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1.
Cells ; 10(8)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34440846

RESUMO

Oxysterols, or cholesterol oxidation products, are naturally occurring lipids which regulate the physiology of cells, including those of the immune system. In contrast to effects that are mediated through nuclear receptors or by epigenetic mechanism, which take tens of minutes to occur, changes in the activities of cell-surface receptors caused by oxysterols can be extremely rapid, often taking place within subsecond timescales. Such cell-surface receptor effects of oxysterols allow for the regulation of fast cellular processes, such as motility, secretion and endocytosis. These cellular processes play critical roles in both the innate and adaptive immune systems. This review will survey the two broad classes of cell-surface receptors for oxysterols (G-protein coupled receptors (GPCRs) and ion channels), the mechanisms by which cholesterol oxidation products act on them, and their presence and functions in the different cell types of the immune system. Overall, this review will highlight the potential of oxysterols, synthetic derivatives and their receptors for physiological and therapeutic modulation of the immune system.


Assuntos
Sistema Imunitário/metabolismo , Oxisteróis/metabolismo , Humanos , Canais Iônicos/metabolismo , Oxisteróis/química , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/metabolismo , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/metabolismo
2.
Front Immunol ; 12: 679856, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34135907

RESUMO

Neutrophil trafficking, homeostatic and pathogen elicited, depends upon chemoattractant receptors triggering heterotrimeric G-protein Gαißγ signaling, whose magnitude and kinetics are governed by RGS protein/Gαi interactions. RGS proteins typically limit Gαi signaling by reducing the duration that Gαi subunits remain GTP bound and able to activate downstream effectors. Yet how in totality RGS proteins shape neutrophil chemoattractant receptor activated responses remains unclear. Here, we show that C57Bl/6 mouse neutrophils containing a genomic knock-in of a mutation that disables all RGS protein-Gαi2 interactions (G184S) cannot properly balance chemoattractant receptor signaling, nor appropriately respond to inflammatory insults. Mutant neutrophils accumulate in mouse bone marrow, spleen, lung, and liver; despite neutropenia and an intrinsic inability to properly mobilize from the bone marrow. In vitro they rapidly adhere to ICAM-1 coated plates, but in vivo they poorly adhere to blood vessel endothelium. Those few neutrophils that cross blood vessels and enter tissues migrate haphazardly. Following Concanavalin-A administration fragmented G184S neutrophils accumulate in liver sinusoids leading to thrombo-inflammation and perivasculitis. Thus, neutrophil Gαi2/RGS protein interactions both limit and facilitate Gαi2 signaling thereby promoting normal neutrophil trafficking, aging, and clearance.


Assuntos
Senescência Celular , Quimiotaxia de Leucócito , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Transdução de Sinais , Animais , Transplante de Medula Óssea , Senescência Celular/genética , Senescência Celular/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Humanos , Imunofenotipagem , Masculino , Camundongos , Neutropenia/etiologia , Neutrófilos/efeitos dos fármacos , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo
3.
Front Immunol ; 12: 625922, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34168640

RESUMO

Low density neutrophils (LDNs) are described in a number of inflammatory conditions, cancers and infections and associated with immunopathology, and a mechanistic role in disease. The role of LDNs at homeostasis in healthy individuals has not been investigated. We have developed an isolation protocol that generates high purity LDNs from healthy donors. Healthy LDNs were identical to healthy normal density neutrophils (NDNs), aside from reduced neutrophil extracellular trap formation. CD66b, CD16, CD15, CD10, CD54, CD62L, CXCR2, CD47 and CD11b were expressed at equivalent levels in healthy LDNs and NDNs and underwent apoptosis and ROS production interchangeably. Healthy LDNs had no differential effect on CD4+ or CD8+ T cell proliferation or IFNγ production compared with NDNs. LDNs were generated from healthy NDNs in vitro by activation with TNF, LPS or fMLF, suggesting a mechanism of LDN generation in disease however, we show neutrophilia in people with Cystic Fibrosis (CF) was not due to increased LDNs. LDNs are present in the neutrophil pool at homeostasis and have limited functional differences to NDNs. We conclude that increased LDN numbers in disease reflect the specific pathology or inflammatory environment and that neutrophil density alone is inadequate to classify discrete functional populations of neutrophils.


Assuntos
Separação Celular , Citometria de Fluxo , Neutrófilos/imunologia , Antígenos CD/metabolismo , Apoptose , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Microambiente Celular , Centrifugação com Gradiente de Concentração , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Armadilhas Extracelulares/metabolismo , Voluntários Saudáveis , Homeostase , Humanos , Interferon gama/metabolismo , Contagem de Leucócitos , Transtornos Leucocíticos/imunologia , Transtornos Leucocíticos/metabolismo , Ativação Linfocitária , Ativação de Neutrófilo , Neutrófilos/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-8B/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
4.
Front Immunol ; 12: 637053, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34108959

RESUMO

Background: Programmed cell death 10 (PDCD10) plays a crucial role in regulating tumor phenotyping, especially in glioblastoma (GBM). Glioma-associated microglia/macrophages (GAMs) in tumor pathological microenvironment contribute to GBM progression. We previously found that the infiltration of GAMs was associated with PDCD10 expression in GBM patients. The present study aims to further explore the regulation of PDCD10 on GAMs in GBM. Methods: Overexpression of PDCD10 in human- and murine-GBM cells was established by lentiviral transduction. Cell behaviors and polarization of primary microglia, microglia- and macrophage-like cells were investigated through indirect co-culture with GBM cells in vitro respectively. The PDCD10-induced release of chemokines was identified by a chemokine protein array. The cross-talk between GBM and microglia as well as macrophages was further studied using selective antagonist SB225002. Finally, an orthotopic homograft mouse model was employed to verify the results of in vitro experiments. Results: Indirect co-culture with PDCD10-overexpressed GBM cells promoted proliferation and migration of microglia- and macrophage-like cells, and stimulated pro-tumorigenic polarization of primary microglia, microglia- and macrophage-like cells. Pdcd10-upregulated GBM cells triggered a nearly 6-fold increase of CXC motif chemokine ligand 2 (CXCL2) release, which in turn activated CXC chemokine receptor 2 (CXCR2) and downstream Erk1/2 and Akt signaling in primary microglia, microglia- and macrophage-like cells. The blockage of CXCR2 signaling with specific inhibitor (SB225002) abolished microglia- and macrophage-like cell migration induced by PDCD10-upregulated GBM cells. Moreover, Pdcd10-upregulated GL261 cells promoted GAMs recruitment and tumor growth in vivo. Conclusion: Our study demonstrates that overexpression of PDCD10 in GBM recruits and activates microglia/macrophages, which in turn promotes tumor progression. CXCL2-CXCR2 signaling mediated by PDCD10 is potentially involved in the crosstalk between GBM cells and GAMs.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Microglia/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL2/antagonistas & inibidores , Quimiocina CXCL2/metabolismo , Técnicas de Cocultura , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Compostos de Fenilureia/farmacologia , Proteínas Proto-Oncogênicas/genética , Células RAW 264.7 , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia
5.
Metabolism ; 121: 154804, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34097917

RESUMO

AIMS/HYPOTHESIS: Inflammation has a major role in diabetic kidney disease. We thus investigated the role of the IL-8-CXCR1/2 axis in favoring kidney damage in diabetes. METHODS: Urinary IL-8 levels were measured in 1247 patients of the Joslin Kidney Study in type 2 diabetes (T2D). The expression of IL-8 and of its membrane receptors CXCR1/CXCR2 was quantified in kidney tissues in patients with T2D and in controls. The effect of CXCR1/2 blockade on diabetic kidney disease was evaluated in db/db mice. RESULTS: IL-8 urinary levels were increased in patients with T2D and diabetic kidney disease, with the highest urinary IL-8 levels found in the patients with the largest decline in glomerular filtration rate, with an increased albumin/creatine ratio and the worst renal outcome. Moreover, glomerular IL-8 renal expression was increased in patients with T2D, as compared to controls. High glucose elicits abundant IL-8 secretion in cultured human immortalized podocytes in vitro. Finally, in diabetic db/db mice and in podocytes in vitro, CXCR1/2 blockade mitigated albuminuria, reduced mesangial expansion, decreased podocyte apoptosis and reduced DNA damage. CONCLUSIONS/INTERPRETATION: The IL-8- CXCR1/2 axis may have a role in diabetic kidney disease by inducing podocyte damage. Indeed, targeting the IL-8-CXCR1/2 axis may reduce the burden of diabetic kidney disease.


Assuntos
Nefropatias Diabéticas/genética , Interleucina-8/fisiologia , Receptores CXCR/fisiologia , Adulto , Animais , Estudos de Casos e Controles , Células Cultivadas , Estudos de Coortes , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Itália , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Podócitos/metabolismo , Podócitos/patologia , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais/fisiologia
6.
Clin Exp Metastasis ; 38(4): 401-410, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34115261

RESUMO

Peritoneal metastasis is an insidious aspect of colorectal cancer. The aim of the present study was to define mechanisms regulating colon cancer cell adhesion and spread to peritoneal wounds after abdominal surgery. Mice was laparotomized and injected intraperitoneally with CT-26 colon carcinoma cells and metastatic noduli in the peritoneal cavity was quantified after treatment with a CXCR2 antagonist or integrin-αV-antibody. CT-26 cells expressed cell surface chemokine receptors CXCR2, CXCR3, CXCR4 and CXCR5. Stimulation with the CXCR2 ligand, CXCL2, dose-dependently increased proliferation and migration of CT-26 cells in vitro. The CXCR2 antagonist, SB225002, dose-dependently decreased CXCL2-induced proliferation and migration of colon cancer cells in vitro. Intraperitoneal administration of CT-26 colon cancer cells resulted in wide-spread growth of metastatic nodules at the peritoneal surface of laparotomized animals. Laparotomy increased gene expression of CXCL2 at the incisional line. Pretreatment with CXCR2 antagonist reduced metastatic nodules by 70%. Moreover, stimulation with CXCL2 increased CT-26 cell adhesion to extracellular matrix (ECM) proteins in a CXCR2-dependent manner. CT-26 cells expressed the αV, ß1 and ß3 integrin subunits and immunoneutralization of αV abolished CXCL2-triggered adhesion of CT-26 to vitronectin, fibronectin and fibrinogen. Finally, inhibition of the αV integrin significantly attenuated the number of carcinomatosis nodules by 69% in laparotomized mice. These results were validated by use of the human colon cancer cell line HT-29 in vitro. Our data show that colon cancer cell adhesion and growth on peritoneal wound sites is mediated by a CXCL2-CXCR2 signaling axis and αV integrin-dependent adhesion to ECM proteins.


Assuntos
Quimiocina CXCL2/metabolismo , Neoplasias do Colo/patologia , Integrina alfaV/metabolismo , Neoplasias Peritoneais/secundário , Receptores de Interleucina-8B/metabolismo , Animais , Proteínas da Matriz Extracelular/metabolismo , Xenoenxertos , Humanos , Camundongos , Transdução de Sinais
7.
Biochem Pharmacol ; 190: 114658, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34146540

RESUMO

Chronic myeloid leukemia (CML) is a reciprocal translocation disorder driven by a breakpoint cluster region (BCR)-Abelson leukemia virus (ABL) fusion gene that stimulates abnormal tyrosine kinase activity. Tyrosine kinase inhibitors (TKIs) are effective in treating Philadelphia chromosome (Ph) + CML patients. However, the appearance of TKI-resistant CML cells is a hurdle in CML treatment. Therefore, it is necessary to identify novel alternative treatments targeting tyrosine kinases. This study was designed to determine whether C-X-C chemokine receptor 2 (CXCR2) could be a novel target for TKI-resistant CML treatment. Interleukin 8 (IL-8), a CXCR2 ligand, was significantly increased in the bone marrow serum of initially diagnosed CML patients and TKI-resistant CML cell conditioned media. CXCR2 antagonists suppressed the proliferation of CML cells via cell cycle arrest in the G2/M phase. CXCR2 inhibition also attenuated mTOR, c-Myc, and BCR-ABL expression, leading to CML cell apoptosis, irrespective of TKI responsiveness. Moreover, SB225002, a CXCR2 antagonist, caused higher cell death in TKI-resistant CML cells than TKIs. Using a mouse xenograft model, we confirmed that SB225002 suppresses tumor growth, with a prominent effect on TKI-resistant CML cells. Our findings demonstrate that IL-8 is a prognostic factor for the progression of CML. Inhibiting the CXCR2-mTOR-c-Myc cascade is a promising therapeutic strategy to overcome TKI-sensitive and TKI-insensitive CML. Thus, CXCR2 blockade is a novel therapeutic strategy to treat CML, and SB225002, a commercially available CXCR2 antagonist, might be a candidate drug that could be used to treat TKI-resistant CML.


Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Mesilato de Imatinib/farmacologia , Interleucina-8/genética , Interleucina-8/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Masculino , Pessoa de Meia-Idade , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores de Interleucina-8B/genética , Triazóis/farmacologia , Adulto Jovem
8.
J Orthop Surg Res ; 16(1): 327, 2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016144

RESUMO

BACKGROUND: Steroid-induced osteonecrosis of the femoral head (ONFH) is a common hip joint disease and is difficult to be diagnosed early. At present, the pathogenesis of steroid-induced ONFH remains unclear, and recognized and effective diagnostic biomarkers are deficient. The present study aimed to identify potentially important genes and signaling pathways involved in steroid-induced ONFH and investigate their molecular mechanisms. METHODS: Microarray data sets GSE123568 (peripheral blood) and GSE74089 (cartilage) were obtained from the Gene Expression Omnibus database, including 34 ONFH samples and 14 control samples. Morpheus software and Venn diagram were used to identify DEGs and co-expressed DEGs, respectively. Besides, we conducted Kyoto Encyclopedia of Genome (KEGG) and gene ontology (GO) pathway enrichment analysis. We construct a protein-protein interaction (PPI) network through GEO2R and used cytoHubba to divide the PPI network into multiple sub-networks. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to verify the bioinformatics analysis results. RESULTS: A total of 118 intersecting DEGs were obtained between the peripheral blood and cartilage samples, including 40 upregulated genes and 78 downregulated genes. Then, GO and KEGG pathway enrichment analysis revealed that upregulated DEGs focused on the signaling pathways related to staphylococcus aureus infection, leishmaniasis, antigen processing, and presentation, as well as asthma and graft-versus-host disease. Downregulated genes were concentrated in the FoxO signaling pathway, AMPK signaling pathway, signaling pathway regulating stem cell pluripotency, and mTOR signaling pathway. Some hub genes with high interactions such as CXCR1, FPR1, MAPK1, FOXO3, FPR2, CXCR2, and TYROBP were identified in the PPI network. The results of qRT-PCR demonstrated that CXCR1, FPR1, and TYROBP were upregulated while MAPK1 was downregulated in peripheral blood of steroid-induced ONFH patients. This was consistent with the bioinformatics analysis. CONCLUSIONS: The present study would provide novel insight into the genes and associated pathways involved in steroid-induced ONFH. CXCR1, FPR1, TYROBP, and MAPK1 may be used as potential drug targets and biomarkers for the diagnosis and prognosis of steroid-induced ONFH.


Assuntos
Biologia Computacional/métodos , Cabeça do Fêmur , Estudos de Associação Genética/métodos , Glucocorticoides/efeitos adversos , Osteonecrose/induzido quimicamente , Osteonecrose/genética , Transdução de Sinais/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Feminino , Proteína Forkhead Box O3/metabolismo , Humanos , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Adulto Jovem
9.
Front Immunol ; 12: 667177, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34025668

RESUMO

Accumulating evidence suggests that tumor-infiltrating immune cells (TICs) in the tumor microenvironment (TME) serve as promising therapeutic targets. CXCL8 (IL-8) may also be a potential therapeutic target in cancer. CXCL8 is a potent chemotactic factor for neutrophils, myeloid-derived suppressor cells (MDSCs) and monocytes, which are considered immunosuppressive components in cancer-bearing hosts. Here, we identified the TME-related gene CXCL8 in a high-ImmuneScore population that contributed to better survival in colorectal cancer (CRC) patients from The Cancer Genome Atlas (TCGA) database. An integrated gene profile and functional analysis of TIC proportions revealed that the dendritic cell (DC) activation markers CD80, CD83, and CD86 were positively correlated with CXCL8 expression, suggesting that CXCL8 may be functional as antitumor immune response status in the TME. The gene signature was further validated in independent GSE14333 and GSE38832 cohorts from the Gene Expression Omnibus (GEO). To test the differential contributions of immune and tumor components to progression, three CRC cell lines, CT26, MC38 and HCT116, were used. In vitro results suggested no significant growth or survival changes following treatment with an inhibitor of the CXCL8 receptor (CXCR1/2) such as reparixin or danirixin. In vivo treatment with danirixin (antagonists of CXCR2) promoted tumor progression in animal models established with CT26 cells. CXCR2 antagonism may function via an immune component, with CXCR2 antagonist treatment in mice resulting in reduced activated DCs and correlating with decreased Interferon gamma (IFN-γ) or Granzyme B expressed CD8+ T cells. Furthermore, CXCL8 induced DC migration in transwell migration assays. Taken together, our data suggested that targeting the CXCL8-CXCR2 axis might impede DC activation or recruitment, and this axis could be considered a favorable factor rather than a target for critical antitumor effects on CRC.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Células Dendríticas/patologia , Interleucina-8/metabolismo , Receptores de Interleucina-8B/metabolismo , Animais , Biomarcadores Tumorais/genética , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Células Dendríticas/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-8/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Receptores de Interleucina-8B/genética , Microambiente Tumoral
10.
Aging (Albany NY) ; 13(10): 13405-13420, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34038868

RESUMO

cDNA microarray data conducted by our group revealed overexpression of CXCL2 and CXCL8 in ovarian cancer (OC) microenvironment. Herein, we have proven that the chemokine receptor, CXCR2, is a pivotal molecule in re-sensitizing OC to cisplatin, and its inhibition decreases cell proliferation, viability, tumor size in cisplatin-resistant cells, as well as reversed the overexpression of mesenchymal epithelium transition markers. Altogether, our study indicates a central effect of CXCR2 in preventing tumor progression, due to acquisition of cisplatin chemoresistant phenotype by tumor cells, and patients' high lethality rate. We found that the overexpression of CXCR2 by OC cells is persistent and anomalously confined to the cellular nuclei, thus pointing to an urge in developing highly lipophilic molecules that promptly permeate cells, bind to and inhibit nuclear CXCR2 to fight OC, instead of relying on the high-cost genetic engineered cells.


Assuntos
Cisplatino/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Receptores de Interleucina-8B/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CXCL2/metabolismo , Embrião de Galinha , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina-8B/metabolismo , Análise de Sobrevida , Serina-Treonina Quinases TOR/metabolismo
11.
Eur J Immunol ; 51(9): 2274-2280, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33963542

RESUMO

In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.


Assuntos
Quimiotaxia/imunologia , Armadilhas Extracelulares/imunologia , Interleucina-8/imunologia , Neutrófilos/imunologia , Acetilcisteína/metabolismo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais/imunologia
12.
Nat Commun ; 12(1): 2547, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953162

RESUMO

Receptors and their ligands are important therapeutic targets for about one third of marketed drugs. Here, we describe an epitope-guided approach for selection of antibodies that modulate cellular signaling of targeted receptors. We chose CXC chemokine receptor 2 (CXCR2) in the G-protein coupled receptor superfamily as receptor and a CXCR2 N-terminal peptide for antibody selection. We obtain a highly selective, tight-binding antibody from a 1011-member antibody library using combinatorial enrichment. Structural and Hydrogen-Deuterium-Exchange mass spectrometry analyses demonstrate antibody interaction with an N-terminal region of CXCR2 that is part of the IL-8 epitope. The antibody strongly inhibits IL-8-induced and CXCR2-mediated neutrophil chemotaxis in vitro and alleviates hCXCR2-dependent experimental autoimmune encephalomyelitis symptoms in mice. As inappropriate neutrophil migration accompanies many diseases including inflammatory bowel disease, glomerulonephritis, allergic asthma, chronic obstructive pulmonary disease, and cancer, this antibody has potential for development as a therapeutic agent, akin to anti-TNF antibodies. However, an important difference here is that the antibody targets the chemokine receptor and competes with natural ligand, rather than targeting the ligand itself.


Assuntos
Anticorpos/farmacologia , Encefalomielite Autoimune Experimental/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Receptores de Interleucina-8B/metabolismo , Animais , Sítios de Ligação , Quimiocinas , Quimiotaxia , Encefalomielite Autoimune Experimental/imunologia , Endocitose , Epitopos , Humanos , Imunoglobulina G , Interleucina-8/metabolismo , Ligantes , Camundongos , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa
13.
Front Immunol ; 12: 659996, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33912188

RESUMO

Tumor associated neutrophils (TANs) are frequently detected in triple-negative breast cancer (TNBC). Recent studies also reveal the importance of neutrophils in promoting tumor progression and metastasis during breast cancer. However, the mechanisms regulating neutrophil trafficking to breast tumors are less clear. We sought to determine whether neutrophil trafficking to breast tumors is determined directly by the malignant potential of cancer cells. We found that tumor conditioned media (TCM) harvested from highly aggressive, metastatic TNBC cells induced a polarized morphology and robust neutrophil migration, while TCM derived from poorly aggressive estrogen receptor positive (ER+) breast cancer cells had no activity. In a three-dimensional (3D) type-I collagen matrix, neutrophils migrated toward TCM from aggressive breast cancer cells with increased velocity and directionality. Moreover, in a neutrophil-tumor spheroid co-culture system, neutrophils migrated with increased directionality towards spheroids generated from TNBC cells compared to ER+ cells. Based on these findings, we next sought to characterize the active factors secreted by TNBC cell lines. We found that TCM-induced neutrophil migration is dependent on tumor-derived chemokines, and screening TCM elution fractions based on their ability to induce polarized neutrophil morphology revealed the molecular weight of the active factors to be around 12 kDa. TCM from TNBC cell lines contained copious amounts of GRO (CXCL1/2/3) chemokines and TGF-ß cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-ß, while the activity remained intact in the presence of either single receptor inhibitor. Together, our findings establish a direct link between the malignant potential of breast cancer cells and their ability to induce neutrophil migration. Our study also uncovers a novel coordinated function of TGF-ß and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.


Assuntos
Neutrófilos/metabolismo , Receptores de Interleucina-8B/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocinas/metabolismo , Quimiocinas/farmacologia , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Citocinas/farmacologia , Feminino , Humanos , Ligantes , Células MCF-7 , Infiltração de Neutrófilos/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Neoplasias de Mama Triplo Negativas/patologia
14.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807899

RESUMO

We aimed to evaluate the angiogenic capacity of CXCL2 and IL8 affecting human endothelial cells to clarify their potential role in glioblastoma (GBM) angiogenesis. Human GBM samples and controls were stained for proangiogenic factors. Survival curves and molecule correlations were obtained from the TCGA (The Cancer Genome Atlas) database. Moreover, proliferative, migratory and angiogenic activity of peripheral (HUVEC) and brain specific (HBMEC) primary human endothelial cells were investigated including blockage of CXCR2 signaling with SB225502. Gene expression analyses of angiogenic molecules from endothelial cells were performed. Overexpression of VEGF and CXCL2 was observed in GBM patients and associated with a survival disadvantage. Molecules of the VEGF pathway correlated but no relation for CXCR1/2 and CXCL2/IL8 was found. Interestingly, receptors of endothelial cells were not induced by addition of proangiogenic factors in vitro. Proliferation and migration of HUVEC were increased by VEGF, CXCL2 as well as IL8. Their sprouting was enhanced through VEGF and CXCL2, while IL8 showed no effect. In contrast, brain endothelial cells reacted to all proangiogenic molecules. Additionally, treatment with a CXCR2 antagonist led to reduced chemokinesis and sprouting of endothelial cells. We demonstrate the impact of CXCR2 signaling on endothelial cells supporting an impact of this pathway in angiogenesis of glioblastoma.


Assuntos
Neoplasias Encefálicas , Quimiocina CXCL2/metabolismo , Glioblastoma , Células Endoteliais da Veia Umbilical Humana/metabolismo , Interleucina-8/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Glioblastoma/patologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
Proc Natl Acad Sci U S A ; 118(8)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33597297

RESUMO

Multiple sclerosis (MS) is a chronic neurological disease of the central nervous system driven by peripheral immune cell infiltration and glial activation. The pathological hallmark of MS is demyelination, and mounting evidence suggests neuronal damage in gray matter is a major contributor to disease irreversibility. While T cells are found in both gray and white matter of MS tissue, they are typically confined to the white matter of the most commonly used mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Here, we used a modified EAE mouse model (Type-B EAE) that displays severe neuronal damage to investigate the interplay between peripheral immune cells and glial cells in the event of neuronal damage. We show that CD4+ T cells migrate to the spinal cord gray matter, preferentially to ventral horns. Compared to CD4+ T cells in white matter, gray matter-infiltrated CD4+ T cells were mostly immobilized and interacted with neurons, which are behaviors associated with detrimental effects to normal neuronal function. T cell-specific deletion of CXCR2 significantly decreased CD4+ T cell infiltration into gray matter in Type-B EAE mice. Further, astrocyte-targeted deletion of TAK1 inhibited production of CXCR2 ligands such as CXCL1 in gray matter, successfully prevented T cell migration into spinal cord gray matter, and averted neuronal damage and motor dysfunction in Type-B EAE mice. This study identifies astrocyte chemokine production as a requisite for the invasion of CD4+T cell into the gray matter to induce neuronal damage.


Assuntos
Astrócitos/patologia , Linfócitos T CD4-Positivos/metabolismo , Substância Cinzenta/patologia , Esclerose Múltipla/patologia , Receptores de Interleucina-8B/metabolismo , Animais , Astrócitos/metabolismo , Linfócitos T CD4-Positivos/patologia , Quimiocina CXCL1/metabolismo , Quimiocina CXCL5/metabolismo , Quimiocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Feminino , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Corno Ventral da Medula Espinal/patologia , Imagem com Lapso de Tempo
17.
Cells ; 10(1)2021 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-33401590

RESUMO

In order to provide a sufficient number of cells for clinical use, mesenchymal stem cells (MSCs) must be cultured for long-term expansion, which inevitably triggers cellular senescence. Although the small size of MSCs is known as a critical determinant of their fate, the main regulators of stem cell senescence and the underlying signaling have not been addressed. Umbilical cord blood-derived MSCs (UCB-MSCs) were obtained using size-isolation methods and then cultured with control or small cells to investigate the major factors that modulate MSC senescence. Cytokine array data suggested that the secretion of interukin-8 (IL-8) or growth-regulated oncogene-alpha (GROa) by senescent cells was markedly inhibited during incubation of small cells along with suppression of cognate receptor (C-X-C motif chemokine receptor2, CXCR2) via blockade of the autocrine/paracrine positive loop. Moreover, signaling via toll-like receptor 2 (TLR2) and TLR5, both pattern recognition receptors, drove cellular senescence of MSCs, but was inhibited in small cells. The activation of TLRs (2 and 5) through ligand treatment induced a senescent phenotype in small cells. Collectively, our data suggest that small cell from UCB-MSCs exhibit delayed cellular senescence by inhibiting the process of TLR signaling-mediated senescence-associated secretory phenotype (SASP) activation.


Assuntos
Tamanho Celular , Senescência Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Receptor 5 Toll-Like/metabolismo , Comunicação Autócrina , Quimiocina CXCL1/metabolismo , Sangue Fetal/citologia , Humanos , Recém-Nascido , Mediadores da Inflamação/metabolismo , Interleucina-8/metabolismo , Fenótipo , Receptores de Interleucina-8B/metabolismo
18.
Anticancer Res ; 41(2): 783-794, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33517283

RESUMO

BACKGROUND/AIM: The inflammatory cytokine IL-8 and its receptor CXCR2 are key signalling pathway molecules in cancer development. We hypothesized that IL-8/CXCR2 signalling promotes tumour progression in oesophageal squamous cell carcinoma (ESCC) patients. MATERIALS AND METHODS: We examined the relationship between IL-8/CXCR2 expression and clinicopathological factors by immunohistochemistry in samples from 63 patients with resectable ESCC. The effects of IL-8/CXCR2 signalling on cell proliferation and gene expression were examined in vitro and in vivo using ESCC cell lines. RESULTS: Increased IL-8/CXCR2 signalling was associated with shorter overall survival (p<0.05) and recurrence-free survival (p<0.05) in ESCC patients. Multivariate analysis identified IL-8/CXCR2 expression as a prognostic factor for surgically treated ESCC (p<0.05). In vitro, IL-8 exposure or over-expression significantly enhanced ESCC cell proliferation. SB225002, a CXCR2-specific antagonist, and IL-8 siRNA significantly suppressed cell proliferation. CONCLUSION: IL-8/CXCR2 expression is an independent prognostic factor for surgically treated ESCC, and IL-8/CXCR2 signalling contributes to ESCC cell proliferation.


Assuntos
Neoplasias Esofágicas/cirurgia , Carcinoma de Células Escamosas do Esôfago/cirurgia , Interleucina-8/metabolismo , Receptores de Interleucina-8B/metabolismo , Regulação para Cima , Idoso , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/antagonistas & inibidores , Interleucina-8/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Compostos de Fenilureia/farmacologia , Prognóstico , RNA Interferente Pequeno/farmacologia , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/genética , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida
19.
Cell Oncol (Dordr) ; 44(1): 77-92, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32910411

RESUMO

PURPOSE: Paracrine interactions with the stromal environment, including fibroblasts, may be important in the pathogenesis of ovarian cancer. Here, we evaluated the effect of conditioned media derived from ovarian fibroblasts (fibroblast-CMs) and their major cytokines on the growth of ovarian cancer cells, as well as the involvement of mitogen-activated protein kinases (MAPKs) and AKT in mediating this effect. METHODS: Ovarian cancer cells were cultured in serum-free media (SF), or conditioned media of fibroblasts derived from normal ovary (CM1) and ovarian tumor tissue (CM2). Cell proliferation was measured by MTT assay. Phosphorylation of MAPKs and AKT was evaluated by Western blotting. Specific inhibitors of MAPKs and AKT were used to evaluate their respective involvement in mediating increased cell growth. Cytokine levels in fibroblast-CMs were measured using Luminex assays. Immunohistochemical staining was conducted for CXCL1, CXCR2 and phosphorylated p38 in primary ovarian tumors. RESULTS: CM1 and CM2 significantly increased the growth of ovarian cancer cells relative to SF. In OVCAR3 and OVCAR4 cells, p38 phosphorylation was strongly induced by fibroblast-CMs, and pre-treatment with a p38 inhibitor prevented the growth increase induced by fibroblast-CMs. Fibroblasts secreted high levels of IL-6, IL-8, MCP1 and CXCL1. Treatment with only CXCL1 (1 µg/ml) increased cell growth and p38 phosphorylation. Treatment with a CXCR2 inhibitor effectively prevented p38 activation and cell growth induced by fibroblast-CMs. High expression of both CXCL1 and CXCR2 correlated with high expression of phosphorylated p38 in primary ovarian tumors. CONCLUSIONS: From our data, we conclude that CXCL1 is a key factor derived from ovarian fibroblasts that is responsible for increased ovarian cancer cell growth in part through p38 activation. Phosphorylated p38 can be used as a biomarker to predict CXCL1-CXCR2 interaction in vivo.


Assuntos
Comunicação Celular , Quimiocina CXCL1/metabolismo , Células Epiteliais/patologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Receptores de Interleucina-8B/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Citocinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/patologia
20.
Biochimie ; 180: 30-42, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33122103

RESUMO

Bidirectional communication between the neuroendocrine stress and immune systems permits classically anti-inflammatory glucocorticoids to exert pro-inflammatory effects in specific cells and tissues. Liver macrophages/Kupffer cells play a crucial role in initiating inflammatory cascades mediated by the release of pro-inflammatory cytokines following tissue injury. However, the effects of repeated acute psychological stress on hepatic inflammatory phenotype and macrophage activation state remains poorly understood. We have utilised a model of repeated acute stress in rodents to observe the changes in hepatic inflammatory phenotype, including anti-inflammatory vitamin D status, in addition to examining markers of classically and alternatively-activated macrophages. Male Wistar rats were subjected to control conditions or 6 h of restraint stress applied for 1 or 3 days (n = 8 per group) after which plasma concentrations of stress hormone, enzymes associated with liver damage, and vitamin D status were examined, in addition to hepatic expression of pro- and anti-inflammatory markers. Stress increased glucocorticoids and active vitamin D levels in addition to expression of glucocorticoid alpha/beta receptor, whilst changes in circulating hepatic enzymes indicated sustained liver damage. A pro-inflammatory response was observed in liver tissues following stress, and inducible nitric oxide synthase being observed within hepatic macrophage/Kupffer cells. Together, this suggests that stress preferentially induces a pro-inflammatory response in the liver.


Assuntos
Hepatite/metabolismo , Hepatite/fisiopatologia , Ativação de Macrófagos/fisiologia , Estresse Psicológico/sangue , Estresse Psicológico/fisiopatologia , Animais , Biomarcadores , Citocinas/metabolismo , Macrófagos do Fígado/metabolismo , Masculino , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , PPAR gama/metabolismo , Ratos Wistar , Receptores de Glucocorticoides/genética , Receptores de Interleucina-8B/metabolismo , Vitamina D/metabolismo
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