RESUMEN
Loss of function mutations in Diaphanous related formin 1 (DIAPH1) are associated with seizures, cortical blindness, and microcephaly syndrome (SCBMS) and are recently linked to combined immunodeficiency. However, the extent of defects in T and innate lymphoid cells (ILCs) remain unexplored. Herein, we characterized the primary T, natural killer (NK) and helper ILCs of six patients carrying two novel loss of function mutation in DIAPH1 and Jurkat cells after DIAPH1 knockdown. Mutations were identified by whole exome sequencing. T-cell immunophenotyping, proliferation, migration, cytokine signaling, survival, and NK cell cytotoxicity were studied via flow cytometry-based assays, confocal microscopy, and real-time qPCR. CD4+ T cell proteome was analyzed by mass spectrometry. p.R351* and p.R322*variants led to a significant reduction in the DIAPH1 mRNA and protein levels. DIAPH1-deficient T cells showed proliferation, activation, as well as TCR-mediated signaling defects. DIAPH1-deficient PBMCs also displayed impaired transwell migration, defective STAT5 phosphorylation in response to IL-2, IL-7 and IL-15. In vitro generation/expansion of Treg cells from naïve T cells was significantly reduced. shRNA-mediated silencing of DIAPH1 in Jurkat cells reduced DIAPH1 protein level and inhibited T cell proliferation and IL-2/STAT5 axis. Additionally, NK cells from patients had diminished cytotoxic activity, function and IL-2/STAT5 axis. Lastly, DIAPH1-deficient patients' peripheral blood contained dramatically reduced numbers of all helper ILC subsets. DIAPH1 deficiency results in major functional defects in T, NK cells and helper ILCs underlining the critical role of formin DIAPH1 in the biology of those cell subsets.
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Forminas , Células Asesinas Naturales , Humanos , Forminas/genética , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Masculino , Células Jurkat , Femenino , Mutación , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Niño , Inmunidad Innata , Preescolar , Citocinas/metabolismo , Transducción de Señal , Inmunofenotipificación , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
BACKGROUND AND AIMS: The inconsistent data on thiamine status in obese subjects necessitates an examination of genes associated with intestinal absorption of thiamine. We aimed to reveal thiamine status in obese subjects and examine the expression of SLC19A2/3 genes encoding thiamine transporters and Sp1 transcription factor. METHODS AND RESULTS: Thirty-five adult obese subjects and 11 healthy controls were included in this cross-sectional study. Small intestine epithelial cells were used for quantitative RT-PCR analysis of the gene expression. The daily thiamine and energy intake were assessed with a food frequency questionnaire. Thiamine phosphate esters were hydrolyzed to free thiamine, and liquid chromatography with a tandem mass spectrometry-based method was used to measure total thiamine in whole blood. Daily energy intake according to body weight and daily carbohydrate intake were not significantly different between groups after adjustment for sex. Although daily thiamine intake was significantly lower in the obesity group (p = 0.015), obese subjects had significantly higher whole blood thiamine levels than controls (44.96 ± 14.6 ng/mL and 33.05 ± 8.6 ng/mL, p = 0.002). There was a significant positive correlation between whole blood thiamine and BMI (r = 0.342, p = 0.020). SLC19A2 gene expression was lower in those with BMI ≥35 kg/m2 (p = 0.036). A significant positive correlation was found between SLC19A2 expression and whole blood thiamine level (r = 0.310, p = 0.038). CONCLUSION: A possible association between intestinal thiamine intake and total thiamine in whole blood was determined. The transcriptional changes of genes encoding the high-affinity membrane thiamine transporters, especially SLC19A2, probably play a role in this relationship.
RESUMEN
The anti-inflammatory and immunosuppressive drugs which are used in the treatment of Graft-versus-Host Disease (GVHD) have limited effects in controlling the severity of the disease. In this study, we aimed to investigate the prophylactic effect of Alantolactone (ALT) in a murine model of experimental GVHD. The study included 4 BALB/c groups as hosts: Naïve (n = 7), Control GVHD (n = 16), ALT-GVHD (n = 16), and Syngeneic transplantation (n = 10). Busulfan (20 mg/kg/day) for 4 days followed by cyclophosphamide (100 mg/kg/day) were administered for conditioning. Allogeneic transplantation was performed with cells collected from mismatched female C57BL/6, and GVHD development was monitored by histological and flow cytometric assays. Additionally, liver biopsies were taken from GVHD patient volunteers between ages 2-18 (n = 4) and non-GVHD patients between ages 2-50 (n = 5) and cultured ex vivo with ALT, and the supernatants were used for ELISA. ALT significantly ameliorated histopathological scores of the GVHD and improved GVHD clinical scores. CD8+ T cells were shown to be reduced after ALT treatment. More importantly, ALT treatment skewed T cells to a more naïve phenotype (CD62L+ CD44-). ALT did not alter Treg cell number or frequency. ALT treatment appears to suppress myeloid cell lineage (CD11c+). Consistent with reduced myeloid lineage, liver and small intestine levels of GM-CSF were reduced in ALT-treated mice. IL-6 gene expression was significantly reduced in the intestinal tissue. Ex vivo ALT-treated liver biopsy samples from GVHD patients showed a trend of decrease in pro-inflammatory cytokines but there was no statistical significance. Collectively, the data indicated that ALT may have immunomodulatory actions in a preclinical murine GVHD model.
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Linfocitos T CD8-positivos , Enfermedad Injerto contra Huésped , Lactonas , Sesquiterpenos de Eudesmano , Humanos , Ratones , Femenino , Animales , Ratones Endogámicos C57BL , Enfermedad Injerto contra Huésped/prevención & control , Trasplante Homólogo , Trasplante de Médula ÓseaRESUMEN
In 15 Turkish LAD-1 patients and controls, we assessed the impact of pathogenic ITGB2 mutations on Th17/Treg differentiation and functions, and innate lymphoid cell (ILC) subsets. The percentage of peripheral blood Treg cells, in vitro-generated induced Tregs differentiated from naive CD4+ T cells were decreased despite the elevated absolute counts of CD4+ cells in LAD-1 patients. Serum IL-23 levels were elevated in LAD-1 patients. Post-curdlan stimulation, LAD-1 patient-derived PBMCs produced more IL-17A. Additionally, the percentages of CD18-deficient Th17 cells expanded from total or naïve CD4+ T cells were higher. The blood ILC3 subset was significantly elevated in LAD-1. Finally, LAD-1 PBMCs showed defects in trans-well migration and proliferation and were more resistant to apoptosis. Defects in de novo generation of Tregs from CD18-deficient naïve T cells and elevated Th17s, and ILC3s in LAD-1 patients' peripheral blood suggest a type 3-skewed immunity and may contribute to LAD-1-associated autoimmune symptoms.
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Síndrome de Deficiencia de Adhesión del Leucocito , Linfocitos T Reguladores , Humanos , Inmunidad Innata , Linfocitos T CD4-Positivos , Células Th17RESUMEN
Insufficient dietary folate intake, hereditary malabsorption, or defects in folate transport may lead to combined immunodeficiency (CID). Although loss of function mutations in the major intestinal folate transporter PCFT/SLC46A1 was shown to be associated with CID, the evidence for pathogenic variants of RFC/SLC19A1 resulting in immunodeficiency was lacking. We report two cousins carrying a homozygous pathogenic variant c.1042 G > A, resulting in p.G348R substitution who showed symptoms of immunodeficiency associated with defects of folate transport. SLC19A1 expression by peripheral blood mononuclear cells (PBMC) was quantified by real-time qPCR and immunostaining. T cell proliferation, methotrexate resistance, NK cell cytotoxicity, Treg cells and cytokine production by T cells were examined by flow cytometric assays. Patients were treated with and benefited from folinic acid. Studies revealed normal NK cell cytotoxicity, Treg cell counts, and naive-memory T cell percentages. Although SLC19A1 mRNA and protein expression were unaltered, remarkably, mitogen induced-T cell proliferation was significantly reduced at suboptimal folic acid and supraoptimal folinic acid concentrations. In addition, patients' PBMCs were resistant to methotrexate-induced apoptosis supporting a functionally defective SLC19A1. This study presents the second pathogenic SLC19A1 variant in the literature, providing the first experimental evidence that functionally defective variants of SLC19A1 may present with symptoms of immunodeficiency.
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Síndromes de Inmunodeficiencia , Leucovorina , Proteína Portadora de Folato Reducido , Humanos , Ácido Fólico/genética , Ácido Fólico/metabolismo , Leucovorina/uso terapéutico , Leucovorina/metabolismo , Leucocitos Mononucleares/metabolismo , Metotrexato/farmacología , Metotrexato/uso terapéutico , Transportador de Folato Acoplado a Protón/genética , Transportador de Folato Acoplado a Protón/metabolismo , Proteína Portadora de Folato Reducido/genética , Síndromes de Inmunodeficiencia/tratamiento farmacológico , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/metabolismoRESUMEN
PROBLEM: Although pregnant women with gestational diabetes (GD), morbidly adherent placenta (MAP), and pregnancy hypertension (pHT) diseases lead to intrauterine growth restriction (IUGR), little is known about their effect on mucosal-associated invariant T (MAIT) and innate lymphoid cells (ILC) in the umbilical cord. This study aimed to quantify and characterize MAIT cells and ILCs in the cord blood of pregnant women with GD, MAP, and pHT diseases. METHOD OF STUDY: Cord blood mononuclear cells (CBMCs) were isolated by Ficoll-Paque gradient. CD3+ TCRVα7.2+ CD161high cells and ILC subsets were quantified by flow cytometry. CBMCs were stimulated with PMA/Ionomycin and Golgi Plug for 4 h and stained for IFN-γ, TNF-α, and granzyme B. The stained cells were analyzed on FACS ARIA III. RESULTS: Compared with healthy pregnancies, in the cord blood of the pHT group, elevated number of lymphocytes was observed. Moreover, the absolute number of IFN-γ producing CD4+ or CD4- subsets of CD3+ TCRVα7.2+ CD161high cells as well as those producing granzyme B were significantly elevated in the pHT group compared to healthy controls suggesting increased MAIT cell activity in the pHT cord blood. Similarly, in the MAP group, the absolute number of total CD3+ TCRVα7.2+ CD161high cells, but not individual CD4+ or negative subsets, were significantly increased compared with healthy controls' cord blood. Absolute numbers of total CD3+ TCRVα7.2+ CD161high cells and their subsets were comparable in the cord blood of the GD group compared with healthy controls. Finally, the absolute number of total ILCs and ILC3 subset were significantly elevated in only pHT cord blood compared with healthy controls. Our data also reveal that IFN-γ+ or granzyme B+ cell numbers negatively correlated with fetal birth weight. CONCLUSIONS: CD3+ TCRVα7.2+ CD161high cells and ILCs show unique expansion and activity in the cord blood of pregnant women with distinct diseases causing IUGR and may play roles in fetal growth restriction.
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Diabetes Gestacional , Hipertensión Inducida en el Embarazo , Placenta Accreta , Subgrupos de Linfocitos T , Diabetes Gestacional/inmunología , Femenino , Sangre Fetal/citología , Sangre Fetal/inmunología , Granzimas , Humanos , Hipertensión Inducida en el Embarazo/inmunología , Inmunidad Innata , Linfocitos , Placenta/patología , Placenta Accreta/inmunología , Embarazo , Subgrupos de Linfocitos T/citologíaRESUMEN
Multiple Sclerosis is an immune-mediated neurodegenerative disease. IL-23-mediated signaling and Th17 cells play critical roles in disease pathogenesis in murine models of disease and humans. Sphingosine 1 phosphate (S1P) regulates migration of several types of immune cells including Th17 cells. S1P analogues (fingolimod (FTY720) and Siponimod (BAF312)) have been approved and currently used for MS treatment. Immunomodulatory roles for FTY720 have been defined, however, how different S1P analogues impact human Th17 and Treg cell generation and cytokine production, and IL-23-mediated signaling have not yet been explored in detail. In the current study, we investigated the effects of S1P receptor 1 (S1P1) specific S1P analogue SEW2871, S1P1 and S1P5 specific BAF312, and non-selective FTY720 on human Th17 and Treg differentiation and IL-23-mediated signaling. All three S1P analogues directly inhibited Th17 cell differentiation ex vivo while increasing Treg differentiation from naive CD4 + T cells. All three S1P analogues suppressed IL-23-mediated STAT4, NF-kB and AKT activation. Lastly, all three S1P analogues also inhibited Dectin-1 expression by both mature and immature monocyte-derived dendritic cells (moDCs) and in turn curdlan-mediated production of IL-23p19, p40, IL-6 and IL-1ß cytokines. Our results provide novel insight into the immunomodulatory roles of different S1P analogues on human Th17 and Treg cell biology.
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Enfermedades Neurodegenerativas , Esfingosina , Animales , Azetidinas , Compuestos de Bencilo , Clorhidrato de Fingolimod/farmacología , Clorhidrato de Fingolimod/uso terapéutico , Humanos , Inmunosupresores/farmacología , Interleucina-23 , Lisofosfolípidos/farmacología , Ratones , Oxadiazoles , Glicoles de Propileno/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Linfocitos T Reguladores/metabolismo , Células Th17/metabolismo , TiofenosRESUMEN
BACKGROUND: Sitosterolemia, also known as phytosterolemia, results from increased intestinal absorption of plant sterols and decreased intestinal and biliary excretion of sterols, resulting in increased levels of plant sterols in the plasma. The most common symptoms include xanthomas, premature atherosclerosis, hemolytic anemia and macrothrombocytopenia, however delayed diagnosis or misdiagnosis also occur. PATIENT AND METHODS: Clinical exome sequencing was performed on a 10-year-old boy whom we followed up with signs of pancytopenia accompanied by macrothrombocytopenia and stomatocytosis. In addition, the blood sterol levels of the patient and his family were studied. RESULTS: A novel homozygous c.904 + 5G > C intronic variant was detected in ABCG5 gene in index case. The mother and father were identified as carriers. The blood plant sterol levels of the patient and his family were studied, and the levels in the patient confirmed Sitosterolemia. Sitosterol levels decreased dramatically with restricted diet and ezetimibe treatment. CONCLUSION: In children, signs of Sitosterolemia may be subtle and the only symptom may be hematological. Therefore, Sitosterolemia should be kept in mind in children with stomatocytosis and macrothrombocytopenia.
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Hipercolesterolemia , Enfermedades Intestinales , Errores Innatos del Metabolismo Lipídico , Pancitopenia , Fitosteroles , Adolescente , Niño , Humanos , Hipercolesterolemia/diagnóstico , Hipercolesterolemia/genética , Enfermedades Intestinales/complicaciones , Enfermedades Intestinales/diagnóstico , Enfermedades Intestinales/genética , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Masculino , Pancitopenia/complicaciones , Fitosteroles/efectos adversos , Fitosteroles/genética , SitoesterolesRESUMEN
BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with large genetic background, but identification of pathogenic variants has proceeded slowly because hundreds of loci are involved in this complex disorder. CC2D1A gene firstly associated with the intellectual disability (ID) in a family with a large deletion. We aimed to contribute to the literature by sequencing this gene and by this way we report novel CC2D1A variations in patients with ASD. METHODS: Forty families who have a child with a diagnosis of ASD were enrolled to the study. DNA samples were obtained from each family member. Bidirectional CC2D1A gene sequencing was performed with CEQ Cycle Sequencing Kit, and the products were analyzed on the Beckman CEQ 8000. All of the genetic analysis was conducted in Erciyes University Genome and Stem Cell Center (GENKOK). RESULTS: According to the sequencing results, we defined new alterations in this gene with two SNPs in exon 15 and 19 (rs747172992 and rs1364074600) in our patients. We found a pathogenic variant in one patient. This variant was located in the acceptor region. Six of the variants were missense mutations. Additionally, six different benign variants were detected in 30 patients; however, they were not associated with ASD. Two patients carried multiple rare variants. CONCLUSION: In vitro and in vivo functional analysis with this gene will help to understand its contribution to ASD pathogenesis. Future studies may help to elucidate the underlying biological mechanisms of these variants leading to the autism phenotype.
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Trastorno del Espectro Autista , Discapacidad Intelectual , Humanos , Trastorno del Espectro Autista/genética , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Mutación Missense , Proteínas de Unión al ADNRESUMEN
Activation of the hypothalamic-pituitary-adrenal (HPA) axis using an insulin tolerance test (ITT) is a medical diagnostic procedure that is frequently used in humans to assess the HPA and growth-hormone (GH) axes. Whether sex differences exist in the response to ITT stress is unknown. Thus, investigations into the analysis of transcripts during activation of the HPA axis in response to hypoglycemia have revealed the underlying influences of sex in signaling pathways that stimulate the HPA axis. We assessed four time points of ITT application in Balb/c mice. After insulin injection, expression levels of 192 microRNAs and 41 mRNAs associated with the HPA, GH and hypothalamic-pituitary-gonadal (HPG) axes were determined by real-time RT-PCR in the hypothalamus, pituitary and adrenal tissues, as well as blood samples (Raw data accession: https://drive.google.com/drive/folders/10qI00NAtjxOepcNKxSJnQbJeBFa6zgHK?usp=sharing ). Although the ITT is commonly used as a gold standard for evaluating the HPA axis, we found completely different responses between males and females with respect to activation of the HPA axis. While activation of several transcripts in the hypothalamus and pituitary was observed after performing the ITT in males within 10 min, females responded via the pituitary and adrenal immediately and durably over 40 min. Additionally, we found that microRNA alterations precede mRNA responses in the HPA axis. Furthermore, robust changes in the levels of several transcripts including Avpr1b and Avpr2 observed at all time points strongly suggest that transcriptional control of these genes occurs mostly via differential signaling in pituitary and blood between males and females. Male and female HPA axis responses to ITT involve a number of sophisticated regulatory signaling pathways of miRNAs and mRNAs. Our results highlight the first robust markers in several layers of HPA, HPG and GH axis involved in ITT/hypoglycemia stress-induced dynamics.
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Hormona de Crecimiento Humana , Hipoglucemia , Animales , Femenino , Hormona de Crecimiento Humana/metabolismo , Hipoglucemia/inducido químicamente , Hipoglucemia/genética , Hipoglucemia/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Insulina/metabolismo , Masculino , Ratones , Sistema Hipófiso-Suprarrenal/metabolismo , Caracteres Sexuales , Transcriptoma/genéticaRESUMEN
IL-22 is an alpha-helical cytokine which belongs to the IL-10 family of cytokines. IL-22 is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ T, iNKT, Th17 and Th22 cells and some granulocytes. IL-22 receptor is expressed primarily by non-haematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knockout mice were previously shown to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), how temporal IL-22 manipulation in adult mice would affect EAE course has not been studied previously. In this study, we overexpressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to explore the therapeutic impact of IL-22 modulation on the EAE course. IL-22 overexpression significantly decreased EAE scores and demyelination, and reduced infiltration of IFN-γ+IL-17A+Th17 cells into the central nervous system (CNS). The neutralization of IL-22 did not alter the EAE pathology significantly. We show that IL-22-mediated protection is independent of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22. Thus, overexpression of Reg3γ significantly exacerbated EAE scores, demyelination and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+Th17 cells to CNS. We also show that Reg3γ may inhibit IL-2-mediated STAT5 signalling and impair expansion of Treg cells in vivo and in vitro. Finally, Reg3γ overexpression dramatically impacted intestinal microbiota during EAE. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptide Reg3γ in the pathogenesis of CNS inflammation in a murine model of MS.
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Encefalomielitis Autoinmune Experimental/inmunología , Interleucinas/metabolismo , Esclerosis Múltiple/inmunología , Proteínas Asociadas a Pancreatitis/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica , Células HEK293 , Humanos , Interleucinas/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Pancreatitis/genética , Receptores de Interleucina/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Interleucina-22RESUMEN
BACKGROUND: Dedicator of cytokinesis 8 (DOCK8) deficiency is the main cause of the autosomal recessive hyper-IgE syndrome (HIES). We previously reported the selective loss of group 3 innate lymphoid cell (ILC) number and function in a Dock8-deficient mouse model. In this study, we sought to test whether DOCK8 is required for the function and maintenance of ILC subsets in humans. METHODS: Peripheral blood ILC1-3 subsets of 16 DOCK8-deficient patients recruited at the pretransplant stage, and seven patients with autosomal dominant (AD) HIES due to STAT3 mutations, were compared with those of healthy controls or post-transplant DOCK8-deficient patients (n = 12) by flow cytometry and real-time qPCR. Sorted total ILCs from DOCK8- or STAT3-mutant patients and healthy controls were assayed for survival, apoptosis, proliferation, and activation by IL-7, IL-23, and IL-12 by cell culture, flow cytometry, and phospho-flow assays. RESULTS: DOCK8-deficient but not STAT3-mutant patients exhibited a profound depletion of ILC3s, and to a lesser extent ILC2s, in their peripheral blood. DOCK8-deficient ILC1-3 subsets had defective proliferation, expressed lower levels of IL-7R, responded less to IL-7, IL-12, or IL-23 cytokines, and were more prone to apoptosis compared with those of healthy controls. CONCLUSION: DOCK8 regulates human ILC3 expansion and survival, and more globally ILC cytokine signaling and proliferation. DOCK8 deficiency leads to loss of ILC3 from peripheral blood. ILC3 deficiency may contribute to the susceptibility of DOCK8-deficient patients to infections.
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Inmunidad Innata , Síndrome de Job , Citocinas , Factores de Intercambio de Guanina Nucleótido , Humanos , Síndrome de Job/genética , Linfocitos , MutaciónRESUMEN
PURPOSE: Interleukin-2-inducible T cell kinase (ITK) is an important mediator of T cell receptor signaling. Loss of function mutations in ITK results in hypogammaglobulinemia and CD4+ T cell loss in humans, and the patients often present with EBV-associated B cell lymphoproliferative syndrome. Itk-deficient mice show loss of T cell naivety, impaired cytolytic activity of CD8+ T cells, and defects in CD4+ T cell lineage choice decisions. In mice, Itk mutations were shown to affect Th17-Treg lineage choice in favor of the latter. In this study, we explored whether human ITK reciprocally regulates Th17-Treg balance as its murine ortholog. METHODS: Whole Exome Sequencing was used to identify the mutation. ITK-deficient peripheral blood lymphocytes were characterized by FACSAria III-based flow cytometric assays with respect to proliferation, apoptosis, cytokine production, and innate lymphoid cell (ILC) frequency. Sorted T cells from healthy donors were exposed to ibrutinib, an irreversible ITK inhibitor, to assess ITK's contribution to Th17 and Treg cell generation and functions. RESULTS: In this study, we report a child with a novel ITK mutation who showed impaired CD3/CD28 induced proliferation in T cells. ITK-mutant cells were more apoptotic irrespective of TCR activation. More importantly, T cells produced less Th17-associated cytokines IL-17A, IL-22, and GM-CSF. Conversely, Th1-associated IFN-γ production was increased. An irreversible inhibitor of ITK, ibrutinib, blocked ex vivo Th17 generation and IL-17A production, conversely augmented FOXP3 expression only at low doses in Treg cultures. Finally, we analyzed peripheral ILC populations and observed a relative decrease in ILC2 and ILC3 frequency in our ITK-deficient patient. CONCLUSIONS: To our knowledge, this is the first report showing that both genetic and chemical inhibition of ITK result in reduced Th17 generation and function in humans. We also report, for the first time, a reduction in ILC2 and ILC3 populations in an ITK-deficient human patient.
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Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Inmunidad Innata , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/deficiencia , Linfocitos T Reguladores/metabolismo , Células Th17/metabolismo , Animales , Apoptosis , Biomarcadores , Proliferación Celular , Preescolar , Consanguinidad , Citocinas/metabolismo , Análisis Mutacional de ADN , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Linaje , Proteínas Tirosina Quinasas/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Células Th17/inmunologíaRESUMEN
Sphingosine-1 phosphate receptor 1 (S1PR1) is expressed by lymphocytes and regulates their egress from secondary lymphoid organs. Innate lymphoid cell (ILC) family has been expanded with the discovery of group 1, 2 and 3 ILCs, namely ILC1, ILC2 and ILC3. ILC3 and ILC1 have remarkable similarity to CD4+ helper T cell lineage members Th17 and Th1, respectively, which are important in the pathology of multiple sclerosis (MS). Whether human ILC subsets express S1PR1 or respond to its ligands have not been studied. In this study, we used peripheral blood/cord blood and tonsil lymphocytes as a source of human ILCs. We show that human ILCs express S1PR1 mRNA and protein and migrate toward S1P receptor ligands. Comparison of peripheral blood ILC numbers between fingolimod-receiving and treatment-free MS patients revealed that, in vivo, ILCs respond to fingolimod, an S1PR1 agonist, resulting in ILC-penia in circulation. Similarly, murine ILCs responded to fingolimod by exiting blood and accumulating in the secondary lymph nodes. Importantly, ex vivo exposure of ILC3 and ILC1 to fingolimod or SEW2871, another S1PR1 antagonist, reduced production of ILC3- and ILC1- associated cytokines GM-CSF, IL-22, IL-17, and IFN-γ, respectively. Surprisingly, despite reduced number of lamina propria-resident ILC3s in the long-term fingolimod-treated mice, ILC3-associated IL-22, IL-17A, GM-CSF and antimicrobial peptides were high in the gut compared to controls, suggesting that its long term use may not compromise mucosal barrier function. To our knowledge, this is the first study to investigate the impact of fingolimod on human ILC subsets in vivo and ex vivo, and provides insight into the impact of long term fingolimod use on ILC populations.
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Clorhidrato de Fingolimod/metabolismo , Linfocitos/efectos de los fármacos , Esclerosis Múltiple/inmunología , Receptores de Esfingosina-1-Fosfato/metabolismo , Animales , Células Cultivadas , Citocinas/metabolismo , Humanos , Inmunidad Innata , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Esfingosina-1-Fosfato/agonistas , Células TH1/inmunología , Células Th17/inmunología , Distribución TisularRESUMEN
Triple-negative breast cancer (TNBC) is associated with poor prognosis owing to its aggressive and heterogeneous nature, and the lack of therapeutic targets. Although Forkhead Box M1 (FOXM1) is one of the most important oncogenes contributing to tumorigenesis, progression, and drug resistance in TNBC, the underlying molecular mechanisms are not well understood. Emerging evidence indicates that autophagy plays a critical role in cell survival and protective mechanism in TNBC. However, signaling pathways that are involved in the regulation of autophagy remain to be elucidated. In the present study, we examined the role of FOXM1 in regulating autophagy in TNBC cells and found that FOXM1 is upregulated during induction of autophagy. We found that inhibition of FOXM1 suppressed starvation and rapamycin-induced autophagy and expression of the major autophagy regulators, LC3 and Beclin-1. Further studies demonstrated that FOXM1 directly binds to the promotors of LC3 and Beclin-1 genes and transcriptionally regulates their expression by chromatin immunoprecipitation (ChIP) and luciferase gene reporter assays. In conclusion, our study provides the first evidence about the role of FOXM1 in regulating expression of LC3 and Beclin-1 and autophagy in TNBC cells. Our findings provide novel insight into the role of FOXM1 regulation of the autophagic survival pathway and potential molecular target for treating TNBC. KEY MESSAGES: ⢠FOXM1 promotes tumorigenesis and progression of TNBC. However, the underlying molecular mechanism by which FOXM1 promotes TNBC tumorigenesis is unclear. The goal of our study was to determine the role of FOXM1 in the regulation of autophagy that plays a role in TNBC progression. Our findings show that FOXM1 binds to promoters of the genes encoding the major autophagy proteins, Beclin and LC3, and provide new insights into the regulation of autophagy, which is being targeted in many clinical trials.
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Beclina-1/genética , Proteína Forkhead Box M1/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Asociadas a Microtúbulos/genética , Neoplasias de la Mama Triple Negativas/genética , Autofagia , Línea Celular Tumoral , Femenino , Humanos , Activación Transcripcional , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Autism spectrum disorder (ASD) is characterized by repetitive stereotypic behaviors, restricted interests, social withdrawal, and communication deficits. Aggression and insensitivity to pain are largely unexplained in these cases. We analyzed nine mRNA expressions of the candidate genes related to aggression and insensitivity to pain in the peripheral blood of patients with ASD. Whole blood samples were obtained from 40 autistic patients (33 boys, 7 girls) and 50 age- and sex-matched controls (37 boys and 13 girls) to isolate RNA. Gene expression was assessed by quantitative Real-Time PCR (qRT-PCR) in the Erciyes University Genome and Stem Cell Center (GENKOK). All of the gene expressions except CRHR1 and SLC6A4 were found to be statistically different between the ASD patients and controls. Gene expression also differed according to gender. Alterations in the mRNA expression patterns of the HTR1E, OPRL1, OPRM1, TACR1, PRKG1, SCN9A and DRD4 genes provide further evidence for a relevant effect of the respective candidate genes on the pathophysiology of ASD. Future studies may determine the sensitivity of these candidate markers in larger samples including further neuropsychiatric diagnosis.
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Trastorno del Espectro Autista/genética , Canal de Sodio Activado por Voltaje NAV1.7/genética , ARN Mensajero/sangre , Receptor de Serotonina 5-HT1A/genética , Receptores de Dopamina D4/genética , Receptores Opioides/genética , Agresión , Trastorno del Espectro Autista/sangre , Trastorno del Espectro Autista/fisiopatología , Biomarcadores/sangre , Preescolar , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Femenino , Humanos , Masculino , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Percepción del Dolor , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , Receptores de Dopamina D4/metabolismo , Receptores Opioides/metabolismoRESUMEN
OBJECTIVE: In this study, we aimed to assess the effects of long- and short-term IL-15 cytokine exposure of human monocyte-derived curdlan-matured dendritic cells (DCs) on the production of Th17 cell-polarizing cytokine IL-23 and subsequent Th17 cell activation. METHODS: Peripheral blood mononuclear cells (PBMCs) were purified using Ficoll-Paque from healthy donors. Monocytes were magnetically selected using CD14 Miltenyi beads and differentiated into DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for five days in the presence or absence of IL-15 (100ng/ml) for long-term exposure experiments. Then, DCs were matured with peptidoglycan (PGN), or curdlan for 24 hours. For short-term exposure experiments, IL-15 was added only during maturation of DCs. Then, DCs were characterized concerning the expression of MHC II and costimulatory molecules, production of cytokine subunits IL-23p19, IL-12p40, IL-12p35 and cytokine IL-23 via flow cytometry or real-time qPCR or ELISA. Finally, the phosphorylation of signaling molecules after curdlan stimulation was assessed using phospho-flow assays. RESULTS: IL-15 exposure suppressed IL-23 production by DCs. As a result, IL-15-exposed DCs suppressed IL-17 production by allogeneic T cells. Importantly, we observed a reduction in the surface Dectin-1 receptor levels by IL-15-exposed DCs. In line with these observations, curdlan stimulation resulted in reduced phosphorylation of ERK1/2, NF-kB p65 and AKT by human DCs exposed to IL-15 compared with controls. These results may explain why IL-15-exposed DCs produce less IL-23 after maturation with curdlan, which is a ligand of Dectin-1. CONCLUSION: Short- or long-term exposure to IL-15 of human DCs during their differentiation or maturation programs DCs against Th17 cell polarization, which suggests that IL-15 availability may affect CD4+ T cell-mediated protective immunity to fungal infections.
RESUMEN
Autophagy is a catabolic process for degrading dysfunctional proteins and organelles, and closely associated with cancer cell survival under therapeutic, metabolic stress, hypoxia, starvation and lack of growth factors, contributing to resistance to therapies. However, the role of autophagy in breast cancer cells is not well understood. In the present study, we investigated the role of autophagy in highly aggressive and metastatic triple negative breast cancer (TNBC) and non-metastatic breast cancer cells and demonstrated that the knockdown of autophagy-related genes (LC3 and Beclin-1) inhibited autophagy and significantly suppressed cell proliferation, colony formation, migration/invasion and induced apoptosis in MDA-MB-231 and BT-549 TNBC cells. Knockdown of LC3 and Beclin-1 led to inhibition of multiple proto-oncogenic signaling pathways, including cyclin D1, uPAR/integrin-ß1/Src, and PARP1. In conclusion, our study suggests that LC3 and Beclin-1 are required for cell proliferation, survival, migration and invasion, and may contribute to tumor growth and progression of highly aggressive and metastatic TNBC cells and therapeutic targeting of autophagy genes may be a potential therapeutic strategy for TNBC in breast cancer.
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Beclina-1/antagonistas & inhibidores , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Terapia Molecular Dirigida/métodos , ARN Interferente Pequeño/farmacología , Neoplasias de la Mama Triple Negativas/patología , Autofagia/genética , Beclina-1/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Ciclina D1/antagonistas & inhibidores , Ciclina D1/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Integrina beta1/metabolismo , Células MCF-7 , Proteínas Asociadas a Microtúbulos/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Neoplasias de la Mama Triple Negativas/genética , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
BACKGROUND: The number of cancer cases around the world has increased according to the World Health Organization (WHO) reports, nearly 14 million new cases and 8.2 million cancer associated mortalities have been reported in 2012. Chemotherapeutic resistance is a major problematic issue in the management of patients with breast tumor. OBJECTIVE: In this study, the apoptotic gene profile of 4T1 mouse breast cancer cells treated with MC-A in combination with cisplatin or epirubicin was evaluated to decipher the possible apoptotic molecular targets. METHODS: The effects of MC-A in combination with cisplatin (CIS) or epirubicin (EPI) on cytotoxicity, cell migration, wound healing, clonogenicity along with enhanced effect of these combinations on 84 apoptosis related genes were tested in 4T1 cancer cells. RESULTS: MC-A in combination with epirubicin or cisplatin robustly induced cytotoxicity in 4T1 cells in vitro. MC-A in combination with cisplatin or epirubicin showed significantly inhibition of cell migration compared to treatment with each agent alone. Genes involved in positive regulation of apoptosis, negative regulator of apoptosis, death-like, mitochondrial apoptotic signaling, induction of apoptosis through DR3 and DR4/5 death receptors, and anti-apoptosis were highly affected in MC-A+cisplatin or MC-A+epirubicin combinations compared to each agent only. CONCLUSIONS: In conclusion, the apoptotic response of 4T1 cancer cells to chemotherapeutic drugs occurs in different ways. MC-A in combination with these chemotherapeutic drugs could modulate the expression of genes involved in both extrinsic and intrinsic pathways of apoptosis, leading to higly effective apoptotic signalling in cancer treatment.