RESUMEN
BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and µMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Microbiota , Enfermedad del Hígado Graso no Alcohólico , Humanos , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Ratones Endogámicos C57BL , Hígado/patología , Fibrosis , Cirrosis Hepática/complicaciones , Ratones Transgénicos , Inmunoglobulina A/metabolismo , Inmunoglobulina A/farmacología , Modelos Animales de Enfermedad , Dieta Alta en Grasa/efectos adversosRESUMEN
The c-Jun N-terminal kinase (JNK) signaling pathway mediates adaptation to stress signals and has been associated with cell death, cell proliferation, and malignant transformation in the liver. However, up to now, its function was experimentally studied mainly in young mice. By generating mice with combined conditional ablation of Jnk1 and Jnk2 in liver parenchymal cells (LPCs) (JNK1/2LPC-KO mice; KO, knockout), we unraveled a function of the JNK pathway in the regulation of liver homeostasis during aging. Aging JNK1/2LPC-KO mice spontaneously developed large biliary cysts that originated from the biliary cell compartment. Mechanistically, we could show that cyst formation in livers of JNK1/2LPC-KO mice was dependent on receptor-interacting protein kinase 1 (RIPK1), a known regulator of cell survival, apoptosis, and necroptosis. In line with this, we showed that RIPK1 was overexpressed in the human cyst epithelium of a subset of patients with polycystic liver disease. Collectively, these data reveal a functional interaction between JNK signaling and RIPK1 in age-related progressive cyst development. Thus, they provide a functional linkage between stress adaptation and programmed cell death (PCD) in the maintenance of liver homeostasis during aging.
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Envejecimiento/metabolismo , Enfermedades de los Conductos Biliares/etiología , Enfermedades de los Conductos Biliares/metabolismo , Caspasa 8/metabolismo , Quistes/etiología , Quistes/metabolismo , Sistema de Señalización de MAP Quinasas , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Animales , Apoptosis , Biopsia , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Inmunohistoquímica , Inmunofenotipificación , Hepatopatías/etiología , Hepatopatías/metabolismo , Ratones , Proteína Quinasa 8 Activada por Mitógenos/deficiencia , NecroptosisRESUMEN
BACKGROUND & AIMS: Intestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice. METHODS: We generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces. RESULTS: Mcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development. CONCLUSION: The antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.
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Carcinoma/patología , Mucosa Intestinal/patología , Neoplasias Intestinales/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Animales , Apoptosis/genética , Apoptosis/inmunología , Carcinogénesis/genética , Carcinogénesis/inmunología , Carcinogénesis/patología , Carcinoma/diagnóstico , Carcinoma/genética , Modelos Animales de Enfermedad , Endoscopía , Células Epiteliales/patología , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/diagnóstico por imagen , Neoplasias Intestinales/diagnóstico , Neoplasias Intestinales/genética , Ratones , Ratones Transgénicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genéticaRESUMEN
Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.
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Inmunidad Mucosa/inmunología , Interleucina-18/inmunología , Mucosa Intestinal/inmunología , Animales , Citocinas/inmunología , Sistema Nervioso Entérico/inmunología , Sistema Nervioso Entérico/metabolismo , Células Epiteliales/inmunología , Femenino , Células Caliciformes/inmunología , Interleucina-18/biosíntesis , Mucosa Intestinal/metabolismo , Intestino Delgado/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/inmunología , Ratas , Ratas Sprague-Dawley , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Transducción de Señal/inmunologíaRESUMEN
Non-alcoholic fatty liver disease ranges from steatosis to non-alcoholic steatohepatitis (NASH), potentially progressing to cirrhosis and hepatocellular carcinoma (HCC). Here, we show that platelet number, platelet activation and platelet aggregation are increased in NASH but not in steatosis or insulin resistance. Antiplatelet therapy (APT; aspirin/clopidogrel, ticagrelor) but not nonsteroidal anti-inflammatory drug (NSAID) treatment with sulindac prevented NASH and subsequent HCC development. Intravital microscopy showed that liver colonization by platelets depended primarily on Kupffer cells at early and late stages of NASH, involving hyaluronan-CD44 binding. APT reduced intrahepatic platelet accumulation and the frequency of platelet-immune cell interaction, thereby limiting hepatic immune cell trafficking. Consequently, intrahepatic cytokine and chemokine release, macrovesicular steatosis and liver damage were attenuated. Platelet cargo, platelet adhesion and platelet activation but not platelet aggregation were identified as pivotal for NASH and subsequent hepatocarcinogenesis. In particular, platelet-derived GPIbα proved critical for development of NASH and subsequent HCC, independent of its reported cognate ligands vWF, P-selectin or Mac-1, offering a potential target against NASH.
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Plaquetas/metabolismo , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Animales , Plaquetas/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Citocinas/metabolismo , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Endotelio/efectos de los fármacos , Endotelio/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones Transgénicos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Recuento de PlaquetasRESUMEN
Recent studies suggest that clear cell renal cell carcinoma (ccRCC) possesses a rare population of cancer stem cells (CSCs) that might contribute to tumor heterogeneity, metastasis and therapeutic resistance. Nevertheless, their relevance for renal cancer is still unclear. In this study, we successfully isolated CSCs from established human ccRCC cell lines. CSCs displayed high expression of the chemokine IL-8 and its receptor CXCR1. While recombinant IL-8 significantly increased CSC number and properties in vitro, CXCR1 inhibition using an anti-CXCR1 antibody or repertaxin significantly reduced these features. After injection into immune-deficient mice, CSCs formed primary tumors that metastasized to the lung and liver. All xenografted tumors in mice expressed high levels of IL-8 and CXCR1. Furthermore, IL-8/CXCR1 expression significantly correlated with decreased overall survival in ccRCC patients. These results suggest that the IL-8/CXCR1 phenotype is associated with CSC-like properties in renal cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Carcinoma de Células Renales/genética , Interleucina-8/genética , Neoplasias Renales/genética , Células Madre Neoplásicas/patología , Receptores de Interleucina-8A/genética , Animales , Línea Celular Tumoral , Proliferación Celular/fisiología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Renales/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Receptores de Interleucina-8B/antagonistas & inhibidoresRESUMEN
Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.
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Carcinogénesis/metabolismo , Carcinogénesis/patología , Caspasa 8/metabolismo , Daño del ADN , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Animales , Apoptosis , Carcinoma Hepatocelular/patología , Proliferación Celular , Senescencia Celular , Enfermedad Crónica , Cruzamientos Genéticos , Reparación del ADN , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Femenino , Inestabilidad Genómica , Hepatectomía , Hepatocitos/patología , Histonas/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/metabolismo , Hígado/patología , Regeneración Hepática , Masculino , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Fosforilación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factores de RiesgoRESUMEN
Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.
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Glucagón/uso terapéutico , Enfermedades Metabólicas/tratamiento farmacológico , Triyodotironina/efectos de los fármacos , Animales , Aterosclerosis/tratamiento farmacológico , Peso Corporal/efectos de los fármacos , Huesos/efectos de los fármacos , Ingeniería Química/métodos , Colesterol/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Modelos Animales de Enfermedad , Combinación de Medicamentos , Sistemas de Liberación de Medicamentos , Sinergismo Farmacológico , Glucagón/efectos adversos , Glucagón/química , Glucagón/farmacología , Hiperglucemia/tratamiento farmacológico , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Terapia Molecular Dirigida , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Triyodotironina/efectos adversos , Triyodotironina/química , Triyodotironina/farmacologíaRESUMEN
Members of the Burkholderia cepacia complex (Bcc) cause chronic opportunistic lung infections in people with cystic fibrosis (CF), resulting in a gradual lung function decline and, ultimately, patient death. The Bcc is a complex of 20 species and is rarely eradicated once a patient is colonized; therefore, vaccination may represent a better therapeutic option. We developed a new proteomics approach to identify bacterial proteins that are involved in the attachment of Bcc bacteria to lung epithelial cells. Fourteen proteins were reproducibly identified by two-dimensional gel electrophoresis from four Bcc strains representative of two Bcc species: Burkholderia cenocepacia, the most virulent, and B. multivorans, the most frequently acquired. Seven proteins were identified in both species, but only two were common to all four strains, linocin and OmpW. Both proteins were selected based on previously reported data on these proteins in other species. Escherichia coli strains expressing recombinant linocin and OmpW showed enhanced attachment (4.2- and 3.9-fold) to lung cells compared to the control, confirming that both proteins are involved in host cell attachment. Immunoproteomic analysis using serum from Bcc-colonized CF patients confirmed that both proteins elicit potent humoral responses in vivo Mice immunized with either recombinant linocin or OmpW were protected from B. cenocepacia and B. multivorans challenge. Both antigens induced potent antigen-specific antibody responses and stimulated strong cytokine responses. In conclusion, our approach identified adhesins that induced excellent protection against two Bcc species and are promising vaccine candidates for a multisubunit vaccine. Furthermore, this study highlights the potential of our proteomics approach to identify potent antigens against other difficult pathogens.
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Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteriocinas/metabolismo , Infecciones por Burkholderia/prevención & control , Complejo Burkholderia cepacia/fisiología , Células Epiteliales/microbiología , Adhesinas Bacterianas/inmunología , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Bacteriocinas/inmunología , Infecciones por Burkholderia/inmunología , Fibrosis Quística/inmunología , Fibrosis Quística/microbiología , Modelos Animales de Enfermedad , Escherichia coli/genética , Escherichia coli/fisiología , Femenino , Expresión Génica , Humanos , Ratones Endogámicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resultado del TratamientoRESUMEN
The immune suppressive and anti-inflammatory capabilities of bone marrow-derived mesenchymal stromal cells (MSCs) represent an innovative new tool in regenerative medicine and immune regulation. The potent immune suppressive ability of MSC over T cells, dendritic cells, and natural killer cells has been extensively characterized, however, the effect of MSC on B cell function has not yet been clarified. In this study, the direct effect of MSC on peripheral blood B cell function is defined and the mechanism utilized by MSC in enhancing B cell survival in vitro identified. Human MSC supported the activation, proliferation, and survival of purified CD19(+) B cells through a cell contact-dependent mechanism. These effects were not mediated through B cell activating factor or notch signaling. However, cell contact between MSC and B cells resulted in increased production of vascular endothelial growth factor (VEGF) by MSC facilitating AKT phosphorylation within the B cell and inhibiting caspase 3-mediated apoptosis. Blocking studies demonstrated that this cell contact-dependent effect was not dependent on signaling through CXCR4-CXCL12 or through the epidermal growth factor receptor (EGFR). These results suggest that direct cell contact between MSC and B cells supports B cell viability and function, suggesting that MSC may not represent a suitable therapy for B cell-mediated disease.
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Apoptosis/inmunología , Linfocitos B/citología , Caspasa 3/metabolismo , Células Madre Mesenquimatosas/citología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Antígenos CD19/inmunología , Linfocitos B/inmunología , Células de la Médula Ósea/citología , Proliferación Celular/fisiología , Humanos , Células Asesinas Naturales/inmunología , Trasplante de Células Madre Mesenquimatosas , Linfocitos T/citología , Activación Transcripcional/inmunología , Regulación hacia ArribaRESUMEN
Mutations that result in loss of function of Nod2, an intracellular receptor for bacterial peptidoglycan, are associated with Crohn's disease. Here we found that the E3 ubiquitin ligase Pellino3 was an important mediator in the Nod2 signaling pathway. Pellino3-deficient mice had less induction of cytokines after engagement of Nod2 and had exacerbated disease in various experimental models of colitis. Furthermore, expression of Pellino3 was lower in the colons of patients with Crohn's disease. Pellino3 directly bound to the kinase RIP2 and catalyzed its ubiquitination. Loss of Pellino3 led to attenuation of Nod2-induced ubiquitination of RIP2 and less activation of the transcription factor NF-κB and mitogen-activated protein kinases (MAPKs). Our findings identify RIP2 as a substrate for Pellino3 and Pellino3 as an important mediator in the Nod2 pathway and regulator of intestinal inflammation.