RESUMO
The tight junction proteins (TJPs) are major determinants of endothelial cells comprising physiological vascular barriers such as the blood-brain barrier, but little is known about their expression and role in immune cells. In this study we assessed TJP expression in human leukocyte subsets, their induction by immune activation and modulation associated with autoimmune disease states and therapies. A consistent expression of TJP complexes was detected in peripheral blood leukocytes (PBLs), predominantly in B and T lymphocytes and monocytes, whereas the in vitro application of various immune cell activators led to an increase of claudin 1 levels, yet not of claudin 5. Claudins 1 and 5 levels were elevated in PBLs of multiple sclerosis (MS) patients in relapse, relative to patients in remission, healthy controls and patients with other neurological disorders. Interestingly, claudin 1 protein levels were elevated also in PBLs of patients with type 1 diabetes (T1D). Following glucocorticoid treatment of MS patients in relapse, RNA levels of JAM3 and CLDN5 and claudin 5 protein levels in PBLs decreased. Furthermore, a correlation between CLDN5 pre-treatment levels and clinical response phenotype to interferon-ß therapy was detected. Our findings indicate that higher levels of leukocyte claudins are associated with immune activation and specifically, increased levels of claudin 5 are associated with MS disease activity. This study highlights a potential role of leukocyte TJPs in physiological states, and autoimmunity and suggests they should be further evaluated as biomarkers for aberrant immune activity and response to therapy in immune-mediated diseases such as MS.
Assuntos
Leucócitos/metabolismo , Proteínas de Membrana/metabolismo , Esclerose Múltipla/metabolismo , Junções Íntimas/metabolismo , Adulto , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The tight junctions (TJs) form continuous intracellular contacts, which help create selective barriers in epithelial and endothelial cell layers. The structures created by the TJs are very dynamic and can be rapidly remodeled in response to physiological and pathological signals. Claudin 5 is a membranal TJ protein which plays a critical role in determining the permeability of endothelial barriers. We describe the regulation of claudin 5 degradation by the ubiquitin-proteasome system (UPS). Our results indicate that claudin 5 has a relatively short half-life and can be polyubiquitinated on lysine 199. This ubiquitination appears to trigger the proteasome-dependent degradation of claudin 5. Other mechanisms also seem to be involved in the post-translational regulation of claudin 5, including a ubiquitin-independent and probably indirect lysosomal-dependent pathway. These findings provide evidence for the involvement of the UPS in the regulation of claudin 5 levels, and set the stage for further research to determine the involvement of this pathway in the modulation of the properties of TJs and cell-layer barriers.
Assuntos
Claudinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação/fisiologia , Linhagem Celular Tumoral , Claudina-5 , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Junções Íntimas/fisiologia , Ubiquitina/metabolismoRESUMO
BACKGROUND: Cancer immunotherapy has revolutionized cancer treatment. However, considering the limited success of immunotherapy to only some cancer types and patient cohorts, there is an unmet need for developing new treatments that will result in higher response rates in patients with cancer. Immunoglobulin-like transcript 2 (ILT2), a LILRB family member, is an inhibitory receptor expressed on a variety of immune cells including T cells, natural killer (NK) cells and different myeloid cells. In the tumor microenvironment, binding of class I MHC (in particular HLA-G) to ILT2 on immune cells mediates a strong inhibitory effect, which manifests in inhibition of antitumor cytotoxicity of T and NK cells, and prevention of phagocytosis of the tumor cells by macrophages. METHODS: We describe here the development and characteristics of BND-22, a novel, humanized monoclonal antibody that selectively binds to ILT2 and blocks its interaction with classical MHC I and HLA-G. BND-22 was evaluated for its binding and blocking characteristics as well as its ability to increase the antitumor activity of macrophages, T cells and NK cells in various in vitro, ex vivo and in vivo systems. RESULTS: Collectively, our data suggest that BND-22 enhances activity of both innate and adaptive immune cells, thus generating robust and comprehensive antitumor immunity. In humanized mice models, blocking ILT2 with BND-22 decreased the growth of human tumors, hindered metastatic spread to the lungs, and prolonged survival of the tumor-bearing mice. In addition, BND-22 improved the antitumor immune response of approved therapies such as anti-PD-1 or anti-EGFR antibodies. CONCLUSIONS: BND-22 is a first-in-human ILT2 blocking antibody which has demonstrated efficient antitumor activity in various preclinical models as well as a favorable safety profile. Clinical evaluation of BND-22 as a monotherapy or in combination with other therapeutics is under way in patients with cancer. TRIAL REGISTRATION NUMBER: NCT04717375.
Assuntos
Antígenos HLA-G , Neoplasias , Animais , Antígenos HLA-G/metabolismo , Humanos , Imunoterapia , Células Matadoras Naturais , Camundongos , Neoplasias/tratamento farmacológico , Linfócitos T , Microambiente TumoralRESUMO
Cathepsins are involved in a variety of physiological processes including antigen processing and presentation and extracellular matrix degradation. In the present study, we evaluated whether expression levels of cathepsins S and B and their inhibitors cystatins B and C are affected by multiple sclerosis (MS) disease state (relapse and remission) and therapies (interferon-ß [IFN-ß] and the glucocorticoid [GC] methylprednisolone), and whether they are associated with the IFN-ß response phenotype. Real-time PCR was employed to compare RNA expression levels in peripheral blood leucocytes (PBLs) and ELISA to determine serum protein levels of MS patients and matched healthy individuals. Cathepsin S RNA was higher in MS patients in the relapse state compared to controls (by 74%, P = 3 × 10(-5), n = 30 versus n = 18) with a similar increase observed in serum (66%, P = 0.002, n = 18 versus n = 20). GC treatment reduced cathepsin S levels in PBL RNA (by 44%, P = 6 × 10(-6), n = 27) and serum proteins (by 27%, P = 1 × 10(-5), n = 26), reduced the serum protein levels of pro-cathepsin B (by 8%, P = 0.0007, n = 23), and in parallel increased the serum levels of their inhibitor cystatin C (by 82%, P = 8 × 10(-6), n = 26). IFN-ß therapy significantly elevated the RNA levels (n = 16) of cathepsin B (by 16%, P = 0.03), cystatin B (44%, P = 0.004) and cystatin C (48%, P = 0.011). In the serum, only cathepsin S levels were reduced by IFN-ß (16%, P = 0.006, n = 25). Interestingly, pre-treatment serum cathepsin S/cystatin C ratio was higher in 'good responders' to IFN-ß therapy compared to patients without a good response (by 94%, P = 0.003). These results suggest that cathepsin S and cystatin C may contribute to disease activity in MS, specifically in a subgroup of patients that are responsive to IFN-ß therapy, and that these proteins should be further evaluated as biomarkers in MS.
Assuntos
Catepsina B/metabolismo , Catepsinas/metabolismo , Cistatina B/metabolismo , Cistatina C/metabolismo , Esclerose Múltipla/metabolismo , Adolescente , Adulto , Biomarcadores , Catepsina B/antagonistas & inibidores , Catepsina B/biossíntese , Catepsinas/antagonistas & inibidores , Catepsinas/biossíntese , Cistatina B/biossíntese , Cistatina B/sangue , Cistatina C/biossíntese , Cistatina C/sangue , Progressão da Doença , Feminino , Humanos , Interferon beta/farmacologia , Leucócitos/citologia , Masculino , Metilprednisolona/farmacologia , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , RNA Mensageiro/biossínteseRESUMO
BACKGROUND: Monocytes, which are key players in innate immunity, are outnumbered by neutrophils and lymphocytes among peripheral white blood cells. The cytokine interferon-ß (IFN-ß) is widely used as an immunomodulatory drug for multiple sclerosis and its functional pathways in peripheral blood mononuclear cells (PBMCs) have been previously described. The aim of the present study was to identify novel, cell-specific IFN-ß functions and pathways in tumor necrosis factor (TNF)-α-activated monocytes that may have been missed in studies using PBMCs. METHODOLOGY/PRINCIPAL FINDINGS: Whole genome gene expression profiles of human monocytes and T cells were compared following in vitro priming to TNF-α and overnight exposure to IFN-ß. Statistical analyses of the gene expression data revealed a cell-type-specific change of 699 transcripts, 667 monocyte-specific transcripts, 21 T cell-specific transcripts and 11 transcripts with either a difference in the response direction or a difference in the magnitude of response. RT-PCR revealed a set of differentially expressed genes (DEGs), exhibiting responses to IFN-ß that are modulated by TNF-α in monocytes, such as RIPK2 and CD83, but not in T cells or PBMCs. Known IFN-ß promoter response elements, such as ISRE, were enriched in T cell DEGs but not in monocyte DEGs. The overall directionality of the gene expression regulation by IFN-ß was different in T cells and monocytes, with up-regulation more prevalent in T cells, and a similar extent of up and down-regulation recorded in monocytes. CONCLUSIONS: By focusing on the response of distinct cell types and by evaluating the combined effects of two cytokines with pro and anti-inflammatory activities, we were able to present two new findings First, new IFN-ß response pathways and genes, some of which were monocytes specific; second, a cell-specific modulation of the IFN-ß response transcriptome by TNF-α.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon beta/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/metabolismo , Análise por Conglomerados , Redes Reguladoras de Genes , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Especificidade de Órgãos , Reprodutibilidade dos Testes , Transdução de Sinais , Transcrição Gênica , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Pathogenic T cells of multiple sclerosis (MS) patients have been suggested to be endowed with an increased resistance to apoptosis, contributing to their increased survival. We report herein increased levels of the anti-apoptotic MCL1 protein and its half-life in activated lymphocytes of MS patients, which were not associated with differences in MCL1 RNA levels or with alterations in the expression levels of the known E3 ligases of MCL1-ß-TrCP and HUWE1. Concomitantly, the expression levels of the pro-apoptotic protein BAK were decreased in MS patients at relapse. These findings suggest the dysregulation of the apoptosis-related proteins MCL1 and BAK in MS.
Assuntos
Apoptose , Esclerose Múltipla/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Linfócitos T/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/biossíntese , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases/biossíntese , Adulto JovemRESUMO
We have previously shown that several phosphodiesterase (PDE) subtypes are up-regulated in muscles and lymph node cells (LNC) of rats with experimental autoimmune myasthenia gravis (EAMG). In the present study we investigated PDE expression during the course of EAMG and experimental allergic encephalomyelitis (EAE) and found that the up-regulated expression of selected PDE subtypes in both experimental models is correlated with disease severity. In EAMG, PDE expression is correlated also with muscle damage. A similar up-regulation of PDE was also observed in the respective human diseases, MG and multiple sclerosis (MS). Our findings suggest that change in PDE expression levels is a general phenomenon in autoimmune diseases and may also be used as a marker for disease severity.