RESUMEN

Unraveling the concentration-dependent spatiotemporal organization of receptors in the plasma membrane is crucial to understand cell signal initiation. A paradigm of this process is the oligomerization of CD95 during apoptosis signaling, with different oligomerization models being discussed. Here, we establish the molecular-sensitive approach cell lifetime Förster resonance energy transfer image spectroscopy to determine CD95 configurations in live cells. These data are corroborated by stimulated emission depletion microscopy, confocal photobleaching step analysis, and fluorescence correlation spectroscopy. We probed CD95 interactions for concentrations of ~10 to 1000 molecules per square micrometer, over nanoseconds to hours, and molecular to cellular scales. Quantitative benchmarking was achieved establishing high-fidelity monomer and dimer controls. While CD95 alone is primarily monomeric (~96%) and dimeric (4%), the addition of ligand induces oligomerization to dimers/trimers (~15%) leading to cell death. This study highlights molecular concentration effects and oligomerization dynamics. It reveals a minimal model, where small CD95 oligomers suffice to efficiently initiate signaling.

Asunto(s)

Transducción de Señal , Receptor fas , Receptor fas/metabolismo , Receptor fas/química , Humanos , Transferencia Resonante de Energía de Fluorescencia/métodos , Multimerización de Proteína , Apoptosis , Proteína Ligando Fas/metabolismo , Proteína Ligando Fas/química , Membrana Celular/metabolismo

RESUMEN

INTRODUCTION: The aim of this present work was to investigate the mechanism of the microRNA (miR)-216a-5p/FASL axis in mice with acute kidney injury (AKI). METHODS: Mice kidney ischemia/reperfusion (I/R) injury was used as AKI models in this study. I/R mice were injected with miR-216a-5p- and FASL-related constructs to investigate potential mechanisms of kidney protection. Kidney function, inflammation, oxidative stress, and kidney cell apoptosis were assessed after 24 h of reperfusion. In vitro, the hypoxia-reoxygenation (H/R) model was used with kidney tubular epithelial cells (TECs) to mimic kidney I/R injury. H/R-treated TECs were transfected with miR-216a-5p- and FASL-related constructs to detect cell viability, inflammation, and oxidative stress. MiR-216a-5p and FASL expression levels in mouse kidney tissues and in H/R-treated TECs were detected. RESULTS: MiR-216a-5p was downregulated and FASL was upregulated in kidney tissues of I/R mice and H/R-treated TECs. Upregulating miR-216a-5p attenuated kidney cell apoptosis and the damage of kidney function, and reduced inflammatory factor levels and oxidative stress response in kidney tissues of I/R mice. Upregulating miR-216a-5p advanced cell viability and reduced inflammatory factor levels and oxidative stress response in H/R-treated TECs. Downregulation of FASL effectively reversed the influences of downregulation of miR-216a-5p on kidney injury in mice and kidney TEC survival. CONCLUSION: Our study reveals that miR-216a-5p reduces I/R-induced pathological kidney damage in AKI via suppressing FASL.

Asunto(s)

Lesión Renal Aguda , Proteína Ligando Fas , MicroARNs , Daño por Reperfusión , Animales , Masculino , Ratones , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/genética , Apoptosis , Modelos Animales de Enfermedad , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Estrés Oxidativo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & control

RESUMEN

Endometriosis, affecting 10% of women, is defined as implantation, survival, and growth of endometrium-like/endometriotic tissue outside the uterine cavity, causing inflammation, infertility, pain, and susceptibility to ovarian cancer. Despite extensive studies, its etiology and pathogenesis are poorly understood and largely unknown. The prevailing view is that the immune system of endometriosis patients fails to clear ectopically disseminated endometrium from retrograde menstruation. Exosomes are small extracellular vesicles that exhibit immunomodulatory properties. We studied the role of endometriotic tissue-secreted exosomes in the pathophysiology of endometriosis. Two exosome-mediated mechanisms known to impair the immune response were investigated: 1) downregulation of NKG2D-mediated cytotoxicity and 2) FasL- and TRAIL-induced apoptosis of activated immune cells. We showed that secreted endometriotic exosomes isolated from supernatants of short-term explant cultures carry the NKG2D ligands MICA/B and ULBP1-3 and the proapoptotic molecules FasL and TRAIL on their surface, i.e., signature molecules of exosome-mediated immune suppression. Acting as decoys, these exosomes downregulate the NKG2D receptor, impair NKG2D-mediated cytotoxicity, and induce apoptosis of activated PBMCs and Jurkat cells through the FasL- and TRAIL pathway. The secreted endometriotic exosomes create an immunosuppressive gradient at the ectopic site, forming a "protective shield" around the endometriotic lesions. This gradient guards the endometriotic lesions against clearance by a cytotoxic attack and creates immunologic privilege by induction of apoptosis in activated immune cells. Taken together, our results provide a plausible, exosome-based mechanistic explanation for the immune dysfunction and the compromised immune surveillance in endometriosis and contribute novel insights into the pathogenesis of this enigmatic disease.

Asunto(s)

Apoptosis , Endometriosis , Endometrio , Exosomas , Subfamilia K de Receptores Similares a Lectina de Células NK , Ligando Inductor de Apoptosis Relacionado con TNF , Humanos , Endometriosis/inmunología , Endometriosis/metabolismo , Endometriosis/patología , Femenino , Exosomas/metabolismo , Exosomas/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Apoptosis/inmunología , Endometrio/inmunología , Endometrio/patología , Endometrio/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Regulación hacia Abajo/inmunología , Proteína Ligando Fas/metabolismo , Proteína Ligando Fas/inmunología , Citotoxicidad Inmunológica , Adulto , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología

RESUMEN

The Tianzhu white yak, a globally rare species, holds immense value as a source for yak materials. While the Fas/FasL pathway is pivotal in granulosa cells apoptosis, its precise molecular workings remain enigmatic. This study endeavours to decipher the role of follicle-stimulating hormone (FSH) in suppressing ovarian granulosa cells (GC) apoptosis in the Tianzhu white yak. Utilizing advanced cell culture techniques, we employed the MTT method, flow cytometry, fluorescence labelling and RT-PCR to investigate the apoptotic effects of FSH on yak GCs. Our results reveal that FSH's inhibitory effect on GC apoptosis follows a normal distribution pattern, peaking at an FSH concentration of 100 ng/mL with an apoptosis inhibition rate of 89.31%. When serum was withdrawn, an FSH concentration of 2 × 106 ng/mL reduced apoptosis by 72.84%. Annexin V-FITC staining revealed membrane invaginations, bubble and protrusion formation on the cell surface, and alterations in membrane structure and cell morphology. Flow cytometry analysis further demonstrated that FSH administration prior to early granulosa cell apoptosis had a more profound effect than during gradual apoptosis, both showing a suppressive effect on early follicular granulosa cell apoptosis. A transcription-level analysis conducted 3 h prior to serum withdrawal, with the addition of 100 ng/mL FSH, revealed intricate regulations in the expression of Fas/FasL. Notably, we observed a gradual increase in FasL expression over time, yet the presence of FSH effectively down-regulated FasL expression to baseline levels, without notable changes in Fas expression. Immunocytochemical analysis further confirmed the presence of both Fas and FasL on the cell membrane, nucleus and cytoplasm, with varying intensities depending on the duration of FSH treatment. Our findings suggest that FSH may suppress the apoptotic pathway in follicular primarily by down-regulating FasL expression, indicating that Fas-regulated mitochondrial pathways play a more prominent role compared to death receptor pathways. This study offers a fresh perspective on the mechanism underlying follicular atresia in Tianzhu white yaks and lays a solid theoretical foundation for the expansion of this endangered species' population.

Asunto(s)

Apoptosis , Proteína Ligando Fas , Hormona Folículo Estimulante , Células de la Granulosa , ARN Mensajero , Receptor fas , Animales , Femenino , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Apoptosis/efectos de los fármacos , Proteína Ligando Fas/metabolismo , Proteína Ligando Fas/genética , Hormona Folículo Estimulante/farmacología , Bovinos , Receptor fas/metabolismo , Receptor fas/genética , ARN Mensajero/metabolismo , Citometría de Flujo/veterinaria

RESUMEN

The pathogenesis of ankylosing spondylitis (AS) remains unclear, and while recent studies have implicated necroptosis in various autoimmune diseases, an investigation of its relationship with AS has not been reported. In this study, we utilized the Gene Expression Omnibus database to compare gene expressions between AS patients and healthy controls, identifying 18 differentially expressed necroptosis-related genes (DENRGs), with 8 upregulated and 10 downregulated. Through the application of three machine learning algorithms-least absolute shrinkage and selection operation, support vector machine-recursive feature elimination and random forest-two hub genes, FASLG and TARDBP, were pinpointed. These genes demonstrated high specificity and sensitivity for AS diagnosis, as evidenced by receiver operating characteristic curve analysis. These findings were further supported by external datasets and cellular experiments, which confirmed the downregulation of FASLG and upregulation of TARDBP in AS patients. Immune cell infiltration analysis suggested that CD4+ T cells, CD8+ T cells, NK cells and neutrophils may be associated with the development of AS. Notably, in the group with high FASLG expression, there was a significant infiltration of CD8+ T cells, memory-activated CD4+ T cells and resting NK cells, with relatively less infiltration of memory-resting CD4+ T cells and neutrophils. Conversely, in the group with high TARDBP expression, there was enhanced infiltration of naïve CD4+ T cells and M0 macrophages, with a reduced presence of memory-resting CD4+ T cells. In summary, FASLG and TARDBP may contribute to AS pathogenesis by regulating the immune microenvironment and immune-related signalling pathways. These findings offer new insights into the molecular mechanisms of AS and suggest potential new targets for therapeutic strategies.

Asunto(s)

Biología Computacional , Necroptosis , Espondilitis Anquilosante , Espondilitis Anquilosante/genética , Espondilitis Anquilosante/patología , Humanos , Biología Computacional/métodos , Necroptosis/genética , Perfilación de la Expresión Génica , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Regulación de la Expresión Génica , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Redes Reguladoras de Genes , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Curva ROC , Bases de Datos Genéticas

RESUMEN

Regular assessment of disease activity in relapsing-remitting multiple sclerosis (RRMS) is required to optimize clinical outcomes. Biomarkers can be a valuable tool for measuring disease activity in multiple sclerosis (MS) if they reflect the pathological processes underlying MS pathogenicity. In this pilot study, we combined multiple biomarkers previously analyzed in RRMS patients into an MS disease activity (MSDA) score to evaluate their ability to predict relapses and treatment response to glatiramer acetate (GA). Response Gene to Complement 32 (RGC-32), FasL, IL-21, SIRT1, phosphorylated SIRT1 (p-SIRT1), and JNK1 p54 levels were used to generate cut-off values for each biomarker. Any value below the cutoff for RGC-32, FasL SIRT1, or p-SIRT1 or above the cutoff for IL-21 or JNK1 p54 was given a +1 value, indicating relapse or lack of response to GA. Any value above the cutoff value for RGC-32, FasL, SIRT1, p-SIRT1 or below that for IL-21 or JNK1 p54 was given a -1 value, indicating clinical stability or response to GA. An MSDA score above +1 indicated a relapse or lack of response to treatment. An MSDA score below -1 indicated clinical stability or response to treatment. Our results showed that the MSDA scores generated using either four or six biomarkers had a higher sensitivity and specificity and significantly correlated with the expanded disability status scale. Although these results suggest that the MSDA test can be useful for monitoring therapeutic response to biologic agents and assessing clinically challenging situations, the present findings need to be confirmed in larger studies.

Asunto(s)

Biomarcadores , Acetato de Glatiramer , Sirtuina 1 , Humanos , Masculino , Adulto , Femenino , Sirtuina 1/metabolismo , Acetato de Glatiramer/uso terapéutico , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Proteína Ligando Fas/metabolismo , Resultado del Tratamiento , Proyectos Piloto , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Interleucinas , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/diagnóstico , Índice de Severidad de la Enfermedad , Inmunosupresores/uso terapéutico

RESUMEN

OBJECTIVE: To investigate the effect of Porphyromonas gingivalis (Pg) infection on immune escape of oesophageal cancer cells and the role of YTHDF2 and Fas in this regulatory mechanism. METHODS: We examined YTHDF2 and Fas protein expressions in esophageal squamous cell carcinoma (ESCC) tissues with and without Pg infection using immunohistochemistry and in Pg-infected KYSE150 cells using Western blotting. The interaction between YTHDF2 and Fas was investigated by co-immunoprecipitation (Co-IP). Pg-infected KYSE150 cells with lentivirus-mediated YTHDF2 knockdown were examined for changes in expression levels of YTHDF2, cathepsin B (CTSB), Fas and FasL proteins, and the effect of E64 (a cathepsin inhibitor) on these proteins were observed. After Pg infection and E64 treatment, KYSE150 cells were co-cultured with human peripheral blood mononuclear cells (PBMCs), and the expressions of T cell-related effector molecules were detected by flow cytometry. RESULTS: ESCC tissues and cells with Pg infection showed significantly increased YTHDF2 expression and lowered Fas expression. The results of Co-IP demonstrated a direct interaction between YTHDF2 and Fas. In Pg-infected KYSE150 cells with YTHDF2 knockdown, the expression of CTSB was significantly reduced while Fas and FasL expressions were significantly increased. E64 treatment of KYSE150 cells significantly decreased the expression of CTSB without affecting YTHDF2 expression and obviously increased Fas and FasL expressions. Flow cytometry showed that in Pg-infected KYSE150 cells co-cultured with PBMCs, the expressions of Granzyme B and Ki67 were significantly decreased while PD-1 expression was significantly enhanced. CONCLUSION: Pg infection YTHDF2-dependently regulates the expression of Fas to facilitate immune escape of esophageal cancer and thus promoting cancer progression, suggesting the key role of YTHDF2 in regulating immune escape of esophageal cancer.

Asunto(s)

Neoplasias Esofágicas , Porphyromonas gingivalis , Proteínas de Unión al ARN , Receptor fas , Humanos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Neoplasias Esofágicas/inmunología , Neoplasias Esofágicas/metabolismo , Línea Celular Tumoral , Receptor fas/metabolismo , Infecciones por Bacteroidaceae/inmunología , Infecciones por Bacteroidaceae/metabolismo , Infecciones por Bacteroidaceae/microbiología , Carcinoma de Células Escamosas de Esófago/inmunología , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/genética , Proteína Ligando Fas/metabolismo , Escape del Tumor

RESUMEN

Rationale: It has been emergingly recognized that apoptosis generates plenty of heterogeneous apoptotic vesicles (apoVs), which play a pivotal role in the maintenance of organ and tissue homeostasis. However, it is unknown whether apoVs influence postnatal ovarian folliculogenesis. Methods: Apoptotic pathway deficient mice including Fas mutant (Fasmut ) and Fas ligand mutant (FasLmut ) mice were used with apoV replenishment to evaluate the biological function of apoVs during ovarian folliculogenesis. Ovarian function was characterized by morphological analysis, biochemical examination and cellular assays. Mechanistical studies were assessed by combinations of transcriptomic and proteomic analysis as well as molecular assays. CYP17A1-Cre; Axin1fl /fl mice was established to verify the role of WNT signaling during ovarian folliculogenesis. Polycystic ovarian syndrome (PCOS) mice and 15-month-old mice were used with apoV replenishment to further validate the therapeutic effects of apoVs based on WNT signaling regulation. Results: We show that systemic administration of mesenchymal stem cell (MSC)-derived apoptotic vesicles (MSC-apoVs) can ameliorate impaired ovarian folliculogenesis, PCOS phenotype, and reduced birth rate in Fasmut and FasLmut mice. Mechanistically, transcriptome analysis results revealed that MSC-apoVs downregulated a number of aberrant gene expression in Fasmut mice, which were enriched by kyoto encyclopedia of genes and genomes (KEGG) pathway analysis in WNT signaling and sex hormone biosynthesis. Furthermore, we found that apoptotic deficiency resulted in aberrant WNT/ß-catenin activation in theca and mural granulosa cells, leading to responsive action of dickkopf1 (DKK1) in the cumulus cell and oocyte zone, which downregulated WNT/ß-catenin expression in oocytes and, therefore, impaired ovarian folliculogenesis via NPPC/cGMP/PDE3A/cAMP cascade. When WNT/ß-catenin was specially activated in theca cells of CYP17A1-Cre; Axin1fl /fl mice, the same ovarian impairment phenotypes observed in apoptosis-deficient mice were established, confirming that aberrant activation of WNT/ß-catenin in theca cells caused the impairment of ovarian folliculogenesis. We firstly revealed that apoVs delivered WNT membrane receptor inhibitor protein RNF43 to ovarian theca cells to balance follicle homeostasis through vesicle-cell membrane integration. Systemically infused RNF43-apoVs down-regulated aberrantly activated WNT/ß-catenin signaling in theca cells, contributing to ovarian functional maintenance. Since aging mice have down-regulated expression of WNT/ß-catenin in oocytes, we used MSC-apoVs to treat 15-month-old mice and found that MSC-apoVs effectively ameliorated the ovarian function and fertility capacity of these aging mice through rescuing WNT/ß-catenin expression in oocytes. Conclusion: Our studies reveal a previously unknown association between apoVs and ovarian folliculogenesis and suggest an apoV-based therapeutic approach to improve oocyte function and birth rates in PCOS and aging.

Asunto(s)

Apoptosis , Células Madre Mesenquimatosas , Folículo Ovárico , Ovario , Síndrome del Ovario Poliquístico , Vía de Señalización Wnt , Animales , Femenino , Síndrome del Ovario Poliquístico/metabolismo , Ratones , Células Madre Mesenquimatosas/metabolismo , Folículo Ovárico/metabolismo , Ovario/metabolismo , Modelos Animales de Enfermedad , Envejecimiento/fisiología , Proteína Ligando Fas/metabolismo , Proteína Ligando Fas/genética

RESUMEN

The transmembrane death receptor Fas transduces apoptotic signals upon binding its ligand, FasL. Although Fas is highly expressed in cancer cells, insufficient cell surface Fas expression desensitizes cancer cells to Fas-induced apoptosis. Here, we show that the increase in Fas microaggregate formation on the plasma membrane in response to the inhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis. We used a clinically accessible Rho-kinase inhibitor, fasudil, that reduces endocytosis dynamics by increasing plasma membrane tension. In combination with exogenous soluble FasL (sFasL), fasudil promoted cancer cell apoptosis, but this collaborative effect was substantially weaker in nonmalignant cells. The combination of sFasL and fasudil prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft mouse model. Our results demonstrate that sFasL has strong potential for apoptosis-directed cancer therapy when Fas microaggregate formation is augmented by mechano-inhibition of endocytosis.

Asunto(s)

Apoptosis , Endocitosis , Proteína Ligando Fas , Receptor fas , Humanos , Endocitosis/efectos de los fármacos , Apoptosis/efectos de los fármacos , Animales , Proteína Ligando Fas/metabolismo , Receptor fas/metabolismo , Ratones , Línea Celular Tumoral , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Glioblastoma/patología , Glioblastoma/metabolismo , Glioblastoma/tratamiento farmacológico

RESUMEN

The clinical application of doxorubicin (DOX) is mainly restricted by its serious side effects, poor drug delivery efficiency, and limited immunogenic death (ICD) effect. To improve DOX-based chemotherapy and ameliorate its adverse effects, we utilized 3LL cell-derived extracellular vesicles to encapsulate DOX and sodium nitroprusside (SNP) to obtain DOX/SNP@CM, which could effectively target the tumor site by harnessing the inherent homologous targeting property of tumor cell membranes. DOX performed its role on chemotherapy, and SNP successfully respond to the intracellular GSH to continuously generate nitric oxide (NO). The in situ-produced NO upregulated the Fas expression on the tumor cell surface, thereby sensitizing the Fas/FasL pathway-mediated tumor cell apoptosis of DOX. Furthermore, NO also boosted the intratumoral infiltration of cytotoxic T cells by promoted ICD effect towards tumor cells. Importantly, the anti-tumor immunity tightly cooperated with Fas/FasL mediated tumor cell apoptosis by NO-mediated manipulation on Fas/FasL interaction, collectively making DOX/SNP@CM exert significant tumor growth inhibition with low-dose DOX. Remarkably, DOX and SNP both are widely used clinical medicines, ensuring DOX/SNP@CM a potential opportunity for future practical applications.

Asunto(s)

Antibióticos Antineoplásicos , Apoptosis , Doxorrubicina , Vesículas Extracelulares , Proteína Ligando Fas , Nitroprusiato , Receptor fas , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Proteína Ligando Fas/metabolismo , Receptor fas/metabolismo , Animales , Nitroprusiato/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Ratones , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/farmacología , Óxido Nítrico/metabolismo , Inmunoterapia/métodos , Ratones Endogámicos C57BL , Femenino , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Neoplasias/inmunología , Humanos , Transducción de Señal/efectos de los fármacos , Ratones Endogámicos BALB C , Sistemas de Liberación de Medicamentos/métodos

RESUMEN

The longevity of grafts remains a major challenge in allogeneic transplantation due to immune rejection. Systemic immunosuppression can impair graft function and can also cause severe adverse effects. Here, we report a local immuno-protective strategy to enhance post-transplant persistence of allografts using a mesenchymal stem cell membrane-derived vesicle (MMV)-crosslinked hydrogel (MMV-Gel). MMVs are engineered to upregulate expression of Fas ligand (FasL) and programmed death ligand 1 (PD-L1). The MMVs are retained within the hydrogel by crosslinking. The immuno-protective microenvironment of the hydrogel protects allografts by presenting FasL and PD-L1. The binding of these ligands to T effector cells, the dominant contributors to graft destruction and rejection, results in apoptosis of T effector cells and generation of regulatory T cells. We demonstrate that implantation with MMV-Gel prolongs the survival and function of grafts in mouse models of allogeneic pancreatic islet cells and skin transplantation.

Asunto(s)

Proteína Ligando Fas , Hidrogeles , Trasplante de Islotes Pancreáticos , Ratones Endogámicos C57BL , Trasplante de Piel , Linfocitos T Reguladores , Trasplante Homólogo , Animales , Hidrogeles/química , Ratones , Proteína Ligando Fas/metabolismo , Proteína Ligando Fas/inmunología , Linfocitos T Reguladores/inmunología , Trasplante de Islotes Pancreáticos/métodos , Trasplante de Piel/métodos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/inmunología , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones Endogámicos BALB C , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Rechazo de Injerto/prevención & control , Rechazo de Injerto/inmunología , Humanos , Masculino , Apoptosis/efectos de los fármacos

RESUMEN

Fas ligand (FasL, CD178) belongs to classical apoptotic molecules, however, recent evidence expands the spectrum of FasL functions into non-apoptotic processes which also applies for the bone. Tgfb subfamily members (Tgfb1, Tgfb2, Tgfb3) represent major components in osteogenic pathways and extracellular matrix. Their possible association with FasL has not yet been investigated but can be postulated. To test such a hypothesis, FasL deficient (gld) calvaria-derived cells were examined with a focus on the expression of Tgfb receptor ligands. The qPCR analysis revealed significantly increased expression of Tgfb1, Tgfb2 and Tgfb3 in gld cells. To check the vice versa effect, the gld cells were stimulated by soluble FasL. As a consequence, a dramatic decrease in expression levels of all three ligands was observed. This phenomenon was also confirmed in IDG-SW3 (osteoblastic cells of endochondral origin). TFLink gateway identified Fosl2 as an exclusive candidate of FasL capable to impact expression of all three Tgfb ligands. However, Fosl2 siRNA did not cause any significant changes in expression of Tgfb ligands. Therefore, the upregulation of the three ligands is likely to occur separately. In this respect, we tested the only exclusive candidate transcription factor for Tgfb3, Prrx1. Additionally, an overlapping candidate for Tgfb1 and Tgfb2, Mef2c capable to modulate expression of sclerostin, was examined. Prrx1 as well as Mef2c were found upregulated in gld samples and their expression decreased after addition of FasL. The same effect of FasL treatment was observed in the IDG-SW3 model. Taken together, FasL deficiency causes an increase in the expression of Tgfb ligands and stimulation by FasL reduces Tgfb expression in osteoblastic cells. The candidates mediating the effect comprise Prrx1 for Tgfb3 and Mef2c for Tgfb1/2. These results indicate FasL as a novel cytokine interfering with Tgfb signaling and thus the complex osteogenic network. The emerging non-apoptotic functions of FasL in bone development and maintenance should also be considered in treatment strategies such as the anti-osteoporotic factor.

Asunto(s)

Proteína Ligando Fas , Osteoblastos , Transducción de Señal , Factor de Crecimiento Transformador beta3 , Proteína Ligando Fas/metabolismo , Osteoblastos/metabolismo , Animales , Ratones , Factor de Crecimiento Transformador beta3/metabolismo , Factor de Crecimiento Transformador beta3/genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta2/farmacología , Factores de Transcripción MEF2/metabolismo , Factores de Transcripción MEF2/genética , Factor de Crecimiento Transformador beta/metabolismo , Línea Celular

RESUMEN

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is a life-threatening disease affecting mostly the ileum of preemies. Intestinal epithelial cell (IEC) apoptosis contributes to NEC pathogenesis. However, how scattered crypt IEC apoptosis leads to NEC with excessive villus epithelial necrosis remains unclear. METHODS: A novel triple-transgenic mouse model, namely, 3xTg-iAPcIEC (inducible apoptosis phenotype in crypt-IEC), was developed to induce IEC-specific overexpression of Fasl transgene using doxycycline (Dox)-inducible tetO-rtTA system and villin-cre technology. The 3-days-old neonatal 3xTg-iAPcIEC mice and their littermate controls were subcutaneously (s.c.) challenged with a single dose of Dox. Intestinal tissues were processed at different time points to examine scattered crypt IEC apoptosis-mediated NEC development. Gene knockout technology, antibody-mediated cell depletion, and antibiotic-facilitated Gram-positive bacteria depletion were used to study mechanisms. RESULTS: Treatment of 3xTg-iAPcIEC mouse pups with Dox induces scattered crypt IEC apoptosis followed by crypt inflammation and excessive villous necrosis resembling NEC. This progression correlated with elevated Ifng, Rip3, CD8+ T cells, and Gram-positive bacteria in the ileum. Mechanistically, IFN-γ and RIP3-activated signals mediate the effect of scattered crypt IEC apoptosis on the induction of intestinal crypt inflammation and villous necrosis. Meanwhile, pathophysiological events of CD8+ T cell infiltration and dysbiosis with Gram-positive bacteria primarily contribute to excessive villous inflammation and necrosis. Notably, blocking any of these events protects against NEC development in 3xTg-iAPcIEC mouse pups, underlining their central roles in NEC pathogenesis. CONCLUSIONS: Scattered crypt IEC apoptosis induces NEC in mouse pups via IFN-γ, RIP3, CD8+ T cells, and Gram-positive bacteria-mediated comprehensive pathophysiological events. Our findings may advance knowledge in the prevention and treatment of NEC.

Asunto(s)

Apoptosis , Modelos Animales de Enfermedad , Enterocolitis Necrotizante , Proteína Ligando Fas , Interferón gamma , Mucosa Intestinal , Ratones Transgénicos , Animales , Enterocolitis Necrotizante/patología , Enterocolitis Necrotizante/metabolismo , Ratones , Mucosa Intestinal/patología , Mucosa Intestinal/microbiología , Mucosa Intestinal/inmunología , Interferón gamma/metabolismo , Proteína Ligando Fas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Células Epiteliales/patología , Células Epiteliales/metabolismo , Linfocitos T CD8-positivos/inmunología , Necrosis , Animales Recién Nacidos , Doxiciclina/farmacología , Humanos , Íleon/patología , Íleon/inmunología

RESUMEN

Idiopathic severe aplastic anemia (SAA) is a disease of bone marrow failure caused by T-cell-induced destruction of hematopoietic stem and progenitor cells (HSPCs), however the mechanism remains unclear. We performed single-cell RNA sequencing of PBMCs and BMMCs from SAA patients and healthy donors and identified a CD8+ T cell subset with a tissue residency phenotype (Trm) in bone marrow that exhibit high IFN-γ and FasL expression and have a higher ability to induce apoptosis in HSPCs in vitro through FasL expression. CD8+ Trm cells were induced by IL-15 presented by IL-15Rα on monocytes, especially CD16+ monocytes, which were increased in SAA patients. CD16+ monocytes contributed to IL-15-induced CD38+CXCR6+ pre-Trm differentiation into CD8+ Trm cells, which can be inhibited by the CD38 inhibitor 78c. Our results demonstrate that IL-15-induced CD8+ Trm cells are pathogenic cells that mediate HSPC destruction in SAA patients and are therapeutic targets for future treatments.

Asunto(s)

Anemia Aplásica , Linfocitos T CD8-positivos , Proteínas Ligadas a GPI , Células Madre Hematopoyéticas , Interleucina-15 , Monocitos , Receptores de IgG , Humanos , Anemia Aplásica/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Interleucina-15/farmacología , Interleucina-15/inmunología , Receptores de IgG/metabolismo , Receptores de IgG/inmunología , Monocitos/inmunología , Monocitos/efectos de los fármacos , Femenino , Masculino , Adulto , Células Madre Hematopoyéticas/inmunología , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/inmunología , Persona de Mediana Edad , Proteína Ligando Fas/metabolismo , Proteína Ligando Fas/inmunología , Adulto Joven , Adolescente , Interferón gamma/inmunología , Interferón gamma/metabolismo , Receptores de Interleucina-15/metabolismo , Receptores de Interleucina-15/inmunología , Apoptosis/efectos de los fármacos , Diferenciación Celular/inmunología

RESUMEN

DNA origami is capable of spatially organizing molecules into sophisticated geometric patterns with nanometric precision. Here we describe a reconfigurable, two-dimensional DNA origami with geometrically patterned CD95 ligands that regulates immune cell signalling to alleviate rheumatoid arthritis. In response to pH changes, the device reversibly transforms from a closed to an open configuration, displaying a hexagonal pattern of CD95 ligands with ~10 nm intermolecular spacing, precisely mirroring the spatial arrangement of CD95 receptor clusters on the surface of immune cells. In a collagen-induced arthritis mouse model, DNA origami elicits robust and selective activation of CD95 death-inducing signalling in activated immune cells located in inflamed synovial tissues. Such localized immune tolerance ameliorates joint damage with no noticeable side effects. This device allows for the precise spatial control of cellular signalling, expanding our understanding of ligand-receptor interactions and is a promising platform for the development of pharmacological interventions targeting these interactions.

Asunto(s)

Artritis Reumatoide , ADN , Tolerancia Inmunológica , Transducción de Señal , Receptor fas , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Animales , ADN/química , ADN/inmunología , Ratones , Receptor fas/metabolismo , Receptor fas/inmunología , Proteína Ligando Fas/metabolismo , Proteína Ligando Fas/inmunología , Humanos

RESUMEN

Protopanaxadiol (PPD), which has a molecular structure similar to cholesterol, is a potent anticancer agent that has been proposed to target the lipid membrane for the pharmacological effects. However, the underlying mechanism by which PPD modulates the cell membrane leading to cancer cell death is not be fully understood. In this work, we used single cell infrared spectroscopy, scanning electron microscopy and confocal microscopy to investigate the effects of PPD on human hepatocellular carcinoma (HepG2) cells, focusing on the change in membrane structure. We found that PPD significantly reduced the number of membrane tubules over the course of treatment. Interestingly, the addition of PPD could promote the formation of lipid raft-like domains (PPD rafts) and even restore the domain disruption caused by methyl-beta-cyclodextrin depletion of membrane cholesterol. In addition, PPD pre-treatment may increase the induction effect of FasL, which impairs cell viability, although it does not appear to be beneficial for Fas clustering in the PPD rafts. Collectively, these results highlight a non-classical mechanism by which PPD induces HepG2 apoptosis by directly affecting the physical properties of the cell membrane, providing a novel insight into understanding membrane-targeted therapy.

Asunto(s)

Apoptosis , Microdominios de Membrana , Sapogeninas , Humanos , Apoptosis/efectos de los fármacos , Células Hep G2 , Microdominios de Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacos , Sapogeninas/farmacología , Sapogeninas/química , Proteína Ligando Fas/metabolismo , Supervivencia Celular/efectos de los fármacos , Colesterol/metabolismo , beta-Ciclodextrinas/farmacología , beta-Ciclodextrinas/química , Antineoplásicos/farmacología , Antineoplásicos/química , Receptor fas/metabolismo

RESUMEN

The dysregulated immune response and inflammation resulting in severe COVID-19 are still incompletely understood. Having recently determined that aberrant death-ligand-induced cell death can cause lethal inflammation, we hypothesized that this process might also cause or contribute to inflammatory disease and lung failure following SARS-CoV-2 infection. To test this hypothesis, we developed a novel mouse-adapted SARS-CoV-2 model (MA20) that recapitulates key pathological features of COVID-19. Concomitantly with occurrence of cell death and inflammation, FasL expression was significantly increased on inflammatory monocytic macrophages and NK cells in the lungs of MA20-infected mice. Importantly, therapeutic FasL inhibition markedly increased survival of both, young and old MA20-infected mice coincident with substantially reduced cell death and inflammation in their lungs. Intriguingly, FasL was also increased in the bronchoalveolar lavage fluid of critically-ill COVID-19 patients. Together, these results identify FasL as a crucial host factor driving the immuno-pathology that underlies COVID-19 severity and lethality, and imply that patients with severe COVID-19 may significantly benefit from therapeutic inhibition of FasL.

Asunto(s)

COVID-19 , Modelos Animales de Enfermedad , Proteína Ligando Fas , SARS-CoV-2 , Animales , Ratones , Líquido del Lavado Bronquioalveolar , COVID-19/patología , COVID-19/inmunología , COVID-19/metabolismo , COVID-19/virología , COVID-19/mortalidad , Proteína Ligando Fas/metabolismo , Inflamación/patología , Inflamación/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Pulmón/patología , Pulmón/virología , Pulmón/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratones Endogámicos C57BL

RESUMEN

Controlled cell death is essential for the regulation of the immune system and plays a role in pathogen defense. It is often altered in pathogenic conditions such as cancer, viral infections and autoimmune diseases. The Fas receptor and its corresponding membrane-bound ligand (FasL) are part of the extrinsic apoptosis pathway activated in these cases. A soluble form of FasL (sFasL), produced by ectodomain shedding, displays a diverse but still elusive set of non-apoptotic functions and sometimes even serves as a pro-survival factor. To gather more knowledge about the characteristics of this protein and the impact N-glycosylations may have, access to homogeneous posttranslationally modified variants of sFasL is needed. Therefore, we developed a flexible strategy to obtain such homogeneously N-glycosylated variants of sFasL by applying chemical protein synthesis. This strategy can be flexibly combined with enzymatic methods to introduce more complex, site selective glycosylations.

Asunto(s)

Proteína Ligando Fas , Apoptosis , Proteína Ligando Fas/metabolismo , Proteína Ligando Fas/química , Receptor fas/metabolismo , Receptor fas/química , Glicosilación , Procesamiento Proteico-Postraduccional , Solubilidad

RESUMEN

Signalling by the Unfolded Protein Response (UPR) or by the Death Receptors (DR) are frequently activated towards pro-tumoral outputs in cancer. Herein, we demonstrate that the UPR sensor IRE1 controls the expression of the DR CD95/Fas, and its cell death-inducing ability. Both genetic and pharmacologic blunting of IRE1 activity increased CD95 expression and exacerbated CD95L-induced cell death in glioblastoma (GB) and Triple-Negative Breast Cancer (TNBC) cell lines. In accordance, CD95 mRNA was identified as a target of Regulated IRE1-Dependent Decay of RNA (RIDD). Whilst CD95 expression is elevated in TNBC and GB human tumours exhibiting low RIDD activity, it is surprisingly lower in XBP1s-low human tumour samples. We show that IRE1 RNase inhibition limited CD95 expression and reduced CD95-mediated hepatic toxicity in mice. In addition, overexpression of XBP1s increased CD95 expression and sensitized GB and TNBC cells to CD95L-induced cell death. Overall, these results demonstrate the tight IRE1-mediated control of CD95-dependent cell death in a dual manner through both RIDD and XBP1s, and they identify a novel link between IRE1 and CD95 signalling.

Asunto(s)

Ribonucleasas , Neoplasias de la Mama Triple Negativas , Animales , Ratones , Humanos , Ribonucleasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Respuesta de Proteína Desplegada , Muerte Celular

RESUMEN

OBJECTIVE: To investigate the anogenital distance from the upper verge of the anus to the posterior fourchette (AGDAF), FASL, and BCL2 combination as a reliable and non-invasive tool for the diagnosis of endometriosis. METHODS: This study included 100 women with endometriosis and 50 women without endometriosis as the control group. All cases underwent history taking, body mass index (BMI) measurement, AGD measurement, and FASL and BCL2 immunohistochemical staining of the eutopic endometrial tissue. RESULTS: This study included 150 women divided into endometriosis and control groups. Endometriosis cases significantly had shorter AGDAF, 22.9 ± 2.6 mm, compared with the control group, 27.3 ± 3.5 mm (P < 0.001). Lower FASL and higher BCL2 expression were associated with endometriosis (P < 0.001). The combined measurement of AGDAF (cut-off point 24.55 mm) with FASL and BCL2 was associated with endometriosis (P < 0.001). The combined diagnostic sensitivity, specificity, positive predictive value, and negative predictive value of AGDAF, FASL, and BCL2 were 83%, 78%, 87.3%, and 69.6%, respectively. The area under the curve was greater for AGDAF, FASL, and BCL2 in combination than for individual measurements. CONCLUSION: Combining short AGDAF with high BCL2 and low FASL is a highly sensitive, non-invasive diagnostic tool for endometriosis.

Asunto(s)

Canal Anal , Endometriosis , Endometrio , Proteína Ligando Fas , Proteínas Proto-Oncogénicas c-bcl-2 , Humanos , Femenino , Endometriosis/diagnóstico , Endometriosis/patología , Adulto , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Endometrio/patología , Endometrio/metabolismo , Canal Anal/patología , Proteína Ligando Fas/metabolismo , Proteína Ligando Fas/análisis , Estudios de Casos y Controles , Sensibilidad y Especificidad , Adulto Joven , Valor Predictivo de las Pruebas