Exosites mediate the anti-inflammatory effects of a multifunctional serpin from the saliva of the tick Ixodes ricinus Pierre-Paul Prevot 1 , Alain Beschin 2,3 , Laurence Lins 4 ,Je ´ ro ˆ me Beaufays 1 , Ame ´ lie Grosjean 5,6 , Le ´ a Bruys 2,3 , Benoı ˆ t Adam 4 , Michel Brossard 5,6 , Robert Brasseur 4 , Karim Zouaoui Boudjeltia 5,6 , Luc Vanhamme 1,7, * and Edmond Godfroid 1, * 1 Laboratoire de Biologie Mole ´ culaire des Ectoparasites, Universite ´ Libre de Bruxelles, Gosselies, Belgium 2 Department of Molecular and Cellular Interactions, VIB, Brussels, Belgium 3 Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Belgium 4 Centre de Biophysique Mole ´ culaire et Nume ´ rique, Faculte ´ Universitaire des Sciences Agronomiques de Gembloux, Belgium 5 Laboratoire de Me ´ decine Expe ´ rimentale, Universite ´ Libre de Bruxelles, Montigny-le-Tilleul, Belgium 6 Institut de Zoologie, Universite ´ de Neucha ˆ tel, Switzerland 7 Parasitologie Mole ´ culaire, Institut de Biologie et de Me ´ decine Mole ´ culaires (IBMM), Universite ´ Libre de Bruxelles, Gosselies, Belgium Ticks are hematophagous arachnid ectoparasites, sec- ond only to mosquitoes as pathogen vectors worldwide [1]. Ixodes ricinus is widely found in Europe and is able to take its blood meal from a variety of verte- brates, ranging from amphibians to mammals, and including domestic animals and humans [2]. It is the one of the vectors of Borrelia burgdorferi, the agent of Lyme disease. I. ricinus is characterized by a long-last- ing blood meal, leaving time for its host to activate defensive reactions such as pain (stimulating scratch- ing) and hemostasis (repairing the wound and involv- ing coagulation), as well as innate, adaptive immune Keywords inflammatory; receptor binding domain; sepsis; serpin; tick Correspondence E. Godfroid, Laboratoire de Biologie Mole ´ culaire des Ectoparasites, Institut de Biologie et de Me ´ decine Mole ´ culaires (IBMM), Universite ´ Libre de Bruxelles, rue des professeurs Jeener et Brachet 12, B-6041 Gosselies, Belgium Fax: +32 2 650 9900 Tel: +32 2 650 9934 E-mail: edmond.godfroid@ulb.ac.be *These authors contributed equally to this work (Received 4 February 2009, revised 2 April 2009, accepted 3 April 2009) doi:10.1111/j.1742-4658.2009.07038.x Serine protease inhibitors (serpins) are a structurally related but function- ally diverse family of ubiquitous proteins. We previously described Ixodes ricinus immunosuppressor (Iris) as a serpin from the saliva of the tick I. ricinus displaying high affinity for human leukocyte elastase. Iris also displays pleotropic effects because it interferes with both the immune response and hemostasis of the host. It thus inhibits lymphocyte prolifera- tion and the secretion of interferon-c or tumor necrosis factor-a by periph- eral blood mononuclear cells, and also platelet adhesion, coagulation and fibrinolysis. Its ability to interfere with coagulation and fibrinolysis, but not platelet adhesion, depends on the integrity of its antiproteolytic reactive center loop domain. Here, we dissect the mechanisms underlying the inter- action of recombinant Iris with peripheral blood mononuclear cells. We show that Iris binds to monocytes ⁄ macrophages and inhibits their ability to secrete tumor necrosis factor-a. Recombinant Iris also has a protective role in endotoxemic shock. The anti-inflammatory ability of Iris does not depend on its antiprotease activity. Moreover, we pinpoint the exosites involved in this activity. Abbreviations CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; IL, interleukin; Iris, Ixodes ricinus immunosuppressor; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; PBMC, peripheral blood mononuclear cell; PDB, Protein Data Bank; RBD, receptor binding domain; RCL, reactive center loop; rIris, recombinant Iris; serpins, serine protease inhibitor; TNF-, tumor necrosis factor. FEBS Journal 276 (2009) 3235–3246 ª 2009 The Authors Journal compilation ª 2009 FEBS 3235 reactions. In response, ticks have evolved a battery of countermeasures [3–7], mainly involving salivary pro- teins, several of which are induced during the feeding process and injected at the site of the wound. Some of these proteins have been identified in several tick spe- cies [8–17]. They comprise a variety of enzymes inter- fering with different physiological processes. We have previously reported the construction and analysis of a cDNA subtractive library which led to the identifica- tion of I. ricinus immunosuppressor (Iris), a protein expressed in the salivary glands and secreted in the saliva during the blood meal [18–20]. Structural analy- sis and site-directed mutagenesis confirmed that Iris is a member of the serine protease inhibitor (serpin) family. The protein structure of serpins is characterized by three b sheets (A, B and C) and eight or nine a helices [21]. A typical feature of serpins is the reac- tive center loop (RCL), a protein motif of 20 amino acids, located near the C-terminus of the protein. This motif contains a scissile bond between the so-called residues P1 and P1¢, which is cleaved by the target pro- tease. This cleavage triggers structural rearrangement of both the protease and the inhibitor in a suicide mechanism that irreversibly complexes and inactivates both interacting partners. All these features have been uncovered in Iris [21]. However, because serpins are involved in a wide variety of physiological processes, prediction of the function of Iris in tick saliva based solely on its belonging to this family was precluded. We previously expressed Iris as a recombinant pro- tein and showed that it inhibits serine proteases such as human leukocyte elastase, tissue plasminogen acti- vator, thrombin and factor Xa. Affinity rate constants and inhibition values indicated that Iris preferentially targets the human leukocyte elastase. [21] In addition, in agreement with its enzymatic activity, Iris was shown to interfere with coagulation and fibrinolysis [21]. These effects were dependent on the protease inhibitory function of the RCL domain of Iris, because the Leu339Ala (P2) mutant devoid of antiprotease activity did not influence coagulation or fibrinolysis. However, Iris also increases the platelet adhesion time [21]; an activity not affected by the mutation. This sug- gests that domains other than the RCL are involved in this distinct protein activity. To add to the multifunc- tionality of this protein, Iris has also been found to modulate cytokine production by human peripheral blood mononuclear cells (PBMC) [18]. Leboulle et al. demonstrated that soluble protein extracts of Chinese hamster ovary (CHO)-KI cells expressing recombinant Iris (rIris) inhibited the production of tumor necrosis factor (TNF-)a, interleukin (IL)-6, IL-8 and inter- feron-c triggered by lipopolysaccharide (LPS). Here, we further address the multifunctional charac- ter of Iris. More precisely, we dissect its anti-inflamma- tory activity. The results indicate that Iris inhibits TNF-a production by direct interaction with the monocyte ⁄ macrophage populations of PBMC. More- over, its ability to interfere with TNF-a production is independent of its antiprotease activity. Finally, we also provide evidence that Iris, as an inhibitor of TNF-a production, may be used as a therapeutic tool against endotoxemic shock. Results Iris inhibits TNF-a production by PBMC We have previously reported that the tick salivary ser- pin Iris could interfere with the immune response. Indeed, total soluble protein extracts from CHO-KI cells expressing rIris–6His inhibited the production of TNF-a by PBMC which had been activated by the Toll-like receptor (TLR)-4 trigger, LPS [18]. In order to dissect the mode of action of Iris, we purified large amounts of the protein expressed in a baculovirus sys- tem. We first investigated the ability of Iris to suppress production of TNF-a by PBMC stimulated with LPS. Because levels of TNF-a production by PBMC vary between healthy donors, prior experiments on multiple donors were performed to determine the appropriate rIris concentration range for these studies. This range was found to be 10–400 nm (results not shown). In all experiments, dexamethasone was used as a control to inhibit LPS-induced TNF-a production [22]. The results shown in Fig. 1 (from three independent *** *** *** *** *** Fig. 1. Effect of rIris on TNF-a production by LPS-stimulated human PBMC. PBMC were activated by LPS (10 lgÆmL )1 ) for 4 h in the presence or absence of the indicated rIris concentrations. TNF-a concentrations were then measured in the harvested super- natants. DEX, dexamethasone (10 l M) was used as positive control for inhibition of TNF-a production; NS, nonstimulated cells; LPS, LPS-stimulated cells. Values are the mean of three experiments (± SD). *P < 0.05, **P < 0.01, ***P < 0.001 compared with controls (one-way ANOVA). Exosites mediate anti-inflammatory effects of Iris P P. Prevot et al. 3236 FEBS Journal 276 (2009) 3235–3246 ª 2009 The Authors Journal compilation ª 2009 FEBS experiments performed on one representative in five donors using the appropriate concentration range) indicate that the pure recombinant protein inhibited TNF-a production by PBMC which had been stimu- lated by LPS. This effect was dose dependent, reaching a maximum at 200–400 nm Iris. Iris also impaired the spontaneous release of TNF-a by PBMC (Fig. 1). Moreover, the ability of Iris to impair TNF-a pro- duction could be inhibited by polyclonal anti-Iris serum [20]. Indeed, as shown in Fig. 2, increasing concentrations of anti-Iris serum progressively restored TNF-a levels in the PBMC supernatant to the values induced by LPS stimulation in the absence of Iris. This effect was presumably related to the specific neutraliza- tion of Iris because the preimmune serum remained without effect. Furthermore, Iris inhibited not only the production of TNF-a by PBMC activated via the TLR4 ⁄ LPS pathway, but also, to varying extents, by peptidogly- can (PGN), poly(I : C) and ODN 2006, which are recognized by TLR2, TLR3 and TLR9, respectively (Table 1). Dexamethasone (10 lm) was used as a positive control and, as expected, inhibited TNF-a production by 95%. Together, these data show that Iris inhibited TNF-a production by PBMC and suggested that this activity was independent of the TLR stimulus. Iris binds to monocytes/macrophages To investigate which PBMC subset population was the target of rIris, flow cytometry experiments were per- formed. Fluorescent-labeled Iris did not bind on CD3) (T lymphocytes), or CD19) (B lymphocytes) positive cells (results not shown). By contrast, Iris binding was detected on both the CD11b+ CD14+ CD16+ and CD11b+ CD14+ CD16) monocyte subsets (Fig. 3). Moreover, the binding of fluorescent-labeled Iris could be competed out by co- or preincubation with anti-Iris serum but not with preimmune sera. Furthermore, bind- ing of fluorescent-labeled Iris to monocytes could be abrogated by co- or preincubation with nonlabeled Iris, but not by co- or preincubation with LPS (not shown). Inhibition of LPS-induced TNF-a release is independent of the antiprotease activity of Iris We next asked whether the inhibiting effect of Iris on TNF-a released by LPS-activated PBMC was depen- dent on its serpin activity. For that purpose, rIris, its cleaved elastase-complexed form or the mutant L339A (the latter two lacking antiprotease activity) were *** *** ** Fig. 2. Polyconal anti-rIris serum neutralize the capacity of Iris to inhibit the release of TNF-a by LPS-activated PBMC. PBMC were incubated for 4 h with or without LPS (10 lgÆmL )1 ) in the presence (+rIris) or absence ()rIris) of 100 n M rIris that had been preincuba- ted for 5 min at 37 °C with the indicated dilutions of anti-Iris serum. TNF-a concentrations were measured in the harvested superna- tants. ⁄ , no antibody added; PI, preimmune serum. Values are the mean of three experiments (± SD). Table 1. Effect of recombinant Ixodes ricinus immunosuppressor (rIris) on production of tumor necrosis factor-alpha (TNF-a) by peripheral blood mononuclear cells (PBMC) stimulated via distinct Toll-like receptors (TLRs). PBMC (2 · 10 5 cellsÆwell )1 ) were incubated with the indicated chemicals [lipopolysaccharide (LPS), 10 lgÆmL )1 ; peptidoglycan (PGN), 10 lgÆmL )1 ; poly(I : C) 10 lgÆmL )1 or ODN 2006, 2 lgÆmL )1 ] in complete RPMI-1640 medium supplemented or lacking rIris (400 n M). Cells were left at 37 °C, 5% CO 2 for 4 or 24 h, as indicated. Culture supernatants were harvested and TNF-a dosed by ELISA. Numbers are the mean of three experiments (± SD) of three individuals per experiment. TNF-a production (pgÆmL )1 ) PBMC stimulation LPS (100 ngÆmL )1 ) PGN (2 lgÆmL )1 ) Poly(I : C) (10 lgÆmL )1 ) ODN 2006 (10 lgÆmL )1 ) After 4 h Iris 295.9 (± 32.4)*** 61.4 (± 2.4)*** 145.9 (± 27.1)* 342.3 (± 43.7)*** Control 1174.1 (± 138.1) 98.6 (± 5.8) 222.7 (± 41.4) 736.4 (± 89.6) After 24 h Iris 701.4 (± 102.4)** 89.1 (± 9.4)*** 409.1 (± 37.1)*** 240.5 (± 23.2)*** Control 1338.2 (± 124.9) 418.6 (± 42.4) 885.5 (± 49.7) 1226.4 (± 101.8) *P < 0.05, **P < 0.01, ***P < 0.001 compared with controls (one-way ANOVA). P P. Prevot et al. Exosites mediate anti-inflammatory effects of Iris FEBS Journal 276 (2009) 3235–3246 ª 2009 The Authors Journal compilation ª 2009 FEBS 3237 added to LPS-stimulated PBMC before evaluating TNF-a production. As illustrated in Fig. 4, all forms of Iris tested, whether retaining the serpin activity (native rIris) or not (L339A mutant, cleaved elastase-complexed Iris), had a similar effect: their addition to LPS-stimulated PBMC resulted in a progressive (dose-dependent) reduction in TNF-a release compared with the cells stimulated with LPS alone. The effect was maximal at 400 nm, reducing TNF-a release to a level lower than that produced by PBMC cultured without LPS stimulation (not shown). These results showed that inhibition of TNF-a release by Iris does not require its serine protease inhibitor activity or integrity of the RCL domain. Exosites mediate the inhibitory effect of Iris on TNF-a production The similar effects of wild-type native, cleaved prote- ase-complexed and RCL mutant forms of Iris on TNF-a production suggested that Iris activity was independent of serpin activity ⁄ domain. Therefore, we sought to predict the domains of Iris distinct from the RCL domain that may potentially CD16 10 4 10 5 Comp-PE-A Comp-FITC-A 10 3 0 CD14 CD14 – CD14 + x 80 100 B C A 60 100 80 60 % of Ma % of Max 40 20 40 20 Comp-FITC-A Comp-FITC-A 0 0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 Fig. 3. Iris interacts with the mono- cyte ⁄ macrophage PBMC population. PBMC were incubated with fluorescent-labeled rIris, CD14 and CD16 antibodies. (A) Expres- sion of CD16 and CD14 on PBMC. (B) CD14) populations gated in (A) were analyzed for Iris binding (dotted line, CD14) ) CD16+ ) cells; tinted line, CD14) CD16)cells). (C) CD14+ populations gated in (A) were analyzed for Iris binding (dotted line, CD14) CD16+ ) cells; bold line, CD14+ CD16+ cells, tinted line, CD14+ CD16) ) cells). FACS profiles are representative of one of five individuals tested in two independent experiments. Numbers in the FACS profiles indicate the percentage of cells within the indicated gates. 0 200 400 600 800 1000 400 200 100 50 25 0 Concentration (n M) TNF-α α concentration (pg·mL –1 ) Iris L339A Iris + Elastase Elastase Fig. 4. Dose-dependent effects of wild-type and mutated rIris on TNF-a release by LPS-stimulated PBMC. PBMC were activated for 4 h by LPS (10 lgÆmL )1 ) in the presence or absence of wild-type rIris (Iris), mutant rIris (L339A), rIris–elastase complex (Iris + elas- tase) or elastase alone (elastase) at the indicated concentrations. TNF-a concentrations were measured in the harvested cell superna- tants. In order to prepare the Iris–elastase complex, the two pro- teins were incubated in equimolar quantities for 1 h at 37 °C. Only samples with a recorded elastase activity < 25% compared with the control were used. Values are the mean of three experiments (± SD). Exosites mediate anti-inflammatory effects of Iris P P. Prevot et al. 3238 FEBS Journal 276 (2009) 3235–3246 ª 2009 The Authors Journal compilation ª 2009 FEBS interact with other proteins by molecular modeling. Two different types of sequence-based methods were used. On the one hand, potential immunogenic domains were predicted, based on sequence analysis of Iris, using a combination of DeLisi & Berzofsky’s [23], Eisenberg et al.’s [24] and HCA [25] methods. This approach, predicting accessible, charged amphipathic fragments [26,27], identified seven putative epi- topes ⁄ immunogenic fragments located between posi- tions 7–21 (ep1), 66–79 (ep2), 85–98 (ep3), 105–120 (ep4), 127–143 (ep5), 290–306 (ep6) and 312–325 (ep7) of Iris (Fig. 5). On the other hand, the RDB method identified eight regions within Iris as putative pro- tein ⁄ protein interaction sites (Fig. 5) located between positions 18–27 [receptor binding domain (RBD)1], 61–71 (RBD2), 91–98 (RBD3), 111–116 (RBD4), 125– 133 (RBD5) 139–150 (RBD6) 190–198 (RBD7) and 223–227 (RBD8), respectively. We relied on a 3D model of Iris that we had established previously [21] (Fig. 5) to address the location and accessibility of these antigenic and RBD domains. Five of the putative interacting domains identified by either method were overlapping (RBD1–RBD5). One (RBD3) was not considered able to form a good interacting domain because of a bad Berzofsky score. The properties of the four remaining selected peptides are summarized in Table 2. The analysis indicated that: (a) domains 62–67 (overlapping antigenic peptide ep2), 128–131 and 142–147 (both overlapping peptide ep5) defined particularly good interacting domains; and (b) antibodies against peptides 2 and 3 should interfere with a common interaction site (Fig. 5). The four peptides described in Table 2 (ep1, ep2, ep4, ep5) were synthesized and used to immunize rab- bits. The resulting antisera were evaluated for their neutralizing effect on the ability of Iris to block LPS- induced TNF-a production in PBMC. The antibody titers of the different sera were similar (not shown). Table 3 shows that antibodies targeting peptides ep2 and ep4 reduced the inhibition of TNF-a production in a dose-dependent manner, the anti-ep4 serum being more potent than the anti-ep2 serum. Conversely, anti- ep1 serum had no effect on the inhibitory action of Iris. Finally, the action of anti-ep5 serum could not be analyzed because it inhibited the production of TNF-a by itself, i.e. in the absence of Iris (data not shown). From these results, we conclude that the interaction site responsible for the anti-inflammatory effects of Iris is a conformational region covering domains RBD2 and RBD4, notably composed of helices D (67–79) Fig. 5. Potential antigenic epitopes and RBD prediction in Iris. The 3D structure of Iris is represented as a blue ribbon and the P1 resi- due important for the antiprotease activity is represented in orange (true volume). The axis of helices D and E and sheet1A is high- lighted by a green, red or pink arrow, respectively. (A) The predicted epitopes are represented in yellow and numbered as fol- lows: (1) 7–21, (2) 66–79, (3) 85–98, (4) 105–120, (5) 127–143, (6) 290–306, (7) 312–325. (B) The predicted RBD are represented in yellow and numbered as follows: (1) 21–25, (2) 62–67, (3) 92–99, (4) 111–115, (5) 128–131, (6) 142–147, (7) 192–196, (8) 223–228. Table 2. Potential amphipathic domains within Ixodes ricinus immunosuppressor (Iris) predicted both as immunogenic epitopes and recep- tor-binding domain. The first column indicates the amino acid positions in the protein sequence. Angle, the calculated angle between the helix axis and the plane of a model membrane. ASA, accessible surface area. +, the peptide has an adequate mean surface accessibility ‡ 30%. Peptide Sequence Total number aa Number positive charge Number negative charge Number polar aa Angle (°) ASA 7–21 NHILNFSVDLYKRLK 15 3 1 4 20 + 66–79 DKIHDHFSSFLCKL 14 2 2 4 0 + 105–120 EYTTLLQKSYDSTIKA 16 2 2 4 0 + 127–143 ADRVRLEVNAWVEEVTR 17 3 4 2 0 + P P. Prevot et al. Exosites mediate anti-inflammatory effects of Iris FEBS Journal 276 (2009) 3235–3246 ª 2009 The Authors Journal compilation ª 2009 FEBS 3239 and E (104–114) and sheet 1A (117–121) (Fig. 5). These domains are distinct from the RCL (amino acids 324–340) involved in the antihemostatic action of Iris [21] and are not affected by the structural rearrange- ment during protease inhibition. Iris delivery inhibits LPS-induced septic shock Because of its ability to interfere with TNF-a release, Iris seemed a good candidate to counteract endo- toxemia. The in vivo half-life of Iris was determined to verify whether it was appropriate for use in an animal model of endotoxemia. Figure 6 shows that the 125 I- labeled Iris concentration in plasma remained stayed similar for at least 20 h after i.p. injection, decreasing to 20% of the maximum observed value 44 h after administration. In order to address the ability of Iris to counteract endotoxemia, we used a model of murine sepsis following LPS injection. Mice were separated into two groups and treated with Iris (30 mgÆkg )1 , i.p.) or NaCl ⁄ P i respectively. Two hours later, endotoxemia was induced by LPS injection (40 mgÆkg )1 , i.p.). As shown in Fig. 7, the mortality rate in the Iris-treated group ( 50%) was significantly lower than in the NaCl ⁄ P i -treated group ( 80%) (P < 0.001). Further- more, mean survival time in the Iris-treated group was 48 h, compared with 24 h in the control group, indi- cating that Iris increased both survival rate and sur- vival time. In order to discount the possibility of a BSA-like effect for rIris, we injected mice with the same amount of ovalbumin, another serpin. As expected, there was a significant difference in mortality rate between rIris- and ovalbumin-treated groups (P = 0.0019), whereas no difference was found between the NaCl ⁄ P i - and ovalbumin-treated groups. However, when administrated after the induction of endotoxemia, Iris remained without effect (results not shown). In addition, Iris had no beneficial effect on caecum ligature puncture-induced sepsis (results not shown). Because Iris was able to reduce TNF-a release in a LPS-activated PBMC culture, we asked whether the protective effect of Iris was related to interference with the cytokine storm usually associated with septic death. Figure 8 shows that the administration of Iris significantly inhibited TNF-a release (P < 0.001), and to a lesser extent the release of monocyte chemo- attractant protein-1 (MCP-1) (P < 0.01) and IL-6 (P < 0.05) in the blood following LPS injection. Table 3. Effect of neutralization of Ixodes ricinus immunosuppres- sor (Iris) activity on tumor necrosis factor-alpha (TNF-a) production induced by lipopolysaccharide (LPS) in peripheral blood mononu- clear cells by polyclonal anti-rIris serum. Results are expressed as percentage of cytokine concentration recorded in the presence of LPS and absence of rIris (+LPS; )rIris). ⁄ , no antibody added; )LPS, no LPS added. TNF-a expression values are expressed in percentage relative to control ()rIris; +LPS) Antibodies dilution TNF-a production +rIris )rIris +LPS +LPS 10 20 40 80 ⁄⁄ 10 Anti-ep1 28 24 24 23 25 100 2 Anti-ep2 56 43 32 27 25 100 3 Anti-ep4 80 45 44 31 25 100 6 Fig. 6. Half-life of 125 I-labled Iris in the blood. 125 I-labled Iris (10 lg; 10 7 c.p.m.) was administrated i.p. Blood samples were collected at the indicated times by cardiac puncture, and platelet-poor plasma was prepared by centrifugation. Aliquots were counted in a gamma counter. Counts per minute per 500-lL aliquots are plotted against time. Values are the mean of three experiments (± SD). 0 20 40 60 80 100 0 20 40 60 80 100 Iris PBS Ovalbumin (h) Survival (%) Fig. 7. Iris treatment protects against LPS-induced toxic shock. Mice were injected i.p. with rIris (30 mgÆkg )1 ) or NaCl ⁄ P i . Two hours later, septic shock was induced by i.p. administration of E. coli serotype O111:B4 LPS (40 mgÆkg )1 ). Survival was recorded and plotted against time as percentage of injected animals surviving (n = 40). Exosites mediate anti-inflammatory effects of Iris P P. Prevot et al. 3240 FEBS Journal 276 (2009) 3235–3246 ª 2009 The Authors Journal compilation ª 2009 FEBS IL-10 production was not affected. This is in agreement with our previous observation that in vitro Iris inhibits the LPS-induced increase in TNF-a, inter- feron-c, IL-6 and IL-8, although IL-10 levels are not affected [18]. The IL-1b concentration remained too low to detect any statistically significant difference and was apparently unaffected during the time course of the experiment (data not shown). Again, this con- firmed our previous in vitro measurements [18]. In summary, Iris is able to increase ⁄ prolong both survival rate and survival time in mice undergoing LPS-induced endotoxemic shock. This correlated with a reduction of endotoxemic cytokine production. Discussion Iris as a multifunctional tick saliva protein The currently documented functions of serpins and tick saliva suggest a role for tick serpin(s) in the modu- lation of immune response, coagulation, fibrinolysis, complement regulation and inflammation or angiogen- esis [28,29]. In particular, the I. ricinus immunosup- pressor protein Iris, which is induced in tick saliva during the blood meal [18–20], was suggested to dis- turb the TH1 ⁄ TH2 balance by inhibiting interferon-c production. It was also shown to preferentially target the human leukocyte elastase or pork pancreatic elas- tase [21] and, to a lesser extent, tissue plasminogen activator, coagulation factor X and thrombin. As such, Iris may act in physiological processes relevant for the tick blood meal, disturbing its serpin activity hemosta- sis through interference with fibrinolysis, contact phase-activated pathway of coagulation and, to a lesser extent, platelet aggregation. Iris may also exert anti-inflammatory activity because soluble protein extracts of CHO-KI cells expressing rIRIS inhibited production of inflammatory cytokines like TNF-a trig- gered by LPS [18]. In this study, we documented that affinity-purified rIris was able to block TNF-a produced by PBMC activated by various TLR agonists, namely LPS (TLR4), poly(I : C) (TLR3), ODN 2006 (TLR9) and PGN (TLR2). Moreover, we indicated how Iris may exert its blocking effect: Iris was found to interact physically with the two major monocyte fractions of PBMC, namely CD11b+ CD14+ CD16) and CD11b+ CD14+ CD16+ cells [30]. Appropriate con- trols discarded the possibility of an action mediated by interference with LPS, reagents used in the dosage, TNF-a itself or TNF-a half-life (results not shown). Finally, we also showed that delivery of Iris in vivo significantly lowered the mortality rate and increased the survival time of mice undergoing LPS-induced sep- tic shock. This effect was not observed upon injection of the same amounts of ovalbumin, another serpin, used as a control, arguing against a BSA-like effect (Fig. 7). The observed protective effect of Iris was correlated with the inhibition of TNF-a, MCP-1 and IL-6 production, all of which participate in the cyto- kine storm associated with LPS endotoxinemia [31,32]. Of note, Iris had no activity in caecum ligature punc- ture-induced sepsis (not shown). This is reminiscent of the lack of action of specific anti-TNF-a IgG in the latter pathology and in agreement with the lack of involvement of TNF-a in this model [33]. However, Iris had a significant beneficial effect only when admin- istrated before LPS-induced endotoxemic shock (not Fig. 8. Effect of rIris on LPS-induced cyto- kine release in vivo. Mice were injected i.p. with rIris (30 mgÆkg )1 ) or NaCl ⁄ P i . Two hours later, septic shock was induced by i.p. administration of E. coli serotype O111:B4 LPS (40 mgÆkg )1 ). TNF-a, IL-6, MCP-1 and IL-10 levels were measured in the platelet-poor plasma collected by cardiac puncture at the indicated times post injec- tion of LPS. Results are expressed as means ± SEM of six mice per group for each time point. *P < 0.05, **P < 0.01, ***P < 0.001 compared with controls (one-way ANOVA). P P. Prevot et al. Exosites mediate anti-inflammatory effects of Iris FEBS Journal 276 (2009) 3235–3246 ª 2009 The Authors Journal compilation ª 2009 FEBS 3241 shown). This may be expected, because maximal TNF- a levels are recorded very shortly (90 min) after LPS- induced endotoxemic shock, suggesting the need for immediate action in order to interfere. Exosites mediate the anti-inflammatory action of Iris There are straightforward connections between the enzymatic function of Iris – a specific inhibitor of leu- kocyte elastase – and some of its putative physiological activities. In this regard, the proinflammatory effects of fragments generated from extracellular matrix deg- radation by elastase are well documented. For exam- ple, degradation products of elastin or heparan sulfate proteoglycan can act as chemoattractants towards inflammatory cells or activate TLR4, respectively [34,35]. TLR4 activation by LPS, responsible for fever, shock and death in sepsis, is thought to be prevented in vivo by the extracellular matrix. By cleaving matrix proteins, elastase liberates TLR4 from this extracellu- lar matrix constraint, favoring its interaction with potential ligands and activation of the inflammatory immune reaction [36]. Leukocyte elastase has also been found to modulate chemokine and cytokine activity, activate cell-surface receptors and cleave the antiadhe- sive coat of neutrophils [34,37]. Therefore, by inhibi- ting leukocyte elastase, Iris could clearly interfere with the inflammatory response. Through its inhibition of tissue plasminogen activator, factor X and thrombin, Iris may also interfere directly with coagulation. The reported data nevertheless suggested that some functions of Iris were independent of its enzymatic inhibitory activity. Indeed, both wild-type Iris and the mutant devoid of serpin activity (L339A; P2) were found to similarly increase platelet adhesion time [21]. This was reminiscent of the biological activity of native alpha-1-antitrypsin, which was shown to be indepen- dent of its inhibitory activity on serine proteases [38]. Moraga et al. [39] demonstrated that cleaved (devoid of activity) alpha-1-antitrypsin still has an effect on IL-6 and TNF-a production by monocytes ⁄ macro- phages. This action was inferred to the presence of exosites within alpha-1-antitrypsin. Similar assump- tions could be made regarding Iris. To test this hypothesis, we first evaluated whether the ability of Iris to inhibit TNF-a released by PBMC activated by LPS was dependent on the activity ⁄ integ- rity of the RCL (anti-proteasic domain). Wild-type Iris and its inactive mutant L339A inhibited release of the inflammatory cytokine to the same extent. This is in sharp contrast to the effect of rIris on fibrinolysis, an activity lost in the RCL mutant [21]. This indepen- dence of the protease inhibitory activity of the anti-inflammatory activity of Iris was further sup- ported using the cleaved ⁄ inactivated Iris protein obtained by incubation with its target serine protease. We observed that an elastase ⁄ Iris complex retains its inhibitory activity on the production of TNF-a. This further confirms that Iris devoid of its inhibitory activ- ity still inhibits TNF-a production. It further indicates that Iris preserves its anti-inflammatory activity even after a conformation change. Because an intact RCL domain seems dispensable for the inhibitory action of Iris on TNF-a production, the contribution of exosites was evaluated. These can be predicted using either the RBD method [26] or by epitope predicting (according to the Berzofsky, Eisen- berg and HCA methods). According to the most strin- gent criteria of the two methods, four antigenic peptides were selected within Iris and synthesized to generate specific antibodies. Antibodies targeting two of these peptides were shown to impair the ability of Iris to inhibit TNF-a by LPS-activated PBMC. Anti- ep2 serum had an activity twice as low as that of anti-ep4 serum. Because the different sera had the same antibody titer and were used at the same dilu- tion, this might translate into a real biological differ- ence and be related to the 3D location of these epitopes. The antigenic fragments are close in the 3D structure and correspond to helices D and E-s1A (posi- tions 2, helix D, and 4, helix E-s1A, in Fig. 5A). Therefore, an interacting site, involved in the anti- inflammatory function of Iris, corresponds to a region involving helices hD and hE. However, it cannot be ruled out that only the helix E-s1A region is implicated in inhibition of TNF-a release, because antibodies tar- geting helix E seem more potent inhibitors (although displaying the same affinity); the effects observed with antibodies targeting helix D may be related to an arti- fact caused by steric hindrance, antibodies masking helix E when binding to helix D (Fig. 8). Further mutational studies could be performed to verify whether the helix D region is truly implicated in the immunomodulatory function of Iris. However, these different mutations (with one or more mutations on a large peptide of 16 amino acids) might weaken the protein structure and distort the functional analysis. To summarize, we showed that Iris exerts an anti- inflammatory action, blocking the release of TNF-a by various TLR agonist-activated monocytes, independent of the RCL and antiprotease activity. This action is mediated by (an) exosite(s) (RBD2 and RBD4), nota- bly composed of helices D (67–79) and E (104–114) and sheet 1A (117–121), likely to mediate direct inter- action with monocytes. Thus, Iris might be beneficial Exosites mediate anti-inflammatory effects of Iris P P. Prevot et al. 3242 FEBS Journal 276 (2009) 3235–3246 ª 2009 The Authors Journal compilation ª 2009 FEBS for the parasite, by interfering with hemostasis [21], and for the host by blocking excessive serine protease activity during acute inflammation and regulating the expression of pro- and anti-inflammatory mediators. This may prove particularly useful when removed from the parasite–host interaction context because it may form the basis of a drug acting in pathological situa- tions involving the overexpression of TNF-a. Materials and methods Preparation of rIris, mutant L339A and the cleaved forms of rIris Purified recombinant wild-type Iris (rIris) (Fig. S1) and mutant L339A were produced in a baculovirus expression system, according to Prevot et al. [21]. The cleaved form of rIris was obtained by incubation for 1 h at 37 °Cinthe presence of equimolar amounts of pancreatic elastase. Puri- fication buffers were prepared with Limulus amoebocyte lysate reagent water (Lonza, Valais, Switzerland). Purified proteins were diluted in NaCl ⁄ P i (pH 7.4) and tested for endotoxin contamination using the QCL-1000 kit (Lonza). Endotoxin levels were < 0.4 enzyme unitsÆmg )1 of protein in all preparations used. Samples containing endotoxin amounts superior to that threshold were loaded on Detoxi- Removal endotoxin gel columns according to the manufac- turer’s instructions (Pierce, Rockford, IL, USA) to ensure the removal of endotoxins. This was followed by dialysis against buffers appropriate for the following experiments. Protein concentrations in the endotoxin-purified batches were determined using a microBCA kit (Pierce) according to the manufacturer’s instructions. In order to control the protein activity of the different forms of rIris, elastase inhibition was assessed, as described by Prevot et al. [20]. Briefly, rIris, L339A mutant and the cleaved form of rIris were incubated with pancreatic elas- tase at an equimolar ratio for 10 min at room temperature, in 0.1 m Tris buffer, pH 7.5. After addition of a chromo- genic elastase substrate [succinyl-(Ala)3-p-nitroanilide; Sigma, St Louis, MO, USA] to a final concentration of 0.5 mm, absorbance was measured at 405 nm for 280 s. Absorbance values used to calculate elastase inhibition were corrected with controls containing buffer and substrate only. Inhibition values were 70% and 0% for rIris and L339A mutant, respectively. Cell isolation and culture PBMC were isolated from buffy coats using Ficoll–Leuco- sep tubes (Greiner Bio One, Stuttgart, Germany) according to the manufacturer’s instructions. Briefly, heparinated blood samples from three healthy human donors were cen- trifuged at 400 g for 35 min, at 18 °C in Leucosep tubes. Cells at the interface were then collected and washed three times in NaCl ⁄ P i . Cell numbers were determined using a Burker counting chamber. PBMC were seeded in 96-well culture plates (2 · 10 5 cellsÆ well )1 ; Falcon, Becton Dickinson, Plymouth, UK) and acti- vated by the indicated stimulus (PGN, 10 lg Æ mL )1 ; ODN 2006, 2 lgÆmL )1 ; poly(I : C), 10 lgÆmL )1 ; LPS, 100 ngÆmL )1 ) in a total volume of 200 lL complete RPMI- 1640 medium in the presence or absence of the different form of rIris (0–400 nm). Cells were incubated at 37 °C, 5% CO 2 for various times (4–24 h) depending on the cytokines to be assayed. Culture supernatants were conserved at – 80 °C before analysis for their contents in cytokine. Dexa- methasone (10 lm; Sigma) was used as a positive control for inhibition of cytokines production. Flow cytometry rIris was labeled with fluorescein isothiocyanate (FITC–Iris) or allophycocyanin (APC–Iris) using the Alexa Fluor Ò 488 and 647 Protein Labeling kits (Molecular Probes, Carlsbad, CA, USA), respectively. Labeled rIris (100 nm) was incu- bated with 5 · 10 5 cells, LPS stimulated (100 ngÆmL )1 ; when indicated), for 30 min at 4 °C in the dark. Cells were then washed in NaCl ⁄ P i before incubation with the appro- priate antibodies (CD3, CD19, CD11b, CD14, CD16) labeled with R-Phycoerythrin or FITC (Biocytex, Marseille, France) for another 30 min at 4°C in the dark. After wash- ing twice with NaCl ⁄ P i , cells were subjected to FACS anal- ysis using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). To ascertain the specificity of the labeled rIris fluorescent signal, we performed a competition test between labeled and unlabeled rIris (both 100 nm). Briefly, the labeled rIris signal on PBMC was measured in presence of equimolar concentration of unlabeled rIris. Cytokine measurements Cytokine production was assessed using ELISA kits (human TNF-a or mouse MCP-1, TNF-a, IL-6, IL-10, and IL-1b) (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Polyclonal anti-rIris serum production Anti-rIris serum was produced as described by Prevot et al. [20]. Briefly, female New Zealand white rabbits (Har- lan, the Netherlands) were injected subcutaneously with 50 lg of purified rIris in NaCl ⁄ P i (pH 7.5), emulsified in Freund’s adjuvant. The first injection was performed in complete Freund’s adjuvant and two boosters in incom- plete Freund’s adjuvant. Injections were performed at 3-week intervals. Animals were bled on the day of injection P P. Prevot et al. Exosites mediate anti-inflammatory effects of Iris FEBS Journal 276 (2009) 3235–3246 ª 2009 The Authors Journal compilation ª 2009 FEBS 3243 and 2 weeks after the last booster. Two rabbits were mock-immunized following the same protocol with NaCl ⁄ P i in Freund’s adjuvant as negative controls. Animal care and experimental procedures were carried out in accordance with the Helsinki Declaration (Publication 85– 23, revised 1985), local institutional guidelines (laboratory license number LA 1500474) and the Belgian law of August 14 th , 1986 as well as the royal decree of November 14 th , 1993 on the protection of laboratory animals. Iris neutralization assays rIris was preincubated with dilutions of immune and con- trol (mock immunized or preimmune sera) sera for 10 min at 37 °C in NaCl ⁄ P i . The effect of these samples on cyto- kine production by PBMCs activated by LPS was then assessed as described above. Prediction of interaction sites The prediction of binding sites from the protein sequence was made using the RBD method [26]. This method is derived from the Eisenberg’s method [24] based on the calcu- lation of the mean hydrophobicity <H> and the mean hydrophobic moment <l> for each amino acid by moving a five-residue window along the sequence. The method pre- dicts accessible and charged domains potentially involved in an interaction and is described in detail in Gallet et al. [26]. Prediction of epitopes The method involves the search for hydrophilic amphipathic helices based on the primary sequence of the protein [27]. Amphipathic domains were predicted using a combination of various available methods, such as DeLisi & Berzofsky’s [23], Eisenberg et al.’s [24] and HCA [25] methods. The first method relies on known antigenic sites, the second allows the detection of residues located at the protein surface, whereas in the latter, amphipathic domains are visualized on a bidimensional representation of the protein sequence. Furthermore, the secondary structure of the protein was also predicted using different algorithms, such as NPSA (http:// npsa-pbil.ibcp.fr), PROF [40] and Psipred [41]. Fragments corresponding to appropriate criteria of anti- genicity, amphipathicity and helicity are then reconstructed in 3D and minimized using hyperchem 6.0 (Hypercube Inc, Gainesville, FL, USA). Their interaction with a model membrane is simulated by the IMPALA method [42]. This step allows an evaluation of the hydrophobic ⁄ hydrophilic segregation (i.e. the amphipathicity) of the helices. The mean surface accessibility of the predicted epitopes on the Iris 3D structures was calculated by averaging the accessible surface area of the peptide residues using the Shrake and Rupley method, as described previously [43]. Anti-peptide serum production Rabbits were immunized by an injection of 0.1 mg of each synthetic antigenic peptide coupled to KLH. Two subsequent boosters were given at a 2-week interval. Sera were collected 1 week after the last booster. The antibody titer was mea- sured by ELISA as described previously [20]. Briefly, 250 ng of rIris in NaCl ⁄ P i was initially coated onto 96-well plates (Nunc, Rochester, NY, USA) overnight at 4 °C. Wells were then saturated for 1 h in NaCl ⁄ P i ⁄ 0.1% Tween 20 ⁄ 1% BSA at room temperature. The coated plates were incubated with various dilutions of immune or preimmune sera for 2 h at room temperature. A secondary biotinylated anti-IgG (dilu- tion 1 : 10 000) was added for 1 h, followed by peroxidase- coupled streptavidin (1 : 10 000) for 30 min at room temper- ature. Finally, the TMB chromogen (Sigma) was added for 10 min. Absorbance was then read at 450 and 630 nm with a Model 680 microplate reader (Bio-Rad, Hercules, CA, USA). Values were expressed as antibody titers as defined by the serum dilution at the inflection point of the curve. Iodination of Iris 125 I-labeled Iris was prepared by iodination with [ 125 I] sodium iodide (Perkin Elmer, Walthman, MA, USA) at 1 mCiÆmg )1 of protein, using Iodo-gen (Pierce; 100 lgÆmg )1 of protein) following the manufacturer’s instructions. Briefly, 2 Iodo-Gen beads were washed in 1 mL NaCl ⁄ P i , incubated for 5 min with 20 lL sodium iodide (10 mCiÆmL )1 ), and then with 200 lg Iris (1 mgÆmL )1 ) for 15 min at room temperature. Free iodide was removed using Zeba Desalt Spin Columns (Pierce) following the manufacturer’s instructions. Determination of 125 I-labeled Iris half-life in rat blood The in vivo blood persistence of 125 I-labeled Iris was evalu- ated after i.p. administration in female Whistar Hanover rats (200 g) of 10 7 c.p.m. (corresponding to 10 lg Iris, resuspended in 200 lL NaCl ⁄ P i ). Blood was collected 3, 20, 40, 60 and 120 h later by cardiac puncture and citrated (13 mm, final concentration). Platelet-poor plasma was then obtained by centrifugation and 500-lL aliquots were placed in glass test tubes. Radioactivity was measured using a gamma counter (LKB, Wallac, Finland). All animals were maintained and handled according to local and national ethical guidelines. Animal model of septic shock Two groups, each containing 40 female NMRI mice (30– 35 g), were injected i.p. with: (a) 500 lL Iris (30 mgÆkg )1 ) dialyzed against NaCl ⁄ P i , or (b) NaCl ⁄ P i alone for the Exosites mediate anti-inflammatory effects of Iris P P. 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This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 276 (2009) 3235–3246 ª 2009 The Authors Journal compilation ª 2009 FEBS . Exosites mediate the anti-inflammatory effects of a multifunctional serpin from the saliva of the tick Ixodes ricinus Pierre-Paul Prevot 1 , Alain Beschin 2,3 ,. the salivary gland of the tick, Ixodes scapularis: identification of fac- tor X and factor Xa as scaffolds for the inhibition of factor VIIa ⁄ tissue factor