RESUMEN
Primary and acquired therapy resistance is a major problem in patients with BRAF-mutant melanomas being treated with BRAF and MEK inhibitors (BRAFI, MEKi). Therefore, development of alternative therapy regimes is still required. In this regard, new drug combinations targeting different pathways to induce apoptosis could offer promising alternative approaches. Here, we investigated the combination of proteasome and Kv1.3 potassium channel inhibition on chemo-resistant, BRAF inhibitor-resistant as well as sensitive human melanoma cells. Our experiments demonstrated that all analyzed melanoma cell lines were sensitive to proteasome inhibitor treatment at concentrations that are not toxic to primary human fibroblasts. To further reduce proteasome inhibitor-associated side effects, and to foster apoptosis, potassium channels, which are other targets to induce pro-apoptotic effects in cancer cells, were blocked. In support, combined exposure of melanoma cells to proteasome and Kv1.3 channel inhibitor resulted in synergistic effects and significantly reduced cell viability. On the molecular level, enhanced apoptosis correlated with an increase of intracellular Kv1.3 channels and pro-apoptotic proteins such as Noxa and Bak and a reduction of anti-apoptotic proteins. Thus, use of combined therapeutic strategies triggering different apoptotic pathways may efficiently prevent the outgrowth of drug-resistant and -sensitive BRAF-mutant melanoma cells. In addition, this could be the basis for an alternative approach to treat other tumors expressing mutated BRAF such as non-small-cell lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Melanoma , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Canal de Potasio Kv1.3/genética , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos , Línea Celular Tumoral , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma/patología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Reguladoras de la Apoptosis/metabolismo , MutaciónRESUMEN
Objective: The formation of large intracellular protein aggregates of the inflammasome adaptor ASC is a hallmark of inflammasome activation and characteristic of autoinflammation. Inflammasome activated cells release the highly proinflammatory cytokine IL-1ß in addition to ASC specks into the extracellular space. Autoinflammatory activity has been demonstrated in systemic JIA, however minimal data exist on the role of inflammasomes in other JIA subtypes. We therefore investigated, if pyroptotic cells are present in the circulation of oligo- and poly-articular JIA. Methods: Peripheral blood of JIA patients (n = 46) was investigated for ASC speck formation, a key step in inflammasome activation, by flow cytometry and immunofluorescence. Free ASC and proinflammatory cytokine levels were determined by ELISA and multiplex assay. Results: Oligo-articular JIA patients showed a significantly increased proportion of ASC speck+ monocytes compared to poly-articular JIA patients. In serum free ASC alone is not sufficient to assess inflammasome activity and does not correlate with ASC speck+ monocytes. Compared to control several cytokines were significantly elevated in samples of JIA patients. JIA serum containing antinuclear antibodies, incubated with ASC specks boosts a secondary inflammation by IL-1ß production in macrophages. Conclusion: For the first time, we detect ex vivo inflammasome activation by ASC speck formation in oligo- and poly-articular JIA patients. Most notably, inflammasome activation was significantly higher in oligo- compared to poly-articular JIA patients. This data suggests that inflammasome derived autoinflammation may have a greater influence in the previously thought autoimmune oligo-articular JIA patients.
RESUMEN
Inflammasome activation is linked to the aggregation of the adaptor protein ASC into a multiprotein complex, known as the ASC speck. Redistribution of cytosolic ASC to this complex has been widely used as a readout for inflammasome activation and precedes the downstream proteolytic release of the proinflammatory cytokines, IL-1ß and IL-18. Although inflammasomes are important for many diseases such as periodic fever syndromes, COVID-19, gout, sepsis, atherosclerosis and Alzheimer's disease, only a little knowledge exists on the precise and cell type specific occurrence of inflammasome activation in patient samples ex vivo. In this report, we provide detailed information about the optimal conditions to reliably identify inflammasome activated monocytes by ASC speck formation using a modified flow cytometric method introduced by Sester et al. in 2015. Since no protocol for optimal sample processing exists, we tested human blood samples for various conditions including anticoagulant, time and temperature, the effect of one freeze-thaw cycle for PBMC storage, and the fast generation of a positive control. We believe that this flow cytometric protocol will help researchers to perform high quality translational research in multicenter studies, and therefore provide a basis for investigating the role of the inflammasome in the pathogenesis of various diseases.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Citometría de Flujo/métodos , Inflamasomas/inmunología , Anticoagulantes , Citometría de Flujo/normas , Humanos , Inflamasomas/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Manejo de Especímenes , Temperatura , Factores de TiempoRESUMEN
BACKGROUND: As one alternative to the Schirmer test, strip meniscometry has been presented as a methodology which also seems to be suitable for quantifying a lack of tear volume. METHODS: In a randomized prospective clinical study 391 eyes from 201 subjects were assigned to 3 groups according to the severity of eye surface deficiency (group 0: 225 healthy eyes, f/mâ¯= 1.25, mean ageâ¯= 50.1⯱ 17.6 years; group 1: 112 eyes with mild keratoconjunctivitis sicca (KCS), f/mâ¯= 1.38, mean ageâ¯= 58.7⯱ 13.9 years; group 2: 54 eyes with manifest KCS, f/mâ¯= 2.375, mean ageâ¯= 52.6⯱ 14.1 years). Objective test parameters used were strip meniscometry, Schirmer's 1 test and the Jones-test. In order to determine the condition of the ocular surface and tear film more precisely, slit-lamp examination, the detection of lid-parallel conjunctival folds (LIPCOF) and optical coherence tomography (OCT) meniscometry were also performed. Subjective discomfort was objectified by using the Ocular Surface Disease Index questionnaire (OSDI). RESULTS: The results of strip meniscometry, Schirmer's 1 test, the Jones test and OCT meniscometry were significantly lower in KCS subjects than in healthy subjects (pâ¯< 0.001), whereas the OSDI score was significantly higher (pâ¯< 0.001). The parameters strip meniscometry, Schirmer's 1 test, the Jones test and OCT meniscometry scores correlated with each other. Sensitivity and specificity of strip meniscometry ranged from 0.79 to 0.89 and from 0.42 to 0.5. CONCLUSION: Strip meniscometry is suitable to detect a lack of tear volume. Advantageous is the rapid performance of the procedure (5 s per eye) and a good agreement with established tests (e.g. Schirmer's test). To improve the specificity, strip meniscometry must be combined with other examination methods.
Asunto(s)
Síndromes de Ojo Seco , Adulto , Anciano , Síndromes de Ojo Seco/diagnóstico , Humanos , Persona de Mediana Edad , Estudios Prospectivos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , LágrimasRESUMEN
BACKGROUND & AIMS: Pancreatitis starts with primarily sterile local inflammation that induces systemic inflammatory response syndrome, followed by compensatory anti-inflammatory response syndrome (CARS). We investigated the mechanisms of these processes in mice and human serum. METHODS: We induced severe acute pancreatitis by partial duct ligation with caerulein stimulation or intraperitoneal injection of l-arginine in mice with deletion of interleukin (IL)12B, NLRP3, or IL18 and in mice given MCC950, a small molecule inhibitor of the NLRP3-inflammasome. Pancreata were collected from mice and analyzed by histology, and cytokine levels were measured in serum samples. We measured activation of adaptive immune responses in mice with pancreatitis by flow cytometry analysis of T cells (CD25 and CD69) isolated from the spleen. Differentiation of T-helper (Th1) cells, Th2 cells, and T-regulatory cells was determined by nuclear staining for TBET, GATA3, and FOXP3. We performed transcriptome analysis of mouse lymph nodes and bone marrow-derived macrophages after incubation with acini. We measured levels of cytokines in serum samples from patients with mild and severe acute pancreatitis. RESULTS: Activation of the adaptive immune response in mice was initiated by macrophage-derived, caspase 1-processed cytokines and required activation of NLRP3 (confirmed in serum samples from patients with pancreatitis). Spleen cells from mice with pancreatitis had increases in Th2 cells but not in Th1 cells. Bone marrow-derived macrophages secreted IL1B and IL18, but not IL12, after co-incubation with pancreatic acini. T-cell activation and severity of acute pancreatitis did not differ significantly between IL12B-deficient and control mice. In contrast, NLRP3- or IL18-deficient mice had reduced activation of T cells and no increase in Th2 cell-mediated responses compared with control mice. The systemic type 2 immune response was mediated by macrophage-derived cytokines of the IL1 family. Specifically, IL18 induced a Th2 cell-mediated response in the absence of IL12. MCC950 significantly reduced neutrophil infiltration, T-cell activation, and disease severity in mice. CONCLUSIONS: In mice with severe pancreatitis, we found systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome developed in parallel. Infiltrating macrophages promote inflammation and simultaneously induce a Th2 cell-mediated response via IL18. Inhibition of NLRP3 reduces systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome and might be used to treat patients with severe pancreatitis.
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Furanos/administración & dosificación , Inflamasomas/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Pancreatitis/inmunología , Sulfonamidas/administración & dosificación , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Células Acinares , Inmunidad Adaptativa , Animales , Arginina/toxicidad , Células Cultivadas , Ceruletida/toxicidad , Citocinas/sangre , Citocinas/inmunología , Modelos Animales de Enfermedad , Compuestos Heterocíclicos de 4 o más Anillos , Humanos , Indenos , Inyecciones Intraperitoneales , Interleucina-18/inmunología , Interleucina-18/metabolismo , Macrófagos/inmunología , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Páncreas/citología , Páncreas/inmunología , Páncreas/patología , Pancreatitis/inducido químicamente , Pancreatitis/tratamiento farmacológico , Cultivo Primario de Células , Sulfonas , Síndrome de Respuesta Inflamatoria Sistémica/sangre , Síndrome de Respuesta Inflamatoria Sistémica/tratamiento farmacológico , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Protein arginine deiminase (PAD) enzymes catalyze the conversion of protein-bound arginine into citrulline, an irreversible posttranslational modification with loss of a positive charge that can influence protein-protein interactions and protein structure. Protein arginine deiminase activity depends on high intracellular calcium concentrations occurring in dying cells. In this study, we demonstrate that protein citrullination is common during pyroptotic cell death in macrophages and that inhibition of PAD enzyme activity by Cl-amidine, a pan-PAD inhibitor, blocks NLRP3 inflammasome assembly and proinflammatory IL-1ß release in macrophages. Genetic deficiency of either PAD2 or PAD4 alone in murine macrophages does not impair IL-1ß release; however, pharmacological inhibition or small interfering RNA knockdown of PAD2 within PAD4-/- macrophages does. Our results suggest that PAD2 and 4 activity in macrophages is required for optimal inflammasome assembly and IL-1ß release, a finding of importance for autoimmune diseases and inflammation.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Desiminasas de la Arginina Proteica/metabolismo , Animales , Citrulinación/fisiología , Humanos , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Piroptosis/fisiologíaRESUMEN
OBJECTIVE: The formation of large intracellular protein aggregates of the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain; also know as PYCARD) is a hallmark of inflammasome activation. ASC speck-forming cells release the highly proinflammatory cytokine IL-1ß in addition to ASC specks into the extracellular space during pyroptotic cell death. There ASC specks can propagate inflammation to other nonactivated cells or tissues. HIV-1 retroviral infection triggers inflammasome activation of abortively infected CD4⺠T cells in secondary lymphatic tissues. However, if pyroptosis occurs in other peripheral blood mononuclear cells (PBMCs) of HIV-1-infected patients is currently unknown. We investigated if ASC speck positive cells are present in the circulation of HIV-1-infected patients. DESIGN AND METHODS: PBMCs or plasma of HIV-1 infected, antiretroviral therapy-naive patients were analyzed for the presence of ASC speck⺠cells or extracellular ASC and compared with healthy controls. Intracellular staining for ASC was employed to detect ASC speck⺠cells within PBMCs by flow cytometry, and ELISA to detect free ASC in the plasma. ASC multimerization was confirmed by immunoblot. RESULTS: Peripheral blood CD14âºâºCD16â» monocytes were ASC speck⺠in HIV patients, but not in healthy controls. In the subgroup analysis, HIV patients with lower CD4⺠T-cell counts and higher viral load had significantly more ASC speck⺠monocytes. ASC speck formation did not correlate with Gag expression, coinfection, lactate dehydrogenase or C-reactive protein. CONCLUSION: Our findings suggest that pyroptotic CD14âºâºCD16â» classical monocytes of HIV-1-infected patients release ASC specks into the blood stream, a phenomenon that may contribute to HIV-1 induced inflammation and immune activation.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/análisis , Infecciones por VIH/patología , Inflamasomas/metabolismo , Monocitos/química , Adulto , Recuento de Linfocito CD4 , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Proteínas Ligadas a GPI/análisis , Humanos , Immunoblotting , Receptores de Lipopolisacáridos/análisis , Masculino , Persona de Mediana Edad , Receptores de IgG/análisis , Coloración y Etiquetado , Carga ViralRESUMEN
Systemic lupus erythematosus (SLE) is a chronic, life-threatening autoimmune disorder, leading to multiple organ pathologies and kidney destruction. Analyses of numerous murine models of spontaneous SLE have revealed a critical role for endosomal TLRs in the production of autoantibodies and development of other clinical disease manifestations. Nevertheless, the corresponding TLR9-deficient autoimmune-prone strains consistently develop more severe disease pathology. Injection of BALB/c mice with 2,6,10,14-tetramethylpentadecane (TMPD), commonly known as pristane, also results in the development of SLE-like disease. We now show that Tlr9(-/-) BALB/c mice injected i.p. with TMPD develop more severe autoimmunity than do their TLR-sufficient cohorts. Early indications include an increased accumulation of TLR7-expressing Ly6C(hi) inflammatory monocytes at the site of injection, upregulation of IFN-regulated gene expression in the peritoneal cavity, and an increased production of myeloid lineage precursors (common myeloid progenitors and granulocyte myeloid precursors) in the bone marrow. TMPD-injected Tlr9(-/-) BALB/c mice develop higher autoantibody titers against RNA, neutrophil cytoplasmic Ags, and myeloperoxidase than do TMPD-injected wild-type BALB/c mice. The TMP-injected Tlr9(-/-) mice, and not the wild-type mice, also develop a marked increase in glomerular IgG deposition and infiltrating granulocytes, much more severe glomerulonephritis, and a reduced lifespan. Collectively, the data point to a major role for TLR7 in the response to self-antigens in this model of experimental autoimmunity. Therefore, the BALB/c pristane model recapitulates other TLR7-driven spontaneous models of SLE and is negatively regulated by TLR9.
Asunto(s)
Linaje de la Célula , Nefritis Lúpica/patología , Células Mieloides/patología , Receptor Toll-Like 9/deficiencia , Animales , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Citometría de Flujo , Inmunosupresores/toxicidad , Nefritis Lúpica/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Células Mieloides/inmunología , Reacción en Cadena de la Polimerasa , Terpenos/toxicidadRESUMEN
OBJECTIVE: Nephrogenic systemic fibrosis (NSF) is a progressive fibrosing disorder that may develop in patients with chronic kidney disease after administration of gadolinium (Gd)-based contrast agents (GBCAs). In the setting of impaired renal clearance of GBCAs, Gd deposits in various tissues and fibrosis subsequently develops. However, the precise mechanism by which fibrosis occurs in NSF is incompletely understood. Because other profibrotic agents, such as silica or asbestos, activate the nucleotide-binding oligomerisation domain (NOD)-like receptor protein 3 (NLRP3) inflammasome and initiate interleukin (IL)-1ß release with the subsequent development of fibrosis, we evaluated the effects of GBCAs on inflammasome activation. METHODS: Bone marrow derived macrophages from C57BL/6, Nlrp3(-/-) and Asc(-/-) mice were incubated with three Gd-containing compounds and IL-1ß activation and secretion was detected by ELISA and western blot analysis. Inflammasome activation and regulation was investigated in IL-4- and interferon (IFN)γ-polarised macrophages by ELISA, quantitative real time (qRT)-PCR and NanoString nCounter analysis. Furthermore, C57BL/6 and Nlrp3(-/-)mice were intraperitoneally injected with GBCA and recruitment of inflammatory cells to the peritoneum was analysed by fluorescence-activated cell sorting (FACS). RESULTS: Free Gd and GBCAs activate the NLRP3 inflammasome and induce IL-1ß secretion in vitro. Gd-diethylenetriaminepentaacetic acid also induces the recruitment of neutrophils and inflammatory monocytes to the peritoneum in vivo. Gd activated IL-4-polarised macrophages more effectively than IFNγ-polarised macrophages, which preferentially expressed genes known to downregulate inflammasome activity. CONCLUSIONS: These data suggest that Gd released from GBCAs triggers a NLRP3 inflammasome-dependent inflammatory response that leads to fibrosis in an appropriate clinical setting. The preferential activation of IL-4-differentiated macrophages is consistent with the predominantly fibrotic presentation of NSF.
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Proteínas Portadoras/efectos de los fármacos , Medios de Contraste/farmacología , Gadolinio/farmacología , Inflamasomas/efectos de los fármacos , Interleucina-1beta/efectos de los fármacos , Macrófagos/efectos de los fármacos , Dermopatía Fibrosante Nefrogénica/inmunología , Peritoneo/efectos de los fármacos , Peritonitis/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Medios de Contraste/efectos adversos , Modelos Animales de Enfermedad , Gadolinio/efectos adversos , Gadolinio DTPA/efectos adversos , Gadolinio DTPA/farmacología , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Dermopatía Fibrosante Nefrogénica/inducido químicamente , Dermopatía Fibrosante Nefrogénica/genética , Compuestos Organometálicos/efectos adversos , Compuestos Organometálicos/farmacología , Peritoneo/inmunología , Peritonitis/inducido químicamente , Peritonitis/genéticaRESUMEN
Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The nucleotide-binding domain and leucine-rich repeat containing family (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in triggering caspase-1-dependent IL-1ß maturation and resistance to fungal dissemination in Candida albicans infection. ß-Glucans are major components of fungal cell walls that trigger IL-1ß secretion in both murine and human immune cells. In this study, we sought to determine the contribution of ß-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-apoptosis-associated speck-like protein containing caspase recruitment domain protein-caspase-1 inflammasome is absolutely critical for IL-1ß production in response to ß-glucans. Interestingly, we also found that both complement receptor 3 (CR3) and dectin-1 play a crucial role in coordinating ß-glucan-induced IL-1ß processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the proapoptotic protease caspase-8 in promoting ß-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1ß maturation. A strong requirement for CR3 and caspase-8 also was found for NLRP3-dependent IL-1ß production in response to heat-killed C. albicans. Taken together, these results define the importance of dectin-1, CR3, and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating ß-glucan- and C. albicans-induced innate responses in dendritic cells. Collectively, these findings establish a novel link between ß-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components.
Asunto(s)
Candida albicans/inmunología , Candidiasis/inmunología , Caspasa 8/inmunología , Polisacáridos Fúngicos/inmunología , Interleucina-1beta/inmunología , Lectinas Tipo C/inmunología , Antígeno de Macrófago-1/inmunología , beta-Glucanos/inmunología , Animales , Candida albicans/genética , Candidiasis/genética , Candidiasis/patología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Caspasa 8/genética , Muerte Celular/genética , Muerte Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/patología , Polisacáridos Fúngicos/genética , Humanos , Interleucina-1beta/genética , Lectinas Tipo C/genética , Antígeno de Macrófago-1/genética , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLRRESUMEN
Microbes or danger signals trigger inflammasome sensors, which induce polymerization of the adaptor ASC and the assembly of ASC specks. ASC specks recruit and activate caspase-1, which induces maturation of the cytokine interleukin 1ß (IL-1ß) and pyroptotic cell death. Here we found that after pyroptosis, ASC specks accumulated in the extracellular space, where they promoted further maturation of IL-1ß. In addition, phagocytosis of ASC specks by macrophages induced lysosomal damage and nucleation of soluble ASC, as well as activation of IL-1ß in recipient cells. ASC specks appeared in bodily fluids from inflamed tissues, and autoantibodies to ASC specks developed in patients and mice with autoimmune pathologies. Together these findings reveal extracellular functions of ASC specks and a previously unknown form of cell-to-cell communication.
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Apoptosis/inmunología , Caspasa 1/inmunología , Proteínas del Citoesqueleto/inmunología , Inflamación/inmunología , Interleucina-1beta/inmunología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Anticuerpos/inmunología , Proteínas Reguladoras de la Apoptosis , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Caspasa 1/genética , Inhibidores de Caspasas/farmacología , Comunicación Celular/inmunología , Proteínas del Citoesqueleto/genética , Humanos , Inflamasomas/inmunología , Lisosomas/patología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Fagocitosis/inmunología , Priones/química , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Transducción de Señal/inmunologíaRESUMEN
Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE-DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo.
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ADN/metabolismo , Neumonía/metabolismo , Neumonía/patología , Receptores Inmunológicos/metabolismo , Animales , Secuencia de Bases , Membrana Celular/metabolismo , Cristalografía por Rayos X , ADN/química , Endocitosis , Endosomas/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligandos , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , FN-kappa B/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/química , Electricidad Estática , Receptor Toll-Like 9/metabolismoRESUMEN
Loss-of-function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces systemic lupus erythematosus-like disease. Although Fas/Fas ligand (FasL) interactions have been strongly implicated in the activation-induced cell death of both lymphocytes and other APCs, FasL can also trigger the production of proinflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase cleavage site in the ectodomain. Matrix metalloproteinase cleavage inactivates membrane-bound FasL and releases a soluble form reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the proapoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation to produce the deleted cleavage site (ΔCS) mouse line. ΔCS mice express higher levels of membrane-bound FasL than do wild-type mice and fail to release soluble FasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and ΔCS BALB/c mice were compared with control TMPD-injected BALB/c mice. We found that FasL deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. In contrast, ΔCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in anti-nuclear Ab specificity, and markedly increased proteinuria and kidney pathology compared with controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL proinflammatory activity.
Asunto(s)
Proteína Ligando Fas/metabolismo , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Animales , Apoptosis/inmunología , Células 3T3 BALB , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Proteína Ligando Fas/inmunología , Citometría de Flujo , Inmunosupresores/toxicidad , Enfermedades Renales/patología , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Terpenos/toxicidad , TranscriptomaRESUMEN
INTRODUCTION: Rheumatoid arthritis (RA) is a systemic disease and the most prevalent of all autoimmune disorders. Here we review recent advances in the development and availability of biologic agents with a focus on monoclonal antibody or smaller formats of targeted engineered therapeutics including novel, non-antibody-based therapeutics. AREAS COVERED: Today an array of biologics blocking either proinflammatory cytokines or lymphocyte activation/survival are available that enable a substantial improvement over conventional disease-modifying antirheumatic drugs (DMARDs). We review the engineering process of antibody-based biologics, their preclinical and clinical application, and current efforts to treat RA by interfering with B-cell function (notable targets covered are CD20, CD38, B-cell activating factor, transmembrane activator and calcium-modulating and cyclophilin interactor), with T-cell function (CD3, CD4, CD28), with bone erosion (RANKL), and with cytokines or growth factors (tumor necrosis factor, interleukin-1 [IL-1], IL-6, IL-17, VEGF). Future treatment choices might encompass the blockade or modulation of danger-associated molecular patterns such as HMGB1, pattern recognition receptors, messenger RNAs or noncoding RNAs, histone acetylation, and inflammasome components. EXPERT OPINION: Although current therapies can reduce the signs and symptoms of RA for many patients, the quest for a cure (or a more complete blockade of the structural damage) in RA is still ongoing and will need treatment approaches, which are not exclusively confined to blocking a particular cytokine, receptor, or autoreactive B or T cell involved in disease progression. To this end exciting treatment alternatives and drug targets are on the horizon that may become available to patients in the future.
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Anticuerpos Monoclonales/uso terapéutico , Artritis Reumatoide/terapia , Productos Biológicos/uso terapéutico , Ensayos Clínicos como Asunto , Evaluación Preclínica de Medicamentos , Humanos , Ingeniería de ProteínasRESUMEN
Fas, a TNF family receptor, is activated by the membrane protein Fas ligand expressed on various immune cells. Fas signaling triggers apoptosis and induces inflammatory cytokine production. Among the Fas-induced cytokines, the IL-1ß family cytokines require proteolysis to gain biological activity. Inflammasomes, which respond to pathogens and danger signals, cleave IL-1ß cytokines via caspase-1. However, the mechanisms by which Fas regulates IL-1ß activation remain unresolved. In this article, we demonstrate that macrophages exposed to TLR ligands upregulate Fas, which renders them responsive to receptor engagement by Fas ligand. Fas signaling activates caspase-8 in macrophages and dendritic cells, leading to the maturation of IL-1ß and IL-18 independently of inflammasomes or RIP3. Hence, Fas controls a novel noncanonical IL-1ß activation pathway in myeloid cells, which could play an essential role in inflammatory processes, tumor surveillance, and control of infectious diseases.
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Caspasa 8/fisiología , Interleucina-18/biosíntesis , Interleucina-1beta/biosíntesis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/fisiología , Receptor fas/fisiología , Animales , Caspasa 8/genética , Caspasa 8/metabolismo , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Activación Enzimática/inmunología , Proteína de Dominio de Muerte Asociada a Fas/deficiencia , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/fisiología , Inflamasomas/metabolismo , Inflamasomas/fisiología , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
ICOS is expressed on activated T cells and particularly on CXCR5+ follicular Th cells in germinal centers (GC). Its deletion leads to a profound deficiency in memory B cell formation and switched Ab response in humans. Here, we show that in ICOS-deficient patients the generation of GCs is severely disturbed, and the numbers of circulating CXCR5+CD45RO+ memory CD4 T cells are significantly reduced, indicating an essential role of ICOS in the differentiation of CXCR5+CD4 T cells. The GC-specific CD57+CXCR5+ subpopulation is virtually absent. In ICOS-/- mice, the decrease of circulating CXCR5+CD4 T cells reflects the reduction of CXCR5+ follicular Th cells in lymph nodes and spleen. Therefore, in concurrence with the absence of CXCR5+ T cells in the blood of CD40L-deficient patients, these data support the hypothesis that circulating CD57+CXCR5+ T cells are GC derived and thus may serve as a surrogate marker for the presence of functional GCs in humans.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD4-Positivos/inmunología , Centro Germinal/citología , Receptores de Citocinas/metabolismo , Subgrupos de Linfocitos T/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos B/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Centro Germinal/inmunología , Humanos , Memoria Inmunológica , Proteína Coestimuladora de Linfocitos T Inducibles , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores CXCR5 , Receptores de Quimiocina , Receptores de Citocinas/inmunologíaRESUMEN
The homozygous deletion of the inducible costimulator (ICOS), an activation-induced member of the CD28 family on T cells, causes an antibody deficiency syndrome in affected humans. The identification of a total of 9 ICOS-deficient patients revealed that this monogenic disease comprises the full clinical phenotype described for common variable immunodeficiency (CVID), including recurrent bacterial infections, adult as well as childhood onset, splenomegaly, autoimmune phenomena (autoimmune neutropenia), intestinal lymphoid hyperplasia, and malignancy (carcinoma of the vulva). All patients exhibited a profound hypogammaglobulinemia and a disturbed B-cell homeostasis. The severe reduction of class-switched memory B cells resulted from poor germinal center formation in the absence of ICOS. The additional decrease of naive B cells was associated with a partial inhibition of the early B-cell development at the pre-B-I stage. T-cell homeostasis seemed not to be affected, but low IL-10 production by ICOS-deficient T cells may contribute to the disturbed germinal center reaction. Human ICOS deficiency is indistinguishable from CVID and thus serves as a monogenic model for this complex syndrome.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/genética , Inmunodeficiencia Variable Común/genética , Inmunodeficiencia Variable Común/patología , Adulto , Agammaglobulinemia/genética , Agammaglobulinemia/inmunología , Agammaglobulinemia/patología , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/patología , Carcinoma/genética , Carcinoma/inmunología , Carcinoma/patología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Preescolar , Inmunodeficiencia Variable Común/inmunología , Femenino , Centro Germinal/inmunología , Centro Germinal/patología , Homocigoto , Humanos , Memoria Inmunológica/genética , Memoria Inmunológica/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles , Interleucina-10/inmunología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Modelos Inmunológicos , Neutropenia/genética , Neutropenia/inmunología , Neutropenia/patología , Linaje , Linfocitos T/inmunología , Linfocitos T/patología , Neoplasias de la Vulva/genética , Neoplasias de la Vulva/inmunología , Neoplasias de la Vulva/patologíaRESUMEN
Acute lymphoblastic leukaemia (ALL) is the commonest form of childhood malignancy, and most cases arise from B-cell clones arrested at the pre-B-cell stage of differentiation. The molecular events that arrest pre-B-cell differentiation in the leukaemic pre-B cells have not been well characterized. Here we show that the differentiation regulator SLP-65 (an adaptor protein also called BLNK or BASH) inhibits pre-B-cell leukaemia in mice. Reconstitution of SLP-65 expression in a SLP-65-/- pre-B-cell line led to enhanced differentiation in vitro and prevented the development of pre-B-cell leukaemia in immune-deficient mice. Tyrosine 96 of SLP-65 was required for this activity. The murine SLP-65-/- pre-B-cell leukaemia resembles human childhood pre-B ALL. Indeed, 16 of the 34 childhood pre-B ALL samples that were tested showed a complete loss or drastic reduction of SLP-65 expression. This loss is probably due to the incorporation of alternative exons into SLP-65 transcripts, leading to premature stop codons. Thus, the somatic loss of SLP-65 and the accompanying block in pre-B-cell differentiation might be one of the primary causes of childhood pre-B ALL.