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1.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445576

RESUMO

CD40 crosslinking plays an important role in regulating cell migration, adhesion and proliferation in renal cell carcinoma (RCC). CD40/CD40L interaction on RCC cells activates different intracellular pathways but the molecular mechanisms leading to cell scattering are not yet clearly defined. Aim of our study was to investigate the main intracellular pathways activated by CD40 ligation and their specific involvement in RCC cell migration. CD40 ligation increased the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH (2)-terminal kinase (JNK) and p38 MAPK. Furthermore, CD40 crosslinking activated different transcriptional factors on RCC cell lines: AP-1, NFkB and some members of the Nuclear Factor of Activated T cells (NFAT) family. Interestingly, the specific inhibition of NFAT factors by cyclosporine A, completely blocked RCC cell motility induced by CD40 ligation. In tumor tissue, we observed a higher expression of NFAT factors and in particular an increased activation and nuclear migration of NFATc4 on RCC tumor tissues belonging to patients that developed metastases when compared to those who did not. Moreover, CD40-CD40L interaction induced a cytoskeleton reorganization and increased the expression of integrin ß1 on RCC cell lines, and this effect was reversed by cyclosporine A and NFAT inhibition. These data suggest that CD40 ligation induces the activation of different intracellular signaling pathways, in particular the NFATs factors, that could represent a potential therapeutic target in the setting of patients with metastatic RCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , Fatores de Transcrição NFATC/metabolismo , Idoso , Apoptose , Biomarcadores Tumorais/genética , Antígenos CD40/genética , Ligante de CD40/genética , Movimento Celular , Proliferação de Células , Reagentes para Ligações Cruzadas , Feminino , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Fatores de Transcrição NFATC/genética , Metástase Neoplásica , Prognóstico , Células Tumorais Cultivadas
2.
Cell Rep ; 36(8): 109570, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34390647

RESUMO

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.


Assuntos
COVID-19/prevenção & controle , Linfócitos T/imunologia , Vacinas Sintéticas/administração & dosagem , Anticorpos Antivirais/sangue , Ligante de CD40/metabolismo , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Interferon gama/metabolismo , Interleucina-2/metabolismo , Peptídeos/imunologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Vacinação , Vacinas Sintéticas/imunologia
3.
Nat Commun ; 12(1): 3754, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145241

RESUMO

Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4+ T cells display impaired Th1 polarization, as reflected by reduced interferon-γ production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c+ dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-γ concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway.


Assuntos
Aterosclerose/patologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Interferon gama/metabolismo , Placa Aterosclerótica/patologia , Animais , Plaquetas/metabolismo , Linfócitos T CD4-Positivos/citologia , Doenças Cardiovasculares/patologia , Células Dendríticas/imunologia , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/citologia , Transdução de Sinais/fisiologia , Trombose/patologia
4.
Cell Rep ; 35(13): 109320, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34146478

RESUMO

Memory B cells seem to be more durable than antibodies and thus crucial for the long-term immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here we investigate SARS-CoV-2 spike-specific memory B cells and their dependence on CD4+ T cell help in different settings of coronavirus disease 2019 (COVID-19). Compared with severely ill individuals, those who recovered from mild COVID-19 develop fewer but functionally superior spike-specific memory B cells. Generation and affinity maturation of these cells is best associated with IL-21+CD4+ T cells in recovered individuals and CD40L+CD4+ T cells in severely ill individuals. The increased activation and exhaustion of memory B cells observed during COVID-19 correlates with CD4+ T cell functions. Intriguingly, CD4+ T cells recognizing membrane protein show a stronger association with spike-specific memory B cells than those recognizing spike or nucleocapsid proteins. Overall, we identify CD4+ T cell subsets associated with the generation of B cell memory during SARS-CoV-2 infection.


Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Antivirais/imunologia , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Reações Cruzadas , Humanos , Memória Imunológica , Interleucinas/imunologia , Interleucinas/metabolismo
5.
Methods Mol Biol ; 2285: 141-152, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928550

RESUMO

The detection and functional characterization of antigen-reactive T helper (Th) cells has been challenging due to their low frequency and functional heterogeneity. Antigen-reactive T cell enrichment (ARTE) allows the in-depth characterization of antigen-specific Th lymphocytes as a prerequisite for better understanding the role of adaptive immune responses in health and disease. ARTE is based on detection of the activation markers CD154 (CD40L) (expressed on all conventional Th cell subsets, Tcons) and CD137 (4-1BB) (expressed on regulatory T cells, Tregs), which are upregulated on the surface of CD4+ T cells upon short-term (7 h) in vitro stimulation with antigens in the presence of antigen-presenting cells (APCs). To substantially increase the sensitivity for the detection of antigen-specific Th cells, ARTE combines magnetic pre-enrichment of rare antigen-reactive T cells with multiparameter flow cytometry. Using CD154 and CD137 in combination allows the parallel detection of reactive Tcons and Tregs, after stimulation with the antigen. Thus, the ARTE technology now enables to characterize antigen-specific T cells with increased sensitivity of detection allowing even the investigation of antigen-specific Th cells in the naive T cell repertoire and regardless of prior knowledge of MHC alleles or antigenic epitopes.


Assuntos
Ligante de CD40/imunologia , Citometria de Fluxo , Separação Imunomagnética , Ativação Linfocitária , Linfócitos T Auxiliares-Indutores/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Ligante de CD40/metabolismo , Epitopos , Humanos , Fenótipo , Projetos de Pesquisa , Linfócitos T Auxiliares-Indutores/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Fluxo de Trabalho
6.
J Immunol ; 206(8): 1793-1805, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33762325

RESUMO

In addition to the membrane-bound form, CD154 also exists as a soluble molecule originating from an intracellular and membrane cleavage. We have previously shown that CD154 cleavage from T cell surface is mediated by CD40 and involves the action of ADAM10/ADAM17 enzymes. In the aim of defining the importance of CD154 maintained on cell surface, we generated a CD154 mutated at the cleavage site. Our data show that the double mutation of E112 and M113 residues of CD154 abolishes its spontaneous release and the CD40-mediated cleavage from cell surface but does not affect its binding to CD40. We also demonstrated that both the release of CD154 from the intracellular milieu and its CD40-mediated cleavage from cell surface are highly dependent on ADAM10/ADAM17 enzymes. The CD154-EM mutant was shown capable of inducing a more prominent apoptotic response in susceptible B cell lines than the wild-type (WT) form of the molecule. In addition, human B cells cultured in the presence of the CD154-EM mutant exhibited upregulated proliferative responses compared with the CD154-WT. The CD154-EM mutant was also shown to trigger differentiation of human B cells, reflected by an increased Ig production, more significantly than CD154-WT. Thus, our data strongly suggest that cleavage-resistant CD154 is a more prominent stimulant than the cleavable form of the molecule. Therefore, a maintained expression of CD154 on cell membrane and a disturbed cleavage of the molecule could be a mechanism by which CD154 is involved in some pathological conditions and should be revisited.


Assuntos
Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Membrana Celular/metabolismo , Espaço Intracelular/metabolismo , Linfócitos T/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Apoptose , Ligante de CD40/genética , Diferenciação Celular , Células HEK293 , Humanos , Imunoglobulinas/metabolismo , Mutagênese Sítio-Dirigida , Mutação/genética , Ligação Proteica , Proteólise , Transdução de Sinais
7.
J Biol Chem ; 296: 100399, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33571526

RESUMO

CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvß3 and α5ß1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4ß1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvß3, α5ß1, and α4ß1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants.


Assuntos
Ligante de CD40/metabolismo , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Receptores de Superfície Celular/química , Linfócitos T/metabolismo , Sítio Alostérico , Animais , Ligante de CD40/imunologia , Linhagem Celular , Cricetinae , Humanos , Integrina alfa4beta1/imunologia , Integrina alfa5beta1/imunologia , Integrina alfaVbeta3/imunologia , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Linfócitos T/imunologia
8.
Toxicol Appl Pharmacol ; 415: 115441, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33556388

RESUMO

The immunotoxicity of zearalenone (ZEA) and deoxynivalenol (DON), two of the most common environmental mycotoxins, has been well investigated. However, due to the complexity of the immune system, especially during bacterial infection, many types of immune cells are involved in invasion resistance and bacterial clearance. Of these, T helper 2 (Th2) cells, which are members of the helper T cell family, assist B cells to activate and differentiate into antibody-secreting cells, participate in humoral immune response, and, ultimately, eliminate pathogens. Thus, it is important to identify the stage at which these toxins affect the immune function, and to clarity the underlying mechanisms. In this study, mice infected with Listeria monocytogenes (Listeria) were used to study the effects of ZEA, DON, and ZEA + DON on Th2 differentiation, Interleukin-4 Receptor (IL-4R) expression, costimulatory molecules expression and cytokine secretion after Listeria infection. Naive CD4+ T cells, isolated from mice, were used to verify the in vivo effects and the associated mechanisms. In vivo experiments showed that these toxins aggravated spleen damage after Listeria infection and reduced the differentiation of Th2 cells by affecting the synthesis of IL-4R of CD4+ T cells. In addition, the level of the costimulatory molecule CD154 decreased. Consistent with this, in vitro studies showed that these toxins inhibited the differentiation of mouse naive CD4+ T cell into Th2 subtype and decreased IL-4R levels. In addition, the levels of costimulatory molecules CD154, CD278 and the Th2 cells secrete cytokines IL-4, IL-6, and IL-10 decreased. Based on our in vivo and in vitro experiments, we suggest that ZEA, DON, and ZEA + DON inhibit the expression of costimulatory molecules on CD4+ T cell, and inhibit the IL-4R-mediated Th2 cell differentiation. This may indicate that the body cannot normally resist or clear the pathogen after mycotoxin poisoning.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Listeria monocytogenes/patogenicidade , Listeriose/induzido quimicamente , Ativação Linfocitária/efeitos dos fármacos , Receptores de Interleucina-4/metabolismo , Baço/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Tricotecenos/toxicidade , Zearalenona/toxicidade , Animais , Ligante de CD40/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/metabolismo , Listeriose/microbiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais , Baço/imunologia , Baço/metabolismo , Baço/microbiologia , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/microbiologia
9.
Arch Pharm Res ; 44(1): 117-132, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33394309

RESUMO

Ulcerative colitis (UC) and Crohn's disease (CD) are chronic and multifactorial diseases that affect the intestinal tract, both characterized by recurrent inflammation of the intestinal mucosa, resulting in abdominal pain, diarrhea, vomiting and, rectal bleeding. Inflammatory bowel diseases (IBD) regroup these two disorders. The exact pathological mechanism of IBD remains ambiguous and poorly known. In genetically predisposed patients, defects in intestinal mucosal barrier are due to an uncontrolled inflammatory response to normal flora. In addition to the genetic predisposition, these defects could be triggered by environmental factors or by a specific lifestyle which is widely accepted as etiological hypothesis. The involvement of the CD40/CD40L platelet complex in the development of IBD has been overwhelmingly demonstrated. CD40L is climacteric in cell signalling in innate and adaptive immunity, the CD40L expression on the platelet cell surface gives them an immunological competence. The IL-1, a major inflammation mediator could be involved in different ways in the development of IBD. Here, we provide a comprehensive review regarding the role of platelet CD40/CD40L in the pathophysiological effect of IL-1 in the development of Crohn's disease (CD). This review could potentially help future approaches aiming to target these two pathways for therapeutic purposes and elucidate the immunological mechanisms driving gut inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Doença de Crohn/imunologia , Interleucina-1/metabolismo , Anti-Inflamatórios/uso terapêutico , Plaquetas/imunologia , Plaquetas/metabolismo , Antígenos CD40/antagonistas & inibidores , Ligante de CD40/antagonistas & inibidores , Doença de Crohn/tratamento farmacológico , Doença de Crohn/patologia , Humanos , Interleucina-1/antagonistas & inibidores , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Ativação Plaquetária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
10.
Int Immunopharmacol ; 91: 107337, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33401206

RESUMO

CD40 ligand (CD40L) and B-cell activating factor (BAFF) play important roles in the function of B cells. However, the difference of their regulatory effects remains obscure. In this study, we used anti-CD40 to imitate CD40L and investigated the different regulatory effects of CD40L and BAFF on the function of B cells. In the functional analyses, both anti-CD40 and BAFF significantly enhanced the survival and differentiation of B cells, and slightly increased the activation and proliferation. However, in the transcriptome analysis, anti-CD40 and BAFF exerted very different regulation on the gene expression profile of B cells. Anti-CD40 upregulated the expression of genes related to the adaptive immune function of B cells, but BAFF enhanced the genes associated with the innate immune function. Furthermore, the effect analysis of the combination of anti-CD40 or BAFF with anti-IgM also demonstrated that anti-CD40 could cooperate with anti-IgM to promote the proliferation of B cells, but BAFF could not do it. The mechanism study revealed that the different effects of anti-CD40 and BAFF on B cells were resulting from the different modulation on NF-кB, ERK1/2, and PI3K-AKT signaling pathways. Collectively, the results suggest that CD40L mainly promotes adaptive immune function of B cells, but BAFF primarily enhances innate immune function.


Assuntos
Anticorpos/farmacologia , Fator Ativador de Células B/farmacologia , Linfócitos B/efeitos dos fármacos , Antígenos CD40/antagonistas & inibidores , Ligante de CD40/metabolismo , Imunidade Adaptativa/efeitos dos fármacos , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Imunidade Inata/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Transdução de Sinais , Baço/citologia , Transcriptoma
11.
J Immunol ; 206(2): 345-354, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33298614

RESUMO

Flagellin is an immunodominant Ag in Crohn disease, with many patients showing anti-flagellin Abs. To study the clonality of flagellin-reactive CD4 cells in Crohn patients, we used a common CD154-based enrichment method following short-term Ag exposure to identify Ag-reactive CD4 cells. CD154 expression and cytokine production following Ag exposure compared with negative control responses (no Ag exposure) revealed that only a small fraction of CD154-enriched cells could be defined by Ag-reactive cytokine responses. This was especially true for low-frequency flagellin-reactive CD4 cells compared with polyclonal stimulation or Candida albicans Ag exposure. Moreover, we found that culture conditions used for the assay contributed to background CD40L (CD154) expression in the CD154-enriched CD4 cells. Using a cut-off rule based on flow cytometry results of the negative control CD154-enriched CD4 cells, we could reliably find the fraction of Ag-reactive cells in the CD154-enriched population. Ag-reactive CD4 cytokine production was restricted to CD4 cells with an effector memory phenotype and the highest levels of induced CD154 expression. This has important implications for identifying Ag-specific T cells of interest for single cell cloning, phenotyping, and transcriptomics.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença de Crohn/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Ligante de CD40/genética , Ligante de CD40/metabolismo , Células Cultivadas , Flagelina/imunologia , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Célula Única , Especificidade do Receptor de Antígeno de Linfócitos T , Adulto Jovem
12.
Cell Mol Life Sci ; 78(3): 1065-1083, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32506167

RESUMO

CD40-activated CD40L reverse signaling is a major physiological regulator of axon and dendrite growth from developing hippocampal pyramidal neurons. Here we have studied how CD40L-mediated reverse signaling promotes the growth of these processes. Cultures of hippocampal pyramidal neurons were established from Cd40-/- mouse embryos to eliminate endogenous CD40/CD40L signaling, and CD40L reverse signaling was stimulated by a CD40-Fc chimera. CD40L reverse signaling increased phosphorylation and hence activation of proteins in the PKC, ERK, and JNK signaling pathways. Pharmacological activators and inhibitors of these pathways revealed that whereas activation of JNK inhibited growth, activation of PKC and ERK1/ERK2 enhanced growth. Experiments using combinations of pharmacological reagents revealed that these signaling pathways regulate growth by functioning as an interconnected and interdependent network rather than acting in a simple linear sequence. Immunoprecipitation studies suggested that stimulation of CD40L reverse signaling generated a receptor complex comprising CD40L, PKCß, and the Syk tyrosine kinase. Our studies have begun to elucidate the molecular network and interactions that promote axon and dendrite growth from developing hippocampal neurons following activation of CD40L reverse signaling.


Assuntos
Axônios/metabolismo , Ligante de CD40/metabolismo , Dendritos/fisiologia , Transdução de Sinais , Animais , Butadienos/farmacologia , Antígenos CD40/deficiência , Antígenos CD40/genética , Células Cultivadas , Dendritos/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/metabolismo
13.
J Leukoc Biol ; 109(2): 309-325, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32441445

RESUMO

Many acute viral infections target tissue Mϕs, yet the mechanisms of Mϕ-mediated control of viruses are poorly understood. Here, we report that CD40 expressed by peritoneal Mϕs restricts early infection of a broad range of RNA viruses. Loss of CD40 expression enhanced virus replication as early as 12-24 h of infection and, conversely, stimulation of CD40 signaling with an agonistic Ab blocked infection. With peritoneal cell populations infected with the filovirus, wild-type (WT) Ebola virus (EBOV), or a BSL2 model virus, recombinant vesicular stomatitis virus encoding Ebola virus glycoprotein (rVSV/EBOV GP), we examined the mechanism conferring protection. Here, we demonstrate that restricted virus replication in Mϕs required CD154/CD40 interactions that stimulated IL-12 production through TRAF6-dependent signaling. In turn, IL-12 production resulted in IFN-γ production, which induced proinflammatory polarization of Mϕs, protecting the cells from infection. These CD40-dependent events protected mice against virus challenge. CD40-/- mice were exquisitely sensitive to intraperitoneal challenge with a dose of rVSV/EBOV GP that was sublethal to CD40+/+ mice, exhibiting viremia within 12 h of infection and rapidly succumbing to infection. This study identifies a previously unappreciated role for Mϕ-intrinsic CD40 signaling in controlling acute virus infection.


Assuntos
Antígenos CD40/metabolismo , Imunidade Inata , Macrófagos/imunologia , Macrófagos/virologia , Vírus de RNA/fisiologia , Transdução de Sinais , Viroses/imunologia , Replicação Viral/fisiologia , Doença Aguda , Animais , Ligante de CD40/metabolismo , Ebolavirus/fisiologia , Glicoproteínas/imunologia , Humanos , Interferon gama/metabolismo , Interleucina-12/biossíntese , Camundongos Endogâmicos C57BL , Modelos Biológicos , Peritônio/patologia , Peritônio/virologia , Fator 6 Associado a Receptor de TNF/metabolismo , Viroses/virologia
14.
Cancer Immunol Immunother ; 70(5): 1451-1464, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33180184

RESUMO

INTRODUCTION: Gastrointestinal (GI) malignancies, such as cholangiocarcinoma, pancreatic carcinoma, and metastatic colorectal carcinoma, have a poor prognosis and effective therapeutic approaches are still challenging. Checkpoint inhibition with PD-1 or PDL-1 antibodies revealed promising results in different tumor entities; however, only few patients with GI tumors can potentially benefit from PD1/PDL1 inhibiting immunotherapy. Further immunotherapeutic strategies for GI malignancies are urgently needed. The aim of this study was to demonstrate that in vitro activation of the immune checkpoint CD40/CD40L can improve DC action towards bile duct, pancreas, and colorectal carcinoma. METHODS: Human DC were isolated from buffy coats from healthy donors, pulsed with tumor lysates and then transduced with adenoviruses encoding human CD40L (Ad-hCD40L). Using transwell assays, the effects of (m)CD40L on DC immunoactivation compared to (s)CD40L were analyzed. Surface marker and cytokine/chemokine expression were measured by flow cytometry, ELISA and cytokine arrays. Capacity of Ad-hCD40L-transduced DC to induce tumor-specific effector cells was tested using MTT proliferation assay and cytotoxicity assays. Apoptosis induction on tumor cells after culturing with supernatants of Ad-hCD40L-transduced DC was analyzed by flow cytometry. RESULTS: Ad-hCD40L transduction induced a high expression of (s)CD40L and (m)CD40L on DC and seemed to induce a strong cellular CD40/CD40L interaction among DC, leading to the formation of cell aggregates. Due to the CD40/CD40L interaction, a significant upregulation of DC maturation markers and a Th1-shift on cytokines/chemokines in the supernatant of DC were achieved. Interestingly, a pure Th1-shift was only achieved, when a cellular CD40/CD40L interaction among DC took place. (s)CD40L induced almost no upregulation of maturation markers and rather resulted in a Th2-cytokine expression, such as IL-10. Correspondingly, (m)CD40L-expressing DC led to significant proliferation and stimulation of tumor-specific effector cells with increased cytotoxicity towards pancreatic, bile duct and colorectal tumor cells. Supernatants of Ad-hCD40L-transduced DC could also induce apoptosis in the different tumor cells in vitro. CONCLUSION: Stimulation of the immune checkpoint CD40L/CD40 by endogenous expression of (m)CD40L provokes a cellular interaction, which increases the immunomodulatory capacity of DC. A Th1 cytokine/chemokine expression is induced, leading to a significant proliferation and enabling cytotoxicity of effector cells towards human bile duct, pancreatic and colorectal tumor cells. The present data point to the promising approach for DC-based immunotherapy of gastrointestinal malignances by activating the CD40/CD40L immune checkpoint.


Assuntos
Colangiocarcinoma/imunologia , Neoplasias Colorretais/imunologia , Células Dendríticas/imunologia , Imunoterapia/métodos , Neoplasias Pancreáticas/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos CD40/genética , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Citotoxicidade Imunológica , Humanos , Ativação Linfocitária , Transdução de Sinais , Células Th1/imunologia , Equilíbrio Th1-Th2 , Células Th2/imunologia
15.
J Immunol ; 206(3): 481-493, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33380497

RESUMO

B cells in the germinal center (GC) are programmed to form plasma cells (PCs) or memory B cells according to signals received by receptors that are translated to carry out appropriate activities of transcription factors. However, the precise mechanism underlying this process to complete the GC reaction is unclear. In this study, we show that both genetic ablation and pharmacological inhibition of glycogen synthase kinase 3 (GSK3) in GC B cells of mice facilitate the cell fate decision toward PC formation, accompanied by acquisition of dark zone B cell properties. Mechanistically, under stimulation with CD40L and IL-21, GSK3 inactivation synergistically induced the transcription factors Foxo1 and c-Myc, leading to increased levels of key transcription factors required for PC differentiation, including IRF4. This GSK3-mediated alteration of transcriptional factors in turn facilitated the dark zone transition and consequent PC fate commitment. Our study thus reveals the upstream master regulator responsible for interpreting external cues in GC B cells to form PCs mediated by key transcription factors.


Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Quinase 3 da Glicogênio Sintase/metabolismo , Plasmócitos/imunologia , Animais , Ligante de CD40/metabolismo , Diferenciação Celular , Células Cultivadas , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interleucinas/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo
16.
Nat Commun ; 11(1): 6357, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311473

RESUMO

The prevailing 'division of labor' concept in cellular immunity is that CD8+ T cells primarily utilize cytotoxic functions to kill target cells, while CD4+ T cells exert helper/inducer functions. Multiple subsets of CD4+ memory T cells have been characterized by distinct chemokine receptor expression. Here, we demonstrate that analogous CD8+ memory T-cell subsets exist, characterized by identical chemokine receptor expression signatures and controlled by similar generic programs. Among them, Tc2, Tc17 and Tc22 cells, in contrast to Tc1 and Tc17 + 1 cells, express IL-6R but not SLAMF7, completely lack cytotoxicity and instead display helper functions including CD40L expression. CD8+ helper T cells exhibit a unique TCR repertoire, express genes related to skin resident memory T cells (TRM) and are altered in the inflammatory skin disease psoriasis. Our findings reveal that the conventional view of CD4+ and CD8+ T cell capabilities and functions in human health and disease needs to be revised.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Receptores de Interleucina-6/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/metabolismo , Diferenciação Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica , Expressão Gênica , Humanos , Imunidade Celular , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-6/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Pele/imunologia , Subpopulações de Linfócitos T/imunologia
17.
Int J Mol Sci ; 21(22)2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-33198327

RESUMO

The CD40-CD40 ligand (CD40L) dyad represents a scientific and clinical field that has raised many controversies in the past and cannot be clearly defined as being an either beneficial or harmful pathway. Being crucially involved in physiological immunological processes as well as pathological inflammatory reactions, the signaling pathway has been recognized as a key player in the development of both autoimmune and cardiovascular disease. Even though the possibilities of a therapeutic approach to the dyad were recognized decades ago, due to unfortunate events, detailed in this review, pharmacological treatment targeting the dyad, especially in patients suffering from atherosclerosis, is not available. Despite the recent advances in the treatment of classical cardiovascular risk factors, such as arterial hypertension and diabetes mellitus, the treatment of the associated low-grade inflammation that accounts for the progression of atherosclerosis is still challenging. Low-grade inflammation can be detected in a significant portion of patients that suffer from cardiovascular disease and it is therefore imperative to develop new therapeutic strategies in order to combat this driver of atherosclerosis. Of note, established cardiovascular drugs such as angiotensin-converting enzyme inhibitors or statins have proven beneficial cardiovascular effects that are also related to their pleiotropic immunomodulatory properties. In this review, we will discuss the setbacks encountered as well as new avenues discovered on the path to a different, inflammation-centered approach for the treatment of cardiovascular disease with the CD40-CD40L axis as a central therapeutic target.


Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Doenças Cardiovasculares/metabolismo , Transdução de Sinais , Animais , Aterosclerose/metabolismo , Doenças Autoimunes/metabolismo , Sistema Cardiovascular , Ensaios Clínicos como Assunto , Inativação Gênica , Humanos , Inflamação , Camundongos , Camundongos Transgênicos , Fatores de Risco
18.
Molecules ; 25(19)2020 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-33050076

RESUMO

The objective of this study was to assess the inhibitory effect of the flavonoid aromadendrin on T cell activity to identify a non-cytotoxic immunosuppressive reagent. Conventional and qualitative PCR, MTT assays, flow cytometry and Western blotting were used to evaluate the effect of aromadendrin on the activity, cell viability and confluency, and proximal signal transduction of activated T cells. Aromadendrin effectively regulated IL-2 and IFNγ production in vitro from activated Jurkat T cells without cytotoxicity. Pre-treatment with aromadendrin also suppressed the expression levels of surface molecules CD69, CD25, and CD40L. Reduced calcium (Ca2+) influx in activated T cells pre-treated with aromadendrin was observed. Western blotting revealed that aromadendrin blocked the dephosphorylation of nuclear factor of activated T (NFAT) cells and its nuclear translocation. Involvement of the NFκB and MAPK pathways in the inhibitory effect of aromadendrin was also demonstrated. Results obtained demonstrated the suppressive effect of aromadendrin on T cell activation by Ca2+ influx regulation through NFAT activity suppression of the activated T cells.


Assuntos
Cálcio/metabolismo , Flavonoides/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Linfócitos T/efeitos dos fármacos , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Ligante de CD40/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Humanos , Imunossupressores/farmacologia , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Células Jurkat , Lectinas Tipo C/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/metabolismo
19.
PLoS One ; 15(9): e0238163, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881885

RESUMO

OBJECTIVE: We evaluated the effects of grape juice (Vitis labrusca L.) on dyslipidemia, resistance to insulin, and left ventricular hypertrophy (LVH) in mice homozygous for the absence of the LDL receptor gene (LDLr -/-) under a hyperlipidemic diet. METHODOLOGY: We divided 30 male mice (3 months old) into three groups (n = 10); the HL group was fed a high-fat diet, the HLU group received a high-fat diet and 2 g/kg/day of grape juice, and the HLS group was fed a high-fat diet and simvastatin (20 mg/kg/day). We assessed the blood pressure profile of the mice. We also determined the levels of C-reactive protein (CRP) and lipid profile, glycemic and insulinemic profiles, and calculated the HOMA-IR. Cardiomyocyte hypertrophy, interstitial collagen deposit, and the expression of CD40 ligand (CD40L) and metalloproteinases 2 and 9 were assessed immunohistologically. RESULTS: After 60 days, the mice treated with grape juice showed similar results as those of the group treated with simvastatin. The use of grape fruit attenuated dyslipidemia and insulin resistance and significantly increased the levels of high cholesterol density lipoproteins (HDLc). The antioxidant potential of phenolic compounds associated with the increase in HDLc levels in the mice of the HLU group prevented the development of LVH and arterial hypertension since it inhibited the inflammatory response induced by the CD40 pathway and its ligand CD40L. Consequently, there was a lower expression of MMP-2 and MMP-9 and lower serum levels of CRP. CONCLUSION: Grape juice has a hypolipidemic and cardiac protective potential, presenting a similar effect as that of simvastatin through a direct antioxidant action of phenolic compounds, or indirectly, via antioxidant action and anti-inflammatory activity of the HDLc. These results suggest that grape juice is a functional food possessing a high potential to prevent cardiovascular diseases.


Assuntos
Dislipidemias/patologia , Sucos de Frutas e Vegetais , Hipertrofia Ventricular Esquerda/prevenção & controle , Vitis/química , Animais , Pressão Sanguínea/efeitos dos fármacos , Proteína C-Reativa/análise , Ligante de CD40/genética , Ligante de CD40/metabolismo , Colágeno/metabolismo , Dieta Hiperlipídica , Dislipidemias/tratamento farmacológico , Sucos de Frutas e Vegetais/análise , Ventrículos do Coração/patologia , Hipertrofia Ventricular Esquerda/patologia , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Substâncias Protetoras/química , Substâncias Protetoras/uso terapêutico , Receptores de LDL/deficiência , Receptores de LDL/genética , Sinvastatina/uso terapêutico , Vitis/metabolismo
20.
Cell Rep ; 32(12): 108180, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32966789

RESUMO

Human dendritic cells (DCs) comprise subsets with distinct phenotypic and functional characteristics, but the transcriptional programs that dictate their identity remain elusive. Here, we analyze global chromatin accessibility profiles across resting and stimulated human DC subsets by means of the assay for transposase-accessible chromatin using sequencing (ATAC-seq). We uncover specific regions of chromatin accessibility for each subset and transcriptional regulators of DC function. By comparing plasmacytoid DC responses to IFN-I-producing and non-IFN-I-producing conditions, we identify genetic programs related to their function. Finally, by intersecting chromatin accessibility with genome-wide association studies, we recognize DC subset-specific enrichment of heritability in autoimmune diseases. Our results unravel the basis of human DC subset heterogeneity and provide a framework for their analysis in disease pathogenesis.


Assuntos
Cromatina/metabolismo , Células Dendríticas/metabolismo , Adulto , Ligante de CD40/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Regulação da Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Proteínas Repressoras/metabolismo , Fatores de Risco , Escleroderma Sistêmico/genética , Transcrição Genética , Adulto Jovem
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