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An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Th17-cells play a key role in the pathogenesis of autoimmune hepatitis (AIH). Dysregulation of Th17-cells in AIH is linked to defective response to aryl-hydrocarbon-receptor (AhR) activation. AhR modulates adaptive immunity and is regulated by aryl-hydrocarbon-receptor-repressor (AHRR), which inhibits AhR transcriptional activity. In this study, we investigated whether defective Th17-cell response to AhR derives from aberrant AHRR regulation in AIH. Th17-cells, obtained from the peripheral blood of AIH patients (n = 30) and healthy controls (n = 30) were exposed to AhR endogenous ligands, and their response assessed in the absence or presence of AHRR silencing. Therapeutic effects of AHRR blockade were tested in a model of Concanavalin-A (Con-A)-induced liver injury in humanized mice. AHRR was markedly upregulated in AIH Th17-cells, following exposure to l-kynurenine, an AhR endogenous ligand. In patients, silencing of AHRR boosted Th17-cell response to l-kynurenine, as reflected by increased levels of CYP1A1, the main gene controlled by AhR; and decreased IL17A expression. Blockade of AHRR limited the differentiation of naïve CD4-cells into Th17 lymphocytes; and modulated Th17-cell metabolic profile by increasing the levels of uridine via ATP depletion or pyrimidine salvage. Treatment with 2'-deoxy-2'-fluoro-d-arabinonucleic acid (FANA) oligonucleotides to silence human AHRR in vivo, reduced ALT levels, attenuated lymphocyte infiltration on histology, and heightened frequencies of regulatory immune subsets in NOD/scid/gamma mice, reconstituted with human CD4 cells, and exposed to Con-A. In conclusion, blockade of AHRR in AIH restores Th17-cell response to AHR, and limits Th17-cell differentiation through generation of uridine. In vivo, silencing of AHRR attenuates liver damage in NOD/scid/gamma mice. Blockade of AHRR might therefore represent a novel therapeutic strategy to modulate effector Th17-cell immunity and restore homeostasis in AIH.
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Hepatite Autoimune , Receptores de Hidrocarboneto Arílico , Animais , Humanos , Camundongos , Hepatite Autoimune/genética , Hidrocarbonetos , Cinurenina , Camundongos Endogâmicos NOD , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Repressoras/genética , Células Th17/metabolismo , UridinaRESUMO
Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.
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Doenças Autoimunes/imunologia , Biopterinas/análogos & derivados , Neoplasias/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Administração Oral , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/metabolismo , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/patologia , Biopterinas/biossíntese , Biopterinas/metabolismo , Biopterinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Coenzimas/metabolismo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Humanos , Hipersensibilidade/imunologia , Ferro/metabolismo , Cinurenina/metabolismo , Cinurenina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
RATIONALE: The increased mortality and morbidity seen in critically injured patients appears associated with systemic inflammatory response syndrome (SIRS) and immune dysfunction, which ultimately predisposes to infection. Mitochondria released by injury could generate danger molecules, for example, ATP, which in turn would be rapidly scavenged by ectonucleotidases, expressed on regulatory immune cells. OBJECTIVE: To determine the association between circulating mitochondria, purinergic signalling and immune dysfunction after trauma. METHODS: We tested the impact of hepatocyte-derived free mitochondria on blood-derived and lung-derived CD8 T cells in vitro and in experimental mouse models in vivo. In parallel, immune phenotypic analyses were conducted on blood-derived CD8 T cells obtained from trauma patients. RESULTS: Isolated intact mitochondria are functional and generate ATP ex vivo. Extracellular mitochondria perturb CD8+ T cells in co-culture, inducing select features of immune exhaustion in vitro. These effects are modulated by scavenging ATP, modelled by addition of apyrase in vitro. Injection of intact mitochondria into recipient mice markedly upregulates the ectonucleotidase CD39, and other immune checkpoint markers in circulating CD8+ T cells. We note that mice injected with mitochondria, prior to instilling bacteria into the lung, exhibit more severe lung injury, characterised by elevated neutrophil influx and by changes in CD8+ T cell cytotoxic capacity. Importantly, the development of SIRS in injured humans, is likewise associated with disordered purinergic signalling and CD8 T cell dysfunction. CONCLUSION: These studies in experimental models and in a cohort of trauma patients reveal important associations between extracellular mitochondria, aberrant purinergic signalling and immune dysfunction. These pathogenic factors with immune exhaustion are linked to SIRS and could be targeted therapeutically.
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Antígenos CD , Linfócitos T CD8-Positivos , Animais , Humanos , Camundongos , Trifosfato de Adenosina/metabolismo , Biomarcadores/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Mitocôndrias , Síndrome de Resposta Inflamatória Sistêmica/metabolismoRESUMO
BACKGROUND AND AIMS: The "gut homing" hypothesis suggests the pathogenesis of primary sclerosing cholangitis (PSC) is driven by aberrant hepatic expression of gut adhesion molecules and subsequent recruitment of gut-derived T cells to the liver. However, inconsistencies lie within this theory including an absence of investigations and comparisons with other chronic liver diseases (CLD). Here, we examine "the gut homing theory" in patients with PSC with associated inflammatory bowel disease (PSC-IBD) and across multiple inflammatory liver diseases. APPROACH AND RESULTS: Expression of MAdCAM-1, CCL25, and E-Cadherin were assessed histologically and using RT-PCR on explanted liver tissue from patients with CLD undergoing OLT and in normal liver. Liver mononuclear cells were isolated from explanted tissue samples and the expression of gut homing integrins and cytokines on hepatic infiltrating gut-derived T cells was assessed using flow cytometry. Hepatic expression of MAdCAM-1, CCL25 and E-Cadherin was up-regulated in all CLDs compared with normal liver. There were no differences between disease groups. Frequencies of α4ß7, αEß7, CCR9, and GPR15 expressing hepatic T cells was increased in PSC-IBD, but also in CLD controls, compared with normal liver. ß7 expressing hepatic T cells displayed an increased inflammatory phenotype compared with ß7 negative cells, although this inflammatory cytokine profile was present in both the inflamed and normal liver. CONCLUSIONS: These findings refute the widely accepted "gut homing" hypothesis as the primary driver of PSC and indicate that aberrant hepatic recruitment of gut-derived T cells is not unique to PSC, but is a panetiological feature of CLD.
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Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Quimiocinas CC/metabolismo , Colangite Esclerosante , Trato Gastrointestinal , Hepatopatias , Fígado , Mucoproteínas/metabolismo , Moléculas de Adesão Celular/isolamento & purificação , Colangite Esclerosante/imunologia , Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/patologia , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Cadeias beta de Integrinas/metabolismo , Fígado/metabolismo , Fígado/patologia , Hepatopatias/classificação , Hepatopatias/metabolismo , Hepatopatias/patologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismoRESUMO
Occipital headache, the perception of pain in the back of the head, is commonly described by patients diagnosed with migraine, tension-type headache, and occipital neuralgia. The greater and lesser occipital nerves play central role in the pathophysiology of occipital headache. In the clinical setup, such headaches are often treated with onabotulinumtoxinA, a neurotoxin capable of disrupting ability of nociceptors to get activated and/or release proinflammatory neuropeptides. Attempting to understand better onabotulinumtoxinA mechanism of action in reducing headache frequency, we sought to determine its effects on expression of inflammatory genes in injected occipital tissues. To achieve this goal, we injected 40 units of onabotulinumtoxinA into four muscle groups (occipitalis, splenius capitis, semispinalis capitis, and trapezius muscles-all located on one side of the occiput) of patients with chronic bilateral occipital headache scheduled for occipital nerve decompression surgery 1 month later. At the time of surgery, we collected discarded muscle, fascia and periosteum tissues from respective locations on both sides of the neck and occiput and performed targeted transcriptome analyses to determine expression level of inflammatory genes in onabotulinumtoxinA-injected and onabotulinumA-uninjected tissues. We found that (i) onabotulinumtoxinA alters expression of inflammatory genes largely in periosteum, minimally in muscle and not at all in fascia; (ii) expression of inflammatory genes in uninjected periosteum and muscle is significantly higher in historical onabotulinumA responders than historical non-responders; (iii) in historical responders' periosteum, onabotulinumA decreases expression of nearly all significantly altered genes, gene sets that define well recognized inflammatory pathways (e.g. pathways involved in adaptive/innate immune response, lymphocyte activation, and cytokine, chemokine, NF-kB, TNF and interferon signalling), and abundance of 12 different immune cell classes (e.g. neutrophils, macrophages, cytotoxic T-, NK-, Th1-, B- and dendritic-cells), whereas in historical non-responders it increases gene expression but to a level that is nearly identical to the level observed in the uninjected periosteum and muscle of historical responders; and surprisingly (iv) that the anti-inflammatory effects of onabotulinumA are far less apparent in muscles and absent in fascia. These findings suggest that in historical responders' periosteum-but not muscle or fascia-inflammation contributes to the pathophysiology of occipital headache, and that further consideration should be given to the possibility that onabotulinumA mechanism of action in migraine prevention could also be achieved through its ability to reduce pre-existing inflammation, likely through localized interaction that lead to reduction in abundance of immune cells in the calvarial periosteum.
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Toxinas Botulínicas Tipo A , Transtornos da Cefaleia , Transtornos de Enxaqueca , Neuralgia , Toxinas Botulínicas Tipo A/farmacologia , Toxinas Botulínicas Tipo A/uso terapêutico , Expressão Gênica , Cefaleia/tratamento farmacológico , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Transtornos de Enxaqueca/tratamento farmacológico , Resultado do TratamentoRESUMO
Macrophages are tissue-resident immune cells that are crucial for the initiation and maintenance of immune responses. Purinergic signaling modulates macrophage activity and impacts cellular plasticity. The ATP-activated purinergic receptor P2X7 (also known as P2RX7) has pro-inflammatory properties, which contribute to macrophage activation. P2X7 receptor signaling is, in turn, modulated by ectonucleotidases, such as CD39 (also known as ENTPD1), expressed in caveolae and lipid rafts. Here, we examined P2X7 receptor activity and determined impacts on ectonucleotidase localization and function in macrophages primed with lipopolysaccharide (LPS). First, we verified that ATP boosts CD39 activity and caveolin-1 protein expression in LPS-primed macrophages. Drugs that disrupt cholesterol-enriched domains - such as nystatin and methyl-ß-cyclodextrin - decreased CD39 enzymatic activity in all circumstances. We noted that CD39 colocalized with lipid raft markers (flotillin-2 and caveolin-1) in macrophages that had been primed with LPS followed by treatment with ATP. P2X7 receptor inhibition blocked these ATP-mediated increases in caveolin-1 expression and inhibited the colocalization with CD39. Further, we found that STAT3 activation is significantly attenuated caveolin-1-deficient macrophages treated with LPS or LPS+BzATP. Taken together, our data suggest that P2X7 receptor triggers the initiation of lipid raft-dependent mechanisms that upregulates CD39 activity and could contribute to limit macrophage responses restoring homeostasis.
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Caveolina 1 , Receptores Purinérgicos P2X7 , Trifosfato de Adenosina , Caveolina 1/genética , Lipopolissacarídeos , Macrófagos , Microdomínios da Membrana , Receptores Purinérgicos P2X7/genéticaRESUMO
BACKGROUND AND AIMS: Genetic predisposition to autoimmune hepatitis (AIH) in adults is associated with possession of human leukocyte antigen (HLA) class I (A*01, B*08) and class II (DRB1*03, -04, -07, or -13) alleles, depending on geographic region. Juvenile autoimmune liver disease (AILD) comprises AIH-1, AIH-2, and autoimmune sclerosing cholangitis (ASC), which are phenotypically different from their adult counterparts. We aimed to define the relationship between HLA profile and disease course, severity, and outcome in juvenile AILD. APPROACH AND RESULTS: We studied 236 children of European ancestry (152 female [64%], median age 11.15 years, range 0.8-17), including 100 with AIH-1, 59 with AIH-2, and 77 with ASC. The follow-up period was from 1977 to June 2019 (median 14.5 years). Class I and II HLA genotyping was performed using PCR/sequence-specific primers. HLA B*08, -DRB1*03, and the A1-B8-DR3 haplotype impart predisposition to all three forms of AILD. Homozygosity for DRB1*03 represented the strongest risk factor (8.8). HLA DRB1*04, which independently confers susceptibility to AIH in adults, was infrequent in AIH-1 and ASC, suggesting protection; and DRB1*15 (DR15) was protective against all forms of AILD. Distinct HLA class II alleles predispose to the different subgroups of juvenile AILD: DRB1*03 to AIH-1, DRB1*13 to ASC, and DRB1*07 to AIH-2. Possession of homozygous DRB1*03 or of DRB1*13 is associated with fibrosis at disease onset, and possession of these two genes in addition to DRB1*07 is associated with a more severe disease in all three subgroups. CONCLUSIONS: Unique HLA profiles are seen in each subgroup of juvenile AILD. HLA genotype might be useful in predicting responsiveness to immunosuppressive treatment and course.
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Colangite Esclerosante/genética , Hepatite Autoimune/genética , População Branca/genética , Adolescente , Criança , Pré-Escolar , Feminino , Predisposição Genética para Doença , Antígenos HLA/genética , Antígeno HLA-A1/genética , Antígeno HLA-B8/genética , Antígeno HLA-DR3/genética , Cadeias HLA-DRB1/genética , Humanos , Lactente , Masculino , Índice de Gravidade de DoençaRESUMO
BACKGROUND & AIMS: In autoimmune hepatitis (AIH), the imbalance between regulatory T cells (Tregs) and T-helper type 17 (Th17) cells has been linked to low levels of CD39, an ectoenzyme that hydrolyses ATP, ultimately generating immunosuppressive adenosine. Upregulation of CD39 results from activation of aryl hydrocarbon receptor (AHR), which mediates toxin responses to modulate T-cell immunity. In this study, we investigated whether altered AHR signalling underlies defective CD39 expression and function in AIH Tregs and Th17 cells, therefore contributing to regulatory/effector cell imbalance. METHODS: Tregs and Th17 cells, obtained from the peripheral blood of 49 patients with AIH and 21 healthy individuals (HI), were tested for response to endogenous and exogenous AHR ligands. RESULTS: When compared to those of HI, AIH-derived Tregs and Th17 cells displayed impaired responses to AHR activation, reflected by impaired upregulation of CD39, delayed increase in ectoenzymatic activity, and defective Treg suppressive function. These impairments resulted, at least in part, from heightened levels of AHRR and Erα in Tregs and high HIF-1α in Th17 cells, and were reverted upon molecular blockade. Importantly, in AIH-derived Tregs, the binding affinity of AHR was higher for Erα than ARNT. CONCLUSIONS: In AIH, high levels of AHRR and HIF-1α inhibit AHR signalling in Tregs and Th17 cells. AHR non-canonical binding to Erα further amplifies the lack of effective CD39 upregulation. Blockade of these inhibitory and/or non-canonical activation pathways represents a potential therapeutic approach to restore CD39 and immunohomeostasis in AIH. LAY SUMMARY: In patients with autoimmune hepatitis, the imbalance between regulatory T cells and T helper type-17 cells is linked to dysfunction of the aryl hydrocarbon receptor pathway, resulting from aberrant inhibition or non-canonical activation. These alterations impair Treg- and Th17 cell-induced upregulation of CD39, an ectoenzyme key to immunoregulation. Blockade of excessive inhibition or non-canonical activation of the aryl hydrocarbon receptor pathway might represent a novel therapeutic strategy to control inflammation while restoring immune balance in autoimmune hepatitis.
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Apirase/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hepatite Autoimune , Fígado , Receptores de Hidrocarboneto Arílico/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/metabolismo , Células Cultivadas , Descoberta de Drogas , Hepatite Autoimune/sangue , Hepatite Autoimune/imunologia , Hepatite Autoimune/terapia , Humanos , Imunidade Celular/imunologia , Imunomodulação , Ligantes , Fígado/imunologia , Fígado/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Regulação para CimaRESUMO
Regulatory T-cells (Tregs) are key players in the maintenance of immune homeostasis by preventing immune responses to self-antigens. Defects in Treg frequency and/or function result in overwhelming CD4 and CD8 T cell immune responses participating in the autoimmune attack. Perpetuation of autoimmune damage is also favored by Treg predisposition to acquire effector cell features upon exposure to a proinflammatory challenge. Treg impairment plays a permissive role in the initiation and perpetuation of autoimmune liver diseases, namely autoimmune hepatitis, primary biliary cholangitis and primary sclerosing cholangitis. In this Review, we outline studies reporting the role of Treg impairment in the pathogenesis of these conditions and discuss methods to restore Treg number and function either by generation/expansion in the test tube or through in vivo expansion upon administration of low dose IL-2. Challenges and caveats of these potential therapeutic strategies are also reviewed and discussed.
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Autoantígenos/imunologia , Autoimunidade , Hepatite Autoimune/etiologia , Hepatite Autoimune/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Biomarcadores , Gerenciamento Clínico , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Regulação da Expressão Gênica , Hepatite Autoimune/diagnóstico , Hepatite Autoimune/terapia , Humanos , Tolerância Imunológica , Imunomodulação , Imunoterapia , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Pregnancy presents the maternal immune system with a unique immunological challenge since it has to defend against pathogens while tolerating paternal allo-antigens expressed by fetal tissues. T helper (Th) cells play a central role in modulating immune responses and recent advances have defined distinct contributions of various Th cell subsets throughout each phase of human pregnancy, while dysregulation in Th responses show association with multiple obstetrical complications. In addition to localized decidual mechanisms, modulation of Th cell immunity during gestation is mediated largely by oscillations in sex hormone concentrations. Aberrant Th cell responses also underlie several autoimmune disorders while pregnancy-induced changes in the balance of Th cell immunity has been shown to exert favorable outcomes in the progression Th1 and Th17 driven autoimmune conditions only to be followed by post-partal exacerbations in disease.
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Doenças Autoimunes/imunologia , Complicações na Gravidez/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Doenças Autoimunes/sangue , Progressão da Doença , Feminino , Hormônios Esteroides Gonadais/sangue , Humanos , Gravidez , Complicações na Gravidez/sangueRESUMO
Cancers are able to grow by subverting immune suppressive pathways, to prevent the malignant cells as being recognized as dangerous or foreign. This mechanism prevents the cancer from being eliminated by the immune system and allows disease to progress from a very early stage to a lethal state. Immunotherapies are newly developing interventions that modify the patient's immune system to fight cancer, by either directly stimulating rejection-type processes or blocking suppressive pathways. Extracellular adenosine generated by the ectonucleotidases CD39 and CD73 is a newly recognized "immune checkpoint mediator" that interferes with anti-tumor immune responses. In this review, we focus on CD39 and CD73 ectoenzymes and encompass aspects of the biochemistry of these molecules as well as detailing the distribution and function on immune cells. Effects of CD39 and CD73 inhibition in preclinical and clinical studies are discussed. Finally, we provide insights into potential clinical application of adenosinergic and other purinergic-targeting therapies and forecast how these might develop in combination with other anti-cancer modalities.
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5'-Nucleotidase/metabolismo , Anticorpos Monoclonais/uso terapêutico , Antígenos CD/metabolismo , Apirase/metabolismo , Imunoterapia/métodos , Neoplasias/terapia , 5'-Nucleotidase/imunologia , Animais , Antígenos CD/imunologia , Apirase/imunologia , Ensaios Clínicos como Assunto , Terapia Combinada , Avaliação Pré-Clínica de Medicamentos , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Neoplasias/imunologia , Evasão Tumoral , Microambiente TumoralRESUMO
Purinergic signaling is important in the activation and differentiation of macrophages, which play divergent roles in the pathophysiology of liver fibrosis. The ectonucleotidase CD39 is known to modulate the immunoregulatory phenotype of macrophages, but whether this specifically impacts cholestatic liver injury is unknown. Here, we investigated the role of macrophage-expressed CD39 on the development of biliary injury and fibrosis in a mouse model of sclerosing cholangitis. Myeloid-specific CD39-deficient mice (LysMCreCd39fl/fl) were generated. Global CD39 null (Cd39-/-), wild-type (WT), LysMCreCd39fl/fl, and Cd39fl/fl control mice were exposed to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce biliary fibrosis. Hepatic hydroxyproline levels, liver histology, immunohistochemistry, mRNA expression levels, and serum biochemistry were then assessed. Following 3 weeks of DDC-feeding, Cd39-/- mice exhibited more severe fibrosis, when compared to WT mice as reflected by morphology and increased liver collagen content. Myeloid-specific CD39 deletion in LysMCreCd39fl/fl mice recapitulated the phenotype of global Cd39-/-, after exposure to DDC, and resulted in similar worsening of liver fibrosis when compared to Cd39fl/fl control animals. Further, DDC-treated LysMCreCd39fl/fl mice exhibited elevated serum levels of transaminases and total bilirubin, as well as increased hepatic expression of the profibrogenic genes Tgf-ß1, Tnf-α, and α-Sma. However, no clear differences were observed in the expression of macrophage-elaborated specific cytokines between LysMCreCd39fl/fl and Cd39fl/fl animals subjected to biliary injury. Our results in the DDC-induced biliary type liver fibrosis model suggest that loss of CD39 expression on myeloid cells largely accounts for the exacerbated sclerosing cholangitis in global CD39 knockouts. These findings indicate that macrophage expressed CD39 protects from biliary liver injury and fibrosis and support a potential therapeutic target for human hepatobiliary diseases.
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Antígenos CD/metabolismo , Apirase/metabolismo , Colangite Esclerosante/metabolismo , Animais , Colangite Esclerosante/induzido quimicamente , Colangite Esclerosante/patologia , Modelos Animais de Doenças , Cirrose Hepática/metabolismo , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piridinas/toxicidadeRESUMO
Exact causes for autoimmune diseases remain unclear and no cures are available. Breakdown of immunotolerance could set the stage for unfettered immune responses that target self-antigens. Impaired regulatory immune mechanisms could have permissive roles in autoreactivity. Abnormal regulatory immune cell function, therefore, might be a major determinant of the pathogenesis of autoimmune disease. All current treatments are associated with some level of clinical toxicity. Treatment to specifically target dysregulated immunity in these diseases would be a great advance. Extracellular adenosine is a signaling mediator that suppresses inflammation through activation of P1 receptors, most active under pathological conditions. Mounting evidence has linked alterations in the generation of adenosine from extracellular nucleotides by ectonucleotidases, and associated perturbations in purinergic signaling, to the immunological disruption and loss of immunotolerance in autoimmunity. Targeted modulation of the purinergic signaling by either targeting ectonucleotidases or modulating P1 purinergic receptors could therefore restore the balance between autoreactive immune responses; and thereby allow reestablishment of immunotolerance. We review the roles of CD39 and CD73 ectoenzymes in inflammatory states and with the dysregulation of P1 receptor signaling in systemic and organ-specific autoimmunity. Correction of such perturbations could be exploited in potential therapeutic applications.
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Antígenos de Neoplasias/metabolismo , Apirase/metabolismo , Doenças Autoimunes/metabolismo , Receptores Purinérgicos P1/metabolismo , Tetraspaninas/metabolismo , Adenosina/metabolismo , Doenças Autoimunes/tratamento farmacológico , Humanos , Especificidade de Órgãos , Transdução de SinaisRESUMO
In Crohn's disease, pathogenic Th17-cells express low levels of CD39 ectonucleotidase and are refractory to the immunosuppressive effects of unconjugated bilirubin (UCB), an endogenous ligand for aryl-hydrocarbon-receptor (AhR). This resistance to AhR ligation might be associated with alterations in responses to hypoxia. Limited exposure to hypoxia appears beneficial in acute tissue injury. However, in protracted inflammation, hypoxemia may paradoxically result in Th17-cell activation. We report here that in vitro exposure of Th17-cells from Crohn's disease patients to hypoxia limits responsiveness to AhR stimulation by UCB, as reflected by lower CD39 levels. Blockade of hypoxia-inducible-factor-1alpha (HIF-1α) upregulates CD39 and favors Th17-cell regulatory responses. Resistance of Th17-cells to AhR signaling results, in part, from HIF-1α-dependent induction of ATP-binding cassette (ABC) transporters: multidrug-resistance-protein-1 (MDR1) and multidrug-resistance-associated-protein-4 (MRP4). Increased ABC transporters promote efflux of suppressive AhR ligands, such as UCB, from Th17-cells. Inhibition of MDR1, MRP4 and/or HIF-1α with ritonavir (RTV) reconstitutes AhR function in Th17-cells, enhancing therapeutic effects of UCB in dextran-sulfate-sodium-induced experimental colitis. Deleterious effects of hypoxia on Th17-cells in Crohn's disease can be ameliorated either by inhibiting HIF-1α or by suppressing ABC transporters to increase UCB availability as an AhR substrate. Targeting HIF-1α-ABC transporters could provide innovative therapeutic pathways for IBD.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Colite/imunologia , Doença de Crohn/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Receptores de Hidrocarboneto Arílico/imunologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/imunologia , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/farmacologia , Apirase/genética , Apirase/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Bilirrubina/imunologia , Bilirrubina/farmacologia , Hipóxia Celular , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/genética , Doença de Crohn/genética , Doença de Crohn/patologia , Sulfato de Dextrana/administração & dosagem , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa/imunologia , Mucosa/patologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Receptores de Hidrocarboneto Arílico/genética , Ritonavir/farmacologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/patologiaRESUMO
Imbalance between T regulatory (Treg) and T effector (Teff) cells is likely to contribute to the induction and perpetuation of liver damage in autoimmune hepatitis (AIH) and autoimmune sclerosing cholangitis (AISC) either through inability of Tregs to restrain proliferation and effector cytokine production by responders or through conversion of Tregs into T helper type 1 (Th1) or type 17 (Th17) effector lymphocytes. We investigated the effect of Treg skewing on the phenotypic and functional properties of CD4+ CD127+ CD25high cells, an activated subset of Teff, in 32 patients with AIH and 20 with AISC and in 36 healthy subjects. In AIH/AISC we noted a substantial increase in peripheral blood-derived CD4+ CD127+ CD25high cells that display a Th1/Th17 phenotypic profile, as reflected by heightened interferon gamma and interleukin 17 (IL-17) production as well as by high levels of T-bet and related orphan receptor 3 expression, which is strongly correlated with disease activity. CD4+ CD127+ CD25high cells are unresponsive to low-dose IL-2 and in patients have marked proliferative ability, further enhanced by stimulation with IL-7. CD4+ CD127+ CD25high cells obtained from CD4+ cells exposed to Treg polarizing conditions display enhanced IL-10 production; up-regulate CD49b and LAG-3, markers of T regulatory 1 cells; and effectively suppress responder cell proliferation in both healthy subjects and AIH/AISC patients through a mechanism which is dependent on interferon gamma and IL-17. The suppressive function of CD4+ CD127+ CD25high cells is maintained upon proinflammatory challenge in healthy subjects but not in AIH/AISC. CONCLUSION: Treg skewing confers activated Teff phenotypic and functional properties of T regulatory 1 cells in health and in AIH/AISC, though suppressive function is lost in patients upon proinflammatory challenge; protracted modulation of the inflammatory environment is required to attenuate the effector potential while boosting immunoregulatory properties in Teff. (Hepatology 2017;66:1570-1584).
Assuntos
Colangite Esclerosante/imunologia , Hepatite Autoimune/imunologia , Linfócitos T Reguladores/fisiologia , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Adulto JovemRESUMO
Several lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4(dim)CD5(bright) phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D-related (DR). In addition, LN-CLL cells have higher expression of α4ß1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4(dim)CD5(bright) with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49d(hi) CLL cells showed an enhanced capacity to activate T cells compared with CD49d(lo) subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4(+) T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells.
Assuntos
Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Endotélio Vascular/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Proteínas de Neoplasias/imunologia , Migração Transendotelial e Transepitelial/imunologia , Linfócitos T CD4-Positivos/patologia , Endotélio Vascular/patologia , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , MasculinoRESUMO
BACKGROUND & AIMS: Vitamin D has immune modulating effects on cytokines. Serum vitamin D levels are associated with the risk of relapse in patients with ulcerative colitis (UC), through unknown mechanisms. We tested the hypothesis that this beneficial role of vitamin D on UC is mediated through anti-inflammatory serum cytokine profiles. METHODS: Serum samples from a prospective cohort of seventy UC patients in clinical remission were collected and baseline histological and endoscopic scores were recorded at enrollment. Clinical relapse events were recorded over the 12-month follow-up period. Serum vitamin D and cytokines levels (IL-6, IL-8, IL-17A, TNF-α, IFN-γ, IL-4, IL-10) were quantified using ELISA. Linear regression was used to determine correlation between vitamin D and cytokine profiles. Logistic regression models were used to determine the association between serum cytokine profiles and baseline histologic mucosal healing and clinical relapse. RESULTS: Higher serum vitamin D levels positively correlated with higher ratios of IL-4â¯+â¯IL-10/IL-17Aâ¯+â¯TNF-α (râ¯=â¯0.37, Pâ¯<â¯.01), and IL-4â¯+â¯IL-10/IL-6â¯+â¯TNF-α (râ¯=â¯0.32, Pâ¯<â¯.01). In multivariate analysis, IL-4â¯+â¯IL-10/IL-17Aâ¯+â¯TNF-α ratios at baseline were associated with the presence of histologic mucosal healing (O.R. 1.29, 95% CI 1.02-1.62, Pâ¯=â¯.03). A higher ratio of serum IL-4â¯+â¯IL-10 to IL-6â¯+â¯TNF-α was associated with a reduced risk of clinical relapse (O.R. 0.72, 95% CI 0.58-0.89, Pâ¯=â¯.003), and longer time to relapse (pâ¯=â¯.03), over the 12-month follow-up period. This ratio during remission had an AUC of 0.7 in predicting later clinical relapse. CONCLUSIONS: Vitamin D is associated with anti-inflammatory serum cytokine profiles. Anti-inflammatory cytokine patterns may mediate the protective effects of higher serum vitamin D levels in patients with ulcerative colitis.
Assuntos
Colite Ulcerativa/sangue , Citocinas/sangue , Vitamina D/sangue , Adulto , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND & AIMS: The severity of sepsis can be linked to excessive inflammatory responses resulting in hepatic injury. P2X7 receptor activation by extracellular ATP (eATP) exacerbates inflammation by augmenting cytokine production; while CD39 (ENTPD1) scavenges eATP to generate adenosine, thereby limiting P2X7 activation and resulting in A2A receptor stimulation. We aim to determine how the functional interaction of P2X7 receptor and CD39 control the macrophage response, and consequently impact on sepsis and liver injury. METHODS: Sepsis was induced by cecal ligation and puncture in C57BL/6 wild-type (WT) and CD39-/- mice. Several in vitro assays were performed using peritoneal or bone marrow derived macrophages to determine CD39 ectonucleotidase activity and its role in sepsis-induced liver injury. RESULTS: CD39 expression in macrophages limits ATP-P2X7 receptor pro-inflammatory signaling. P2X7 receptor paradoxically boosts CD39 activity. Inhibition and/or deletion of P2X7 receptor in LPS-primed macrophages attenuates cytokine production and inflammatory signaling as well as preventing ATP-induced increases in CD39 activity. Septic CD39-/- mice exhibit higher levels of inflammatory cytokines and show more pronounced liver injury than WT mice. Pharmacological P2X7 blockade largely prevents tissue damage, cell apoptosis, cytokine production, and the activation of inflammatory signaling pathways in the liver from septic WT, while only attenuating these outcomes in CD39-/- mice. Furthermore, the combination of P2X7 blockade with adenosine A2A receptor stimulation completely inhibits cytokine production, the activation of inflammatory signaling pathways, and protects septic CD39-/- mice against liver injury. CONCLUSIONS: CD39 attenuates sepsis-associated liver injury by scavenging eATP and ultimately generating adenosine. We propose boosting of CD39 would suppress P2X7 responses and trigger adenosinergic signaling to limit systemic inflammation and restore liver homeostasis during the acute phase of sepsis. Lay summary: CD39 expression in macrophages limits P2X7-mediated pro-inflammatory responses, scavenging extracellular ATP and ultimately generating adenosine. CD39 genetic deletion exacerbates sepsis-induced experimental liver injury. Combinations of a P2X7 antagonist and adenosine A2A receptor agonist are hepatoprotective during the acute phase of abdominal sepsis.
Assuntos
Antígenos CD/metabolismo , Apirase/metabolismo , Fígado/imunologia , Fígado/lesões , Receptores Purinérgicos P2X7/metabolismo , Sepse/imunologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/genética , Apirase/deficiência , Apirase/genética , Citocinas/biossíntese , Modelos Animais de Doenças , Interleucina-1beta/biossíntese , Fígado/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/deficiência , Receptores Purinérgicos P2X7/genética , Fator de Transcrição STAT3/metabolismo , Sepse/terapia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
BACKGROUND & AIMS: T-helper-type 17 (Th17) cells are involved in autoimmune tissue damage. CD39 is an ectonucleotidase that catalyzes extracellular ATP/ADP hydrolysis, culminating in the generation of immunosuppressive adenosine. Functional CD39 expression confers immunosuppressive properties upon immune cells. As the proportion of CD39 lymphocytes is decreased in juvenile autoimmune liver disease (AILD), we have explored whether decreased CD39 expression is present on Th17 cells and whether this phenomenon is associated with heightened effector function and inflammation. METHODS: Thirty-eight patients with juvenile AILD (22 autoimmune hepatitis and 16 autoimmune sclerosing cholangitis), 8 disease controls (DC) and 16 healthy subjects (HS) were studied. Peripheral blood cell phenotype was determined by flow cytometry; ability to suppress by inhibition of cell proliferation/effector cytokine production; ectoenzymatic activity by thin layer chromatography; expression of adenosine receptor, adenosine deaminase (ADA) and phosphodiesterases (PDE) by quantitative real-time PCR or by Western Blot. RESULTS: CD39(+) Th17 (Th17(CD39+)) cells from HS appear activated and contain high frequencies of lymphocytes producing regulatory cytokines. In AILD, however, Th17(CD39+) cells are markedly diminished and fail to generate AMP/adenosine, thereby limiting control of both target cell proliferation and IL-17 production. When compared to HS, Th17 cells from AILD patients also show lower A2A adenosine receptor expression while displaying similar levels of PDE4A, PDE4B and ADA. Only rare Th17(CD39+) cells are observed by liver immunohistochemistry. CONCLUSIONS: Th17(CD39+) cells in juvenile AILD are both quantitatively decreased and qualitatively deficient. Low levels CD39 and A2A expression may contribute to the perpetuation of Th17 cell effector properties and unfettered inflammation in this disease.