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1.
Immunohorizons ; 5(2): 70-80, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542028

RESUMO

Tyrosine kinase 2 (TYK2) is a member of the JAK family of nonreceptor tyrosine kinase, together with JAK1, JAK2, and JAK3. JAKs are important signaling mediators of many proinflammatory cytokines and represent compelling pharmacological targets for autoimmune and inflammatory diseases. Pan-acting small-molecule JAK inhibitors were approved for the treatment of rheumatoid arthritis and ulcerative colitis. However, their limited selectivity among JAK members have led to undesirable side effects, driving a search toward specific JAK inhibitors. Recently, TYK2 has emerged as a target of choice for the treatment of autoimmune diseases and severe COVID-19 with an optimum balance between efficacy and safety, based on observations from human genetics studies and clinical outcomes of several agents targeting cytokine pathways for which TYK2 plays an essential role. In this article, we address selective targeting of TYK2 from the genetic sequence space through development of antisense oligonucleotides (ASOs) against TYK2 mRNA. Potent ASO candidates were identified from the screening of over 200 ASOs using locked nucleic acid gapmer design. The lead ASOs exhibited potent and selective knockdown of TYK2 mRNA and protein across a panel of model human cell lines in a dose-dependent manner, showing no reduction in the mRNA and protein expression levels of other JAK paralogs. In agreement with the depletion of TYK2 proteins, several TYK2-mediated cytokine signaling pathways, including IFN-α and IL-12, were inhibited upon ASO treatment. Our results established the TYK2 ASOs as investigational tool compound and potential therapeutic agent for the treatment of autoimmune diseases and severe COVID-19.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Inibidores de Janus Quinases/uso terapêutico , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/genética , TYK2 Quinase/genética , Progressão da Doença , Técnicas de Silenciamento de Genes , Humanos , Interferon-alfa/metabolismo , Interleucina-12/metabolismo , Células Jurkat , Terapia de Alvo Molecular , Oligonucleotídeos Antissenso/uso terapêutico , Transdução de Sinais
2.
Nat Commun ; 12(1): 301, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436596

RESUMO

Macrophages are innate immune cells that contribute to fighting infections, tissue repair, and maintaining tissue homeostasis. To enable such functional diversity, macrophages resolve potentially conflicting cues in the microenvironment via mechanisms that are unclear. Here, we use single-cell RNA sequencing to explore how individual macrophages respond when co-stimulated with inflammatory stimuli LPS and IFN-γ and the resolving cytokine IL-4. These co-stimulated macrophages display a distinct global transcriptional program. However, variable negative cross-regulation between some LPS + IFN-γ-specific and IL-4-specific genes results in cell-to-cell heterogeneity in transcription. Interestingly, negative cross-regulation leads to mutually exclusive expression of the T-cell-polarizing cytokine genes Il6 and Il12b versus the IL-4-associated factors Arg1 and Chil3 in single co-stimulated macrophages, and single-cell secretion measurements show that these specialized functions are maintained for at least 48 h. This study suggests that increasing functional diversity in the population is one strategy macrophages use to respond to conflicting environmental cues.


Assuntos
Polaridade Celular , Macrófagos/citologia , Animais , Arginase/metabolismo , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Aprendizado de Máquina , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Redes Neurais de Computação , Razão de Chances , Análise de Célula Única , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos dos fármacos
3.
BMC Infect Dis ; 20(1): 828, 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33176707

RESUMO

BACKGROUND: Severe and disseminated non-tuberculous mycobacterial (NTM) infections are frequently linked to a genetic predisposition but acquired defects of the interferon gamma (IFNγ) / interleukin 12 (IL-12) pathway need to be considered in adult patients with persistent or recurrent infections. Neutralizing anti-IFNγ autoantibodies disrupting IFNγ signalling have been identified as the cause of a severe and unique acquired immunodeficiency syndrome with increased susceptibility to NTM and other intracellular pathogens. CASE PRESENTATION: An adult Asian female with a previous history of recurrent NTM infections presented with persistent diarrhea, abdominal pain, night sweats and weight loss. Severe colitis due to a simultaneous infection with cytomegalovirus (CMV) and Salmonella typhimurium was diagnosed, with both pathogens also detectable in blood samples. Imaging studies further revealed thoracic as well as abdominal lymphadenopathy and a disseminated Mycobacterium intracellulare infection was diagnosed after a lymph node biopsy. Further diagnostics revealed the presence of high-titer neutralizing anti-IFNγ autoantibodies, allowing for the diagnosis of adult-onset immunodeficiency with anti-IFNγ autoantibodies (AIIA). CONCLUSIONS: We here present a severe case of acquired immunodeficiency with anti-IFNγ autoantibodies with simultaneous, disseminated infections with both viral and microbial pathogens. The case illustrates how the diagnosis can cause considerable difficulties and is often delayed due to unusual presentations. Histological studies in our patient give further insight into the pathophysiological significance of impaired IFNγ signalling. B-cell-depleting therapy with rituximab offers a targeted treatment approach in AIIA.


Assuntos
Autoanticorpos/imunologia , Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/isolamento & purificação , Síndromes de Imunodeficiência/diagnóstico , Interferon gama/imunologia , Linfadenopatia/diagnóstico , Complexo Mycobacterium avium/isolamento & purificação , Infecção por Mycobacterium avium-intracellulare/diagnóstico , Infecções por Salmonella/diagnóstico , Salmonella typhimurium/isolamento & purificação , Adulto , Antibacterianos/uso terapêutico , Antivirais/uso terapêutico , Autoanticorpos/sangue , Biópsia , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/virologia , Diagnóstico Tardio , Feminino , Seguimentos , Humanos , Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Interferon gama/metabolismo , Interleucina-12/metabolismo , Linfadenopatia/complicações , Linfadenopatia/tratamento farmacológico , Linfadenopatia/patologia , Infecção por Mycobacterium avium-intracellulare/complicações , Infecção por Mycobacterium avium-intracellulare/tratamento farmacológico , Infecção por Mycobacterium avium-intracellulare/microbiologia , Infecções por Salmonella/complicações , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/microbiologia , Resultado do Tratamento
4.
Proc Natl Acad Sci U S A ; 117(35): 21557-21567, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817415

RESUMO

Multiple sclerosis (MS) is the most common human demyelinating disease of the central nervous system. The IL-12 family of cytokines has four members, which are IL-12 (p40:p35), IL-23 (p40:p19), the p40 monomer (p40), and the p40 homodimer (p402). Since all four members contain p40 in different forms, it is important to use a specific monoclonal antibody (mAb) to characterize these molecules. Here, by using such mAbs, we describe selective loss of p40 in serum of MS patients as compared to healthy controls. Similarly, we also observed decrease in p40 and increase in IL-12, IL-23, and p402 in serum of mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS, as compared to control mice. Interestingly, weekly supplementation of mouse and human recombinant p40 ameliorated clinical symptoms and disease progression of EAE. On the other hand, IL-12, IL-23, and p402 did not exhibit such inhibitory effect. In addition to EAE, p40 also suppressed collagen-induced arthritis in mice. Using IL-12Rß1-/-, IL-12Rß2-/-, and IL-12Rß1+/-/IL-12Rß2-/- mice, we observed that p40 required IL-12Rß1, but not IL-12Rß2, to suppress EAE. Interestingly, p40 arrested IL-12-, IL-23-, or p402-mediated internalization of IL-12Rß1, but neither IL-12Rß2 nor IL-23R, protected regulatory T cells, and suppressed Th1 and Th17 biasness. These studies identify p40 as an anti-autoimmune cytokine with a biological role different from IL-12, IL-23, and p402 in which it attenuates autoimmune signaling via suppression of IL-12Rß1 internalization, which may be beneficial in patients with MS and other autoimmune disorders.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Subunidade p40 da Interleucina-12/imunologia , Subunidade p40 da Interleucina-12/farmacologia , Receptores de Interleucina-12/antagonistas & inibidores , Adulto , Animais , Células Cultivadas , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Feminino , Humanos , Interleucina-12/imunologia , Interleucina-12/metabolismo , Interleucina-23/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/sangue , Esclerose Múltipla/imunologia , Ligação Proteica , Receptores de Interleucina-12/imunologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Células Th17/efeitos dos fármacos , Células Th17/imunologia
5.
Mol Immunol ; 126: 120-128, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32823236

RESUMO

The interleukin 12 (IL-12) family of cytokines regulates T cell functions and is key for the orchestration of immune responses. Each heterodimeric IL-12 family member is a glycoprotein. However, the impact of glycosylation on biogenesis and function of the different family members has remained incompletely defined. Here, we identify glycosylation sites within human IL-12 family subunits that become modified upon secretion. Building on these insights, we show that glycosylation is dispensable for secretion of human IL-12 family cytokines except for IL-35. Furthermore, our data show that glycosylation differentially influences IL-12 family cytokine functionality, with IL-27 being most strongly affected. Taken together, our study provides a comprehensive analysis of how glycosylation affects biogenesis and function of a key human cytokine family and provides the basis for selectively modulating their secretion via targeting glycosylation.


Assuntos
Interleucina-12/metabolismo , Interleucinas/metabolismo , Glicosilação , Células HEK293 , Humanos , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-23/genética , Interleucina-23/imunologia , Interleucina-23/metabolismo , Interleucinas/genética , Interleucinas/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
6.
Nat Commun ; 11(1): 3366, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632165

RESUMO

CD4+ T lymphocytes consist of naïve, antigen-specific memory, and memory-phenotype (MP) cell compartments at homeostasis. We recently showed that MP cells exert innate-like effector function during host defense, but whether MP CD4+ T cells are functionally heterogeneous and, if so, what signals specify the differentiation of MP cell subpopulations under homeostatic conditions is still unclear. Here we characterize MP lymphocytes as consisting of T-bethigh, T-betlow, and T-bet- subsets, with innate, Th1-like effector activity exclusively associated with T-bethigh cells. We further show that the latter population depends on IL-12 produced by CD8α+ type 1 dendritic cells (DC1) for its differentiation. Finally, our data demonstrate that this tonic IL-12 production requires TLR-MyD88 signaling independent of foreign agonists, and is further enhanced by CD40-CD40L interactions between DC1 and CD4+ T lymphocytes. We propose that optimal differentiation of T-bethigh MP lymphocytes at homeostasis is driven by self-recognition signals at both the DC and Tcell levels.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Homeostase/imunologia , Memória Imunológica/imunologia , Proteínas com Domínio T/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Comunicação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-12/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Transdução de Sinais/imunologia , Proteínas com Domínio T/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo
7.
Exp Parasitol ; 217: 107934, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32698075

RESUMO

The inadequacy of available treatments for leishmaniasis has presented up to 40% therapeutic failure. This fact suggests an urgency in the discovery of new drugs or alternative approaches for treating this disease. The objective of this study was to evaluate the antileishmanial activity of combined therapy between crotamine (CTA) from Crotalus durissus terrificus and the pentavalent antimonial Glucantime® (GLU). The assays were in vitro performed measuring the inhibition of Leishmania amazonensis amastigotes, followed by the evaluation of cellular production of cytokines and nitrites. After that, analytical methods were performed in order to characterize the molecules involved in the study by Mass Spectrometry, molecular affinity through an in silico assay and Surface Plasmon Resonance. In vivo experiments with BALB/c mice were performed by analyzing parasitemia, lesion size and immunological mediators. In the in vitro experiments, the pharmacological association improved the inhibition of the amastigotes, modulated the production of cytokines and nitric oxide. The therapy improved the effectiveness of the GLU, demonstrating a decreased parasitemia in the infected tissues. Altogether, the results suggest that the combined approach with CTA and GLU may be a promising alternative for the treatment of cutaneous leishmaniasis.


Assuntos
Antiprotozoários/uso terapêutico , Venenos de Crotalídeos/uso terapêutico , Crotalus , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Antimoniato de Meglumina/uso terapêutico , Animais , Antiprotozoários/farmacologia , Venenos de Crotalídeos/farmacologia , Combinação de Medicamentos , Interleucina-12/sangue , Interleucina-12/metabolismo , Leishmania mexicana/isolamento & purificação , Linfonodos/parasitologia , Macrófagos Peritoneais , Espectrometria de Massas , Antimoniato de Meglumina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Óxido Nítrico/metabolismo , Nitritos/análise , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismo
8.
Sci Rep ; 10(1): 12232, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699419

RESUMO

Ferritin is an iron-binding molecule, which comprises 24 subunits, heavy (FeH) and light (FeL) subunits, suggested to have a pathogenic role by the 'hyperferritinemic syndrome'. In this work, we tested (1) FeH and FeL in bone marrow (BM) and sera in patients with macrophage activation syndrome (MAS); (2) pro-inflammatory effects of ferritin, FeL, and FeH on macrophages; (3) ability of FeH-stimulated macrophages to stimulate the proliferation of peripheral blood mononuclear cells (PBMCs); (4) production of mature IL-1ß and IL-12p70 in extracellular compartments of FeH-stimulated macrophages. Immunofluorescence analysis and liquid chromatography mass spectrometry (LC-MS/MS) based proteomics were performed to identify FeL and FeH in BM and sera, respectively, in the same patients. Macrophages were stimulated with ferritin, FeH, and FeL to assess pro-inflammatory effects by RT-PCR and western blot. The proliferation of co-cultured PBMCs with FeH-stimulated macrophages was tested. Immunofluorescence showed an increased FeH expression in BMs, whereas LC-MS/MS identified that FeL was mainly represented in sera. FeH induced a significant increase of gene expressions of IL-1ß, IL-6, IL-12, and TNF-α, more marked with FeH, which also stimulated NLRP3. FeH-stimulated macrophages enhanced the proliferation of PBMCs. The ELISA assays showed that mature form of IL-1ß and IL-12p70 were increased, in extracellular compartments of FeH-stimulated macrophages. Our results showed FeH in BM biopsies of MAS patients, whereas, LC-MS/MS identified FeL in the sera. FeH showed pro-inflammatory effects on macrophages, stimulated NLRP3, and increased PBMCs proliferation.


Assuntos
Apoferritinas/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Células Cultivadas , Expressão Gênica/fisiologia , Humanos , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
9.
N Engl J Med ; 382(24): 2337-2343, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32521134

RESUMO

We describe a case of life-threatening disseminated coccidioidomycosis in a previously healthy child. Like most patients with disseminated coccidioidomycosis, this child had no genomic evidence of any known, rare immune disease. However, comprehensive immunologic testing showed exaggerated production of interleukin-4 and reduced production of interferon-γ. Supplementation of antifungal agents with interferon-γ treatment slowed disease progression, and the addition of interleukin-4 and interleukin-13 blockade with dupilumab resulted in rapid resolution of the patient's clinical symptoms. This report shows that blocking of type 2 immune responses can treat infection. This immunomodulatory approach could be used to enhance immune clearance of refractory fungal, mycobacterial, and viral infections. (Supported by the Jeffrey Modell Foundation and the National Institutes of Health.).


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antifúngicos/uso terapêutico , Coccidioidomicose/tratamento farmacológico , Interferon gama/uso terapêutico , Encéfalo/diagnóstico por imagem , Pré-Escolar , Coccidioidomicose/imunologia , Progressão da Doença , Quimioterapia Combinada , Humanos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Interleucina-13/antagonistas & inibidores , Interleucina-4/antagonistas & inibidores , Interleucina-4/metabolismo , Imagem por Ressonância Magnética , Masculino , Isoformas de Proteínas , Receptores de Interleucina-12/química , Receptores de Interleucina-12/genética , Coluna Vertebral/diagnóstico por imagem , Células Th1/imunologia
10.
Nat Commun ; 11(1): 2295, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385253

RESUMO

Cytomegalovirus-based vaccine vectors offer interesting opportunities for T cell-based vaccination purposes as CMV infection induces large numbers of functional effector-like cells that accumulate in peripheral tissues, a process termed memory inflation. Maintenance of high numbers of peripheral CD8 T cells requires continuous replenishment of the inflationary T cell pool. Here, we show that the inflationary T cell population contains a small subset of cells expressing the transcription factor Tcf1. These Tcf1+ cells resemble central memory T cells and are proliferation competent. Upon sensing viral reactivation events, Tcf1+ cells feed into the pool of peripheral Tcf1- cells and depletion of Tcf1+ cells hampers memory inflation. TCR repertoires of Tcf1+ and Tcf1- populations largely overlap, with the Tcf1+ population showing higher clonal diversity. These data show that Tcf1+ cells are necessary for sustaining the inflationary T cell response, and upholding this subset is likely critical for the success of CMV-based vaccination approaches.


Assuntos
Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Muromegalovirus/fisiologia , Fator 1 de Transcrição de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/virologia , Animais , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Células Clonais , Memória Imunológica , Interferon Tipo I/metabolismo , Interleucina-12/metabolismo , Camundongos Endogâmicos C57BL , Fenótipo
11.
Biochem Pharmacol ; 175: 113928, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32217101

RESUMO

The cytokines interleukin-12 (IL-12) and IL-23 share a common IL-12/IL-23p40 subunit in structure and play a central role in T cell-mediated responses in inflammation. Over-activated IL-12 and IL-23 signaling drives aberrant T helper (Th) 1 and Th17 immune responses and contributes to immune-mediated diseases. Evidence from genome-wide association studies has shown that genetic alterations in the IL-12/IL-23 signaling pathways have significant links with chronic inflammation. In addition, accumulating evidence from animal models and clinical trials has provided insights into the effectiveness of blocking the IL-12/IL-23 pathways in immune regulation, broadening the clinical indications of IL-12/IL-23 pathway effectors in immune-mediated diseases. More recently, it has been addressed that the balance between IL and 12 and IL-23 is also critical in carcinogenesis. IL-12- and IL-23-driven T cell cytokines are especially important in controlling tumor initiation, growth, and metastasis, and thus, the IL-12/IL-23 pathway may be a promising target for immunotherapy. This review focuses on IL-12/IL-23 signal transduction and biological functionality in autoimmunity and oncoimmunology. We discuss the therapeutic rationale for targeting these cytokines to treat immune-mediated diseases and issues regarding their inadvertent consequences in the balance of host defense and tumor surveillance and summarize their recent clinical applications in immune-mediated diseases.


Assuntos
Doenças Autoimunes/imunologia , Interleucina-12/antagonistas & inibidores , Interleucina-12/imunologia , Interleucina-23/antagonistas & inibidores , Interleucina-23/imunologia , Neoplasias/imunologia , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/imunologia , Fatores Imunológicos/metabolismo , Imunoterapia/métodos , Imunoterapia/tendências , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
12.
Tokai J Exp Clin Med ; 45(1): 10-17, 2020 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-32219804

RESUMO

OBJECTIVE: To distinguish between first wheeze and asthma in early childhood, we investigated respiratory viruses and cytokine/chemokine profiles among patients with first wheeze and established asthma. METHODS: We enrolled children with acute exacerbations of wheezing (17 first wheeze and 32 asthma) and 11 controls (no wheezing) aged between 10 months and 6 years. Nasal aspirates were obtained, and virus detection was performed with antigenic assay kits and/or RT-PCR. Serum 27 cytokines/chemokines were assayed by a multi-cytokine detection system. RESULTS: Rhinovirus and respiratory syncytial (RS) virus were dominant in acute exacerbations of asthma. However, many types of viruses were isolated in first wheeze. Serum IL-8 and IL-12 values were significantly higher in first wheeze than in acute asthma or the controls. IL-5 and IP-10 levels in acute asthma and first wheeze cases were higher than in the controls. Both of them were significantly higher in cases of acute asthma than in convalescence stage of asthma cases. Only IP-10 was significantly higher in first wheeze than in convalescence stage of first wheeze cases. CONCLUSIONS: Different profiles in virus detection and production of IL-8 and IL-12 might distinguish between first wheeze and childhood asthma.


Assuntos
Asma/metabolismo , Asma/virologia , Interleucina-12/metabolismo , Interleucina-8/metabolismo , Sons Respiratórios , Vírus Sinciciais Respiratórios/isolamento & purificação , Rhinovirus/isolamento & purificação , Criança , Pré-Escolar , Progressão da Doença , Eosinófilos , Feminino , Humanos , Lactente , Contagem de Leucócitos , Masculino , Neutrófilos
13.
J Immunol ; 204(7): 1869-1880, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32132181

RESUMO

Leishmaniases are neglected tropical diseases. The treatment of leishmaniasis relies exclusively on chemotherapy including amphotericin B (AmB), miltefosine (hexadecylphosphocholine), and pentamidine. Besides the fact that these molecules are harmful for patients, little is known about the impact of such antileishmanial drugs on primary human cells in relation to immune function. The present study demonstrates that all antileishmanial drugs inhibit CD4 and CD8 T cell proliferation at the doses that are not related to increased cell death. Our results highlight that antileishmanial drugs have an impact on monocytes by altering the expression of IL-12 induced by LPS, whereas only AmB induced IL-10 secretion; both cytokines are essential in regulating Th1 cell-mediated immunity. Interestingly, IL-12 and anti-IL-10 Abs improved T cell proliferation inhibited by AmB. Furthermore, our results show that in contrast to hexadecylphosphocholine and pentamidine, AmB induced gene expression of the inflammasome pathway. Thus, AmB induced IL-1ß and IL-18 secretions, which are reduced by specific inhibitors of caspase activation (Q-VD) and NLRP3 activation (MCC950). Our results reveal previously underestimated effects of antileishmanial drugs on primary human cells.


Assuntos
Antiparasitários/farmacologia , Inflamassomos/efeitos dos fármacos , Interleucina-12/metabolismo , Leishmania/genética , Leishmaniose/tratamento farmacológico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-10/metabolismo , Leishmania/metabolismo , Leishmaniose/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos
14.
J Leukoc Biol ; 107(4): 663-671, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32017227

RESUMO

This study tests the hypothesis that activation of MAPK by physiologically relevant concentrations of IL-33 contributes to enhanced cytokine expression by IL-12 stimulated human NK cells. While IL-33 canonically triggers type 2 cytokine responses, this cytokine can also synergize with type 1 cytokines like IL-12 to provoke IFN-γ. We show that picogram concentrations of IL-12 and IL-33 are sufficient to promote robust secretion of IFN-γ by human NK cells that greatly exceeds resposes to either cytokine alone. Nanogram doses of IL-33, potentially consistent with levels in tissue microenvironments, synergize with IL-12 to induce secretion of additional cytokines, including TNF and GM-CSF. IL-33-induced activation of the p38 MAPK pathway in human NK cells is crucial for enhanced release of IFN-γ and TNF in response to IL-12. Mechanistically, IL-33-induced p38 MAPK signaling enhances stability of IFNG transcripts and triggers A disintegrin and metalloproteinase domain 17 (ADAM17) mediated cleavage of TNF from the cell surface. These data support our hypothesis and suggest that altered sensitivity of NK cells to IL-12 in the presence of IL-33 may have important consequences in diseases associated with mixed cytokine milieus, like asthma and chronic obstructive pulmonary disease.


Assuntos
Citocinas/metabolismo , Interleucina-33/metabolismo , Células Matadoras Naturais/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteína ADAM17/metabolismo , Linhagem Celular , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-12/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT4/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
15.
Infect Immun ; 88(4)2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32014892

RESUMO

Rodents are critical for the transmission of Toxoplasma gondii to the definitive feline host via predation, and this relationship has been extensively studied as a model for immune responses to parasites. Neospora caninum is a closely related coccidian parasite of ruminants and canines but is not naturally transmitted by rodents. We compared mouse innate immune responses to N. caninum and T. gondii and found marked differences in cytokine levels and parasite growth kinetics during the first 24 h postinfection (hpi). N. caninum-infected mice produced significantly higher levels of interleukin-12 (IL-12) and interferon gamma (IFN-γ) by as early as 4 hpi, but the level of IFN-γ was significantly lower or undetectable in T. gondii-infected mice during the first 24 hpi. "Immediate" IFN-γ and IL-12p40 production was not detected in MyD88-/- mice. However, unlike IL-12p40-/- and IFN-γ-/- mice, MyD88-/- mice survived N. caninum infections at the dose used in this study. Serial measures of parasite burden showed that MyD88-/- mice were more susceptible to N. caninum infections than wild-type (WT) mice, and control of parasite burdens correlated with a pulse of serum IFN-γ at 3 to 4 days postinfection in the absence of detectable IL-12. Immediate IFN-γ was partially dependent on the T. gondii mouse profilin receptor Toll-like receptor 11 (TLR11), but the ectopic expression of N. caninum profilin in T. gondii had no impact on early IFN-γ production or parasite proliferation. Our data indicate that T. gondii is capable of evading host detection during the first hours after infection, while N. caninum is not, and this is likely due to the early MyD88-dependent recognition of ligands other than profilin.


Assuntos
Coccidiose/imunologia , Fatores Imunológicos/metabolismo , Interferon gama/metabolismo , Neospora/imunologia , Doenças dos Roedores/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Animais , Interferon gama/deficiência , Interleucina-12/deficiência , Interleucina-12/metabolismo , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/metabolismo , Neospora/crescimento & desenvolvimento , Análise de Sobrevida , Fatores de Tempo , Toxoplasma/crescimento & desenvolvimento
16.
Nat Commun ; 11(1): 608, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001704

RESUMO

Rewiring cellular sensors to trigger non-natural responses is fundamental for therapeutic cell engineering. Current designs rely on engineered receptors that are limited to single inputs, and often suffer from high leakiness and low fold induction. Here, we present Generalized Engineered Activation Regulators (GEARs) that overcome these limitations by being pathway-specific rather than input-specific. GEARs consist of the MS2 bacteriophage coat protein fused to regulatory or transactivation domains, and work by rerouting activation of the NFAT, NFκB, MAPK or SMAD pathways to dCas9-directed gene expression from genomic loci. This system enables membrane depolarization-induced activation of insulin expression in ß-mimetic cells and IL-12 expression in activated Jurkat cells, as well as IL-12 production in response to the immunomodulatory cytokines TGFß and TNFα in HEK293T cells. Engineered cells with the ability to reinterpret the extracellular milieu have potential for applications in immunotherapy and in the treatment of metabolic diseases.


Assuntos
Reprogramação Celular/genética , Genoma , Transdução de Sinais/genética , Proteína 9 Associada à CRISPR/metabolismo , Sinalização do Cálcio , Engenharia Genética , Células HEK293 , Humanos , Imunomodulação , Inflamação/genética , Inflamação/patologia , Insulina/metabolismo , Interleucina-12/metabolismo , Células Jurkat , Ativação Linfocitária/imunologia , Potenciais da Membrana , Linfócitos T/imunologia , Transgenes
17.
J Immunol ; 204(7): 1724-1735, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32086386

RESUMO

IL-23 and IL-12, two structurally related heterodimeric cytokines sharing a common subunit, divergently promote Th cell development and expansion. Both cytokines have been implicated in the pathogenesis of thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves disease. In TAO, CD34+ fibrocytes, putatively derived from bone marrow, can be identified in the orbit. There they masquerade as CD34+ orbital fibroblasts (OF) (CD34+ OF) and cohabitate with CD34- OF in a mixed fibroblast population (GD-OF). Slit2, a neural axon repellent, is expressed and released by CD34- OF and dampens the inflammatory phenotype of fibrocytes and CD34+ OF. In this study we report that thyrotropin (TSH) and the pathogenic, GD-specific monoclonal autoantibody, M22, robustly induce IL-23 in human fibrocytes; however, IL-12 expression is essentially undetectable in these cells under basal conditions or following TSH-stimulation. In contrast, IL-12 is considerably more inducible in GD-OF, cells failing to express IL-23. This divergent expression and induction of cytokines appears to result from cell type-specific regulation of both gene transcription and mRNA stabilities. It appears that the JNK pathway activity divergently attenuates IL-23p19 expression while enhancing that of IL-12p35. The shift from IL-23p19 expression in fibrocytes to that of IL-12p35 in their derivative CD34+ OF results from the actions of Slit2. Thus, Slit2 might represent a molecular determinant of balance between IL-23 and IL-12 expression, potentially governing immune responses in TAO.


Assuntos
Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Proteínas do Tecido Nervoso/imunologia , Tireotropina/metabolismo , Antígenos CD34/metabolismo , Células Cultivadas , Citocinas/metabolismo , Doença de Graves/metabolismo , Oftalmopatia de Graves/metabolismo , Humanos , Órbita/metabolismo , Estabilidade de RNA/fisiologia , Receptores da Tireotropina/metabolismo , Transdução de Sinais/fisiologia
18.
Mediators Inflamm ; 2020: 3019378, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32104147

RESUMO

CD19+CD24hiCD38hi B cells are immature transitional B cells that, in normal individuals, exert suppressive effects by IL-10 production but are quantitatively altered and/or functionally impaired in individuals with various autoimmune diseases. Primary biliary cholangitis (PBC), an autoimmune disease, clinically presents as chronic cholestasis and nonsuppurative destructive cholangitis. A role for CD19+CD24hiCD38hi B cells in PBC is unknown. This study investigated the frequency and functional variation of circulating CD19+CD24hiCD38hi B cells in PBC patients. Flow cytometry was employed to quantify the percentage of CD19+CD24hiCD38hi B cells in peripheral blood samples. Correlations between CD19+CD24hiCD38hi B cells and routine laboratory parameters were assessed. Levels of IL-10, TNF-α, IL-6 and IL-12, and Tim-1 in CD19+CD24hiCD38hi B cells from PBC patients were analyzed. The effect of CD19+CD24hiCD38hi B cells on CD4+T cell differentiation was evaluated. The percentage of CD19+CD24hiCD38hi B cells in PBC patients was significantly higher than in healthy controls and was positively correlated with liver cholestasis. After activation by anti-B cell receptor and CpG, the production of IL-10 was decreased and the production of IL-6 and IL-12 was increased in CD19+CD24hiCD38hi B cells from PBC patients. Moreover, Tim-1 levels were significantly downregulated in CD19+CD24hiCD38hi B cells from PBC patients. Coculture showed that PBC-derived CD19+CD24hiCD38hi B cells were less capable of CD4+T cell inhibition, but promoted Th1 cell differentiation. In conclusion, PBC patients have expanded percentages, but impaired CD19+CD24hiCD38hi B cells, which correlate with disease damage. In PBC patients, this B cell subset has a skewed proinflammatory cytokine profile and a decreased capacity to suppress immune function, which may contribute to the pathogenesis of PBC.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Antígeno CD24/metabolismo , Cirrose Hepática Biliar/imunologia , Cirrose Hepática Biliar/patologia , Idoso , Diferenciação Celular/fisiologia , Feminino , Citometria de Fluxo , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Cirrose Hepática Biliar/metabolismo , Masculino , Pessoa de Meia-Idade , Células-Tronco de Sangue Periférico/imunologia , Células-Tronco de Sangue Periférico/metabolismo , Células-Tronco de Sangue Periférico/patologia , Fator de Necrose Tumoral alfa/metabolismo
20.
Parasitol Res ; 119(3): 1023-1033, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32065264

RESUMO

Toxoplasma gondii is an important zoonotic protozoan worldwide which infects most of warm-blooded mammals and birds, including human, and cause toxoplasmosis. As an intracellular parasite, T. gondii must evade host immune surveillance, such as IL-12 and IFN-γ, in order to survive and multiply in macrophages and other host cells. By delaying IL-12 secretion of host macrophages within 24 h after infection, T. gondii ensures not only self-survival but also the establishment of chronic infection of host cells. MicroRNA plays an important role in regulating gene transcription and translation. The mechanisms of IL-12 production during T. gondii infection are still unknown. Thus, understanding how the parasites manipulate IL-12 production by host macrophage is critical for the effective prevention and therapy of T. gondii infection. In the present study, regulation of delayed macrophage IL-12 production during T. gondii infection was explored. We found that the production of IL-12 after T. gondii infection was inhibited during the first 24 h and then resumed. The expression pattern of miR-187 production was consistent with the production pattern of IL-12 during T. gondii infection. The downregulation of miR-187 promoted Akt and P65 phosphorylation and delayed IL-12 production at late stage (after 24 h) of T. gondii infection. Dual-luciferase reporter assay indicated that MiR-187 targeted the NFKBIZ gene. Our results suggested that the delayed IL-12 production in mouse macrophages during T. gondii infection was regulated by miR-187.


Assuntos
Interleucina-12/metabolismo , Macrófagos/metabolismo , MicroRNAs/genética , Toxoplasma/imunologia , Toxoplasmose/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Regulação para Baixo , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo
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