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1.
J Autoimmun ; 120: 102633, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33932829

RESUMO

Naturally-occurring autoantibodies to certain components of autophagy processes have been described in a few autoimmune diseases, but their fine specificity, their relationships with clinical phenotypes, and their potential pathogenic functions remain elusive. Here, we explored IgG autoantibodies reacting with a panel of cytoplasmic endosomal/lysosomal antigens and individual heat-shock proteins, all of which share links to autophagy. Sera from autoimmune patients and from MRL/lpr and NZB/W lupus-prone mice reacted with the C-terminal residues of lysosome-associated membrane glycoprotein (LAMP)2A. No cross-reaction was observed with LAMP2B or LAMP2C variants, with dsDNA or mononucleosomes, or with heat-shock protein A8. Moreover, administering chromatography-purified LAMP2A autoantibodies to MRL/lpr mice accelerated mortality. Furthermore, flow cytometry revealed elevated cell-surface expression of LAMP2A on MRL/lpr B cells. These findings reveal the involvement of a new class of autoantibodies targeting the C-terminus of LAMP2A, a receptor for cytosolic proteins targeted for degradation via chaperone-mediated autophagy. These autoantibodies could affect the autophagy process, which is abnormally upregulated in lupus. The data presented support a novel connection between autophagy dysregulation, autoimmune processes and pathophysiology in lupus.


Assuntos
Antígenos/imunologia , Suscetibilidade a Doenças/imunologia , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/metabolismo , Lisossomos/imunologia , Animais , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoimunidade , Autofagia/imunologia , Biomarcadores , Estudos de Casos e Controles , Modelos Animais de Doenças , Endossomos/imunologia , Endossomos/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteínas de Choque Térmico/imunologia , Humanos , Imunoglobulina G/imunologia , Lúpus Eritematoso Sistêmico/patologia , Proteína 2 de Membrana Associada ao Lisossomo/imunologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Peptídeos/imunologia
2.
J Autoimmun ; 90: 132-145, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29486915

RESUMO

Sjögren's syndrome is a multifactorial systemic autoimmune disorder characterized by lymphocytic infiltrates in exocrine organs. Patients present with sicca symptoms, such as extensive dry eyes and dry mouth, and parotid enlargement. Other serious complications include profound fatigue, chronic pain, major organ involvement, neuropathies and lymphomas. Current treatments only focus on relieving symptoms and do not target the origin of the disease, which is largely unknown. The question we addressed here was whether some defects exist in autophagy processes in Sjögren's syndrome and if they can be corrected or minimized using an appropriate mechanism-driven treatment targeting this central survival pathway. Using a recognized murine model of secondary Sjögren's syndrome, we identified molecular alterations of autophagy occurring in the salivary glands of MRL/lpr mice, and discovered that opposite (up- or down-regulated) autophagy events can arise in distinct organs of the same mouse strain, here in lymphoid organs and salivary glands. We showed further that the therapeutic P140 peptide, known to directly act on chaperone-mediated autophagy, rescued MRL/lpr mice from cellular infiltration and autophagy defects occurring in salivary glands. Our findings provide a proof-of-concept that targeting autophagy might represent a promising therapeutic strategy for treating patients with Sjögren's syndrome.


Assuntos
Autofagia , Fragmentos de Peptídeos/uso terapêutico , Glândulas Salivares/fisiologia , Síndrome de Sjogren/terapia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr
3.
J Autoimmun ; 92: 114-125, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29884340

RESUMO

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an autoimmune disease of the peripheral nerves evolving with diffuse sensory and motor symptoms. Although it is claimed that in neurodegenerative pathologies, a common feature is the failure of proteolytic systems to adequately eliminate aggregated or misfolded proteins, it has not been addressed whether autophagy, a central "clearance" system delivering damaged intracellular components to lysosomes, is affected in CIDP. The focus of the present investigation was therefore to determine if some defects exist in autophagy processes in this setting and if they can be corrected or minimized using an appropriate treatment targeting this survival pathway. Experiments were performed using a rat model mimicking human CIDP, also known as chronic experimental autoimmune neuritis (c-EAN), the disease establishment and development of which was followed at both the clinical and biological levels (indices of disease severity, histopathological alteration, cytokines and antibodies rates). Based on immunofluorescence and western immunoblotting experiments on sciatic nerves and spleen cells from c-EAN rats, we demonstrate that both, macroautophagy and chaperone-mediated autophagy (CMA), are significantly altered in non-neuronal cells of the peripheral nervous system. We show further that a 21-mer synthetic phosphopeptide called P140, known to target CMA and successfully used in pathological settings where CMA markers are overexpressed, considerably ameliorates the clinical and biological course of the disease in c-EAN rats. P140 displayed prophylactic and therapeutic effects, both in terms of disease intensity and chronicity, and preserved sciatic nerves from disease-related damages. Our findings uncover new disrupted molecular pathways in a c-EAN model and provide a proof-of-concept that targeting CMA might represent a promising therapeutic strategy for treating inflammatory neuropathies for which no disease-specific treatment is currently available.


Assuntos
Produtos Biológicos/uso terapêutico , Neurite Autoimune Experimental/terapia , Fragmentos de Peptídeos/uso terapêutico , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/terapia , Nervo Isquiático/fisiologia , Animais , Autofagia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Masculino , Atividade Motora , Deficiências na Proteostase , Ratos , Ratos Endogâmicos Lew , Nervo Isquiático/efeitos dos fármacos
4.
Angiogenesis ; 19(1): 39-52, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26419779

RESUMO

OBJECTIVES: Inflammation and angiogenesis are two tightly linked processes in arthritis, and therapeutic targeting of pro-angiogenic factors may contribute to control joint inflammation and synovitis progression. In this work, we explored whether vaccination against vascular endothelial growth factor (VEGF) ameliorates collagen-induced arthritis (CIA). METHODS: Anti-VEGF vaccines were heterocomplexes consisting of the entire VEGF cytokine (or a VEGF-derived peptide) linked to the carrier protein keyhole limpet hemocyanin (KLH). Two kinds of vaccines were separately tested in two independent experiments of CIA. In the first, we tested a kinoid of the murine cytokine VEGF (VEGF-K), obtained by conjugating VEGF-A to KLH. For the second, we selected two VEGF-A-derived peptide sequences to produce heterocomplexes (Vpep1-K and Vpep2-K). DBA/1 mice were immunized with either VEGF-K, Vpep1-K, or Vpep2-K, before CIA induction. Clinical and histological scores of arthritis, anti-VEGF, anti-Vpep Ab titers, and anti-VEGF Abs neutralizing capacity were determined. RESULTS: Both VEGF-K and Vpep1-K significantly ameliorated clinical arthritis scores and reduced synovial inflammation and joint destruction at histology. VEGF-K significantly reduced synovial vascularization. None of the vaccines reduced anti-collagen Ab response in mice. Both VEGF-K and Vpep1-K induced persistently high titers of anti-VEGF Abs capable of inhibiting VEGF-A bioactivity. CONCLUSION: Vaccination against the pro-angiogenic factor VEGF-A leads to the production of anti-VEGF polyclonal Abs and has a significant anti-inflammatory effect in CIA. Restraining Ab response to a single peptide sequence (Vpep1) with a peptide vaccine effectively protects immunized mice from joint inflammation and destruction.


Assuntos
Artrite Experimental/imunologia , Inflamação/patologia , Articulações/patologia , Terapia de Alvo Molecular , Vacinas/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos/imunologia , Artrite Experimental/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunidade Humoral/imunologia , Imunização , Masculino , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Membrana Sinovial/irrigação sanguínea , Membrana Sinovial/patologia , Fator A de Crescimento do Endotélio Vascular/química
5.
Biochim Biophys Acta ; 1842(7): 916-26, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24631654

RESUMO

Production of high titer of antibodies against nuclear components is a hallmark of systemic lupus erythematosus, an autoimmune disease characterized by the progressive chronic inflammation of multiple joints and organs. Organ damage and dysfunction such as renal failure are typical clinical features in lupus. Cell hypermetabolism and hypertrophy can accelerate organ dysfunction. In this study we focus on a specific murine model of lupus, the MRL/lpr strain, and investigated the role of cyclic guanosine monophosphate (cGMP) catabolism in organ remodeling of main target tissues (kidney, spleen and liver) in comparison with age-matched control mice. In MRL/lpr-prone mice, the cGMP-phosphodiesterase (PDE) activities were significantly increased in the kidney (3-fold, P<0.001), spleen (2-fold, P<0.001) and liver (1.6-fold, P<0.05). These raised activity levels were paralleled by both an increased activity of PDE1 in the kidney (associated with nephromegaly) and in the liver, and PDE2 in the spleen of lupus-prone mice. The up-regulation of PDE1 and PDE2 activities were associated with a decrease in intracellular cGMP levels. This underlines an alteration of cGMP-PDE signaling in the kidney, spleen and liver targeting different PDEs according to organs. In good agreement with these findings, a single intravenous administration to MRL/lpr mice of nimodipine (PDE1 inhibitor) but not of EHNA (PDE2 inhibitor) was able to significantly lower peripheral hypercellularity (P=0.0401), a characteristic feature of this strain of lupus-prone mice. Collectively, our findings are important for generating personalized strategies to prevent certain forms of the lupus disease as well as for understanding the role of PDEs and cGMP in the pathophysiology of lupus.


Assuntos
GMP Cíclico/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Animais , Feminino , Lúpus Eritematoso Sistêmico/genética , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos MRL lpr , Regulação para Cima
6.
FASEB J ; 28(7): 2840-51, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24671707

RESUMO

Autoantibodies to nuclear antigens arise in human autoimmune diseases, but a unifying pathogenetic mechanism remains elusive. Recently we reported that exposure of neutrophils to inflammatory conditions induces the citrullination of core histones by peptidylarginine deiminase 4 (PAD4) and that patients with autoimmune disorders produce autoantibodies that recognize such citrullinated histones. Here we identify histone H1 as an additional substrate of PAD4, localize H1 within neutrophil extracellular traps, and detect autoantibodies to citrullinated H1 in 6% of sera from patients with systemic lupus erythematosus and Sjögren's syndrome. No preference for deiminated H1 was observed in healthy control sera and sera from patients with scleroderma or rheumatoid arthritis. We map binding to the winged helix of H1 and determine that citrulline 53 represents a key determinant of the autoantibody epitope. In addition, we quantitate RNA for H1 histone subtypes in mature human neutrophils and identify citrulline residues by liquid chromatography and tandem mass spectrometry. Our results indicate that deimination of linker histones generates new autoantibody epitopes with enhanced potential for stimulating autoreactive human B cells.-Dwivedi, N., Neeli, I., Schall, N., Wan, H., Desiderio, D. M., Csernok, E., Thompson, P. R., Dali, H., Briand, J.-P., Muller, S., Radic, M. Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity.


Assuntos
Autoanticorpos/imunologia , Autoimunidade/imunologia , Histonas/imunologia , Neutrófilos/imunologia , Sequência de Aminoácidos , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Epitopos/imunologia , Humanos , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Alinhamento de Sequência
7.
J Autoimmun ; 39(3): 143-53, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22727561

RESUMO

After a long period where the potential of therapeutic peptides was let into oblivion and even dismissed, there is a revival of interest in peptides as potential drug candidates. Novel strategies for limiting metabolism and improve their bioavailability, and alternative routes of administration have emerged. This resulted in a large number of peptide-based drugs that are now being marketed in different indications. Regarding autoimmunity, successful data have been reported in numerous mouse models of autoimmune inflammation, yet relatively few clinical trials based on synthetic peptides are currently underway. This review reports on peptides that show much promises in appropriate mouse models of autoimmunity and describes in more detail clinical trials based on peptides for treating autoimmune patients. A particular emphasis is given to the 21-mer peptide P140/Lupuzor that has completed successfully phase I, phase IIa and phase IIb clinical trials for systemic lupus erythematosus.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Fatores Imunológicos/farmacocinética , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Fragmentos de Peptídeos/farmacocinética , Sequência de Aminoácidos , Animais , Doenças Autoimunes/imunologia , Autoimunidade/efeitos dos fármacos , Disponibilidade Biológica , Ensaios Clínicos como Assunto , Vias de Administração de Medicamentos , Esquema de Medicação , Humanos , Fatores Imunológicos/administração & dosagem , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/mortalidade , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/administração & dosagem , Taxa de Sobrevida
8.
Front Immunol ; 13: 904669, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35720371

RESUMO

In systemic lupus erythematosus, T cells display multiple abnormalities. They are abnormally activated, secrete pro-inflammatory cytokines, help B cells to generate pathogenic autoantibodies, and provoke the accumulation of autoreactive memory T cells. P140, a synthetic peptide evaluated in phase-III clinical trials for lupus, binds HSPA8/HSC70 chaperone protein. In vitro and in vivo, it interferes with hyperactivated chaperone-mediated autophagy, modifying overexpression of major histocompatibility complex class II molecules and antigen presentation to autoreactive T cells. Here, we show that in P140-treated lupus mice, abnormalities affecting T and B cells are no longer detectable in secondary lymphoid tissue and peripheral blood. Data indicate that P140 acts by depleting hyper-activated autoreactive T and B cells and restores normal immune homeostasis. Our findings suggest that P140 belongs to a new family of non-immunosuppressive immunoregulators that do not correct T and B cell abnormalities but rather contribute to the clearance of deleterious T and B cells.


Assuntos
Lúpus Eritematoso Sistêmico , Fragmentos de Peptídeos , Animais , Apresentação de Antígeno , Linfócitos B , Proteínas de Choque Térmico HSC70 , Camundongos , Fragmentos de Peptídeos/metabolismo
9.
Ann Rheum Dis ; 70(5): 837-43, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21173017

RESUMO

BACKGROUND: The P140 phosphopeptide issued from the spliceosomal U1-70K small nuclear ribonucleoprotein protein displays protective properties in MRL/lpr lupus-prone mice. It binds both major histocompatibility class II (MHCII) and HSC70/Hsp73 molecules. P140 peptide increases MRL/lpr peripheral blood lymphocyte apoptosis and decreases autoepitope recognition by T cells. OBJECTIVE: To explore further the mode of action of P140 peptide on HSC70+ antigen-presenting cells. METHODS: P140 biodistribution was monitored in real time using an imaging system and by fluorescence and electron microscopy. Fluorescence activated cell sorting and Western blotting experiments were used to evaluate the P140 effects on autophagic flux markers. RESULTS: P140 fluorescence accumulated especially in the lungs and spleen. P140 peptide reduced the number of peripheral and splenic T and B cells without affecting these cells in normal mice. Remaining MRL/lpr B cells responded normally to mitogens. P140 peptide decreased the expression levels of HSC70/Hsp73 chaperone and stable MHCII dimers, which are both increased in MRL/lpr splenic B cells. It impaired refolding properties of chaperone HSC70. In MRL/lpr B cells, it increased the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulated lysosomal degradation during autophagic flux. CONCLUSION: The study results suggest that after P140 peptide binding to HSC70, the endogenous (auto)antigen processing might be greatly affected in MRL/lpr antigen-presenting B cells, leading to the observed decrease of autoreactive T-cell priming and signalling via a mechanism involving a lysosomal degradation pathway. This unexpected mechanism might explain the beneficial effect of P140 peptide in treated MRL/lpr mice.


Assuntos
Autofagia/efeitos dos fármacos , Proteínas de Choque Térmico HSC70/antagonistas & inibidores , Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/patologia , Fragmentos de Peptídeos/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos B/patologia , Proteínas de Choque Térmico HSC70/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos MRL lpr , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual
10.
Cells ; 10(9)2021 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-34572117

RESUMO

The involvement of autophagy and its dysfunction in asthma is still poorly documented. By using a murine model of chronic house dust mite (HDM)-induced airway inflammation, we tested the expression of several autophagy markers in the lung and spleen of asthma-like animals. Compared to control mice, in HDM-sensitized and challenged mice, the expression of sequestosome-1/p62, a multifunctional adaptor protein that plays an important role in the autophagy machinery, was raised in the splenocytes. In contrast, its expression was decreased in the neutrophils recovered from the bronchoalveolar fluid, indicating that autophagy was independently regulated in these two compartments. In a strategy of drug repositioning, we treated allergen-sensitized mice with the therapeutic peptide P140 known to target chaperone-mediated autophagy. A single intravenous administration of P140 in these mice resulted in a significant reduction in airway resistance and elastance, and a reduction in the number of neutrophils and eosinophils present in the bronchoalveolar fluid. It corrected the autophagic alteration without showing any suppressive effect in the production of IgG1 and IgE. Collectively, these findings show that autophagy processes are altered in allergic airway inflammation. This cellular pathway may represent a potential therapeutic target for treating selected patients with asthma.


Assuntos
Asma/complicações , Hipersensibilidade/complicações , Inflamação/prevenção & controle , Pulmão/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Pyroglyphidae/patogenicidade , Animais , Asma/patologia , Autofagia , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Modelos Animais de Doenças , Eosinófilos/imunologia , Feminino , Hipersensibilidade/patologia , Imunoglobulina E/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pyroglyphidae/química , Proteína Sequestossoma-1/metabolismo
11.
Front Pharmacol ; 12: 752194, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34744730

RESUMO

Dysregulation of CXCL12/SDF-1-CXCR4/CD184 signaling is associated with inflammatory diseases and notably with systemic lupus erythematosus. Issued from the lead molecule chalcone-4, the first neutraligand of the CXCL12 chemokine, LIT-927 was recently described as a potent analogue with improved solubility and stability. We aimed to investigate the capacity of LIT-927 to correct immune alterations in lupus-prone MRL/lpr mice and to explore the mechanism of action implemented by this small molecule in this model. We found that in contrast to AMD3100, an antagonist of CXCR4 and agonist of CXCR7, LIT-927 reduces the excessive number of several B/T lymphocyte subsets occurring in the blood of sick MRL/lpr mice (including CD3+/CD4-/CD8-/B220+ double negative T cells). In vitro, LIT-927 downregulated the overexpression of several activation markers on splenic MRL/lpr lymphocytes. It exerted effects on the CXCR4 pathway in MRL/lpr CD4+ T spleen cells. The results underline the importance of the CXCL12/CXCR4 axis in lupus pathophysiology. They indicate that neutralizing CXCL12 by the neutraligand LIT-927 can attenuate hyperactive lymphocytes in lupus. This mode of intervention might represent a novel strategy to control a common pathophysiological mechanism occurring in inflammatory diseases.

12.
Sci Rep ; 8(1): 16820, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30429537

RESUMO

The nuclear translocation of endogenous heat shock cognate protein HSPA8 is a requisite for cell survival during oxidative and heat shock stress. Upon these events, cytoplasmic HSPA8 is thought to concentrate within the nucleus and nucleolus. When the situation returns to normal, HSPA8 is released from its nuclear/nucleolar anchors and redistributes into the cytoplasm. By using different stress conditions and a 21-mer phosphopeptide tool called P140, which binds HSPA8 and hampers its chaperone properties, we deciphered the cellular and molecular effects arising during this vital cytoplasmic-nuclear-cytoplasmic shuttling process. Using the non-metastatic fibroblastoid cell line MRL/N-1 derived from a MRL/MpTn-gld/gld lupus-prone mouse, we discovered that P140 treatment neutralized the egress of HSPA8 from nucleus to cytoplasm in the cell recovery phase. This lack of relocation of HSPA8 into the cytoplasm of heat-shocked MRL/N-1 cells altered the ability of these cells to survive when a second mild oxidative stress mimicking inflammatory conditions was applied. Crosslinking experiments followed by proteomics studies showed that P140 binds regions close to nuclear import and export signal sequences encompassed within the HSPA8 structure. These data are consistent with HSPA8 having a crucial cell protective role against reactive oxygen species (ROS) production by mitochondria during inflammatory conditions.


Assuntos
Transporte Ativo do Núcleo Celular , Sobrevivência Celular , Proteínas de Choque Térmico HSC70/metabolismo , Resposta ao Choque Térmico , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Camundongos , Estresse Oxidativo , Ligação Proteica , Sinais Direcionadores de Proteínas
13.
Arthritis Rheumatol ; 70(7): 1077-1088, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29499102

RESUMO

OBJECTIVE: Phosphopeptide P140 (Lupuzor) is an inhibitor of autophagy currently being evaluated in late-stage clinical trials for the treatment of lupus. This study was undertaken to investigate the effect of P140 ex vivo on human T and B cells. METHODS: Human B cells, T cells, and dendritic cells were analyzed by flow cytometry and cellular assays. The expression of autophagy markers was evaluated by immunoblotting and flow cytometry. The levels of B cell receptor (BCR) signaling markers and HLA molecules were assessed by flow cytometry. Toll-like receptor ligands were screened using an assay with transfected HEK 293 cells. P140 cell entry and trafficking were measured by immunofluorescence in the presence of various inhibitors of endosomal pathways. RESULTS: As was previously observed after intravenous injection of the peptide in a mouse model of lupus, P140 entered human B cells by a clathrin coat-dependent endocytosis process and homed into lysosomes. The peptide displayed no direct effect on BCR signaling of memory, naive mature, transitional, and B1 cells. However, it strongly reduced the overexpression of HLA class II molecules on lupus B cells that were acting as antigen-presenting cells, down-regulated the maturation and differentiation of B cells into plasma cells, and decreased IgG secretion. CONCLUSION: These findings show that P140 down-regulates HLA class II overexpression in human lupus B cells, and also that P140 hampers the differentiation of B cells into autoantibody-secreting plasma cells, likely due to the resulting lack of T cell signaling and activation. This mechanism appears to switch off the downstream events leading to secretion of pathogenic autoantibodies, thus explaining the highly promising results obtained in clinical trials of P140 (Lupuzor) for the treatment of lupus.


Assuntos
Linfócitos B/fisiologia , Diferenciação Celular/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Fragmentos de Peptídeos/farmacologia , Linfócitos T/fisiologia , Autofagia/efeitos dos fármacos , Células HEK293 , Humanos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/efeitos dos fármacos
14.
Cells ; 6(3)2017 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-28657591

RESUMO

Autophagy is a tightly regulated mechanism that allows cells to renew themselves through the lysosomal degradation of proteins, which are misfolded or produced in excess, and of damaged organelles. In the context of immunity, recent research has specially attempted to clarify its roles in infection, inflammation and autoimmunity. Autophagy has emerged as a spotlight in several molecular pathways and trafficking events that participate to innate and adaptive immunity. Deregulation of autophagy has been associated to several autoimmune diseases, in particular to systemic lupus erythematosus. Nowadays, however, experimental data on the implication of autophagy in animal models of autoimmunity or patients remain limited. In our investigations, we use Murphy Roths Large (MRL)/lymphoproliferation (lpr) lupus-prone mice as a mouse model for lupus and secondary Sjögren's syndrome, and, herein, we describe methods applied routinely to analyze different autophagic pathways in different lymphoid organs and tissues (spleen, lymph nodes, salivary glands). We also depict some techniques used to analyze autophagy in lupus patient's blood samples. These methods can be adapted to the analysis of autophagy in other mouse models of autoinflammatory diseases. The understanding of autophagy implication in autoimmune diseases could prove to be very useful for developing novel immunomodulatory strategies. Our attention should be focused on the fact that autophagy processes are interconnected and that distinct pathways can be independently hyper-activated or downregulated in distinct organs and tissues of the same individual.

15.
Cells ; 6(3)2017 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-28927011

RESUMO

The rat sciatic nerve has attracted widespread attention as an excellent model system for studying autophagy alterations in peripheral neuropathies. In our laboratory, we have developed an original rat model, which we used currently in routine novel drug screening and to evaluate treatment strategies for chronic inflammatory demyelinating polyneuropathy (CIDP) and other closely related diseases. Lewis rats injected with the S-palmitoylated P0(180-199) peptide develop a chronic, sometimes relapsing-remitting type of disease. Our model fulfills electrophysiological criteria of demyelination with axonal degeneration, confirmed by immunohistopathology and several typical features of CIDP. We have set up a series of techniques that led us to examine the failures of autophagy pathways in the sciatic nerve of these model rats and to follow the possible improvement of these defects after treatment. Based on these newly introduced methods, a novel area of investigation is now open and will allow us to more thoroughly examine important features of certain autophagy pathways occurring in sciatic nerves.

16.
Front Immunol ; 8: 16, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28191006

RESUMO

During inflammatory reaction, neutrophils exhibit numerous cellular and immunological functions, notably the formation of neutrophil extracellular traps (NETs) and autophagy. NETs are composed of decondensed chromatin fibers coated with various antimicrobial molecules derived from neutrophil granules. NETs participate in antimicrobial defense and can also display detrimental roles and notably trigger some of the immune features of systemic lupus erythematosus (SLE) and other autoimmune diseases. Autophagy is a complex and finely regulated mechanism involved in the cell survival/death balance that may be connected to NET formation. To shed some light on the connection between autophagy and NET formation, we designed a number of experiments in human neutrophils and both in normal and lupus-prone MRL/lpr mice to determine whether the synthetic peptide P140, which is capable of selectively modulating chaperone-mediated autophagy (CMA) in lymphocytes, could alter NET formation. P140/Lupuzor™ is currently being evaluated in phase III clinical trials involving SLE patients. Overall our in vitro and in vivo studies established that P140 does not influence NET formation, cytokine/chemokine production, or CMA in neutrophils. Thus, the beneficial effect of P140/Lupuzor™ in SLE is apparently not directly related to modulation of neutrophil function.

17.
Autophagy ; 11(3): 472-86, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25719862

RESUMO

The P140 peptide, a 21-mer linear peptide (sequence 131-151) generated from the spliceosomal SNRNP70/U1-70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired.


Assuntos
Autofagia , Chaperonas Moleculares/química , Fragmentos de Peptídeos/química , Fosfopeptídeos/química , Ribonucleoproteína Nuclear Pequena U1/química , Animais , Autoimunidade , Linfócitos B/imunologia , Endocitose , Endossomos/metabolismo , Proteínas de Choque Térmico HSC70/química , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Lisossomos/química , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Células NIH 3T3 , Fosforilação , Serina/química , Linfócitos T/imunologia
18.
Methods Mol Biol ; 1134: 173-92, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24497362

RESUMO

Synthetic peptides are attracting increasing attention as therapeutics. Despite their potential, however, only a few selected peptides have been able to enter in clinical trials for chronic autoimmune diseases and systemic lupus erythematosus (SLE) in particular. Here, we describe and discuss a series of assays, which may help in characterizing valuable candidate peptides that were applied in our laboratory to develop the lupus P140 peptide program. The different steps of selection include the choice of the initial autoantigen, the design, synthesis and purification of peptides, their preliminary screen by measuring cytokines produced ex vivo by T cells and their binding to major histocompatibility complex class II (MHCII) molecules, their capacity to lower peripheral cell hyperproliferation in lupus-prone MRL/lpr mice, and, as a final step, their ability to slow down the development of lupus disease in model animals.


Assuntos
Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Peptídeos/uso terapêutico , Animais , Autoantígenos/química , Autoantígenos/imunologia , Citocinas/biossíntese , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica/imunologia
19.
Autophagy ; 7(5): 539-40, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21282971

RESUMO

The P140 phosphopeptide encompassing residues 131-151 of the spliceosomal U1-70K snRNP protein displays protective properties in lupus patients and MRL/lpr mice. It increases peripheral blood lymphocyte apoptosis via a mechanism involving γδ T cells. After intravenous administration, P140 accumulates in the lungs and spleen. It binds both the HSC70/Hsp73 chaperone and MHC class II (MHCII) molecules, which colocalize in splenic MRL/lpr B cells. Expression of HSC70 and MHCII, which is increased in MRL/lpr splenic B cells, is diminished after P140 administration. P140 impairs refolding properties of HSC70 and alters expression of stable MHCII molecules in B lymphocytes. In MRL/lpr B cells, P140 increases the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulation of autophagic flux. Our study reveals a very unique property of P140 peptide that alters the autophagy pathway leading to a defect of endogenous (auto)antigen processing in MRL/lpr antigen-presenting B cells and a decrease of T cell priming and signaling.


Assuntos
Autofagia/efeitos dos fármacos , Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Peptídeos/farmacologia , Animais , Autofagia/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Proteínas de Choque Térmico HSC70/metabolismo , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Lúpus Eritematoso Sistêmico/terapia , Camundongos , Camundongos Endogâmicos MRL lpr , Peptídeos/imunologia , Peptídeos/uso terapêutico , Estabilidade Proteica/efeitos dos fármacos
20.
PLoS One ; 4(4): e5273, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19390596

RESUMO

The phosphopeptide P140 issued from the spliceosomal U1-70K snRNP protein is recognized by lupus CD4(+) T cells, transiently abolishes T cell reactivity to other spliceosomal peptides in P140-treated MRL/lpr mice, and ameliorates their clinical features. P140 modulates lupus patients' T cell response ex vivo and is currently included in phase IIb clinical trials. Its underlying mechanism of action remains elusive. Here we show that P140 peptide binds a unique cell-surface receptor, the constitutively-expressed chaperone HSC70 protein, known as a presenting-protein. P140 induces apoptosis of activated MRL/lpr CD4(+) T cells. In P140-treated mice, it increases peripheral blood lymphocyte apoptosis and decreases B cell, activated T cell, and CD4(-)CD8(-)B220(+) T cell counts via a specific mechanism strictly depending on gammadelta T cells. Expression of inflammation-linked genes is rapidly regulated in CD4(+) T cells. This work led us to identify a powerful pathway taken by a newly-designed therapeutic peptide to immunomodulate lupus autoimmunity.


Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Fragmentos de Peptídeos/farmacologia , Linfócitos T Reguladores/imunologia , Animais , Apoptose , Linfócitos B/metabolismo , Sítios de Ligação , Regulação para Baixo , Imunofluorescência , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/terapia , Camundongos , Camundongos Endogâmicos MRL lpr , Modelos Biológicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/análise , Ribonucleoproteína Nuclear Pequena U1/imunologia , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ressonância de Plasmônio de Superfície , Linfócitos T/imunologia
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