La calcineurina en macrófagos: polarización y modulación de la respuesta inflamatoria.

Tesis doctoral inédita, leída en Universidad Autónoma de Madrad, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25/04/2014La Calcineurina (CN) es una proteína fosfatasa dependiente de calcio y calmodulina (CaM) que defosforila residuos fosforilados de serina y treonina. Fue identificada como la diana de las drogas inmunosupresoras CsA y FK506, y desde entonces se ha estudiado ampliamente su función en la homeostasis del sistema inmune. La mayoría de estudios se han centrado en elucidar el papel de esta fosfatasa en las células T, sin embargo, los trabajos que han analizado su implicación en la función de los macrófagos son escasos. Los macrófagos, como mediadores clave del sistema inmune innato, determinan el curso de las respuestas inflamatorias; contribuyen al ambiente inflamatorio secretando citoquinas y quimioquinas y además pueden polarizarse influenciados por el contexto inflamatorio. Los macrófagos son células plásticas que pueden adoptar una gran variedad de fenotipos en respuesta a diferentes estímulos in vivo. In vitro, se han clasificado en dos fenotipos extremos: macrófagos M1, que presentan propiedades pro-inflamatorias y macrófagos M2, que ejercen funciones inmunomoduladoras o inmunosupresoras. En este trabajo de tesis, estudiamos el papel de la CN en la polarización de los macrófagos y analizamos su potencial como diana terapéutica para el tratamiento de enfermedades inflamatorias. Utilizando estrategias para el bloqueo específico de la señalización mediada por la CN, revelamos un papel de esta fosfatasa en la polarización de los macrófagos que no había sido descrito con anterioridad probablemente debido al uso de la CsA y el FK506 como inhibidores de la CN. Mostramos que la inhibición de la CN por el peptido LxVP o su deleción genética induce un fenotipo anti-inflamatorio en los macrófagos. Por el contrario,mostramos que el tratamiento con la CsA o el FK506 no provoca los mismos efectos. Investigamos la divergencia entre las diferentes estrategias de inhibición de la CN y encontramos que el LxVP y la deleción genética de la CN, pero no la CsA ni el FK506, inducen la fosforilación de la MAPK p38 a través de la inhibición de la expresión de la fosfatasa MKP-1. Además, evidenciamos el potencial antiinflamatorio del bloqueo específico de la CN en los macrófagos en los modelos murinos de artritis inducida por colágeno e hipersensibilidad por contacto inducida por oxazolona. Realizamos experimentos de terapia génica con lentivirus codificantes del peptido LxVP y también experimentos de terapia celular con macrófagos deficientes en la CN. Ambos tratamientos tuvieron un claro efecto terapéutico en el establecimiento y progreso de la respuesta inflamatoria. En este trabajo describimos una nueva función de la CN en la polarización de los macrófagos e identificamos una asociación entre la CN y la MAPK p38 en este proceso. También proponemos una estrategia alternativa de inhibición específica de la CN con potencial uso en el tratamiento de enfermedades inflamatorias.Calcineurin (CN) is a calcium dependent serine/threonine phosphatase. It was identified as the target of the immunosuppressants cyclosporin A and FK506 and since then, its involvement in the homeostasis of the immune system has been extensively analyzed. Most studies have been focused on the role of this phosphatase in T cells, however little is known about its implication in macrophage functionality. Macrophages, as key players of the innate immune system, determine the course of an inflammatory response; they contribute to the inflammatory environment by secreting cytokines and chemokines and also can be polarized influenced by the inflammatory context. Macrophages are plastic cells that adopt a large variety of phenotypes in response to different stimuli in vivo. In vitro, they have been classified into two extreme phenotypes: M1 macrophages, which present pro-inflammatory properties, and M2 macrophages which exert immunomodulatory and immunosuppresor effects. Here, we study the role of CN in macrophage polarization and also analyze the potential of CN as a macrophage-specific target for the treatment of inflammatory diseases. By using specific CN-targeting strategies we reveal a key role for CN in macrophage polarization that had not been previously revealed by the use of CsA or FK506 as CN-inhibitors. We show that CN-inhibition by the LxVP peptide or CN-gene deletion induces an antiinflammatory phenotype in macrophages. However, this does not occur upon CsA or FK506 treatment. We investigate this divergence between the different CN-targeting approaches and find that LxVP- and CN-gene deletion, but not CsA and FK506, induce p38 MAPK activation through the inhibition of MKP-1 expression, which in turn mediates the anti-inflammatory macrophage polarization. We also evidence the antiinflammatory potential of specifically targeting CN in macrophages in the murine models of collagen-induced arthritis and oxazolone-induced contact hypersensitivity. We perform gene therapy experiments with lentiviruses encoding the LxVP peptide and also cell therapy experiments with CN-inhibited macrophages. Both treatments result in a significant therapeutic effect in the onset or progression of the inflammatory responses. In this study we describe a novel role for CN in macrophage polarization and identify a link between CN and p38 MAPK in this process. We also propose alternative and more specific CN-targeting strategies for the treatment of inflammatory diseases

Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody–envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires

Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody–envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires

IL-10 released by a new inflammation-regulated lentiviral system efficiently attenuates zymosan-induced arthritis

We thank Dr Filip Lim for critical reading of the manuscript, and Dr S. Bartlett for English editing and helpful discussions. We also thank Drs. David Sancho and M. A. del Pozo for providing us with DCs and immortalized MEFs, respectively. AR is supported by Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I) and Instituto de Salud Carlos III (FIS; PI060122), the Spanish Ministry of Science and Innovation (MICINN;SAF2009-10691) and the Comunidad Autónoma de Madrid (S2006/BIO-0236 and S2010/BMD-2312). JMR is supported by MICINN (RECAVA RD06/0014/005) and by from Fundació La Marató de TV3 (Grant 080731

Antibody elicited by HIV-1 immunogen vaccination in macaques displaces Env fusion peptide and destroys a neutralizing epitope

HIV-1 vaccine design aims to develop an immunogen that elicits broadly neutralizing antibodies against a desired epitope, while eliminating responses to off-target regions of HIV-1 Env. Here we report isolation and characterization of Ab1245, an off-target antibody against the Env gp120-gp41 interface, from V3-glycan patch immunogen-primed and boosted macaques. A 3.7 Å cryo-EM structure of an Ab1245-Env complex reveals one Ab1245 Fab binding asymmetrically to Env trimer at the gp120-gp41 interface using its long CDRH3 to mimic regions of gp41. The mimicry includes positioning of a CDRH3 methionine into the gp41 tryptophan clasp, resulting in displacement of the fusion peptide and fusion peptide-proximal region. Despite fusion peptide displacement, Ab1245 is non-neutralizing even at high concentrations, implying that only two fusion peptides per trimer are required for viral–host membrane fusion. These structural analyses facilitate immunogen design to prevent elicitation of Ab1245-like antibodies that block neutralizing antibodies against the fusion peptide

Engineering HIV Immunogens to Elicit IOMA-like Antibodies Targeting the CD4 Binding Site

Background: The most effective way to control the AIDS epidemic would be through active vaccination. The current focus of HIV vaccine design is induction of broadly neutralizing antibodies (bNAbs) against the HIV Envelope protein (Env) that neutralize the majority of circulating viral strains. Due to their breadth and potency, the most desirable bNAbs to elicit through immunization are the VRC01-class bNAbs that target the conserved host receptor (CD4) binding site (CD4bs). VRC01-class bNAbs mimic CD4 binding to share a common mode of gp120 binding and glycan accommodation using a VH1-2*02-derived variable heavy (VH) domain. While attractive candidates for immunogen design, features of VRC01-class bNAbs such as a high degree of somatic hypermutation (SHM) and a short (5-residue) light chain (LC) complementarity determining region 3 (CDRL3) (found in only 1% of human LCs) suggest they might be difficult to elicit through vaccination. We recently published a structural characterization of a novel VH1-2*02-derived CD4bs bNAb, named IOMA, that is an attractive target for immunogen design due to its relatively low levels of SHMs and normal-length CDRL3 (8 residues). Here we describe our work to design an immunogen that elicits IOMA-like antibodies by engineering a gp120 from the 426c Env. Methods: We used a yeast display approach coupled with multiple rounds of enrichment by fluorescent-activated cell sorting (FACS) to select gp120 variants that bind to the inferred germline of IOMA (IOMA iGL). Results: Binding studies using enzyme-linked immunosorbent assays (ELISAs) and surface plasmon resonance (SPR) demonstrated that our novel gp120 immunogens bind to IOMA iGL with micromolar affinity. Conclusions: We will present the results of immunization experiments using these novel immunogens in mouse models

Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques.

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires

A broadly neutralizing macaque monoclonal antibody against the HIV-1 V3-Glycan patch

A small fraction of HIV-1- infected humans develop broadly neutralizing antibodies (bNAbs) against HIV-1 that protect macaques from simian immunodeficiency HIV chimeric virus (SHIV). Similarly, a small number of macaques infected with SHIVs develop broadly neutralizing serologic activity, but less is known about the nature of simian antibodies. Here, we report on a monoclonal antibody, Ab1485, isolated from a macaque infected with SHIVAD8 that developed broadly neutralizing serologic activity targeting the V3-glycan region of HIV-1 Env. Ab1485 neutralizes 38.1% of HIV-1 isolates in a 42-pseudovirus panel with a geometric mean IC50 of 0.055 µg/mLl and SHIVAD8 with an IC50 of 0.028 µg/mLl. Ab1485 binds the V3-glycan epitope in a glycan-dependent manner. A 3.5 Å cryo-electron microscopy structure of Ab1485 in complex with a native-like SOSIP Env trimer showed conserved contacts with the N332gp120 glycan and gp120 GDIR peptide motif, but in a distinct Env-binding orientation relative to human V3/N332gp120 glycan-targeting bNAbs. Intravenous infusion of Ab1485 protected macaques from a high dose challenge with SHIVAD8. We conclude that macaques can develop bNAbs against the V3-glycan patch that resemble human V3-glycan bNAbs

Structural characterization of a highly-potent V3-glycan broadly neutralizing antibody bound to natively-glycosylated HIV-1 envelope

Broadly neutralizing antibodies (bNAbs) isolated from HIV-1-infected individuals inform HIV-1 vaccine design efforts. Developing bNAbs with increased efficacy requires understanding how antibodies interact with the native oligomannose and complex-type N-glycan shield that hides most protein epitopes on HIV-1 envelope (Env). Here we present crystal structures, including a 3.8-Å X-ray free electron laser dataset, of natively glycosylated Env trimers complexed with BG18, the most potent V3/N332_(gp120) glycan-targeting bNAb reported to date. Our structures show conserved contacts mediated by common D gene-encoded residues with the N332_(gp120) glycan and the gp120 GDIR peptide motif, but a distinct Env-binding orientation relative to PGT121/10-1074 bNAbs. BG18’s binding orientation provides additional contacts with N392_(gp120) and N386_(gp120) glycans near the V3-loop base and engages protein components of the V1-loop. The BG18-natively-glycosylated Env structures facilitate understanding of bNAb–glycan interactions critical for using V3/N332_(gp120) bNAbs therapeutically and targeting their epitope for immunogen design