RESUMO
BACKGROUND: Mesenchymal stromal cells (MSCs) tropism for tumours allows their use as carriers of antitumoural factors and in vitro transcribed mRNA (IVT mRNA) is a promising tool for effective transient expression without insertional mutagenesis risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with antitumor properties by stimulating the specific immune response. The aim of this work was to generate modified MSCs by IVT mRNA transfection to overexpress GM-CSF and determine their therapeutic effect alone or in combination with doxorubicin (Dox) in a murine model of hepatocellular carcinoma (HCC). METHODS: DsRed or GM-CSF IVT mRNAs were generated from a cDNA template designed with specific primers followed by reverse transcription. Lipofectamine was used to transfect MSCs with DsRed (MSC/DsRed) or GM-CSF IVT mRNA (MSC/GM-CSF). Gene expression and cell surface markers were determined by flow cytometry. GM-CSF secretion was determined by ELISA. For in vitro experiments, the J774 macrophage line and bone marrow monocytes from mice were used to test GM-CSF function. An HCC model was developed by subcutaneous inoculation (s.c.) of Hepa129 cells into C3H/HeN mice. After s.c. injection of MSC/GM-CSF, Dox, or their combination, tumour size and mouse survival were evaluated. Tumour samples were collected for mRNA analysis and flow cytometry. RESULTS: DsRed expression by MSCs was observed from 2 h to 15 days after IVT mRNA transfection. Tumour growth remained unaltered after the administration of DsRed-expressing MSCs in a murine model of HCC and MSCs expressing GM-CSF maintained their phenotypic characteristic and migration capability. GM-CSF secreted by modified MSCs induced the differentiation of murine monocytes to dendritic cells and promoted a proinflammatory phenotype in the J774 macrophage cell line. In vivo, MSC/GM-CSF in combination with Dox strongly reduced HCC tumour growth in C3H/HeN mice and extended mouse survival in comparison with individual treatments. In addition, the tumours in the MSC/GM-CSF + Dox treated group exhibited elevated expression of proinflammatory genes and increased infiltration of CD8 + T cells and macrophages. CONCLUSIONS: Our results showed that IVT mRNA transfection is a suitable strategy for obtaining modified MSCs for therapeutic purposes. MSC/GM-CSF in combination with low doses of Dox led to a synergistic effect by increasing the proinflammatory tumour microenvironment, enhancing the antitumoural response in HCC.
Assuntos
Carcinoma Hepatocelular , Doxorrubicina , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Neoplasias Hepáticas , Células-Tronco Mesenquimais , RNA Mensageiro , Animais , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Linhagem Celular Tumoral , Transplante de Células-Tronco Mesenquimais/métodos , Humanos , Camundongos Endogâmicos C3H , TransfecçãoRESUMO
Monocytes can differentiate into macrophages (Mo-Macs) or dendritic cells (Mo-DCs). The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the differentiation of monocytes into Mo-Macs, while the combination of GM-CSF/interleukin (IL)-4 is widely used to generate Mo-DCs for clinical applications and to study human DC biology. Here, we report that pharmacological inhibition of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) in the presence of GM-CSF and the absence of IL-4 induces monocyte differentiation into Mo-DCs. Remarkably, we find that simultaneous inhibition of PPARγ and the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) induces the differentiation of Mo-DCs with stronger phenotypic stability, superior immunogenicity, and a transcriptional profile characterized by a strong type I interferon (IFN) signature, a lower expression of a large set of tolerogenic genes, and the differential expression of several transcription factors compared with GM-CSF/IL-4 Mo-DCs. Our findings uncover a pathway that tailors Mo-DC differentiation with potential implications in the fields of DC vaccination and cancer immunotherapy.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos , Monócitos , Humanos , Monócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , PPAR gama/metabolismo , Interleucina-4/farmacologia , Interleucina-4/metabolismo , Células Dendríticas/metabolismo , Diferenciação Celular/fisiologia , Células CultivadasRESUMO
AIMS: In this study we evaluated the effect of pharmacological treatment with AnxA1-derived peptide Ac2-26 in an experimental model of toxicity induced by cisplatin. MAIN METHODS: Male rats were divided into Sham (control), Cisplatin (received intraperitoneal injections of 10â¯mg/kg/day of cisplatin for 3â¯days) and Ac2-26 (received intraperitoneal injections of 1â¯mg/kg/day of peptide, 15â¯min before cisplatin) groups. KEY FINDINGS: After 6â¯h of the last dose of cisplatin, an acute inflammatory response was observed characterized by a marked increase in the number of neutrophils and GM-CSF, IL-ß, IL-6, IL-10 and TNF-α plasma levels. These findings were associated with increased AnxA1 protein levels in liver and kidneys, as well as positive AnxA1/Fpr2 circulating leukocytes. Treatment with Ac2-26 produced higher levels of GM-CSF, corroborating the high numbers of neutrophils, and the anti-inflammatory cytokine IL-4. Ac2-26 preserved the morphology of liver structures and increased Fpr1 expression, preventing the damage caused by cisplatin. In the kidneys, Ac2-26 caused downregulation of renal Fpr1 and Fpr2 levels and abrogated the increased levels of the CLU and KIM-1 biomarkers of kidney damage induced by cisplatin. However, no effect of peptide treatment was detected in cisplatin-induced kidney morphology injury. SIGNIFICANCE: Despite activation of the anti-inflammatory AnxA1/Fpr axis during cisplatin administration, treatment with Ac2-26 did not efficiently prevent its deleterious effects on the liver and kidneys.
Assuntos
Anexina A1 , Animais , Anexina A1/química , Anexina A1/metabolismo , Anexina A1/farmacologia , Anti-Inflamatórios/farmacologia , Cisplatino/metabolismo , Cisplatino/toxicidade , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Rim/metabolismo , Fígado/metabolismo , Masculino , Peptídeos/química , RatosRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine that regulates inflammatory responses in various autoimmune and inflammatory disorders. OBJECTIVE: The purpose of this study was to analyze the gingival crevicular fluid (GCF) for GM-CSF, interleukin-1 beta (IL-1ß), and macrophage inflammatory protein-1 alpha (MIP-1α) levels in patients with stage I, stage II, stage III, and stage IV periodontitis (SI-P, SII-P, SIII-P, and SIV-P). METHODOLOGY: A total of 126 individuals were recruited for this study, including 21 periodontal healthy (PH), 21 gingivitis (G), 21 SI-P, 21 SII-P, 21 SIII-P, and 21 SIV-P patients. Plaque index (PI), gingival index (GI), presence of bleeding on probing (BOP), probing depth (PD), and attachment loss (AL) were used during the clinical periodontal assessment. GCF samples were obtained and analyzed by an enzyme-linked immunosorbent assay (ELISA). RESULTS: GCF GM-CSF, MIP-1α, and IL-1ß were significantly higher in SII-P and SIII-P groups than in PH, G, and SI-P groups (p<0.05). There was no significant difference among the PH, G, and SI-P groups in IL-1ß, GM-CSF, and MIP-1α levels (p>0.05). CONCLUSIONS: These results show that GM-CSF expression was increased in SII-P, SIII-P, and SIV-P. Furthermore, GM-CSF levels may have some potential to discriminate between early and advanced stages of periodontitis.
Assuntos
Gengivite , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Periodontite , Líquido do Sulco Gengival , Gengivite/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Índice Periodontal , Periodontite/metabolismoRESUMO
Background: Limited data are available regarding the differences between immunological, biochemical, and cellular contents of human colostrum following maternal infection during pregnancy with coronavirus 2 disease (COVID-19). Objective: To investigate whether maternal COVID-19 infection may affect immunological, biochemical, and cellular contents of human colostrum. Methods: Using a case-control study design, we collected colostrum from 14 lactating women with a previous diagnosis of COVID-19 during pregnancy and 12 without a clear diagnosis during September 2020 to May 2021. Colostrum samples were analysed for some enzymes and non-enzymatic oxidative stress markers (SOD, CAT, GPx, MDA, GSH, GSSG, H2O2, MPO) and for IL-1ß, IL-6, tumour necrosis factor (TNF)-α, protein induced by interferon gamma (IP)-10, IL-8, IFN-λ1, IL12p70, IFN-α2, IFN-λ2/3, granulocyte macrophage colony stimulating factor (GM-CSF), IFN-ß, IL-10 and IFN-γ, along with IgA and IgG for the SARS-CoV-2 S protein. We perform immunophenotyping to assess the frequency of different cell types in the colostrum. Results: Colostrum from the COVID-19 symptomatic group in pregnancy contained reduced levels of H2O2, IFN-α2, and GM-CSF. This group had higher levels of GSH, and both NK cell subtypes CD3-CD56brightCD16-CD27+IFN-γ+ and CD3-CD56dimCD16+CD27- were also increased. Conclusion: The present results reinforce the protective role of colostrum even in the case of mild SARS-Cov-2 infection, in addition to demonstrating how adaptive the composition of colostrum is after infections. It also supports the recommendation to encourage lactating women to continue breastfeeding after COVID-19 illness.
Assuntos
COVID-19 , Complicações Infecciosas na Gravidez , Gravidez , Feminino , Humanos , Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Colostro/metabolismo , COVID-19/metabolismo , Estudos de Casos e Controles , Peróxido de Hidrogênio/metabolismo , Lactação , SARS-CoV-2 , Interferon gama/metabolismo , Complicações Infecciosas na Gravidez/metabolismoRESUMO
Oligozoospermia is a common infertility disease, and the incidence rate is increasing year by year. Cuscuta chinensis is a commonly used medicine for the treatment of oligozoospermia in Chinese medicine. Flavonoids are its main component. GM-CSF is a multifunctional cytokine that plays an important role in the inflammatory response. In this paper, we performed HE staining and immunohistochemical staining on the testis of rats with oligozoospermia. We intend to study the expression changes of GM-CSF in rats with oligospermia and the effect of flavonoids on the expression of GM-CSF in testis of rats with oligozoospermia.
La oligozoospermia es una enfermedad común de infertilidad, con una tasa de incidencia que aumenta año tras año. Cuscuta chinensis es un medicamento de uso común para el tratamiento de la oligozoospermia en la medicina china. Los flavonoides son su componente principal. GM-CSF es una citocina multifuncional que tiene un rol importante en la respuesta inflamatoria. En este trabajo, realizamos tinción con hematoxilina y eosina y tinción inmunohistoquímica en testículos de ratas con oligozoospermia. TNuestro objetivo fue estudiar los cambios de expresión de GM-CSF en ratas con oligozoospermia y el efecto de los flavonoides en la expresión de GM-CSF en testículos de ratas con oligozoospermia.
Assuntos
Animais , Masculino , Ratos , Oligospermia/metabolismo , Oligospermia/tratamento farmacológico , Flavonoides/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Cuscuta , Testículo/efeitos dos fármacos , Testículo/metabolismo , Imuno-Histoquímica , Ratos Sprague-DawleyRESUMO
The larval stage of the cestode Echinococcus granulosus causes cystic echinococcosis in humans and livestock. This larva is protected by the millimeter-thick, mucin-based laminated layer (LL), from which materials have to be shed to allow parasite growth. We previously reported that dendritic cells (DCs) respond to microscopic pieces of the mucin gel of the LL (pLL) with unconventional maturation phenotypes, in the absence or presence of Toll-like receptor (TLR) agonists, including lipopolysaccharide (LPS). We also reported that the presence of pLL inhibited the activating phosphorylation of the phosphatidylinositol 3-kinase (PI3K) effector Akt induced by granulocyte-macrophage colony-stimulating factor or interleukin-4. We now show that the inhibitory effect of pLL extends to LPS as a PI3K activator, and results in diminished phosphorylation of GSK3 downstream from Akt. Functionally, the inhibition of Akt and GSK3 phosphorylation are linked to the blunted upregulation of CD40, a major feature of the unconventional maturation phenotype. Paradoxically, all aspects of unconventional maturation induced by pLL depend on PI3K class I. Additional components of the phagocytic machinery are needed, but phagocytosis of pLL particles is not required. These observations hint at a DC response mechanism related to receptor-independent mechanisms proposed for certain crystalline and synthetic polymer-based particles; this would fit the previously reported lack of detection of molecular-level motifs necessary of the effects of pLL on DCs. Finally, we report that DCs exposed to pLL are able to condition DCs not exposed to the material so that these cannot upregulate CD40 in full in response to LPS.
Assuntos
Antígenos CD40/biossíntese , Células Dendríticas/imunologia , Echinococcus granulosus/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Células Cultivadas , Equinococose/imunologia , Equinococose/parasitologia , Equinococose/patologia , Ativação Enzimática/imunologia , Quinase 3 da Glicogênio Sintase/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-4/metabolismo , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/fisiologia , Fosforilação , Transdução de Sinais/imunologiaRESUMO
Reprogramming of neutrophils by malignant cells is well-described for many types of solid tumors, but data remain scarce for hematological diseases. Chronic lymphocytic leukemia (CLL) is characterized for a deep immune dysregulation mediated by leukemic cells that compromises patient's outcome. Murine models of CLL highlight the relevance of myeloid cells as tumor-driven reprogramming targets. In our study, we evaluated neutrophil reprogramming by CLL cells. We first show that the proportion of the CD16high CD62Ldim neutrophil subset in peripheral blood of CLL patients is increased compared to age-matched healthy donors (HD). In vitro, neutrophils from HD cultured in the presence of CLL cells or conditioned media (CM) from CLL cells exhibited a longer lifespan. Depletion of G-CSF and GM-CSF from CM partially reversed the protective effect. In addition, the proportion of viable neutrophils that displayed a CD16high CD62Ldim phenotype was increased in the presence of CM from CLL cells, being TGF-ß/IL-10 responsible for this effect. Altogether, our results describe a novel mechanism through which CLL cells can manipulate neutrophils.
Assuntos
Diferenciação Celular/fisiologia , Tolerância Imunológica/fisiologia , Selectina L/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Neutrófilos/patologia , Receptores de IgG/metabolismo , Idoso , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Eikenella corrodens is a gram-negative bacterium, and although primarily associated with periodontal infections or infective endocarditis, it has been identified in coronary atheromatous plaques. The effect of its lipopolysaccharide (LPS) on human coronary artery endothelial cells (HCAECs) is unknown. Our aim was to examine the mechanism underlying the inflammatory response in HCAECs stimulated with E. corrodens-LPS and to evaluate monocyte adhesion. Endothelial responses were determined by measuring the levels of chemokines and cytokines using flow cytometry. The surface expression of intercellular adhesion molecule 1 (ICAM-1) was determined using a cell-based ELISA, and the adhesion of THP-1 monocytes to HCAECs was also monitored. The involvement of toll-like receptors (TLRs) 2 and 4 was examined using TLR-neutralizing antibodies, and activation of extracellular signal-regulated kinase (ERK)1/2 and nuclear factor-kappa B (NF-κB) p65 were measured by western blotting and ELISA, respectively. Eikenella corrodens-LPS increased secretion of interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and expression of ICAM-1 on the surface of HCAECs, consistent with the increased adhesion of THP-1 cells. Moreover, E. corrodens-LPS interacted with TLR4, a key receptor able to maintain the levels of IL-8, MCP-1, and GM-CSF in HCAECs. Phosphorylation of ERK1/2 and activation of NF-κB p65 were also increased. The results indicate that E. corrodens-LPS activates HCAECs through TLR4, ERK, and NF-κB p65, triggering a pro-atherosclerotic endothelial response and enhancing monocyte adhesion.
Assuntos
Doença da Artéria Coronariana/induzido quimicamente , Doença da Artéria Coronariana/imunologia , Vasos Coronários/efeitos dos fármacos , Eikenella corrodens/metabolismo , Células Endoteliais/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/imunologia , Monócitos/efeitos dos fármacos , Anticorpos Neutralizantes , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , Células THP-1/imunologia , Receptor 2 Toll-Like , Receptor 4 Toll-Like/efeitos dos fármacosRESUMO
BACKGROUND: A simple blood test for detecting active tuberculosis (TB) could be key to this epidemic containment, given that a large proportion of patients are unable to produce sputum for testing. Currently available interferon-γ release assays (IGRAs) are inadequate to diagnose active TB, with reported pooled sensitivity and specificity both under 81%. OBJECTIVE: To explore whether cytokines/chemokines other than interferon-γ in response to long-term cell stimulation could improve the ability to distinguish between different TB infection status. METHODS: We prospectively enrolled subjects with newly diagnosed pulmonary TB and their household contacts in Santiago. All contacts were tested with IGRA. Peripheral blood mononuclear cells were obtained and antigen-specific stimulated for 72â¯h before collecting their culture supernatants. RESULTS: Subjects with active TB displayed markedly low cytokines/chemokines secretion upon PBMC stimulation, with lower GM-CSF being the best differentiator from IGRA(+) contacts, with 71% (95% CI 53-85) sensitivity, 86% (95% CI 65-97) specificity and AUC = 0.79 (p = 0.0003). On the other hand, when compared to the uninfected IGRA(-) contacts, higher level of IL-2 secretion was the best indicator of active TB, with 73.5% (95% CI 56-87) sensitivity, 85% (95% CI 66-96) specificity and AUCâ¯=â¯0.79 (pâ¯=â¯0.0001). No single cytokine/chemokine released upon stimulation could accurately differentiate between active TB and all TB contacts grouped together. CONCLUSION: GM-CSF and IL-2 provided the best yield to differentiate active TB from latent TB and from TB uninfected, respectively, with higher specificities than that reported for IGRAs. However, none of both resulted sensitive enough to be used as a stand-alone biomarker for active TB.