RESUMEN
The spatial arrangement of immune cells within the tumor microenvironment (TME) and their interactions play critical roles in the initiation and development of cancer. Several advanced technologies such as imaging mass cytometry (IMC) providing the immunological landscape of the TME with single-cell resolution. In this study, we develop a new method to quantify the spatial proximity between different cell types based on single-cell spatial data. Using this method on IMC data from 416 lung adenocarcinoma patients, we show that the proximity between different cell types is more correlated with patient prognosis compared to the traditional features such immune cell density and fractions. Consistent with previous reports, our results validate that proximity of T helper (Th) and B cells to cancer cells is associated with survival benefits. More importantly, we discover that the proximity of M2 macrophages to multiple immune cells is associated with poor prognosis. When Th/B cells are stratified into M2-distal and M2-proximal, the abundance of the former but not the latter category of Th/B cells is correlated with enhanced patient survival. Additionally, the abundance of M2-distal and M2-proximal cytotoxic T cells (Tc) is respectively associated with good and poor prognosis. Our results indicate that the prognostic effect of Th, Tc, and B cells in the tumor microenvironment is modulated by the nearby M2 macrophages. The proposed new method proposed can be readily applied to all single-cell spatial data for revealing functional impact of immune cell interactions.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Linfocitos Infiltrantes de Tumor , Macrófagos , Microambiente Tumoral , Humanos , Pronóstico , Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/mortalidad , Microambiente Tumoral/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/mortalidad , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Macrófagos/inmunología , Macrófagos/patología , Macrófagos/metabolismo , Linfocitos B/inmunología , Linfocitos B/patología , Linfocitos B/metabolismo , Análisis de la Célula Individual/métodosRESUMEN
AIMS: Cardiac fibrosis is characterized by aberrant collagen deposition in the heart. Macrophage polarization or infiltration is the main reason to accelerate the collagen deposition. We attempted to investigate the involvement of MKL1 in macrophages during the development of cardiac fibrosis. MATERIALS AND METHODS: Cardiac fibrosis is induced by myocardial infarction (MI). The MKL1f/f mice were crossed to the Lyz2-cre mice to generate macrophage conditional MKL1 knockout mice (MKL1ΔMφ). In addition, macrophage conditional MKL1 overexpression mice (MKL1MÏ-OE) were constructed by crossing Lyz2-cre mice to MKL1ΔN200-Rosa26 mice. KEY FINDINGS: MKL1 expression was significantly increased in macrophages of both ischemic cardiomyopathy (ICM) patients and mice induced to develop myocardial infarction. Deletion of MKL1 in macrophages improved the heart function after MI-induced cardiac fibrosis. Consistently, MKL1MÏ-OE mice displayed more severe cardiac fibrosis and worsened heart function than the control mice after MI. Moreover, administration of a small-molecule MKL1 inhibitor CCG-1423 also decreased the collagen deposition after MI. SIGNIFICANCE: Our data demonstrate that MKL1 in macrophages contributes to cardiac fibrosis pathogenesis and reinforce the notion that targeting MKL1 may yield effective antifibrotic therapeutics in the heart.
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Modelos Animales de Enfermedad , Fibrosis , Macrófagos , Infarto del Miocardio , Transactivadores , Animales , Humanos , Masculino , Ratones , Colágeno/metabolismo , Fibrosis/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/patología , Infarto del Miocardio/metabolismo , Miocardio/patología , Miocardio/metabolismo , Transactivadores/metabolismo , Transactivadores/genéticaRESUMEN
The malignant behavior and immune escape ability of cancer cells lead to therapeutic failure and poor prognosis for patients with various cancers, including colon cancer. Plexin domain containing 1 (PLXDC1) was initially identified to exert key roles in tumor by regulating angiogenesis and has recently proved to be involved in cell proliferation and migration of glioblastoma and gastric cancer cells. However, its roles in colon cancer remain unclear. In this study, the online bioinformatics databases confirmed high expression of PLXDC1 in colon cancer specimens, which was associated with cancer stages and nodal metastasis. Similarly, the increased expression of PLXDC1 was also validated in our collected samples and colon cancer cells. Moreover, patients with high expression of PLXDC1 had shorter survival, indicating that PLXDC1 might be a potential prognostic predictor for colon cancer patients. Notably, targeting PLXDC1 inhibited cancer cell viability and invasion, and enhanced cell apoptosis. Intriguingly, Tumor Immune Estimation Resource database confirmed that PLXDC1 expression was related to various tumor-infiltrating immune cells in colon adenocarcinoma including macrophages, and its expression was also correlated with M2-like macrophage markers. In vitro, colon cancer cells with PLXDC1 downregulation had a reduced ability to recruit and polarize macrophage towards M2 phenotype by decreasing the percentage of CD206+ cells and M2-like markers (CD206, CD163, arginase1, and interleukin 10 [IL-10]). Moreover, PLXDC1 knockdown attenuated M2 macrophage-mediated promotion in cancer cell viability and invasion. Mechanically, inhibition of PLXDC1 suppressed activation of the IL-6/Signal transducer and activator of transcription 3 (STAT3) signaling. Reactivating the above pathway by transfection with IL-6 plasmids reversed the suppressive effects of PLXDC1 knockdown on cancer cell malignant behaviors, macrophage recruitment and M2-like polarization. Thus, PLXDC1 downregulation may inhibit the malignancy of colon cancer cells and their ability to recruit and polarize macrophages towards M2 phenotype by blocking the IL-6/STAT3 pathway. Together, targeting PLXDC1 may attenuate the progression of colon cancer by direct roles in cancer cells and indirect roles in macrophage polarization, representing a promising therapeutic target for colon cancer patients.
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Biomarcadores de Tumor , Neoplasias del Colon , Macrófagos , Humanos , Neoplasias del Colon/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/genética , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Pronóstico , Macrófagos/metabolismo , Macrófagos/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/genética , Masculino , Femenino , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
BACKGROUND: CT-detected Extramural venous invasion (EMVI) is known as an independent risk factor for distant metastasis in patients with advanced gastric cancer (GC). However, the molecular basis is not clear. In colorectal cancer, M2 macrophages plays a vital role in determining EMVI. This study aimed to investigate the relationship between CT-detected EMVI and the M2 macrophages as well as prognosis predictionusing a radiogenomic approach. METHOD: We utilized EMVI-related genes (from mRNA sequencing of 13 GC samples correlated with EMVI score by spearman analysis, P < 0.01) to overlap the co-expression genes of WGCNA module and M2 macrophages related genes (from mRNA data of 371 GC patients in TCGA database), generating a total of 136 genes. An EMVI-M2-prognosis-related hub gene signature was constructed by COX and least absolute shrinkage and selection operator (LASSO) analysis from a training cohort TCGA database (n = 371) and validated it in a validation cohort from GEO database (n = 357). High- and low-risk groups were divided by hub gene (EGFLAM and GNG11) signature-derived risk scores. We assessed its predictive ability through Kaplan-Meier (K-M) curve and COX analysis. Furthermore, we utilized ESTIMATE to detect tumor mutation burden (TMB) and evaluate sensitivity to immune checkpoint inhibitors (ICIs). Expression of hub genes was tested using western blotting and immunohistochemistry (IHC) analysis. RESULTS: The overall survival (OS) was significantly reduced in the high-risk group (Training/Validation: AUC = 0.701/0.620; P < 0.001/0.003). Furthermore, the risk score was identified as an independent predictor of OS in multivariate COX regression analyses (Training/Validation: HR = 1.909/1.928; 95% CI: 1.225-2.974/1.308-2.844). The low-risk group exhibited significantly higher TMB levels (P = 1.6e- 07) and greater sensitivity to ICIs. Significant higher expression of hub-genes was identified on multiple GC cell lines and original samples. Hub-genes knockdown in gastric cancer cell lines inhibited their proliferation, metastatic and invasive capacity to varying degrees. In vivo experiments indicate that EGFLAM, as one of the hub genes, its high expression can serve as a biomarker for low response to immunotherapy. CONCLUSION: Our study demonstrated EMVI-M2 gene signature could effectively predict the prognosis of GC tissue, reflecting the relationship between EMVI and M2 macrophages.
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Regulación Neoplásica de la Expresión Génica , Macrófagos , Invasividad Neoplásica , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Femenino , Pronóstico , Persona de Mediana Edad , Tomografía Computarizada por Rayos X , Estimación de Kaplan-Meier , Análisis de Supervivencia , Transcriptoma/genética , Animales , Línea Celular Tumoral , Perfilación de la Expresión Génica , Reproducibilidad de los Resultados , AncianoRESUMEN
Macrophages are a dynamic cell type of the immune system implicated in the pathophysiology of vascular diseases and are a major contributor to pathological inflammation. Excessive macrophage accumulation, activation, and polarization is observed in aortic aneurysm (AA), atherosclerosis, and pulmonary arterial hypertension. In general, macrophages become activated and polarized to a pro-inflammatory phenotype, which dramatically changes cell behavior to become pro-inflammatory and infiltrative. These cell types become cumbersome and fail to be cleared by normal mechanisms such as autophagy. The result is a hyper-inflammatory environment causing the recruitment of adjacent cells and circulating immune cells to further augment the inflammatory response. In AA, this leads to excessive ECM degradation and chemokine secretion, ultimately causing macrophages to dominate the immune cell landscape in the aortic wall. In atherosclerosis, monocytes are recruited to the vascular wall, where they polarize to the pro-inflammatory phenotype and induce inflammatory pathway activation. This leads to the development of foam cells, which significantly contribute to neointima and necrotic core formation in atherosclerotic plaques. Pro-inflammatory macrophages, which affect other vascular diseases, present with fragmented mitochondria and corresponding metabolic dysfunction. Targeting macrophage mitochondrial dynamics has proved to be an exciting potential therapeutic approach to combat vascular disease. This review will summarize mitochondrial and metabolic mechanisms of macrophage activation, polarization, and accumulation in vascular diseases.
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Metabolismo Energético , Macrófagos , Mitocondrias , Fenotipo , Enfermedades Vasculares , Humanos , Animales , Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patología , Enfermedades Vasculares/fisiopatología , Enfermedades Vasculares/inmunología , Transducción de Señal , Activación de Macrófagos , Mediadores de Inflamación/metabolismo , Dinámicas MitocondrialesRESUMEN
Acute lymphoblastic leukemia (ALL) is the most common type of malignancy in children. ALL prognosis after initial diagnosis is generally good; however, patients suffering from relapse have a poor outcome. The tumor microenvironment is recognized as an important contributor to relapse, yet the cell-cell interactions involved are complex and difficult to study in traditional experimental models. In the present study, we established an innovative larval zebrafish xenotransplantation model, that allows the analysis of leukemic cells (LCs) within an orthotopic niche using time-lapse microscopic and flow cytometric approaches. LCs homed, engrafted and proliferated within the hematopoietic niche at the time of transplant, the caudal hematopoietic tissue (CHT). A specific dissemination pattern of LCs within the CHT was recorded, as they extravasated over time and formed clusters close to the dorsal aorta. Interactions of LCs with macrophages and endothelial cells could be quantitatively characterized. This zebrafish model will allow the quantitative analysis of LCs in a functional and complex microenvironment, to study mechanisms of niche mediated leukemogenesis, leukemia maintenance and relapse development.
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Leucemia-Linfoma Linfoblástico de Células Precursoras , Microambiente Tumoral , Pez Cebra , Animales , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Humanos , Modelos Animales de Enfermedad , Comunicación Celular , Xenoinjertos , Nicho de Células Madre , Línea Celular Tumoral , Células Endoteliales/patología , Macrófagos/patología , Macrófagos/metabolismo , Trasplante HeterólogoRESUMEN
The proneural-mesenchymal (PN-MES) transformation of glioma stem cells (GSCs) can significantly increase proliferation, invasion, chemotherapy tolerance, and recurrence. M2-like polarization of tumor-associated macrophages (TAMs) has a strong immunosuppressive effect, promoting tumor malignancy and angiogenesis. There is limited understanding on the interactions between GSCs and TAMs as well as their associated molecular mechanisms. In the present study, bioinformatics analysis, GSC and TAM co-culture, determination of TAM polarization phenotypes, and other in vitro experiments confirmed that CCL2 secreted by MES-GSCs promotes TAM-M2 polarization via the IKZF1-CD84-SHP2 pathway and PN-MES transformation of GSCs via the IKZF1-LRG1 pathway in TAMs. IKZF1 inhibitors could significantly reduce tumor volumes in animal glioma models and improve survival, as well as suppress TAM-M2 polarization and the GSC malignant phenotype. The results of this study indicate the important interaction between TAMs and GSCs in the glioma microenvironment as well as its role in tumor progression. The findings also suggest a novel target for follow-up clinical transformation research on the regulation of TAM function and GSCs malignant phenotype.
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Quimiocina CCL2 , Glioma , Factor de Transcripción Ikaros , Células Madre Neoplásicas , Microambiente Tumoral , Macrófagos Asociados a Tumores , Glioma/patología , Glioma/genética , Glioma/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CCL2/genética , Animales , Macrófagos Asociados a Tumores/metabolismo , Macrófagos Asociados a Tumores/patología , Humanos , Ratones , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/metabolismo , Microambiente Tumoral/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Transducción de Señal/genética , Línea Celular Tumoral , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Antígenos CD/metabolismo , Antígenos CD/genética , Regulación Neoplásica de la Expresión Génica , Macrófagos/metabolismo , Macrófagos/patologíaRESUMEN
The salivary glands are often damaged during head and neck cancer radiotherapy. This results in chronic dry mouth, which adversely affects quality of life and for which there is no long-term cure. Mouse models of salivary gland injury are routinely used in regenerative research. However, there is no clear consensus on the radiation regime required to cause injury. Here, we analysed three regimes of γ-irradiation of the submandibular salivary gland. Transcriptional analysis, immunofluorescence and flow cytometry was used to profile DNA damage, gland architecture and immune cell changes 3â days after single doses of 10 or 15â Gy or three doses of 5â Gy. Irrespective of the regime, radiation induced comparable levels of DNA damage, cell cycle arrest, loss of glandular architecture, increased pro-inflammatory cytokines and a reduction in tissue-resident macrophages, relative to those observed in non-irradiated submandibular glands. Given these data, coupled with the fact that repeated anaesthetic can negatively affect animal welfare and interfere with saliva secretion, we conclude that a single dose of 10â Gy irradiation is the most refined method of inducing acute salivary gland injury in a mouse model.
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Daño del ADN , Fraccionamiento de la Dosis de Radiación , Ratones Endogámicos C57BL , Glándulas Salivales , Animales , Glándulas Salivales/efectos de la radiación , Glándulas Salivales/patología , Glándula Submandibular/efectos de la radiación , Glándula Submandibular/patología , Rayos gamma/efectos adversos , Citocinas/metabolismo , Ratones , Masculino , Macrófagos/efectos de la radiación , Macrófagos/patología , Macrófagos/metabolismo , Puntos de Control del Ciclo Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , FemeninoRESUMEN
Macrophages in atherosclerotic lesions exhibit a spectrum of behaviours or phenotypes. The phenotypic distribution of monocyte-derived macrophages (MDMs), its correlation with MDM lipid content, and relation to blood lipoprotein densities are not well understood. Of particular interest is the balance between low density lipoproteins (LDL) and high density lipoproteins (HDL), which carry bad and good cholesterol respectively. To address these issues, we have developed a mathematical model for early atherosclerosis in which the MDM population is structured by phenotype and lipid content. The model admits a simpler, closed subsystem whose analysis shows how lesion composition becomes more pathological as the blood density of LDL increases relative to the HDL capacity. We use asymptotic analysis to derive a power-law relationship between MDM phenotype and lipid content at steady-state. This relationship enables us to understand why, for example, lipid-laden MDMs have a more inflammatory phenotype than lipid-poor MDMs when blood LDL lipid density greatly exceeds HDL capacity. We show further that the MDM phenotype distribution always attains a local maximum, while the lipid content distribution may be unimodal, adopt a quasi-uniform profile or decrease monotonically. Pathological lesions exhibit a local maximum in both the phenotype and lipid content MDM distributions, with the maximum at an inflammatory phenotype and near the lipid content capacity respectively. These results illustrate how macrophage heterogeneity arises in early atherosclerosis and provide a framework for future model validation through comparison with single-cell RNA sequencing data.
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Aterosclerosis , Lipoproteínas HDL , Lipoproteínas LDL , Macrófagos , Conceptos Matemáticos , Fenotipo , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Aterosclerosis/patología , Aterosclerosis/metabolismo , Aterosclerosis/sangre , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/sangre , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Modelos Cardiovasculares , Metabolismo de los Lípidos , Lipoproteínas/metabolismo , Lipoproteínas/sangre , Simulación por ComputadorRESUMEN
The formation of macrophage-derived foam cells has been recognized as the pathological hallmark of atherosclerotic diseases. However, the pathological evolution dynamics and underlying regulatory mechanisms remain largely unknown. Herein, we introduce a single-particle rotational microrheology method for pathological staging of macrophage foaming and antiatherosclerotic explorations by probing the dynamic changes of lysosomal viscous feature over the pathological evolution progression. The principle of this method involves continuous monitoring of out-of-plane rotation-caused scattering brightness fluctuations of the gold nanorod (AuNR) probe-based microrheometer and subsequent determination of rotational relaxation time to analyze the viscous feature in macrophage lysosomes. With this method, we demonstrated the lysosomal viscous feature as a robust pathological reporter and uncovered three distinct pathological stages underlying the evolution dynamics, which are highly correlated with a pathological stage-dependent activation of the NLRP3 inflammasome-involved positive feedback loop. We also validated the potential of this positive feedback loop as a promising therapeutic target and revealed the time window-dependent efficacy of NLRP3 inflammasome-targeted drugs against atherosclerotic diseases. To our knowledge, the pathological staging of macrophage foaming and the pathological stage-dependent activation of the NLRP3 inflammasome-involved positive feedback mechanism have not yet been reported. These findings provide insights into in-depth understanding of evolutionary features and regulatory mechanisms of macrophage foaming, which can benefit the analysis of effective therapeutical drugs as well as the time window of drug treatment against atherosclerotic diseases in preclinical studies.
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Aterosclerosis , Células Espumosas , Oro , Proteína con Dominio Pirina 3 de la Familia NLR , Aterosclerosis/patología , Animales , Oro/química , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Células Espumosas/patología , Células Espumosas/metabolismo , Macrófagos/patología , Macrófagos/metabolismo , Humanos , Lisosomas/metabolismo , Inflamasomas/metabolismo , Nanotubos/química , ReologíaRESUMEN
Chronic obstructive pulmonary disease (COPD) is a complex syndrome with poorly understood mechanisms driving its early progression (GOLD stages 1-2). Elucidating the genetic factors that influence early-stage COPD, particularly those related to airway inflammation and remodeling, is crucial. This study analyzed lung tissue sequencing data from patients with early-stage COPD (GSE47460) and smoke-exposed mice. We employed Weighted Gene Co-Expression Network Analysis (WGCNA) and machine learning to identify potentially pathogenic genes. Further analyses included single-cell sequencing from both mice and COPD patients to pinpoint gene expression in specific cell types. Cell-cell communication and pseudotemporal analyses were conducted, with findings validated in smoke-exposed mice. Additionally, Mendelian randomization (MR) was used to confirm the association between candidate genes and lung function/COPD. Finally, functional validation was performed in vitro using cell cultures. Machine learning analysis of 30 differentially expressed genes identified 8 key genes, with CLEC5A emerging as a potential pathogenic factor in early-stage COPD. Bioinformatics analyses suggested a role for CLEC5A in macrophage-mediated inflammation during COPD. Two-sample Mendelian randomization linked CLEC5A single nucleotide polymorphisms (SNPs) with Forced Expiratory Volume in One Second (FEV1), FEV1/Forced Vital Capacity (FVC) and early/later on COPD. In vitro, the knockdown of CLEC5A led to a reduction in inflammatory markers within macrophages. Our study identifies CLEC5A as a critical gene in early-stage COPD, contributing to its pathogenesis through pro-inflammatory mechanisms. This discovery offers valuable insights for developing early diagnosis and treatment strategies for COPD and highlights CLEC5A as a promising target for further investigation.
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Progresión de la Enfermedad , Inflamación , Lectinas Tipo C , Macrófagos , Polimorfismo de Nucleótido Simple , Enfermedad Pulmonar Obstructiva Crónica , Receptores de Superficie Celular , Animales , Humanos , Masculino , Ratones , Inflamación/genética , Inflamación/patología , Inflamación/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Pulmón/patología , Pulmón/metabolismo , Aprendizaje Automático , Macrófagos/metabolismo , Macrófagos/patología , Análisis de la Aleatorización Mendeliana , Ratones Endogámicos C57BL , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Gallbladder carcinoma (GBC) is a malignant hepatobiliary cancer characterized by an intricate tumor microenvironments (TME) and heterogeneity. The traditional GBC 2D culture models cannot faithfully recapitulate the characteristics of the TME. Three-dimensional (3D) bioprinting enables the establishment of high-throughput and high-fidelity multicellular GBC models. In this study, we designed a concentric cylindrical tetra-culture model to reconstitute the spatial distribution of cells in tumor tissue, with the inner portion containing GBC cells, and the outer ring containing a mixture of endothelial cells, fibroblasts, and macrophages. We confirmed the survival, proliferation, biomarker expression and gene expression profiles of GBC 3D tetra-culture models. Hematoxylin-eosin (HE) and immunofluorescence staining verified the morphology and robust expression of GBC/endothelial/fibroblast/macrophage biomarkers in GBC 3D tetra-culture models. Single-cell RNA sequencing revealed two distinct subtypes of GBC cells within the model, glandular epithelial and squamous epithelial cells, suggesting the mimicry of intratumoral heterogeneity. Comparative transcriptome profile analysis among variousin vitromodels revealed that cellular interactions and the TME in 3D tetra-culture models reshaped the biological processes of tumor cells to a more aggressive phenotype. GBC 3D tetra-culture models restored the characteristics of the TME as well as intratumoral heterogeneity. Therefore, this model is expected to have future applications in tumor biology research and antitumor drug development.
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Bioimpresión , Neoplasias de la Vesícula Biliar , Impresión Tridimensional , Microambiente Tumoral , Humanos , Neoplasias de la Vesícula Biliar/patología , Neoplasias de la Vesícula Biliar/metabolismo , Línea Celular Tumoral , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos/citología , Proliferación CelularRESUMEN
Sepsis has a high mortality rate and leads to multi-organ failure, including lung injury. Inactive rhomboid protease family protein (iRhom2) has been identified as accountable for the release of TNF-α, a crucial mediator in the development of sepsis. This study aimed to evaluate the role of iRhom2 in sepsis and sepsis-induced acute lung injury (ALI). TNF-α and IL-6 secretion in vitro by peritoneal macrophages from wild-type (WT) and iRhom2 knoukout (KO) mice was assessed by enzyme-linked immunosorbent assay. Cecal ligation and puncture (CLP)-induced murine sepsis model was used for in vivo experiments. To evaluate the role of iRhom2 deficiency on survival during sepsis, both WT and iRhom2 KO mice were monitored for 8 consecutive days following the CLP. For histologic and biochemical examination, the mice were killed 18 h after CLP. iRhom2 deficiency improved the survival of mice after CLP. iRhom2 deficiency decreased CD68+ macrophage infiltration in lung tissues. Multiplex immunohistochemistry revealed that the proportion of Ki-67+ CD68+ macrophages was significantly lower in iRhom2 KO mice than that in WT mice after CLP. Moreover, CLP-induced release of TNF-α and IL-6 in the serum were significantly inhibited by iRhom2 deficiency. iRhom2 deficiency reduced NF-kB p65 and IκBα phosphorylation after CLP. iRhom2 deficiency reduces sepsis-related mortality associated with attenuated macrophage infiltration and proliferation in early lung injury. iRhom2 may play a pivotal role in the pathogenesis of sepsis and early stage of sepsis-induced ALI. Thus, iRhom2 may be a potential therapeutic target for the management of sepsis and sepsis-induced ALI.
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Ratones Endogámicos C57BL , Ratones Noqueados , Sepsis , Animales , Sepsis/metabolismo , Sepsis/patología , Ratones , Masculino , Proteínas Portadoras/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Macrófagos/metabolismo , Macrófagos/patología , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patologíaRESUMEN
Macrophages and osteocytes are important regulators of inflammation, osteogenesis and osteoclastogenesis. However, their interactions under adverse conditions, such as biomaterial-associated infection (BAI) are not fully understood. We aimed to elucidate how factors released from macrophages modulate osteocyte responses in an in vitro indirect 3D co-culture model. Human monocyte-derived macrophages were cultured on etched titanium disks and activated with either IL-4 cytokine (anti-inflammatory M2 phenotype) or Staphylococcus aureus secreted virulence factors to simulate BAI (pro-inflammatory M1 phenotype). Primary osteocytes in collagen gels were then stimulated with conditioned media (CM) from these macrophages. The osteocyte response was analyzed by gene expression, protein secretion, and immunostaining. M1 phenotype macrophages were confirmed by IL-1ß and TNF-α secretion, and M2 macrophages by ARG-1 and MRC-1.Osteocytes receiving M1 CM revealed bone inhibitory effects, denoted by reduced secretion of bone formation osteocalcin (BGLAP) and increased secretion of the bone inhibitory sclerostin (SOST). These osteocytes also downregulated the pro-mineralization gene PHEX and upregulated the anti-mineralization gene MEPE. Additionally, exhibited pro-osteoclastic potential by upregulating pro-osteoclastic gene RANKL expression. Nonetheless, M1-stimulated osteocytes expressed a higher level of the potent pro-osteogenic factor BMP-2 in parallel with the downregulation of the bone inhibitor genes DKK1 and SOST, suggesting a compensatory feedback mechanisms. Conversely, M2-stimulated osteocytes mainly upregulated anti-osteoclastic gene OPG expression, suggesting an anti-catabolic effect. Altogether, our findings demonstrate a strong communication between M1 macrophages and osteocytes under M1 (BAI)-simulated conditions, suggesting that the BAI adverse effects on osteoblastic and osteoclastic processes in vitro are partly mediated via this communication. STATEMENT OF SIGNIFICANCE: Biomaterial-associated infections are major challenges and the underlying mechanisms in the cellular interactions are missing, especially among the major cells from the inflammatory side (macrophages as the key cell in bacterial clearance) and the regenerative side (osteocyte as main regulator of bone). We evaluated the effect of macrophage polarization driven by the stimulation with bacterial virulence factors on the osteocyte function using an indirect co-culture model, hence mimicking the scenario of a biomaterial-associated infection. The results suggest that at least part of the adverse effects of biomaterial associated infection on osteoblastic and osteoclastic processes in vitro are mediated via macrophage-to-osteocyte communication.
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Comunicación Celular , Macrófagos , Osteocitos , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos/microbiología , Osteocitos/metabolismo , Osteocitos/efectos de los fármacos , Técnicas de Cocultivo , Infecciones Relacionadas con Prótesis/patología , Infecciones Relacionadas con Prótesis/metabolismo , Infecciones Relacionadas con Prótesis/microbiología , Medios de Cultivo Condicionados/farmacología , Modelos Biológicos , Osteogénesis/efectos de los fármacosRESUMEN
Cell volume as a characteristic of changes in response to external environmental cues has been shown to control the fate of stem cells. However, its influence on macrophage behavior and macrophage-mediated inflammatory responses have rarely been explored. Herein, through mediating the volume of macrophages by adding polyethylene glycol (PEG), we demonstrated the feasibility of fine-tuning cell volume to regulate macrophage polarization towards anti-inflammatory phenotypes, thereby enabling to reverse macrophage-mediated inflammation response. Specifically, lower the volume of primary macrophages can induce both resting macrophages (M0) and stimulated pro-inflammatory macrophages (M1) to up-regulate the expression of anti-inflammatory factors and down-regulate pro-inflammatory factors. Further mechanistic investigation revealed that macrophage polarization resulting from changing cell volume might be mediated by JAK/STAT signaling pathway evidenced by the transcription sequencing analysis. We further propose to apply this strategy for the treatment of arthritis via direct introduction of PEG into the joint cavity to modulate synovial macrophage-related inflammation. Our preliminary results verified the credibility and effectiveness of this treatment evidenced by the significant inhibition of cartilage destruction and synovitis at early stage. In general, our results suggest that cell volume can be a biophysical regulatory factor to control macrophage polarization and potentially medicate inflammatory response, thereby providing a potential facile and effective therapy for modulating macrophage mediated inflammatory responses. STATEMENT OF SIGNIFICANCE: Cell volume has recently been recognized as a significantly important biophysical signal in regulating cellular functionalities and even steering cell fate. Herein, through mediating the volume of macrophages by adding polyethylene glycol (PEG), we demonstrated the feasibility of fine-tuning cell volume to induce M1 pro-inflammatory macrophages to polarize towards anti-inflammatory M2 phenotype, and this immunomodulatory effect may be mediated by the JAK/STAT signaling pathway. We also proposed the feasible applications of this PEG-induced volume regulation approach towards the treatment of osteoarthritis (OA), wherein our preliminary results implied an effective alleviation of early synovitis. Our study on macrophage polarization mediated by cell volume may open up new pathways for immune regulation through microenvironmental biophysical clues.
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Inflamación , Quinasas Janus , Macrófagos , Factores de Transcripción STAT , Transducción de Señal , Macrófagos/metabolismo , Macrófagos/patología , Transducción de Señal/efectos de los fármacos , Animales , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Inflamación/patología , Ratones , Polietilenglicoles/farmacología , Ratones Endogámicos C57BL , MasculinoRESUMEN
BACKGROUND: Pericardial fluid (PF) contains cells, proteins, and inflammatory mediators, such as cytokines, chemokines, growth factors, and matrix metalloproteinases. To date, we lack an adequate understanding of the inflammatory response that acute injury elicits in the pericardial space. OBJECTIVE: To characterize the inflammatory profile in the pericardial space acutely after ischemia/reperfusion. METHODS: Pigs were used to establish a percutaneous ischemia/reperfusion injury model. PF was removed from pigs at different time points postanesthesia or postischemia/reperfusion. Flow cytometry was used to characterize the immune cell composition of PF, while multiplex analysis was performed on the acellular portion of PF to determine the concentration of inflammatory mediators. There was a minimum of 3 pigs per group. RESULTS: While native PF mainly comprises macrophages, we show that neutrophils are the predominant inflammatory cell type in the pericardial space after injury. The combination of acute ischemia/reperfusion (IR) and repeatedly accessing the pericardial space significantly increases the concentration of interleukin-1 beta (IL-1ß) and interleukin-1 receptor antagonist (IL-1ra). IR significantly increases the pericardial concentration of TGFß1 but not TGFß2. We observed that repeated manipulation of the pericardial space can also drive a robust pro-inflammatory response, resulting in a significant increase in immune cells and the accumulation of potent inflammatory mediators in the pericardial space. CONCLUSION: In the present study, we show that both IR and surgical manipulation can drive robust inflammatory processes in the pericardial space, consisting of an increase in inflammatory cytokines and alteration in the number and composition of immune cells.
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Modelos Animales de Enfermedad , Mediadores de Inflamación , Animales , Mediadores de Inflamación/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/inmunología , Daño por Reperfusión Miocárdica/metabolismo , Porcinos , Líquido Pericárdico/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Sus scrofa , Pericardio/patología , Pericardio/inmunología , Pericardio/metabolismo , Macrófagos/inmunología , Macrófagos/patología , Macrófagos/metabolismo , Factores de Tiempo , Citocinas/metabolismoRESUMEN
The systemic inflammatory response syndrome (SIRS) represents a self-amplifying cascade of inflammatory reactions and pathophysiological states triggered by infectious or non-infectious factors. The identification of disease targets and differential proteins in the liver (the unique and important immune organ) of SIRS mice treated with the lead compound D1 was conducted using the Genecards database and proteomic analysis, respectively. Subsequently, NOTCH1 was identified as the potential hub target via an intersection analysis between the aforementioned differentially expressed proteins and disease targets. Based on our previous research on the structure-activity relationship, we designed and synthesized a series of SIRS-related derivatives, wherein butyl, halogen, and ester groups were incorporated into benzophenone, aiming at exploring the anti-inflammatory protective action from the perspective of macrophage polarization. Notably, these derivatives exhibited a direct binding capability to the O-glucosylation site (SER496) or its vicinities (such as SER492, VAL485) of NOTCH1 using docking, SPR, DARTS, and CETSA techniques. Mechanistically, derivative D6 exerted anti-inflammatory effects via the dual NOTCH pathway. Firstly, it could inhibit NOTCH1 nuclear transcriptional activity, attenuate the interaction between NICD and RBPJK, concurrently suppress NF-κB and NLRP3 inflammasome (NLRP3, ASC, and cleaved CASP1) activation, and promote NICD (NOTCH1 active fragments) ubiquitination metabolism (the nuclear transcriptional pathway). Secondly, it might possess the ability to increase PGC1α level, subsequently, enhance ATP and MMP levels, mitigate ROS production, increase mitochondrial numbers, and ameliorate mitochondrial inflammatory damage (the mitochondrial pathway). Importantly, the activator Jagged1 could effectively reverse the aforementioned effects, while the inhibitor DAPT exhibited a synergistic effect, suggesting that the nuclear transcriptional regulation and mitochondrial regulation were both in a NOTCH1-dependent manner. Subsequently, it effectively alleviated the inflammatory response and preserved organ function as evidenced by up-regulating M2-type macrophage-related anti-inflammatory cytokines (IL10, TGFß, CD206, and ARG1) and down-regulating M1-type macrophage-related pro-inflammatory cytokines (NO, IL6, IL18, iNOS, TNFα, CD86, and IL1ß). In a word, derivative D6 modulated macrophage polarization and effectively mitigated SIRS by targeting inhibition of the dual NOTCH pathway.
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Benzofenonas , Mitocondrias , Receptor Notch1 , Transducción de Señal , Síndrome de Respuesta Inflamatoria Sistémica , Animales , Benzofenonas/farmacología , Benzofenonas/química , Ratones , Receptor Notch1/metabolismo , Receptor Notch1/genética , Transducción de Señal/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Síndrome de Respuesta Inflamatoria Sistémica/tratamiento farmacológico , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Antiinflamatorios/farmacología , Antiinflamatorios/química , Humanos , Masculino , Simulación del Acoplamiento Molecular , Inflamasomas/metabolismo , Inflamasomas/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Modelos Animales de Enfermedad , Células RAW 264.7 , Transcripción Genética/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Intestinal ischemia-reperfusion (IR) injury is a common gastrointestinal disease that induces severe intestinal dysfunction. Intestinal myenteric neurons participate in maintaining the intestinal function, which will be severely injured by IR. Macrophages are widely reported to be involved in the pathogenesis of organ IR injury, including intestine, which is activated by NLRP3 signaling. Lonicerin (LCR) is a natural extracted monomer with inhibitory efficacy against the NLRP3 pathway in macrophages. The present study aims to explore the potential protective function of LCR in intestinal IR injury. Myenteric neurons were extracted from mice. RAW 264.7 cells were stimulated by H/R with or without 10 µM and 30 µM LCR. Remarkable increased release of IL-6, MCP-1, and TNF-α were observed in H/R treated RAW 264.7 cells, along with an upregulation of NLRP3, cleaved-caspase-1, IL-1ß, and EZH2, which were sharply repressed by LCR. Myenteric neurons were cultured with the supernatant collected from each group. Markedly decreased neuron number and shortened length of neuron axon were observed in the H/R group, which were signally reversed by LCR. RAW 264.7 cells were stimulated by H/R, followed by incubated with 30 µM LCR with or without pcDNA3.1-EZH2. The inhibition of LCR on NLRP3 signaling in H/R treated RAW 264.7 cells was abolished by EZH2 overexpression. Furthermore, the impact of LCR on neuron number and neuron axon length in myenteric neurons in the H/R group was abated by EZH2 overexpression. Collectively, LCR alleviated intestinal myenteric neuron injury induced by H/R treated macrophages via downregulating EZH2.
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Regulación hacia Abajo , Proteína Potenciadora del Homólogo Zeste 2 , Macrófagos , Neuronas , Daño por Reperfusión , Animales , Ratones , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Células RAW 264.7 , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/patología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Regulación hacia Abajo/efectos de los fármacos , Intestinos/patología , Intestinos/efectos de los fármacos , Plexo Mientérico/metabolismo , Plexo Mientérico/patología , Masculino , Ratones Endogámicos C57BLRESUMEN
Cardiac myxoma is a commonly encountered tumor within the heart that has the potential to be life-threatening. However, the cellular composition of this condition is still not well understood. To fill this gap, we analyzed 75,641 cells from cardiac myxoma tissues based on single-cell sequencing. We defined a population of myxoma cells, which exhibited a resemblance to fibroblasts, yet they were distinguished by an increased expression of phosphodiesterases and genes associated with cell proliferation, differentiation, and adhesion. The clinical relevance of the cell populations indicated a higher proportion of myxoma cells and M2-like macrophage infiltration, along with their enhanced spatial interaction, were found to significantly contribute to the occurrence of embolism. The immune cells surrounding the myxoma exhibit inhibitory characteristics, with impaired function of T cells characterized by the expression of GZMK and TOX, along with a substantial infiltration of tumor-promoting macrophages expressed growth factors such as PDGFC. Furthermore, in vitro co-culture experiments showed that macrophages promoted the growth of myxoma cells significantly. In summary, this study presents a comprehensive single-cell atlas of cardiac myxoma, highlighting the heterogeneity of myxoma cells and their collaborative impact on immune cells. These findings shed light on the complex pathobiology of cardiac myxoma and present potential targets for intervention.
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Neoplasias Cardíacas , Mixoma , Microambiente Tumoral , Humanos , Mixoma/patología , Mixoma/genética , Mixoma/inmunología , Microambiente Tumoral/inmunología , Microambiente Tumoral/genética , Neoplasias Cardíacas/genética , Neoplasias Cardíacas/patología , Neoplasias Cardíacas/inmunología , Macrófagos/inmunología , Macrófagos/patología , Proliferación Celular/genética , Masculino , FemeninoRESUMEN
Hypoxia is a critical factor contributing to a poor prognosis and challenging glioma therapy. Previous studies have indicated that hypoxia drives M2 polarization of macrophages and promotes cancer progression in various solid tumors. However, the more complex and diverse mechanisms underlying this process remain to be elucidated. Here, we aimed to examine the functions of hypoxia in gliomas and preliminarily investigate the underlying mechanisms of M2 macrophage polarization caused by hypoxia. We found that hypoxia significantly enhances the malignant phenotypes of U87 and U251 cells by regulating glycolysis. In addition, hypoxia mediated accumulation of the glycolysis product [lactic acid (LA)], which is subsequently absorbed by macrophages to induce its M2 polarization, and this process is reverted by both the glycolysis inhibitor and silenced monocarboxylate transporter (MCT-1) in macrophages, indicating that M2 macrophage polarization is associated with the promotion of glycolysis by hypoxia. Interestingly, we also found that hypoxia mediated LA accumulation in glioma cells upon uptake by macrophages upregulates H3K18La expression and promotes tumor necrosis factor superfamily member 9 (TNFSF9) expression in a histone-lactylation-dependent manner based on the results of chromatin immunoprecipitation sequencing (ChIP seq) enrichment analysis. Subsequent in vitro and in vivo experiments further indicated that TNFSF9 facilitated glioma progression. Mechanistically, hypoxia-mediated LA accumulation in glioma cells is taken up by macrophages and then induces its M2 macrophage polarization by regulating TNFSF9 expression via MCT-1/H3K18La signaling, thus facilitating the malignant progression of gliomas.NEW & NOTEWORTHY Our study revealed that hypoxia induces the production of LA accumulation through glycolysis in glioma cells, which is subsequently absorbed by macrophages and leads to its M2 polarization via the MCT-1/H3K18La/TNFSF9 axis, ultimately significantly promoting the malignant progression of glioma cells. These findings are novel and noteworthy as they provide insights into the connection between energy metabolism and epigenetics in gliomas.