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1.
Oncoimmunology ; 10(1): 1962591, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34408924

RESUMO

Interferon-gamma (IFN-γ) is a major effector molecule of immunity and a common feature of tumors responding to immunotherapy. Active IFN-γ signaling can directly trigger apoptosis and cell cycle arrest in human cancer cells. However, the mechanisms underlying these actions remain unclear. Here, we report that IFN-γ rapidly increases protein synthesis and causes the unfolded protein response (UPR), as evidenced by the increased expression of glucose-regulated protein 78, activating transcription factor-4, and c/EBP homologous protein (CHOP) in cells treated with IFN-γ. The JAK1/2-STAT1 and AKT-mTOR signaling pathways are required for IFN-γ-induced UPR. Endoplasmic reticulum (ER) stress promotes autophagy and restores homeostasis. Surprisingly, in IFN-γ-treated cells, autophagy was impaired at the step of autophagosome-lysosomal fusion and caused by a significant decline in the expression of lysosomal membrane protein-1 and -2 (LAMP-1/LAMP-2). The ER stress inhibitor 4-PBA restored LAMP expression in IFN-γ-treated cells. IFN-γ stimulation activated the protein kinase-like ER kinase (PERK)-eukaryotic initiation factor 2a subunit (eIF2α) axis and caused a reduction in global protein synthesis. The PERK inhibitor, GSK2606414, partially restored global protein synthesis and LAMP expression in cells treated with IFN-γ. We further investigated the functional consequences of IFN-γ-induced ER stress. We show that inhibition of ER stress significantly prevents IFN-γ-triggered apoptosis. CHOP knockdown abrogated IFN-γ-mediated apoptosis. Inhibition of ER stress also restored cyclin D1 expression in IFN-γ-treated cells. Thus, ER stress and the UPR caused by IFN-γ represent novel mechanisms underlying IFN-γ-mediated anticancer effects. This study expands our understanding of IFN-γ-mediated signaling and its cellular actions in tumor cells.


Assuntos
Apoptose/imunologia , Autofagia/imunologia , Estresse do Retículo Endoplasmático/imunologia , Interferon gama/imunologia , Neoplasias Pulmonares/imunologia , Resposta a Proteínas não Dobradas/imunologia , Humanos , Interferon gama/farmacologia , eIF-2 Quinase/metabolismo
2.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281268

RESUMO

Macrophages (Mφs) are instrumental regulators of the immune response whereby they acquire diverse functional phenotypes following their exposure to microenvironmental cues that govern their differentiation from monocytes and their activation. The complexity and diversity of the mycobacterial cell wall have empowered mycobacteria with potent immunomodulatory capacities. A heat-killed (HK) whole-cell preparation of Mycobacterium obuense (M. obuense) has shown promise as an adjunctive immunotherapeutic agent for the treatment of cancer. Moreover, HK M. obuense has been shown to trigger the differentiation of human monocytes into a monocyte-derived macrophage (MDM) type named Mob-MDM. However, the transcriptomic profile and functional properties of Mob-MDMs remain undefined during an activation state. Here, we characterized cytokine/chemokine release patterns and transcriptomic profiles of lipopolysaccharide (LPS)/interferon γ (IFNγ)-activated human MDMs that were differentiated with HK M. obuense (Mob-MDM(LPS/IFNγ)), macrophage colony-stimulating factor M-MDM(LPS/IFNγ)), or granulocyte/macrophage colony-stimulating factor (GM-MDM(LPS/IFNγ)). Mob-MDM(LPS/IFNγ) demonstrated a unique cytokine/chemokine release pattern (interleukin (IL)-10low, IL-12/23p40low, IL-23p19/p40low, chemokine (C-x-C) motif ligand (CXCL)9low) that was distinct from those of M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ). Furthermore, M-MDM(LPS/IFNγ) maintained IL-10 production at significantly higher levels compared to GM-MDM(LPS/IFNγ) and Mob-MDM(LPS/IFNγ) despite being activated with M1-Mφ-activating stimuli. Comparative RNA sequencing analysis pointed to a distinct transcriptome profile for Mob-MDM(LPS/IFNγ) relative to both M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ) that comprised 417 transcripts. Functional gene-set enrichment analysis revealed significant overrepresentation of signaling pathways and biological processes that were uniquely related to Mob-MDM(LPS/IFNγ). Our findings lay a foundation for the potential integration of HK M. obuense in specific cell-based immunotherapeutic modalities such as adoptive transfer of Mφs (Mob-MDM(LPS/IFNγ)) for cancer treatment.


Assuntos
Quimiocinas/metabolismo , Citocinas/metabolismo , Macrófagos/imunologia , Micobactérias não Tuberculosas/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacocinética , Humanos , Fatores Imunológicos/farmacologia , Técnicas In Vitro , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Transcriptoma
3.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208434

RESUMO

Skullcapflavone II (SFII), a flavonoid derived from Scutellaria baicalensis, has been reported to have anti-inflammatory properties. However, its therapeutic potential for skin inflammatory diseases and its mechanism are unknown. Therefore, this study aimed to investigate the effect of SFII on TNF-α/IFN-γ-induced atopic dermatitis (AD)-associated cytokines, such as thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). Co-stimulation with TNF-α/IFN-γ in HaCaT cells is a well-established model for induction of pro-inflammatory cytokines. We treated cells with SFII prior to TNF-α/IFN-γ-stimulation and confirmed that it significantly inhibited TARC and MDC expression at the mRNA and protein levels. Additionally, SFII also inhibited the expression of cathepsin S (CTSS), which is associated with itching in patients with AD. Using specific inhibitors, we demonstrated that STAT1, NF-κB, and p38 MAPK mediate TNF-α/IFN-γ-induced TARC and MDC, as well as CTSS expression. Finally, we confirmed that SFII significantly suppressed TNF-α/IFN-γ-induced phosphorylation of STAT1, NF-κB, and p38 MAPK. Taken together, our study indicates that SFII inhibits TNF-α/IFN-γ-induced TARC, MDC, and CTSS expression by regulating STAT1, NF-κB, and p38 MAPK signaling pathways.


Assuntos
Catepsinas/biossíntese , Quimiocina CCL17/biossíntese , Quimiocina CCL22/biossíntese , Flavonoides/farmacologia , Interferon gama/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Catepsinas/genética , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL17/genética , Quimiocina CCL22/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HaCaT , Humanos , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
4.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34198981

RESUMO

Recently, adipose-derived stem cells (ADSCs) are considered to be ideal for application in cell therapy or tissue regeneration, mainly due to their wide availability and easy access. In this study, we examined the anti-inflammatory effects of membrane-free stem cell extract (MFSC-Ex) derived from ADSCs against lipopolysaccharide (LPS)/interferon-gamma (IFN-γ) on RAW 264.7 macrophage cells. Exposure of RAW macrophages to LPS and IFN-γ stimuli induced high levels of nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) production. However, pretreatment with MFSC-Ex inhibited LPS/IFN-γ-induced these pro-inflammatory mediators. To clarify the molecular mechanisms underlying the anti-inflammatory property of MFSC-Ex, we analyzed nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) protein expressions by Western blotting. Our study showed that treatment of MFSC-Ex significantly down-regulated inducible nitric oxide synthase (iNOS) and COX-2 protein expressions. Furthermore, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 was also blocked by treatment with MFSC-Ex, indicating that inhibitory effect of MFSC-Ex on MAPK signaling cascade may attribute to inactivation of NF-κB. From these findings, we suggest that MFSC-Ex exert anti-inflammatory activities, which suppressed LPS/IFN-γ-induced production of NO, COX-2 and PGE2 by regulation of NF-κB and MAPK signaling pathway in RAW 264.7 macrophages. In conclusion, MFSC-Ex might provide a new therapeutic opportunity to treatment of inflammatory-related diseases.


Assuntos
Tecido Adiposo/citologia , Anti-Inflamatórios/farmacologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Células-Tronco/metabolismo , Animais , Anti-Inflamatórios/química , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Camundongos , Modelos Biológicos , Substâncias Protetoras/metabolismo , Células RAW 264.7
5.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203758

RESUMO

Synovial fluid contains cytokines, growth factors and resident mesenchymal stem cells (MSCs). The present study aimed to (1) determine the effects of autologous and allogeneic synovial fluid on viability, proliferation and chondrogenesis of equine bone marrow MSCs (BMMSCs) and (2) compare the immunomodulatory properties of equine synovial fluid MSCs (SFMSCs) and BMMSCs after stimulation with interferon gamma (INF-γ). To meet the first aim of the study, the proliferation and viability of MSCs were evaluated by MTS and calcein AM staining assays. To induce chondrogenesis, MSCs were cultured in a medium containing TGF-ß1 or different concentrations of synovial fluid. To meet the second aim, SFMSCs and BMMSCs were stimulated with IFN-γ. The concentration of indoleamine-2,3-dioxygenase (IDO) and nitric oxide (NO) were examined. Our results show that MSCs cultured in autologous or allogeneic synovial fluid could maintain proliferation and viability activities. Synovial fluid affected chondrocyte differentiation significantly, as indicated by increased glycosaminoglycan contents, compared to the chondrogenic medium containing 5 ng/mL TGF-ß1. After culturing with IFN-γ, the conditioned media of both BMMSCs and SFMSCs showed increased concentrations of IDO, but not NO. Stimulating MSCs with synovial fluid or IFN-γ could enhance chondrogenesis and anti-inflammatory activity, respectively, suggesting that the joint environment is suitable for chondrogenesis.


Assuntos
Condrogênese/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Interferon gama/farmacologia , Células-Tronco Mesenquimais/imunologia , Líquido Sinovial/metabolismo , Animais , Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Cavalos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Óxido Nítrico/metabolismo
6.
Nat Commun ; 12(1): 4447, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290243

RESUMO

Tryptophan catabolism is a major metabolic pathway utilized by several professional and non-professional antigen presenting cells to maintain immunological tolerance. Here we report that 3-hydroxy-L-kynurenamine (3-HKA) is a biogenic amine produced via an alternative pathway of tryptophan metabolism. In vitro, 3-HKA has an anti-inflammatory profile by inhibiting the IFN-γ mediated STAT1/NF-κΒ pathway in both mouse and human dendritic cells (DCs) with a consequent decrease in the release of pro-inflammatory chemokines and cytokines, most notably TNF, IL-6, and IL12p70. 3-HKA has protective effects in an experimental mouse model of psoriasis by decreasing skin thickness, erythema, scaling and fissuring, reducing TNF, IL-1ß, IFN-γ, and IL-17 production, and inhibiting generation of effector CD8+ T cells. Similarly, in a mouse model of nephrotoxic nephritis, besides reducing inflammatory cytokines, 3-HKA improves proteinuria and serum urea nitrogen, overall ameliorating immune-mediated glomerulonephritis and renal dysfunction. Overall, we propose that this biogenic amine is a crucial component of tryptophan-mediated immune tolerance.


Assuntos
Aminas Biogênicas/farmacologia , Imunomodulação/efeitos dos fármacos , Cinurenina/análogos & derivados , Animais , Aminas Biogênicas/metabolismo , Aminas Biogênicas/uso terapêutico , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Células Endoteliais , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Inflamação , Interferon gama/farmacologia , Cinurenina/metabolismo , Cinurenina/farmacologia , Cinurenina/uso terapêutico , Camundongos , NF-kappa B/metabolismo , Nefrite/tratamento farmacológico , Nefrite/imunologia , Psoríase/tratamento farmacológico , Psoríase/imunologia , Triptofano/metabolismo
7.
Exp Cell Res ; 406(1): 112738, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34270981

RESUMO

We determined the role of time in adipose-derived stem/stromal cell (ASC) response to a model inflammatory environment. ASCs and other mesenchymal stem/stromal cells exhibit immune plasticity. We evaluated the persistence of pro- and anti-inflammatory phenotypes for ASCs exposed to a sustained or pulse inflammatory stimulus. Using qPCR, flow cytometry, and immunocytochemistry, we monitored the temporal expression and up-regulation patterns of a pro-inflammatory gene (caspase 1), a pleiotropic gene/protein (interleukin 6, IL-6), and an anti-inflammatory gene/protein (indoleamine 2, 3-dioxygenase, IDO1) after exposing ASCs to the cytokines tumor necrosis factor-α and interferon-γ. In response to sustained cytokine stimulation, we discovered that time played a role in the balance of pro- and anti-inflammatory ASC phenotypes. IL-6 was present at all time points for both cytokine-stimulated and non-stimulated conditions, whereas IDO1 was heterogeneously up-regulated in stimulated conditions at later time points. After a pulse stimulus, ASC immunoresponse remained consistent for 96-168 h. As a final measure of immune plasticity, we cultured cytokine-stimulated ASCs with blood-derived macrophages to observe macrophage polarization. While the presence of ASCs altered macrophage phenotype, there was no dependency on the length of ASC cytokine exposure time.


Assuntos
Caspase 1/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Interferon gama/farmacologia , Interleucina-6/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Caspase 1/imunologia , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Regulação da Expressão Gênica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Interleucina-6/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Cultura Primária de Células , Transdução de Sinais , Fatores de Tempo
8.
Molecules ; 26(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065200

RESUMO

Ribes nigrum L. (blackcurrant) leaf extracts, due to high levels of flavonols and anthocyanins, have been shown to exhibit beneficial effects in inflammatory diseases. However, whereas their traditional use has been investigated and validated in several models of inflammation and oxidative stress, the possible impact on skin disorders is still largely unknown. The purpose of this work was to elucidate the effects of R. nigrum leaf extract (RNLE) on keratinocyte-derived inflammatory mediators, elicited by a Th1 or Th2 cytokine milieu. HaCaT cells were challenged with TNF-α, either alone or in combination with the costimulatory cytokines IFN-γ or IL-4, and the release of proinflammatory cytokines and mediators (IL-8, IL-6, s-ICAM-1, and TSLP) was evaluated. The results showed that RNLE preferentially interferes with IFN-γ signaling, demonstrating only negligible activity on TNF-α or IL-4. This effect was attributed to flavonols, which might also account for the ability of RNLE to impair TNF-α/IL-4-induced TSLP release in a cAMP-independent manner. These results suggest that RNLE could have an antiallergic effect mediated in keratinocytes via mechanisms beyond histamine involvement. In conclusion, the discovery of RNLE preferential activity against IFN-γ-mediated inflammation suggests potential selectivity against Th1 type response and the possible use in Th1 inflammatory diseases.


Assuntos
Inflamação/induzido quimicamente , Interferon gama/farmacologia , Queratinócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Folhas de Planta/química , Ribes/química , Linhagem Celular , Citocinas/administração & dosagem , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/administração & dosagem , Mediadores da Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Quempferóis/farmacologia , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Quercetina/farmacologia
9.
Int J Biol Macromol ; 185: 813-820, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34186122

RESUMO

The stability of IFN-γ as a therapeutic protein can play a key role on its anticancer effects. Herein, we explored the thermodynamic parameters and conformational stability of IFN-γ in the presence of calycosin, the main active compound of Radix astragali, by different biophysical and theoretical analysis. Afterwards, the improved anticancer effects of IFN-γ-calycosin interaction relative to IFN-γ alone were assessed on hepatocellular carcinoma (HepG2) cell line by MTT and caspase assays. ITC data indicated that upon interaction of calycosin with IFN-γ the binding and thermodynamic parameters were as follows: Kd = 1.9 µM, ΔG° = -32.45 kJ/mol, ΔH° = -11.91 kJ/mol, and TΔS° = 20.54 kJ/mol. ANS/synchronous fluorescence, CD and UV-Vis spectroscopy studies indicated that the interaction between calycosin and IFN-γ caused the folding of the IFN-γ backbone in to a more packed structure with enhanced α-helix content and higher melting temperature (Tm) value. The spectroscopic outcomes were then verified by molecular docking and molecular dynamic analysis. It was also shown that after incubation of the IFN-γ samples at 50 °C for 60 min in the presence of calycosin (5 µM), the IFN-γ-calycosin system showed a significant antiproliferative effects against hepatocellular carcinoma (HepG2) cells through caspase-9/3 activation and this anticancer effect was more pronounced than free IFN-γ. This data may provide useful information about the development of IFN-γ-based therapeutic platforms.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Interferon gama/farmacologia , Isoflavonas/química , Neoplasias Hepáticas/metabolismo , Antineoplásicos/química , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Estabilidade de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Interferon gama/química , Neoplasias Hepáticas/tratamento farmacológico , Dobramento de Proteína/efeitos dos fármacos , Termodinâmica
10.
Cancer Sci ; 112(9): 3455-3468, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34159686

RESUMO

Clinical reports indicate that gastric cancer (GC) has a high mortality rate, but its pathological mechanism remains poorly understood. This work integrated bioinformatics analysis with experimental verification to explore novel biomarkers of gastric cancer. First, weighted gene coexpression network analysis was applied to screen significant genes correlated with GC development. Gene set enrichment analysis was also used to unearth the most relevant biological functions of significant genes. As a result, we discovered homeobox C9 (HOXC9) as a novel oncogene in GC, primarily through negatively regulating immune response. High expression of HOXC9 predicted a poor prognosis in GC patients, and knocking down HOXC9 efficiently enhanced the interferon-gamma (IFNγ)-dependent apoptosis in two GC cell lines as well as organoids from patients. Furthermore, cleaved caspase-3/7 and phosphorylated signal transducer and activator of transcription 1 (p-STAT1) were also significantly enhanced in HOXC9 knockdown cells and organoids treated with IFNγ. Mechanistically, we found that HOXC9 inhibited the death-associated protein kinase 1 (DAPK1) and its downstream retinoic acid-inducible gene-I (RIG1) to generate GC IFNγ resistance. In summary, we identified and confirmed that HOXC9 generates IFNγ resistance in GC by inhibiting the DAPK1/RIG1/p-STAT1 axis.


Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Homeodomínio/metabolismo , Interferon gama/farmacologia , Receptores do Ácido Retinoico/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/genética , Neoplasias Gástricas/metabolismo , Regulação para Cima/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Estudos de Coortes , Bases de Dados Genéticas , Proteínas Quinases Associadas com Morte Celular/genética , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Oncogenes , Receptores do Ácido Retinoico/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Neoplasias Gástricas/patologia , Transfecção
11.
Mol Med Rep ; 24(1)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33982762

RESUMO

3,4,5­Trihydroxycinnamic acid (THCA) exhibits anti­inflammatory activity in acute or chronic inflammatory disorders, such as acute lung injury and asthma. The present study investigated the anti­inflammatory activity of THCA in a tumor necrosis factor­α/interferon­Î³ (TI) mixture­stimulated human keratinocyte cell line. The results of ELISA and reverse transcription­quantitative PCR revealed that THCA reduced the secretion and mRNA expression levels of interleukin (IL)­6; IL­8; thymus and activation­regulated chemokine; macrophage­derived chemokine; regulated upon activation, normal T cell expressed and secreted; and monocyte chemoattractant protein­1 in TI mixture­stimulated HaCaT cells. In addition, the results of western blot analysis demonstrated that THCA exerted inhibitory activity on the activation of AKT, ERK and nuclear factor­κB in TI mixture­stimulated HaCaT cells. Furthermore, THCA upregulated the expression levels of heme oxygenase­1 and NAD(P)H:quinone oxidoreductase 1, and the activation of nuclear factor erythroid 2­related factor 2 in HaCaT cells. These results demonstrated that THCA may exhibit anti­inflammatory activity in activated HaCaT cells.


Assuntos
Anti-Inflamatórios/farmacologia , Cinamatos/farmacologia , Interferon gama/farmacologia , Queratinócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Quimiocina CCL17/genética , Quimiocina CCL17/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Células HaCaT , Heme Oxigenase-1/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição RelA/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
12.
Front Immunol ; 12: 648946, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936069

RESUMO

Background: Vascular endothelial cells (EC) are critical for regulation of local immune responses, through coordination of leukocyte recruitment from the blood and egress into the tissue. Growing evidence supports an additional role for endothelium in activation and costimulation of adaptive immune cells. However, this function remains somewhat controversial, and the full repertoire and durability of an enhanced endothelial costimulatory phenotype has not been wholly defined. Methods: Human endothelium was stimulated with continuous TNFα or IFNγ for 1-48hr; or primed with TNFα or IFNγ for only 3hr, before withdrawal of stimulus for up to 45hr. Gene expression of cytokines, costimulatory molecules and antigen presentation molecules was measured by Nanostring, and publicly available datasets of EC stimulation with TNFα or IFNγ were leveraged to further corroborate the results. Cell surface protein expression was detected by flow cytometry, and secretion of cytokines was assessed by Luminex and ELISA. Key findings were confirmed in primary human endothelial cells from 4-6 different vascular beds. Results: TNFα triggered mostly positive immune checkpoint molecule expression on endothelium, including CD40, 4-1BB, and ICOSLG but in the context of only HLA class I and immunoproteasome subunits. IFNγ promoted a more tolerogenic phenotype of high PD-L1 and PD-L2 expression with both HLA class I and class II molecules and antigen processing genes. Both cytokines elicited secretion of IL-15 and BAFF/BLyS, with TNFα stimulated EC additionally producing IL-6, TL1A and IL-1ß. Moreover, endothelium primed for a short period (3hr) with TNFα mostly failed to alter the costimulatory phenotype 24-48hr later, with only somewhat augmented expression of HLA class I. In contrast, brief exposure to IFNγ was sufficient to cause late expression of antigen presentation, cytokines and costimulatory molecules. In particular HLA class I, PD-1 ligand and cytokine expression was markedly high on endothelium two days after IFNγ was last present. Conclusions: Endothelia from multiple vascular beds possess a wide range of other immune checkpoint molecules and cytokines that can shape the adaptive immune response. Our results further demonstrate that IFNγ elicits prolonged signaling that persists days after initiation and is sufficient to trigger substantial gene expression changes and immune phenotype in vascular endothelium.


Assuntos
Células Endoteliais/imunologia , Endotélio Vascular/imunologia , Interferon gama/imunologia , Fator de Necrose Tumoral alfa/imunologia , Imunidade Adaptativa/genética , Imunidade Adaptativa/imunologia , Apresentação do Antígeno/genética , Apresentação do Antígeno/imunologia , Células Cultivadas , Quimiocinas/genética , Quimiocinas/imunologia , Quimiocinas/metabolismo , Análise por Conglomerados , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Perfilação da Expressão Gênica/métodos , Humanos , Interferon gama/farmacologia , Fenótipo , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologia
13.
Front Immunol ; 12: 645770, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33968037

RESUMO

Peptide vaccination remains a viable approach to induce T-cell mediated killing of tumors. To identify potential T-cell targets for Triple-Negative Breast Cancer (TNBC) vaccination, we examined the effect of the pro-inflammatory cytokine interferon-γ (IFNγ) on the transcriptome, proteome, and immunopeptidome of the TNBC cell line MDA-MB-231. Using high resolution mass spectrometry, we identified a total of 84,131 peptides from 9,647 source proteins presented by human leukocyte antigen (HLA)-I and HLA-II alleles. Treatment with IFNγ resulted in a remarkable remolding of the immunopeptidome, with only a 34% overlap between untreated and treated cells across the HLA-I immunopeptidome, and expression of HLA-II only detected on treated cells. IFNγ increased the overall number, diversity, and abundance of peptides contained within the immunopeptidome, as well increasing the coverage of individual source antigens. The suite of peptides displayed under conditions of IFNγ treatment included many known tumor associated antigens, with the HLA-II repertoire sampling 17 breast cancer associated antigens absent from those sampled by HLA-I molecules. Quantitative analysis of the transcriptome (10,248 transcripts) and proteome (6,783 proteins) of these cells revealed 229 common proteins and transcripts that were differentially expressed. Most of these represented downstream targets of IFNγ signaling including components of the antigen processing machinery such as tapasin and HLA molecules. However, these changes in protein expression did not explain the dramatic modulation of the immunopeptidome following IFNγ treatment. These results demonstrate the high degree of plasticity in the immunopeptidome of TNBC cells following cytokine stimulation and provide evidence that under pro-inflammatory conditions a greater variety of potential HLA-I and HLA-II vaccine targets are unveiled to the immune system. This has important implications for the development of personalized cancer vaccination strategies.


Assuntos
Apresentação do Antígeno/efeitos dos fármacos , Antígenos HLA/imunologia , Interferon gama/farmacologia , Neoplasias de Mama Triplo Negativas/imunologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Peptídeos/imunologia , Proteômica , Transcriptoma
14.
Front Immunol ; 12: 662443, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936100

RESUMO

All nucleated mammalian cells express major histocompatibility complex (MHC) proteins that present peptides on cell surfaces for immune surveillance. These MHC-presented peptides (pMHC) are necessary for directing T-cell responses against cells harboring non-self antigens derived from pathogens or from somatic mutations. Alterations in tumor-specific antigen repertoires - particularly novel MHC presentation of mutation-bearing peptides (neoantigens) - can be potent targets of anti-tumor immune responses. Here we employed an integrated genomic and proteomic antigen discovery strategy aimed at measuring how interferon gamma (IFN-γ) alters antigen presentation, using a human lymphoma cell line, GRANTA-519. IFN-γ treatment resulted in 126 differentially expressed proteins (2% of all quantified proteins), which included components of antigen presentation machinery and interferon signaling pathways, and MHC molecules themselves. In addition, several proteasome subunits were found to be modulated, consistent with previous reports of immunoproteasome induction by IFN-γ exposure. This finding suggests that a modest proteomic response to IFN-γ could create larger alteration to cells' antigen/epitope repertoires. Accordingly, MHC immunoprecipitation followed by mass spectrometric analysis of eluted peptide repertoires revealed exclusive signatures of IFN-γ induction, with 951 unique peptides reproducibly presented by MHC-I and 582 presented by MHC-II. Furthermore, an additional set of pMHCs including several candidate neoantigens, distinguished control and the IFN-γ samples by their altered relative abundances. Accordingly, we developed a classification system to distinguish peptides which are differentially presented due to altered expression from novel peptides resulting from changes in antigen processing. Taken together, these data demonstrate that IFN-γ can re-shape antigen repertoires by identity and by abundance. Extending this approach to models with greater clinical relevance could help develop strategies by which immunopeptide repertoires are intentionally reshaped to improve endogenous or vaccine-induced anti-tumor immune responses and potentially anti-viral immune responses.


Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/isolamento & purificação , Genômica , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma , Proteômica , Apresentação do Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Epitopos/imunologia , Humanos , Interferon gama/farmacologia , Linfoma , Linfócitos T/imunologia
15.
Front Immunol ; 12: 666356, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054836

RESUMO

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.


Assuntos
Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunoglobulinas/metabolismo , Interferon gama/imunologia , Glicoproteínas de Membrana/metabolismo , Salmo salar/imunologia , Baço/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD/genética , Antígenos CD/imunologia , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Biomarcadores/metabolismo , Doenças dos Peixes/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Interferon gama/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Piscirickettsia , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/veterinária , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo
16.
Nat Commun ; 12(1): 2866, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001890

RESUMO

Unlike HIV infection, which progresses to AIDS absent suppressive anti-retroviral therapy, nonpathogenic infections in natural hosts, such African green monkeys, are characterized by a lack of gut microbial translocation and robust secondary lymphoid natural killer cell responses resulting in an absence of chronic inflammation and limited SIV dissemination in lymph node B-cell follicles. Here we report, using the pathogenic model of antiretroviral therapy-treated, SIV-infected rhesus macaques that sequential interleukin-21 and interferon alpha therapy generate terminally differentiated blood natural killer cells (NKG2a/clowCD16+) with potent human leukocyte antigen-E-restricted activity in response to SIV envelope peptides. This is in contrast to control macaques, where less differentiated, interferon gamma-producing natural killer cells predominate. The frequency and activity of terminally differentiated NKG2a/clowCD16+ natural killer cells correlates with a reduction of replication-competent SIV in lymph node during antiretroviral therapy and time to viral rebound following analytical treatment interruption. These data demonstrate that African green monkey-like natural killer cell differentiation profiles can be rescued in rhesus macaques to promote viral clearance in tissues.


Assuntos
Antirretrovirais/farmacologia , Interferon gama/farmacologia , Interleucinas/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Animais , Antivirais/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Feminino , Células Matadoras Naturais/virologia , Ativação Linfocitária/efeitos dos fármacos , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Carga Viral/efeitos dos fármacos , Viremia/sangue , Viremia/tratamento farmacológico
17.
Nat Commun ; 12(1): 2537, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953170

RESUMO

Metastasis accounts for 90% of cancer-related deaths and, currently, there are no effective clinical therapies to block the metastatic cascade. A need to develop novel therapies specifically targeting fundamental metastasis processes remains urgent. Here, we demonstrate that Salmonella YB1, an engineered oxygen-sensitive strain, potently inhibits metastasis of a broad range of cancers. This process requires both IFN-γ and NK cells, as the absence of IFN-γ greatly reduces, whilst depletion of NK cells in vivo completely abolishes, the anti-metastatic ability of Salmonella. Mechanistically, we find that IFN-γ is mainly produced by NK cells during early Salmonella infection, and in turn, IFN-γ promotes the accumulation, activation, and cytotoxicity of NK cells, which kill the metastatic cancer cells thus achieving an anti-metastatic effect. Our findings highlight the significance of a self-regulatory feedback loop of NK cells in inhibiting metastasis, pointing a possible approach to develop anti-metastatic therapies by harnessing the power of NK cells.


Assuntos
Interferon gama/metabolismo , Interferon gama/farmacologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/efeitos dos fármacos , Metástase Neoplásica/imunologia , Infecções por Salmonella/metabolismo , Salmonella/genética , Animais , Citocinas/metabolismo , Feminino , Imunidade Inata , Imunoterapia/métodos , Interferon gama/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Infecções por Salmonella/tratamento farmacológico
18.
J Biol Chem ; 296: 100218, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33839686

RESUMO

Rare sequence variants in the microglial cell surface receptor TREM2 have been shown to increase the risk for Alzheimer's disease (AD). Disease-linked TREM2 mutations seem to confer a partial loss of function, and increasing TREM2 cell surface expression and thereby its function(s) might have therapeutic benefit in AD. However, druggable targets that could modulate microglial TREM2 surface expression are not known. To identify such targets, we conducted a screen of small molecule compounds with known pharmacology using human myeloid cells, searching for those that enhance TREM2 protein at the cell surface. Inhibitors of the kinases MEK1/2 displayed the strongest and most consistent increases in cell surface TREM2 protein, identifying a previously unreported pathway for TREM2 regulation. Unexpectedly, inhibitors of the downstream effector ERK kinases did not have the same effect, suggesting that noncanonical MEK signaling regulates TREM2 trafficking. In addition, siRNA knockdown experiments confirmed that decreased MEK1 and MEK2 were required for this recruitment. In iPSC-derived microglia, MEK inhibition increased cell surface TREM2 only modestly, so various cytokines were used to alter iPSC microglia phenotype, making cells more sensitive to MEK inhibitor-induced TREM2 recruitment. Of those tested, only IFN-gamma priming prior to MEK inhibitor treatment resulted in greater TREM2 recruitment. These data identify the first known mechanisms for increasing surface TREM2 protein and TREM2-regulated function in human myeloid cells and are the first to show a role for MEK1/MEK2 signaling in TREM2 activity.


Assuntos
Membrana Celular/metabolismo , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Glicoproteínas de Membrana/genética , Microglia/metabolismo , Receptores Imunológicos/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Benzimidazóis/farmacologia , Benzotiazóis/farmacologia , Membrana Celular/efeitos dos fármacos , Colchicina/farmacologia , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Interferon gama/farmacologia , Interleucinas/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Nitrilas/farmacologia , Cultura Primária de Células , Piridonas/farmacologia , Pirimidinonas/farmacologia , Quinazolinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais , Células THP-1 , Fator de Crescimento Transformador beta/farmacologia , Zearalenona/análogos & derivados , Zearalenona/farmacologia
19.
Front Immunol ; 12: 634127, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33828550

RESUMO

Sepsis is associated with a dysregulated inflammatory response to infection. Despite the activation of inflammation, an immune suppression is often observed, predisposing patients to secondary infections. Therapies directed at restoration of immunity may be considered but should be guided by the immune status of the patients. In this paper, we described the use of a high-dimensional flow cytometry (HDCyto) panel to assess the immunophenotype of patients with sepsis. We then isolated peripheral blood mononuclear cells (PBMCs) from patients with septic shock and mimicked a secondary infection by stimulating PBMCs for 4 h in vitro with lipopolysaccharide (LPS) with or without prior exposure to either IFN-γ, or LAG-3Ig. We evaluated the response by means of flow cytometry and high-resolution clustering cum differential analysis and compared the results to PBMCs from healthy donors. We observed a heterogeneous immune response in septic patients and identified two major subgroups: one characterized by hypo-responsiveness (Hypo) and another one by hyper-responsiveness (Hyper). Hypo and Hyper groups showed significant differences in the production of cytokines/chemokine and surface human leukocyte antigen-DR (HLA-DR) expression in response to LPS stimulation, which were observed across all cell types. When pre-treated with either interferon gamma (IFN-γ) or lymphocyte-activation gene 3 (LAG)-3 recombinant fusion protein (LAG-3Ig) prior to LPS stimulation, cells from the Hypo group were shown to be more responsive to both immunostimulants than cells from the Hyper group. Our results demonstrate the importance of patient stratification based on their immune status prior to any immune therapies. Once sufficiently scaled, this approach may be useful for prescribing the right immune therapy for the right patient at the right time, the key to the success of any therapy.


Assuntos
Antígenos CD/farmacologia , Citometria de Fluxo , Imunofenotipagem , Interferon gama/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Monitorização Imunológica , Choque Séptico/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Células Cultivadas , Citocinas/sangue , Antígenos HLA-DR/sangue , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Fenótipo , Valor Preditivo dos Testes , Choque Séptico/sangue , Choque Séptico/diagnóstico , Fluxo de Trabalho
20.
Exp Eye Res ; 207: 108574, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33848524

RESUMO

PURPOSE: Chronic corneal endothelial cell (CEC) loss results in corneal edema and vision loss in conditions such as pseudophakic bullous keratopathy (PBK), Fuchs' dystrophy, and corneal graft failure. Low CEC density has been associated with an elevation of intraocular pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (INF)-γ. These cytokines are capable of triggering pyroptosis, a programmed cell death mechanism mediated by the inflammasome, prompting the activation of the pro-inflammatory cytokine interleukin (IL)-1ß, the perpetuation of inflammation, and subsequent damage of corneal endothelial tissue. Therefore, the purpose of this study was to determine the deleterious contribution of the inflammasome and pyroptosis to CEC loss. METHODS: CECs from human donor corneas were treated ex vivo with TNF-α and IFN-γ for 48 h. Levels of caspase-1 and IL-1ß were then assayed by ELISA, and the expression of caspase-1 and gasdermin-D (GSDM-D) were confirmed by immunofluorescence. Endothelial cell damage was analyzed by a lactate dehydrogenase (LDH) release assay, and oxidative stress was determined by measuring the levels of reactive oxygen species (ROS) in the culture media. RESULTS: Inflammasome activation and oxidative stress were elevated in CECs following exposure to TNF-α and IFN-γ, which resulted in cell death by pyroptosis as determined by LDH release which was inhibited by the caspase-1 inhibitor Ac-YVAD-cmk. CONCLUSION: CEC death is induced by the pro-inflammatory cytokines TNF-α and IFN-γ, which contribute to inflammasome activation. Moreover, the inflammasome is a promising therapeutic target for the treatment of chronic CEC loss.


Assuntos
Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/patologia , Inflamassomos/metabolismo , Interferon gama/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Idoso , Caspase 1/metabolismo , Morte Celular , Endotélio Corneano/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Estresse Oxidativo , Proteínas de Ligação a Fosfato/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Doadores de Tecidos , Adulto Jovem
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