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1.
Nat Commun ; 13(1): 4468, 2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35918309

RESUMO

Bacteria-based tumor therapy has recently attracted wide attentions due to its unique capability in targeting tumors and preferentially colonizing the core area of the tumor. Various therapeutic genes are also harbored into these engineering bacteria to enhance their anti-tumor efficacy. However, it is difficult to spatiotemporally control the expression of these inserted genes in the tumor site. Here, we engineer an ultrasound-responsive bacterium (URB) which can induce the expression of exogenous genes in an ultrasound-controllable manner. Owing to the advantage of ultrasound in tissue penetration, an acoustic remote control of bacterial gene expression can be realized by designing a temperature-actuated genetic switch. Cytokine interferon-γ (IFN-γ), an important immune regulatory molecule that plays a significant role in tumor immunotherapy, is used to test the system. Our results show that brief hyperthermia induced by focused ultrasound promotes the expression of IFN-γ gene, improving anti-tumor efficacy of URB in vitro and in vivo. Our study provides an alternative strategy for bacteria-mediated tumor immunotherapy.


Assuntos
Interferon gama , Neoplasias , Bactérias/metabolismo , Citocinas , Humanos , Imunoterapia/métodos , Interferon gama/genética , Interferon gama/metabolismo , Neoplasias/genética , Neoplasias/terapia
2.
Int J Immunopathol Pharmacol ; 36: 3946320221117933, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35932160

RESUMO

OBJECTIVES: T helper 17 (Th17) cells are involved in the inflammatory response of atherosclerosis. However, their heterogeneity in the atherosclerotic aorta remains elusive. This study was designed to identify aortic Th17 subsets. METHODS: The surface markers and transcription factors of aortic interleukin-17A (IL-17A)-expressing T cells were determined by flow cytometry in an ApoE-deficient mouse atherosclerotic model. Viable aortic IL-17A-expressing T cell subsets were isolated by flow cytometry on the basis of surface markers, followed by characterizing their transcription factors by either flow cytometry or real-time RT-PCR. The effect of aortic IL-17A-expressing T cell subsets on aortic endothelial cells was determined in vitro. RESULTS: C-X-C Motif Chemokine Receptor 3 (CXCR3), interleukin-17 receptor E (IL-17RE), CD200, and C-C Motif Chemokine Receptor 4 (CCR4) marked three subsets of aortic IL-17A-expressing T cells: CXCR3+IL-17RElowCD200+CCR4- T cells expressing T-box protein expressed in T cells (T-bet) and interferon-gamma (IFN-γ), CXCR3+IL-17RElowCD200+CCR4+ T cells expressing T-bet but fewer IFN-γ, and CXCR3-IL-17REhighCD200+CCR4+ T cells expressing very low T-bet and no IFN-γ. Based on these markers, viable aortic Th17 cells, Th17.1 cells, and transitional Th17.1 cells were identified. Both Th17.1 cells and transitional Th17.1 cells were more proliferative than Th17 cells. Compared with Th17 cells, Th17.1 cells plus transitional Th17.1 cells induced higher expression of C-X-C motif chemokine ligand 1 (CXCL1), C-C motif chemokine ligand 2 (CCL2), C-X-C motif chemokine 5 (CXCL5), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in aortic endothelial cells. CONCLUSION: IL-17A-expressing CD4+ T cells were heterogeneous in atherosclerotic aortas.


Assuntos
Aterosclerose , Interleucina-17 , Animais , Aorta/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Ligantes , Camundongos , Receptores de Quimiocinas/metabolismo , Células Th17/metabolismo , Fatores de Transcrição/metabolismo
3.
Nat Commun ; 13(1): 4605, 2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941154

RESUMO

Dogma holds that Toxoplasma gondii persists in neurons because neurons cannot clear intracellular parasites, even with IFN-γ stimulation. As several recent studies questioned this idea, here we use primary murine neuronal cultures from wild type and transgenic mice in combination with IFN-γ stimulation and parental and transgenic parasites to reassess IFN-γ dependent neuronal clearance of intracellular parasites. We find that neurons respond to IFN-γ and that a subset of neurons clear intracellular parasites via immunity regulated GTPases. Whole neuron reconstructions from mice infected with parasites that trigger neuron GFP expression only after full invasion reveal that ~50% of these T. gondii-invaded neurons no longer harbor parasites. Finally, IFN-γ stimulated human pluripotent stem cell derived neurons show an ~50% decrease in parasite infection rate when compared to unstimulated cultures. This work highlights the capability of human and murine neurons to mount cytokine-dependent anti-T. gondii defense mechanisms in vitro and in vivo.


Assuntos
Parasitos , Toxoplasma , Animais , GTP Fosfo-Hidrolases/metabolismo , Humanos , Interferon gama/metabolismo , Camundongos , Neurônios/metabolismo , Parasitos/metabolismo , Toxoplasma/metabolismo
4.
BMC Cancer ; 22(1): 864, 2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941558

RESUMO

BACKGROUND: IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. METHODS: In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. RESULTS: The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. CONCLUSIONS: These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.


Assuntos
Arginina , Neoplasias da Mama , Animais , Arginina/metabolismo , Argininossuccinato Sintase/metabolismo , Mama/metabolismo , Neoplasias da Mama/metabolismo , Bovinos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Interferon gama/metabolismo
5.
Clin Transl Med ; 12(8): e999, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35917405

RESUMO

BACKGROUND: T helper cells in patients with autoimmune disease of idiopathic inflammatory myopathies (IIM) are characterized with the proinflammatory phenotypes. The underlying mechanisms remain unknown. METHODS: RNA sequencing was performed for differential expression genes. Gene expression in CD4+ T-cells was confirmed by quantitative real-time PCR. CD4+ T-cells from IIM patients or healthy controls were evaluated for metabolic activities by Seahorse assay. Glucose uptake, T-cell proliferation and differentiation were evaluated and measured by flow cytometry. Human CD4+ T-cells treated with iron chelators or Pfkfb4 siRNA were measured for glucose metabolism, proliferation and differentiation. Signalling pathway activation was evaluated by western blot and flow cytometry. Mouse model of experimental autoimmune myositis (EAM) were induced and treated with iron chelator or rapamycin. CD4+ T-cell differentiation and muscle inflammation in the EAM mice were evaluated. RESULTS: RNA-sequencing analysis revealed that iron was involved with glucose metabolism and CD4+ T-cell differentiation. IIM patient-derived CD4+ T-cells showed enhanced glycolysis and mitochondrial respiration, which was inhibited by iron chelation. CD4+ T-cells from patients with IIM was proinflammatory and iron chelation suppressed the differentiation of interferon gamma (IFNγ)- and interleukin (IL)-17A-producing CD4+ T-cells, which resulted in an increased percentage of regulatory T (Treg) cells. Mechanistically, iron promoted glucose metabolism by an upregulation of PFKFB4 through AKT-mTOR signalling pathway. Notably, the knockdown of Pfkfb4 decreased glucose influx and thus suppressed the differentiation of IFNγ- and IL-17A-producing CD4+ T-cells. In vivo, iron chelation inhibited mTOR signalling pathway and reduced PFKFB4 expression in CD4+ T-cells, resulting in reduced proinflammatory IFNγ- and IL-17A-producing CD4+ T-cells and increased Foxp3+ Treg cells, leading to ameliorated muscle inflammation. CONCLUSIONS: Iron directs CD4+ T-cells into a proinflammatory phenotype by enhancing glucose metabolism. Therapeutic targeting of iron metabolism should have the potential to normalize glucose metabolism in CD4+ T-cells and reverse their proinflammatory phenotype in IIM.


Assuntos
Doenças Autoimunes , Miosite , Animais , Glucose , Humanos , Inflamação , Interferon gama/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Ferro , Quelantes de Ferro , Camundongos , Miosite/tratamento farmacológico , Fosfofrutoquinase-2 , Linfócitos T Auxiliares-Indutores/metabolismo , Serina-Treonina Quinases TOR/genética , Virulência
6.
Front Immunol ; 13: 917790, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35924240

RESUMO

Development of standardized metrics to support manufacturing and regulatory approval of mesenchymal stromal cell (MSC) products is confounded by heterogeneity of MSC populations. Many reports describe fundamental differences between MSCs from various tissues and compare unstimulated and activated counterparts. However, molecular information comparing biological profiles of activated MSCs across different origins and donors is limited. To better understand common and source-specific mechanisms of action, we compared the responses of 3 donor populations each of human umbilical cord (UC) and bone marrow (BM) MSCs to TNF-α, IL-1ß or IFN-γ. Transcriptome profiles were analysed by microarray and select secretome profiles were assessed by multiplex immunoassay. Unstimulated (resting) UC and BM-MSCs differentially expressed (DE) 174 genes. Signatures of TNF-α-stimulated BM and UC-MSCs included 45 and 14 new DE genes, respectively, while all but 7 of the initial 174 DE genes were expressed at comparable levels after licensing. After IL-1ß activation, only 5 of the 174 DE genes remained significantly different, while 6 new DE genes were identified. IFN-γ elicited a robust transcriptome response from both cell types, yet nearly all differences (171/174) between resting populations were attenuated. Nine DE genes predominantly corresponding to immunogenic cell surface proteins emerged as a BM-MSC signature of IFN-γ activation. Changes in protein synthesis of select analytes correlated modestly with transcript levels. The dynamic responses of licensed MSCs documented herein, which attenuated heterogeneity between unstimulated populations, provide new insight into common and source-imprinted responses to cytokine activation and can inform strategic development of meaningful, standardized assays.


Assuntos
Células-Tronco Mesenquimais , Fator de Necrose Tumoral alfa , Humanos , Interferon gama/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transcriptoma , Fator de Necrose Tumoral alfa/metabolismo , Cordão Umbilical
7.
Int J Med Sci ; 19(8): 1265-1274, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35928722

RESUMO

Objective: To investigate the efficiency and potential mechanisms of exosomes from dendritic cells (DCs) transfected with Forkhead box protein P3 (FOXP3) in the development of experimental autoimmune encephalomyelitis (EAE). Method: Mouse bone marrow-derived immature DCs were loaded with adenovirus carrying FOXP3 gene, and exosomes were generated. Then the exosomes with FOXP3 (FOXP3-EXOs) were co-cultured with CD4+T cell in vitro to evaluate their potential on CD4+T cell proliferation and differentiation, and injected into EAE mice to assess their effects on the development of EAE. Result: FOXP3-EXOs were effective to inhibit the CD4+T cell proliferation and the production of Interferon gamma (IFN-γ), interleukin (IL)-6, and IL-17, while they promoted the production of IL-10 in vitro. Moreover, FOXP3-EXOs treatment significantly decreased the neurological scores, reduced the infiltration of inflammatory cells into the spinal cord, and decreased demyelination in comparison to saline and Con-EXOs treated EAE mice. Moreover, the FOXP3-EXOs treatment resulted in obvious increases in the levels of regulatory T (Treg) cells and IL-10, whereas levels of T helper 1 (Th1) cells, Th17 cells, IFN-γ, IL-6, and IL-17 decreased significantly in the splenocyte culture of EAE mice. Conclusion: The present study preliminarily investigated the effects and potential mechanisms of FOXP3-EXOs in EAE and revealed that the FOXP3-EXOs could inhibit the production of Th1 and Th17 cells and promote the production of Treg cells as well as ameliorate the development of EAE. The neuroprotective effects of FOXP3-EXOs on EAE are likely due to the regulation of Th/Treg balance.


Assuntos
Encefalomielite Autoimune Experimental , Exossomos , Animais , Células Dendríticas , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/terapia , Exossomos/genética , Exossomos/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores , Células Th17
8.
Theranostics ; 12(11): 5086-5102, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35836797

RESUMO

Background: The up-regulation of PD-L1 is recognized as an adaption of cancer cells to evade immune surveillance and attack. However, the intrinsic mechanisms of the induction of PD-L1 by interferon-γ (IFN-γ) in tumor microenvironment remain incompletely characterized. Ubiquitin ligase E3 component N-recognition protein 5 (UBR5) has a critical role in tumorigenesis of triple negative breast cancer (TNBC) by triggering specific immune responses to the tumor. Dual targeting of UBR5 and PD-L1 exhibited superior therapeutic benefits in a preclinical TNBC model in short term. Methods: The regulation of UBR5 to PD-L1 upon IFN-γ stimulation was evaluated through in UBR5 deficiency, reconstitution or overexpression cell line models by quantitative PCR, immunohistochemistry and RNA-seq. The effects of PD-L1 regulation by UBR5 and double blockade of both genes were evaluated in mouse TNBC model. Luciferase reporter assay, chromatin immunoprecipitation-qPCR and bioinformatics analysis were performed to explore the transcription factors involved in the regulation of UBR5 to PD-L1. Results: E3 ubiquitin ligase UBR5 plays a key role in IFN-γ-induced PDL1 transcription in TNBC in an E3 ubiquitination activity-independent manner. RNA-seq-based transcriptomic analyses reveal that UBR5 globally affects the genes in the IFN-γ-induced signaling pathway. Through its poly adenylate binding (PABC) domain, UBR5 enhances the transactivation of PDL1 by upregulating protein kinase RNA-activated (PKR), and PKR's downstream factors including signal transducers and activators of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1). Restoration of PD-L1 expression in UBR5-deficient tumor cells recoups their malignancy in vivo, whereas CRISPR/Cas9-mediated simultaneous abrogation of UBR5 and PD-L1 expression yields synergistic therapeutic benefits than either blockade alone, with a strong impact on the tumor microenvironment. Conclusions: This study identifies a novel regulator of PDL1 transcription, elucidates the underlying molecular mechanisms and provides a strong rationale for combination cancer immunotherapies targeting UBR5 and PD-L1.


Assuntos
Antígeno B7-H1 , Neoplasias de Mama Triplo Negativas , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Interferon gama/metabolismo , Camundongos , Neoplasias de Mama Triplo Negativas/patologia , Evasão Tumoral/genética , Microambiente Tumoral/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Viruses ; 14(7)2022 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-35891518

RESUMO

Natural killer (NK) cells mount an immune response against hepatitis C virus (HCV) infection and can be activated by several cytokines, including interleukin-2 (IL-2), IL-15, and interferon-alpha (IFN-α). By exploiting the Huh7.5 hepatoma cell line infected with the HCV JFH1 genome, we provide novel insights into the antiviral effector functions of human primary NK cells after cytokine stimulation. NK cells activated with IFN-α (IFNα-NKs) had enhanced contact-dependent and -independent responses as compared with NK cells activated with IL-2/IL-15 (IL2/IL15-NKs) and could inhibit HCV replication both in vitro and in vivo. Importantly, IFN-α, but not IL-2/IL-15, protected NK cells from the functional inhibition exerted by HCV. By performing flow cytometry, multiplex cytokine profiling, and mass-spectrometry-based proteomics, we discovered that IFNα-NKs secreted high levels of galectin-9 and interferon-gamma (IFN-γ), and by conducting neutralization assays, we confirmed the major role of these molecules in HCV suppression. We speculated that galectin-9 might act extracellularly to inhibit HCV binding to host cells and downstream infection. In silico approaches predicted the binding of HCV envelope protein E2 to galectin-9 carbohydrate-recognition domains, and co-immunoprecipitation assays confirmed physical interaction. IFN-γ, on the other hand, triggered the intracellular expressions of two antiviral gate-keepers in target cells, namely, myxovirus-1 (MX1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1). Collectively, our data add more complexity to the antiviral innate response mediated by NK cells and highlight galectin-9 as a key molecule that might be exploited to neutralize productive viral infection.


Assuntos
Hepacivirus , Hepatite C , Antivirais/uso terapêutico , Citocinas/metabolismo , Galectinas/metabolismo , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Humanos , Interferon-alfa/metabolismo , Interferon gama/metabolismo , Interleucina-15 , Células Matadoras Naturais
10.
Cells ; 11(14)2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35883685

RESUMO

Gamma-interferon (γ-IFN) significantly inhibits infection by replication-defective viral vectors derived from the human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) but the underlying mechanism remains unclear. Previously we reported that knockdown of γ-IFN-inducible lysosomal thiolreductase (GILT) abrogates the antiviral activity of γ-IFN in TE671 cells but not in HeLa cells, suggesting that other γ-IFN-inducible host factors are involved in its antiviral activity in HeLa cells. We identified cellular factors, the expression of which are induced by γ-IFN in HeLa cells, using a microarray, and analyzed the effects of 11 γ-IFN-induced factors on retroviral vector infection. Our results showed that the exogenous expression of FAT10, IFI6, or IDO1 significantly inhibits both HIV-1- and MLV-based vector infections. The antiviral activity of γ-IFN was decreased in HeLa cells, in which the function of IDO1, IFI6, FAT10, and GILT were simultaneously inhibited. IDO1 is an enzyme that metabolizes an essential amino acid, tryptophan. However, IDO1 did not restrict retroviral vector infection in Atg3-silencing HeLa cells, in which autophagy did not occur. This study found that IDO1, IFI6, FAT10, and GILT are involved in the antiviral activity of γ-IFN, and IDO1 inhibits retroviral infection by inducing autophagy.


Assuntos
Infecções por HIV , HIV-1 , Infecções por Retroviridae , Antirretrovirais/farmacologia , Antivirais/farmacologia , Autofagia , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama/metabolismo , Interferon gama/farmacologia , Vírus da Leucemia Murina , Proteínas Mitocondriais , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Ubiquitinas/farmacologia
11.
Front Cell Infect Microbiol ; 12: 828439, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35873142

RESUMO

Objectives: The host immune response towards Mycobacterium tuberculosis (M. tb) is known to vary with the virulence of mycobacterial species. While the majority of M. tb-exposed individuals develop latent TB infection (LTBI), a small proportion develops active TB disease. The milieu of understudied immune factors is believed to play an important role against host immune response towards mycobacteria. Here, we investigate the role of antiviral factors of the interferon-induced proteins with tetracopeptides (IFITs) family, which, in our previous research, have shown to be upregulated in response to pathogenic M. tb, but as yet have no established role in host response to bacterial infections. Methods: We performed vector-driven overexpression and siRNA-mediated downregulation of IFITs in THP-1 cells infected with different mycobacterial species. Also, we investigated the mRNA levels of IFITs in the LTBI and active-TB cases. Results: Overexpression of IFITs reduces CFUs by ~32% (30%-43%) [Median (IQR)] across three different mycobacterial strains, while knock-down increases CFUs by ~57% (41%-78%). Compared to IFN-γ, treatment of infected THP-1 cells with IFN-ß significantly increases the expression of IFITs, while the overexpression of IFITs had higher mRNA expression of IFN-ß than IFN-γ. Cytokines like IDO-1, IL-6, IL-23, and IFN- γ are observed to play key roles in mycobacterial survival upon IFITs intervention. mRNA expression levels of IFITs were higher in LTBI cases as compared to active TB. Conclusion: Higher expression levels of IFITs reduce in vitro survival of different drug-susceptible and drug-resistant mycobacteria and correlates with latent TB infection in infected individuals, hence emerging as an immuno-therapeutic target against M. tb.


Assuntos
Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose , Antígenos de Bactérias , Antivirais , Citocinas , Humanos , Interferon gama/metabolismo , Interferons , RNA Mensageiro
12.
Cell Signal ; 97: 110400, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35820543

RESUMO

Expression of the immune checkpoint programmed death ligand-1 (PD-L1) is increased in ovarian cancer (OC) and correlates with poor prognosis. Interferon-γ (IFNγ) induces PD-L1 expression in OC cells, resulting in their increased proliferation and tumor growth, but the mechanisms that regulate the PD-L1 expression in OC remain unclear. Here, we show that the IFNγ-induced PD-L1 expression in OC cells is associated with increased levels of STAT1, Tyr-701 pSTAT1 and Ser-727 pSTAT1. Suppression of JAK1 and STAT1 significantly decreases the IFNγ-induced PD-L1 expression in OC cells, and STAT1 overexpression increases the IFNγ-induced PD-L1 expression. In addition, IFNγ induces expression of the transcription factor interferon regulatory factor 1 (IRF1) and IRF1 suppression attenuates the IFNγ-induced gene and protein levels of PD-L1. Chromatin immunoprecipitation results show that IFNγ induces PD-L1 promoter acetylation and recruitment of STAT1, Ser-727 pSTAT1 and IRF1 in OC cells. Together, these findings demonstrate that the IFNγ-induced PD-L1 expression in OC cells is regulated by JAK1, STAT1, and IRF1 signaling, and suggest that targeting the JAK1/ STAT1/IRF1 pathway may provide a leverage to regulate the PD-L1 levels in ovarian cancer.


Assuntos
Antígeno B7-H1 , Neoplasias Ovarianas , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Interferon gama/metabolismo , Interferon gama/farmacologia , Janus Quinase 1/metabolismo , Neoplasias Ovarianas/genética , Fator de Transcrição STAT1/metabolismo
13.
Proc Natl Acad Sci U S A ; 119(31): e2205042119, 2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35881799

RESUMO

Dimethyl fumarate (DMF) is an immunomodulatory treatment for multiple sclerosis (MS). Despite its wide clinical use, the mechanisms underlying clinical response are not understood. This study aimed to reveal immune markers of therapeutic response to DMF treatment in MS. For this purpose, we prospectively collected peripheral blood mononuclear cells (PBMCs) from a highly characterized cohort of 44 individuals with MS before and at 12 and 48 wk of DMF treatment. Single cells were profiled using high-dimensional mass cytometry. To capture the heterogeneity of different immune subsets, we adopted a bioinformatic multipanel approach that allowed cell population-cluster assignment of more than 50 different parameters, including lineage and activation markers as well as chemokine receptors and cytokines. Data were further analyzed in a semiunbiased fashion implementing a supervised representation learning approach to capture subtle longitudinal immune changes characteristic for therapy response. With this approach, we identified a population of memory T helper cells expressing high levels of neuroinflammatory cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon γ [IFNγ]) as well as CXCR3, whose abundance correlated with treatment response. Using spectral flow cytometry, we confirmed these findings in a second cohort of patients. Serum neurofilament light-chain levels confirmed the correlation of this immune cell signature with axonal damage. The identified cell population is expanded in peripheral blood under natalizumab treatment, substantiating a specific role in treatment response. We propose that depletion of GM-CSF-, IFNγ-, and CXCR3-expressing T helper cells is the main mechanism of action of DMF and allows monitoring of treatment response.


Assuntos
Biomarcadores Farmacológicos , Citocinas , Fumarato de Dimetilo , Imunossupressores , Esclerose Múltipla , Linfócitos T Auxiliares-Indutores , Biomarcadores Farmacológicos/metabolismo , Citocinas/metabolismo , Fumarato de Dimetilo/farmacologia , Fumarato de Dimetilo/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Interferon gama/metabolismo , Depleção Linfocítica , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Análise de Célula Única , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia
14.
Dev Cell ; 57(15): 1847-1865.e9, 2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35803280

RESUMO

Immune surveillance is critical to prevent tumorigenesis. Gliomas evade immune attack, but the underlying mechanisms remain poorly understood. We show that glioma cells can sustain growth independent of immune system constraint by reducing Notch signaling. Loss of Notch activity in a mouse model of glioma impairs MHC-I and cytokine expression and curtails the recruitment of anti-tumor immune cell populations in favor of immunosuppressive tumor-associated microglia/macrophages (TAMs). Depletion of T cells simulates Notch inhibition and facilitates tumor initiation. Furthermore, Notch-depleted glioma cells acquire resistance to interferon-γ and TAMs re-educating therapy. Decreased interferon response and cytokine expression by human and mouse glioma cells correlate with low Notch activity. These effects are paralleled by upregulation of oncogenes and downregulation of quiescence genes. Hence, suppression of Notch signaling enables gliomas to evade immune surveillance and increases aggressiveness. Our findings provide insights into how brain tumor cells shape their microenvironment to evade immune niche control.


Assuntos
Neoplasias Encefálicas , Glioma , Animais , Neoplasias Encefálicas/metabolismo , Transformação Celular Neoplásica , Citocinas , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Humanos , Evasão da Resposta Imune , Interferon gama/metabolismo , Camundongos , Microambiente Tumoral/fisiologia
15.
J Cancer Res Ther ; 18(3): 704-711, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35900543

RESUMO

Background: Programmed death-1 (PD-1) and T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) are two major immune checkpoint receptors expressed on immune cells and their expression is related to the exhaustion phenotype. In the present in vitro study, blocking of PD-1 and Tim-3 molecules was performed on isolated natural killer (NK) cells from patients with chronic lymphocytic leukemia (CLL) to restore their functional properties. Materials and Methods: NK cells fraction was positively isolated from fresh peripheral blood of 18 CLL patients, treated with anti-PD-1 and anti-Tim-3 blocking monoclonal antibodies and co-cultured with K562 target cells to evaluate their apoptosis induction by Annexin V-PI method. Blocked NK cells were also incubated with anti-CD107a antibody to assess their degranulation properties by flow cytometry. The level of secreted tumor node factor-alpha (TNF-α) and interferon-gamma (IFN-γ) by NK cells was also measured by ELISA. Results: Our results showed similar functional properties in terms of degranulation and apoptosis of K562 target cells by isolated NK cells from CLL patients in PD-1/Tim-3 blocked and control groups. It was also shown that blocking of PD-1 and Tim-3 could not improve the production of pro-inflammatory TNF-α and IFN-γ cytokines by isolated NK cells from CLL patients. Conclusion: Altogether, our results indicated that pretreatment of NK cells with anti-PD-1 and anti-Tim-3 blocking antibodies in CLL patients at early clinical stages cannot improve their functional properties. Besides many other malignancies, the application of checkpoint inhibitors in CLL needs more investigations and complementary studies.


Assuntos
Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Leucemia Linfocítica Crônica de Células B , Receptor de Morte Celular Programada 1/metabolismo , Humanos , Interferon gama/metabolismo , Células K562 , Células Matadoras Naturais , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
16.
Comput Intell Neurosci ; 2022: 5492602, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35814563

RESUMO

Glioblastoma is the most malignant primary glioma. Conventional treatment methods that include surgery, radiotherapy, and chemotherapy have a limited curative effect on the tumor. With the deepening of molecular biology research, molecular targeted therapy has opened a new era of tumor therapy. Programmed death ligand 1 (PD-L1) has been proved to play a pivotal role in the tumor immune evasion process. Previous studies have confirmed the specific expression of PD-L1 in glioblastoma tissues and cells, but there are few studies on inflammation regulating PD-L1 in glioblastoma. In this study, real-time PCR, flow cytometry, and western blot were applied to detect PD-L1 in glioblastoma cells. Short hairpin RNA was used to knock down PD-L1 in glioblastoma cells. Cell counting kit-8 experiment and wound-healing assay were used to detect the proliferation and migration of glioblastoma cells. Here we demonstrated that PD-L1 was overexpressed in glioblastoma cells, and interferon-gamma (IFN-γ) induces PD-L1 in glioblastoma cells via activating p38/JNK/ERK signaling pathways. To summarize, PD-L1 promotes the occurrence and development of glioblastoma. IFN-γ counteracts the tumor-promoting effects mediated by PD-L1 in glioblastoma. IFN-γ regulates PD-L1 through multiple signaling pathways, but the total effect of IFN-γ-mediated inflammatory signals still need to be further explored in glioblastoma. PD-L1 enhances the proliferation and migration of glioblastoma cells by regulating CDK4, CDK6, MMP-2, and vimentin molecules. Most importantly, targeting PD-L1 can be applied in the treatment of glioblastoma. We speculate that IFN-γ may affect glioblastoma through other pathways, and we will continue to further explore the mechanisms in the future.


Assuntos
Antígeno B7-H1 , Glioblastoma , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Interferon gama/metabolismo , Interferon gama/farmacologia , Transdução de Sinais
17.
Int J Mol Sci ; 23(13)2022 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-35806463

RESUMO

Cardiovascular disease (CVD) is causing high mortality worldwide (World Health Organization-WHO, 2015). Atherosclerosis, the hardening and narrowing of arteries caused by the accumulation of fatty acids and lipids (cholesterol plaques), is a main reason of stroke, myocardial infarction, and angina. Present therapies for cardiovascular disease basically use statins such as ß-Hydroxy ß-methylglutaryl-CoA, with <70% efficacy and multiple side effects. An in vitro investigation was conducted to evaluate the impact of kaempferol, a natural medication, in an atherosclerotic cell model. We used cytotoxicity assays, Boyden chamber invasion assays, and quantitative PCR. Affymetrix microarrays were used to profile the entire transcriptome of kaempferol-treated cell lines, and Partek Genomic Suite was used to interpret the results. Kaempferol was not cytotoxic to THP-1 macrophages. In comparison to the control, kaempferol reduced monocyte migration mediated by monocyte chemotactic protein 1 (MCP-1) by 80%. The qPCR results showed a 73.7-fold reduction in MCP-1 and a 2.5-fold reduction in intercellular adhesion molecule 1 (ICAM-1) expression in kaempferol-treated cells. In interferon gamma (IFN-γ) without kaempferol and IFN-γ with kaempferol treated cells, we found 295 and 168 differentially expressed genes (DEGs), respectively. According to DEG pathway analysis, kaempferol exhibits anti-atherosclerosis and anti-inflammatory characteristics. Kaempferol is an effective and safe therapy for atherosclerosis.


Assuntos
Aterosclerose , Doenças Cardiovasculares , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Doenças Cardiovasculares/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Colesterol/metabolismo , Humanos , Interferon gama/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo
18.
Sci Rep ; 12(1): 11185, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35778468

RESUMO

T cell-dendritic cell (DC) interactions contribute to reciprocal stimulation leading to DC maturation that results in production of interleukin-12 (IL-12) and interferon-gamma (IFN-γ). Both cytokines have been implicated in autoimmune diseases while being necessary for effective immune responses against foreign antigens. We describe a lipidic peptide, designated IK14004, that modifies crosstalk between T cells and DCs resulting in suppression of IL-12p40/IFN-γ production. T cell production of interleukin-2 (IL-2) and IFN-γ is uncoupled and IL-12p70 production is enhanced. IK14004 induces expression of activating co-receptors in CD8+ T cells and increases the proportion of Foxp3-expressing CD4+ T regulatory cells. The potential for IK14004 to impact on signalling pathways required to achieve a balanced immune response upon stimulation of DCs and T cells is highlighted. This novel compound provides an opportunity to gain further insights into the complexity of T cell-DC interactions relevant to autoimmunity associated with malignancies and may have therapeutic benefit.


Assuntos
Células Dendríticas , Linfócitos T Reguladores , Citocinas/metabolismo , Interferon gama/metabolismo , Interleucina-12/metabolismo , Linfócitos T Reguladores/metabolismo
19.
Nat Commun ; 13(1): 4047, 2022 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-35831295

RESUMO

Signal transducer and activator of transcription (STAT) proteins communicate from cell-surface receptors to drive transcription of immune response genes. The parasite Toxoplasma gondii blocks STAT1-mediated gene expression by secreting the intrinsically disordered protein TgIST that traffics to the host nucleus, binds phosphorylated STAT1 dimers, and occupies nascent transcription sites that unexpectedly remain silenced. Here we define a core region within internal repeats of TgIST that is necessary and sufficient to block STAT1-mediated gene expression. Cellular, biochemical, mutational, and structural data demonstrate that the repeat region of TgIST adopts a helical conformation upon binding to STAT1 dimers. The binding interface is defined by a groove formed from two loops in the STAT1 SH2 domains that reorient during dimerization. TgIST binding to this newly exposed site at the STAT1 dimer interface alters its conformation and prevents the recruitment of co-transcriptional activators, thus defining the mechanism of blocked transcription.


Assuntos
Proteínas Intrinsicamente Desordenadas , Toxoplasma , Interferon gama/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Fosforilação , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Domínios de Homologia de src
20.
Int J Mol Sci ; 23(14)2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35887351

RESUMO

Specific T cells are crucial to control SARS-CoV-2 infection, avoid reinfection and confer protection after vaccination. We have studied patients with severe or moderate COVID-19 pneumonia, compared to patients who recovered from a severe or moderate infection that had occurred about 4 months before the analyses. In all these subjects, we assessed the polyfunctionality of virus-specific CD4+ and CD8+ T cells by quantifying cytokine production after in vitro stimulation with different SARS-CoV-2 peptide pools covering different proteins (M, N and S). In particular, we quantified the percentage of CD4+ and CD8+ T cells simultaneously producing interferon-γ, tumor necrosis factor, interleukin (IL)-2, IL-17, granzyme B, and expressing CD107a. Recovered patients who experienced a severe disease display high proportions of antigen-specific CD4+ T cells producing Th1 and Th17 cytokines and are characterized by polyfunctional SARS-CoV-2-specific CD4+ T cells. A similar profile was found in patients experiencing a moderate form of COVID-19 pneumonia. No main differences in polyfunctionality were observed among the CD8+ T cell compartments, even if the proportion of responding cells was higher during the infection. The identification of those functional cell subsets that might influence protection can thus help in better understanding the complexity of immune response to SARS-CoV-2.


Assuntos
COVID-19 , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Humanos , Interferon gama/metabolismo , SARS-CoV-2
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