Chemokine-binding Proteins Research Articles

The N-terminal domain of a tick evasin is critical for chemokine binding and neutralization and confers specific binding activity to other evasins

Tick chemokine-binding proteins (evasins) are an emerging class of biologicals that target multiple chemokines and show anti-inflammatory activities in preclinical disease models. Using yeast surface display, we identified a CCL8-binding evasin, P672, from the tick Rhipicephalus pulchellus. We found that P672 binds CCL8 and eight other CC-class chemokines with a Kd < 10 nm and four other CC chemokines with a Kd between 10 and 100 nm and neutralizes CCL3, CCL3L1, and CCL8 with an IC50 < 10 nm. The CC chemokine–binding profile was distinct from that of evasin 1 (EVA1), which does not bind CCL8. We also show that P672's binding activity can be markedly modulated by the location of a StrepII-His purification tag. Combining native MS and bottom-up proteomics, we further demonstrated that P672 is glycosylated and forms a 1:1 complex with CCL8, disrupting CCL8 homodimerization. Homology modeling of P672 using the crystal structure of the EVA1 and CCL3 complex as template suggested that 44 N-terminal residues of P672 form most of the contacts with CCL8. Replacing the 29 N-terminal residues of EVA1 with the 44 N-terminal residues of P672 enabled this hybrid evasin to bind and neutralize CCL8, indicating that the CCL8-binding properties of P672 reside, in part, in its N-terminal residues. This study shows that the function of certain tick evasins can be manipulated simply by adding a tag. We conclude that homology modeling helps identify regions with transportable chemokine-binding functions within evasins, which can be used to construct hybrid evasins with altered properties.

Development of Novel Promiscuous Anti-Chemokine Peptibodies for Treating Autoimmunity and Inflammation.

Chemokines and their receptors play critical roles in the progression of autoimmunity and inflammation. Typically, multiple chemokines are involved in the development of these pathologies. Indeed, targeting single chemokines or chemokine receptors has failed to achieve significant clinical benefits in treating autoimmunity and inflammation. Moreover, the binding of host atypical chemokine receptors to multiple chemokines as well as the binding of chemokine-binding proteins secreted by various pathogens can serve as a strategy for controlling inflammation. In this work, promiscuous chemokine-binding peptides that could bind and inhibit multiple inflammatory chemokines, such as CCL2, CCL5, and CXCL9/10/11, were selected from phage display libraries. These peptides were cloned into human mutated immunoglobulin Fc-protein fusions (peptibodies). The peptibodies BKT120Fc and BKT130Fc inhibited the ability of inflammatory chemokines to induce the adhesion and migration of immune cells. Furthermore, BKT120Fc and BKT130Fc also showed a significant inhibition of disease progression in a variety of animal models for autoimmunity and inflammation. Developing a novel class of antagonists that can control the courses of diseases by selectively blocking multiple chemokines could be a novel way of generating effective therapeutics.

Ticks from diverse genera encode chemokine-inhibitory evasin proteins

To prolong residence on their hosts, ticks secrete many salivary factors that target host defense molecules. In particular, the tick Rhipicephalus sanguineus has been shown to produce three salivary glycoproteins named "evasins," which bind to host chemokines, thereby inhibiting the recruitment of leukocytes to the location of the tick bite. Using sequence similarity searches, we have identified 257 new putative evasin sequences encoded by the genomes or salivary or visceral transcriptomes of numerous hard ticks, spanning the genera Rhipicephalus, Amblyomma, and Ixodes of the Ixodidae family. Nine representative sequences were successfully expressed in Escherichia coli, and eight of the nine candidates exhibited high-affinity binding to human chemokines. Sequence alignments enabled classification of the evasins into two subfamilies: C8 evasins share a conserved set of eight Cys residues (four disulfide bonds), whereas C6 evasins have only three of these disulfide bonds. Most of the identified sequences contain predicted secretion leader sequences, N-linked glycosylation sites, and a putative site of tyrosine sulfation. We conclude that chemokine-binding evasin proteins are widely expressed among tick species of the Ixodidae family, are likely to play important roles in subverting host defenses, and constitute a valuable pool of anti-inflammatory proteins for potential future therapeutic applications.

The Role of Atypical Chemokine Receptor CCXCKR (CCRL1) in Human Diseases

The role of chemokines and their receptors have been identified in many biological activities such as immune response and angiogenesis; however, their regulatory ways are under investigation. In recent years, homologous proteins’ typical chemokine receptors have been identified. Despite the structural similarity due to changes in a particular motif, they are not able to create signals through G proteins within the cell; therefore, these chemokine receptors are called silent or unusual. Through binding and internalization and degradation of the chemokines, these receptors regulate the level of ligand in the environment. In this paper, about fifty articles published in the field of the regulatory role of CCX-CKR receptor in some human diseases such as cancer and inflammatory diseases were reviewed. CCX-CKR is one of the chemokine binding proteins that like other silent chemokine receptors, is not able to induce intracellular signaling pathways. Since chemokine network plays a major role in many diseases such as cancer and autoimmune diseases through the impact of CCX-CKR on the level of chemokine environment, preventing intracellular signal creation by these proteins can be considered as a therapeutic target.

Evasins: Therapeutic Potential of a New Family of Chemokine-Binding Proteins from Ticks.

Blood-sucking parasites, such as ticks, remain attached to their hosts for relatively long periods of time in order to obtain their blood meal without eliciting an immune response. One mechanism used to avoid rejection is the inhibition of the recruitment of immune cells, which can be achieved by a class of chemokine-binding proteins (CKBPs) known as Evasins. We have identified three distinct Evasins produced by the salivary glands of the common brown dog tick, Rhipicephalus sanguineus. They display different selectivities for chemokines, the first two identified show a narrow selectivity profile, while the third has a broader binding spectrum. The Evasins showed efficacy in animal models of inflammatory disease. Here, we will discuss the potential of their development for therapeutic use, addressing both the advantages and disadvantages that this entails.

Chemokine binding proteins: An immunomodulatory strategy going viral

Chemokines are chemotactic cytokines whose main function is to direct cell migration. The chemokine network is highly complex and its deregulation is linked to several diseases including immunopathology, cancer and chronic pain. Chemokines also play essential roles in the antiviral immune response. Viruses have therefore developed several counter strategies to modulate chemokine activity. One of these is the expression of type I transmembrane or secreted proteins with the ability to bind chemokines and modulate their activity. These proteins, termed viral chemokine binding proteins (vCKBP), do not share sequence homology with host proteins and are immunomodulatory in vivo. In this review we describe the discovery and characterization of vCKBP, explain their role in the context of infection in vivo and discuss relevant novel findings.

Effects of the secretory/excretory products of lepeophtheirus salmonis on atlantic salmon leukocyte migration

Lepeophtheirus salmonis evades its host, Atlantic salmon, by inhibiting inflammatory responses; however, the mechanisms of this activity are not well characterized. Studies from other arthropod ectoparasites have identified chemokine-binding proteins in the saliva and salivary gland extracts that inhibit neutrophil recruitment and activation in vitro and in vivo by binding to chemokines such as leukotriene B4 (LTB4). To determine if L. salmonis is using a similar mechanism to evade the host’s immune response the effects of L. salmonis secretory/excretory products (SEPs) on LTB4 stimulated leukocytes in vitro were examined. Results showed an increase in leukocyte recruitment in response to LTB4 immune stimulation; yet, pre-incubation of LTB4 with SEPs for 1 hour significantly reduced cell migration compared to LTB4 stimulation alone. This data suggests that L. salmonis inhibits inflammatory responses in Atlantic salmon by secreting proteins with chemokine inhibiting activity. Furthermore, we measured the effects of SEPs on the host’s response to chitin, a component of the exoskeleton of arthropods known to induce inflammation and IL-8 production, in vivo. Histological analysis of epithelial tissues collected 6 hours post chitin exposure showed a significant increase in leukocyte recruitment to chitin + dopamine seawater (CDSW) treatment compared to the no injection control. SEPs + chitin and SEPs only treatments were similar, with fewer leukocytes present at injections sites. These treatments were not significantly different from positive controls. To gain a better understanding of the chemotactic pathways disrupted by SEPs, the expression of genes involved in neutrophil activation and cellular chemotaxis in epithelial tissues collected from the in vivo chitin challenge was assessed. Data showed CDSW significantly upregulated IL-8 gene expression relative to the vehicle control at 24h post challenge. When SEPs were administered in combination with chitin, IL-8 expression was not significantly different from the CDSW and chitin only treatments. Results suggest SEPs are not inhibiting IL-8 transcription in response to chitin. While histologically fewer leukocytes are migrating in the chitin + SEP and SEP only treatments, transcriptionally it was shown there is no change in IL-8 expression between chitin and chitin + SEP treatments. The in vivo data suggests SEPs are directly interacting with leukocytes inhibiting the release of IL-8, or are directly interacting with IL-8 inhibiting its ability to interact with immune cells and stimulate an inflammatory response in Atlantic salmon.

Methods for the Recognition of GAG-Bound Chemokines.

Chemokines play a pivotal role in the multistep cascade of cellular recruitment, where they provide the directional signal. They activate cells through a high-affinity interaction with their receptors, members of the large family of heptahelical G protein-coupled receptors. In order to provide the directional signal, they bind to cell surface proteoglycans through a low-affinity interaction with the glycosaminoglycan (GAG) moiety. While several methods have been described to measure the chemokine-GAG interaction, this chapter describes methods to identify whether anti-chemokine antibodies or chemokine-binding proteins recognize the GAG-bound chemokine.

Mutagenesis by Phage Display.

Chemokines are small chemoattractant proteins involved in the recruitment of leukocytes to the site of inflammation. Due to their prominent role in the inflammatory process, chemokine inhibitors have been developed by parasites to remain undetected not only by the host immune system but also by various laboratories to develop anti-inflammatory compounds. Taking advantage of the small size of natural chemokine-binding proteins, we report here several methods to facilitate their characterization using phage display to identify the chemokine-binding site and to modulate the selectivity of such inhibitors. Interestingly, these methods could be adapted to display the natural inhibitors of other cytokines or even cytokines on phage surface.

Immune modulation by virus-encoded secreted chemokine binding proteins

Chemokines are chemoattractant cytokines that mediate the migration of immune cells to sites of infection which play an important role in innate and adaptive immunity. As an immune evasion strategy, large DNA viruses (herpesviruses and poxviruses) encode soluble chemokine binding proteins that bind chemokines with high affinity, even though they do not show sequence similarity to cellular chemokine receptors. This review summarizes the different secreted viral chemokine binding proteins described to date, with special emphasis on the diverse mechanisms of action they exhibit to interfere with chemokine function and their specific contribution to virus pathogenesis.

Identification of the Pharmacophore of the CC Chemokine-binding Proteins Evasin-1 and -4 Using Phage Display

To elucidate the ligand-binding surface of the CC chemokine-binding proteins Evasin-1 and Evasin-4, produced by the tick Rhipicephalus sanguineus, we sought to identify the key determinants responsible for their different chemokine selectivities by expressing Evasin mutants using phage display. We first designed alanine mutants based on the Evasin-1·CCL3 complex structure and an in silico model of Evasin-4 bound to CCL3. The mutants were displayed on M13 phage particles, and binding to chemokine was assessed by ELISA. Selected variants were then produced as purified proteins and characterized by surface plasmon resonance analysis and inhibition of chemotaxis. The method was validated by confirming the importance of Phe-14 and Trp-89 to the inhibitory properties of Evasin-1 and led to the identification of a third crucial residue, Asn-88. Two amino acids, Glu-16 and Tyr-19, were identified as key residues for binding and inhibition of Evasin-4. In a parallel approach, we identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8. It therefore appears that Evasin-1 and -4 use different pharmacophores to bind CC chemokines, with the principal binding occurring through the C terminus of Evasin-1, but through the N-terminal region of Evasin-4. However, both proteins appear to target chemokine N termini, presumably because these domains are key to receptor signaling. The results also suggest that phage display may offer a useful approach for rapid investigation of the pharmacophores of small inhibitory binding proteins.

Genome-wide association study of CSF levels of 59 alzheimer's disease candidate proteins: significant associations with proteins involved in amyloid processing and inflammation.

Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10−10) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.

Research resource: Gene profiling of G protein-coupled receptors in the arcuate nucleus of the female.

The hypothalamic arcuate nucleus controls many critical homeostatic functions including energy homeostasis, reproduction, and motivated behavior. Although G protein-coupled receptors (GPCRs) are involved in the regulation of these functions, relatively few of the GPCRs have been identified specifically within the arcuate nucleus. Here, using TaqMan low-density arrays we quantified the mRNA expression of nonolfactory GPCRs in mouse arcuate nucleus. An unprecedented number of GPCRs (total of 292) were found to be expressed, of which 183 were known and 109 were orphan GPCRs. The known GPCR genes expressed were classified into several functional clusters including hormone/neurotransmitter, growth factor, angiogenesis and vasoactivity, inflammation and immune system, and lipid messenger receptors. The plethora of orphan genes expressed in the arcuate nucleus were classified into 5 structure-related classes including class A (rhodopsin-like), class B (adhesion), class C (other GPCRs), nonsignaling 7-transmembrane chemokine-binding proteins, and other 7-transmembrane proteins. Therefore, for the first time, we provide a quantitative estimate of the numerous GPCRs expressed in the hypothalamic arcuate nucleus. Finally, as proof of principle, we documented the expression and function of one of these receptor genes, the glucagon-like peptide 1 receptor (Glp1r), which was highly expressed in the arcuate nucleus. Single-cell RT-PCR revealed that Glp1r mRNA was localized in proopiomelanocortin neurons, and using whole-cell recording we found that the glucagon-like peptide 1-selective agonist exendin-4 robustly excited proopiomelanocortin neurons. Thus, the quantitative GPCR data emphasize the complexity of the hypothalamic arcuate nucleus and furthermore provide a valuable resource for future neuroendocrine/endocrine-related experiments.

International Union of Basic and Clinical Pharmacology. LXXXIX. Update on the Extended Family of Chemokine Receptors and Introducing a New Nomenclature for Atypical Chemokine Receptors

Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, and Power CA (2000) Pharmacol Rev 52:145-176]. Since then, several new receptors have been discovered, and major advances have been made for the others in many areas, including structural biology, signal transduction mechanisms, biology, and pharmacology. New and diverse roles have been identified in infection, immunity, inflammation, development, cancer, and other areas. The first two drugs acting at chemokine receptors have been approved by the U.S. Food and Drug Administration (FDA), maraviroc targeting CCR5 in human immunodeficiency virus (HIV)/AIDS, and plerixafor targeting CXCR4 for stem cell mobilization for transplantation in cancer, and other candidates are now undergoing pivotal clinical trials for diverse disease indications. In addition, a subfamily of atypical chemokine receptors has emerged that may signal through arrestins instead of G proteins to act as chemokine scavengers, and many microbial and invertebrate G protein-coupled chemokine receptors and soluble chemokine-binding proteins have been described. Here, we review this extended family of chemokine receptors and chemokine-binding proteins at the basic, translational, and clinical levels, including an update on drug development. We also introduce a new nomenclature for atypical chemokine receptors with the stem ACKR (atypical chemokine receptor) approved by the Nomenclature Committee of the International Union of Pharmacology and the Human Genome Nomenclature Committee.

Evasin‐4, a tick‐derived chemokine‐binding protein with broad selectivity can be modified for use in preclinical disease models

Rhipicephalus sanguineus, the common brown dog tick, produces several chemokine-binding proteins which are secreted into the host in its saliva to modulate the host response during feeding. Two of these demonstrate very restricted selectivity profiles. Here, we describe the characterization of the third, which we named Evasin-4. Evasin-4 was difficult to produce recombinantly using its native signal peptide in HEK cells, but expressed very well using the urokinase-type plasminogen activator signal peptide. Using SPR, Evasin-4 was shown to bind most CC chemokines. Investigation of the neutralization properties by inhibition of chemokine-induced chemotaxis showed that binding and neutralization did not correlate in all cases. Two major anomalies were observed: no binding was observed to CCL2 and CCL13, yet Evasin-4 was able to inhibit chemotaxis induced by these chemokines. Conversely, binding to CCL25 was observed, but Evasin-4 did not inhibit CCL25-induced chemotaxis. Size-exclusion chromatography confirmed that Evasin-4 forms a complex with CCL2 and CCL18. In accordance with the standard properties of unmodified small proteins, Evasin-4 was rapidly cleared following in vivo administration. To enhance the in vivo half-life and optimize its potential as a therapeutic agent, Fc fusions of Evasin-4 were created. Both the N- and C-terminal fusions were shown to retain binding activity, with the C-terminal fusion showing a modest reduction in potency.

Novel Anti-Inflammatory Strategies Through Protein Engineering of Evasins

Event Abstract Back to Event Novel Anti-Inflammatory Strategies Through Protein Engineering of Evasins Pauline Bonvin1, 2, Jeffrey Shaw2, Christine A. Power2 and Amanda E. Proudfoot1, 2* 1 NovImmune S.A., Switzerland 2 Merck Serono S.A., Switzerland We have identified 3 chemokine binding proteins (ChBP) from a tick salivary gland library which were named Evasin-1, -3 and -4. Evasin-1 and -4 bind CC-chemokines, the first being highly selective whilst the second has a broad pattern of selectivity. Evasin-3 binds the neutrophil chemoattractant ELR CXC-chemokines. In order to create therapeutic modalities for the treatment of chronic inflammatory diseases, the Evasins have been fused to Fc to produce molecules with a long half-life. The fusions have been made both at the N- and the C-terminus of the Evasins. Both Evasin-3 fusions showed similar activity whereas Fc-Evasin-4 was superior to Evasin-4-Fc. We then took advantage of this property to create a bi-specific molecule by fusion of Evasin-4 to Enbrel®, a commercialized anti-TNF in order to inhibit simultaneously cytokine and chemokine pathways. As both Evasin-4 and Enbrel® have been shown to be effective in murine models but still also allow space for improved efficacy, this combination will provide an answer to the potential synergistic effect of the two strategies. We have characterized both their binding properties and their neutralizing activities in vitro. The strategy to test the efficacy of these Evasin/Fc fusions in vivo will be discussed. Acknowledgements The research leading to these results has received funding from the European Union Seventh Framework Programme [FP7-2007-2013] under grant agreement n° HEALTH-F4-2011-281608 (TIMER). References M. Deruaz, A. Frauenschuh, A.L. Alessandri, J.M. Dias, F.M. Coelho, R.C. Russo, B.R. Ferreira, G.J. Graham, J.P. Shaw, T.N. Wells, M.M. Teixeira, C.A. Power, A.E. Proudfoot, Ticks produce highly selective chemokine binding proteins with antiinflammatory activity. J.Exp.Med. 205 (2008) 2019-2031. A. Frauenschuh, C.A. Power, M. Deruaz, B.R. Ferreira, J.S. Silva, M.M. Teixeira, J.M. Dias, T. Martin, T.N. Wells, A.E. Proudfoot, Molecular cloning and characterization of a highly selective chemokine-binding protein from the tick Rhipicephalus sanguineus. J.Biol.Chem. 282 (2007) 27250-27258. M. Déruaz, P. Bonvin, I.C. Severin, Z. Johnson, P. Malinge, S. Krohn, C.A. Power, A.E. Proudfoot, Characterization of Evasin-4, a tick-derived chemokine binding protein with broad selectivity, and its Fc fusions. Manuscript submitted for publication Keywords: Chemokines, Ticks, Evasin, Chemokine-binding protein, Fc fusion, Chronic inflammatory disease Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Bonvin P, Shaw J, Power CA and Proudfoot AE (2013). Novel Anti-Inflammatory Strategies Through Protein Engineering of Evasins. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01109 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Amanda E Proudfoot, NovImmune S.A., Plan-Les-Ouates, Switzerland, 361869@frontiersin.org Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Pauline Bonvin Jeffrey Shaw Christine A Power Amanda E Proudfoot Google Pauline Bonvin Jeffrey Shaw Christine A Power Amanda E Proudfoot Google Scholar Pauline Bonvin Jeffrey Shaw Christine A Power Amanda E Proudfoot PubMed Pauline Bonvin Jeffrey Shaw Christine A Power Amanda E Proudfoot Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Treatment with the CC chemokine-binding protein Evasin-4 improves post-infarction myocardial injury and survival in mice

Chemokines trigger leukocyte trafficking and are implicated in cardiovascular disease pathophysiology. Chemokine-binding proteins, called "Evasins" have been shown to inhibit both CC and CXC chemokine-mediated bioactivities. Here, we investigated whether treatment with Evasin-3 (CXC chemokine inhibitor) and Evasin-4 (CC chemokine inhibitor) could influence post-infarction myocardial injury and remodelling. C57Bl/6 mice were submitted in vivo to left coronary artery permanent ligature and followed up for different times (up to 21 days). After coronary occlusion, three intraperitoneal injections of 10 μg Evasin-3, 1 μg Evasin-4 or equal volume of vehicle (PBS) were performed at 5 minutes, 24 hours (h) and 48 h after ischaemia onset. Both anti-chemokine treatments were associated with the beneficial reduction in infarct size as compared to controls. This effect was accompanied by a decrease in post-infarction myocardial leukocyte infiltration, reactive oxygen species release, and circulating levels of CXCL1 and CCL2. Treatment with Evasin-4 induced a more potent effect, abrogating the inflammation already at one day after ischaemia onset. At days 1 and 21 after ischaemia onset, both anti-chemokine treatments failed to significantly improve cardiac function, remodelling and scar formation. At 21-day follow-up, mouse survival was exclusively improved by Evasin-4 treatment when compared to control vehicle. In conclusion, we showed that the selective inhibition of CC chemokines (i.e. CCL5) with Evasin-4 reduced cardiac injury/inflammation and improved survival. Despite the inhibition of CXC chemokine bioactivities, Evasin-3 did not affect mouse survival. Therefore, early inhibition of CC chemokines might represent a promising therapeutic approach to reduce the development of post-infarction heart failure in mice.

Structural mechanism of ER retrieval of MHC class I by cowpox.

Author SummaryViruses encode a wide array of proteins whose principle function is to disable the surveillance and effector functions of the immune system. A common viral target is the MHC class I antigen processing and presentation pathway, which is a potent mechanism used by the host for the detection and killing of virally infected cells. In this study we have delineated the immune evasion mechanism of the cowpox-encoded CPXV203 protein, which is known to potently block the normal trafficking of MHCI from the endoplasmic reticulum (ER) to the plasma membrane. CPXV203 does this by highjacking an ER-retrieval system that usually serves to capture defective, chaperone complexed MHCI proteins in the Golgi and send them to the ER. We show that CPXV203 adopts a compact beta-sandwich structure and engages evolutionarily conserved MHCI determinants that are located under the peptide-binding platform. The viral protein binds a variety of different MHCI proteins weakly at the pH found in the ER, but the affinity and half-life are significantly augmented at the more acidic conditions found in the Golgi. Together these data suggest that CPXV203 works cooperatively with the endogenous ER retrieval process to promiscuously target fully assembled MHCI, thereby preventing T-cell killing of cowpox infected cells.

Subversion of cytokine networks by virally encoded decoy receptors.

During the course of evolution, viruses have captured or created a diverse array of open reading frames, which encode for proteins that serve to evade and sabotage the host innate and adaptive immune responses that would otherwise lead to their elimination. These viral genomes are some of the best textbooks of immunology ever written. The established arsenal of immunomodulatory proteins encoded by viruses is large and growing, and includes specificities for virtually all known inflammatory pathways and targets. The focus of this review is on herpes and poxvirus-encoded cytokine and chemokine-binding proteins that serve to undermine the coordination of host immune surveillance. Structural and mechanistic studies of these decoy receptors have provided a wealth of information, not only about viral pathogenesis but also about the inner workings of cytokine signaling networks.

O043 Treatment with chemokine-binding protein Evasin-3 reduces atherosclerotic vulnerability for ischemic stroke in mice

Introduction The presence and rupture of a carotid plaque inducing a severe arterial lumen stenosis have been associated with a consequent acute ischemic stroke in the brain. Treatment with chemokine-binding proteins isolated from tick salivary glands (also called “Evasins”) has been shown to reduce leukocyte recruitment in animal models of several diseases, such as acute myocardial infarction and rheumatoid arthritis. In the present study, we investigated the potential benefits of the selective inhibition of neutrophil recruitment (mediated by Evasin-3 treatment) in both primary and secondary prevention of ischemic stroke. Methods Two mouse models of shear stress-induced carotid atherosclerosis and cerebral ischemia and reperfusion were used (for ischemic stroke primary and secondary prevention strategies, respectively). In the first model, preventive chronic treatment with Evasin-3 (5 daily injections [at 1 microg/mouse] per week for total 3-week treatment period) was tested as compared with control vehicle (PBS) in ApoE−/− mice submitted to the implantation of “cast” carotid device. In the second model, C57Bl/6 mice aged from 8 to 12 weeks were used. The left middle cerebral artery (MCA) was occluded for 45 min with an intraluminal Dafilon 6.0 nylon suture. Then, blood flow was re-established and mice were followed up for 24 h of reperfusion. Five minutes after MCA occlusion, mice received one intraperitoneal injection of Evasin-3 (10 microg/mouse) or control vehicle (PBS). Leukocyte infiltration, infarct size and blood brain barrier (BBB) permeability were determined at 24 h of reperfusion. Results In atherosclerotic plaques of ApoE−/− mice, primary prevention treatment with Evasin-3 was associated with the reduction of neutrophils and MMP-9 content in aortic root and carotid plaques as compared to control vehicle. Secondary prevention treatment approach (after ischemic stroke) with Evasin-3 was associated with a significant reduction in neutrophil infiltration in the ischemic brain as compared to control vehicle. However, this anti-inflammatory effect was not associated with any improvements in cerebral infarct size, edema and BBB permeability. Conclusion Primary prevention strategy of Evasin-3 treatment reduced atherosclerotic plaque vulnerability for ischemic stroke. Acute Evasin-3 administration after cerebral ischemia onset significantly reduced brain inflammation, but failed to improve post-stroke outcomes. References None.