The Macrophage Soluble Receptor AIM/Api6/CD5L Displays a Broad Pathogen Recognition Spectrum and Is Involved in Early Response to Microbial Aggression

RESEARCH ARTICLE

The macrophage soluble receptor AIM/Api6/CD5L displays a broad pathogen recognition spectrum and is involved in early response to microbial aggression

Vanesa G. Martinez1,6, Cristina Escoda-Ferran1,6, Ineˆs Tadeu Simo˜es1, Satoko Arai2, Marc Orta Mascaro´ 1, Esther Carreras1, Mario Martı´nez-Florensa1, Jose´ Yelamos3, Toru Miyazaki2 and Francisco Lozano1,4,5

Apoptosis inhibitor of macrophages (AIMs), a homologue of human Spa, is a mouse soluble member of the scavenger receptor cysteine-rich superfamily (SRCR-SF). This family integrates a group of proteins expressed by innate and adaptive immune cells for which no unifying function has yet been described. Pleiotropic functions have been ascribed to AIM, from viability support in lymphocytes during thymic selection to lipid metabolism and anti-inflammatory effects in autoimmune pathologies. In the present report, the pathogen binding properties of AIM have been explored. By using a recombinant form of AIM (rAIM) expressed in mammalian cells, it is shown that this protein is able to bind and aggregate Gram-positive and Gram-negative bacteria, as well as pathogenic and saprophytic fungal species. Importantly, endogenous AIM from mouse serum also binds to microorganisms and secretion of AIM was rapidly induced in mouse spleen macrophages following exposure to conserved microbial cell wall components. Cytokine release induced by well-known bacterial and fungal Toll-like receptor (TLR) ligands on mouse splenocytes was also inhibited in the presence of rAIM. Furthermore, mouse models of pathogen-associated molecular patterns (PAMPs)-induced septic shock of bacterial and fungal origin showed that serum AIM levels changed in a time-dependent manner. Altogether, these data suggest that AIM plays a general homeostatic role by supporting innate humoral defense during pathogen aggression. Cellular & Molecular Immunology (2014) 11, 343–354; doi:10.1038/cmi.2014.12; published online 3 March 2014

Keywords: innate immunity; pattern recognition receptor; scavenger receptor

INTRODUCTION components essential for pathogen survival and not shared by The innate immune system represents the first line of host the host; examples of PAMPs include lipopolysaccharide (LPS) defense for multicellular organisms to maintain homeostasis from Gram-negative bacteria, lipotheicoic acid (LTA) and pep- of the internal environment against foreign pathogens (micro- tydoglycan (PGN) from Gram-positive bacteria, and mannan organisms, chemicals).1 Both the humoral and the cellular and b-glucan from fungi. Recognition of PAMPs by PRRs us- arms of the innate immune system are equipped with a set of ually initiates an inflammatory cascade that involves recruitment germ-line encoded receptors named pattern recognition recep- of leukocytes to the site of infection, activation of antimicrobial tors (PRRs), which recognize a relatively small spectrum of effector mechanisms and induction of adaptive immune res- highly conserved and broadly distributed structures of protein, ponses that promote clearance of infection and long-term saccharide, lipid and nucleic acid nature, of exogenous as well immune memory.2 PRRs can be either secreted molecules that as endogenous origin.2 Typically, PRRs recognize pathogen- circulate in blood and lymph, or cell-surface receptors present associated molecular patterns (PAMPs), which are conserved on haemopoietic, endothelial and/or epithelial cells. Examples of

1Grup d’Immunoreceptors del Sistema Innat i Adaptatiu, Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 2Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Tokyo; 3Cancer Research Program, Hospital del Mar Medical Research Institute and Department of Immunology, Hospital del Mar, Barcelona, Spain; 4Servei d’Immunologia, Centre de Diagno`stic Biome`dic, Hospital Clı´nic Barcelona, Barcelona, Spain and 5Departament de Biologia Cellular, Immunologia i Neurocie`ncies, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain 6 Authors contributed equally to this work. Correspondence: Dr F Lozano, Centre Esther Koplowitz, Rossello´149-153, 08036 Barcelona, Spain. E-mail: [email protected] Received: 30 October 2013; Revised: 4 February 2014; Accepted: 6 February 2014 AIM/Api6/CD5L and pathogen recognition VG Martinez et al 344

PRRs include C-type lectins (i.e., Dectin-1), Toll-like receptors Infections are thought to be at the basis of the atherogenesis (i.e., TLR1–11) and scavenger receptors (i.e., SR-AI/II, CD36, process by promoting chronic inflammation and atherogenic MARCO). lipid profiles.29 Thus, a possible role of AIM/Api6/CD5L in The scavenger receptor cysteine-rich superfamily (SRCR-SF) is pathogen recognition as well as on the in vivo innate immune an ancient and highly conserved group of membrane-bound and/ response against microbial aggression was investigated in the or soluble proteins mostly reported in the animal kingdom, from present work. Although a previous report has shown the ability 10 low invertebrates to mammals.3,4 Members of the SRCR-SF are of Spa to bind and aggregate bacteria, very little evidence if any characterized by the presence of one or several repeats of a highly is available for AIM/Api6/CD5L in this regard, apart from the conserved cysteine-rich extracellular SRCR domain (of about description that it enhances the phagocytic function of macro- 30 100–110 aa in size) first identified in the type A macrophage phages in Corynebacterium parvum-induced hepatitis. The scavenger receptor 1.5 Despite the overall structure conservation results herein reported demonstrate that AIM/Api6/CD5L binds of the SRCR domains across different species, no unifying bio- to different PAMPs present not only on bacterial (either Gram- logical function has been reported for them. They do not possess negative or Gram-positive), but also fungal (either saprophytic enzymatic activity, although some SRCR domains have been or pathogenic) cell surfaces and support a role for AIM/Api6/ involved in protein–protein interactions, the best studied exam- CD5L as a component of the humoral arm of the innate ples being those of CD6 with CD166/ALCAM,6 and of CD163 immune system during early phases of pathogen aggression. with the haptoglobin–haemoglobin complex.7 In recent years, a number of studies also support the recognition of PAMPs by some MATERIALS AND METHODS SRCR-SF members such as MARCO,8 DMBT1/SAG/gp340,9 Ethical and biosafety approval Spa,10 CD6,11 CD5,12 S5D-SRCRB,13 SCARA514 and CD163.15 This study was approved by the ethical research committee of The apoptosis inhibitor of macrophages (AIMs), also known the Fundacio´ Clı´nic per a la Recerca Biome`dica. In vivo studies as Api6 (for apoptosis inhibitor 6) or CD5L (for CD5-like), is a were carried out under protocols approved by the Ethics mouse soluble member of the SRCR-SF which is secreted by Committee for Animal Research of the University of mature tissue macrophages. AIM/Api6/CD5L was initially Barcelona. All efforts were made to minimize animal suffering. reported as an apoptosis inhibitor supporting the survival of Cell lines macrophages and also other myeloid and lymphoid cell types The Human Embryonic Kidney cell line HEK293-EBNA, which against various apoptosis-inducing stimuli.16–18 AIM/Api6/ stably expresses the EBNA-1 antigen from Epstein - Barr virus, CD5L is considered the mouse homologue of human Spa19 was kindly provided by Takako Sasaki (Max-Planck-Institut with which it shares an overall 73% amino acid identity.20 fu¨r Biochemie, Martinsried, Germany). HEK293 cells stably However, differences in the glycosylation and tissue expression expressing TLR2 (HEK293-TLR2) were a kind gift from Dr patterns of these two proteins could account for functional Golenbock (University of Massachusetts Medical School, discrepancies between them.18,20 As secreted molecules, they Worcester, MA, USA). Both HEK293-EBNA and HEK293- m are readily detected (at ng g/ml level) in human and mouse TLR2 cells were grown in DMEM/F12 (Gibco Life Science, serum20,21 with Spa levels varying in different clinical situa- 16,22–25 Grand Island, NY, USA) supplemented with antibiotics tions. RNA analysis indicates that both molecules are (100 U/ml penicillin, 100 mg/ml streptomycin), 10% heat-inac- expressed in lymphoid tissues (thymus, spleen fetal liver, bone tivated fetal calf serum (FCS) (Walkersville, Biowhittaker, MD, marrow), but a more detailed immunohistochemical analysis USA) and 250 mg/ml Geneticin (Gibco, NY, USA). on AIM/Api6/CD5L expression showed that it is restricted to 18 only a fraction of tissue macrophages. Interestingly, increased Expression of recombinant AIM/Api6/CD5L AIM mRNA levels were observed in thioglycollate-induced A recombinant form of AIM/Api6/CD5L (rAIM) was peritoneal exudate macrophages, which could not be re- expressed into HEK293-EBNA cells by using an episomal induced by treatment with PMA, LPS and cytokines like IFN- eukaryotic system (pCEP-Pu/BM40)31 in a way similar to that 18 c or interleukins. Taken together, all these data suggest that previously reported for Spa.21 Briefly, AIM/Api6/CD5L cDNA AIM/Api6/CD5L expression requires specific microenviron- was obtained by reverse transcription of mouse peritoneal ments as well as that this molecule is involved in both regu- exudate macrophage mRNA isolated using the Ultraspec lation of the inflammatory response and in the development RNA isolation system (Biotecx Laboratories, Houston, TX, 18,19 and maintenance of the lymphoid compartment. USA) from healthy C57Bl/6 mice and subsequent polymerase Recent accumulating evidence has revealed the involvement chain reaction amplification with the 59-GCCCGGCTAGC- of AIM/Api6/CD5L in obesity-related metabolic disorders.26 GTCTCCAACCAAAGTGCAG-39 (forward) and 59-CGCG- Accordingly, AIM/Api6/CD5L was found expressed in foam GATCCTCACACATCAAAGTCTGTGCA-39 (reverse) AIM- cells at atherosclerotic lesions, where it supports the survival specific primers, which incorporated NheI and BamHI restric- of macrophages causing inflammatory responses and athero- tion sites (underlined), respectively. The amplified polymerase sclerosis development.27 Moreover, in adipose tissue, AIM/ chain reaction product (1013 bp) was cloned into appropri- Api6/CD5L is endocytosed and stimulates lipolysis through ately restricted pCEP-Pu/BM-40 vector such that the AIM inhibition of fatty acid synthase activity.28 protein sequence starting at Ser23 (from the immature

Cellular & Molecular Immunology AIM/Api6/CD5L and pathogen recognition VG Martinez et al 345 unprocessed protein) and ending at Val351 was fused in frame grown at 28 uC in yeast peptone dextrose medium with aeration, just after the BMP-40 signal peptide in the final construct. The then harvested by centrifugation at 3500 r.p.m. for 10 min and resulting plasmid (20 mg) was transfected into HEK293-EBNA resuspended in PBS to a final density of 1010 colony forming cells (106 cells in 10-cm culture dishes) using the calcium phos- units (cfu)/ml (for bacteria) or 108 cfu/ml (for fungi). Quanti- phate method. Stable transfectants were cultured in DMEM/ fication was performed by plating bacteria and fungi dilutions on F12 plus 10% FCS and 250 mg/ml Geneticin and selected agar. For microbial binding assays, aliquots of ,53107 bacteria with 1 mg/ml of puromycin (Sigma, St Louis, MO, USA) until and ,53106 fungi were incubated o/n at 4 uC under gentle confluence. To determine rAIM protein expression, 1 ml of orbital rotation with different amounts of biotin-labelled rAIM serum-free culture supernatant was precipitated in 14% tri- in Tris-buffered saline (TBS) plus 3% BSA and 5 mM CaCl2 to a chloroacetic acid and 0.14% Triton-X and then centrifuged final volume of 0.5 ml, in the presence or absence of different at 13 400 g for 30 min at 4 uC. The pellet was washed once with competitors (LPS and LTA at 0, 10, 50 or 100 mg, and rSpa at 0, cold acetone, resolved on SDS–PAGE under reducing condi- 0.5, 1 and 2 mg). Following incubation, microbial cells were tions and stained with Coomassie blue. pelletedandwashedoncewith0.5mlofTBSplusBSAand When cells reached confluence, complete growth medium Ca21 and twice with 0.5 ml of TBS alone to remove nonspecifi- was replaced with serum-free DMEM/F12 plus Geneticin and cally bound proteins. The washed pellets were resuspended in puromycin; cells were then grownforafurther6–9days,with reducing Laemmli’s sample buffer for 15 min at 100 uCand rAIM-containing supernatant being collected every 3 days. rAIM- separated on 8% SDS–PAGE gels, followed by electrotransfer containing supernatant was then concentrated in Amicon Ultra- to a nitrocellulose membrane (BioRad, CA, USA). Membranes 15 Centrifugal Filter Units (Millipore,MA,USA),thenresolved were subjected to western blot analysis as described above, using on SDS–PAGE and stained with Coomassie blue for purity assess- HRP-labelled streptavidin (Roche,Basel,Switzerland)orrabbit ment and quantification by band densitometry using known polyclonal anti-AIM/Spa antiserum plus HRP-labelled goat anti- amounts of bovine seroalbumin (BSA) as standard. rSpa was rabbit IgG (GE Healthcare, Buckinghamshire, UK). affinity-purified as previously described.10 For bacterial and fungal binding assays of AIM present in Protein biotinylation was performed with EZ-Link PEO- serum, E. coli (53107 cfu/ml) and C. albicans (53106 cfu/ml) maleimide-activated biotin (Pierce/Perbio Science, Cheshire, were incubated in a 100 ml volume of binding buffer with 5 ml UK) following the manufacturer’s instructions and monitored mouse serum for 1 h at 4 uC under gentle orbital rotation. by western blotting. Bacteria and fungi were then pelleted at 5000 r.p.m. for 5 min and the supernatant incubated again with more bacteria or Generation of anti-AIM/Api6/CD5L antibodies fungi under the same conditions. This process was repeated A rabbit polyclonal serum against Spa was obtained in our for a total of eight times, with the final incubation being o/n laboratory by immunizing rabbits fortnightly with four intra- at 4 uC. A small aliquot of supernatant was subjected to western muscular injections of trichloroacetic acid-precipitated serum- blotting for detection of AIM as described before, using rabbit free Spa supernatant (50 mg total protein each) in complete polyclonal anti-AIM/Spa antiserum. (first) and incomplete (following doses) Freund’s adjuvant (Life Technologies, Gaithersburg, MD, USA). Cross-reactivity Aggregation assays of this polyclonal antiserum for AIM/Api6/CD5L was tested by Aggregation assays were performed by using fresh o/n cultures western blot analysis of mouse serum (Supplementary Figure of E. coli, S. aureus, Candida albicans and S. cerevisiae harvested 1). To this end, diluted mouse serum pools (1 : 50 and 1 : 100 in by centrifugation at 3500 r.p.m. for 10 min and then resus- phosphate-buffered saline. (PBS)) as well as rAIM and rSpa pended in PBS to a final density of 1010 bacteria or 108 fungi (used as positive controls) were run on SDS–PAGE in dupli- per milliltre. Fluorescein isothiocyanate (FITC) (Sigma) was cate, transferred to nitrocellulose membranes and analyzed dissolved in PBS to a concentration of 10 mM and then added with the rabbit polyclonal antiserum diluted in blocking buffer to the bacterial/fungal suspensions to a final concentration of (2% BSA in PBS, left panel) or blocking buffer plus rAIM 1 mM for 30 min at room temperature (RT). Following removal supernatant (1 : 500) (right panel), followed by horseradish of excess unbound FITC by extensive washing with PBS, 10 mlof peroxidase (HRP)-labelled anti-rat IgG. FITC-labelled bacterial/fungal suspension (53107 bacteria and 6 5310 fungi, respectively) in TBS containing 5 mM CaCl2 were Bacterial and fungal protein binding assays incubated o/n at RT with 20 mg/ml of concentrated rAIM or Escherichia coli ATCC25922 and Staphylococcus aureus undiluted supernatant from AIM-producing HEK293-EBNA ATCC29213 were obtained from the American Type Culture transfectants. Then 10 ml of the fresh samples were examined Collection. Saccharomyces pombe and Saccharomyces cerevisiae by fluorescence microscopy (OptiPhot-2; Nikon, Tokyo, Japan). were kindly provided by the Cell Biology Unit of the University of Barcelona. The rest of the bacterial and fungal strains used in ELISA assays this study are clinical isolates characterized by the Department Interaction of rAIM with PAMPs was analyzed by coating 96- of Microbiology of the Hospital Clinic of Barcelona, using well microtiter plates (Nunc, Roskilde, Denmark) o/n at 4 uC standard biochemical procedures. Bacteria were grown over- with 2 mg/well of purified LPS from E. coli K12 (InvivoGen, night (o/n) at 37 uC in Luria Bertoni medium and fungi were San Diego, CA, USA), PGN (cat# 77140; Sigma), LTA from S.

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aureus (cat# 2515; Sigma), Zymosan A (cat# z4250; Sigma), for 1 h at RT in blocking solution (PBS plus 3% BSA). AIM was Glucan from Baker’s yeast (cat# g5011; Sigma), b(1R3)-glu- detected by incubation with a rabbit anti-AIM/Spa polyclonal can (cat# 89862; Sigma), b-D-glucan (cat# g6513; Sigma) or antiserum generated in our laboratory, followed by incubation Mannan (cat# m7504; Sigma) in coating buffer (100 mM with a 1 : 2500 dilution of HRP-labelled goat anti-rabbit IgG (GE NaHCO3, pH 9.5). Nonspecific binding to plastic wells was Healthcare). Between each incubation step, excess unbound prevented by incubation for 1 h at RT in blocking solution protein was washed off three times with PBS 0.01% Tween 20. (PBS plus 3% BSA). Serum-free rAIM supernatant was then Colour was developed by adding TMB liquid substrate and the added to the wells and incubated for 2 h at RT. After extensive absorbance was measured at 450 nm. The assay was repeated at washing, 1 mg of biotin-labelled rSpa was added for 1 h at RT least twice with similar results. followed by addition of HRP-labelled streptavidin at a 1 : 1000 dilution for a further 30 min. Between each incubation step, Immunofluorescence studies excess unbound proteins were washed off three times with PBS The presence of AIM was studied in plastic-adherent splenocytes 0.01% Tween 20. Colour was developed by adding 3,39,5,59- cultured on fibronectin-coated coverslips (50 mg/ml in cell cul- tetramethylbenzidine (TMB) liquid substrate (Sigma) and the ture media). Cells adhered to the coverslip were washed with absorbance was measured at 450 nm. The assay was repeated at PBS and blocked with 10% FCS in PBS for 1 h at RT. They were least twice with similar results. then incubated with a 1 : 25 dilution of polyclonal anti-AIM antiserum for 1 h at RT, followed by incubation with Alexa Cytokine release assays 488-conjugated donkey anti-rabbit IgG (Invitrogen, CA, USA). Parental HEK293 cells and HEK293-TLR2 transfectants were Coverslips were mounted with diamidino-2-phenylindole, seeded on a 96-well plate at a density of 53104 cells per well for visualized in a fluorescence microscope, and images were ana- 24 h and then cultured in FCS-free DMEM/F12 media for a lyzed by ImageJ software ImageJ.net. Approximately 100 cells further 24 h. Cells were then pulsed with 20 mg/ml PGN for 24 h were counted for each condition and the staining intensity cal- in the presence of FCS-free supernatants from HEK293-EBNA culated as the total intensity of the image divided by the number transfectants expressing rAIM, rSpa or untransfected HEK- of cells in the field. Results are expressed as arbitrary units. EBNA as negative control. After this time, culture supernatant samples (25 ml) were collected and assayed for human IL-8 by Mouse models of PAMP-induced septic shock ELISA (BD OptEIA, Human IL-8 ELISA Set; BD Biosciences, Two models of LPS-induced septic shock were used where the San Diego, CA, USA) following manufacturer’s instructions. dose of LPS was considered non-lethal (1 mg/kg) as previously For TNF-a release, plastic-adherent splenocytes from C57Bl/ determined by preliminary experiments. LPS was injected i.p. 6 mice were seeded on a 48-well plate at a density of 2.53105 to 10-week-old C57Bl/6 mice (Charles River, Barcelona, Spain) cells per well, grown for 24 h and then cultured in RPMI serum- and serum was obtained 2, 6 and 24 h following LPS injection free media for a further 24 h. Cells were then pulsed with 1 mg (n53 for each time point, where three different mice were LPS or b-D-Glucan for 24 h in the presence or absence of FCS- killed and bled at each time point). Zymosan-induced septic free supernatants from HEK293-EBNA transfectants expres- shock models were carried out in the same way, with a non- sing rSpa or rAIM. TNF-a was then determined in culture lethal (500 mg/kg) dose used, as empirically determined. supernatant samples (100 mL) by ELISA (BD OptEIA, Mouse Serum samples were diluted (1 : 100) and boiled in reducing TNF-a ELISA Set; BD Biosciences) following manufacturer’s Laemmli’s sample buffer for 15 min. They were then separated instructions. on 8% SDS–PAGE gels, followed by electrotransfer to a nitrocellulose membrane. Membranes were subjected to western Macrophage AIM/Api6/CD5L release assays blot analysis using a rabbit anti-AIM/Spa polyclonal antiserum Spleen cells from C57Bl/6 mice were dissociated through a 10 mm followed by incubation with 1 : 2500 diluted HRP-labelled goat filter and incubated in a Petri dish for 1 h at 37 uCinRPMI anti-rabbit IgG. Bands were revealed by using Amersham ECL medium supplemented with 10% FCS, plus antibiotics. The Prime WB Reagent (GE Healthcare). plate was washed and the plastic-adherent cells were recovered with 10 ml of CellMates Accutase Cell Detachment Statistical analysis Solution (Miltenyi Biotech, Cologne, Germany) for 5 min at Statistical significance of differences between result groups was 37 uC,andwerethenculturedatadensityof83105 cells/ml determined using Student’s t-test. In all experiments, differences in a 48-well culture plate (Nunc, Denmark, Roskilde, Denmark) were considered statistically significant when P was ,0.05. with RPMI 1640 medium supplemented with 1.2 mg/ml Gentamicin (Braun, Kronbeg, Germany). The following day, RESULTS paraformaldehyde-fixed whole bacterial cell suspensions (E. coli Bacterial binding and aggregating properties of AIM/Api6/ and S. aureus,108 cfu/ml) or PAMPs (LPS or LTA, 3 mg/ml) CD5L were added to the culture, incubated at 37 uC, and the super- The microbial binding properties of AIM/Api6/CD5L were natants recovered at different time points. ELISA plates were assessed in vitro by using a recombinant form of this receptor coated with 100 ml of the culture supernatants o/n at 4 uC. (rAIM) devoid of any protein tag and expressed by using a Nonspecific binding to plastic wells was prevented by incubation mammalian cell episomal system (pCEP-Pu/HEK293-EBNA).

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In these experiments, whole live bacterial cell suspensions were order to test whether the microbial binding properties of AIM incubated with biotin-labelled serum-free rAIM supernatants, could be extended to fungi, protein binding experiments were and the presence of cell-bound protein was further detected by carried out with whole fungal cells. As shown in Figure 2a, biotin- western blot analysis. As shown by Figure 1a, rAIM could be labelled rAIM supernatant bound to all the fungal cells analyzed, detected in cell pellets from both Gram-negative (E. coli)and either saprophytic (S. cerevisiae, S. pombe)orpathogenic(C. Gram-positive (S. aureus) bacteria in a dose-dependent man- albicans, C. neoformans). Again, AIM from serum was also shown ner. Importantly, serum AIM from C57Bl/6 mice was also to bind to whole fungal cells, as shown by progressive depletion shown to bind to whole bacterial cells. As illustrated by by several rounds (38) of adsorption to fungal cell suspensions Figure 1b, depletion of serum AIM was achieved after several (Figure 2b). Interestingly, the fungal binding properties were also rounds (38) of adsorption to bacterial cell pellets of Gram- shared by biotin-labelled rSpa (Figure 2c), a protein for which negative origin (E. coli). Moreover, binding of biotin-labelled only bacterial binding properties have been reported so far.10 rAIM to both Gram-negative and Gram-positive bacteria was As illustrated by competitive ELISA assays shown in competed in a dose-dependent manner by unlabelled affinity- Figure 2d, direct binding of both rAIM and rSpa to the fungal purified rSpa, the homologous human protein for which bac- particle Zymosan as well as to purified conserved fungal cell 10 terial binding properties have been previously reported wall constituents such as mannan and b-D-glucan was (Figure 1c). observed. The fact that the addition of rAIM and rSpa is To better identify the precise nature of the cell wall compon- sequential again indicates that the observed competitive effect ent/s responsible for the bacterial binding ability of rAIM, fur- can be ascribed to direct binding of rAIM to fungal PAMPs. ther competitive bacterial binding and ELISA assays were Moreover, serum-free rAIM-containing supernatants were carried out. As shown in Figure 1d, the binding of biotin- also able to aggregate whole fungal cell suspensions in a manner labelled rAIM supernatant to both Gram-positive and Gram- dependent on the presence of divalent cations (Figure 2e). negative bacteria was competed by purified bacterial PAMPs Taken together, the results indicate that AIM and its human such as LPS or LTA, which are in turn derived from Gram- counterpart Spa can bind to a number of different microbia, negative and Gram-positive bacteria, respectively. This is in both pathogenic and saprophytic. contrast with data from Spa, which show that binding of this protein to E. coli was competed by LPS but not LTA.10 Inhibition of PAMP-induced cytokine release by AIM/Api6/ In a further set of competitive ELISA assays, the ability of CD5L rAIM to interfere with the previously reported binding of Spa To further analyze the biological consequences of the inter- to conserved bacterial cell wall components was investigated. action of AIM/Api6/CD5L with bacterial and fungal PAMPs, To this end, ELISA plates were coated with purified PAMPs the effect of rAIM on PAMP-induced cytokine release was from both Gram-negative (LPS) and Gram-positive (LTA, investigated in different cellular systems. To this end, PGN) bacteria and then incubated with unlabelled rAIM HEK293 cells stably transfected with TLR2 32 were exposed to supernatants. After extensive washing, biotin-labelled affinity PGN, a well-known TLR2 ligand, in the presence or absence of purified rSpa was added to the wells. As shown by Figure 1e, serum-free rAIM-containing culture supernatants, and the unlabelled rAIM competed the binding of biotinylated rSpa to IL-8 levels monitored 18 h later. As shown in Figure 3a, the all the bacterial PAMPs tested in a dose-dependent manner. PGN-induced release of IL-8 was inhibited by undiluted rAIM- Because of the sequential addition of rAIM and rSpa, competi- containing supernatants. No inhibition was observed when tive effect can be ascribed to direct binding of rAIM to PAMPs using either diluted (by half) rAIM-containing supernatants as opposed to rAIM binding to rSpa. or supernatants from untransfected HEK293 cells. No IL-8 In agreement with its pathogen-binding properties, it was release was detected following exposure of parental untrans- also investigated whether rAIM was able to induce bacterial fected HEK293 cells to PGN. When the same experiments aggregation, a simple and effective mechanism to control were performed in the presence or absence of serum-free microbial dissemination and infection. As shown in Figure 1f, rSpa-containing supernatants, similar inhibitory effects were addition of rAIM-containing supernatants induced divalent observed (Figure 3b). Moreover, undiluted rAIM- and rSpa- cation-dependent aggregation of different bacterial cell suspen- containing supernatants were also able to reduce the in vitro sions of both Gram-positive (S. aureus) and Gram-negative (E. TNF-a production by spleen macrophages in response to bac- coli, A. baumannii, S. typhimurium) origin, in a way similar to terial LPS, a well-known TLR4 ligand, as well as to fungal b-D- that previously reported for rSpa,10 here used as a positive glucan, a well-known Dectin-1 ligand (Figure 3c and d). These control. No aggregation was observed following addition of results indicate that by binding to bacterial and fungal ligands of an unrelated protein such as BSA (Figure 1f). TLRs and/or other PRRs, AIM (and Spa) would interfere with the subsequent cytokine-mediated inflammatory response. Fungal binding and aggregating properties of AIM/Api6/ CD5L AIM is released by macrophages in vitro in response to PRRs are able to recognize conserved structural components PAMPs from a wide variety of pathogens, thus often being able to bind Previous reports had shown that AIM mRNA was not induced in not only bacterial but also fungal, viral or parasitic cell walls. In response to LPS.18 However, given the potential PAMP-binding

Cellular & Molecular Immunology AIM/Api6/CD5L and pathogen recognition VG Martinez et al 348

a E. coli S. aureus b rAIM 0x 1x 8x

rAIMb (μg) 1 0.50 0.25 0.12 0.06 1 0.50 0.25 0.12 0.06 c E. coli S. aureus rSpα (μg) 0 0.5 11220 0.5

50 kDa

d e E. coli S. aureus 1.0 * ** * ** rAIM (μg) PAMP (μg) 0 10 50 100 0 10 50 100 * ** * ** 0 LPS O55 0.5 0.5 1

LTA Absorbance 450nm 2 0.0 LPS LTA PGN

f BSA rSPα SN rAIM SN rSPα SN + EDTA AIM SN + EDTA

E. coli

S. aureus

A. baumannii

S. typhimurium

Figure 1 rAIM binds to and aggregates whole bacteria through recognition of conserved cell wall components. (a) Increasing amounts of biotin- 10 labelled rAIM were incubated o/n at 4 uC with whole live bacteria cell suspensions (1310 cfu/ml; 200 ml) in binding buffer (TBS plus 5 mM CaCl2 and 1% BSA). Extensively washed bacterial pellets were separated by SDS–PAGE for further western blot analysis using HRP-labelled streptavidin and developed by chemiluminescence. Figure shows a representative experiment out of three performed. (b) Mouse serum (10 ml) was incubated for 1 h at 4 uC with E. coli (53107 cfu) in 100 ml binding buffer. Bacteria were then pelleted and the serum supernatant re-incubated with fresh bacteria for a new immunoprecipitation round. An aliquot of supernatant before (03) and after 31 and 38 rounds was analyzed by western blot using a polyclonal anti-AIM antiserum followed by HRP-labelled anti-rabbit IgG. Figure shows a representative experiment out of two performed. (c) Bacteria (131010 cfu/ml; 200 ml) were incubated o/n at 4 uC with biotin-labelled rAIM (1 mg) in the presence of increasing amounts of unlabelled rSpa, and then processed as in a. Figure shows a representative experiment out of three performed. (d) Bacteria (131010 cfu/ml; 200 ml) were incubated o/n at 4 uC with biotin-labelled rAIM (1 mg) in the presence of the indicated increasing amounts of LPS or LTA. Washed bacterial pellets were separated by SDS–PAGE and analyzed as in a. Figure shows a representative experiment out of three performed. (e) ELISA plates coated with LPS, LTA or PGN (2 mg/well, each) were incubated with increasing amounts of unlabelled rAIM for 2 h at RT. After extensive washing, biotin-labelled rSpa (1 mg) was added for 1 h at RT followed by addition of HRP-labelled streptavidin (1 : 1000 dilution) for a further 30 min. Colour was developed with TMB and absorbance read at 450 nm. Absorbance is represented relative to absorbance when no rAIM supernatant has been added. Results represent averaged values from three independent experiments. *P,0.05, **P,0.01. (f) FITC-labelled Gram-negative (A. baumannii, S. typhimurium, E. coli) and Gram-positive (S. aureus) bacteria (131010 cfu/ml; 10 ml) were incubated o/n at RT with rAIM (1 mg) in binding buffer in the presence (1EDTA) or absence of 5 mM EDTA. Fresh samples were then visualized in a fluorescence microscope. Magnification, 3100. Figure shows a representative experiment out of four performed. AIM, apoptosis inhibitor of macrophage; BSA, bovine seroalbumin; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; LPS, lipopolysaccharide; LTA, lipotheicoic acid; PGN, peptydoglycan; RT, room temperature; TBS, Tris-buffered saline; TMB, 3,39,5,59-tetramethylbenzidine.

Cellular & Molecular Immunology AIM/Api6/CD5L and pathogen recognition VG Martinez et al 349

a b rAIM 0x 1x 8x

pombecerevisae. albicans. neoformans rAIMb S. S. C C

d 0.6

c * * 0.4 AIM SN (μl) αb * pombe cerevisae. albicans. neoformans * * S. S. C C rSp * 0 * * * 0.2 * 25 50

Absorbance 450nm 100 0.0 200 Mannan βD-glucan Zymosan

e HEK293rSPα SN rAIM SNrSPα SN + EDTA AIM SN + EDTA

C. albicans

S. cerevisae

C. neoformans

S. pombe

Figure 2 rAIM binds to and aggregates whole live fungal cells through recognition of conserved cell wall components. (a) Biotin-labelled rAIM (1 mg) was incubated o/n at 4 uC with whole live saprophytic (S. pombe, S. cerevisiae) or pathogenic (C. albicans, C. neoformans) fungal cells (13108 cfu/ ml; 200 ml) in binding buffer (TBS plus 5 mM CaCl2 and 2% BSA). The cell pellets were extensively washed and then analyzed by western blot with HRP-labelled streptavidin and developed by chemiluminescence. Figure shows a representative experiment out of three performed. (b) Fungi (53106 cfu/ml) were incubated with 10 ml mouse serum in 100 ml binding buffer for 1 h at 4 uC; fungal cells were then pelleted and the supernatant was re-incubated with fresh fungi for a new round. An aliquot of supernatant before (03) and after 13 and 83 rounds was analyzed by western blot using a polyclonal anti-AIM antibody followed by anti-rabbit-HRP. Figure shows a representative experiment out of two performed. (c) The same assays as in a were performed by using biotin-labelled rSpa (1 mg). Figure shows a representative experiment out of three performed. (d) ELISA plates were coated with mannan, b-D-glucan or zymosan (2 mg each/well) o/n at 4 uC, and then incubated with increasing amounts (in ml) of serum- free supernatant from rAIM-expressing HEK293-EBNA cells for 2 h at RT. This was followed by incubation with biotin-labelled rSpa (1 mg) for 1 h at RT and further streptavidin-HRP for an additional 30 min. Colour was developed with TMB and absorbance read at 450 nm. Results represent averaged absorbance values from three independent experiments. *P,0.05. (e) FITC-labelled fungal cells (13107 cfu/ml; 50 ml) were incubated o/n at RT with serum-free supernatant from rAIM-expressing HEK293-EBNA cells (50 ml) in binding buffer in the presence (1EDTA) or absence of 5 mM EDTA. Fresh samples were then visualized in a fluorescence microscope. Magnification, 3100. Figure shows a representative experiment out of four performed. AIM, apoptosis inhibitor of macrophage; BSA, bovine seroalbumin; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; RT, room temperature; TMB, 3,39,5,59-tetramethylbenzidine. and anti-inflammatory properties of AIM during PAMP expo- Figure 4a, both Gram-negative (E. coli) and Gram-positive (S. sure, it was further investigated whether the presence of PAMPs aureus) bacteria increased AIM levels in culture supernatants at would induce the release of AIM. To confirm this, plastic-adher- 1 h post-stimulation. A similar increase in AIM levels was ent mouse splenocytes were exposed to paraformaldehyde-fixed observed following exposure of plastic-adherent spleen cells to whole bacteria (E. coli and S. aureus) and the levels of AIM purified bacterial (LPS, PGN and LTA) and fungal (Zymosan measured in culture supernatant by direct ELISA. As shown in and b-glucan) PAMPs (Figure 4b and c). Interestingly, the

Cellular & Molecular Immunology AIM/Api6/CD5L and pathogen recognition VG Martinez et al 350

ab* 2000 HEK 293 400 * HEK TLR2 HEK 293 1500 300 HEK TLR2

1000 200 IL-8 (pg/ml) IL-8 (pg/ml) 500 100

0 0

SN Basal PGN Basal PGN α α SN 1:2

PGN+ AIM SN PGN+Sp PGN+ AIM SN 1:2 PGN+Sp PGN+ HEK 293 SN PGN+HEK 293 SN cd*** 150 *** 800 *** *** 600 100 (pg/ml)

(pg/ml) 400 α α 50 TNF TNF 200 ND ND ND ND 0 0

SN LPS SN Basal α Basal α βGlucan

LPS+Sp LPS+AIM SN βGlucan+Sp βGlucan+AIM SN

Figure 3 Immunomodulating properties of rSpa and rAIM. (a and b) IL-8 release assay. HEK293 and HEK293 TLR2 cells were seeded on a 96-well plate at a density of 53104 cells per well, grown for 24 h and then cultured in serum-free media for a further 24 h. Cells were then pulsed with 20 mg/ ml PGN for 24 h in the presence of serum-free supernatants (undiluted and diluted in half in serum-free media) from HEK293-EBNA transfectants expressing rAIM (a) or rSpa (b), or undiluted supernatant from HEK293-untransfected cells as control. IL-8 was then determined in culture supernatant samples by ELISA. Figure shows averaged results from three independent experiments. *P,0.05. (c and d) TNF-a release assay. Plastic-adherent splenocytes were seeded on a 48-well plate at a density of 2.53105 cells per well, grown for 24 h and then cultured in serum-free media for a further 24 h. Cells were then pulsed with 1 mg b-D-Glucan or LPS for 24 h in the presence or absence of supernatants from HEK293- EBNA transfectants expressing rSpa or rAIM. TNF-a was then determined in culture supernatant samples by ELISA. Figure shows averaged results from two independent experiments. ***P,0.005. AIM, apoptosis inhibitor of macrophage; ND, not detected; LPS, lipopolysaccharide; PGN, peptidoglycan; TLR, Toll-like receptor.

increase peaked as soon as 1 h post-stimulation and did not spleen cells with a polyclonal anti-AIM antiserum after treat- increase any further, suggesting a rapid release of the pre- ment with LTA or vehicle for 1 h at 37 uC. The fluorescence formed protein from cellular deposits. Indeed, when LTA intensity for cells treated with vehicle or LTA was then deter- was washed out from spleen cell cultures after 1 h of stimu- mined, as the total fluorescence measured divided by the num- lation and replaced with fresh media, AIM levels detected after ber of cells in the field. As shown in Figure 4f, fluorescence a further 8 h incubation showed no additional increase intensity for AIM staining was decreased in LTA-treated cells, (Figure 4d). Similar results were obtained with Spa when confirming the release of AIM into the culture supernatant in stimulating plastic-adherent cells derived from human response to LTA treatment. PBMCs (data not shown). In a further set of experiments, pre-incubation of plastic-adherent spleen cells with inhibitors In vivo PAMP challenge decreases serum AIM levels of transcription (actinomycin D), protein synthesis (cyclohexi- As previously mentioned, AIM is an abundant serum protein mide) or protein transport into Golgi (brefeldin) before expo- with PAMP- binding ability. Accordingly, it was further inves- sure to LTA for 1 h, only slightly inhibited AIM release into cell tigated whether infusion of bacterial and/or fungal PAMPs culture media (Figure 4e). Although this inhibition was signifi- would be able to affect circulating AIM levels during in vivo cant, the low magnitude and the fact that all three different challenge. To this end, the serum levels of AIM were monitored compounds induced a similar level of inhibition suggest that over time following i.p. administration of lethal or sublethal this was due to a general toxic effect of the compounds used. doses of LPS or Zymosan to C57Bl/6 mice, as empirically deter- Support for the putative release of AIM from preformed sto- mined. Serum samples were obtained from mice 2, 6 and 24 h rage was also obtained by staining permeabilized plastic-adherent post-PAMP injection and the levels of AIM were determined by

Cellular & Molecular Immunology AIM/Api6/CD5L and pathogen recognition VG Martinez et al 351

a b 0.25 *** 0.8 LPS 0111 0.20 ** 0.6 LTA 0.15 PGN 0.4 0.10 0.2 0.05 Absorbance 450nm Absorbance 450nm NI 0.00 0.0 NI E. coli S. aureus 124 8 4 Hours post-stimulus cd 0.8 Zymosan 0.6 *** 0.6 BG 0.4 0.4

0.2 0.2 Absorbance 450nm

NI Absorbance 450nm 0.0 0.0 124 8 4 1h1h refresh media Hours post-stimulus

ef 0.6 2.5 ***

2.0 * * * .U.) 0.4 1.5

1.0 0.2 Intensity (A 0.5 Absorbance 450nm 0.0 0.0 0 0 3 mg/ml LTA Brefeldin

Actinomycin D Cycloheximide

Figure 4 Secretion of AIM is stimulated by the presence of bacterial and fungal PAMPs. (a–c) Plastic-adherent cells (2.53105) from C57Bl/6 mouse spleens were cultured in RPMI media and stimulated for 1 h with 108 cfu/ml E. coli or S. aureus (a) or for different time periods with 3 mg/ml of LPS O111, LTA and PGN (b), as well as Zymosan and b-D-glucan (c). AIM levels were then determined in an ELISA assay, coating plates with supernatant from the splenocyte cultures and detecting the presence of AIM with an anti-AIM polyclonal antibody, followed by HRP-conjugated anti-rabbit IgG. Figure shows a representative experiment out of three performed. (d) Plastic-adherent cells (2.53105) from C57Bl/6 mouse spleens were cultured in RPMI media and stimulated with 3 mg/ml of LTA. After a 1-h incubation, the media was removed and replaced by fresh media. AIM levels were then determined in an ELISA assay as above. Figure shows a representative experiment out of three performed. (e) Plastic- adherent splenocytes from murine spleens were cultured and stimulated with LTA as above in the presence of 1 mg/ml of brefeldin, cycloheximide or actinomycin D. AIM was measured in cell culture supernatants as previously described. Figure shows a representative experiment out of two performed. *P,0.05. (f) Plastic-adherent cells from murine spleens were grown on fibronectin-coated coverslips and stained with anti-AIM polyclonal antibody, followed by an Alexa-488-conjugated anti-rabbit IgG antibody. Cells were then photographed in a fluorescence microscope and the total fluorescence intensity divided by the number of cells in the field. At least three fields were counted for each treatment. Results are expressed as arbitrary units (A.U.). AIM, apoptosis inhibitor of macrophage; HRP, horseradish peroxidase; LPS, lipopolysaccharide; LTA, lipotheicoic acid; PAMP, pathogen-associated molecular pattern; PGN, peptydoglycan. western blotting. The results (Figure 5) show that serum AIM AIM levels behaved in a similar manner to above reported levels are decreased, as compared to basal levels, 2 and 6 h post- for sublethal doses, except that they remained below basal levels injection of sublethal doses of both LPS (1 mg/kg) and at 24 h post-challenge (data not shown). Since the dose is lethal, Zymosan (500 mg/kg), and that they recover and even surpass almost no mice survive more than 24 h, so it is not possible to those observed in basal conditions after 24 h. This recovery in know whether AIM levels recover at a later time. These results AIM levels was paralleled by an improvement in mice well- suggest that AIM can act as a soluble PRR, which can bind to being. Moreover, when higher (lethal) doses of the two PAMPs, and consequently, its serum levels are affected during PAMPs were used (10 and 700 mg/kg, respectively), serum microbial challenge.

Cellular & Molecular Immunology AIM/Api6/CD5L and pathogen recognition VG Martinez et al 352

a b 150000 Hours post-LPS injection

rAIM .U.) 0 2 624 100000

50000 Intensity (A

0 02462 Hours post-LPS injection cd 80000 Hours post-Zymosan injection 60000 .U.)

0 2 6 24 rAIM 40000

Intensity (A 20000

0 02462 Hours post-Zymosan injection

Figure 5 AIM serum levels in vivo following PAMP challenge. (a)Mice(n53 for each time point) were injected i.p. with 1 mg/kg LPS and serum was obtained in basal conditions and 2, 6 and 24 h following LPS injection. AIM serum levels were determined by western blotting using a polyclonal anti-AIM antibody, followed by an HRP-conjugated anti-rabbit IgG. (c) Densitometric measurement of the bands in a expressed in arbitrary units (A.U.). (c)The same as in a,exceptthatmice(n53 for each time point) were injected i.p. with 500 mg/kg Zymosan. (d) Densitometric measurement of the bands in c. AIM, apoptosis inhibitor of macrophage; HRP, horseradish peroxidase; LPS, lipopolysaccharide; PAMP, pathogen-associated molecular pattern.

DISCUSSION cause tissue damage (and ultimately septic shock) through The present report shows that AIM/Api6/CD5L, a soluble their pro-inflammatory activity.33 Binding of free PAMPs member of the SRCR superfamily found in mouse serum, acts could thus neutralize the pro-inflammatory effect of these as a pattern-recognition receptor for PAMPs present in bac- compounds and protect tissues from damage. The results pre- teria and fungi. This might not seem surprising, considering sented herein show that AIM/Api6/CD5L and Spa have the that Spa, the human homologue of AIM, has been found to act potential to reduce pro-inflammatory cytokine and chemokine as a PRR for LPS and LTA.10 However, although both proteins secretion by mouse and human cells, respectively, thus suggest- share approximately 70% homology, their glycosylation pat- ing a role for these proteins in tissue protection during expo- tern is very different, which might account for functional dif- sure to PAMPs. ferences. Indeed, even though binding of LPS and LTA by rSpa The fact that AIM/Api6/CD5L and Spa appear to be useful in was found to be mediated by two fully independent sites, bind- fighting infection and protecting tissue from inflammation- ing of both PAMPs by rAIM has now been found to depend on related damage could lead one to believe that expression of a single or two closely adjacent sites, judging by how LPS is able these proteins might be induced following pathogen infection to inhibit LTA binding to rAIM and vice versa. This study also or by the presence of PAMPs. Previous studies, however, have describes the expanded spectrum of rSpa PAMP binding, reported lack of AIM mRNA induction in macrophages by including now not only bacteria but also pathogenic and sap- PAMPs such as LPS.18 Our results show that AIM and Spa rophytic fungi; these binding properties are also shared by levels are increased in the culture medium of mouse and rAIM. It should be noted that both rAIM and rSpa also have human monocyte/macrophages, respectively, following treat- the ability to aggregate as well as to bind bacteria and fungi, ment with whole bacteria and several bacterial and fungal which could be a defensive mechanism used by these proteins PAMPs. The reason for the apparent discrepancy could be that in preventing single cell invasion of tissues by microorganisms, AIM and Spa accumulate in cytoplasmic stores ready for rapid as well as facilitating their elimination. The ability to induce release, which is triggered by the presence of PAMPs, indepen- aggregation is easily explained by the structure of these pro- dently from mRNA transcription. Indeed, the time frame of teins, consisting of three tandem repeats of highly homologous AIM/Api6/CD5L release into the culture medium is not con- SRCR domains. sistent with de novo protein synthesis by monocyte/macro- Both AIM/Api6/CD5L and Spa were shown by our results to phages, peaking as early as 1 h following PAMP exposure. bind to whole bacteria and fungi through recognition of well- Moreover, release of AIM/Api6/CD5L into the culture medium known bacterial (LPS, LTA, and PGN) and fungal (mannan was not substantially affected by inhibiting transcription, pro- and b-D-glucans) PAMPs. It is known that PAMPs alone, with- tein synthesis or transport into the Golgi, supporting the out accompanying infection by microorganisms, are able to notion that AIM/Api6/CD5L is mostly preformed in cells and

Cellular & Molecular Immunology AIM/Api6/CD5L and pathogen recognition VG Martinez et al 353 its release is triggered by PAMPs. Storing these proteins inside as the ability to impair PAMP-induced pro-inflammatory cyto- cells ready for release would be of great use as a rapid reaction kine production, which could be useful in protecting tissues against infection, whereby their release would be triggered by from local inflammation damage, and also from more genera- the presence of pathogen compounds which could quickly be lized effects of PAMPs such as sepsis and septic shock syndrome. neutralized, protecting the tissue. Aggregation of pathogens could also impair the spread of infection and might even facili- COMPETING FINANCIAL INTERESTS tate the phagocytic elimination of microorganisms. The authors declare no financial interests. The importance of these results lies in the fact that, to date, the only known inducers of AIM/Spa mRNA expression are ACKNOWLEDGEMENTS LXR ligands.34 Triggering of AIM release by PAMPs could We would like to thank Dr Rosa Aligue´ (Cell Biology Unit of the therefore be a novel mechanism through which the innate University of Barcelona) and Dr. Jordi Vila (Department of immune system attempts to control infection and reduce Microbiology of the Hospital Clinic of Barcelona) for their kind help with microbial specimens. This study was supported by grants from inflammatory damage to tissues. the Spanish Ministerio de Economı´a y Competitividad (SAF2010- To test this idea, an in vivo challenge with PAMPs was carried 19717), Generalitat de Catalunya (2009SGR1101) and Instituto de out and a time-dependent decrease in AIM serum levels in LPS Salud Carlos III (Spanish Network for Research in Infectious Diseases, and Zymosan-treated mice was observed. Interestingly, when REIPI, RD12/0015/0018). VGM is recipient of a fellowship from animals were treated with low (non-lethal) doses of PAMPs, Ministerio de Economia y Competitividad (JCI-2010-06378). CE-F is the decrease of AIM levels in serum was followed by a sharp recipient of a fellowship from Spanish Ministerio de Economı´ay increase, which even surpassed the basal levels. This suggests Competitividad (BES-2008-005544). that AIM is spent in situations where circulating PAMPs are present, presumably because it binds to PAMPs and contri- Supplementary Information accompanies the paper on Cellular & Molecular Immunology’s website. butes to their elimination; however, these levels can recover relatively quickly. Moreover, the recovery of AIM serum levels paralleled the improvement in mice wellbeing. A recent report shows that transfection with AIM enhances macrophage bac- 1 Gordon S. Pattern recognition receptors: doubling up for the innate tericidal ability in a model of infection by Mycobacterium tuber- immune response. Cell 2002; 111: 927–930. culosis.35 Interestingly, AIM levels increased early in mice 2 Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002; 20: 197–216. serum (24 h) following inoculation of M. tuberculosis, which 3 Resnick D, Pearson A, Krieger M. The SRCR superfamily: a family is in agreement with our results. This increase in AIM levels reminiscent of the Ig superfamily. Trends Biochem Sci 1994; 19:5–8. could, at least in part, be ascribed to PAMP-mediated trigger- 4 Sarrias MR, Gronlund J, Padilla O, Madsen J, Holmskov U, Lozano F. ing of AIM release into the circulation; however, the release The Scavenger Receptor Cysteine-Rich (SRCR) domain: an ancient and highly conserved protein module of the innate immune system. observed in vitro is much faster than that observed in the in vivo Crit Rev Immunol 2004; 24: 1–37. experiments. This suggests that other, still unknown, inflam- 5 Freeman M, Ashkenas J, Rees DJ, Kingsley DM, Copeland NG, Jenkins mation-related mechanisms might induce AIM expression NA et al. An ancient, highly conserved family of cysteine-rich protein and/or release. Indeed, it has been reported that AIM mRNA domains revealed by cloning type I and type II murine macrophage scavenger receptors. Proc Natl Acad Sci USA 1990; 87: 8810–8814. is induced in peritoneal macrophages following thyoglycollate- 6 Aruffo A, Bowen MA, Patel DD, Haynes BF, Starling GC, Gebe JA et al. induced inflammation.18 CD6–ligand interactions: a paradigm for SRCR domain function? Further studies will need to be carried out in order to deter- Immunol Today 1997; 18: 498–504. 7 Kristiansen M, Graversen JH, Jacobsen C, Sonne O, Hoffman HJ, Law mine the true importance of AIM expression levels in situations SK et al. Identification of the haemoglobin scavenger receptor. Nature of infection or PAMP-induced sepsis and septic shock syn- 2001; 409: 198–201. drome. It should be noted that as well as a pattern recognition 8 Brannstrom A, Sankala M, Tryggvason K, Pikkarainen T. Arginine receptor, AIM is also a survival factor for macrophages, key residues in domain V have a central role for bacteria-binding activity 18,36–39 of macrophage scavenger receptor MARCO. Biochem Biophys Res contributors to inflammation. Future experiments need Commun 2002; 290: 1462–1469. to assess which of the dual functions of AIM predominates in 9 Bikker FJ, Ligtenberg AJ, End C, Renner M, Blaich S, Lyer S et al. the early immune response against pathogens. Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains. In summary, we have identified AIM as a PRR with similar J Biol Chem 2004; 279: 47699–47703. bindingpropertiesasthoseofitshumanhomologueSpa,ca- 10 Sarrias MR, Rosello S, Sanchez-Barbero F, Sierra JM, Vila J, Yelamos pable of binding and aggregating a broad spectrum of microbial J et al. A role for human Sp alpha as a pattern recognition receptor. agents (bacteria and fungi), thus playing a role in the early J Biol Chem 2005; 280: 35391–35398. 11 Sarrias MR, Farnos M, Mota R, Sanchez-Barbero F, Ibanez A, response to microbial aggression. These properties are in line Gimferrer I et al. CD6 binds to pathogen-associated molecular with those displayed by other members of the SRCR superfamily patterns and protects from LPS-induced septic shock. Proc Natl such as MARCO,40 SAG/DMBT-1,9,41 sCD611 and sCD163,42 Acad Sci USA 2007; 104: 11724–11729. and reinforces the notion that the PRR function could be one 12 Vera J, Fenutria R, Canadas O, Figueras M, Mota R, Sarrias MR et al. The CD5 ectodomain interacts with conserved fungal cell wall unifying trait shared by this superfamily’s members. AIM/Api6/ components and protects from zymosan-induced septic shock-like CD5L has also shown other immunomodulating properties such syndrome. Proc Natl Acad Sci USA 2009; 106: 1506–1511.

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