RESUMO
Extracellular vesicles (EVs), characterized by low immunogenicity, high biocompatibility and targeting specificity along with excellent blood-brain barrier permeability, are increasingly recognized as promising drug delivery vehicles for treating a variety of diseases, such as cancer, inflammation and viral infection. However, recent findings demonstrate that the intracellular delivery efficiency of EVs fall short of expectations due to phagocytic clearance mediated by the host mononuclear phagocyte system through Fcγ receptors, complement receptors as well as non-opsonic phagocytic receptors. In this text, we investigate a range of bacterial virulence proteins that antagonize host phagocytic machinery, aiming to explore their potential in engineering EVs to counteract phagocytosis. Special emphasis is placed on IdeS secreted by Group A Streptococcus and ImpA secreted by Pseudomonas aeruginosa, as they not only counteract phagocytosis but also bind to highly upregulated surface biomarkers αVß3 on cancer cells or cleave the tumor growth and metastasis-promoting factor CD44, respectively. This suggests that bacterial anti-phagocytic proteins, after decorated onto EVs using pre-loading or post-loading strategies, can not only improve EV-based drug delivery efficiency by evading host phagocytosis and thus achieve better therapeutic outcomes but also further enable an innovative synergistic EV-based cancer therapy approach by integrating both phagocytosis antagonism and cancer targeting or deactivation.
Assuntos
Vesículas Extracelulares , Fagocitose , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Fagocitose/imunologia , Humanos , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/metabolismo , Integrina alfaVbeta3/metabolismo , Integrina alfaVbeta3/imunologia , Receptores de Hialuronatos/metabolismo , Receptores de Hialuronatos/imunologia , Pseudomonas aeruginosa/imunologiaRESUMO
Plasmacytoid dendritic cells (pDCs) are strongly implicated as a major source of IFN-I in systemic lupus erythematosus (SLE), triggered through TLR-mediated recognition of nucleic acids released from dying cells. However, relatively little is known about how TLR signaling and IFN-I production are regulated in pDCs. In this article, we describe a role for integrin αvß3 in regulating TLR responses and IFN-I production by pDCs in mouse models. We show that αv and ß3-knockout pDCs produce more IFN-I and inflammatory cytokines than controls when stimulated through TLR7 and TLR9 in vitro and in vivo. Increased cytokine production was associated with delayed acidification of endosomes containing TLR ligands, reduced LC3 conjugation, and increased TLR signaling. This dysregulated TLR signaling results in activation of B cells and promotes germinal center (GC) B cell and plasma cell expansion. Furthermore, in a mouse model of TLR7-driven lupus-like disease, deletion of αvß3 from pDCs causes accelerated autoantibody production and pathology. We therefore identify a pDC-intrinsic role for αvß3 in regulating TLR signaling and preventing activation of autoreactive B cells. Because αvß3 serves as a receptor for apoptotic cells and cell debris, we hypothesize that this regulatory mechanism provides important contextual cues to pDCs and functions to limit responses to self-derived nucleic acids.
Assuntos
Autoimunidade , Células Dendríticas , Integrina alfaVbeta3 , Lúpus Eritematoso Sistêmico , Camundongos Knockout , Transdução de Sinais , Receptor 7 Toll-Like , Animais , Camundongos , Células Dendríticas/imunologia , Integrina alfaVbeta3/imunologia , Integrina alfaVbeta3/metabolismo , Autoimunidade/imunologia , Receptor 7 Toll-Like/imunologia , Receptor 7 Toll-Like/metabolismo , Receptor 7 Toll-Like/genética , Lúpus Eritematoso Sistêmico/imunologia , Transdução de Sinais/imunologia , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Citocinas/imunologia , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismo , Linfócitos B/imunologia , Autoanticorpos/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Ativação Linfocitária/imunologia , Modelos Animais de DoençasRESUMO
Precise and effective manipulation of protein functions still faces tremendous challenges. Herein we report a programmable peptide molecule, consisted of targeting and self-assembly modules, that enables specific and highly efficient assembly governed by targeting receptor proteins. Upon binding to the cell membrane receptor, peptide conformation is somewhat stabilized along with decreased self-assembly activation energy, promoting peptide-protein complex oligomerization. We first design a GNNQQNY-RGD peptide (G7-RGD) to recognize integrin αV ß3 receptor for proof-of-concept study. In the presence of αV ß3 protein, the critical assembly concentration of free G7-RGD decreases from 525 to 33â µM and the resultant G7-RGD cluster drives integrin receptor oligomerization. Finally, a bispecific assembling peptide antiCD3-G7-RGD is rationally designed for cancer immunotherapy, which validates CD3 oligomerization and concomitant T cell activation, leading to T cell-mediated cancer cell cytolysis.
Assuntos
Imunoterapia , Integrina alfaVbeta3/análise , Neoplasias/terapia , Peptídeos/química , Humanos , Integrina alfaVbeta3/imunologia , Neoplasias/imunologia , Peptídeos/imunologiaRESUMO
CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvß3 and α5ß1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4ß1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvß3, α5ß1, and α4ß1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants.
Assuntos
Ligante de CD40/metabolismo , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Receptores de Superfície Celular/química , Linfócitos T/metabolismo , Sítio Alostérico , Animais , Ligante de CD40/imunologia , Linhagem Celular , Cricetinae , Humanos , Integrina alfa4beta1/imunologia , Integrina alfa5beta1/imunologia , Integrina alfaVbeta3/imunologia , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Linfócitos T/imunologiaRESUMO
Cyclic RGD (cRGD) peptide-conjugated boronated albumin was developed to direct toward integrin αvß3, which overexpresses on many cancer cells. A stepwise conjugation of c[RGDfK(Mal)] and maleimide-conjugated closo-dodecaborate (MID) to bovine serum albumin (BSA) afforded cRGD-MID-BSA, which was noncytotoxic toward both U87MG and A549 cells. As compared with l-BPA, selective antitumor activity of cRGD-MID-BSA toward U87MG cells overexpressing integrin αvß3 was identified after thermal neutron irradiation. In vivo fluorescence live imaging of Cy5-conjugated cRGD-MID-BSA and MID-BSA revealed that both cRGD-MID-BSA and MID-BSA similarly reached the maximum accumulation during 8-12 h after injection. The selective accumulation and retention of Cy5-cRGD-MID-BSA was more pronounced than Cy5-MID-BSA after 24 h. An in vivo boron neutron capture therapy (BNCT) study revealed that the cRGD peptide ligand combination enhanced accumulation of MID-BSA into tumor cells in U87MG xenograft models. The significant tumor growth suppression was observed in U87MG xenograft models at a dose of 7.5 mg [10B]/kg after neutron irradiation.
Assuntos
Terapia por Captura de Nêutron de Boro/métodos , Boro/administração & dosagem , Portadores de Fármacos/química , Integrina alfaVbeta3/metabolismo , Isótopos/administração & dosagem , Neoplasias/radioterapia , Animais , Boro/química , Compostos de Boro/administração & dosagem , Compostos de Boro/química , Linhagem Celular Tumoral , Feminino , Humanos , Integrina alfaVbeta3/imunologia , Microscopia Intravital , Isótopos/química , Camundongos , Neoplasias/diagnóstico por imagem , Neoplasias/imunologia , Neoplasias/patologia , Peptídeos Cíclicos/química , Soroalbumina Bovina/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Expression of the matricellular protein CCN1 (CYR61) is associated with inflammation and is required for successful wound repair. Here, we show that CCN1 binds bacterial pathogen-associated molecular patterns including peptidoglycans of Gram-positive bacteria and lipopolysaccharides of Gram-negative bacteria. CCN1 opsonizes methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa and accelerates their removal by phagocytosis and increased production of bactericidal reactive oxygen species in macrophages through the engagement of integrin αvß3. Mice with myeloid-specific Ccn1 deletion and knock-in mice expressing CCN1 unable to bind αvß3 are more susceptible to infection by S. aureus or P. aeruginosa, resulting in increased mortality and organ colonization. Furthermore, CCN1 binds directly to TLR2 and TLR4 to activate MyD88-dependent signaling, cytokine expression and neutrophil mobilization. CCN1 is therefore a pattern recognition receptor that opsonizes bacteria for clearance and functions as a damage-associated molecular pattern to activate inflammatory responses, activities that contribute to wound healing and tissue repair.
Assuntos
Proteína Rica em Cisteína 61/metabolismo , Proteínas Opsonizantes/metabolismo , Infecções por Pseudomonas/imunologia , Infecções Estafilocócicas/imunologia , Receptores Toll-Like/metabolismo , Animais , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/imunologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Humanos , Integrina alfaVbeta3/imunologia , Integrina alfaVbeta3/metabolismo , Masculino , Staphylococcus aureus Resistente à Meticilina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Opsonizantes/genética , Moléculas com Motivos Associados a Patógenos/metabolismo , Fagocitose/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/imunologia , Células Sf9 , Transdução de Sinais/imunologia , Infecções Estafilocócicas/microbiologia , Receptores Toll-Like/imunologiaRESUMO
BACKGROUND: Fetal/neonatal alloimmune thrombocytopenia (FNAIT) results from maternal alloantibodies (abs) reacting with fetal platelets expressing paternal human platelet antigens (HPAs), mostly HPA-1a. Anti-HPA-1a abs, are the most frequent cause of severe thrombocytopenia and intracranial hemorrhage (ICH). OBJECTIVES: Titration of anti-HPA-1a in maternal serum using standard National Institute for Biological Standards and Control (NIBSC) 03/152 is one diagnostic approach to predict the severity of FNAIT. Recently, we found three anti-HPA-1a subtypes reacting with the ß3 subunit independently or dependently from complexes with αIIb and αv. Endothelial cell-reactive anti-αvß3 abs were found predominantly in cases with ICH. Our aim was to assess whether available standard material represents all anti-HPA-1a subtypes. MATERIALS AND METHODS: In this study, anti-HPA-1a sera (NIBSC 03/152) and human monoclonal antibodies (moabs) against HPA-1a (moabs 26.4 and 813) were evaluated using transfected cell lines expressing αIIbß3, αvß3 or monomeric cß3. RESULTS: Flow cytometry analyses with well-characterized murine moabs recognizing αIIbß3, αvß3, or ß3 alone demonstrated that AP3 reacts compound-independently, whereas compound-dependent moabs Gi5 and 23C6 reacted only with complexes. NIBSC 03/152, moabs 26.4, and 813 against HPA-1a reacted like AP3, same results were obtained with monomeric cß3 in immunoblotting. Antigen capture assay targeting endothelial cells showed anti-HPA-1a reactivity disappearance after cß3 beads adsorption. Furthermore, in contrast to anti-HPA-1a abs from ICH cases, none of NIBSC 03/152, 26.4, and 813 inhibited tube formation. CONCLUSION: These results suggest that current anti-HPA-1a standard material contains only the anti-ß3 subtype. The absence of anti-αvß3 makes NIBSC 03/152 less suitable as standard to predict the severity of FNAIT.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos de Plaquetas Humanas/imunologia , Testes Imunológicos , Integrina alfaVbeta3/imunologia , Integrina beta3/imunologia , Isoanticorpos/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Trombocitopenia Neonatal Aloimune/diagnóstico , Células Endoteliais/imunologia , Células HEK293 , Humanos , Isoanticorpos/sangue , Neovascularização Fisiológica , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Trombocitopenia Neonatal Aloimune/sangue , Trombocitopenia Neonatal Aloimune/imunologiaRESUMO
Osteoarthritis (OA) is the leading cause of joint failure, yet the underlying mechanisms remain elusive, and no approved therapies that slow progression exist. Dysregulated integrin function was previously implicated in OA pathogenesis. However, the roles of integrin αVß3 and the integrin-associated receptor CD47 in OA remain largely unknown. Here, transcriptomic and proteomic analyses of human and murine osteoarthritic tissues revealed dysregulated expression of αVß3, CD47, and their ligands. Using genetically deficient mice and pharmacologic inhibitors, we showed that αVß3, CD47, and the downstream signaling molecules Fyn and FAK are crucial to OA pathogenesis. MicroPET/CT imaging of a mouse model showed elevated ligand-binding capacities of integrin αVß3 and CD47 in osteoarthritic joints. Further, our in vitro studies demonstrated that chondrocyte breakdown products, derived from articular cartilage of individuals with OA, induced αVß3/CD47-dependent expression of inflammatory and degradative mediators, and revealed the downstream signaling network. Our findings identify a central role for dysregulated αVß3 and CD47 signaling in OA pathogenesis and suggest that activation of αVß3 and CD47 signaling in many articular cell types contributes to inflammation and joint destruction in OA. Thus, the data presented here provide a rationale for targeting αVß3, CD47, and their signaling pathways as a disease-modifying therapy.
Assuntos
Antígeno CD47/metabolismo , Cartilagem Articular/patologia , Integrina alfaVbeta3/metabolismo , Osteoartrite/imunologia , Transdução de Sinais/imunologia , Animais , Antígeno CD47/genética , Antígeno CD47/imunologia , Cartilagem Articular/citologia , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/imunologia , Células Cultivadas , Condrócitos , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/imunologia , Masculino , Camundongos , Osteoartrite/diagnóstico por imagem , Osteoartrite/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Cultura Primária de Células , Proteômica , Membrana Sinovial/citologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Sinoviócitos , Microtomografia por Raio-XRESUMO
Tumor-associated macrophages (TAM) are highly expressed within the tumor microenvironment of a wide range of cancers, where they exert a protumor phenotype by promoting tumor cell growth and suppressing antitumor immune function. Here, we show that TAM accumulation in human and mouse tumors correlates with tumor cell expression of integrin αvß3, a known driver of epithelial cancer progression and drug resistance. A monoclonal antibody targeting αvß3 (LM609) exploited the coenrichment of αvß3 and TAMs to not only eradicate highly aggressive drug-resistant human lung and pancreas cancers in mice, but also to prevent the emergence of circulating tumor cells. Importantly, this antitumor activity in mice was eliminated following macrophage depletion. Although LM609 had no direct effect on tumor cell viability, it engaged macrophages but not natural killer (NK) cells to induce antibody-dependent cellular cytotoxicity (ADCC) of αvß3-expressing tumor cells despite their expression of the CD47 "don't eat me" signal. In contrast to strategies designed to eliminate TAMs, these findings suggest that anti-αvß3 represents a promising immunotherapeutic approach to redirect TAMs to serve as tumor killers for late-stage or drug-resistant cancers. SIGNIFICANCE: Therapeutic antibodies are commonly engineered to optimize engagement of NK cells as effectors. In contrast, LM609 targets αvß3 to suppress tumor progression and enhance drug sensitivity by exploiting TAMs to trigger ADCC.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Integrina alfaVbeta3/imunologia , Macrófagos/imunologia , Neoplasias Epiteliais e Glandulares/imunologia , Animais , Antineoplásicos/farmacologia , Progressão da Doença , Humanos , Camundongos , Neoplasias Epiteliais e Glandulares/patologia , Fagocitose/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Early precise diagnosis of cancers is crucial to realize more effective therapeutic interventions with minimal toxic effects. Cancer phenotypes may also alter greatly among patients and within individuals over the therapeutic process. The identification and characterization of specific biomarkers expressed on tumor cells are in high demand for diagnosis and treatment, but they are still a challenge. Herein, we designed three new bioorthogonal surface-enhanced Raman scattering (SERS) nanoprobes and successfully applied the cocktail of them for MDA-MB-231 and MCF-7 breast cancer multiplex phenotype detection. The SERS nanoprobes containing Raman reporters with diynl, azide, or cyano moieties demonstrated apparent Raman shift peaks in 2205, 2120, and 2230 cm-1, respectively, in the biologically Raman-silent region. Three target ligands, including oligonucleotide aptamer (AS1411), arginine-glycine-aspatic acid (RGD) peptide, and homing cell adhesion molecule antibody (anti-CD44), were separately conjugated to the nanoprobes for active recognition capability. The cocktail of the nanoprobes manifested minimal cytotoxicity and simultaneously multiplex phenotype imaging of MDA-MB-231 and MCF-7 cells. Quantitative measurement of cellular uptake by inductively coupled plasma mass spectrometry (ICPMS) verified that MDA-MB-231 cells harbored a much higher expression level of CD44 receptor than MCF-7 cells. For in vivo SERS detection, Raman shift peaks of 2120, 2205, and 2230 cm-1 in the micro-tumor were clearly observed, representing the existence of three specific biomarkers of nucleolin, integrin αvß3, and CD44 reporter, which could be used for early cancer phenotype identification. The biodistribution results indicated that target ligand modified nanoprobes exhibited much more accumulation in tumors than those nanoprobes without target ligands. The multicolor cocktail of bioorthogonal SERS nanoprobes offers an attractive and insightful strategy for early cancer multiplex phenotype targeting and diagnosis in vivo that is noninvasive and has low cross-talk, unique spectral-molecular signature, high sensitivity, and negligible background interference.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico por imagem , Imunoconjugados/química , Sondas Moleculares/química , Nanopartículas/química , Análise Espectral Raman/métodos , Animais , Anticorpos/química , Aptâmeros de Nucleotídeos/química , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Feminino , Expressão Gênica , Xenoenxertos , Humanos , Receptores de Hialuronatos/antagonistas & inibidores , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Imunoconjugados/administração & dosagem , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/imunologia , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Sondas Moleculares/administração & dosagem , Nanopartículas/administração & dosagem , Oligopeptídeos/química , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Espectrofotometria Atômica , NucleolinaRESUMO
Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5ß1 and αvß3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvß3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Hidroxicolesteróis/metabolismo , Imunidade Inata/imunologia , Integrina alfa5beta1/imunologia , Integrina alfaVbeta3/imunologia , Macrófagos/imunologia , Animais , Adesões Focais , Inflamação , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Interleucina-6/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Soluble urokinase receptor (suPAR) is a circulatory molecule that activates αvß3 integrin on podocytes, causes foot process effacement, and contributes to proteinuric kidney disease. While active integrin can be targeted by antibodies and small molecules, endogenous inhibitors haven't been discovered yet. Here we report what we believe is a novel renoprotective role for the inducible costimulator ligand (ICOSL) in early kidney disease through its selective binding to podocyte αvß3 integrin. Contrary to ICOSL's immune-regulatory role, ICOSL in nonhematopoietic cells limited the activation of αvß3 integrin. Specifically, ICOSL contains the arginine-glycine-aspartate (RGD) motif, which allowed for a high-affinity and selective binding to αvß3 and modulation of podocyte adhesion. This binding was largely inhibited either by a synthetic RGD peptide or by a disrupted RGD sequence in ICOSL. ICOSL binding favored the active αvß3 rather than the inactive form and showed little affinity for other integrins. Consistent with the rapid induction of podocyte ICOSL by inflammatory stimuli, glomerular ICOSL expression was increased in biopsies of early-stage human proteinuric kidney diseases. Icosl deficiency in mice resulted in an increased susceptibility to proteinuria that was rescued by recombinant ICOSL. Our work identified a potentially novel role for ICOSL, which serves as an endogenous αvß3-selective antagonist to maintain glomerular filtration.
Assuntos
Ligante Coestimulador de Linfócitos T Induzíveis , Integrina alfaVbeta3 , Falência Renal Crônica , Podócitos , Proteinúria , Motivos de Aminoácidos , Animais , Taxa de Filtração Glomerular/efeitos dos fármacos , Taxa de Filtração Glomerular/genética , Taxa de Filtração Glomerular/imunologia , Humanos , Ligante Coestimulador de Linfócitos T Induzíveis/genética , Ligante Coestimulador de Linfócitos T Induzíveis/imunologia , Ligante Coestimulador de Linfócitos T Induzíveis/farmacologia , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/imunologia , Falência Renal Crônica/tratamento farmacológico , Falência Renal Crônica/genética , Falência Renal Crônica/imunologia , Falência Renal Crônica/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Podócitos/imunologia , Podócitos/patologia , Proteinúria/tratamento farmacológico , Proteinúria/genética , Proteinúria/imunologia , Proteinúria/patologiaRESUMO
Macrophages are a predominant immune cell population that drive inflammatory responses and exhibit transitions in phenotype and function during tissue remodeling in disease and repair. Thus, engineering an immunomodulatory biomaterial has significant implications for resolving inflammation. Here, a biomimetic and photoresponsive hyaluronan (HA) hydrogel nanocomposite with tunable 3D extracellular matrix (ECM) adhesion sites for dynamic macrophage immunomodulation is engineered. Photodegradative alkoxylphenacyl-based polycarbonate (APP) nanocomposites are exploited to permit user-controlled Arg-Gly-Asp (RGD) adhesive peptide release and conjugation to a HA-based ECM for real-time integrin activation of macrophages encapsulated in 3D HA-APP nanocomposite hydrogels. It is demonstrated that photocontrolled 3D ECM-RGD peptide conjugation can activate αvß3 integrin of macrophages, and periodic αvß3 integrin activation can enhance anti-inflammatory M2 macrophage polarization. Altogether, an emerging use of biomimetic, photoresponsive, and bioactive HA-APP nanocomposite hydrogel is highlighted to command 3D cell-ECM interactions for modulating macrophage polarization, which may shed light on cell-ECM interactions in innate immunity and inspire new biomaterial-based immunomodulatory therapies.
Assuntos
Ácido Hialurônico , Hidrogéis , Imunomodulação , Macrófagos/imunologia , Nanocompostos/química , Raios Ultravioleta , Animais , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Hidrogéis/química , Hidrogéis/farmacologia , Imunomodulação/efeitos dos fármacos , Imunomodulação/efeitos da radiação , Integrina alfaVbeta3/imunologia , Camundongos , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Células RAW 264.7RESUMO
Essentials The pathogenesis of immune thrombocytopenia (ITP) has not been fully clarified. We analyzed the role of anti-αvß3 autoantibody in the pathogenesis of ITP in patients. Anti-αvß3 autoantibody impeded megakaryocyte migration and adhesion to the vascular niche. Anti-αv ß3 autoantibody potentially contributes to the pathogenesis of refractory ITP. SUMMARY: Background The pathogenesis of immune thrombocytopenia (ITP) has not been fully clarified. Anti-αvß3 integrin autoantibody is detected in chronic ITP patients, but its contribution to ITP is still unclear. Objectives To clarify the potential role of anti-αvß3 integrin autoantibody in chronic ITP and the related mechanism. Methods Relationship between levels of anti-αvß3 autoantibody and platelets in chronic ITP patients was evaluated. The influence of anti-αvß3 antibody on megakaryocyte (MK) survival, differentiation, migration and adhesion was assessed, and the associated signal pathways were investigated. Platelet recovery and MKs' distribution were observed in an ITP mouse model pretreated with different antibodies. Result In this study, we showed that the anti-αvß3 autoantibody usually coexists with anti-αIIbß3 autoantibody in chronic ITP patients, and patients with both autoantibodies have lower platelets. In in vitro studies, we showed that the anti-αvß3 antibody had no significant effect on the survival and proliferation of MKs, whereas it decreased formations of proplatelet significantly. Anti-αvß3 antibody impeded stromal cell derived facor-1 alpha (SDF-1α)- mediated migration and inhibited the phosphorylation of protein kinase B. Anti-αvß3 antibody significantly inhibited MKs' adhesion to endothelial cells and Fibrogen. The phosphorylation of focal adhesion kinase and proto-oncogene tyrosine-protein kinase Src induced by adhesion was inhibited when MKs were pretreated with anti-αvß3 antibody. In in vivo studies, we showed that injection with anti-αv antibody delayed platelet recovery in a mouse model of ITP. Conclusions These findings demonstrate that the autoantibody against integrin αv ß3 may aggravate thrombocytopenia in ITP patients by impeding MK migration and adhesion to the vascular niche, which provides new insights into the pathogenesis of ITP.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Integrina alfaVbeta3/imunologia , Megacariócitos/imunologia , Púrpura Trombocitopênica Idiopática/imunologia , Adolescente , Adulto , Idoso , Animais , Adesão Celular , Movimento Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Células Endoteliais/metabolismo , Feminino , Sangue Fetal/citologia , Humanos , Masculino , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Fosforilação , Contagem de Plaquetas , Glicoproteína IIb da Membrana de Plaquetas/imunologia , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Púrpura Trombocitopênica Idiopática/sangue , Células Estromais/metabolismo , Trombopoese , Adulto JovemRESUMO
Intestinal macrophages are highly mobile cells with extraordinary plasticity and actively contribute to cytokine-mediated epithelial cell damage. The mechanisms triggering macrophage polarization into a proinflammatory phenotype are unknown. Here, we report that during inflammation macrophages enhance its intercellular adhesion properties in order to acquire a M1-phenotype. Using in vitro and in vivo models we demonstrate that intercellular adhesion is mediated by integrin-αVß3 and relies in the presence of the unconventional class I myosin 1F (Myo1F). Intercellular adhesion mediated by αVß3 stimulates M1-like phenotype in macrophages through hyperactivation of STAT1 and STAT3 downstream of ILK/Akt/mTOR signaling. Inhibition of integrin-αVß3, Akt/mTOR, or lack of Myo1F attenuated the commitment of macrophages into a pro-inflammatory phenotype. In a model of colitis, Myo1F deficiency strongly reduces the secretion of proinflammatory cytokines, decreases epithelial damage, ameliorates disease activity, and enhances tissue repair. Together our findings uncover an unknown role for Myo1F as part of the machinery that regulates intercellular adhesion and polarization in macrophages.
Assuntos
Colite Ulcerativa/imunologia , Integrina alfaVbeta3/metabolismo , Ativação de Macrófagos , Macrófagos/imunologia , Miosina Tipo I/metabolismo , Animais , Linhagem Celular Tumoral , Colite Ulcerativa/induzido quimicamente , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Humanos , Integrina alfaVbeta3/imunologia , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miosina Tipo I/genética , Miosina Tipo I/imunologia , Cultura Primária de Células , Células RAW 264.7RESUMO
The LM609 antibody specifically recognizes αVß3 integrin and inhibits angiogenesis, bone resorption, and viral infections in an arginine-glycine-aspartate-independent manner. LM609 entered phase II clinical trials for the treatment of several cancers and was also used for αVß3-targeted radioimmunotherapy. To elucidate the mechanisms of recognition and inhibition of αVß3 integrin, we solved the structure of the LM609 antigen-binding fragment by X-ray crystallography and determined its binding affinity for αVß3. Using single-particle electron microscopy, we show that LM609 binds at the interface between the ß-propeller domain of the αV chain and the ßI domain of the ß3 chain, near the RGD-binding site, of all observed integrin conformational states. Integrating these data with fluorescence size-exclusion chromatography, we demonstrate that LM609 sterically hinders access of large ligands to the RGD-binding pocket, without obstructing it. This work provides a structural framework to expedite future efforts utilizing LM609 as a diagnostic or therapeutic tool.
Assuntos
Anticorpos Monoclonais/química , Antígenos/química , Fragmentos Fab das Imunoglobulinas/química , Integrina alfaVbeta3/química , Oligopeptídeos/química , Motivos de Aminoácidos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/imunologia , Inibidores da Angiogênese/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos/genética , Antígenos/imunologia , Antivirais/química , Antivirais/imunologia , Antivirais/metabolismo , Sítios de Ligação , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/imunologia , Conservadores da Densidade Óssea/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/imunologia , Ligantes , Modelos Moleculares , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
In tuberculosis (TB), the innate inflammatory immune response drives tissue destruction, morbidity, and mortality. Monocytes secrete matrix metalloproteinases (MMPs), which have key roles in local tissue destruction and cavitation. We hypothesized that integrin signaling might regulate monocyte MMP secretion in pulmonary TB during cell adhesion to the extracellular matrix (ECM). Adhesion to type I collagen and fibronectin by Mycobacterium tuberculosis-stimulated monocytes increased MMP-1 gene expression by 2.6-fold and 4.3-fold respectively, and secretion by 60% (from 1208.1 ± 186 to 1934.4 ± 135 pg/ml; p < 0.0001) and 63% (1970.3 ± 95 pg/ml; p < 0.001). MMP-10 secretion increased by 90% with binding to type I collagen and 55% with fibronectin, whereas MMP-7 increased 57% with collagen. The ECM did not affect the secretion of tissue inhibitors of metalloproteinases-1 or -2. Integrin αVß3 surface expression was specifically upregulated in stimulated monocytes and was further increased after adhesion to type I collagen. Binding of either ß3 or αV integrin subunits increased MMP-1/10 secretion in M. tuberculosis-stimulated monocytes. In a cohort of TB patients, significantly increased integrin ß3 mRNA accumulation in induced sputum was detected, to our knowledge, for the first time, compared with control subjects (p < 0.05). Integrin αVß3 colocalized with areas of increased and functionally active MMP-1 on infected monocytes, and αVß3 blockade markedly decreased type I collagen breakdown, and impaired both monocyte adhesion and leukocyte migration in a transwell system (p < 0.0001). In summary, our data demonstrate that M. tuberculosis stimulation upregulates integrin αVß3 expression on monocytes, which upregulates secretion of MMP-1 and -10 on adhesion to the ECM. This leads to increased monocyte recruitment and collagenase activity, which will drive inflammatory tissue damage.
Assuntos
Adesão Celular , Movimento Celular , Matriz Extracelular/metabolismo , Integrina alfaVbeta3/genética , Monócitos/imunologia , Mycobacterium tuberculosis/imunologia , Colágeno Tipo I/metabolismo , Colagenases/metabolismo , Matriz Extracelular/efeitos dos fármacos , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Humanos , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/imunologia , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 10 da Matriz/imunologia , Metaloproteinase 10 da Matriz/metabolismo , Metaloproteinase 7 da Matriz/imunologia , Metaloproteinase 7 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Monócitos/microbiologia , Monócitos/fisiologia , Transdução de Sinais , Escarro/química , Regulação para CimaRESUMO
BACKGROUND AND AIMS: Diabetes is a major risk factor for the development of atherosclerosis. Hyperglycemia stimulates vascular smooth muscle cells (VSMC) to secrete ligands that bind to the αVß3 integrin, a receptor that regulates VSMC proliferation and migration. This study determined whether an antibody that had previously been shown to block αVß3 activation and to inhibit VSMC proliferation and migration in vitro, inhibited the development of atherosclerosis in diabetic pigs. METHODS: Twenty diabetic pigs were maintained on a high fat diet for 22 weeks. Ten received injections of anti-ß3 F(ab)2 and ten received control F(ab)2 for 18 weeks. RESULTS: The active antibody group showed reduction of atherosclerosis of 91 ± 9% in the left main, 71 ± 11%, in left anterior descending, 80 ± 10.2% in circumflex, and 76 ± 25% in right coronary artery, (p < 0.01 compared to lesions areas from corresponding control treated arteries). There were significant reductions in both cell number and extracellular matrix. Histologic analysis showed neointimal hyperplasia with macrophage infiltration, calcifications and cholesterol clefts. Antibody treatment significantly reduced number of macrophages contained within lesions, suggesting that this change contributed to the decrease in lesion cellularity. Analysis of the biochemical changes within the femoral arteries that received the active antibody showed a 46 ± 12% (p < 0.05) reduction in the tyrosine phosphorylation of the ß3 subunit of αVß3 and a 40 ± 14% (p < 0.05) reduction in MAP kinase activation. CONCLUSIONS: Blocking ligand binding to the αVß3 integrin inhibits its activation and attenuates increased VSMC proliferation that is induced by chronic hyperglycemia. These changes result in significant decreases in atherosclerotic lesion size in the coronary arteries. The results suggest that this approach may have efficacy in treating the proliferative phase of atherosclerosis in patients with diabetes.
Assuntos
Doença da Artéria Coronariana/prevenção & controle , Diabetes Mellitus Experimental/tratamento farmacológico , Angiopatias Diabéticas/prevenção & controle , Fragmentos Fab das Imunoglobulinas/farmacologia , Integrina alfaVbeta3/antagonistas & inibidores , Músculo Liso Vascular/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/metabolismo , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/metabolismo , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/metabolismo , Artéria Femoral/patologia , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Injeções Subcutâneas , Integrina alfaVbeta3/imunologia , Integrina alfaVbeta3/metabolismo , Ligantes , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Neointima , Fosforilação , Placa Aterosclerótica , Ligação Proteica , Sus scrofaRESUMO
Secreted phospholipase A2 type IIA (sPLA2-IIA) is a well-established pro-inflammatory protein and has been a major target for drug discovery. However, the mechanism of its signaling action has not been fully understood. We previously found that sPLA2-IIA binds to integrins αvß3 and α4ß1 in human and that this interaction plays a role in sPLA2-IIA's signaling action. Our recent studies found that sPLA2-IIA activates integrins in an allosteric manner through direct binding to a newly identified binding site of integrins (site 2), which is distinct from the classical RGD-binding site (site 1). The sPLA2-IIA-induced integrin activation may be related to the signaling action of sPLA2-IIA. Since sPLA2-IIA is present in normal human tears in addition to rheumatoid synovial fluid at high concentrations the sPLA2-IIA-mediated integrin activation on leukocytes may be involved in immune responses in normal and pathological conditions.
Assuntos
Fosfolipases A2 do Grupo II/química , Integrina alfa4beta1/química , Integrina alfaVbeta3/química , Transdução de Sinais/imunologia , Regulação Alostérica , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Sítios de Ligação , Regulação da Expressão Gênica , Fosfolipases A2 do Grupo II/genética , Fosfolipases A2 do Grupo II/imunologia , Humanos , Integrina alfa4beta1/genética , Integrina alfa4beta1/imunologia , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/imunologia , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Líquido Sinovial/química , Líquido Sinovial/imunologia , Lágrimas/química , Lágrimas/imunologiaRESUMO
Peptide-conjugated polysaccharide matrices using bioactive laminin-derived peptides are useful biomaterials for tissue and cell engineering. Here, we demonstrate an easy handling preparation method for peptide-polysaccharide matrices using polyion complex with both alginate and chitosan. First, aldehyde-alginate was synthesized by oxidization of alginate using NaIO4 , and then, reacted with Cys-peptides. Next, the peptide-alginate solution was added to a chitosan-coated plate, and the peptide-polyion complex matrices (peptide-PCMs) were prepared. The peptide-PCMs using an integrin αvß3-binding peptide (A99a: ALRGDN, mouse laminin α1 chain 1145-1150) and an integrin α2ß1-binding peptide (EF1XmR: RLQLQEGRLHFXFD, X = Nle, mouse laminin α1 chain 2751-2763) showed strong cell attachment activity in a dose-dependent manner. When we examined the effect of various spacers on the biological activity of A99a-PCM, hydrophobic and long spacers enhanced the cell attachment activity. Further, the A99a-PCM with the spacers strongly promoted neurite outgrowth. The polyion complex method is an easy way to obtain insolubilized matrix and is widely applicable for various polysaccharides. The peptide-PCM is useful as a biomaterial for cell and tissue engineering.