RESUMO
Homeostasis of the immune system depends on the proper function of regulatory T cells (T(reg) cells). Compromised suppressive activity of T(reg) cells leads to autoimmune disease and graft rejection and promotes anti-tumor immunity. Here we report a previously unrecognized requirement for the serine-threonine phosphatase PP2A in the function of T(reg) cells. T(reg) cells exhibited high PP2A activity, and T(reg) cell-specific ablation of the PP2A complex resulted in a severe, multi-organ, lymphoproliferative autoimmune disorder. Mass spectrometry revealed that PP2A associated with components of the mTOR metabolic-checkpoint kinase pathway and suppressed the activity of the mTORC1 complex. In the absence of PP2A, T(reg) cells altered their metabolic and cytokine profile and were unable to suppress effector immune responses. Therefore, PP2A is required for the function of T(reg) cells and the prevention of autoimmunity.
Assuntos
Doenças Autoimunes/imunologia , Transtornos Linfoproliferativos/imunologia , Proteína Fosfatase 2/imunologia , Linfócitos T Reguladores/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Autoimunidade/genética , Autoimunidade/imunologia , Células Cultivadas , Ceramidas/imunologia , Ceramidas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Células Jurkat , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Fosforilação/imunologia , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Reguladores/metabolismo , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
We had shown previously that the protein phosphatase 2A regulatory subunit PPP2R2D suppresses IL-2 production, and PPP2R2D deficiency in T cells potentiates the suppressive function of regulatory T (Treg) cells and alleviates imiquimod-induced lupus-like pathology. In this study, in a melanoma xenograft model, we noted that the tumor grew in larger sizes in mice lacking PPP2R2D in T cells (LckCreR2Dfl/fl) compared with wild type (R2Dfl/fl) mice. The numbers of intratumoral T cells in LckCreR2Dfl/fl mice were reduced compared with R2Dfl/fl mice, and they expressed a PD-1+CD3+CD44+ exhaustion phenotype. In vitro experiments confirmed that the chromatin of exhaustion markers PD-1, LAG3, TIM3, and CTLA4 remained open in LckCreR2Dfl/fl CD4 T conventional compared with R2Dfl/fl T conventional cells. Moreover, the percentage of Treg cells (CD3+CD4+Foxp3+CD25hi) was significantly increased in the xenografted tumor of LckCreR2Dfl/fl mice compared with R2Dfl/fl mice probably because of the increase in the percentage of IL-2-producing LckCreR2Dfl/fl T cells. Moreover, using adoptive T cell transfer in mice xenografted with melanoma, we demonstrated that PPP2R2D deficiency in T cells enhanced the inhibitory effect of Treg cells in antitumor immunity. At the translational level, analysis of publicly available data from 418 patients with melanoma revealed that PPP2R2D expression levels correlated positively with tumor-infiltration level of CD4 and CD8 T cells. The data demonstrate that PPP2R2D is a negative regulator of immune checkpoint receptors, and its absence exacerbates effector T cell exhaustion and promotes Treg cell expansion. We conclude that PPP2R2D protects against melanoma growth, and PPP2R2D-promoting regimens can have therapeutic value in patients with melanoma.
Assuntos
Melanoma , Linfócitos T Reguladores , Animais , Proliferação de Células , Humanos , Interleucina-2/metabolismo , Melanoma/metabolismo , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Proteína Fosfatase 2/metabolismoRESUMO
Interleukin 27 has both pro-inflammatory and anti-inflammatory properties in autoimmunity. The anti-inflammatory effects of IL-27 are linked with inhibition of Th17 differentiation but the IL-27 effect on myeloid cells is less studied. Herein we demonstrate that IL-27 inhibits IL-23-induced inflammation associated not only with Th17 cells but also with myeloid cell infiltration in the joints and splenic myeloid populations of CD11b+ GR1+ and CD3-CD11b+CD11c-GR1- cells. The IL-27 anti-inflammatory response was associated with reduced levels of myeloid cells in the spleen and bone marrow. Overall, our data demonstrate that IL-27 has an immunosuppressive role that affects IL-23-dependent myelopoiesis in the bone marrow and its progression to inflammatory arthritis and plays a crucial role in controlling IL-23 driven joint inflammation by negatively regulating the expansion of myeloid cell subsets.
Assuntos
Artrite Experimental , Interleucina-27 , Animais , Citocinas , Inflamação , Interleucina-23 , Células Th17RESUMO
OBJECTIVE: To investigate the role of calcium/calmodulin-dependent protein kinase IV (CaMK4) in the development of joint injury in a mouse model of arthritis and patients with RA. METHODS: Camk4-deficient, Camk4flox/floxLck-Cre, and mice treated with CaMK4 inhibitor KN-93 or KN-93 encapsulated in nanoparticles tagged with CD4 or CD8 antibodies were subjected to collagen-induced arthritis (CIA). Inflammatory cytokine levels, humoral immune response, synovitis, and T-cell activation were recorded. CAMK4 gene expression was measured in CD4+ T cells from healthy participants and patients with active RA. Micro-CT and histology were used to assess joint pathology. CD4+ and CD14+ cells in patients with RA were subjected to Th17 or osteoclast differentiation, respectively. RESULTS: CaMK4-deficient mice subjected to CIA displayed improved clinical scores and decreased numbers of Th17 cells. KN-93 treatment significantly reduced joint destruction by decreasing the production of inflammatory cytokines. Furthermore, Camk4flox/floxLck-Cre mice and mice treated with KN93-loaded CD4 antibody-tagged nanoparticles developed fewer Th17 cells and less severe arthritis. CaMK4 inhibition mitigated IL-17 production by CD4+ cells in patients with RA. The number of in vitro differentiated osteoclasts from CD14+ cells in patients with RA was significantly decreased with CaMK4 inhibitors. CONCLUSION: Using global and CD4-cell-targeted pharmacologic approaches and conditionally deficient mice, we demonstrate that CaMK4 is important in the development of arthritis. Using ex vivo cell cultures from patients with RA, CaMK4 is important for both Th17 generation and osteoclastogenesis. We propose that CaMK4 inhibition represents a new approach to control the development of arthritis.
Assuntos
Artrite Experimental , Osteogênese , Animais , Camundongos , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/uso terapêutico , Células Th17 , Citocinas/metabolismo , Artrite Experimental/metabolismo , Diferenciação CelularRESUMO
Protein phosphatase 2A (PP2A) composed of a scaffold subunit, a catalytic subunit, and multiple regulatory subunits is a ubiquitously expressed serine/threonine phosphatase. We have previously shown that the PP2A catalytic subunit is increased in T cells from patients with systemic lupus erythematosus and promotes IL-17 production by enhancing the activity of Rho-associated kinase (ROCK) in T cells. However, the molecular mechanism whereby PP2A regulates ROCK activity is unknown. In this study, we show that the PP2A regulatory subunit PPP2R2A is increased in T cells from people with systemic lupus erythematosus and binds to, dephosphorylates, and activates the guanine nucleotide exchange factor GEF-H1 at Ser885, which in turn increases the levels of RhoA-GTP and the activity of ROCK in T cells. Genetic PPP2R2A deficiency in murine T cells reduced Th1 and Th17, but not regulatory T cell differentiation and mice with T cell-specific PPP2R2A deficiency displayed less autoimmunity when immunized with myelin oligodendrocyte glycoprotein peptide. Our studies indicate that PPP2R2A is the regulatory subunit that dictates the PP2A-directed enhanced Th1 and Th17 differentiation, and therefore, it represents a therapeutic target for pathologies linked to Th1 and Th17 cell expansion.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Proteína Fosfatase 2/metabolismo , Células Th1/imunologia , Células Th17/imunologia , Animais , Hidrolases de Éster Carboxílico/genética , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Lúpus Eritematoso Sistêmico/genética , Ativação Linfocitária , Camundongos , Camundongos Knockout , Proteína Fosfatase 2/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Transplant glomerulopathy (TG) is a major cause of late allograft loss. Increased urine podocin/creatinine ratio in TG signifies accelerated podocyte loss. The mechanisms that lead to podocyte injury in TG remain unclear. We report that IgG from kidney transplant recipients with TG, but not from those without TG, cause a reduction in the expression of nephrin, significant podocyte actin cytoskeleton, and motility changes. These changes are preceded by increased expression of calcium/calmodulin kinase IV (CAMK4). Mechanistically, we found that CAMK4 phosphorylates GSK3ß (glycogen synthase kinase 3 beta), activates the Wnt pathway and stabilizes the nephrin transcriptional repressor SNAIL. Silencing neonatal Fc Receptor (FcRn) or CAMK4 prevented the podocyte-damaging effects of IgG from patients with TG. Furthermore, we show that removal of N-linked glycosyl residues from these IgG did not interfere with its entry into the podocytes but eliminated its ability to upregulate CAMK4 and cause podocyte injury. The translational value of these findings is signified by the fact that CAMK4 is increased in podocytes of patients with TG but not in those without TG despite other forms of renal dysfunction. Our results offer novel considerations to limit podocyte injury in patients with kidney transplants, which may lead to eventual glomerular destabilization and transplant glomerulopathy.
Assuntos
Transplante de Rim , Podócitos , Cálcio , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Humanos , Imunoglobulina G , Recém-Nascido , Transplante de Rim/efeitos adversosRESUMO
Acute and chronic kidney failure is common in hospitalized patients with COVID-19, yet the mechanism of injury and predisposing factors remain poorly understood. We investigated the role of complement activation by determining the levels of deposited complement components (C1q, C3, FH, C5b-9) and immunoglobulin along with the expression levels of the injury-associated molecules spleen tyrosine kinase (Syk), mucin-1 (MUC1) and calcium/calmodulin-dependent protein kinase IV (CaMK4) in the kidney tissues of people who succumbed to COVID-19. We report increased deposition of C1q, C3, C5b-9, total immunoglobulin, and high expression levels of Syk, MUC1 and CaMK4 in the kidneys of COVID-19 patients. Our study provides strong rationale for the expansion of trials involving the use of inhibitors of these molecules, in particular C1q, C3, Syk, MUC1 and CaMK4 to treat patients with COVID-19.
Assuntos
COVID-19/metabolismo , Proteínas do Sistema Complemento/metabolismo , Rim/metabolismo , Mucina-1/metabolismo , SARS-CoV-2 , Quinase Syk/metabolismo , Idoso , Idoso de 80 Anos ou mais , COVID-19/patologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas do Sistema Complemento/genética , Evolução Fatal , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mucina-1/genética , Quinase Syk/genéticaRESUMO
Lung inflammation and damage is prominent in people infected with SARS-Cov-2 and a major determinant of morbidity and mortality. We report the deposition of complement components in the lungs of people who succumbed to COVID-19 consistent with the activation of the classical and the alternative pathways. Our study provides strong rationale for the expansion of trials involving the use of complement inhibitors to treat patients with COVID-19.
Assuntos
COVID-19/imunologia , Ativação do Complemento/imunologia , Via Alternativa do Complemento/imunologia , Lesão Pulmonar/imunologia , Idoso , Idoso de 80 Anos ou mais , COVID-19/complicações , Inativadores do Complemento/farmacologia , Inativadores do Complemento/uso terapêutico , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Imuno-Histoquímica , Pulmão/diagnóstico por imagem , Pulmão/imunologia , Pulmão/patologia , Lesão Pulmonar/complicações , Lesão Pulmonar/patologia , Lesão Pulmonar/virologia , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND/OBJECTIVES: Rhabdomyosarcoma (RMS) is characterized by the expression of the myogenic regulatory protein MYOD1. Histologic types include alveolar, embryonal (ERMS), and spindle cell sclerosing RMS (SRMS). SRMS harbors MYOD1 mutations in a subset of adult cases in association with poor prognosis. DESIGN/METHODS: To study the level of MYOD1 protein expression and its clinical significance, we have analyzed variable numbers of pediatric (<18 years of age) and adult (age range ≥18 to 35 years) ERMS and SRMS cases for presence or absence of MYOD1 immunoreactivity in correlation with clinical outcome and MYOD1 L122R mutations. RESULTS: Lack of MYOD1 immunoreactivity, identified in 23.8% of nonalveolar RMS (non-ARMS) cases, was more prevalent in SRMS (44%) than ERMS (17.2%) and was significantly associated with low overall survival and unfavorable tumor sites (p < .05). Lack of MYOD1 immunoreactivity was not associated with MYOD1 L122R mutations, which were identified in 3/37 (8%) cases including only two of 31 (6.5%) pediatric cases, one of 11 or 9% pediatric SRMS, and one case of infant ERMS. CONCLUSION: These studies highlight the prognostic role of MYOD1 in non-ARMS. Lack of MYOD1 immunoreactivity is associated with poor prognosis in ERMS and SRMS. MYOD1 gene mutations are generally infrequent in pediatric RMS. Although mutations are predominant in SRMS, they may exceptionally occur in infantile ERMS.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma Embrionário , Rabdomiossarcoma , Adolescente , Adulto , Criança , Humanos , Lactente , Mutação , Proteína MyoD/genética , Prognóstico , Rabdomiossarcoma/genética , Adulto JovemRESUMO
Th17 cells favor glycolytic metabolism, and pyruvate dehydrogenase (PDH) is the key bifurcation enzyme, which in its active dephosphorylated form advances the oxidative phosphorylation from glycolytic pathway. The transcriptional factor, inducible cAMP early repressor/cAMP response element modulator (ICER/CREM), has been shown to be induced in Th17 cells and to be overexpressed in CD4+ T cells from the patients with systemic lupus erythematosus (SLE). We found that glycolysis and lactate production in in vitro Th17-polarized T cells was reduced and that the expression of pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2), an enzyme that converts the inactive PDH to its active form, and PDH enzyme activity were increased in Th17 cells from ICER/CREM-deficient animals. ICER was found to bind to the Pdp2 promoter and suppress its expression. Furthermore, forced expression of PDP2 in CD4+ cells reduced the in vitro Th17 differentiation, whereas shRNA-based suppression of PDP2 expression increased in vitro Th17 differentiation and augmented experimental autoimmune encephalomyelitis. At the translational level, PDP2 expression was decreased in memory Th17 cells from patients with SLE and forced expression of PDP2 in CD4+ T cells from lupus-prone MRL/lpr mice and patients with SLE suppressed Th17 differentiation. These data demonstrate the direct control of energy production during Th17 differentiation in health and disease by the transcription factor ICER/CREM at the PDH metabolism bifurcation level.
Assuntos
Diferenciação Celular , Regulação Enzimológica da Expressão Gênica , Fosfoproteínas Fosfatases/biossíntese , Elementos de Resposta , Células Th17/enzimologia , Animais , Domínio Catalítico , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/imunologia , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Masculino , Camundongos , Camundongos Knockout , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/imunologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
Current technologies and available scaffold materials do not support long-term cell viability, differentiation and maintenance of podocytes, the ultra-specialized kidney resident cells that are responsible for the filtration of the blood. We developed a new platform which imitates the native kidney microenvironment by decellularizing fibroblasts grown on surfaces with macromolecular crowding. Human immortalized podocytes cultured on this platform displayed superior viability and metabolic activity up to 28 days compared to podocytes cultured on tissue culture plastic surfaces. The new platform displayed a softer surface and an abundance of growth factors and associated molecules. More importantly it enabled podocytes to display molecules responsible for their structure and function and a superior development of intercellular connections/interdigitations, consistent with maturation. The new platform can be used to study podocyte biology, test drug toxicity and determine whether sera from patients with podocytopathies are involved in the expression of glomerular pathology.
RESUMO
Intestinal ischemia followed by reperfusion leads to local and remote organ injury attributed to inflammatory response during the reperfusion phase. The extent to which ischemia contributes to ischemia/reperfusion injury has not been thoroughly studied. After careful evaluation of intestinal tissue following 30 min of ischemia, we noticed significant local mucosal injury in wild-type mice. This injury was drastically reduced in C3-deficient mice, suggesting C3 involvement. Depletion of circulating complement with cobra venom factor eliminated, as expected, injury recorded at the end of the reperfusion phase but failed to eliminate injury that occurred during the ischemic phase. Immunohistochemical studies showed that tissue damage during ischemia was associated with increased expression of C3/C3 fragments primarily in the intestinal epithelial cells, suggesting local involvement of complement. In vitro studies using Caco2 intestinal epithelial cells showed that in the presence of LPS or exposure to hypoxic conditions the cells produce higher C3 mRNA as well as C3a fragment. Caco2 cells were also noted to produce cathepsins B and L, and inhibition of cathepsins suppressed the release of C3a. Finally, we found that mice treated with a cathepsin inhibitor and cathepsin B-deficient mice suffer limited intestinal injury during the ischemic phase. To our knowledge, our findings demonstrate for the first time that significant intestinal injury occurs during ischemia prior to reperfusion and that this is due to activation of C3 within the intestinal epithelial cells in a cathepsin-dependent manner. Modulation of cathepsin activity may prevent injury of organs exposed to ischemia.
Assuntos
Complemento C3/metabolismo , Isquemia Mesentérica/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Western Blotting , Células CACO-2 , Catepsinas/metabolismo , Complemento C3/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Isquemia Mesentérica/imunologia , Isquemia Mesentérica/patologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologiaRESUMO
Ischemia-reperfusion (IR) injury to the small intestine following clamping of the superior mesenteric artery results in an intense local inflammatory response that is characterized by villous damage and neutrophil infiltration. IL-17A, a cytokine produced by a variety of cells in response to inflammatory cytokines released following tissue injury, has been implicated in IR injury. Using Il17a-/- , Il23r-/- , and Rorc-/- mice and administration of anti-IL-17A and anti-IL-23 neutralizing Abs to wild-type mice, we demonstrate that intestinal IR injury depends on IL-17A and that IL-17A is downstream of the binding of autoantibody to ischemia-conditioned tissues and subsequent complement activation. Using bone marrow chimeras, we demonstrate that the IL-17A required for intestinal IR injury is derived from hematopoietic cells. Finally, by transferring autoantibody-rich sera into Rag2γc-/- and Rag2-/- mice, we demonstrate that innate lymphoid cells are the main producers of IL-17A in intestinal IR injury. We propose that local production of IL-17A by innate lymphoid cells is crucial for the development of intestinal IR injury and may provide a therapeutic target for clinical exploitation.
Assuntos
Interleucina-17/metabolismo , Intestino Delgado/imunologia , Intestino Delgado/patologia , Linfócitos/imunologia , Traumatismo por Reperfusão/imunologia , Animais , Anticorpos Bloqueadores/administração & dosagem , Autoanticorpos/metabolismo , Células Cultivadas , Ativação do Complemento , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Humanos , Imunidade Inata , Interleucina-17/genética , Artéria Mesentérica Superior/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Receptores de Interleucina/genéticaRESUMO
We have found that calcium calmodulin kinase IV is increased in T cells, podocytes, and mesangial cells from patients with systemic lupus erythematosus, as well as in lupus-prone mice, podocytes of patients with focal segmental glomerulosclerosis, and in mice injected with doxorubicin. We showed that this accounts for aberrant T cell function and glomerular damage. Using nanoparticles (nlg) loaded with a small drug inhibitor of calcium calmodulin kinase IV and tagged with antibodies directed to CD4 we have been able to show inhibition of autoimmunity and lupus nephritis. Also, using nlg tagged with antibodies to nephrin, we showed suppression of nephritis in lupus-prone mice and of glomerular damage in mice exposed to doxorubicin. We propose the development of approaches to deliver drugs to cells in a targeted and precise manner.
Assuntos
Benzilaminas/administração & dosagem , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Nefrite Lúpica/tratamento farmacológico , Nanopartículas , Inibidores de Proteínas Quinases/administração & dosagem , Sulfonamidas/administração & dosagem , Linfócitos T/imunologia , Animais , Antibióticos Antineoplásicos/toxicidade , Benzilaminas/uso terapêutico , Antígenos CD4 , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Metilação de DNA , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Sistemas de Liberação de Medicamentos , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/imunologia , Humanos , Lúpus Eritematoso Sistêmico , Nefrite Lúpica/imunologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos MRL lpr , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/uso terapêutico , Sulfonamidas/uso terapêutico , Linfócitos T Reguladores/imunologia , Células Th17/imunologiaRESUMO
Ablepharon macrostomia syndrome (AMS) and Barber-Say syndrome (BSS) are rare congenital ectodermal dysplasias characterized by similar clinical features. To establish the genetic basis of AMS and BSS, we performed extensive clinical phenotyping, whole exome and candidate gene sequencing, and functional validations. We identified a recurrent de novo mutation in TWIST2 in seven independent AMS-affected families, as well as another recurrent de novo mutation affecting the same amino acid in ten independent BSS-affected families. Moreover, a genotype-phenotype correlation was observed, because the two syndromes differed based solely upon the nature of the substituting amino acid: a lysine at TWIST2 residue 75 resulted in AMS, whereas a glutamine or alanine yielded BSS. TWIST2 encodes a basic helix-loop-helix transcription factor that regulates the development of mesenchymal tissues. All identified mutations fell in the basic domain of TWIST2 and altered the DNA-binding pattern of Flag-TWIST2 in HeLa cells. Comparison of wild-type and mutant TWIST2 expressed in zebrafish identified abnormal developmental phenotypes and widespread transcriptome changes. Our results suggest that autosomal-dominant TWIST2 mutations cause AMS or BSS by inducing protean effects on the transcription factor's DNA binding.
Assuntos
Anormalidades Múltiplas/genética , Anormalidades do Olho/genética , Doenças Palpebrais/genética , Hirsutismo/genética , Hipertelorismo/genética , Hipertricose/genética , Macrostomia/genética , Modelos Moleculares , Fenótipo , Proteínas Repressoras/genética , Anormalidades da Pele/genética , Proteína 1 Relacionada a Twist/genética , Anormalidades Múltiplas/patologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , Exoma/genética , Anormalidades do Olho/patologia , Doenças Palpebrais/patologia , Células HeLa , Hirsutismo/patologia , Humanos , Hipertelorismo/patologia , Hipertricose/patologia , Macrostomia/patologia , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Conformação Proteica , Proteínas Repressoras/química , Análise de Sequência de DNA , Anormalidades da Pele/patologia , Proteína 1 Relacionada a Twist/química , Peixe-ZebraRESUMO
Isolated methylmalonic acidemia (MMA), caused by deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT), is often complicated by end stage renal disease that is resistant to conventional therapies, including liver transplantation. To establish a viable model of MMA renal disease, Mut was expressed in the liver of Mut(-/-) mice as a stable transgene under the control of an albumin (INS-Alb-Mut) promoter. Mut(-/-);Tg(INS-Alb-Mut) mice, although completely rescued from neonatal lethality that was displayed by Mut(-/-) mice, manifested a decreased glomerular filtration rate (GFR), chronic tubulointerstitial nephritis and ultrastructural changes in the proximal tubule mitochondria associated with aberrant tubular function, as demonstrated by single-nephron GFR studies. Microarray analysis of Mut(-/-);Tg(INS-Alb-Mut) kidneys identified numerous biomarkers, including lipocalin-2, which was then used to monitor the response of the GFR to antioxidant therapy in the mouse model. Renal biopsies and biomarker analysis from a large and diverse patient cohort (ClinicalTrials.gov identifier: NCT00078078) precisely replicated the findings in the animals, establishing Mut(-/-);Tg(INS-Alb-Mut) mice as a unique model of MMA renal disease. Our studies suggest proximal tubular mitochondrial dysfunction is a key pathogenic mechanism of MMA-associated kidney disease, identify lipocalin-2 as a biomarker of increased oxidative stress in the renal tubule, and demonstrate that antioxidants can attenuate the renal disease of MMA.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Antioxidantes/farmacologia , Modelos Animais de Doenças , Túbulos Renais Proximais/fisiopatologia , Metilmalonil-CoA Mutase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Animais , Antioxidantes/uso terapêutico , Biomarcadores/metabolismo , Western Blotting , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Fluoresceína-5-Isotiocianato , Genótipo , Taxa de Filtração Glomerular/genética , Humanos , Imuno-Histoquímica , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Camundongos , Camundongos Knockout , Análise em Microsséries , Microscopia Eletrônica de Transmissão , Nefrite Intersticial/genética , Reação em Cadeia da Polimerase em Tempo Real , Transgenes/genética , Ubiquinona/farmacologiaRESUMO
BACKGROUND: The development and evaluation of new therapeutic approaches for malignant mesothelioma has been sparse due, in part, to lack of suitable tumor models. METHODS: We established primary mesothelioma cultures from pleural and ascitic fluids of five patients with advanced mesothelioma. Electron microscopy and immunohistochemistry (IHC) confirmed their mesothelial origin. Patient derived xenografts were generated by injecting the cells in nude or SCID mice, and malignant potential of the cells was analyzed by soft agar colony assay. Molecular profiles of the primary patient tumors, early passage cell cultures, and patient derived xenografts were assessed using mutational analysis, fluorescence in situ hybridization (FISH) analysis and IHC. RESULTS: Primary cultures from all five tumors exhibited morphologic and IHC features consistent to those of mesothelioma cells. Mutations of BAP1 and CDKN2A were each detected in four tumors. BAP1 mutation was associated with the lack of expression of BAP1 protein. Three cell cultures, all of which were derived from BAP1 mutant primary tumors, exhibited anchorage independent growth and also formed tumors in mice, suggesting that BAP1 loss may enhance tumor growth in vivo. Both early passage cell cultures and mouse xenograft tumors harbored BAP1 mutations and CDKN2A deletions identical to those found in the corresponding primary patient tumors. CONCLUSIONS: The mesothelioma patient derived tumor xenografts with mutational alterations that mimic those observed in patient tumors which we established can be used for preclinical development of novel drug regimens and for studying the functional aspects of BAP1 biology in mesothelioma.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Mutação , Neoplasias Pleurais/patologia , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Idoso , Animais , Técnicas de Cultura de Células , Feminino , Humanos , Neoplasias Pulmonares/genética , Masculino , Mesotelioma/genética , Mesotelioma Maligno , Camundongos , Camundongos Nus , Camundongos SCID , Pessoa de Meia-Idade , Transplante de Neoplasias , Neoplasias Experimentais , Neoplasias Pleurais/genética , Células Tumorais Cultivadas , Adulto JovemRESUMO
OBJECTIVE: Complement system is activated in patients with trauma. Although complement activation is presumed to contribute to organ damage and constitutional symptoms, little is known about the involved mechanisms. Because complement components may deposit on RBCs, we asked whether complement deposits on the surface of RBC in trauma and whether such deposition alters RBC function. DESIGN: A prospective experimental study. SETTING: Research laboratory. SUBJECTS: Blood samples collected from 42 trauma patients and 21 healthy donors. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: RBC and sera were collected from trauma patients and control donors. RBCs from trauma patients (n = 40) were found to display significantly higher amounts of C4d on their surface by flow cytometry compared with RBCs from control (n = 17) (p < 0.01). Increased amounts of iC3b were found in trauma sera (n = 27) (vs 12 controls, p < 0.01) by enzyme-linked immunosorbent assay. Incubation of RBC from universal donors (type O, Rh negative) with trauma sera (n = 10) promoted C4d deposition on their surface (vs six controls, p< 0.05). Complement-decorated RBC (n = 6) displayed limited their deformability (vs six controls, p < 0.05) in two-dimensional microchannel arrays. Incubation of RBC with trauma sera (n = 10) promoted the phosphorylation of band 3, a cytoskeletal protein important for the function of the RBC membrane (vs eight controls, p < 0.05), and also accelerated calcium influx (n = 9) and enhanced nitric oxide production (n = 12) (vs four and eight controls respectively, p < 0.05) in flow cytometry. CONCLUSIONS: Our study found the presence of extensive complement activation in trauma patients and presents new evidence in support of the hypothesis that complement activation products deposit on the surface of RBC. Such deposition could limit RBC deformability and promote the production of nitric oxide. Our findings suggest that RBC in trauma patients malfunctions, which may explain organ damage and constitutional symptoms that is not accounted for otherwise by previously known pathophysiologic mechanisms.
Assuntos
Cálcio/sangue , Ativação do Complemento/fisiologia , Eritrócitos/metabolismo , Óxido Nítrico/sangue , Fragmentos de Peptídeos/sangue , Ferimentos e Lesões/sangue , Adulto , Idoso , Estudos de Casos e Controles , Complemento C3b/análise , Complemento C4b , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Escala de Gravidade do Ferimento , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Ferimentos e Lesões/complicaçõesRESUMO
The protein phosphatase 2A (PP2A) regulatory subunit PPP2R2A is involved in the regulation of immune response. We report that lupus-prone mice with T cells deficient in PPP2R2A display less autoimmunity and nephritis. PPP2R2A deficiency promotes NAD+ biosynthesis through the nicotinamide riboside (NR)-directed salvage pathway in T cells. NR inhibits murine Th17 and promotes Treg cell differentiation, in vitro, by PΑRylating histone H1.2 and causing its reduced occupancy in the Foxp3 loci and increased occupancy in the Il17a loci, leading to increased Foxp3 and decreased Il17a transcription. NR treatment suppresses disease in MRL.lpr mice and restores NAD+-dependent poly [ADP-ribose] polymerase 1 (PARP1) activity in CD4 T cells from patients with systemic lupus erythematosus (SLE), while reducing interferon (IFN)-γ and interleukin (IL)-17 production. We conclude that PPP2R2A controls the level of NAD+ through the NR-directed salvage pathway and promotes systemic autoimmunity. Translationally, NR suppresses lupus nephritis in mice and limits the production of proinflammatory cytokines by SLE T cells.
Assuntos
Autoimunidade , Diferenciação Celular , Lúpus Eritematoso Sistêmico , NAD , Proteína Fosfatase 2 , Animais , Feminino , Humanos , Camundongos , Fatores de Transcrição Forkhead/metabolismo , Histonas/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/patologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , NAD/metabolismo , NAD/biossíntese , Niacinamida/análogos & derivados , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteína Fosfatase 2/metabolismo , Compostos de Piridínio , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Células Th17/metabolismoRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulated B cell compartment responsible for the production of autoantibodies. Here, we show that T cell-specific expression of calcium/calmodulin-dependent protein kinase IV (CaMK4) leads to T follicular helper (Tfh) cells expansion in models of T-dependent immunization and autoimmunity. Mechanistically, CaMK4 controls the Tfh-specific transcription factor B cell lymphoma 6 (Bcl6) at the transcriptional level through the cAMP responsive element modulator α (CREMα). In the absence of CaMK4 in T cells, germinal center formation and humoral immunity is impaired in immunized mice, resulting in reduced anti-dsDNA titres, as well as IgG and complement kidney deposition in the lupus-prone B6.lpr mouse. In human Tfh cells, CaMK4 inhibition reduced BCL6 expression and IL-21 secretion ex vivo, resulting in impaired plasmablast formation and IgG production. In patients with SLE, CAMK4 mRNA levels in Tfh cells correlated with those of BCL6. In conclusion, we identify CaMK4/CREMα as a driver of T cell-dependent B cell dysregulation in autoimmunity.