RESUMEN
During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.
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Interleucinas , Neoplasias Hepáticas , Células T Asesinas Naturales , Animales , Ratones , Células Endoteliales/metabolismo , Interleucinas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/secundario , Ratones Endogámicos C57BL , Células T Asesinas Naturales/metabolismo , Neoplasias Colorrectales/metabolismo , Interleucina-22RESUMEN
Autoreactive B cells are considered pathogenic drivers in many autoimmune diseases; however, it is not clear whether autoimmune B cells are invariably pathogenic or whether they can also arise as bystanders of T cell-driven autoimmune pathology. Here, we studied the B cell response in an autoantigen- and CD4+ T cell-driven model of autoimmune hepatitis (AIH), the Alb-iGP_Smarta mouse in which expression of a viral model antigen (GP) in hepatocytes and its recognition by GP-specific CD4+ T cells causes spontaneous AIH-like disease. T cell-driven AIH in Alb-iGP_Smarta mice was marked by autoantibodies and hepatic infiltration of plasma cells and B cells, particularly of isotype-switched memory B cells, indicating antigen-driven selection and activation. Immunosequencing of B cell receptor repertoires confirmed B cell expansion selectively in the liver, which was most likely driven by the hepatic GP model antigen, as indicated by branched networks of connected sequences and elevated levels of IgG antibodies to GP. However, intrahepatic B cells did not produce increased levels of cytokines and their depletion with anti-CD20 antibody did not alter the CD4+ T cell response in Alb-iGP_Smarta mice. Moreover, B cell depletion did not prevent spontaneous liver inflammation and AIH-like disease in Alb-iGP_Smarta mice. In conclusion, selection and isotype-switch of liver-infiltrating B cells was dependent on the presence of CD4+ T cells recognizing liver antigen. However, recognition of hepatic antigen by CD4+ T cells and CD4+ T cell-mediated hepatitis was not dependent on B cells. Thus, autoreactive B cells can be bystanders and need not be drivers of liver inflammation in AIH.
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Hepatitis Autoinmune , Linfocitos T , Ratones , Animales , Autoantígenos , Hígado , Inflamación/patologíaRESUMEN
BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a progressive cholangiopathy characterised by fibrotic stricturing and inflammation of bile ducts, which seems to be driven by a maladaptive immune response to bile duct injury. The histological finding of dendritic cell expansion in portal fields of patients with PSC prompted us to investigate the role of dendritic cells in orchestrating the immune response to bile duct injury. METHODS: Dendritic cell numbers and subtypes were determined in different mouse models of cholangitis by flow cytometry based on lineage-imprinted markers. Findings were confirmed by immunofluorescence microscopy of murine livers, and liver samples from patients with PSC were compared to control samples from bariatric surgery patients. Using genetic tools, selected dendritic cell subsets were depleted in murine cholangitis. The dendritic cell response to bile duct injury was determined by single-cell transcriptomics. RESULTS: Cholangitis mouse models were characterised by selective intrahepatic expansion of type 2 conventional dendritic cells, whereas plasmacytoid and type 1 conventional dendritic cells were not expanded. Expansion of type 2 conventional dendritic cells in human PSC lesions was confirmed by histology. Depletion studies revealed a proinflammatory role of type 2 conventional dendritic cells. Single-cell transcriptomics confirmed inflammatory maturation of the intrahepatic type 2 conventional dendritic cells and identified dendritic cell-derived inflammatory mediators. CONCLUSIONS: Cholangitis is characterised by intrahepatic expansion and inflammatory maturation of type 2 conventional dendritic cells in response to biliary injury. Therefore, type 2 conventional dendritic cells and their inflammatory mediators might be potential therapeutic targets for the treatment of PSC. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease of the bile ducts for which there is no effective treatment. Herein, we show that the inflammatory immune response to bile duct injury is organised by a specific subtype of immune cell called conventional type 2 dendritic cells. Our findings suggest that this cell subtype and the inflammatory molecules it produces are potential therapeutic targets for PSC.
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Sistema Biliar , Colangitis Esclerosante , Colangitis , Humanos , Ratones , Animales , Colangitis/metabolismo , Sistema Biliar/patología , Modelos Animales de Enfermedad , Células Dendríticas/metabolismo , Mediadores de Inflamación/metabolismoRESUMEN
Autoimmune diseases are caused by adaptive immune responses to self-antigens. The development of antigen-specific therapies that suppress disease-related, but not unrelated immune responses in general, is an important goal of biomedical research. We have previously shown that delivery of myelin peptides to liver sinusoidal endothelial cells (LSECs) using LSEC-targeting nanoparticles provides effective protection from CD4 T-cell-driven autoimmune encephalomyelitis. Here, we investigated whether this methodology might also serve antigen-specific treatment of a CD8 T-cell-driven autoimmune disease. As a model for CD8 T-cell-mediated autoimmunity, we used OT-1 T-cell-driven cholangitis in K14-OVAp mice expressing the cognate MHC I-restricted SIINFEKL peptide in cholangiocytes. To study whether peptide delivery to LSECs could modulate cholangitis, SIINFEKL peptide-conjugated nanoparticles were administered intravenously one day before transfer of OT-1 T cells; five days after cell transfer, liver pathology and hepatic infiltrates were analysed. SIINFEKL peptide-conjugated nanoparticles were rapidly taken up by LSECs in vivo, which effectively cross-presented the delivered peptide on MHC I molecules. Intriguingly, K14-OVAp mice receiving SIINFEKL-loaded nanoparticles manifested significantly reduced liver damage compared with vehicle-treated K14-OVAp mice. Mechanistically, treatment with LSEC-targeting SIINFEKL-loaded nanoparticles significantly reduced the number of liver-infiltrating OT-1 T cells, which up-regulated expression of the co-inhibitory receptor PD-1 and down-regulated cytotoxic effector function and inflammatory cytokine production. These findings show that tolerogenic LSECs can effectively internalize circulating nanoparticles and cross-present nanoparticle-bound peptides on MHC I molecules. Therefore, nanoparticle-mediated autoantigen peptide delivery to LSECs might serve the antigen-specific treatment of CD8 T-cell-driven autoimmune disease.
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Autoantígenos/administración & dosificación , Enfermedades Autoinmunes/inmunología , Linfocitos T CD8-positivos/inmunología , Colangitis/inmunología , Células Endoteliales/inmunología , Inmunoterapia/métodos , Hígado/patología , Nanopartículas Magnéticas de Óxido de Hierro/administración & dosificación , Ovalbúmina/administración & dosificación , Linfocitos T Reguladores/inmunología , Animales , Autoantígenos/química , Enfermedades Autoinmunes/terapia , Células Cultivadas , Colangitis/terapia , Reactividad Cruzada , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Humanos , Terapia de Inmunosupresión , Hígado/irrigación sanguínea , Nanopartículas Magnéticas de Óxido de Hierro/química , Ratones , Ratones Transgénicos , Ovalbúmina/química , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/química , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
BACKGROUND & AIMS: IL-17A-producing T cells are present in autoimmune cholestatic liver diseases; however, little is known about the contribution of IL-17 to periductal immune responses. Herein, we investigated the role of IL-17 produced by antigen-specific CD8+ T cells in a mouse model of cholangitis and in vitro in human cholangiocyte organoids. METHODS: K14-OVAp mice express a major histocompatibility complex I-restricted ovalbumin (OVA) peptide sequence (SIINFEKL) on cholangiocytes. Cholangitis was induced by the adoptive transfer of transgenic OVA-specific ovalbumin transgene (OT)-1 CD8+ T cells that either had OT-1wt or lacked IL-17A/F (OT-1IL17ko). The response of mouse and human cholangiocytes/organoids to IL-17A was assessed in vitro. RESULTS: Transfer of OVA-specific OT-1IL17ko cells significantly aggravated periductal inflammation in K14-OVAp recipient mice compared with transfer of OT-1wt T cells. OT-1IL17ko T cells were highly activated in the liver and displayed increased cytotoxicity and proliferation. IL-17A/F produced by transferred OT-1wt CD8+ T cells induced upregulation of the inhibitory molecule programmed cell death ligand 1 (PD-L1) on cholangiocytes, restricting cholangitis by limiting cytotoxicity and proliferation of transferred cells. In contrast, OT-1IL17ko T cells failed to induce PD-L1 on cholangiocytes, resulting in uncontrolled expansion of cytotoxic CD8+ T cells and aggravated cholangitis. Blockade of PD-L1 after transfer of OT-1wt T cells with anti-PD-L1 antibody also resulted in aggravated cholangitis. Using human cholangiocyte organoids, we were able to confirm that IL-17A induces PD-L1 expression in cholangiocytes. CONCLUSIONS: We demonstrate that by upregulating PD-L1 on cholangiocytes, IL-17 has an important role in restricting cholangitis and protecting against CD8+ T cell-mediated inflammatory bile duct injury. Caution should be exercised when targeting IL-17 for the treatment of cholangitis. LAY SUMMARY: IL-17 is assumed to be a driver of inflammation in several autoimmune diseases, such as psoriasis. IL-17 is also present in inflammatory diseases of the bile duct, but its role in these conditions is not clear, as the effects of IL-17 depend on the context of its expression. Herein, we investigated the role of IL-17 in an experimental autoimmune cholangitis mouse model, and we identified an important protective effect of IL-17 on cholangiocytes, enabling them to downregulate bile duct inflammation via checkpoint inhibitor PD-L1.
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Antígeno B7-H1/metabolismo , Conductos Biliares/inmunología , Colangitis , Interleucina-17/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Linfocitos T CD8-positivos/inmunología , Colangitis/inmunología , Colangitis/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Transgénicos , Organoides , Ovalbúmina/genética , Fragmentos de Péptidos/genéticaRESUMEN
BACKGROUND & AIMS: Interleukin (IL)-6 cytokine family members contribute to inflammatory and regenerative processes. Engagement of the signaling receptor subunit gp130 is common to almost all members of the family. In the liver, all major cell types respond to IL-6-type cytokines, making it difficult to delineate cell type-specific effects. We therefore generated mouse models for liver cell type-specific analysis of IL-6 signaling. METHODS: We produced mice with a Cre-inducible expression cassette encoding a designed pre-dimerized constitutive active gp130 variant. We bred these mice to different Cre-drivers to induce transgenic gp130 signaling in distinct liver cell types: hepatic stellate cells, cholangiocytes/liver progenitor cells or hepatocytes. We phenotyped these mice using multi-omics approaches, immunophenotyping and a bacterial infection model. RESULTS: Hepatocyte-specific gp130 activation led to the upregulation of innate immune system components, including acute-phase proteins. Consequently, we observed peripheral mobilization and recruitment of myeloid cells to the liver. Hepatic myeloid cells, including liver-resident Kupffer cells were instructed to adopt a bactericidal phenotype which ultimately conferred enhanced resistance to bacterial infection in these mice. We demonstrate that persistent hepatocyte-specific gp130 activation resulted in amyloid A amyloidosis in aged mice. In contrast, we did not observe overt effects of hepatic stellate cell- or cholangiocyte/liver progenitor cell-specific transgenic gp130 signaling. CONCLUSIONS: Hepatocyte-specific gp130 activation alone is sufficient to trigger a robust innate immune response in the absence of NF-κB activation. We therefore conclude that gp130 engagement, e.g. by IL-6 trans-signaling, represents a safe-guard mechanism in innate immunity. LAY SUMMARY: Members of the interleukin-6 cytokine family signal via the receptor subunit gp130 and are involved in multiple processes in the liver. However, as several liver cell types respond to interleukin-6 family cytokines, it is difficult to delineate cell type-specific effects. Using a novel mouse model, we provide evidence that hepatocyte-specific gp130 activation is sufficient to trigger a robust systemic innate immune response.
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Receptor gp130 de Citocinas/metabolismo , Hepatocitos/metabolismo , Inmunidad Innata/fisiología , Interleucina-6/inmunología , Hígado , Factor de Transcripción STAT3/metabolismo , Reacción de Fase Aguda/inmunología , Animales , Hepatocitos/clasificación , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Transgénicos , Modelos Animales , Transducción de Señal/inmunologíaRESUMEN
BACKGROUND AND AIMS: T cells from patients with primary sclerosing cholangitis (PSC) show a prominent interleukin (IL)-17 response upon stimulation with bacteria or fungi, yet the reasons for this dominant T-helper 17 (Th17) response in PSC are not clear. Here, we analyzed the potential role of monocytes in microbial recognition and in skewing the T-cell response toward Th17. APPROACH AND RESULTS: Monocytes and T cells from blood and livers of PSC patients and controls were analyzed ex vivo and in vitro using transwell experiments with cholangiocytes. Cytokine production was measured using flow cytometry, enzyme-linked immunosorbent assay, RNA in situ hybridization, and quantitative real-time PCR. Genetic polymorphisms were obtained from ImmunoChip analysis. Following ex vivo stimulation with phorbol myristate acetate/ionomycin, PSC patients showed significantly increased numbers of IL-17A-producing peripheral blood CD4+ T cells compared to PBC patients and healthy controls, indicating increased Th17 differentiation in vivo. Upon stimulation with microbes, monocytes from PSC patients produced significantly more IL-1ß and IL-6, cytokines known to drive Th17 cell differentiation. Moreover, microbe-activated monocytes induced the secretion of Th17 and monocyte-recruiting chemokines chemokine (C-C motif) ligand (CCL)-20 and CCL-2 in human primary cholangiocytes. In livers of patients with PSC cirrhosis, CD14hiCD16int and CD14loCD16hi monocytes/macrophages were increased compared to alcoholic cirrhosis, and monocytes were found to be located around bile ducts. CONCLUSIONS: PSC patients show increased Th17 differentiation already in vivo. Microbe-stimulated monocytes drive Th17 differentiation in vitro and induce cholangiocytes to produce chemokines mediating recruitment of Th17 cells and more monocytes into portal tracts. Taken together, these results point to a pathogenic role of monocytes in patients with PSC.
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Colangitis Esclerosante/inmunología , Monocitos/fisiología , Células Th17/citología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Adaptadoras de Señalización CARD/genética , Diferenciación Celular , Quimiocinas/biosíntesis , Femenino , Humanos , Interleucina-1beta/fisiología , Interleucinas/genética , Cirrosis Hepática/inmunología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND & AIMS: T cells are central mediators of liver inflammation and represent potential treatment targets in cholestatic liver disease. Whereas emerging evidence shows that bile acids (BAs) affect T cell function, the role of T cells for the regulation of BA metabolism is unknown. In order to understand this interplay, we investigated the influence of T cells on BA metabolism in a novel mouse model of cholangitis. METHODS: Mdr2-/- mice were crossed with transgenic K14-OVAp mice, which express an MHC class I restricted ovalbumin peptide on biliary epithelial cells (Mdr2-/-xK14-OVAp). T cell-mediated cholangitis was induced by the adoptive transfer of antigen-specific CD8+ T cells. BA levels were quantified using a targeted liquid chromatography-mass spectrometry-based approach. RESULTS: T cell-induced cholangitis resulted in reduced levels of unconjugated BAs in the liver and significantly increased serum and hepatic levels of conjugated BAs. Genes responsible for BA synthesis and uptake were downregulated and expression of the bile salt export pump was increased. The transferred antigen-specific CD8+ T cells alone were able to induce these changes, as demonstrated using Mdr2-/-xK14-OVAp recipient mice on the Rag1-/- background. Mechanistically, we showed by depletion experiments that alterations in BA metabolism were partly mediated by the proinflammatory cytokines TNF and IFN-γ in an FXR-dependent manner, a process that in vitro required cell contact between T cells and hepatocytes. CONCLUSION: Whereas it is known that BA metabolism is dysregulated in sepsis and related conditions, we have shown that T cells are able to control the synthesis and metabolism of BAs, a process which depends on TNF and IFN-γ. Understanding the effect of lymphocytes on BA metabolism will help in the design of combined treatment strategies for cholestatic liver diseases. LAY SUMMARY: Dysregulation of bile acid metabolism and T cells can contribute to the development of cholangiopathies. Before targeting T cells for the treatment of cholangiopathies, it should be determined whether they exert protective effects on bile acid metabolism. Herein, we demonstrate that T cell-induced cholangitis resulted in decreased levels of harmful unconjugated bile acids. T cells were able to directly control synthesis and metabolism of bile acids, a process which was dependent on the proinflammatory cytokines TNF and IFN-γ. Understanding the effect of lymphocytes on bile acid metabolism will help in the design of combined treatment strategies for cholestatic liver diseases.
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Ácidos y Sales Biliares , Colangitis , Interferón gamma/inmunología , Linfocitos T , Factor de Necrosis Tumoral alfa/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Ácidos y Sales Biliares/biosíntesis , Ácidos y Sales Biliares/metabolismo , Vías Biosintéticas/inmunología , Colangitis/inmunología , Colangitis/metabolismo , Colangitis/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Animales , Serpinas/genética , Linfocitos T/metabolismo , Linfocitos T/patología , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide with rising incidence. The inflammatory cytokine, interleukin-6 (IL-6), is a critical mediator of HCC development. It can signal through two distinct pathways: the IL-6 classic and the IL-6 trans-signaling pathway. Whereas IL-6 classic signaling is important for innate and acquired immunity, IL-6 trans-signaling has been linked to accelerated liver regeneration and several chronic inflammatory pathologies. However, its implication in liver tumorigenesis has not been addressed yet. Here, we show that IL-6 trans-signaling, but not IL-6 classic signaling, is essential to promote hepatocellular carcinogenesis by two mechanisms: First, it prevents DNA-damage-induced hepatocyte apoptosis through suppression of p53 and enhances ß-catenin activation and tumor proliferation. Second, IL-6 trans-signaling directly induces endothelial cell proliferation to promote tumor angiogenesis. Consequently, soluble gp130 fused to Fc transgenic mice lacking IL-6 trans-signaling are largely protected from tumor formation in a diethylnitrosamine/3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene model of HCC. CONCLUSION: IL-6 trans-signaling, and not IL-6 classic signaling, is mandatory for development of hepatocellular carcinogenesis. Therefore, specific inhibition of IL-6 trans-signaling, rather than total inhibition of IL-6 signaling, is sufficient to blunt tumor initiation and impair tumor progression without compromising IL-6 classic signaling-driven protective immune responses. (Hepatology 2017;65:89-103).
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Carcinoma Hepatocelular/etiología , Interleucina-6/fisiología , Neoplasias Hepáticas/etiología , Animales , Masculino , Ratones , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: Reduced numbers of regulatory T cells (Treg) have been reported in patients with primary sclerosing cholangitis (PSC); therefore, Treg expansion might serve as a therapeutic approach. Here, we explored whether treatment with IL-2/IL-2 monoclonal antibody complex (IL-2/IL-2Ab complex) could provide in vivo Treg expansion and treatment of experimental sclerosing cholangitis. METHODS: Treg were expanded by repeated injection of IL-2/IL-2Ab complex in mouse models of cholangitis (Mdr2-/-, DDC) or colitis (dextran sulfate sodium [DSS]) as control. In vitro suppressive capacity and gene expression were analyzed in isolated hepatic and splenic Treg. RESULTS: In vivo expansion resulted in a 5-fold increase in hepatic Treg, which localized within the inflamed portal tracts. However, although Treg expansion was associated with reduced pro-inflammatory IL-17 and increased anti-inflammatory IL-10 production by hepatic lymphocytes, the severity of cholangitis was not reduced. In contrast, DSS-induced colitis could be improved by Treg expansion, suggesting a selectively reduced functionality of intrahepatic Treg. Indeed, hepatic Treg manifested reduced Foxp3 expression and reduced suppressive capacity compared to splenic Treg. Hepatic Treg dysfunction could be linked to increased IL-12 signaling due to an upregulation of the IL-12 receptor. Accordingly, IL-12 receptor beta 2 knockout mice (IL-12rb2-/-) were able to maintain hepatic Treg functionality. CONCLUSIONS: Hepatic Treg expanded in vivo failed to improve the course of cholangitis, which was related to the effects of hepatic IL-12 on Treg. Therefore, neutralization of IL-12 should be considered as part of treatment strategies targeting Treg in sclerosing cholangitis. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is associated with a paucity of regulatory T cells (Treg) that have a particular ability to control immune responses; therefore, in vivo expansion of Treg might serve as a treatment of cholangitis. However, in a mouse model of PSC, we show that Treg enrichment in the liver was not sufficient to provide effective control of cholangitis, as the suppressive functionality of hepatic Treg was significantly limited by IL-12 signals. Thus, neutralization of IL-12 should be considered as part of treatment strategies to improve the efficacy of Treg-based treatments for liver diseases. Data accession number: GSE 87898.
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Colangitis Esclerosante/inmunología , Interleucina-12/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Colangitis Esclerosante/genética , Colangitis Esclerosante/patología , Femenino , Factores de Transcripción Forkhead/metabolismo , Interleucina-2/metabolismo , Hígado/inmunología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Interleucina-12/deficiencia , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismo , Transducción de Señal , Linfocitos T Reguladores/metabolismo , Regulación hacia ArribaRESUMEN
Autoimmune liver diseases predominantly affect women. In this study, we aimed to elucidate how sex affects autoimmune hepatic inflammation. Acute experimental cholangitis was induced by adoptive transfer of OVA-specific CD8(+) T cells into mice, which express the cognate Ag on cholangiocytes. In contrast to previous mouse models of cholangitis, this model displayed a strong sexual dimorphism: female mice developed marked cholangitis, whereas male mice were resistant to cholangitis induction. The recruitment of endogenous CD4(+) T cells, but not transferred CD8(+) T cells into female livers was strongly increased. These cells expressed higher amounts of the proinflammatory cytokine IL-17, which was at least in part responsible for the liver inflammation observed. The recruitment of endogenous CD4(+) T cells was associated with increased expression of the chemokines CXCL-9 and CXCL-10 in female livers. The sex-specific factor responsible for the observed differences was found to be testosterone: male mice could be rendered susceptible to liver inflammation by castration, and testosterone treatment was sufficient to completely suppress liver inflammation in female mice. Accordingly, testosterone treatment of female mice significantly reduced the expression of IL-17A, CXCL-9, and CXCL-10 within the liver. Serum testosterone levels of untreated mice negatively correlated with the IL-17, CXCL-9, and CXCL-10 expression in the liver, further supporting a role for testosterone in hepatic immune homeostasis. In conclusion, testosterone was found to be the major determinant of the observed sexual dimorphism. Further study into the role of testosterone for liver inflammation could lead to novel treatment targets in human autoimmune liver diseases.
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Quimiocina CXCL10/genética , Quimiocina CXCL9/genética , Colangitis/prevención & control , Hepatitis/prevención & control , Interleucina-17/metabolismo , Testosterona/farmacología , Enfermedad Aguda , Traslado Adoptivo , Andrógenos/farmacología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/trasplante , Colangitis/genética , Colangitis/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Femenino , Citometría de Flujo , Hepatitis/genética , Hepatitis/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/genética , Ovalbúmina/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores Sexuales , Testosterona/sangreAsunto(s)
Autoinmunidad , Hepatitis Autoinmune , Autoantígenos/inmunología , Autoinmunidad/efectos de los fármacos , Autoinmunidad/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Descubrimiento de Drogas , Hepatitis Autoinmune/tratamiento farmacológico , Hepatitis Autoinmune/inmunología , Hepatitis Autoinmune/patología , Humanos , Factores de RiesgoRESUMEN
BACKGROUND & AIMS: It is well-known that the liver can induce immune tolerance, yet this knowledge could, thus far, not be translated into effective treatments for autoimmune diseases. We have previously shown that liver sinusoidal endothelial cells (LSECs) could substantially contribute to hepatic tolerance through their ability to induce CD4+ Foxp3+ regulatory T cells (Tregs). Here, we explored whether the Treg-inducing potential of LSECs could be harnessed for the treatment of autoimmune disease. METHODS: We engineered a polymeric nanoparticle (NP) carrier for the selective delivery of autoantigen peptides to LSECs in vivo. In the well-characterized autoimmune disease model of experimental autoimmune encephalomyelitis (EAE), we investigated whether administration of LSEC-targeting autoantigen peptide-loaded NPs could protect mice from autoimmune disease. RESULTS: We demonstrate that NP-based autoantigen delivery to LSECs could completely and permanently prevent the onset of clinical EAE. More importantly, in a therapeutic approach, mice with already established EAE improved rapidly and substantially following administration of a single dose of autoantigen peptide-loaded NPs, whereas the control group deteriorated. Treatment efficacy seemed to depend on Tregs. The Treg frequencies in the spleens of mice treated with autoantigen peptide-loaded NPs were significantly higher than those in vehicle-treated mice. Moreover, NP-mediated disease control was abrogated after Treg depletion by repeated administration of Treg-depleting antibody. CONCLUSION: Our findings provide proof of principle that the selective delivery of autoantigen peptides to LSECs by NPs can induce antigen-specific Tregs and enable effective treatment of autoimmune disease. These findings highlight the importance of Treg induction by LSECs for immune tolerance.
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Autoantígenos/administración & dosificación , Enfermedades Autoinmunes/prevención & control , Hígado/citología , Hígado/inmunología , Linfocitos T Reguladores/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Autoinmunidad , Sistemas de Liberación de Medicamentos , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Células Endoteliales/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Básica de Mielina/administración & dosificación , Proteína Básica de Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Glicoproteína Mielina-Oligodendrócito/inmunología , Nanopartículas/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunologíaRESUMEN
BACKGROUND & AIMS: CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) have a profound ability to control immune responses. We have previously shown that the liver is a major source of peripherally induced Tregs. Here, we investigate the liver cell types and molecular mechanisms responsible for hepatic Treg induction. METHODS: To assess the Treg-inducing potential of liver resident antigen-presenting cell types, we studied the conversion of Foxp3(-) non-Tregs into Foxp3(+) Tregs induced by liver dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), or Kupffer cells (KCs). The dependency of Treg induction on TGF-ß was tested in Treg conversion assays using T cells with reduced TGF-ß sensitivity. The suppressive potential of liver cell-induced Tregs was assessed by an in vitro suppression assay and in vivo, in the model of experimental autoimmune encephalomyelitis (EAE). RESULTS: All tested liver cell types were capable of inducing Foxp3(+) Tregs; however, LSECs were most efficient in inducing Tregs. Treg-induction was antigen-specific and depended on TGF-ß. LSECs featured membrane-bound LAP/TGF-ß and the anchor molecule GARP, which is required for tethering LAP/TGF-ß to the cell membrane. LSEC-induced Tregs suppressed proliferation and cytokine secretion of effector T cells in vitro. LSEC-induced Tregs were also functional suppressors in vivo, as neuroantigen-specific Tregs induced by LSECs were able to suppress EAE. CONCLUSIONS: We demonstrate that LSECs are the major liver cell type responsible for TGF-ß dependent hepatic Treg induction. The extraordinary capacity of LSECs to induce Tregs was associated with their unique ability to tether TGF-ß to their membrane.
Asunto(s)
Comunicación Celular/efectos de los fármacos , Factores de Transcripción Forkhead/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Hígado/patología , Linfocitos T Reguladores/patología , Factor de Crecimiento Transformador beta/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/patología , Endotelio/efectos de los fármacos , Endotelio/patología , Técnicas In Vitro , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/patología , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Mutantes , Modelos Animales , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismoRESUMEN
BACKGROUND & AIMS: The liver has a distinct capacity to induce immune tolerance to hepatic antigens. Although liver tolerance can be advantageous for preventing autoimmune and inflammatory diseases, it also can be detrimental by preventing immune surveillance of infected or malignant cells. Here, we investigated the immune mechanisms that establish hepatic tolerance. METHODS: Tolerance was investigated in C-reactive protein (CRP)-myelin basic protein (MBP) mice expressing the neuroantigen MBP in hepatocytes, providing profound resistance to MBP-induced neuroinflammation. Tolerance induction was studied after transfer of MBP-specific CD4 T cells into CRP-MBP mice, and tolerance mechanisms were tested using depleting or blocking antibodies. RESULTS: Although tolerant CRP-MBP mice display increased numbers of forkhead box P3+ regulatory T cells, we here found them not essential for the maintenance of hepatic tolerance. Instead, upon MBP recognition in the liver, MBP-specific T cells became activated to produce interferon (IFN)γ, which, in turn, induced local up-regulation of recruitment molecules, including Chemokine (C-X-C motif) ligand9 and its receptor C-X-C motif chemokine receptor3, facilitating endothelial translocation and redirection of MBP-specific T cells into the hepatic parenchyma. There, the translocated MBP-specific CD4 T cells partly converted into interleukin 10-producing type 1 regulatory T cells, and significantly up-regulated the expression of immune checkpoint molecules, notably cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Intriguingly, although liver tolerance was not affected by impairment of interleukin 10 signaling, concomitant blockade of IFNγ and CTLA-4 abrogated hepatic tolerance induction to MBP, resulting in neuroinflammatory autoimmune disease in these mice. CONCLUSIONS: IFNγ-mediated redirection of autoreactive CD4 T cells into the liver and up-regulation of checkpoint molecules, including CTLA-4, were essential for tolerance induction in the liver, hence representing a potential treatment target for boosting or preventing liver tolerance.
Asunto(s)
Linfocitos T CD4-Positivos , Encefalomielitis Autoinmune Experimental , Animales , Ratones , Autoinmunidad , Quimiocinas , Antígeno CTLA-4 , Encefalomielitis Autoinmune Experimental/prevención & control , Tolerancia Inmunológica , Interleucina-10 , HígadoRESUMEN
BACKGROUND & AIMS: The liver can mitigate the inflammatory activity of infiltrating T cells by mechanisms that are not entirely clear. Here we investigated the role of liver sinusoidal endothelial cells (LSECs) in regulating the activity of inflammatory CD4 T cells. METHODS: Interactions between T helper (Th) 1 or Th17 cells and LSEC were studied by intravital microscopy and by in vitro stimulation assays. RESULTS: Circulating CD4 T cells established lasting and repeated interactions with liver endothelium in vivo. Stimulation of Th1 and Th17 cells by LSEC greatly inhibited their capacity to secrete interferon-γ or interleukin-17 in vitro; in contrast, stimulation by dendritic cells (DCs) resulted in considerable secretion of both cytokines. Cytokine release by Th1 or Th17 cells seemed to be actively suppressed by LSEC, as indicated by the inhibition of cytokine secretion even in the presence of Th1- and Th17-promoting DC. This inhibition of CD4 T cell effector function seemed to depend on the dominance of inhibitory over activating co-stimulatory signals on LSEC, since (1) cytokine secretion could be restored by increased CD28 co-activation; (2) LSEC from interleukin-10(-/-) mice, which manifest increased activating signals, such as MHC II, and decreased inhibitory signals, such as PD-L1, failed to suppress cytokine secretion; and (3) cytokine secretion by Th1 or Th17 cells that lacked PD-1, the ligand for inhibitory PD-L1, could not be suppressed by LSEC. CONCLUSIONS: LSEC inhibit inflammatory cytokine secretion of Th1 and Th17 effector CD4 T cells in dependence of interleukin-10 and PD-1.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Comunicación Celular/inmunología , Células Endoteliales/inmunología , Hígado/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Diferenciación Celular/inmunología , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Endoteliales/citología , Femenino , Tolerancia Inmunológica/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Hígado/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Transducción de Señal/inmunología , Células TH1/citología , Células TH1/inmunología , Células Th17/citología , Células Th17/inmunologíaRESUMEN
The chemokine receptor CXCR3 is preferentially expressed by Th1 cells and critically involved in their recruitment to inflamed tissue. In a mouse model of immune-mediated liver injury inducible by Con A, we investigated the role of CXCR3 in acute IFN-γ-mediated hepatitis as well as in tolerance induction, which has been shown to depend on IL-10-producing CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Induction of Con A hepatitis resulted in increased intrahepatic expression of the CXCR3 ligands CXCL9, CXCL10, and CXCL11. CXCR3(-/-) mice developed a more severe liver injury with higher plasma transaminase activities and a more pronounced Th1/Th17 response compared with wild-type (wt) animals upon Con A injection. Moreover, CXCR3(-/-) mice did not establish tolerance upon Con A restimulation, although Tregs from CXCR3(-/-) mice were still suppressive in an in vitro suppression assay. Instead, Tregs failed to accumulate in livers of CXCR3(-/-) mice upon Con A restimulation in contrast to those from wt animals. Con A-tolerant wt mice harbored significantly increased numbers of intrahepatic CXCR3(+)T-bet(+) Tregs that produced IL-10 compared with nontolerant animals. IFN-γ deficiency or anti-IFN-γ Ab treatment demonstrated that conversion to CXCR3(+)T-bet(+) Tregs depended on a Th1 response. Accordingly, in an immunotherapeutic approach, CD4(+)CD25(+)Foxp3(+) Tregs from Con A-pretreated CXCR3-deficient mice failed to protect against Con A-induced hepatitis, whereas Tregs from Con A-tolerant wt mice allowed CXCR3-deficient mice to recover from Con A hepatitis. In summary, CXCR3(+)T-bet(+)IL-10(+) Tregs are generated in the liver in dependence of IFN-γ, then disseminated into the organism and specifically migrate into the liver, where they limit immune-mediated liver damage.
Asunto(s)
Hepatitis/inmunología , Tolerancia Inmunológica/inmunología , Receptores CXCR3/deficiencia , Linfocitos T Reguladores/inmunología , Animales , Separación Celular , Concanavalina A/toxicidad , Citometría de Flujo , Interferón gamma/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitógenos/toxicidad , Receptores CXCR3/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Autoimmune diseases develop when the adaptive immune system attacks the body's own antigens leading to tissue damage. At least 80 different conditions are believed to have an autoimmune aetiology, including rheumatoid arthritis, type I diabetes, multiple sclerosis or systemic lupus erythematosus. Collectively, autoimmune diseases are a leading cause of severe health impairment along with substantial socioeconomic costs. Current treatments are mostly symptomatic and non-specific, and it is typically not possible to cure these diseases. Thus, the development of more causative treatments that suppress only the pathogenic immune responses, but spare general immunity is of great biomedical interest. The liver offers considerable potential for development of such antigen-specific immunotherapies, as it has a distinct physiological capacity to induce immune tolerance. Indeed, the liver has been shown to specifically suppress autoimmune responses to organ allografts co-transplanted with the liver or to autoantigens that were transferred to the liver. Liver tolerance is established by a unique microenvironment that facilitates interactions between liver-resident antigen-presenting cells and lymphocytes passing by in the low blood flow within the hepatic sinusoids. Here, we summarise current concepts and mechanisms of liver immune tolerance, and review present approaches to harness liver tolerance for antigen-specific immunotherapy.
Asunto(s)
Enfermedades Autoinmunes , Tolerancia Inmunológica , Células Presentadoras de Antígenos , Autoantígenos , Autoinmunidad , Humanos , HígadoRESUMEN
BACKGROUND: The contamination of ecosystem compartments by microplastics (MPs) is an ubiquitous problem. MPs have been observed in mice tissues, and recently in human blood, stool and placenta. However, two aspects remain unclear: whether MPs accumulate in peripheral organs, specifically in the liver, and if liver cirrhosis favours this process. We aimed to examine human liver tissue samples to determine whether MPs accumulate in the liver. METHODS: This proof-of-concept case series, conducted in Germany, Europe, analyzed tissue samples of 6 patients with liver cirrhosis and 5 individuals without underlying liver disease. A total of 17 samples (11 liver, 3 kidney and 3 spleen samples) were analyzed according to the final protocol. A reliable method for detection of MP particles from 4 to 30 µm in human tissue was developed. Chemical digestion of tissue samples, staining with Nile red, subsequent fluorescent microscopy and Raman spectroscopy were performed. Morphology, size and composition of MP polymers were assessed. FINDINGS: Considering the limit of detection, all liver, kidney and spleen samples from patients without underlying liver disease tested negative for MPs. In contrast, MP concentrations in cirrhotic liver tissues tested positive and showed significantly higher concentrations compared to liver samples of individuals without underlying liver disease. Six different microplastic polymers ranging from 4 to 30 µm in size were detected. INTERPRETATION: This proof-of-concept case series assessed the presence of MPs in human liver tissue and found six different MP polymers in the liver of individuals with liver cirrhosis, but not in those without underlying liver disease. Future studies are needed to evaluate whether hepatic MP accumulation represents a potential cause in the pathogenesis of fibrosis, or a consequence of cirrhosis and portal hypertension. FUNDING: No funding was received for conducting this investigator driven study.
Asunto(s)
Microplásticos , Contaminantes Químicos del Agua , Animales , Ecosistema , Monitoreo del Ambiente , Humanos , Cirrosis Hepática , Ratones , Plásticos/química , Polímeros , Contaminantes Químicos del Agua/análisisRESUMEN
The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²5I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²5I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²5I), declined (-33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²5I) decreased (-29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²5I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.