RESUMO
The aberrant protein disulfide isomerase A5 (PDIA5) expression was relevant to the poor prognosis of patients with human cancers. However, its relationship with the epigenetic and genetic alterations and its effect on tumor immunity is still lacking. In the present study, we comprehensively analyzed the immune infiltration role of PDIA5 in human cancers based on large-scale bioinformatics analyses and in vitro experiments. Obvious DNA methylation and moderate alteration frequency of PDIA5 were observed in human cancers. The expression level of PDIA5 was significantly correlated with infiltrated immune cells, immune pathways, and other immune signatures. We found that cancer cells and macrophages exhibited high PDIA5 expression in human cancers using the single-cell RNA sequencing analysis. We also demonstrated the interaction between PDIA5 and immune cells in glioblastoma multiforme (GBM). Multiplex immunofluorescence staining showed the upregulated expression level of PDIA5 and the increased number of M2 macrophage markers-CD163 positive cells in pan-cancer samples. Notably, PDIA5 silencing resulted in upregulated expression of PD-L1 and SPP1 in U251 cells. Silencing of PDIA5 in hepG2 cells, U251 cells, and PC3 cells contributed to a decline in their ability of proliferation, clone formation, and invasion and inhibited the migration of cocultured M2 macrophages. Additionally, PDIA5 also displayed predictive value in the immunotherapy response of both murine and human cancer cohorts. Overall, our findings indicated that PDIA5 might be a potential target for immunotherapies in cancers.
Assuntos
Glioblastoma , Isomerases de Dissulfetos de Proteínas , Animais , Metilação de DNA , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Imunoterapia/métodos , Macrófagos , Camundongos , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologiaRESUMO
A diet rich in saturated fat and carbohydrates causes low-grade chronic inflammation in several organs, including the liver, ultimately driving nonalcoholic steatohepatitis. In this setting, environment-driven lipotoxicity and glucotoxicity induce liver damage, which promotes dendritic cell activation and generates a major histocompatibility complex class II (MHC-II) immunopeptidome enriched with peptides derived from proteins involved in cellular metabolism, oxidative phosphorylation, and the stress responses. Here, we demonstrated that lipotoxicity and glucotoxicity, as driven by a high-fat and high-fructose (HFHF) diet, promoted MHC-II presentation of nested T and B cell epitopes from protein disulfide isomerase family A member 3 (PDIA3), which is involved in immunogenic cell death. Increased MHC-II presentation of PDIA3 peptides was associated with antigen-specific proliferation of hepatic CD4+ immune infiltrates and isotype switch of anti-PDIA3 antibodies from IgM to IgG3, indicative of cellular and humoral PDIA3 autoreactivity. Passive transfer of PDIA3-specific T cells or PDIA3-specific antibodies also exacerbated hepatocyte death, as determined by increased hepatic transaminases detected in the sera of mice subjected to an HFHF but not control diet. Increased humoral responses to PDIA3 were also observed in patients with chronic inflammatory liver conditions, including autoimmune hepatitis, primary biliary cholangitis, and type 2 diabetes. Together, our data indicated that metabolic insults caused by an HFHF diet elicited liver damage and promoted pathogenic immune autoreactivity driven by T and B cell PDIA3 epitopes.
Assuntos
Autoimunidade , Diabetes Mellitus Tipo 2 , Fígado , Isomerases de Dissulfetos de Proteínas , Animais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epitopos , Antígenos de Histocompatibilidade Classe II , Fígado/patologia , Camundongos , Peptídeos , Isomerases de Dissulfetos de Proteínas/imunologia , Isomerases de Dissulfetos de Proteínas/metabolismoRESUMO
Miscarriage frequently occurs in euthyroid women with thyroid autoimmunity (TAI), but its mechanisms remain unclear. Our previous study has found that the serum level of anti-protein disulfide isomerase A3 autoantibody (PDIA3Ab) was significantly increased in mice with TAI. This study was aimed to explore whether there could be an association between the expression of PDIA3Ab and the occurrence of miscarriage in euthyroid TAI women. It was found that the serum level of PDIA3Ab was significantly increased in euthyroid TAI women as compared with that of non-TAI controls. Especially, serum PDIA3Ab level was markedly higher in euthyroid TAI women with miscarriage than the ones without miscarriage. Furthermore, binary logistic regression analysis showed that the serum PDIA3Ab level was an independent risk factor for spontaneous abortion in euthyroid TAI women with an odds ratio of 13.457 (95% CI, 2.965-61.078). The receiver operating characteristic (ROC) analysis of serum PDIA3Ab expression for predicting the miscarriage in euthyroid TAI women showed that the area under the curve was 0.707 ± 0.05 (P < 0.001). The optimal cut-off OD450 value of serum PDIA3Ab was 0.7129 with a sensitivity of 52.5% and specificity of 86.3% in euthyroid TAI women. Trend test showed that the prevalence of spontaneous abortion was markedly increased with the rise of serum PDIA3Ab level among TAI women in a titer-dependent manner. In conclusion, serum PDIA3Ab expression may imply an increased risk of spontaneous abortion in euthyroid TAI women, and it can be used as a new predictive bio-marker.
Assuntos
Aborto Espontâneo/sangue , Autoanticorpos/sangue , Isomerases de Dissulfetos de Proteínas/imunologia , Doenças da Glândula Tireoide/sangue , Aborto Espontâneo/imunologia , Adulto , Autoantígenos/imunologia , Autoimunidade , Feminino , Humanos , Iodeto Peroxidase/imunologia , Proteínas de Ligação ao Ferro/imunologia , Estudos Retrospectivos , Fatores de Risco , Tireoglobulina/imunologia , Doenças da Glândula Tireoide/imunologia , Tireotropina/sangue , Tiroxina/sangueRESUMO
Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme catalyzing the crosslinking between Gln and Lys residues and involved in various pathophysiological events. Besides this crosslinking activity, TG2 functions as a deamidase, GTPase, isopeptidase, adapter/scaffold, protein disulfide isomerase, and kinase. It also plays a role in the regulation of hypusination and serotonylation. Through these activities, TG2 is involved in cell growth, differentiation, cell death, inflammation, tissue repair, and fibrosis. Depending on the cell type and stimulus, TG2 changes its subcellular localization and biological activity, leading to cell death or survival. In normal unstressed cells, intracellular TG2 exhibits a GTP-bound closed conformation, exerting prosurvival functions. However, upon cell stimulation with Ca2+ or other factors, TG2 adopts a Ca2+-bound open conformation, demonstrating a transamidase activity involved in cell death or survival. These functional discrepancies of TG2 open form might be caused by its multifunctional nature, the existence of splicing variants, the cell type and stimulus, and the genetic backgrounds and variations of the mouse models used. TG2 is also involved in the phagocytosis of dead cells by macrophages and in fibrosis during tissue repair. Here, we summarize and discuss the multifunctional and controversial roles of TG2, focusing on cell death/survival and fibrosis.
Assuntos
Aminoaciltransferases/genética , Carbono-Nitrogênio Liases/genética , Fibrose/enzimologia , Proteínas de Ligação ao GTP/genética , Inflamação/enzimologia , Isomerases de Dissulfetos de Proteínas/genética , Transglutaminases/genética , Processamento Alternativo , Aminoaciltransferases/imunologia , Animais , Cálcio/imunologia , Cálcio/metabolismo , Carbono-Nitrogênio Liases/imunologia , Morte Celular , Sobrevivência Celular , Fibrose/genética , Fibrose/imunologia , Fibrose/patologia , Proteínas de Ligação ao GTP/imunologia , Expressão Gênica , Guanosina Trifosfato/imunologia , Guanosina Trifosfato/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Isoenzimas/genética , Isoenzimas/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Fagocitose/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/imunologiaRESUMO
Borrelia burgdorferi causes Lyme disease, the most common tick-transmitted illness in North America. When Ixodes scapularis feed on an infected vertebrate host, spirochetes enter the tick gut along with the bloodmeal and colonize the vector. Here, we show that a secreted tick protein, I. scapularisprotein disulfide isomerase A3 (IsPDIA3), enhances B. burgdorferi colonization of the tick gut. I. scapularis ticks in which ispdiA3 has been knocked down using RNA interference have decreased spirochete colonization of the tick gut after engorging on B. burgdorferi-infected mice. Moreover, administration of IsPDIA3 antiserum to B. burgdorferi-infected mice reduced the ability of spirochetes to colonize the tick when feeding on these animals. We show that IsPDIA3 modulates inflammatory responses at the tick bite site, potentially facilitating spirochete survival at the vector-host interface as it exits the vertebrate host to enter the tick gut. These data provide functional insights into the complex interactions between B. burgdorferi and its arthropod vector and suggest additional targets to interfere with the spirochete life cycle.
Assuntos
Borrelia burgdorferi/fisiologia , Ixodes/metabolismo , Doença de Lyme/transmissão , Isomerases de Dissulfetos de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Vetores Aracnídeos/microbiologia , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Imunidade Humoral , Inflamação/enzimologia , Inflamação/genética , Inflamação/metabolismo , Ixodes/enzimologia , Ixodes/genética , Proteínas de Membrana/metabolismo , Camundongos , Filogenia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Interferência de RNA , Proteínas Recombinantes , Alinhamento de Sequência , Spirochaetales/fisiologiaRESUMO
BACKGROUND: Hemagglutinin (HA), as the surface immunogenic protein, is the most important component of influenza viruses. Previous studies showed that the stability of HA was significant for HA's immunogenicity, and many efforts have been made to stabilize the expressed HA proteins. METHODS: In this study, the protein disulfide isomerases (PDIs) were investigated for the ability to improve the stability of HA protein. Two members of the PDIs family, PDI and ERp57, were over-expressed or down-expressed in 293 T cells. The expression of H3 HA and PDIs were investigated by real-time qPCR, western-blot, immunofluorescence assay, and flow cytometry. The stability of HA was investigated by western-blot under non-reducing condition. Moreover, BALB/c mice were immunized subcutaneously twice with the vaccine that contained HA proteins from the ERp57-overexpressed and conventional 293 T cells respectively to investigate the impact of ERp57 on the immunogenicity of H3N2 HA. RESULTS: The percentage of the disulfide-bonded HA trimers increased significantly in the PDIs-overexpressed 293 T cells, and ERp57 was more valid to the stability of HA than PDI. The knockdown of ERp57 by small interfering RNA significantly decreased the percentage of the disulfide-bonded HA trimers. HA proteins from ERp57-overexpressed 293 T cells stimulated the mice to generate significantly higher HA-specific IgG against H1N1 and H3N2 viruses than those from the conventional cells. The mice receiving H3 HA from ERp57-overexpressed 293 T cells showed the better resistance against H1N1 viruses and the higher survival rate than the mice receiving H3 HA from the conventional cells. CONCLUSION: ERp57 could improve the stability and immunogenicity of H3N2 HA.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Isomerases de Dissulfetos de Proteínas/genética , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Feminino , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Humanos , Imunogenicidade da Vacina , Vírus da Influenza A Subtipo H1N1/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Estabilidade Proteica , VacinaçãoRESUMO
In a forward genetic screen of N-ethyl-N-nitrosourea (ENU)-induced mutant mice for aberrant immune function, we identified mice with a syndromic disorder marked by growth retardation, diabetes, premature death, and severe lymphoid and myeloid hypoplasia together with diminished T cell-independent (TI) antibody responses. The causative mutation was in Pdia6, an essential gene encoding protein disulfide isomerase A6 (PDIA6), an oxidoreductase that functions in nascent protein folding in the endoplasmic reticulum. The immune deficiency caused by the Pdia6 mutation was, with the exception of a residual T cell developmental defect, completely rescued in irradiated wild-type recipients of PDIA6-deficient bone marrow cells, both in the absence or presence of competition. The viable hypomorphic allele uncovered in these studies reveals an essential role for PDIA6 in hematopoiesis, but one extrinsic to cells of the hematopoietic lineage. We show evidence that this role is in the proper folding of Wnt3a, BAFF, IL-7, and perhaps other factors produced by the extra-hematopoietic compartment that contribute to the development and lineage commitment of hematopoietic cells.
Assuntos
Linfócitos/imunologia , Células Mieloides/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Animais , Fator Ativador de Células B/imunologia , Linhagem Celular , Feminino , Células HEK293 , Hematopoese/imunologia , Humanos , Interleucina-7/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Proteína Wnt3A/imunologiaRESUMO
Autoimmune thyroiditis (AIT)-related brain damage is one of most severe extrathyroidal manifestations of AIT, but the mechanism remains unclear. In this study, we confirmed that protein disulfide-isomerase A3 (PDIA3) is expressed in both thyroid and brain tissues of mouse, and found the significantly increased serum levels of anti-PDIA3 antibody (PDIA3Ab) in classical mouse models of thyroiditis. In addition, we investigated the PDIA3-specific autoimmune reaction in thyroid and brain tissues in a mouse model with high-serum PDIA3Ab induced by immunization with recombinant PDIA3 protein. PDIA3-immunized mice had elevated serum thyrotropin and impaired learning and memory. PDIA3-expressing cells had IgG deposition, and IgG colocalized with C3 in the thyroid and brain tissues of PDIA3-immunized mice, resulting in membrane attack complex formation. Our results suggest that PDIA3 protein may be a common autoantigen shared by the thyroid and brain tissues and involve in the thyroidal and intracerebral damage through activating complement system.
Assuntos
Autoanticorpos/imunologia , Encéfalo/imunologia , Encefalite/imunologia , Doença de Hashimoto/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Glândula Tireoide/imunologia , Tireoidite Autoimune/imunologia , Animais , Apoptose/imunologia , Autoantígenos/imunologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Encéfalo/ultraestrutura , Modelos Animais de Doenças , Encefalite/patologia , Encefalite/fisiopatologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Doença de Hashimoto/patologia , Doença de Hashimoto/fisiopatologia , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto , Camundongos , Glândula Tireoide/patologia , Tireoidite Autoimune/patologia , Tireoidite Autoimune/fisiopatologiaRESUMO
Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPß chain (DP84GGPM87). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP84GGPM87 for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP84GGPM87 to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4+ T cells.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos HLA-DP/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Chaperonas Moleculares/imunologia , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/imunologia , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Calnexina/genética , Calnexina/imunologia , Calreticulina/genética , Calreticulina/imunologia , Linhagem Celular , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Chaperonas Moleculares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologiaRESUMO
Protein disulfide isomerase A3 (PDIA3) is a chaperone protein that supports the folding and processing of synthesized proteins. Its expression is associated with the prognosis of laryngeal cancer, hepatocellular carcinoma, diffuse glioma and uterine cervical cancer. In the present study, the expression levels of PDIA3 and its clinicopathological association were examined in 52 cases of gastric cancer (GC). The expression of PDIA3 was examined by immunohistochemistry and scored using a semi-quantitative method. According to the score, GC samples were classified into PDIA3High and PDIA3Low GC. PDIA3High GC samples were predominantly of the intestinal type. Multivariate survival analysis indicated that PDIA3 expression and cancer stage were independent factors. The overall survival of PDIA3High GC cases was significantly favorable compared with that of PDIA3Low GC cases, and this was more evident in cases at an advanced stage. In GC cell cultures, the PDIA3 and major histocompatibility complex (MHC) class I proteins were expressed in three out of the four assessed cell lines according to western blot analysis. Notably, the expression of MHC class I was increased by the stimulation of interferon γ. Coimmunoprecipitation assays suggested the formation of a PDIA3 and MHC class I complex. The findings suggested that PDIA3 may be involved in the immune response of carcinoma cells. The improved prognosis in PDIA3High GC may be accounted for, in part, by sufficient antigen processing and expression of MHC class I, which can be mediated by PDIA3. It was suggested that PDIA3 serves an important role in the pathobiology of GC, and that PDIA3 is a useful marker for the prediction of prognosis.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma/patologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Neoplasias Gástricas/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/imunologia , Carcinoma/imunologia , Carcinoma/mortalidade , Linhagem Celular Tumoral , Feminino , Seguimentos , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Isomerases de Dissulfetos de Proteínas/imunologia , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/mortalidade , Análise de SobrevidaRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV), a virulent pathogen of swine, suppresses the innate immune response and induces persistent infection. One mechanism used by viruses to evade the immune system is to cripple the antigen-processing machinery in monocyte-derived dendritic cells (MoDCs). In this study, we show that MoDCs infected by PRRSV express lower levels of the major histocompatibility complex (MHC)-peptide complex proteins TAP1 and ERp57 and are impaired in their ability to stimulate T cell proliferation and increase their production of CD83. Neutralization of sCD83 removes the inhibitory effects of PRRSV on MoDCs. When MoDCs are incubated with exogenously added sCD83 protein, TAP1 and ERp57 expression decreases and T lymphocyte activation is impaired. PRRSV nonstructural protein 1α (Nsp1α) enhances CD83 promoter activity. Mutations in the ZF domain of Nsp1α abolish its ability to activate the CD83 promoter. We generated recombinant PRRSVs with mutations in Nsp1α and the corresponding repaired PRRSVs. Viruses with Nsp1α mutations did not decrease levels of TAP1 and ERp57, impair the ability of MoDCs to stimulate T cell proliferation, or increase levels of sCD83. We show that the ZF domain of Nsp1α stimulates the secretion of CD83, which in turn inhibits MoDC function. Our study provides new insights into the mechanisms of immune suppression by PRRSV.IMPORTANCE PRRSV has a severe impact on the swine industry throughout the world. Understanding the mechanisms by which PRRSV infection suppresses the immune system is essential for a robust and sustainable swine industry. Here, we demonstrated that PRRSV infection manipulates MoDCs by interfering with their ability to produce proteins in the MHC-peptide complex. The virus also impairs the ability of MoDCs to stimulate cell proliferation, due in large part to the enhanced release of soluble CD83 from PRRSV-infected MoDCs. The viral nonstructural protein 1 (Nsp1) is responsible for upregulating CD83 promoter activity. Amino acids in the ZF domain of Nsp1α (L5-2A, rG45A, G48A, and L61-6A) are essential for CD83 promoter activation. Viruses with mutations at these sites no longer inhibit MoDC-mediated T cell proliferation. These findings provide novel insights into the mechanism by which the adaptive immune response is suppressed during PRRSV infection.
Assuntos
Antígenos CD/imunologia , Células Dendríticas/imunologia , Imunoglobulinas/imunologia , Glicoproteínas de Membrana/imunologia , Monócitos/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Linfócitos T/imunologia , Proteínas não Estruturais Virais/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Antígenos CD/genética , Proliferação de Células , Imunoglobulinas/genética , Glicoproteínas de Membrana/genética , Síndrome Respiratória e Reprodutiva Suína/genética , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Domínios Proteicos , Suínos , Proteínas não Estruturais Virais/genética , Antígeno CD83RESUMO
The upregulated expression of thioredoxin domain-containing protein 5 (TXNDC5) is associated with rheumatoid arthritis in patients and model mice. However, the underlying mechanism by which TXNDC5 influences the pathological activation of rheumatoid arthritis synovial fibroblasts (RASFs) remains unknown. In this study, we show that TXNDC5 expression in RASFs and their cytokine production are significantly upregulated in response to LPS, TNF-α and IL-6, but suppressed by transfection with TXNDC5-siRNA. TXNDC5 is further validated as the direct target of NF-κB signaling. Mechanistically, TXNDC5 directly interacts with heat shock cognate 70 protein (HSC70) to sequester it in the cytoplasm, and HSC70 silencing exerts the same effects as TXNDC5 on the biological activity of RASFs (for example, decreased cell viability, invasion and cytokine production). Furthermore, HSC70 activates NF-κB signaling by destabilizing IκBß protein in the absence of LPS or facilitating its nuclear translocation in the presence of LPS. Importantly, TXNDC5 can also regulate the activity of NF-κB signaling in a HSC70-IκBß-dependent manner. Taken together, by linking HSC70 and NF-κB signaling, TXNDC5 plays a pro-inflammatory role in RASFs, highlighting a potential approach to treat RA by blocking the TXNDC5/HSC70 interaction.
Assuntos
Artrite Reumatoide/imunologia , Fibroblastos/imunologia , Proteínas de Choque Térmico HSC70/imunologia , NF-kappa B/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Transdução de Sinais/imunologia , Membrana Sinovial/imunologia , Artrite Reumatoide/patologia , Feminino , Fibroblastos/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Masculino , Membrana Sinovial/patologiaRESUMO
Essentials ERp72 is a thiol isomerase enzyme. ERp72 levels increase at the platelet surface during platelet activation. We generated a humanized monoclonal antibody which blocks ERp72 enzyme activity (anti-ERp72). Anti-ERp72 inhibits platelet functional responses and thrombosis. SUMMARY: Background Within the endoplasmic reticulum, thiol isomerase enzymes modulate the formation and rearrangement of disulfide bonds in newly folded proteins entering the secretory pathway to ensure correct protein folding. In addition to their intracellular importance, thiol isomerases have been recently identified to be present on the surface of a number of cell types where they are important for cell function. Several thiol isomerases are known to be present on the resting platelet surface, including PDI, ERp5 and ERp57, and levels are increased following platelet activation. Inhibition of the catalytic activity of these enzymes results in diminished platelet function and thrombosis. Aim We previously determined that ERp72 is present at the resting platelet surface and levels increase upon platelet activation; however, its functional role on the cell surface was unclear. We aimed to investigate the role of ERp72 in platelet function and its role in thrombosis. Methods Using HuCAL technology, fully humanized Fc-null anti-ERp72 antibodies were generated. Eleven antibodies were screened for their ability to inhibit ERp72 activity and the most potent inhibitory antibody (anti-ERp72) selected for further testing in platelet functional assays. Results and conclusions Anti-ERp72 inhibited platelet aggregation, granule secretion, calcium mobilisation and integrin activation, revealing an important role for extracellular ERp72 in the regulation of platelet activation. Consistent with this, infusion of anti-ERp72 into mice protected against thrombosis.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Plaquetas/efeitos dos fármacos , Fibrinolíticos/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Trombose/prevenção & controle , Animais , Plaquetas/enzimologia , Plaquetas/imunologia , Cálcio/sangue , Modelos Animais de Doenças , Fibrinogênio/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/imunologia , Camundongos Endogâmicos C57BL , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Isomerases de Dissulfetos de Proteínas/sangue , Isomerases de Dissulfetos de Proteínas/imunologia , Transdução de Sinais/efeitos dos fármacos , Trombose/sangue , Trombose/enzimologiaRESUMO
Changes in the T cell surface redox environment regulate critical cell functions, such as cell migration, viral entry and cytokine production. Cell surface protein disulfide isomerase (PDI) contributes to the regulation of T cell surface redox status. Cell surface PDI can be released into the extracellular milieu or can be internalized by T cells. We have found that galectin-9, a soluble lectin expressed by T cells, endothelial cells and dendritic cells, binds to and retains PDI on the cell surface. While endogenous galectin-9 is not required for basal cell surface PDI expression, exogenous galectin-9 mediated retention of cell surface PDI shifted the disulfide/thiol equilibrium on the T cell surface. O-glycans on PDI are required for galectin-9 binding, and PDI recognition appears to be specific for galectin-9, as galectin-1 and galectin-3 do not bind PDI. Galectin-9 is widely expressed by immune and endothelial cells in inflamed tissues, suggesting that T cells would be exposed to abundant galectin-9, in cis and in trans, in infectious or autoimmune conditions.
Assuntos
Membrana Celular/metabolismo , Galectina 1/metabolismo , Galectinas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Linfócitos T/metabolismo , Sítios de Ligação , Linhagem Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Galectina 1/genética , Galectina 3/genética , Galectina 3/metabolismo , Galectinas/antagonistas & inibidores , Galectinas/genética , Galectinas/farmacologia , Expressão Gênica , Regulação da Expressão Gênica , Glicosilação , Humanos , Modelos Moleculares , Oxirredução , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais , Linfócitos T/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)-dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3-/- mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5-/- mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells.
Assuntos
Plaquetas/imunologia , Complemento C3/genética , Complemento C5/genética , Hemostasia/imunologia , Trombose/imunologia , Veia Cava Inferior/imunologia , Animais , Plaquetas/patologia , Ativação do Complemento , Complemento C3/metabolismo , Complemento C5/metabolismo , Armadilhas Extracelulares/genética , Armadilhas Extracelulares/imunologia , Fibrina/genética , Fibrina/imunologia , Expressão Gênica , Humanos , Hidrolases/genética , Hidrolases/imunologia , Imunidade Inata , Leucócitos/imunologia , Leucócitos/patologia , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/patologia , Fosfatidilserinas/metabolismo , Ativação Plaquetária/imunologia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Proteína-Arginina Desiminase do Tipo 4 , Tromboplastina/genética , Tromboplastina/imunologia , Trombose/sangue , Trombose/genética , Trombose/patologia , Veia Cava Inferior/metabolismo , Veia Cava Inferior/patologiaRESUMO
In the present study, the efficacy of Leishmania donovani protein disulfide isomerase (LdPDI) as a DNA vaccine was evaluated in BALB/C mice. Mice immunized with the LdPDI-DNA construct were found to be the most immuno-reactive, as the construct induced higher T-cell proliferation. The increased T-cell proliferation was associated with a substantial rise in Th1 and Th17+ CD4 cell response and triggered a higher proportion of CD8+ T cells for the release of interferon-gamma along with a reduced splenic parasite load on Days20 and 60 post challenge (PC). Furthermore, the vaccine construct triggered increased interferon (IFN)-γ, interleukin(IL)-17A, and IL-22 release accompanied by decreased extracellular signal-regulated kinases (ERK) 1/2 signaling and increased mitogen-activated protein kinase (MAPK) signaling coinciding with an increase in the amount of nitrite and reactive oxygen species (ROS)in vaccinating the splenocyts. We summarize from our data that the PDI-DNA construct of Leishmania donovani has the potential to elicit protective immunity through the pro-inflammatory cytokines of CD8+ and CD4+(Th1 and Th17) following an intervention in the downstream signaling event of ERK1/2 (probably through p38MAPK signaling). Therefore, the study suggests a new control against visceral leishmaniasis in the future.
Assuntos
Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Proteínas de Protozoários/imunologia , Animais , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Leishmania donovani , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais/imunologia , Células Th1/imunologia , Células Th17/imunologia , Vacinas de DNA/imunologiaRESUMO
Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin ß subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine ß3, and human ß1 and ß2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of ß3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant ß3 subunit. We further developed mouse anti-mouse ß3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbß3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbß3 endogenous PDI-like activity reduced αIIbß3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.
Assuntos
Cadeias beta de Integrinas/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Domínio Catalítico , Moléculas de Adesão Celular , Humanos , Camundongos , Proteínas do Tecido Nervoso , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Proteínas Recombinantes , Semaforinas , Trombose/prevenção & controleRESUMO
Protein disulphide isomerase (PDI) is one of the key enzymes essential for the survival of Leishmania donovani in the host. Our study suggested that PDI is associated with the generation of Th1-type of cellular responses in treated Visceral leishmaniasis (VL) subjects. The stimulation of Peripheral blood mononuclear cells (PBMCs) with recombinant Protein Disulphide Isomerase upregulated the reactive oxygen species generation, Nitric oxide release, IL12 and IFN-γ production indicating its pivotal role in protective immune response. Further, a pre-stimulation of PBMCs with Protein disulphide isomerase induced a strong IFN-γ response through CD8+ T cells in treated VL subjects. These findings also supported through the evidence that this antigen was processed and presented by major histocompatibility complex class I (MHC-1) dependent pathway and had an immunoprophylactic potential which can induce CD8+ T cell protective immune response in MHC class I dependent manner against VL. To find out the possible epitopes that might be responsible for CD8+ T cell specific IFN-γ response, computational approach was adopted. Six novel promiscuous epitopes were predicted to be highly immunogenic and can be presented by 32 different HLA allele to CD8+ T cells. Further investigation will explore more about their immunological relevance and usefulness as vaccine candidates.
Assuntos
Epitopos de Linfócito T/química , Antígenos de Histocompatibilidade Classe I/química , Leishmania donovani/enzimologia , Isomerases de Dissulfetos de Proteínas/química , Adolescente , Adulto , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Epitopos de Linfócito T/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunomodulação , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Masculino , Isomerases de Dissulfetos de Proteínas/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Adulto JovemRESUMO
Evidence continues to increase linking autoimmunity and other complex diseases to the chemicals commonly found in our environment. Bisphenol A (BPA) is a synthetic monomer used widely in many forms, from food containers to toys, medical products and many others. The potential for BPA to participate as a triggering agent for autoimmune diseases is likely due to its known immunological influences. The goal of this research was to determine if immune reactivity to BPA has any correlation with neurological antibodies. BPA binds to a target enzyme called protein disulfide isomerase (PDI). Myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) are neuronal antigens that are target sites for neuroinflammation and neuroautoimmunity. We determined the co-occurrence of anti-MBP and anti-MOG antibodies with antibodies made against BPA bound to human serum albumin in 100 healthy human subjects. Correlation between BPA to PDI, BPA to MOG, BPA to MBP, PDI to MBP and PDI to MOG were all highly statistically significant (P < 0.0001). The outcome of our study suggests that immune reactivity to BPA-human serum albumin and PDI has a high degree of statistical significance with substantial correlation with both MBP and MOG antibody levels. This suggests that BPA may be a trigger for the production of antibodies against PDI, MBP and MOG. Immune reactivity to BPA bound to human tissue proteins may be a contributing factor to neurological autoimmune disorders. Further research is needed to determine the exact relationship of these antibodies with neuroautoimmunities. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd.
Assuntos
Anticorpos Bloqueadores/biossíntese , Anticorpos/metabolismo , Compostos Benzidrílicos/antagonistas & inibidores , Compostos Benzidrílicos/imunologia , Neurônios/imunologia , Fenóis/antagonistas & inibidores , Fenóis/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Adolescente , Adulto , Idoso , Anticorpos/farmacologia , Anticorpos Bloqueadores/análise , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/imunologia , Autoimunidade/efeitos dos fármacos , Autoimunidade/imunologia , Humanos , Pessoa de Meia-Idade , Proteína Básica da Mielina/biossíntese , Proteína Básica da Mielina/genética , Glicoproteína Mielina-Oligodendrócito/biossíntese , Glicoproteína Mielina-Oligodendrócito/genética , Doenças do Sistema Nervoso/induzido quimicamente , Doenças do Sistema Nervoso/imunologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Adulto JovemRESUMO
Autoimmune polyendocrine syndrome type 1 (APS1) is a monogenic disorder that features multiple autoimmune disease manifestations. It is caused by mutations in the Autoimmune regulator (AIRE) gene, which promote thymic display of thousands of peripheral tissue antigens in a process critical for establishing central immune tolerance. We here used proteome arrays to perform a comprehensive study of autoimmune targets in APS1. Interrogation of established autoantigens revealed highly reliable detection of autoantibodies, and by exploring the full panel of more than 9000 proteins we further identified MAGEB2 and PDILT as novel major autoantigens in APS1. Our proteome-wide assessment revealed a marked enrichment for tissue-specific immune targets, mirroring AIRE's selectiveness for this category of genes. Our findings also suggest that only a very limited portion of the proteome becomes targeted by the immune system in APS1, which contrasts the broad defect of thymic presentation associated with AIRE-deficiency and raises novel questions what other factors are needed for break of tolerance.