RESUMEN

We previously reported that leukotriene B4 (LTB4) contained in Trichomonas vaginalis-derived secretory products (TvSP) play an essential role in interleukin-8 (IL-8) production in human mast cell line (HMC-1 cells) via LTB4 receptor (BLT)-mediated Nuclear Factor-kappa B (NF-кB) activation. Dynamin, a GTPase, has been known to be involved in endocytosis of receptors for signaling of production of cytokine or chemokines. In the present study, we investigated the role of dynamin-mediated BLT1 endocytosis in TvSP-induced IL-8 production. When HMC-1 cells were transfected with BLT1 or BLT2 siRNA, TvSP-induced IL-8 production was significantly inhibited compared with that in cells transfected with control siRNA. In addition, pretreatment of HMC-1 cells with a dynamin inhibitor (Dynasore) reduced IL-8 production induced by TvSP or LTB4. TvSP- or LTB4- induced phosphorylation of NF-кB was also attenuated by pretreatment with Dynasore. After exposing HMC-1 cells to TvSP or LTB4, BLT1 was translocated from the intracellular compartments to the plasma membrane within 30 min. At 60 min after stimulation with TvSP or LTB4, BLT1 remigrated from the cell surface to intracellular areas. Pretreatment of HMC-1 cells with dynamin-2 siRNA blocked internalization of BLT1 induced by TvSP or LTB4. Co-immunoprecipitation experiments revealed that dynamin-2 strongly interacted with BLT1 60 min after stimulation with TvSP or LTB4. These results suggest that T. vaginalis-secreted LTB4 induces IL-8 production in HMC-1 cells via dynamin 2-mediated endocytosis of BLT1 and phosphorylation of NF-кB.

Asunto(s)

Dinamina II , Endocitosis , Interleucina-8 , Receptores de Leucotrieno B4 , Trichomonas vaginalis , Humanos , Interleucina-8/metabolismo , Interleucina-8/genética , Receptores de Leucotrieno B4/metabolismo , Receptores de Leucotrieno B4/genética , Endocitosis/efectos de los fármacos , Dinamina II/metabolismo , Dinamina II/genética , Línea Celular , Trichomonas vaginalis/metabolismo , Leucotrieno B4/metabolismo , Mastocitos/metabolismo , Mastocitos/inmunología , FN-kappa B/metabolismo , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/genética

RESUMEN

Trichomoniasis is caused by a sexually transmitted flagellate protozoan parasite Trichomonas vaginalis. T. vaginalis-derived secretory products (TvSP) contain lipid mediators such as leukotriene B4 (LTB4) and various cysteinyl leukotrienes (CysLTs) which included LTC4, LTD4, and LTE4. However, the signaling mechanisms by which T. vaginalis-induced CysLTs stimulate interleukin (IL)-8 production in human mast cells remain unclear. In this study, we investigated these mechanisms in human mast cells (HMC-1). Stimulation with TvSP resulted in increased intracellular reactive oxygen species (ROS) generation and NADPH oxidase 2 (NOX2) activation compared to unstimulated cells. Pre-treatment with NOX2 inhibitors such as diphenyleneiodonium chloride (DPI) or apocynin significantly reduced ROS production in TvSP-stimulated HMC-1 cells. Additionally, TvSP stimulation increased NOX2 protein expression and the translocation of p47phox from the cytosol to the membrane. Pretreatment of HMC-1 cells with PI3K or PKC inhibitors reduced TvSP-induced p47phox translocation and ROS generation. Furthermore, NOX2 inhibitors or NOX2 siRNA prevented CREB phosphorylation and IL-8 gene expression or protein secretion induced by TvSP. Pretreatment with a CysLTR antagonist significantly inhibited TvSP-induced ROS production, CREB phosphorylation, and IL-8 production. These results indicate that CysLT-mediated activation of NOX2 plays a crucial role in ROS-dependent IL-8 production in human mast cells stimulated by T. vaginalis-secreted CysLTs. These findings enhance our understanding of the inflammatory response in trichomoniasis and may inform the development of targeted therapies to mitigate this response.

Asunto(s)

Interleucina-8 , Mastocitos , NADPH Oxidasa 2 , Especies Reactivas de Oxígeno , Receptores de Leucotrienos , Trichomonas vaginalis , Humanos , Trichomonas vaginalis/efectos de los fármacos , Trichomonas vaginalis/metabolismo , Interleucina-8/metabolismo , Interleucina-8/genética , Especies Reactivas de Oxígeno/metabolismo , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Mastocitos/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/parasitología , Mastocitos/inmunología , Línea Celular , Receptores de Leucotrienos/metabolismo , Receptores de Leucotrienos/genética , NADPH Oxidasas/metabolismo , Transducción de Señal/efectos de los fármacos , Leucotrienos/metabolismo

RESUMEN

Background: Cervical cancer (CC) is the fourth most common malignancy among women globally and serves as the main cause of cancer-related deaths among women in developing countries. The early symptoms of CC are often not apparent, with diagnoses typically made at advanced stages, which lead to poor clinical prognoses. In recent years, numerous studies have shown that there is a close relationship between mast cells (MCs) and tumor development. However, research on the role MCs played in CC is still very limited at that time. Thus, the study conducted a single-cell multi-omics analysis on human CC cells, aiming to explore the mechanisms by which MCs interact with the tumor microenvironment in CC. The goal was to provide a scientific basis for the prevention, diagnosis, and treatment of CC, with the hope of improving patients' prognoses and quality of life. Method: The present study acquired single-cell RNA sequencing data from ten CC tumor samples in the ArrayExpress database. Slingshot and AUCcell were utilized to infer and assess the differentiation trajectory and cell plasticity of MCs subpopulations. Differential expression analysis of MCs subpopulations in CC was performed, employing Gene Ontology, gene set enrichment analysis, and gene set variation analysis. CellChat software package was applied to predict cell communication between MCs subpopulations and CC cells. Cellular functional experiments validated the functionality of TNFRSF12A in HeLa and Caski cell lines. Additionally, a risk scoring model was constructed to evaluate the differences in clinical features, prognosis, immune infiltration, immune checkpoint, and functional enrichment across various risk scores. Copy number variation levels were computed using inference of copy number variations. Result: The obtained 93,524 high-quality cells were classified into ten cell types, including T_NK cells, endothelial cells, fibroblasts, smooth muscle cells, epithelial cells, B cells, plasma cells, MCs, neutrophils, and myeloid cells. Furthermore, a total of 1,392 MCs were subdivided into seven subpopulations: C0 CTSG+ MCs, C1 CALR+ MCs, C2 ALOX5+ MCs, C3 ANXA2+ MCs, C4 MGP+ MCs, C5 IL32+ MCs, and C6 ADGRL4+ MCs. Notably, the C2 subpopulation showed close associations with tumor-related MCs, with Slingshot results indicating that C2 subpopulation resided at the intermediate-to-late stage of differentiation, potentially representing a crucial transition point in the benign-to-malignant transformation of CC. CNVscore and bulk analysis results further confirmed the transforming state of the C2 subpopulation. CellChat analysis revealed TNFRSF12A as a key receptor involved in the actions of C2 ALOX5+ MCs. Moreover, in vitro experiments indicated that downregulating the TNFRSF12A gene may partially inhibit the development of CC. Additionally, a prognosis model and immune infiltration analysis based on the marker genes of the C2 subpopulation provided valuable guidance for patient prognosis and clinical intervention strategies. Conclusions: We first identified the transformative tumor-associated MCs subpopulation C2 ALOX5+ MCs within CC, which was at a critical stage of tumor differentiation and impacted the progression of CC. In vitro experiments confirmed the inhibitory effect of knocking down the TNFRSF12A gene on the development of CC. The prognostic model constructed based on the C2 ALOX5+MCs subset demonstrated excellent predictive value. These findings offer a fresh perspective for clinical decision-making in CC.

Asunto(s)

Araquidonato 5-Lipooxigenasa , Progresión de la Enfermedad , Mastocitos , Análisis de la Célula Individual , Microambiente Tumoral , Neoplasias del Cuello Uterino , Humanos , Mastocitos/inmunología , Mastocitos/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/patología , Femenino , Análisis de la Célula Individual/métodos , Microambiente Tumoral/inmunología , Microambiente Tumoral/genética , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Análisis de Secuencia de ARN , Biomarcadores de Tumor/genética

RESUMEN

Chronic wasting disease (CWD) is a transmissible and fatal prion disease that affects cervids. While both oral and nasal routes of exposure to prions cause disease, the spatial and temporal details of how prions enter the central nervous system (CNS) are unknown. Carotid bodies (CBs) are structures that are exposed to blood-borne prions and are densely innervated by nerves that are directly connected to brainstem nuclei, known to be early sites of prion neuroinvasion. All CBs examined contained mast cells expressing the prion protein which is consistent with these cells playing a role in neuroinvasion following prionemia.

Asunto(s)

Cuerpo Carotídeo , Ciervos , Ganglios Linfáticos , Mastocitos , Priones , Animales , Mastocitos/metabolismo , Mastocitos/patología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Priones/metabolismo , Cuerpo Carotídeo/metabolismo , Cuerpo Carotídeo/patología , Ganglios/metabolismo , Ganglios/patología , Enfermedad Debilitante Crónica/metabolismo , Enfermedad Debilitante Crónica/patología

RESUMEN

Rationale: Food allergy is a prevalent disease in the U.S., affecting nearly 30 million people. The primary management strategy for this condition is food avoidance, as limited treatment options are available. The elevation of pathologic IgE and over-reactive mast cells/basophils is a central factor in food allergy anaphylaxis. This study aims to comprehensively evaluate the potential therapeutic mechanisms of a small molecule compound called formononetin in regulating IgE and mast cell activation. Methods: In this study, we determined the inhibitory effect of formononetin on the production of human IgE from peripheral blood mononuclear cells of food-allergic patients using ELISA. We also measured formononetin's effect on preventing mast cell degranulation in RBL-2H3 and KU812 cells using beta-hexosaminidase assay. To identify potential targets of formononetin in IgE-mediated diseases, mast cell disorders, and food allergies, we utilized computational modeling to analyze mechanistic targets of formononetin from various databases, including SEA, Swiss Target Prediction, PubChem, Gene Cards, and Mala Cards. We generated a KEGG pathway, Gene Ontology, and Compound Target Pathway Disease Network using these targets. Finally, we used qRT-PCR to measure the gene expression of selected targets in KU812 and U266 cell lines. Results: Formononetin significantly decreased IgE production in IgE-producing human myeloma cells and PBMCs from food-allergic patients in a dose-dependent manner without cytotoxicity. Formononetin decreased beta-hexosaminidase release in RBL-2H3 cells and KU812 cells. Formononetin regulates 25 targets in food allergy, 51 in IgE diseases, and 19 in mast cell diseases. KEGG pathway and gene ontology analysis of targets showed that formononetin regulated disease pathways, primary immunodeficiency, Epstein-Barr Virus, and pathways in cancer. The biological processes regulated by formononetin include B cell proliferation, differentiation, immune response, and activation processes. Compound target pathway disease network identified NFKB1, NFKBIA, STAT1, STAT3, CCND1, TP53, TYK2, and CASP8 as the top targets regulated at a high degree by formononetin. TP53, STAT3, PTPRC, IL2, and CD19 were identified as the proteins mostly targeted by formononetin. qPCR validated genes of Formononetin molecular targets of IgE regulation in U266 cells and KU812 cells. In U266 cells, formononetin was found to significantly increase the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. In basophils KU812 cells, formononetin significantly increased the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK, TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. Conclusion: These findings comprehensively present formononetin's mechanisms in regulating IgE production in plasma cells and degranulation in mast cells.

Asunto(s)

Hipersensibilidad a los Alimentos , Inmunoglobulina E , Isoflavonas , Quinasas Janus , Leucocitos Mononucleares , Mastocitos , Factores de Transcripción STAT , Transducción de Señal , Isoflavonas/farmacología , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Mastocitos/inmunología , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción STAT/metabolismo , Quinasas Janus/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/inmunología , Hipersensibilidad a los Alimentos/inmunología , Hipersensibilidad a los Alimentos/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Femenino , Adulto , Degranulación de la Célula/efectos de los fármacos , Animales , Persona de Mediana Edad

RESUMEN

Arteriogenesis is an inflammatory driven mechanism, describing the growth of a natural bypass from pre-existing collateral arteries to compensate for an occluded artery. The complement system component C3 is a potent natural inflammatory activator. Here, we investigated its impact on the process of collateral artery growth using C3-deficient (C3 -/-) and wildtype control mice in a murine hindlimb model of arteriogenesis. Induction of arteriogenesis by unilateral femoral artery ligation resulted in decreased perfusion recovery in C3 -/- mice on day 7 as shown by Laser Doppler imaging. Immunofluorescence staining revealed a reduced vascular cell proliferation in C3 -/- mice. Gene expression analysis displayed a significant reduction in monocyte chemoattractant protein-1 (MCP-1) expression in C3 -/- mice. Interestingly, 3 days after induction of arteriogenesis, the number of macrophages (CD68+) recruited to growing collaterals was not affected by C3 deficiency. However, a significant reduction in inflammatory M1-like polarized macrophages (CD68+/MRC1-) was noted. Forced mast cell activation by Compound 48/80 as well as exogenous MCP-1 application rescued the number of M1-like polarized macrophages along with perfusion recovery in C3 -/- mice. In summary, this study demonstrates that complement C3 influences arteriogenesis by mediating MCP-1 expression, which is essential for the induction and enhancement of sterile inflammation.

Asunto(s)

Circulación Colateral , Complemento C3 , Inflamación , Animales , Inflamación/patología , Ratones , Complemento C3/metabolismo , Complemento C3/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL2/genética , Macrófagos/metabolismo , Neovascularización Fisiológica/genética , Ratones Endogámicos C57BL , Miembro Posterior/irrigación sanguínea , Ratones Noqueados , Arteria Femoral/patología , Arterias/crecimiento & desarrollo , Arterias/metabolismo , Masculino , Proliferación Celular , Mastocitos/metabolismo

RESUMEN

Chronic spontaneous urticaria (CSU) is associated with skin mast cell activation, and its triggering mechanisms are not completely elucidated. Evidence suggests an autoimmune component of CSU. Our aim was to assess the usefulness of an autoimmune mast cell activation test (aiMAT) for diagnosing and differentiating CSU into different subtypes. We enrolled 43 patients with active, uncontrolled CSU before starting treatment with omalizumab and 15 controls. Patients were evaluated based on omalizumab response. aiMATs were performed using non-IgE-sensitized (NS) or myeloma IgE-sensitized (S) LAD2 cells, which were then stimulated with CSU/control sera (25 µL and 10 µL). The expression of CD63 was assessed with flow cytometry. CD63 response on NS-LAD2 was significantly increased in CSU patients compared to controls after the stimulation with 25 µL CSU/control sera (p = 0.0007) and with 10 µL CSU/control sera (p = 0.0001). The ROC curve analysis demonstrated an area under the curve (AUC) of 0.82. The cutoff for autoimmune-non-IgE-sensitized-MAT was 40.3% CD63+ LAD2, which resulted in 73.3% sensitivity and 81.4% specificity. CD63 response on S-LAD2 was significantly increased in CSU patients compared to controls after the stimulation with 25 µL CSU/control sera (p = 0.03). The ROC curve analysis demonstrated an AUC of 0.66. The cutoff for the autoimmune-myeloma IgE-sensitized-MAT was 58.4% CD63+ cells, which resulted in 62.8% sensitivity and 66.7% specificity. Overall, 36 out of 43 (84%) patients responded to omalizumab, and 7 (16%) were nonresponders. We found no differences between LAD2 CD63 response and response to omalizumab. In conclusion, aiMAT could represent a new diagnostic tool in CSU. Additional studies are needed to evaluate the potential benefits during omalizumab therapy.

Asunto(s)

Urticaria Crónica , Mastocitos , Tetraspanina 30 , Humanos , Urticaria Crónica/diagnóstico , Urticaria Crónica/tratamiento farmacológico , Urticaria Crónica/inmunología , Urticaria Crónica/sangre , Femenino , Mastocitos/inmunología , Mastocitos/metabolismo , Masculino , Persona de Mediana Edad , Adulto , Tetraspanina 30/metabolismo , Omalizumab/uso terapéutico , Anciano , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Curva ROC , Estudios de Casos y Controles

RESUMEN

Inflammatory Bowel Diseases (IBD), which encompass ulcerative colitis (UC) and Crohn's disease (CD), are characterized by chronic inflammation and tissue damage of the gastrointestinal tract. This study aimed to uncover novel disease-gene signatures, dysregulated pathways, and the immune cell infiltration landscape of inflamed tissues. Eight publicly available transcriptomic datasets, including inflamed and non-inflamed tissues from CD and UC patients were analyzed. Common differentially expressed genes (DEGs) were identified through meta-analysis, revealing 180 DEGs. DEGs were implicated in leukocyte transendothelial migration, PI3K-Akt, chemokine, NOD-like receptors, TNF signaling pathways, and pathways in cancer. Protein-protein interaction network and cluster analysis identified 14 central IBD players, which were validated using eight external datasets. Disease module construction using the NeDRex platform identified nine out of 14 disease-associated genes (CYBB, RAC2, GNAI2, ITGA4, CYBA, NCF4, CPT1A, NCF2, and PCK1). Immune infiltration profile assessment revealed a significantly higher degree of infiltration of neutrophils, activated dendritic cells, plasma cells, mast cells (resting/activated), B cells (memory/naïve), regulatory T cells, and M0 and M1 macrophages in inflamed IBD tissue. Collectively, this study identified the immune infiltration profile and nine disease-associated genes as potential modulators of IBD pathogenesis, offering insights into disease molecular mechanisms, and highlighting potential disease modulators and immune cell dynamics.

Asunto(s)

Biología Computacional , Mapas de Interacción de Proteínas , Humanos , Biología Computacional/métodos , Mapas de Interacción de Proteínas/genética , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Transcriptoma , Colitis Ulcerosa/genética , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Perfilación de la Expresión Génica , Enfermedad de Crohn/genética , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Redes Reguladoras de Genes , Neutrófilos/inmunología , Neutrófilos/metabolismo , Transducción de Señal/genética , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , NADPH Oxidasas

RESUMEN

Sickle cell disease (SCD) is the most common inherited monogenetic disorder. Chronic and acute pain are hallmark features of SCD involving neural and vascular injury and inflammation. Mast cells reside in the vicinity of nerve fibers and vasculature, but how they influence these structures remains unknown. We therefore examined the mechanism of mast cell activation in a sickle microenvironment replete with cell-free heme and inflammation. Mast cells exposed to this environment showed an explosion of nuclear contents with the release of citrullinated histones, suggestive of mast cell extracellular trap (MCET) release. MCETs interacted directly with the vasculature and nerve fibers, a cause of vascular and neural injury in sickle cell mice. MCET formation was dependent upon peptidylarginine deiminase 4 (PAD4). Inhibition of PAD4 ameliorated vasoocclusion, chronic and acute hyperalgesia, and inflammation in sickle mice. PAD4 activation may also underlie neutrophil trap formation in SCD, thus providing a novel target to treat the sequelae of vascular and neural injury in SCD.

Asunto(s)

Anemia de Células Falciformes , Trampas Extracelulares , Hiperalgesia , Mastocitos , Arginina Deiminasa Proteína-Tipo 4 , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/metabolismo , Animales , Hiperalgesia/metabolismo , Hiperalgesia/etiología , Trampas Extracelulares/metabolismo , Ratones , Mastocitos/metabolismo , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Humanos , Masculino , Modelos Animales de Enfermedad , Inflamación/metabolismo , Ratones Endogámicos C57BL , Neutrófilos/metabolismo

RESUMEN

Marfan syndrome (MFS) is a hereditary condition accompanied by disorders in the structural and regulatory properties of connective tissue, including elastic fibers, due to a mutation in the gene encodes for fibrillin-1 protein (FBN1 gene) and the synthesis of abnormal fibrillin-1 glycoprotein. Despite the high potential of mast cells (MCs) to remodel the extracellular matrix (ECM), their pathogenetic significance in MFS has not been considered yet. The group of patients with Marfan syndrome included two mothers and five children (three girls aged 4, 11, and 11 and two boys aged 12 and 13). Normal skin was examined in two children aged 11 and 12. Histochemical, monoplex, and multiplex immunohistochemical techniques; combined protocols of simultaneous histochemical and immunohistochemical staining (the results of staining were assessed using light, epifluorescence, and confocal microscopy); and bioinformatics algorithms for the quantitative analysis of detected targets were used to evaluate mast cells and their relationship with other cells from extracellular structures in the skin dermis. Analysis of the skin MC population in children with Marfan syndrome revealed a considerably increased number of intra-organic populations with the preservation of the specific Tryptase+Chymase+CPA3+ protease profile typical of the skin. The features of the MC histotopography phenotype in MFS consisted of closer colocalization with elastic fibers, smooth muscle cells, and fibroblasts. MCs formed many intradermal clusters that synchronized the activity of cell functions in the stromal landscape of the tissue microenvironment with the help of spatial architectonics, including the formation of cell chains and the creation of fibrous niches. In MCs, the expression of specific proteases, TGF-ß, and heparin increased, with targeted secretion of biologically active substances relative to the dermal elastic fibers, which had specific structural features in MFS, including abnormal variability in thickness along their entire length, alternating thickened and thinned areas, and uneven surface topography. This paper discusses the potential role of MCs in strain analysis (tensometry) of the tissue microenvironment in MFS. Thus, the quantitative and qualitative rearrangements of the skin MC population in MFS are aimed at altering the stromal landscape of the connective tissue. The results obtained should be taken into account when managing clinical signs of MFS manifested in other pathogenetically critical structures of internal organs, including the aorta, tendons, cartilage, and parenchymal organs.

Asunto(s)

Dermis , Tejido Elástico , Síndrome de Marfan , Mastocitos , Humanos , Síndrome de Marfan/metabolismo , Síndrome de Marfan/patología , Síndrome de Marfan/genética , Mastocitos/metabolismo , Mastocitos/patología , Niño , Masculino , Femenino , Tejido Elástico/metabolismo , Tejido Elástico/patología , Preescolar , Dermis/patología , Dermis/metabolismo , Adolescente , Fibrilina-1/metabolismo , Fibrilina-1/genética , Piel/metabolismo , Piel/patología , Matriz Extracelular/metabolismo , Adipoquinas

RESUMEN

BACKGROUND/AIMS: Lemons (Citrus limon ) contain various nutrients and are among the most popular citrus fruit. Besides their antioxidant, anticancer, antibacterial, and anti-inflammatory properties, clinical studies have indicated their anti-allergic properties. METHODS: Using the differential-interference contrast (DIC) microscopy, we examined the effects of lemon juice and peel constituents, such as citric acid, ascorbic acid, hesperetin and eriodictyol, on the degranulation from rat peritoneal mast cells. Using fluorescence imaging with a water-soluble dye, Lucifer Yellow, we also examined their effects on the deformation of the plasma membrane. RESULTS: Lemon juice dose-dependently decreased the number of degranulated mast cells. At concentrations equal to or higher than 0.25 mM, citric acid, hesperetin, and eriodictyol significantly reduced the number of degranulating mast cells in a dose-dependent manner, while ascorbic acid required much higher doses to exert significant effects. At 1 mM, citric acid, hesperetin, and eriodictyol almost completely inhibited exocytosis and washed out the Lucifer Yellow trapped on the mast cell surface, while ascorbic acid did not. CONCLUSION: This study provides in vitro evidence for the first time that lemon constituents, such as citric acid, hesperetin, and eriodictyol, potently exert mast cell-stabilizing properties. These properties are attributable to their inhibitory effects on plasma membrane deformation in degranulating mast cells.

Asunto(s)

Ácido Ascórbico , Citrus , Flavanonas , Hesperidina , Mastocitos , Animales , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Citrus/química , Ratas , Ácido Ascórbico/farmacología , Masculino , Hesperidina/farmacología , Hesperidina/química , Flavanonas/farmacología , Flavanonas/química , Ácido Cítrico/farmacología , Ácido Cítrico/química , Degranulación de la Célula/efectos de los fármacos , Jugos de Frutas y Vegetales/análisis , Peritoneo/citología , Ratas Sprague-Dawley , Exocitosis/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Frutas/química , Isoquinolinas

RESUMEN

Mast cells serve as crucial effector cells within the innate immune system and are predominantly localized in the skin, airways, gastrointestinal tract, urinary and reproductive tracts, as well as in the brain. Under physiological conditions, brain-resident mast cells secrete a diverse array of neuro-regulatory mediators to actively participate in neuroprotection. Meanwhile, as the primary source of molecules causing brain inflammation, mast cells also function as the "first responders" in brain injury. They interact with neuroglial cells and neurons to facilitate the release of numerous inflammatory mediators, proteases, and reactive oxygen species. This process initiates and amplifies immune-inflammatory responses in the brain, thereby contributing to the regulation of neuroinflammation and blood-brain barrier permeability. This article provides a comprehensive overview of the potential mechanisms through which mast cells in the brain may modulate neuroprotection and their pathological implications in various neurological disorders. It is our contention that the inhibition of mast cell activation in brain disorders could represent a novel avenue for therapeutic breakthroughs.

Asunto(s)

Mastocitos , Humanos , Mastocitos/inmunología , Mastocitos/metabolismo , Animales , Encéfalo/inmunología , Encéfalo/patología , Encéfalo/metabolismo , Encefalopatías/inmunología , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/metabolismo , Neuronas/inmunología , Neuronas/metabolismo , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/patología

RESUMEN

Mast cells are multifaceted cells localized in tissues and possess various surface receptors that allow them to respond to inner and external threat signals. Interleukin-33 (IL-33) is a cytokine released by structural cells in response to parasitic infections, mechanical damage, and cell death. IL-33 can activate mast cells, causing them to release an array of mediators. This study aimed to identify the different cytokines released by human cord blood-derived mast cells (hCBMCs) in response to acute and prolonged stimulation with IL-33. For this purpose, a hCBMC model was established and stimulated with 10 ng and 20 ng of recombinant human IL-33 (rhIL-33) for 6 h and 24 h. Total RNA was hybridized using a high-density oligonucleotide microarray. A multiplex assay was performed to assess the released cytokines. Acute exposure to rhIL-33 increased the expression of IL-1α, IL-1ß, IL-6, and IL-13, whereas prolonged exposure increased the expression of IL-5 and IL-10, and cytokines were detected in the culture supernatant. WebGestalt analysis revealed that rhIL-33 induces pathways and biological processes related to the immune system and the acute inflammatory response. This study demonstrates that rhIL-33 can activate hCBMCs to release pro- and anti-inflammatory cytokines, eliciting distinct acute and prolonged responses unique to hCBMCs.

Asunto(s)

Citocinas , Sangre Fetal , Interleucina-33 , Mastocitos , Humanos , Interleucina-33/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Sangre Fetal/citología , Citocinas/metabolismo , Células Cultivadas , Proteínas Recombinantes/farmacología , Perfilación de la Expresión Génica

RESUMEN

Ursolic acid (UA), a pentacyclic triterpene, exhibits diverse pharmacological effects, including potential treatment for allergic diseases. It downregulates thymic stromal lymphopoietin (TSLP) and disrupts mast cell signaling pathways. However, the exact molecular mechanism by which UA interferes with mast cell action remains unclear. Therefore, the current study aimed to uncover molecular entities underlying the effect of UA on mast cells and its potential antipruritic effect, specifically investigating its modulation of key molecules such as TRPV4, PAR2, and MRGPRX2, which are involved in TSLP regulation and sensation. Calcium imaging experiments revealed that UA pretreatment significantly suppressed MRGPRX2 activation (and its mouse orthologue MrgprB2), a G protein-coupled receptor predominantly expressed in mast cells. Molecular docking predictions suggested potential interactions between UA and MRGPRX2/MrgprB2. UA pretreatment also reduced mast cell degranulation through MRGPRX2 and MrgprB2-dependent mechanisms. In a dry skin mouse model, UA administration decreased tryptase and TSLP production in the skin, and diminished TSLP response in the sensory neurons. While PAR2 and TRPV4 activation enhances TSLP production, UA did not inhibit their activity. Notably, UA attenuated compound 48/80-induced scratching behaviors in mice and suppressed spontaneous scratching in a dry skin model. The present study confirms the effective inhibition of UA on MRGPRX2/MrgprB2, leading to reduced mast cell degranulation and suppressed scratching behaviors. These findings highlight the potential of UA as an antipruritic agent for managing various allergy- or itch-related conditions.

Asunto(s)

Degranulación de la Célula , Citocinas , Mastocitos , Receptores Acoplados a Proteínas G , Linfopoyetina del Estroma Tímico , Triterpenos , Ácido Ursólico , Animales , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Degranulación de la Célula/efectos de los fármacos , Citocinas/metabolismo , Ratones , Triterpenos/farmacología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Canales Catiónicos TRPV/metabolismo , Prurito/tratamiento farmacológico , Prurito/metabolismo , Simulación del Acoplamiento Molecular , Receptores de Neuropéptido/metabolismo , Masculino , Piel/efectos de los fármacos , Piel/metabolismo

RESUMEN

Pterygium is often associated with chronic ultraviolet (UV) radiation exposure and characterized by the overgrowth of conjunctiva and extracellular matrix (ECM) remodeling. Notably, several studies in the skin have demonstrated that chronic UV radiation can upregulate Granzyme B (GrB) expression and increase ECM degradation. The aim of this study was to compare GrB expression between pterygium and healthy controls and to further link this GrB expression to mast cells. Post-mortem pterygium tissues and conjunctival tissues from age-matched controls were used to assess GrB expression via immunofluorescence and microscopy. We found a significantly higher density of GrB+ cells from pterygium specimens compared to healthy controls. Furthermore, many of the GrB+ cells in pterygium specimens co-expressed tryptase, a mast cell marker. These findings suggest a role for conjunctival mast cell-secreted GrB in the pathogenesis of pterygium and highlight GrB as a possible therapeutic target in delaying or halting pterygium progression.

Asunto(s)

Conjuntiva , Granzimas , Pterigion , Humanos , Pterigion/metabolismo , Pterigion/patología , Granzimas/metabolismo , Conjuntiva/metabolismo , Conjuntiva/patología , Masculino , Femenino , Persona de Mediana Edad , Anciano , Mastocitos/metabolismo , Adulto , Estudios de Casos y Controles , Anciano de 80 o más Años , Triptasas/metabolismo

RESUMEN

Introduction: The Janus kinase (JAK) family includes four cytoplasmic tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) constitutively bound to several cytokine receptors. JAKs phosphorylate downstream signal transducers and activators of transcription (STAT). JAK-STAT5 pathways play a critical role in basophil and mast cell activation. Previous studies have demonstrated that inhibitors of JAK-STAT pathway blocked the activation of mast cells and basophils. Methods: In this study, we investigated the in vitro effects of ruxolitinib, a JAK1/2 inhibitor, on IgE- and IL-3-mediated release of mediators from human basophils, as well as substance P-induced mediator release from skin mast cells (HSMCs). Results: Ruxolitinib concentration-dependently inhibited IgE-mediated release of preformed (histamine) and de novo synthesized mediators (leukotriene C4) from human basophils. Ruxolitinib also inhibited anti-IgE- and IL-3-mediated cytokine (IL-4 and IL-13) release from basophils, as well as the secretion of preformed mediators (histamine, tryptase, and chymase) from substance P-activated HSMCs. Discussion: These results indicate that ruxolitinib, inhibiting the release of several mediators from human basophils and mast cells, is a potential candidate for the treatment of inflammatory disorders.

Asunto(s)

Basófilos , Janus Quinasa 1 , Janus Quinasa 2 , Mastocitos , Nitrilos , Pirazoles , Pirimidinas , Humanos , Basófilos/efectos de los fármacos , Basófilos/inmunología , Basófilos/metabolismo , Pirimidinas/farmacología , Nitrilos/farmacología , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/metabolismo , Pirazoles/farmacología , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/metabolismo , Janus Quinasa 2/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Células Cultivadas , Inhibidores de las Cinasas Janus/farmacología , Citocinas/metabolismo , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Inhibidores de Proteínas Quinasas/farmacología

RESUMEN

The high-affinity IgE receptor, FcεRI, is typically associated with type 2 effectors such as mast cells (MC). The relatively unique expression profile of FcεRI and accumulating evidence from pre-clinical and clinical settings, such as MC interactions with tumors, have led us to study MCs as a potential therapeutic target in breast cancer. Our work identified MCs interacting with tumor cells at primary sites using the 4T1 (BALB/c) adenocarcinoma model in vivo. However, this analysis was complicated by a surprising finding that the tumor cells intrinsically and strongly expressed FcεRI. We further studied the expression and function of FcεRI in breast cancer cells in vitro. The 4T1 cells expressed FcεRI to a level similar to mouse bone marrow-derived MC (BMMC). Additionally, two established breast cancer cultures derived from human T-47D cells, one estrogen-dependent (E3) and the other estrogen-withdrawn (EWD8), also expressed FcεRI with EWD8 cells showing the greatest abundance. Functional analyses indicated that IgE-mediated antigen stimulation did not elicit classic Ca2+ flux in breast cancer cells as seen in the respective species' MCs; however, FcεRI crosslinking could stimulate IL-6 production from the T-47D derivatives. Preliminary analysis of primary breast cancer biopsy datasets using R2: Genomics Analysis and Visualization Platform was discordant with our in vivo model and in vitro observations. Indeed, FcεRI mRNA abundance declined in metastatic breast cancers compared to non-cancerous breast tissue. Altogether, we report a previously unidentified and immunologically substantive difference between breast cancer models and human primary tumors. Investigators pursuing FcεRI-relevant therapeutics in this context should be aware of this translational barrier.

Asunto(s)

Neoplasias de la Mama , Receptores de IgE , Receptores de IgE/metabolismo , Receptores de IgE/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Humanos , Femenino , Línea Celular Tumoral , Animales , Ratones , Mastocitos/metabolismo , Ratones Endogámicos BALB C , Regulación Neoplásica de la Expresión Génica , Interleucina-6/metabolismo , Inmunoglobulina E/metabolismo

RESUMEN

Immunotherapy is a promising alternative treatment for canine mast cell tumour (MCT). However, evasion of immune recognition by downregulating major histocompatibility complex (MHC) molecules might decline treatment efficiency. Enhancing MHC expression through interferon-gamma (IFN-γ) is crucial for effective immunotherapy. In-house and reference canine MCT cell lines derived from different tissue origins were used. The impacts of IFN-γ treatment on cell viability, expression levels of MHC molecules, as well as cell apoptosis were evaluated through the MTT assay, RT-qPCR and flow cytometry. The results revealed that IFN-γ treatment significantly influenced the viability of canine MCT cell lines, with varying responses observed among different cell lines. Notably, IFN-γ treatment increased the expression of MHC I and MHC II, potentially enhancing immune recognition and MCT cell clearance. Flow cytometry analysis in PBMCs-mediated cytotoxicity assays showed no significant differences in overall apoptosis between IFN-γ treated and untreated canine MCT cell lines across various target-to-effector ratios. However, a trend towards higher percentages of late and total apoptotic cells was observed in the IFN-γ treated C18 and CMMC cell lines, but not in the VIMC and CoMS cell lines. These results indicate a variable response to IFN-γ treatment among different canine MCT cell lines. In summary, our study suggests IFN-γ's potential therapeutic role in enhancing immune recognition and clearance of MCT cells by upregulating MHC expression and possibly promoting apoptosis, despite variable responses across different cell lines. Further investigations are necessary to elucidate the underlying mechanisms and evaluate IFN-γ's efficacy in in vivo models.

Asunto(s)

Apoptosis , Interferón gamma , Leucocitos Mononucleares , Animales , Perros , Interferón gamma/metabolismo , Interferón gamma/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/metabolismo , Mastocitos/efectos de los fármacos , Complejo Mayor de Histocompatibilidad , Mastocitoma/veterinaria , Mastocitoma/inmunología , Enfermedades de los Perros/inmunología , Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/genética

RESUMEN

Canine atopic dermatitis (AD) arises from hypersensitive immune reactions. AD symptoms entail severe pruritus and skin inflammation, with frequent relapses. Consequently, AD patients require continuous management, imposing financial burdens and mental fatigue on pet owners. In this study, we aimed to investigate the therapeutic relevance of secretome from canine adipose tissue-derived mesenchymal stem cells (MSCs), especially after encapsulation in nano-villi chitosan microspheres (CS-MS) to expect improved efficacy. Conditioned media (CM) from MSCs significantly inhibited the proliferation of splenocytes, induced the generation of regulatory T cells, and decreased mast cell degranulation. We found that beneficial soluble factors known to reduce AD symptoms, including transforming growth factor-beta 1, were detectable after sequential concentration and lyophilization of CM. The CS-MS, developed by a phase inversion regeneration method, showed high loading and sustained release of the secretome. Local injection of secretome-loaded CS-MS (ST/SC-MS) effectively reduced clinical severity compared to groups treated with secretome. Histological analysis revealed that ST/SC-MS potently suppressed epidermal hyperplasia, immunocyte infiltration and mast cell activation in the lesion. Taken together, this study presents a novel therapeutic approach exhibiting more potent and prolonged immunoregulatory efficacy of MSC secretome for canine AD treatment.

Asunto(s)

Quitosano , Dermatitis Atópica , Células Madre Mesenquimatosas , Microesferas , Secretoma , Dermatitis Atópica/terapia , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/patología , Animales , Perros , Quitosano/química , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Proliferación Celular/efectos de los fármacos , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Mastocitos/inmunología , Medios de Cultivo Condicionados/farmacología , Preparaciones de Acción Retardada

RESUMEN

BACKGROUND: Altered biosynthesis of eicosanoids is linked to type 2 inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP), but their role in recalcitrant NPs is unclear. OBJECTIVES: We sought to identify endotypes that are linked to recalcitrant CRSwNP, based on eicosanoids, their biosynthetic enzymes, and receptors as well as cytokines and the presence of eosinophils and mast cells in recurrent NPs. METHODS: Mucosal tissue collected at the time of sinus surgery from 54 patients with CRSwNP and 12 non-CRS controls were analysed for leukotriene (LT) E4, prostaglandin (PG) D2, 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and 17 cytokines with ELISAs and Bio-Plex immunoassays. Patient subgroups were identified by cluster analysis and the probability of NP recurrence were tested with logistic regression analyses. Gene expressions were analysed with qPCR. Tryptase and eosinophil-derived neurotoxin (EDN) were measured with ELISAs as indications of the presence of mast cells and eosinophils, respectively. RESULTS: Clustering of patients showed that an inflammatory signature characterised by elevated LTE4, PGD2, 15(S)-HETE and IL-13 was associated with NP recurrence. Previous NP surgery as well as aspirin-exacerbated respiratory disease were significantly more common among these patients. Expression of cyclooxygenase 1 was the only gene associated with NP recurrence. Levels of EDN, but not tryptase, were significantly higher in patients with recurrent NPs. CONCLUSION: Distinguishing endotypes that include LTE4, PGD2, 15HETE and conventional biomarkers of type 2 inflammation could help predict recurrent nasal polyposis and thus identify cases of recalcitrant CRSwNP.

Asunto(s)

Biomarcadores , Ácidos Hidroxieicosatetraenoicos , Leucotrieno E4 , Pólipos Nasales , Prostaglandina D2 , Recurrencia , Rinitis , Sinusitis , Humanos , Sinusitis/metabolismo , Sinusitis/patología , Sinusitis/cirugía , Sinusitis/diagnóstico , Pólipos Nasales/metabolismo , Pólipos Nasales/patología , Pólipos Nasales/cirugía , Pólipos Nasales/genética , Femenino , Masculino , Leucotrieno E4/metabolismo , Persona de Mediana Edad , Enfermedad Crónica , Ácidos Hidroxieicosatetraenoicos/metabolismo , Adulto , Rinitis/metabolismo , Rinitis/patología , Rinitis/diagnóstico , Rinitis/cirugía , Biomarcadores/metabolismo , Prostaglandina D2/metabolismo , Pronóstico , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Eosinófilos/metabolismo , Eosinófilos/patología , Mastocitos/metabolismo , Mastocitos/patología , Rinosinusitis