2,3 on human PTX3 Respiratory secretions contain a complex mixture of innate pro- (Antonio Inforzato, unpublished observations). Neutralizing ␥ in- teins, including PTX3 and members of the collectin family. Early hibitors such as PTX3 (and perhaps SP-A) bearing SA␣2,3Gal and late strains of H3N2 subtype viruses differ in their degree of Downloaded from may provide additional selective pressure to drive evolution of glycosylation and therefore in their sensitivity to collectins, with human viruses toward a decreased affinity for SA␣2,3Gal. Such heavily glycosylated strains isolated after 1977 being particularly evolution would not be expected compromise the fitness of virus sensitive to SP-D (Ref. 3 and Fig. 3). PTX3 and SP-D may play because SA␣2,6Gal is the predominant receptor type present on distinct but cooperative roles in innate host defense against H3N2 epithelial cells lining the human respiratory tract. subtype viruses; PTX3 sensitivity was a feature of early virus PTX3 may interfere with influenza virus infection in vivo in a strains (1968–1975) whereas SP-D sensitivity was a feature of http://www.jimmunol.org/ later (1977–1993) and currently circulating strains. Studies using number of ways. Neutralization of virus infectivity may result SP-DϪ/Ϫ mice (9, 11) have demonstrated the importance of SP-D from direct blocking of virus attachment or entry into host cells, in limiting disease in mice infected with late H3N2 subtype viruses whereas inhibition of viral NA by PTX3 may inhibit the release of that we have shown to be resistant to PTX3. Together, innate in- newly formed virus particles from the surface of infected cells in hibitors may play a cooperative role in limiting initial virus repli- a manner analogous to that of NA-specific Abs (41). Incubation of cation and spread but the predominant inhibitor acting to limit influenza virus with other sialylated inhibitors, such as SP-A, virus growth may be different for particular virus strains. gp340, and mucin, induces viral aggregation (2) which could re- duce levels of infectious virus and promote clearance by muco- Acknowledgments by guest on September 26, 2021 ciliary and phagocytic mechanisms. We have also found that PTX3 We thank Dr. Marica Sassano from Tecnogen Societa`Per Azioni for the binds to influenza virus-infected epithelial cells (Fig. 4C), suggest- supply of PTX3 for the in vivo studies, Ian Barr and Robert Shaw from ing the potential for destruction before the release of newly syn- World Health Organization Collaborating Centre for Reference and Re- thesized virions. To this end, PTX3 has been shown to act directly search on Influenza (Melbourne, Australia) for coordinating testing of ad- as an opsonin, increasing the phagocytic activity of macrophages ditional type A H1N1 and H3N2 subtype viruses and type B influenza for particular yeasts (17, 18). Given that PTX3 binding to apopto- viruses, and Dr. Robert Webster, St. Jude Children’s Research Hospital tic cells enhanced deposition of complement components C1q and (Memphis, TN) for provision of the plasmid vector used to create the C3 (42), opsonization of virus-infected cells with complement reverse engineered viruses for this study. components may further promote further uptake and destruction of virus-infected cells by professional APCs. Disclosures Our in vivo data describe a role for both human and murine The authors have no financial conflict of interest. PTX3 in limiting the growth of PTX3S virus strains in the respi- ratory tract of influenza virus-infected mice. In addition, we report References 1. Hartshorn, K. L., M. R. White, T. Mogues, T. Ligtenberg, E. Crouch, and that therapeutic treatment of C57BL/6 mice with human PTX3 U. Holmskov. 2003. Lung and salivary scavenger receptor glycoprotein-340 con- also reduces the growth of the resistant PR8 strain (Fig. 6D). Al- tribute to the host defense against influenza A viruses. Am. J. Physiol. 285: though therapeutic treatment of uninfected B6 mice with PTX3 L1066–L1076. 2. White, M. R., E. Crouch, M. van Eijk, M. Hartshorn, L. Pemberton, I. Tornoe, does not enhance serum cytokine levels (data not shown), others U. Holmskov, and K. L. Hartshorn. 2005. Cooperative anti-influenza activities of have reported PTX3 to facilitate production of cytokines in an respiratory innate immune proteins and neuraminidase inhibitor. Am. J. Physiol. 288: L831–L840. inflammatory or infectious environment (43, 44). Future studies 3. Reading, P. C., L. S. Morey, E. C. Crouch, and E. M. Anders. 1997. Collectin- will determine the effect of therapeutic PTX3 treatment on the mediated antiviral host defense of the lung: evidence from influenza virus infec- production of proinflammatory cytokines in the airways during tion of mice. J. Virol. 71: 8204–8212. S R 4. Hartshorn, K. L., M. R. White, V. Shepherd, K. Reid, J. C. Jensenius, and infection with PTX3 /PTX3 virus strains; cytokines such as E. C. Crouch. 1997. Mechanisms of anti-influenza activity of surfactant proteins TNF-␣ have been reported to modulate influenza virus-induced A and D: comparison with serum collectins. Am. J. Physiol. 273: L1156–L1166. 5. Hartshorn, K. L., K. B. Reid, M. R. White, J. C. Jensenius, S. M. Morris, inflammation and to mediate direct antiviral activity (45, 46). The A. I. Tauber, and E. Crouch. 1996. Neutrophil deactivation by influenza A vi- PTX3R PR8 virus did, however, grow to similar titers in mouse ruses: mechanisms of protection after viral opsonization with collectins and hem- lung in PTX3ϩ/ϩ and PTX3Ϫ/Ϫ mice (Fig. 7B) and C57BL/6 mice agglutination-inhibiting antibodies. Blood 87: 3450–3461. 6. Hartshorn, K. L., E. C. Crouch, M. R. White, P. Eggleton, A. I. Tauber, D. Chang, were equally susceptible to PR8-induced disease despite PTX3 and K. Sastry. 1994. Evidence for a protective role of pulmonary surfactant pro- treatment (Fig. 6C), arguing that the direct antiviral role of PTX3 tein D (SP-D) against influenza A viruses. J. Clin. Invest. 94: 311–319. 7. Hartley, C. A., D. C. Jackson, and E. M. Anders. 1992. Two distinct serum may be the most important in protection against influenza infection mannose-binding lectins function as ␤ inhibitors of influenza virus: identification in vivo. of bovine serum ␤ inhibitor as conglutinin. J. Virol. 66: 4358–4363. 3398 ANTIVIRAL ACTIVITY OF PTX3 AGAINST INFLUENZA

8. Anders, E. M., C. A. Hartley, and D. C. Jackson. 1990. Bovine and mouse serum 26. Lambre, C. R., H. Terzidis, A. Greffard, and R. G. Webster. 1990. Measurement ␤ inhibitors of influenza A viruses are mannose-binding lectins. Proc. Natl. Acad. of anti-influenza neuraminidase antibody using a peroxidase-linked lectin and Sci. USA 87: 4485–4489. microtitre plates coated with natural substrates. J. Immunol. Methods 135: 49–57. 9. Hawgood, S., C. Brown, J. Edmondson, A. Stumbaugh, L. Allen, J. Goerke, 27. Salustri, A., C. Garlanda, E. Hirsch, M. De Acetis, A. Maccagno, B. Bottazzi, H. Clark, and F. Poulain. 2004. Pulmonary collectins modulate strain-specific A. Doni, A. Bastone, G. Mantovani, P. Beck Peccoz, et al. 2004. PTX3 plays a influenza a virus infection and host responses. J. Virol. 78: 8565–8572. key role in the organization of the cumulus oophorus extracellular matrix and in 10. LeVine, A. M., K. Hartshorn, J. Elliott, J. Whitsett, and T. Korfhagen. 2002. in vivo fertilization. Development 131: 1577–1586. Absence of SP-A modulates innate and adaptive defense responses to pulmonary 28. Caton, A. J., G. G. Brownlee, J. W. Yewdell, and W. Gerhard. 1982. The anti- influenza infection. Am. J. Physiol. 282: L563–L572. genic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). 11. LeVine, A. M., J. A. Whitsett, K. L. Hartshorn, E. C. Crouch, and Cell 31: 417–427. T. R. Korfhagen. 2001. Surfactant protein D enhances clearance of influenza A 29. Nakamura, K., and R. W. Compans. 1979. Host cell- and virus strain-dependent virus from the lung in vivo. J. Immunol. 167: 5868–5873. differences in oligosaccharides of hemagglutinin glycoproteins of influenza A 12. Li, G., J. Siddiqui, M. Hendry, J. Akiyama, J. Edmondson, C. Brown, L. Allen, viruses. Virology 95: 8–23. S. Levitt, F. Poulain, and S. Hawgood. 2002. Surfactant protein-A-deficient mice 30. Benne, C. A., C. A. Kraaijeveld, J. A. van Strijp, E. Brouwer, M. Harmsen, display an exaggerated early inflammatory response to a ␤-resistant strain of J. Verhoef, L. M. van Golde, and J. F. van Iwaarden. 1995. Interactions of sur- influenza A virus. Am. J. Respir. Cell Mol. Biol. 26: 277–282. factant protein A with influenza A viruses: binding and neutralization. J. Infect. 13. Hanaoka, K., T. J. Pritchett, S. Takasaki, N. Kochibe, S. Sabesan, J. C. Paulson, Dis. 171: 335–341. and A. Kobata. 1989. 4-O-acetyl-N-acetylneuraminic acid in the N-linked car- 31. Hennet, T., H. J. Ziltener, K. Frei, and E. Peterhans. 1992. A kinetic study of bohydrate structures of equine and guinea pig ␣ 2-macroglobulins, potent inhib- immune mediators in the lungs of mice infected with influenza A virus. J. Im- itors of influenza virus infection. J. Biol. Chem. 264: 9842–9849. munol. 149: 932–939. 14. Pritchett, T. J., and J. C. Paulson. 1989. Basis for the potent inhibition of influ- 32. Herbert, J., W. L. Hutchinson, J. Carr, J. Ives, R. Jakob-Roetne, K. Yamamura, enza virus infection by equine and guinea pig ␣ 2-macroglobulin. J. Biol. Chem. M. Suzuki, and M. B. Pepys. 2002. Influenza virus infection is not affected by 264: 9850–9858. serum amyloid P component. Mol. Med. 8: 9–15. 15. Bottazzi, B., V. Vouret-Craviari, A. Bastone, L. De Gioia, C. Matteucci, G. Peri, 33. Garlanda, C., B. Bottazzi, A. Bastone, and A. Mantovani. 2005. Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and fe-

F. Spreafico, M. Pausa, C. D’Ettorre, E. Gianazza, et al. 1997. Multimer forma- Downloaded from tion and ligand recognition by the long pentraxin PTX3: similarities and differ- male fertility. Annu. Rev. Immunol. 23: 337–366. ences with the short pentraxins C-reactive protein and serum amyloid P compo- 34. Krizanova, O., and V. Rathova. 1969. Serum inhibitors of myxoviruses. Curr. nent. J. Biol. Chem. 272: 32817–32823. Top. Microbiol. Immunol. 47: 125–151. 35. Gottschalk, A., G. Belyavin, and F. Biddle. 1972. Glycoproteins as influenza 16. Bottazzi, B., C. Garlanda, G. Salvatori, P. Jeannin, A. Manfredi, and virus hemagglutinin inhibitors and as cellular receptors. In Glycoproteins: Their A. Mantovani. 2006. Pentraxins as a key component of innate immunity. Curr. Composition, Structure and Function. A. Gottschalk, ed. Elsevier Publishing Opin. Immunol. 18: 10–15. Company, New York, pp. 1082–1096. 17. Diniz, S. N., R. Nomizo, P. S. Cisalpino, M. M. Teixeira, G. D. Brown, 36. Matrosovich, M., P. Gao, and Y. Kawaoka. 1998. Molecular mechanisms of A. Mantovani, S. Gordon, L. F. Reis, and A. A. Dias. 2004. PTX3 function as an

serum resistance of human influenza H3N2 virus and their involvement in virus http://www.jimmunol.org/ opsonin for the dectin-1-dependent internalization of zymosan by macrophages. adaptation in a new host. J. Virol. 72: 6373–6380. J. Leukocyte Biol. 75: 649–656. 37. Ryan-Poirier, K. A., and Y. Kawaoka. 1993. ␣ 2-macroglobulin is the major 18. Garlanda, C., E. Hirsch, S. Bozza, A. Salustri, M. De Acetis, R. Nota, neutralizing inhibitor of influenza A virus in pig serum. Virology 193: 974–976. A. Maccagno, F. Riva, B. Bottazzi, G. Peri, et al. 2002. Non-redundant role of the 38. Rogers, G. N., J. C. Paulson, R. S. Daniels, J. J. Skehel, I. A. Wilson, and long pentraxin PTX3 in anti-fungal innate immune response. Nature 420: D. C. Wiley. 1983. Single amino acid substitutions in influenza haemagglutinin 182–186. change receptor binding specificity. Nature 304: 76–78. 19. Gaziano, R., S. Bozza, S. Bellocchio, K. Perruccio, C. Montagnoli, L. Pitzurra, 39. Rogers, G. N., T. J. Pritchett, J. L. Lane, and J. C. Paulson. 1983. Differential G. Salvatori, R. De Santis, P. Carminati, A. Mantovani, and L. Romani. 2004. sensitivity of human, avian, and equine influenza A viruses to a glycoprotein Anti- efficacy of pentraxin 3 alone and in combination with inhibitor of infection: selection of receptor specific variants. Virology 131: antifungals. Antimicrob. Agents Chemother. 48: 4414–4421. 394–408. 20. Jeannin, P., B. Bottazzi, M. Sironi, A. Doni, M. Rusnati, M. Presta, V. Maina, 40. Ryan-Poirier, K., Y. Suzuki, W. J. Bean, D. Kobasa, A. Takada, T. Ito, and G. Magistrelli, J. F. Haeuw, G. Hoeffel, et al. 2005. Complexity and comple- Y. Kawaoka. 1998. Changes in H3 influenza A virus receptor specificity during by guest on September 26, 2021 mentarity of outer membrane protein A recognition by cellular and humoral in- replication in humans. Virus Res. 56: 169–176. nate immunity receptors. Immunity 22: 551–560. 41. Kilbourne, E. D., W. G. Laver, J. L. Schulman, and R. G. Webster. 1968. An- 21. Bozza, S., F. Bistoni, R. Gaziano, L. Pitzurra, T. Zelante, P. Bonifazi, tiviral activity of antiserum specific for an influenza virus neuraminidase. J. Virol. K. Perruccio, S. Bellocchio, M. Neri, A. M. Iorio, et al. 2006. Pentraxin 3 protects 2: 281–288. from MCMV infection and reactivation through TLR sensing pathways leading 42. Nauta, A. J., B. Bottazzi, A. Mantovani, G. Salvatori, U. Kishore, to IRF3 activation. Blood 108: 3387–3396. W. J. Schwaeble, A. R. Gingras, S. Tzima, F. Vivanco, J. Egido, et al. 2003. 22. Andersen, O., K. Vilsgaard Ravn, I. Juul Sorensen, G. Jonson, E. Holm Nielsen, Biochemical and functional characterization of the interaction between pentraxin and S. E. Svehag. 1997. Serum amyloid P component binds to influenza A virus 3 and C1q. Eur. J. Immunol. 33: 465–473. haemagglutinin and inhibits the virus infection in vitro. Scand. J. Immunol. 46: 43. Soares, A. C., D. G. Souza, V. Pinho, A. T. Vieira, J. R. Nicoli, F. Q. Cunha, 331–337. A. Mantovani, L. F. Reis, A. A. Dias, and M. M. Teixeira. 2006. Dual function 23. Horvath, A., I. Andersen, K. Junker, B. Lyck Fogh-Schultz, E. Holm Nielsen, of the long pentraxin PTX3 in resistance against pulmonary infection with Kleb- S. Gizurarson, O. Andersen, J. Karman, E. Rajnavolgyi, A. Erdei, and siella pneumoniae in transgenic mice. Microbes Infect. 8: 1321–1329. S. E. Svehag. 2001. Serum amyloid P component inhibits influenza A virus in- 44. Dias, A. A., A. R. Goodman, J. L. Dos Santos, R. N. Gomes, A. Altmeyer, fections: in vitro and in vivo studies. Antiviral Res. 52: 43–53. P. T. Bozza, M. F. Horta, J. Vilcek, and L. F. Reis. 2001. TSG-14 transgenic mice 24. Neumann, G., T. Watanabe, H. Ito, S. Watanabe, H. Goto, P. Gao, M. Hughes, have improved survival to endotoxemia and to CLP-induced . J. Leukocyte D. R. Perez, R. Donis, E. Hoffmann, et al. 1999. Generation of influenza A Biol. 69: 928–936. viruses entirely from cloned cDNAs. Proc. Natl. Acad. Sci. USA 96: 9345–9350. 45. Peper, R. L., and H. Van Campen. 1995. as a mediator of 25. Inforzato, A., G. Peri, A. Doni, C. Garlanda, A. Mantovani, A. Bastone, inflammation in influenza A viral pneumonia. Microb. Pathog. 19: 175–183. A. Carpentieri, A. Amoresano, P. Pucci, A. Roos, et al. 2006. Structure and 46. Hussell, T., A. Pennycook, and P. J. Openshaw. 2001. Inhibition of tumor ne- function of the long pentraxin PTX3 glycosidic moiety: fine-tuning of the inter- crosis factor reduces the severity of virus-specific lung immunopathology. Eur. action with C1q and complement activation. Biochemistry 45: 11540–11551. J. Immunol. 31: 2566–2573.