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PURPOSE: Human trophoblast cell surface antigen 2 (Trop-2) is a protein highly expressed in urothelial cancer (UC). Sacituzumab govitecan (SG) is a Trop-2-directed antibody drug conjugate with a hydrolysable linker and a potent SN-38 payload. This study explored Trop-2 expression in tumors treated with SG in cohorts 1 to 3 (C1-3) from the TROPHY-U-01 study and evaluated whether efficacy was associated with Trop-2 expression. PATIENTS AND METHODS: TROPHY-U-01 (NCT03547973) is an open-label phase II study that assessed the efficacy and safety of SG (alone or in combinations) in patients with unresectable locally advanced or metastatic UC (mUC). Archival tumor samples collected at enrollment for C1-3 were analyzed for Trop-2 membrane expression by considering histological scores (H-scores; scale 0-300) and the percentage of membrane positive tumor cells at low magnification (4×). The association of Trop-2 with clinical endpoints [objective response rate (ORR), progression-free survival (PFS), and overall survival (OS)] was evaluated. RESULTS: In C1-3, tissue was collected from 158 (82%) of 192 treated patients, and 146 (76%) had evaluable Trop-2 data. Trop-2 was highly expressed in tumor samples. The median [interquartile range (IQR)] Trop-2 H-score was 215 (180-246), and the median (IQR) percentage of membrane positive tumor cells was 91% (80-98). Trop-2 expression at any level was observed in 98% of patients. Furthermore, ORR, PFS, and OS benefits were observed across all Trop-2 expression levels. CONCLUSIONS: Trop-2 protein is highly expressed in UC, as confirmed by examining tumors from patients enrolled in the TROPHY-U-01 trial. The results indicate that SG demonstrates efficacy in mUC across Trop-2 expression levels.
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Anticuerpos Monoclonales Humanizados , Antígenos de Neoplasias , Camptotecina , Moléculas de Adhesión Celular , Inmunoconjugados , Humanos , Moléculas de Adhesión Celular/metabolismo , Anticuerpos Monoclonales Humanizados/uso terapéutico , Femenino , Masculino , Anciano , Persona de Mediana Edad , Camptotecina/análogos & derivados , Camptotecina/uso terapéutico , Inmunoconjugados/uso terapéutico , Anciano de 80 o más Años , Adulto , Biomarcadores de Tumor/metabolismo , Neoplasias Urológicas/tratamiento farmacológico , Neoplasias Urológicas/patología , Neoplasias Urológicas/mortalidad , Neoplasias Urológicas/metabolismo , Resultado del Tratamiento , Estadificación de NeoplasiasRESUMEN
Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.
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Virus de la Hepatitis B , Hepatitis B Crónica , Hepatocitos , Humanos , Hepatocitos/virología , Hepatocitos/efectos de los fármacos , Animales , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Ratones , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Proteínas del Núcleo Viral/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , Antígenos del Núcleo de la Hepatitis B/metabolismo , Cápside/metabolismo , Cápside/efectos de los fármacos , Hígado/virología , Hígado/efectos de los fármacos , Hígado/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Ensamble de Virus/efectos de los fármacos , Apoptosis/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
AIMS: Treatment options for advanced urothelial carcinoma (aUC) rapidly evolved: besides immunomodulative therapeutic options and inhibitors targeting Fibroblast growth factor receptor (FGFR) alterations, two new antibody-drug conjugates (ADC), sacituzumab govitecan (SG) and enfortumab vedotin (EV), have been approved. However, little is known about the associations of specific aUC properties and the surface target expression of TROP2 and NECTIN-4. Our aim was to characterize associations of TACSTD2/TROP2 and NECTIN-4/NECTIN-4 protein and gene expression with morphomolecular and clinicopathological characteristics of aUC in two large independent cohorts. METHODS AND RESULTS: The TCGA BLCA (n = 405) and the CCC-EMN (n = 247) cohorts were retrospectively analysed. TROP2/TACSTD2 and NECTIN-4/NECTIN-4 are highly expressed at the protein and transcript level in aUC, and their expression status did not correlate with patient survival in both cohorts. NECTIN-4/NECTIN-4 expression was higher in luminal tumours and reduced in squamous aUCs. NECTIN-4 was negative in 10.6% of samples, and 18.4% of samples had low expression (H-score <15). The TROP2 negativity rate amounted to 6.5%. TACSTD2 and NECTIN-4 expression was reduced in neuroendocrine-like and/or protein-based double-negative tumours. TROP2- and NECTIN-4-negative tumours included one sarcomatoid and four neuroendocrine aUC. FGFR3 alterations and PD-L1 expression on tumour and immune cells did not associate with TROP2 or NECTIN-4 expression. CONCLUSIONS: TACSTD2/TROP2 and NECTIN-4/NECTIN-4 are widely expressed in aUC, independent of FGFR3 alterations or PD-L1 expression, thus representing a suitable target for ADC treatment in the majority of aUC. The expression loss was associated with aggressive morphomolecular aUC subtypes, i.e. neuroendocrine(-like) and sarcomatoid aUC.
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Carcinoma de Células Transicionales , Neoplasias de la Vejiga Urinaria , Humanos , Neoplasias de la Vejiga Urinaria/genética , Nectinas/genética , Antígeno B7-H1 , Estudios Retrospectivos , Moléculas de Adhesión Celular/metabolismo , Antígenos de Neoplasias/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
Background & Aims: Patterns of liver HBV antigen expression have been described but not quantified at single-cell resolution. We applied quantitative techniques to liver biopsies from individuals with chronic hepatitis B and evaluated sampling heterogeneity, effects of disease stage, and nucleos(t)ide (NUC) treatment, and correlations between liver and peripheral viral biomarkers. Methods: Hepatocytes positive for HBV core and HBsAg were quantified using a novel four-plex immunofluorescence assay and image analysis. Biopsies were analysed from HBeAg-positive (n = 39) and HBeAg-negative (n = 75) participants before and after NUC treatment. To evaluate sampling effects, duplicate biopsies collected at the same time point were compared. Serum or plasma samples were evaluated for levels of HBV DNA, HBsAg, hepatitis B core-related antigen (HBcrAg), and HBV RNA. Results: Diffusely distributed individual HBV core+ cells and foci of HBsAg+ cells were the most common staining patterns. Hepatocytes positive for both HBV core and HBsAg were rare. Paired biopsies revealed large local variation in HBV staining within participants, which was confirmed in a large liver resection. NUC treatment was associated with a >100-fold lower median frequency of HBV core+ cells in HBeAg-positive and HBeAg-negative participants, whereas reductions in HBsAg+ cells were not statistically significant. The frequency of HBV core+ hepatocytes was lower in HBeAg-negative participants than in HBeAg-positive participants at all time points evaluated. Total HBV+ hepatocyte burden correlated with HBcrAg, HBV DNA, and HBV RNA only in baseline HBeAg-positive samples. Conclusions: Reductions in HBV core+ hepatocytes were associated with HBeAg-negative status and NUC treatment. Variation in HBV positivity within individual livers was extensive. Correlations between the liver and the periphery were found only between biomarkers likely indicative of cccDNA (HBV core+ and HBcrAg, HBV DNA, and RNA). Impact and Implications: HBV infects liver hepatocyte cells, and its genome can exist in two forms that express different sets of viral proteins: a circular genome called cccDNA that can express all viral proteins, including the HBV core and HBsAg proteins, or a linear fragment that inserts into the host genome typically to express HBsAg, but not HBV core. We used new techniques to determine the percentage of hepatocytes expressing the HBV core and HBsAg proteins in a large set of liver biopsies. We find that abundance and patterns of expression differ across patient groups and even within a single liver and that NUC treatment greatly reduces the number of core-expressing hepatocytes.
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BACKGROUND AND AIMS: The progression of chronic liver diseases towards liver cirrhosis is accompanied by drastic tissue changes. This study combines elaborate transcriptomic and histological methods aiming at spatially resolving the hepatic immune microenvironment in NAFLD (including NASH, primary sclerosing cholangitis, primary biliary cholangitis, and severe alcoholic hepatitis). APPROACH AND RESULTS: Human liver samples were subjected to RNA-sequencing (n=225) and imaging cytometry (n=99) across 3 independent patient cohorts. Liver samples from alcoholic hepatitis and primary biliary cholangitis patients were used for comparison. Myeloid populations were further characterized in corresponding mouse models. Imaging, clinical, and phenotypical data were combined for multidimensional analysis. NAFLD/NASH and primary sclerosing cholangitis disease stages were associated with loss of parenchymal areas, increased ductular cell accumulation, and infiltration of immune cells. NASH patients predominantly exhibited myeloid cell accumulation, whereas primary sclerosing cholangitis patients additionally had pronounced lymphoid cell responses. Correlating to disease stage, both etiologies displayed intense IBA1 + CD16 low CD163 low macrophage aggregation in nonparenchymal areas, with a distinct spatial proximity to ductular cells. Mouse models revealed that disease-associated IBA1 + hepatic macrophages originated from bone marrow-derived monocytes. Using an unbiased, machine learning-based algorithm, IBA1 in combination with hepatocyte and ductular cell immunostaining-predicted advanced cirrhosis in human NASH, primary sclerosing cholangitis, and alcoholic hepatitis. CONCLUSIONS: Loss of hepatocytes and increased ductular reaction are tightly associated with monocyte-derived macrophage accumulation and represent the most prominent common immunological feature revealing the progression of NAFLD, primary sclerosing cholangitis, primary biliary cholangitis, and alcoholic hepatitis, suggesting IBA1 + CD163 low macrophages are key pathogenic drivers of human liver disease progression across diverse etiologies.
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Colangitis Esclerosante , Hepatitis Alcohólica , Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Humanos , Enfermedad del Hígado Graso no Alcohólico/patología , Colangitis Esclerosante/patología , Hepatitis Alcohólica/patología , Hígado/patología , Cirrosis Hepática/complicaciones , Macrófagos , Modelos Animales de EnfermedadRESUMEN
BACKGROUND AND AIMS: Pruritus is associated with multiple liver diseases, particularly those with cholestasis, but the mechanism remains incompletely understood. Our aim was to evaluate serum IL-31 as a putative biomarker of pruritus in clinical trials of an farnesoid X receptor (FXR) agonist, cilofexor, in patients with NASH, primary sclerosing cholangitis (PSC), and primary biliary cholangitis (PBC). APPROACH AND RESULTS: Serum IL-31 was measured in clinical studies of cilofexor in NASH, PSC, and PBC. In patients with PSC or PBC, baseline IL-31 was elevated compared to patients with NASH and healthy volunteers (HVs). IL-31 correlated with serum bile acids among patients with NASH, PBC, and PSC. Baseline IL-31 levels in PSC and PBC were positively correlated with Visual Analog Scale for pruritus and 5-D itch scores. In patients with NASH, cilofexor dose-dependently increased IL-31 from Week (W)1 to W24. In patients with NASH receiving cilofexor 100 mg, IL-31 was higher in those with Grade 2-3 pruritus adverse events (AEs) than those with Grade 0-1 pruritus AEs. IL-31 weakly correlated with C4 at baseline in patients with NASH, and among those receiving cilofexor 100 mg, changes in IL-31 and C4 from baseline to W24 were negatively correlated. IL-31 messenger RNA (mRNA) was elevated in hepatocytes from patients with PSC and NASH compared to HVs. In a humanized liver murine model, obeticholic acid increased IL-31 mRNA expression in human hepatocytes and serum levels of human IL-31. CONCLUSIONS: IL-31 levels correlate with pruritus in patients with cholestatic disease and NASH, with FXR agonist therapy resulting in higher serum levels in the latter group. IL-31 appears to derive in part from increased hepatocyte expression. These findings have therapeutic implications for patients with liver disease and pruritus.
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Colestasis , Cirrosis Hepática Biliar , Enfermedades Metabólicas , Enfermedad del Hígado Graso no Alcohólico , Humanos , Animales , Ratones , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Colestasis/complicaciones , Colestasis/tratamiento farmacológico , Biomarcadores , Enfermedades Metabólicas/complicaciones , Prurito/tratamiento farmacológico , Prurito/etiología , Cirrosis Hepática Biliar/complicaciones , Cirrosis Hepática Biliar/tratamiento farmacológicoRESUMEN
BACKGROUND & AIMS: Chronic HBV is clinically categorized into 4 phases by a combination of serum HBV DNA levels, HBeAg status and alanine aminotransferase (ALT): immunotolerant (IT), immune-active (IA), inactive carrier (IC) and HBeAg-negative hepatitis (ENEG). Immune and virological measurements in the blood have proven useful but are insufficient to explain the interrelation between the immune system and the virus since immune dynamics differ in the blood and liver. Furthermore, the inflammatory response in the liver and parenchymal cells cannot be fully captured in blood. METHODS: Immunological composition and transcriptional profiles of core needle liver-biopsies in chronic HBV phases were compared to those of healthy controls by multiplex immunofluorescence and RNA-sequencing (n = 37 and 78, respectively) analyses. RESULTS: Irrespective of the phase-specific serological profiles, increased immune-gene expression and frequency was observed in chronic HBV compared to healthy livers. Greater transcriptomic deregulation was seen in IA and ENEG (172 vs. 243 DEGs) than in IT and IC (13 vs. 35 DEGs) livers. Interferon-stimulated genes, immune-activation and exhaustion genes (ICOS, CTLA4, PDCD1) together with chemokine genes (CXCL10, CXCL9) were significantly induced in IA and ENEG livers. Moreover, distinct immune profiles associated with ALT elevation and a more accentuated immune-exhaustion profile (CTLA4, TOX, SLAMF6, FOXP3) were observed in ENEG, which set it apart from the IA phase (LGALS9, PDCD1). Interestingly, all HBV phases showed downregulation of metabolic pathways vs. healthy livers (fatty and bile acid metabolism). Finally, increased leukocyte infiltrate correlated with serum ALT, but not with HBV DNA or viral proteins. CONCLUSION: Our comprehensive multi-parametric analysis of human livers revealed distinct inflammatory profiles and pronounced differences in intrahepatic gene profiles across all chronic HBV phases in comparison to healthy liver. LAY SUMMARY: Immunological studies on chronic HBV remain largely restricted to assessment of peripheral responses due to the limited access to the site of infection, the liver. In this study, we comprehensively analyzed livers from a well-defined cohort of patients with chronic HBV and uninfected controls with state-of-the-art techniques, and evaluated the differences in gene expression profiles and inflammation characteristics across distinct disease phases in patients with chronic HBV.
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Hepatitis B Crónica , Hepatitis B , Antígeno CTLA-4 , ADN Viral/genética , Antígenos e de la Hepatitis B , Virus de la Hepatitis B/genética , Humanos , Inflamación/genéticaRESUMEN
BACKGROUND & AIMS: We aim to describe the liver immune microenvironment by analyzing liver biopsies from patients with chronic HBV infection (CHB). Host immune cell signatures and their corresponding localization were characterized by analyzing the intrahepatic transcriptome in combination with a custom multiplex immunofluorescence panel. METHOD: Matching FFPE and fresh frozen liver biopsies were collected from immune active patients within the open-label phase IV study GS-US-174-0149. RNA-Seq was conducted on 53 CHB liver biopsies from 46 patients. Twenty-eight of the 53 samples had matched FFPE biopsies and were stained with a 12-plex panel including cell segmentation, immune and viral biomarkers. Corresponding serum samples were screened using the MSD Human V-plex Screen Service to identify peripheral correlates for the immune microenvironment. RESULTS: Using unsupervised clustering of the transcriptome, we reveal two unique liver immune signatures classified as immune high and immune low based on the quantification of the liver infiltrate gene signatures. Multiplex immunofluorescence analysis demonstrated large periportal lymphoid aggregates in immune high samples consisting of CD4 and CD8 T cells, B cells and macrophages. Differentiation of the high and low immune microenvironments was independent of HBeAg status and peripheral viral antigen levels. In addition, longitudinal analysis indicates that treatment and normalization of ALT correlates with a decrease in liver immune infiltrate and inflammation. Finally, we screened a panel of peripheral biomarkers and identified ICAM-1 and CXCL10 as biomarkers that strongly correlate with these unique immune microenvironments. CONCLUSION: These data provide a description of immune phenotypes in patients with CHB and show that immune responses are downregulated in the liver following nucleotide analogue treatment. This may have important implications for both the safety and efficacy of immune modulator programs aimed at HBV cure. LAY SUMMARY: Liver biopsies from patients with chronic hepatitis B were submitted to RNA-Seq and multiplex immunofluorescence and identified two different liver immune microenvironments: immune high and immune low. Immune high patients showed elevated immune pathways, including interferon signaling pathways, and increase presence of immune cells. Longitudinal analysis of biopsies from treatment experienced patients showed that treatment correlates with a marked decrease in inflammation and these findings may have important implications for both safety and efficacy of immune modulator programs for HBV cure.
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Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.
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Homeostasis/inmunología , Inflamación/inmunología , Interleucinas/inmunología , Factor Estimulante de Colonias de Macrófagos/inmunología , Macrófagos/inmunología , Neoplasias/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Modelos Animales de Enfermedad , Homeostasis/genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos NZB , Ratones Noqueados , Células Mieloides/inmunología , Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Geographic atrophy (GA), the advanced form of dry age-related macular degeneration (AMD), is characterized by progressive loss of retinal pigment epithelium cells and photoreceptors in the setting of characteristic extracellular deposits and remains a serious unmet medical need. While genetic predisposition to AMD is dominated by polymorphisms in complement genes, it remains unclear how complement activation contributes to retinal atrophy. Here we demonstrate that complement is activated on photoreceptor outer segments (POS) in the retina peripheral to atrophic lesions associated with GA. When exposed to human serum following outer blood-retinal barrier breakdown, POS act as potent activators of the classical and alternative complement pathway. In mouse models of retinal degeneration, classical and alternative pathway complement activation on photoreceptors contributed to the loss of photoreceptor function. This was dependent on C5a-mediated recruitment of peripheral blood monocytes but independent of resident microglia. Genetic or pharmacologic inhibition of both classical and alternative complement C3 and C5 convertases was required to reduce progressive degeneration of photoreceptor rods and cones. Our study implicates systemic classical and alternative complement proteins and peripheral blood monocytes as critical effectors of localized retinal degeneration with potential relevance for the contribution of complement activation to GA.
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Activación de Complemento/genética , Atrofia Geográfica/fisiopatología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Atrofia/patología , Activación de Complemento/fisiología , Complemento C3/genética , Complemento C3/fisiología , Complemento C4/genética , Complemento C4/fisiología , Atrofia Geográfica/genética , Humanos , Degeneración Macular/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Interleukin (IL)-22 plays protective roles in infections and in inflammatory diseases that have been linked to its meditation of innate immunity via multiple mechanisms. IL-22 binds specifically to its heterodimeric receptor, which is expressed on a variety of epithelial tissues. UTTR1147A is a recombinant fusion protein that links the human cytokine IL-22 with the Fc portion of human immunoglobulin (Ig) G4. Here, we report extensive in vitro and in vivo nonclinical studies that were conducted to characterize the pharmacological activity of UTTR1147A. The in vitro activity and potency of UTTR1147A were analyzed using primary human hepatocytes and human colonic epithelial cell lines. Assessment of in vivo efficacy was performed in a mouse colitis model and by measuring relevant pharmacodynamic biomarkers, including antimicrobial peptides REG3A/ß, serum amyloid protein A (SAA) and lipopolysaccharide binding protein (LBP). The pharmacokinetic and pharmacodynamic characteristics of UTTR1147A were assessed in healthy mice, rats and cynomolgus monkeys. UTTR1147A induced STAT3 activation through binding to IL-22 receptor expressed in primary human hepatocytes and human colon cell lines. In both, activation occurred in a concentration-dependent manner with similar potencies. In the mouse colitis model, murine IL-22Fc- (muIL-22Fc) treated groups at doses of 1.25⯵g and above had statistically lower average histologic colitis scores compared to the control treated group. Administration of muIL-22Fc or UTTR1147A was associated with a dose-dependent induction of PD markers REG3ß and SAA in rodents as well as REG3A, SAA and LBP in cynomolgus monkeys. The combined data confirm pharmacological activity of IL-22Fc and support potential regenerative and protective mechanisms in epithelial tissues.
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Inmunoglobulina G/metabolismo , Interleucinas/metabolismo , Animales , Área Bajo la Curva , Línea Celular , Colitis/inducido químicamente , Colitis/terapia , Citocinas , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes , Interleucina-22RESUMEN
Given the frequency with which MAP kinase signaling is dysregulated in cancer, much effort has been focused on inhibiting RAS signaling for therapeutic benefit. KSR1, a pseudokinase that interacts with RAF, is a potential target; it was originally cloned in screens for suppressors of constitutively active RAS, and its deletion prevents RAS-mediated transformation of mouse embryonic fibroblasts. In this work, we used a genetically engineered mouse model of pancreatic cancer to assess whether KSR1 deletion would influence tumor development in the setting of oncogenic RAS. We found that Ksr1-/- mice on this background had a modest but significant improvement in all-cause morbidity compared to Ksr1+/+ and Ksr1+/- cohorts. Ksr1-/- mice, however, still developed tumors, and precursor pancreatic intraepithelial neoplastic (PanIN) lesions were detected within a similar timeframe compared to Ksr1+/+ mice. No significant differences in pERK expression or in proliferation were noted. RNA sequencing also did not reveal any unique genetic signature in Ksr1-/- tumors. Further studies will be needed to determine whether and in what settings KSR inhibition may be clinically useful.
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Eliminación de Gen , Homocigoto , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Quinasas/deficiencia , Proteínas Quinasas/genética , Proteínas ras/metabolismo , Adenocarcinoma/complicaciones , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Carcinogénesis/genética , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Morbilidad , Neoplasias Pancreáticas/complicaciones , Neoplasias Pancreáticas/metabolismo , Prolapso Rectal/complicaciones , Prolapso Rectal/prevención & control , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND AND AIMS: The αEß7 integrin is crucial for retention of T lymphocytes at mucosal surfaces through its interaction with E-cadherin. Pathogenic or protective functions of these cells during human intestinal inflammation, such as ulcerative colitis [UC], have not previously been defined, with understanding largely derived from animal model data. Defining this phenotype in human samples is important for understanding UC pathogenesis and is of translational importance for therapeutic targeting of αEß7-E-cadherin interactions. METHODS: αEß7+ and αEß7- colonic T cell localization, inflammatory cytokine production and expression of regulatory T cell-associated markers were evaluated in cohorts of control subjects and patients with active UC by immunohistochemistry, flow cytometry and real-time PCR of FACS-purified cell populations. RESULTS: CD4+αEß7+ T lymphocytes from both healthy controls and UC patients had lower expression of regulatory T cell-associated genes, including FOXP3, IL-10, CTLA-4 and ICOS in comparison with CD4+αEß7- T lymphocytes. In UC, CD4+αEß7+ lymphocytes expressed higher levels of IFNγ and TNFα in comparison with CD4+αEß7- lymphocytes. Additionally the CD4+αEß7+ subset was enriched for Th17 cells and the recently described Th17/Th1 subset co-expressing both IL-17A and IFNγ, both of which were found at higher frequencies in UC compared to control. CONCLUSION: αEß7 integrin expression on human colonic CD4+ T cells was associated with increased production of pro-inflammatory Th1, Th17 and Th17/Th1 cytokines, with reduced expression of regulatory T cell-associated markers. These data suggest colonic CD4+αEß7+ T cells are pro-inflammatory and may play a role in UC pathobiology.
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Linfocitos T CD4-Positivos/inmunología , Colitis Ulcerosa/inmunología , Colon/citología , Integrinas/inmunología , Adulto , Anciano , Estudios de Casos y Controles , Colitis Ulcerosa/metabolismo , Colon/inmunología , Citocinas/metabolismo , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
Age-related macular degeneration (AMD), a leading cause of vision impairment in the ageing population, is characterized by irreversible loss of retinal pigment epithelial (RPE) cells and photoreceptors and can be associated with choroidal neovascularization. Mononuclear phagocytes are often present in AMD lesions, but the processes that direct myeloid cell recruitment remain unclear. Here, we identify IL-33 as a key regulator of inflammation and photoreceptor degeneration after retina stress or injury. IL-33(+) Müller cells were more abundant and IL-33 cytokine was elevated in advanced AMD cases compared with age-matched controls with no AMD. In rodents, retina stress resulted in release of bioactive IL-33 that in turn increased inflammatory chemokine and cytokine expression in activated Müller cells. Deletion of ST2, the IL-33 receptor α chain, or treatment with a soluble IL-33 decoy receptor significantly reduced release of inflammatory mediators from Müller cells, inhibited accumulation of mononuclear phagocytes in the outer retina, and protected photoreceptor rods and cones after a retina insult. This study demonstrates a central role for IL-33 in regulating mononuclear phagocyte recruitment to the photoreceptor layer and positions IL-33 signaling as a potential therapeutic target in macular degenerative diseases.
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Inmunidad Innata , Interleucina-33/metabolismo , Degeneración Macular/inmunología , Anciano , Anciano de 80 o más Años , Animales , Estudios de Casos y Controles , Núcleo Celular/inmunología , Citocinas/metabolismo , Células Ependimogliales/inmunología , Células Ependimogliales/patología , Femenino , Humanos , Técnicas In Vitro , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33/química , Interleucina-33/deficiencia , Interleucina-33/genética , Mácula Lútea/inmunología , Mácula Lútea/patología , Degeneración Macular/genética , Degeneración Macular/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Procesamiento Proteico-Postraduccional , Ratas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Interleucina/deficiencia , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/patologíaRESUMEN
BACKGROUND & AIMS: Etrolizumab is a humanized monoclonal antibody against the ß7 integrin subunit that has shown efficacy vs placebo in patients with moderate to severely active ulcerative colitis (UC). Patients with colon tissues that expressed high levels of the integrin αE gene (ITGAE) appeared to have the best response. We compared differences in colonic expression of ITGAE and other genes between patients who achieved clinical remission with etrolizumab vs those who did. METHODS: We performed a retrospective analysis of data collected from 110 patients with UC who participated in a phase 2 placebo-controlled trial of etrolizumab, as well as from 21 patients with UC or without inflammatory bowel disease (controls) enrolled in an observational study at a separate site. Colon biopsies were collected from patients in both studies and analyzed by immunohistochemistry and gene expression profiling. Mononuclear cells were isolated and analyzed by flow cytometry. We identified biomarkers associated with response to etrolizumab. In the placebo-controlled trial, clinical remission was defined as total Mayo Clinic Score ≤2, with no individual subscore >1, and mucosal healing was defined as endoscopic score ≤1. RESULTS: Colon tissues collected at baseline from patients who had a clinical response to etrolizumab expressed higher levels of T-cell-associated genes than patients who did not respond (P < .05). Colonic CD4(+) integrin αE(+) cells from patients with UC expressed higher levels of granzyme A messenger RNA (GZMA mRNA) than CD4(+) αE(-) cells (P < .0001); granzyme A and integrin αE protein were detected in the same cells. Of patients receiving 100 mg etrolizumab, a higher proportion of those with high levels of GZMA mRNA (41%) or ITGAE mRNA (38%) than those with low levels of GZMA (6%) or ITGAE mRNA (13%) achieved clinical remission (P < .05) and mucosal healing (41% GZMA(high) vs 19% GZMA(low) and 44% ITGAE(high) vs 19% ITGAE(low)). Compared with ITGAE(low) and GZMA(low) patients, patients with ITGAE(high) and GZMA(high) had higher baseline numbers of epithelial crypt-associated integrin αE(+) cells (P < .01 for both), but a smaller number of crypt-associated integrin αE(+) cells after etrolizumab treatment (P < .05 for both). After 10 weeks of etrolizumab treatment, expression of genes associated with T-cell activation and genes encoding inflammatory cytokines decreased by 40%-80% from baseline (P < .05) in patients with colon tissues expressing high levels of GZMA at baseline. CONCLUSIONS: Levels of GZMA and ITGAE mRNAs in colon tissues can identify patients with UC who are most likely to benefit from etrolizumab; expression levels decrease with etrolizumab administration in biomarker(high) patients. Larger, prospective studies of markers are needed to assess their clinical value.
Asunto(s)
Antiinflamatorios/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos CD/metabolismo , Colitis Ulcerosa/tratamiento farmacológico , Colon/efectos de los fármacos , Fármacos Gastrointestinales/uso terapéutico , Granzimas/metabolismo , Cadenas alfa de Integrinas/metabolismo , Antígenos CD/genética , Biopsia , Ensayos Clínicos Fase II como Asunto , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/enzimología , Colitis Ulcerosa/genética , Colon/enzimología , Colon/patología , Perfilación de la Expresión Génica/métodos , Granzimas/genética , Humanos , Inmunohistoquímica , Cadenas alfa de Integrinas/genética , Valor Predictivo de las Pruebas , ARN Mensajero/metabolismo , Ensayos Clínicos Controlados Aleatorios como Asunto , Inducción de Remisión , Estudios Retrospectivos , Factores de Tiempo , Resultado del Tratamiento , Cicatrización de Heridas/efectos de los fármacosRESUMEN
T helper 1 (Th1) cell-associated immunity exacerbates ileitis induced by oral Toxoplasma gondii infection. We show here that attenuated ileitis observed in interleukin-22 (IL-22)-deficient mice was associated with reduced production of Th1-cell-promoting IL-18. IL-22 not only augmented the expression of Il18 mRNA and inactive precursor protein (proIL-18) in intestinal epithelial cells after T. gondii or Citrobacter rodentium infection, but also maintained the homeostatic amount of proIL-18 in the ileum. IL-22, however, did not induce the processing to active IL-18, suggesting a two-step regulation of IL-18 in these cells. Although IL-18 exerted pathogenic functions during ileitis triggered by T. gondii, it was required for host defense against C. rodentium. Conversely, IL-18 was required for the expression of IL-22 in innate lymphoid cells (ILCs) upon T. gondii infection. Our results define IL-18 as an IL-22 target gene in epithelial cells and describe a complex mutual regulation of both cytokines during intestinal infection.
Asunto(s)
Infecciones por Enterobacteriaceae/inmunología , Interleucina-18/inmunología , Interleucinas/inmunología , Mucosa Intestinal/inmunología , Toxoplasmosis/inmunología , Animales , Células Cultivadas , Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/microbiología , Células Epiteliales/inmunología , Ileítis/inmunología , Ileítis/microbiología , Ileítis/parasitología , Íleon/inmunología , Íleon/microbiología , Íleon/parasitología , Inflamación/inmunología , Interferón gamma/biosíntesis , Interleucina-18/biosíntesis , Interleucinas/genética , Mucosa Intestinal/microbiología , Mucosa Intestinal/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Técnicas de Cultivo de Órganos , ARN Mensajero/biosíntesis , Células TH1/inmunología , Toxoplasma/inmunología , Toxoplasmosis/parasitología , Regulación hacia Arriba , Interleucina-22RESUMEN
T-helper type 17 (TH17) cells that produce the cytokines interleukin-17A (IL-17A) and IL-17F are implicated in the pathogenesis of several autoimmune diseases. The differentiation of TH17 cells is regulated by transcription factors such as RORγt, but post-translational mechanisms preventing the rampant production of pro-inflammatory IL-17A have received less attention. Here we show that the deubiquitylating enzyme DUBA is a negative regulator of IL-17A production in T cells. Mice with DUBA-deficient T cells developed exacerbated inflammation in the small intestine after challenge with anti-CD3 antibodies. DUBA interacted with the ubiquitin ligase UBR5, which suppressed DUBA abundance in naive T cells. DUBA accumulated in activated T cells and stabilized UBR5, which then ubiquitylated RORγt in response to TGF-ß signalling. Our data identify DUBA as a cell-intrinsic suppressor of IL-17 production.