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1.
J Vis Exp ; (155)2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32065164

RESUMO

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-α, IFN-γ, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

2.
Int Immunopharmacol ; 77: 105948, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31629216

RESUMO

OBJECTIVE: The role of iNKT cells was investigated in chronic adipose tissue inflammation in obese mice after administration of α-GalCer in different pathways. METHODS: C57BL/6J mice were fed high-fat diet (HFD) for 12 weeks to establish the obese mouse model. The pathology of adipose tissue was observed by H&E staining. The rates of iNKT cells, macrophages and cell subsets in adipose tissue were detected by FCM. Cytokine levels in serum and adipose tissue lymphocyte-stimulated supernatants were assessed with the CBA kit. The expression levels of related transcription factor in adipose tissue were detected by Western blot. RESULTS: The proportions of iNKT cells, iNKT10 cells and M2 macrophages were decreased, while those of iNKT1 and M1 macrophages were increased in adipose tissue of HFD-fed mice. The expression levels of the related transcriptional proteins E4BP4 and Arg-1 were decreased while iNOS expression was increased in adipose tissue. Administration of α-GalCer by subcutaneous injection resulted in increased rates of iNKT10 cells and M2 macrophages, and decreased amounts of M1 macrophages in adipose tissue of HFD-fed mice. The expression of E4BP4 and Arg-1 were up-regulated, but iNOS was down-regulated. Meanwhile, infiltration of inflammatory cells into adipose tissue was further reduced. CONCLUSION: The imbalance between the proportions of iNKT1 and iNKT10 cells may be involved in the development of chronic inflammation in obese adipose tissue. Administration of α-GalCer by subcutaneous injection in HFD-fed mice activates adipose tissue iNKT10 cells, which promote M2 macrophage polarization and improve chronic inflammation in obese adipose tissue.

3.
Immunogenetics ; 71(7): 489-499, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31297569

RESUMO

Epigenetic modifications have been shown to be important for immune cell differentiation by regulating gene transcription. However, the role and mechanism of histone methylation in the development and differentiation of iNKT cells in rheumatoid arthritis (RA) mice have yet to be deciphered. The DBA/1 mouse RA model was established by using a modified GPI mixed peptide. We demonstrated that total peripheral blood, thymus, and spleen iNKT cells in RA mice decreased significantly, while iNKT1 in the thymus and spleen was increased significantly. PLZF protein and PLZF mRNA levels were significantly decreased in thymus DP T cells, while T-bet protein and mRNA were significantly increased in thymus iNKT cells. We found a marked accumulation in H3K27me3 around the promoter regions of the signature gene Zbtb16 in RA mice thymus DP T cells, and an accumulation of H3K4me3 around the promoters of the Tbx21 gene in iNKT cells. The expression levels of UTX in the thymus of RA mice were significantly reduced. The changes in the above indicators were particularly significant in the progressive phase of inflammation (11 days after modeling) and the peak phase of inflammation (14 days after modeling) in RA mice. Developmental and differentiation defects of iNKT cells in RA mice were associated with abnormal methylation levels (H3K27me3 and H3K4me3) in the promoters of key genes Zbtb16 (encoding PLZF) and Tbx21 (encoding T-bet). Decreased UTX of thymus histone demethylase levels resulted in the accumulation of H3K27me3 modification.


Assuntos
Artrite Reumatoide/patologia , Lisina/metabolismo , Células T Matadoras Naturais/patologia , Regiões Promotoras Genéticas , Timo/fisiologia , Animais , Artrite Experimental/patologia , Diferenciação Celular , Epigênese Genética , Regulação da Expressão Gênica , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Metilação , Camundongos Endogâmicos DBA , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Baço/patologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
4.
Int Immunopharmacol ; 74: 105727, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284229

RESUMO

The existence of association between the subpopulation of iNKT cells with different functions and nonalcoholic fatty liver disease has not been confirmed. To investigative the role of iNKT cells in the pathogenesis of nonalcoholic fatty liver disease, we established a non-alcoholic fatty liver model by feeding C57BL/6J mice for 12 weeks with a high-fat diet and injecting α-GalCer through different routes to activate hepatic iNKT cells. The liver of the mice fed a high-fat diet (HFD) had severe hepatic steatosis appearance, elevated pro-inflammatory cytokines and reduced anti-inflammatory cytokines in the liver, and high serum levels of TC, LDL, HDL, and ALT. Our results showed that the percentage of iNKT cells in the liver of the HFD-fed mice was lower than that of the control mice. The expression levels of the related transcription factor of T-bet increased but that of GATA-3 decreased in the HFD-fed mice. The administration of α-GalCer by intraperitoneal injection resulted in increasing of hepatic iNKT and iNKT2 cells but decreasing of hepatic iNKT1 cells, and the expression of GATA-3 and anti-inflammatory cytokine (IL-4) was increased in the liver, and hepatic steatosis was ameliorated in the HFD-fed mice. The administration of α-GalCer by subcutaneous injection resulted in a decrease in hepatic iNKT and iNKT2 and an augmentation of hepatic iNKT1 cells. However, hepatic steatosis was not significantly improved. We concluded that the intraperitoneal injection with α-GalCer effectively improved hepatic steatosis, according to increasing the number of hepatic iNKT2 cells. The precise mechanism requires further exploration.

5.
Int Immunopharmacol ; 67: 427-440, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30586666

RESUMO

BACKGROUND: The therapeutic effect of adoptive infusion of specific thymus-derived invariant natural killer T (iNKT) cells in a mouse model of rheumatoid arthritis (RA) was observed, and the mechanism of cellular immunotherapy was preliminarily explored. METHODS: Thymus-derived iNKT cells were infused to RA model mice, with α-GalCer as a positive control. Then, ankle swelling was examined, as well as inflammatory cell infiltration to the joint tissue (hematoxylin-eosin [H&E] staining). Flow cytometry (FCM) was used to assess iNKT cell and helper T lymphocyte (Th) subsets. Serum cytokine levels were determined with cytometric bead array (CBA), with protein expression levels of related transcription factors assessed by Western blot. RESULTS: The joint swelling in RA model animals were significantly improved in the cell therapy and α-GalCer positive control groups (P < 0.05). In addition, iNKT frequencies in peripheral blood, the thymus and spleen were increased significantly (P < 0.05). Meanwhile, iNKT1 subset frequencies in the thymus and spleen were decreased, as well as splenic Th1 and Th17 cell subset rates, and serum TNF-α, IFN-γ and IL-6 levels. The rates of iNKT2 and Th2 subsets as well as IL-4 and IL-10 levels were increased (P < 0.05). Thymus GATA-3 and splenic PLZF protein levels were increased (P < 0.05). CONCLUSIONS: Adoptive infusion of thymus-derived iNKT cells exerts therapeutic effects in RA mice by increasing iNKT frequency, altering the proportions of iNKT cell subsets, correcting Th cell subset imbalance and reducing the amounts of inflammatory cytokines.


Assuntos
Artrite Reumatoide/terapia , Imunoterapia Adotiva , Células T Matadoras Naturais , Timo/citologia , Animais , Artrite Reumatoide/induzido quimicamente , Masculino , Camundongos , Células T Matadoras Naturais/classificação , Peptídeos/toxicidade , Distribuição Aleatória , Baço/citologia
6.
Sci Rep ; 7: 43220, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28230213

RESUMO

Achondroplasia (ACH), the most common genetic dwarfism in human, is caused by a gain-of function mutation in fibroblast growth factor receptor 3 (FGFR3). Currently, there is no effective treatment for ACH. The development of an appropriate human-relevant model is important for testing potential therapeutic interventions before human clinical trials. Here, we have generated an ACH mouse model in which the endogenous mouse Fgfr3 gene was replaced with human FGFR3G380R (FGFR3ACH) cDNA, the most common mutation in human ACH. Heterozygous (FGFR3ACH/+) and homozygous (FGFR3ACH/ACH) mice expressing human FGFR3G380R recapitulate the phenotypes observed in ACH patients, including growth retardation, disproportionate shortening of the limbs, round head, mid-face hypoplasia at birth, and kyphosis progression during postnatal development. We also observed premature fusion of the cranial sutures and low bone density in newborn FGFR3G380R mice. The severity of the disease phenotypes corresponds to the copy number of activated FGFR3G380R, and the phenotypes become more pronounced during postnatal skeletal development. This mouse model offers a tool for assessing potential therapeutic approaches for skeletal dysplasias related to over-activation of human FGFR3, and for further studies of the underlying molecular mechanisms.


Assuntos
Acondroplasia/patologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Proteínas Mutantes/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Acondroplasia/genética , Animais , Dosagem de Genes , Heterozigoto , Homozigoto , Humanos , Camundongos , Proteínas Mutantes/genética , Mutação de Sentido Incorreto
7.
J Microbiol Immunol Infect ; 49(5): 636-645, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25070282

RESUMO

BACKGROUND/PURPOSE: Helicobacter pylori colonizes the human stomach and contributes to chronic inflammation of the gastric mucosa. H. pylori persistence occurs because of insufficient eradication by phagocytic cells. A key factor of H. pylori, cholesterol-α-glucosyltransferase encoded by capJ that extracts host cholesterol and converts it to cholesteryl glucosides, is important to evade host immunity. Here, we examined whether phagocytic trafficking in macrophages was perturbed by capJ-carrying H. pylori. METHODS: J774A.1 cells were infected with H. pylori at a multiplicity of infection of 50. Live-cell imaging and confocal microscopic analysis were applied to monitor the phagocytic trafficking events. The viability of H. pylori inside macrophages was determined by using gentamicin colony-forming unit assay. The phagocytic routes were characterized by using trafficking-intervention compounds. RESULTS: Wild type (WT) H. pylori exhibited more delayed entry into macrophages and also arrested phagosome maturation more than did capJ knockout mutant. Pretreatment of genistein and LY294002 prior to H. pylori infection reduced the internalization of WT but not capJ-knockout H. pylori in macrophages. CONCLUSION: Cholesterol glucosylation by H. pylori interferes with phagosome trafficking via a lipid-raft and PI3K-dependent manner, which retards engulfment of bacteria for prolonged intracellular survival of H. pylori.


Assuntos
Colesterol/análogos & derivados , Colesterol/metabolismo , Glucosiltransferases/metabolismo , Helicobacter pylori/imunologia , Evasão da Resposta Imune/imunologia , Macrófagos/imunologia , Fagossomos/imunologia , Animais , Linhagem Celular , Glucosiltransferases/genética , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos , Microscopia Confocal , Fagocitose/imunologia , Fagossomos/microbiologia , Fosfatidilinositol 3-Quinases/metabolismo
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