RESUMEN
A function-impairing mutation (feeble) or genomic deletion of SLC15A4 abolishes responses of nucleic acidsensing endosomal toll-like receptors (TLRs) and significantly reduces disease in mouse models of lupus. Here, we demonstrate disease reduction in homozygous and even heterozygous Slc15a4 feeble mutant BXSB male mice with a Tlr7 gene duplication. In contrast to SLC15A4, a function-impairing mutation of SLC15A3 did not diminish type I interferon (IFN-I) production by TLR-activated plasmacytoid dendritic cells (pDCs), indicating divergence of function between these homologous SLC15 family members. Trafficking to endolysosomes and function of SLC15A4 were dependent on the Adaptor protein 3 (AP-3) complex. Importantly, SLC15A4 was required for trafficking and colocalization of nucleic acidsensing TLRs and their ligands to endolysosomes and the formation of the LAMP2+VAMP3+ hybrid compartment in which IFN-I production is initiated. Collectively, these findings define mechanistic processes by which SLC15A4 controls endosomal TLR function and suggest that pharmacologic intervention to curtail the function of this transporter may be a means to treat lupus and other endosomal TLR-dependent diseases.
Asunto(s)
Ácidos Nucleicos , Animales , Endosomas/metabolismo , Ligandos , Lisosomas/metabolismo , Proteínas de Transporte de Membrana/genética , Ratones , Receptores Toll-Like/metabolismoRESUMEN
BACKGROUND: Dysregulation of airway smooth muscle cells (ASM) is central to the severity of asthma. Which molecules dominantly control ASM in asthma is unclear. High levels of the cytokine LIGHT (aka TNFSF14) have been linked to asthma severity and lower baseline predicted FEV1 percentage, implying that signals through its receptors might directly control ASM dysfunction. OBJECTIVE: Our study sought to determine whether signaling via lymphotoxin beta receptor (LTßR) or herpesvirus entry mediator from LIGHT dominantly drives ASM hyperreactivity induced by allergen. METHODS: Conditional knockout mice deficient for LTßR or herpesvirus entry mediator in smooth muscle cells were used to determine their role in ASM deregulation and airway hyperresponsiveness (AHR) in vivo. Human ASM were used to study signals induced by LTßR. RESULTS: LTßR was strongly expressed in ASM from normal and asthmatic subjects compared to several other receptors implicated in smooth muscle deregulation. Correspondingly, conditional deletion of LTßR only in smooth muscle cells in smMHCCreLTßRfl/fl mice minimized changes in their numbers and mass as well as AHR induced by house dust mite allergen in a model of severe asthma. Intratracheal LIGHT administration independently induced ASM hypertrophy and AHR in vivo dependent on direct LTßR signals to ASM. LIGHT promoted contractility, hypertrophy, and hyperplasia of human ASM in vitro. Distinguishing LTßR from the receptors for IL-13, TNF, and IL-17, which have also been implicated in smooth muscle dysregulation, LIGHT promoted NF-κB-inducing kinase-dependent noncanonical nuclear factor kappa-light-chain enhancer of activated B cells in ASM in vitro, leading to sustained accumulation of F-actin, phosphorylation of myosin light chain kinase, and contractile activity. CONCLUSIONS: LTßR signals directly and dominantly drive airway smooth muscle hyperresponsiveness relevant for pathogenesis of airway remodeling in severe asthma.
Asunto(s)
Asma , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Humanos , Ratones , Animales , Receptor beta de Linfotoxina/genética , Asma/patología , Músculo Liso , Miocitos del Músculo Liso/patología , Ratones Noqueados , Alérgenos , Pulmón/patologíaRESUMEN
Double-positive CD4+CD8αß+ (DP) cells are thought to reside as T cell progenitors exclusively within the thymus. We recently discovered an unexpected CD4+ and CD8αß+ immune cell population in healthy and atherosclerotic mice by single-cell RNA sequencing. Transcriptomically, these cells resembled thymic DPs. Flow cytometry and three-dimensional whole-mount imaging confirmed DPs in thymus, mediastinal adipose tissue, and aortic adventitia, but nowhere else. Deep transcriptional profiling revealed differences between DP cells isolated from the three locations. All DPs were dependent on RAG2 expression and the presence of the thymus. Mediastinal adipose tissue DPs resided in close vicinity to invariant NKT cells, which they could activate in vitro. Thymus transplantation failed to reconstitute extrathymic DPs, and frequencies of extrathymic DPs were unaltered by pharmacologic inhibition of S1P1, suggesting that their migration may be locally confined. Our results define two new, transcriptionally distinct subsets of extrathymic DPs that may play a role in aortic vascular homeostasis.
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Tejido Adiposo/inmunología , Aorta Torácica/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Timo/inmunología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/inmunologíaRESUMEN
Atherosclerosis is initiated by subendothelial retention of lipoproteins and cholesterol, which triggers a non-resolving inflammatory process that over time leads to plaque progression in the artery wall. Myeloid cells and in particular macrophages are the primary drivers of the inflammatory response and plaque formation. Several immune cells including macrophages, T cells and B cells secrete the anti-inflammatory cytokine IL-10, known to be essential for the atherosclerosis protection. The cellular source of IL-10 in natural atherosclerosis progression is unknown. This study aimed to determine the main IL10-producing cell type in atherosclerosis. To do so, we crossed VertX mice, in which IRES-green fluorescent protein (eGFP) was placed downstream of exon 5 of the Il10 gene, with atherosclerosis-prone Apoe-/- mice. We found that myeloid cells express high levels of IL-10 in VertX Apoe-/- mice in both chow and western-diet fed mice. By single cell RNA sequencing and flow cytometry analysis, we identified resident and inflammatory macrophages in atherosclerotic plaques as the main IL-10 producers. To address whether IL-10 secreted by myeloid cells is essential for the protection, we utilized LyzMCre+Il10fl/fl mice crossed into the Apoe-/- background and confirmed that macrophages were unable to secrete IL-10. Chow and western diet-fed LyzMCre+Il10fl/fl Apoe-/- mice developed significantly larger atherosclerotic plaques as measured by en face morphometry than LyzMCre-Il10 fl/flApoe-/-. Flow cytometry and cytokine measurements suggest that the depletion of IL-10 in myeloid cells increases Th17 cells with elevated CCL2, and TNFα in blood plasma. We conclude that macrophage-derived IL-10 is critical for limiting atherosclerosis in mice.
Asunto(s)
Aterosclerosis , Interleucina-10 , Placa Aterosclerótica , Animales , Ratones , Aterosclerosis/genética , Aterosclerosis/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Interleucina-10/genética , Interleucina-10/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Placa Aterosclerótica/metabolismo , Ratones Noqueados para ApoERESUMEN
AIMS/HYPOTHESIS: We aimed to characterise and quantify the expression of HLA class II (HLA-II) in human pancreatic tissue sections and to analyse its induction in human islets. METHODS: We immunostained human pancreatic tissue sections from non-diabetic (n = 5), autoantibody positive (Aab+; n = 5), and type 1 diabetic (n = 5) donors, obtained from the Network of Pancreatic Organ Donors (nPOD), with HLA-II, CD68 and insulin. Each tissue section was acquired with a widefield slide scanner and then analysed with QuPath software. In total, we analysed 7415 islets that contained 338,480 cells. Widefield microscopy was further complemented by high resolution imaging of 301 randomly selected islets, acquired using a Zeiss laser scanning confocal (LSM880) to confirm our findings. Selected beta cells were acquired in enhanced resolution using LSM880 with an Airyscan detector. Further, we cultured healthy isolated human islets and reaggregated human islet microtissues with varying concentrations of proinflammatory cytokines (IFN-γ, TNF-α and IL-1ß). After proinflammatory cytokine culture, islet function was measured by glucose-stimulated insulin secretion, and HLA-I and HLA-II expression was subsequently evaluated with immunostaining or RNA sequencing. RESULTS: Insulin-containing islets (ICIs) of donors with type 1 diabetes had a higher percentage of HLA-II positive area (24.31%) compared with type 1 diabetic insulin-deficient islets (IDIs, 0.67%), non-diabetic (3.80%), and Aab+ (2.31%) donors. In ICIs of type 1 diabetic donors, 45.89% of the total insulin signal co-localised with HLA-II, and 27.65% of the islet beta cells expressed both HLA-II and insulin, while in non-diabetic and Aab+ donors 0.96% and 0.59% of the islet beta cells, respectively, expressed both markers. In the beta cells of donors with type 1 diabetes, HLA-II was mostly present in the cell cytoplasm, co-localising with insulin. In the experiments with human isolated islets and reaggregated human islets, we observed changes in insulin secretion upon stimulation with proinflammatory cytokines, as well as higher expression of HLA-II and HLA-I when compared with controls cultured with media, and an upregulation of HLA-I and HLA-II RNA transcripts. CONCLUSIONS/INTERPRETATION: After a long-standing controversy, we provide definitive evidence that HLA-II can be expressed by pancreatic beta cells from patients with type 1 diabetes. Furthermore, this upregulation can be induced in vitro in healthy isolated human islets or reaggregated human islets by treatment with proinflammatory cytokines. Our findings support a role for HLA-II in type 1 diabetes pathogenesis since HLA-II expressing beta cells can potentially become a direct target of autoreactive CD4+ lymphocytes.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Células Secretoras de Insulina/metabolismo , Adolescente , Adulto , Autoanticuerpos/sangre , Células Cultivadas , Niño , Femenino , Glucosa/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Donantes de Tejidos , Regulación hacia Arriba , Adulto JovenRESUMEN
Human leukocyte antigens of class-I (HLA-I) molecules are hyper-expressed in insulin-containing islets (ICI) of type 1 diabetic (T1D) donors. This study investigated the HLA-I expression in autoantibody positive (AAB+) donors and defined its intra-islet and intracellular localization as well as proximity to infiltrating CD8 T cells with high-resolution confocal microscopy. We found HLA-I hyper-expression had already occurred prior to clinical diagnosis of T1D in islets of AAB+ donors. Interestingly, throughout all stages of disease, HLA-I was mostly expressed by alpha cells. Hyper-expression in AAB+ and T1D donors was associated with intra-cellular accumulation in the Golgi. Proximity analysis showed a moderate but significant correlation between HLA-I and infiltrating CD8 T cells only in ICI of T1D donors, but not in AAB+ donors. These observations not only demonstrate a very early, islet-intrinsic immune-independent increase of HLA-I during diabetes pathogenesis, but also point towards a role for alpha cells in T1D.
Asunto(s)
Expresión Génica , Células Secretoras de Glucagón/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Estado Prediabético/etiología , Estado Prediabético/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Autoinmunidad , Biomarcadores , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/metabolismo , Susceptibilidad a Enfermedades/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Transporte de Proteínas , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
PURPOSE: IL-17 is an important effector cytokine driving immune-mediated destruction in autoimmune diseases such as psoriasis. Blockade of the IL-17 pathway after the initiation of insulitis was effective in delaying or preventing the onset of type 1 diabetes (T1D) in rodent models. Expression of IL-17 transcripts in islets from a donor with recent-onset T1D has been reported, however, studies regarding IL-17 protein expression are lacking. We aimed to study whether IL-17 is being expressed in the islets of diabetic donors. METHODS: We stained human pancreatic tissues from non-diabetic (n = 5), auto-antibody positive (aab+) (n = 5), T1D (n = 6) and T2D (n = 5) donors for IL-17, Insulin, and Glucagon, and for CD45 in selected cases. High resolution images were acquired with Zeiss laser scanning confocal microscope LSM780 and analyzed with Zen blue 2.3 software. Cases stained for CD45 were also acquired with widefield slide scanner and analyzed with QuPath software. RESULTS: We observed a clear cytoplasmic staining for IL-17 in insulin-containing islets of donors with T1D and T2D, accounting for an average of 7.8 ± 8.4% and 14.9 ± 16.8% of total islet area, respectively. Both beta and alpha cells were sources of IL-17, but CD45+ cells were not a major source of IL-17 in those donors. Expression of IL-17 was reduced in islets of non-diabetic donors, aab+ donors and in insulin-deficient islets of donors with T1D. CONCLUSION: Our finding that IL-17 is expressed in islets of donors with T1D or T2D is quite intriguing and warrants further mechanistic studies in human islets to understand the role of IL-17 in the context of metabolic and immune stress in beta cells.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/inmunología , Células Secretoras de Glucagón/inmunología , Células Secretoras de Insulina/inmunología , Interleucina-17/análisis , Donantes de Tejidos , Adolescente , Adulto , Preescolar , Femenino , Humanos , Masculino , Adulto JovenRESUMEN
Human herpesvirus-6 (HHV-6) is a ubiquitous pathogen associated with nervous and endocrine autoimmune disorders. The aim of this study was to investigate the presence of HHV-6 in pancreatic tissue sections from non-diabetic, auto-antibody positive (AAB+), and donors with type 1 diabetes (T1D) and explore whether there is any association between HHV-6 and MHC class I hyperexpression and CD8 T cell infiltration. HHV-6 DNA was detected by PCR and its protein was examined by indirect immunofluorescence assay followed by imaging using high-resolution confocal microscopy. Viral DNA (U67) was found in most pancreata of non-diabetic (3 out of 4), AAB+ (3 out of 5) and T1D donors (6 out of 7). Interestingly, HHV-6 glycoprotein B (gB) was more expressed in islets and exocrine pancreas of donors with T1D. However, gB expression was not directly associated with other pathologies. Out of 20 islets with high gB expression, only 3 islets (15%) showed MHC class I hyperexpression. Furthermore, no correlation was found between gB expression and CD8 T cell infiltration on a per-islet basis in any of the groups. Our observations indicate that HHV-6 DNA and protein are present in the pancreas of non-diabetic subjects but gB expression is higher in the pancreas of donors with T1D. The possible role of HHV-6 as a contributory factor for T1D should therefore be further investigated.
Asunto(s)
Diabetes Mellitus Tipo 1/etiología , Susceptibilidad a Enfermedades , Herpesvirus Humano 6 , Páncreas/virología , Infecciones por Roseolovirus/complicaciones , Autoinmunidad , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/metabolismo , Expresión Génica , Herpesvirus Humano 6/genética , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/virología , Páncreas/inmunología , Páncreas/metabolismo , Infecciones por Roseolovirus/virologíaRESUMEN
The interleukin-1 system (IL-1) is a prominent pro-inflammatory pathway responsible for the initiation and regulation of immune responses. Human genetic and preclinical studies suggest a critical role for IL-1ß signaling in ethanol drinking and dependence, but little is known about the effects of chronic ethanol on the IL-1 system in addiction-related brain regions such as the central amygdala (CeA). In this study, we generated naïve, non-dependent (Non-Dep) and dependent (Dep) male mice using a paradigm of chronic-intermittent ethanol vapor exposure interspersed with two-bottle choice to examine 1) the expression of IL-1ß, 2) the role of the IL-1 system on GABAergic transmission, and 3) the potential interaction with the acute effects of ethanol in the CeA. Immunohistochemistry with confocal microscopy was used to assess expression of IL-1ß in microglia and neurons in the CeA, and whole-cell patch clamp recordings were obtained from CeA neurons to measure the effects of IL-1ß (50â¯ng/ml) or the endogenous IL-1 receptor antagonist (IL-1ra; 100â¯ng/ml) on action potential-dependent spontaneous inhibitory postsynaptic currents (sIPSCs). Overall, we found that IL-1ß expression is significantly increased in microglia and neurons of Dep compared to Non-Dep and naïve mice, IL-1ß and IL-1ra bi-directionally modulate GABA transmission through both pre- and postsynaptic mechanisms in all three groups, and IL-1ß and IL-1ra do not alter the facilitation of GABA release induced by acute ethanol. These data suggest that while ethanol dependence induces a neuroimmune response in the CeA, as indicated by increased IL-1ß expression, this does not significantly alter the neuromodulatory role of IL-1ß on synaptic transmission.
Asunto(s)
Núcleo Amigdalino Central/efectos de los fármacos , Etanol/administración & dosificación , Interleucina-1beta/biosíntesis , Ácido gamma-Aminobutírico/metabolismo , Animales , Núcleo Amigdalino Central/metabolismo , Depresores del Sistema Nervioso Central/farmacología , Etanol/efectos adversos , Etanol/toxicidad , Neuronas GABAérgicas/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Receptores de GABA-A/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
Blunting immunopathology without abolishing host defense is the foundation for safe and effective modulation of infectious and autoimmune diseases. Sphingosine 1-phosphate receptor 1 (S1PR1) agonists are effective in treating infectious and multiple autoimmune pathologies; however, mechanisms underlying their clinical efficacy are yet to be fully elucidated. Here, we uncover an unexpected mechanism of convergence between S1PR1 and interferon alpha receptor 1 (IFNAR1) signaling pathways. Activation of S1PR1 signaling by pharmacological tools or endogenous ligand sphingosine-1 phosphate (S1P) inhibits type 1 IFN responses that exacerbate numerous pathogenic conditions. Mechanistically, S1PR1 selectively suppresses the type I IFN autoamplification loop in plasmacytoid dendritic cells (pDCs), a specialized DC subset, for robust type I IFN release. S1PR1 agonist suppression is pertussis toxin-resistant, but inhibited by an S1PR1 C-terminal-derived transactivating transcriptional activator (Tat)-fusion peptide that blocks receptor internalization. S1PR1 agonist treatment accelerates turnover of IFNAR1, suppresses signal transducer and activator of transcription 1 (STAT1) phosphorylation, and down-modulates total STAT1 levels, thereby inactivating the autoamplification loop. Inhibition of S1P-S1PR1 signaling in vivo using the selective antagonist Ex26 significantly elevates IFN-α production in response to CpG-A. Thus, multiple lines of evidence demonstrate that S1PR1 signaling sets the sensitivity of pDC amplification of IFN responses, thereby blunting pathogenic immune responses. These data illustrate a lipid G-protein coupled receptor (GPCR)-IFNAR1 regulatory loop that balances effective and detrimental immune responses and elevated endogenous S1PR1 signaling. This mechanism will likely be advantageous in individuals subject to a range of inflammatory conditions.
Asunto(s)
Células Dendríticas/metabolismo , Interferón-alfa/metabolismo , Receptor de Interferón alfa y beta/metabolismo , Receptores de Lisoesfingolípidos/fisiología , Animales , Ratones , Ratones Noqueados , Proteolisis , Receptor de Interferón alfa y beta/genéticaRESUMEN
Deleterious mutations in the serine lipase DDHD2 are a causative basis of complex hereditary spastic paraplegia (HSP, subtype SPG54) in humans. We recently found that DDHD2 is a principal triglyceride hydrolase in the central nervous system (CNS) and that genetic deletion of this enzyme in mice leads to ectopic lipid droplet (LD) accumulation in neurons throughout the brain. Nonetheless, how HSP-related mutations in DDHD2 relate to triglyceride metabolism and LD formation remains poorly understood. Here, we have characterized a set of HSP-related mutations in DDHD2 and found that they disrupt triglyceride hydrolase activity in vitro and impair the capacity of DDHD2 to protect cells from LD accumulation following exposure to free fatty acid, an outcome that was also observed with a DDHD2-selective inhibitor. We furthermore isolated and characterized LDs from brain tissue of DDHD2-/- mice, revealing that they contain both established LD-associated proteins identified previously in other organs and CNS-enriched proteins, including several proteins with genetic links to human neurological disease. These data, taken together, indicate that the genetic inactivation of DDHD2, as caused by HSP-associated mutations, substantially perturbs lipid homeostasis and the formation and content of LDs, underscoring the importance of triglyceride metabolism for normal CNS function and the key role that DDHD2 plays in this process.
Asunto(s)
Proteínas Asociadas a Gotas Lipídicas/análisis , Gotas Lipídicas/química , Proteínas del Tejido Nervioso/fisiología , Fosfolipasas/fisiología , Animales , Química Encefálica , Dominio Catalítico/efectos de los fármacos , Ácidos Grasos/metabolismo , Homeostasis , Humanos , Ratones , Ratones Noqueados , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Especificidad de Órganos , Fosfolipasas/antagonistas & inhibidores , Fosfolipasas/genética , Fosfolipasas A1/deficiencia , Proteínas Recombinantes/metabolismo , Paraplejía Espástica Hereditaria/genética , Triglicéridos/metabolismoRESUMEN
The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in Ctns-/- cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Cistinosis/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Sustitución de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Cistinosis/genética , Cistinosis/patología , Activadores de Enzimas/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Lisosomas/genética , Ratones , Ratones Noqueados , Mutación Puntual , Transporte de Proteínas/genética , Proteínas de Unión al GTP rab/biosíntesis , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.
Asunto(s)
Endocitosis , Endosomas/metabolismo , Exocitosis , Proteínas de la Membrana/metabolismo , Neutrófilos/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Sustitución de Aminoácidos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/citología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Organismos Libres de Patógenos Específicos , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/genéticaRESUMEN
The molecular pharmacology of the G protein-coupled receptors for sphingosine 1-phosphate (S1P) provides important insight into established and new therapeutic targets. A new, potent bitopic S1P3 antagonist, SPM-354, with in vivo activity, has been used, together with S1P3-knockin and S1P3-knockout mice to define the spatial and functional properties of S1P3 in regulating cardiac conduction. We show that S1P3 is a key direct regulator of cardiac rhythm both in vivo and in isolated perfused hearts. 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol in vivo and S1P in isolated hearts induced a spectrum of cardiac effects, ranging from sinus bradycardia to complete heart block, as measured by a surface electrocardiogram in anesthetized mice and in volume-conducted Langendorff preparations. The agonist effects on complete heart block are absent in S1P3-knockout mice and are reversed in wild-type mice with SPM-354, as characterized and described here. Homologous knockin of S1P3-mCherry is fully functional pharmacologically and is strongly expressed by immunohistochemistry confocal microscopy in Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4)-positive atrioventricular node and His-Purkinje fibers, with relative less expression in the HCN4-positive sinoatrial node. In Langendorff studies, at constant pressure, SPM-354 restored sinus rhythm in S1P-induced complete heart block and fully reversed S1P-mediated bradycardia. S1P3 distribution and function in the mouse ventricular cardiac conduction system suggest a direct mechanism for heart block risk that should be further studied in humans. A richer understanding of receptor and ligand usage in the pacemaker cells of the cardiac system is likely to be useful in understanding ventricular conduction in health, disease, and pharmacology.
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Bloqueo Cardíaco/tratamiento farmacológico , Bloqueo Cardíaco/genética , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/genética , Animales , Cardiotónicos/farmacología , Cardiotónicos/uso terapéutico , Bloqueo Cardíaco/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Esfingosina-1-FosfatoRESUMEN
OBJECTIVE: In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) ß-target gene, PTPRK, promotes RA FLS invasiveness. METHODS: Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The RPTPκ substrate was identified by pull-down assays. RESULTS: PTPRK expression was higher in FLS from patients with RA versus patients with osteoarthritis, resulting from increased TGFB1 expression in RA FLS. RPTPκ knockdown impaired RA FLS spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumour necrosis factor and interleukin 1 stimulation. Furthermore, RPTPκ deficiency impaired the in vivo invasiveness of RA FLS. Molecular analysis revealed that RPTPκ promoted RA FLS migration by dephosphorylation of the inhibitory residue Y527 of SRC. CONCLUSIONS: By regulating phosphorylation of SRC, RPTPκ promotes the pathogenic action of RA FLS, mediating cross-activation of growth factor and inflammatory cytokine signalling by TGFß in RA FLS.
Asunto(s)
Artritis Reumatoide/patología , Fibroblastos/patología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/fisiología , Membrana Sinovial/patología , Factor de Crecimiento Transformador beta1/fisiología , Animales , Artritis Reumatoide/metabolismo , Movimiento Celular/genética , Movimiento Celular/fisiología , Fibroblastos/metabolismo , Fibroblastos/fisiología , Fibroblastos/trasplante , Regulación Enzimológica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Ratones Desnudos , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , ARN Mensajero/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Membrana Sinovial/metabolismo , Membrana Sinovial/trasplante , Regulación hacia ArribaRESUMEN
OBJECTIVE: Aging-associated changes in articular cartilage represent a main risk factor for osteoarthritis (OA). Autophagy is an essential cellular homeostasis mechanism. Aging-associated or experimentally induced defects in autophagy contribute to organismal- and tissue-specific aging, while enhancement of autophagy may protect against certain aging-related pathologies such as OA. The objective of this study was to determine whether glucosamine can activate autophagy. METHODS: Chondrocytes from normal human articular cartilage were treated with glucosamine (0.1- 10 mM). Autophagy activation and phosphorylation levels of Akt, FoxO3, and ribosomal protein S6 were determined by Western blotting. Autophagosome formation was analyzed by confocal microscopy. Reporter mice systemically expressing green fluorescent protein (GFP) fused to light chain 3 (LC3) (GFP-LC3-transgenic mice) were used to assess changes in autophagy in response to starvation and glucosamine treatment. RESULTS: Glucosamine treatment of chondrocytes activated autophagy, as indicated by increased LC3-II levels, formation of LC3 puncta, and increased LC3 turnover. This was associated with glucosamine-mediated inhibition of the Akt/FoxO3/mammalian target of rapamycin pathway. Administration of glucosamine to GFP-LC3-transgenic mice markedly activated autophagy in articular cartilage. CONCLUSION: Glucosamine modulates molecular targets of the autophagy pathway in vitro and in vivo, and the enhancement of autophagy is mainly dependent on the Akt/FoxO/mTOR pathway. These findings suggest that glucosamine is an effective autophagy activator and should motivate future studies on the efficacy of glucosamine in modifying aging-related cellular changes and supporting joint health.
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Autofagia/efectos de los fármacos , Cartílago Articular/citología , Condrocitos/efectos de los fármacos , Glucosamina/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Condrocitos/fisiología , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/efectos de los fármacos , Factores de Transcripción Forkhead/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Transgénicos , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína S6 Ribosómica/efectos de los fármacos , Proteína S6 Ribosómica/metabolismo , Serina-Treonina Quinasas TOR/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
OBJECTIVE: The fibroblast-like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage-degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS. METHODS: A comparative screening of the expression of the PTPome in FLS from patients with RA and patients with osteoarthritis (OA) was conducted. The functional effect on RA FLS of SH2 domain-containing phosphatase 2 (SHP-2), a PTP that was up-regulated in RA, was then analyzed by knockdown using cell-permeable antisense oligonucleotides. RESULTS: PTPN11 was overexpressed in RA FLS compared to OA FLS. Knockdown of PTPN11, which encodes SHP-2, reduced the invasion, migration, adhesion, spreading, and survival of RA FLS. Additionally, signaling in response to growth factors and inflammatory cytokines was impaired by SHP-2 knockdown. RA FLS that were deficient in SHP-2 exhibited decreased activation of focal adhesion kinase and mitogen-activated protein kinases. CONCLUSION: These findings indicate that SHP-2 has a novel role in mediating human FLS function and suggest that it promotes the invasiveness and survival of RA FLS. Further investigation may reveal SHP-2 to be a candidate therapeutic target for RA.
Asunto(s)
Artritis Reumatoide/enzimología , Fibroblastos/enzimología , Osteoartritis/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Membrana Sinovial/enzimología , Artritis Reumatoide/genética , Línea Celular , Movimiento Celular , Fibroblastos/patología , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Oligonucleótidos Antisentido/farmacología , Osteoartritis/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteínas Tirosina Fosfatasas/genética , Transducción de Señal , Membrana Sinovial/patología , Regulación hacia ArribaRESUMEN
Endosomal Toll-like receptors (eTLRs) are essential for the sensing of non-self through RNA and DNA detection. Here, using spatiotemporal analysis of vesicular dynamics, super-resolution microscopy studies, and functional assays, we show that endomembrane defects associated with the deficiency of the small GTPase Rab27a cause delayed eTLR ligand recognition, defective early signaling, and impaired cytokine secretion. Rab27a-deficient neutrophils show retention of eTLRs in amphisomes and impaired ligand internalization. Extracellular signal-regulated kinase (ERK) signaling and ß2-integrin upregulation, early responses to TLR7 and TLR9 ligands, are defective in Rab27a deficiency. CpG-stimulated Rab27a-deficient neutrophils present increased tumor necrosis factor alpha (TNF-α) secretion and decreased secretion of a selected group of mediators, including interleukin (IL)-10. In vivo, CpG-challenged Rab27a-null mice show decreased production of type I interferons (IFNs) and IFN-γ, and the IFN-α secretion defect is confirmed in Rab27a-null plasmacytoid dendritic cells. Our findings have significant implications for immunodeficiency, inflammation, and CpG adjuvant vaccination.
RESUMEN
CD8+ T cells drive anti-cancer immunity in response to antigen-presenting cells such as dendritic cells and subpopulations of monocytes and macrophages. While CD14+ classical monocytes modulate CD8+ T cell responses, the contributions of CD16+ nonclassical monocytes to this process remain unclear. Herein we explored the role of nonclassical monocytes in CD8+ T cell activation by utilizing E2-deficient (E2-/-) mice that lack nonclassical monocytes. During early metastatic seeding, modeled by B16F10-OVA cancer cells injected into E2-/- mice, we noted lower CD8+ effector memory and effector T cell frequencies within the lungs as well as in lung-draining mediastinal lymph nodes in the E2-/- mice. Analysis of the myeloid compartment revealed that these changes were associated with depletion of MHC-IIloLy6Clo nonclassical monocytes within these tissues, with little change in other monocyte or macrophage populations. Additionally, nonclassical monocytes preferentially trafficked to primary tumor sites in the lungs, rather than to the lung-draining lymph nodes, and did not cross-present antigen to CD8+ T cells. Examination of the lung microenvironment in E2-/- mice revealed reduced CCL21 expression in endothelial cells, which is chemokine involved in T cell trafficking. Our results highlight the previously unappreciated importance of nonclassical monocytes in shaping the tumor microenvironment via CCL21 production and CD8+ T cell recruitment.
Asunto(s)
Monocitos , Neoplasias , Ratones , Animales , Linfocitos T CD8-positivos , Células Endoteliales , Pulmón , Neoplasias/metabolismo , Microambiente TumoralRESUMEN
Receptor-type protein phosphatase α (RPTPα) promotes fibroblast-dependent arthritis and fibrosis, in part, by enhancing the activation of the kinase SRC. Synovial fibroblasts lining joint tissue mediate inflammation and tissue damage, and their infiltration into adjacent tissues promotes disease progression. RPTPα includes an ectodomain and two intracellular catalytic domains (D1 and D2) and, in cancer cells, undergoes inhibitory homodimerization, which is dependent on a D1 wedge motif. Through single-molecule localization and labeled molecule interaction microscopy of migrating synovial fibroblasts, we investigated the role of RPTPα dimerization in the activation of SRC, the migration of synovial fibroblasts, and joint damage in a mouse model of arthritis. RPTPα clustered with other RPTPα and with SRC molecules in the context of actin-rich structures. A known dimerization-impairing mutation in the wedge motif (P210L/P211L) and the deletion of the D2 domain reduced RPTPα-RPTPα clustering; however, it also unexpectedly reduced RPTPα-SRC association. The same mutations also reduced recruitment of RPTPα to actin-rich structures and inhibited SRC activation and cellular migration. An antibody against the RPTPα ectodomain that prevented the clustering of RPTPα also inhibited RPTPα-SRC association and SRC activation and attenuated fibroblast migration and joint damage in arthritic mice. A catalytically inactivating RPTPα-C469S mutation protected mice from arthritis and reduced SRC activation in synovial fibroblasts. We conclude that RPTPα clustering retains it to actin-rich structures to promote SRC-mediated fibroblast migration and can be modulated through the extracellular domain.