RESUMEN
We previously reported that NOD.c3c4 mice develop spontaneous autoimmune biliary disease (ABD) with anti-mitochondrial Abs, histopathological lesions, and autoimmune T lymphocytes similar to human primary biliary cholangitis. In this article, we demonstrate that ABD in NOD.c3c4 and related NOD ABD strains is caused by a chromosome 1 region that includes a novel mutation in polycystic kidney and hepatic disease 1 (Pkhd1). We show that a long terminal repeat element inserted into intron 35 exposes an alternative polyadenylation site, resulting in a truncated Pkhd1 transcript. A novel NOD congenic mouse expressing aberrant Pkhd1, but lacking the c3 and c4 chromosomal regions (NOD.Abd3), reproduces the immunopathological features of NOD ABD. RNA sequencing of NOD.Abd3 common bile duct early in disease demonstrates upregulation of genes involved in cholangiocyte injury/morphology and downregulation of immunoregulatory genes. Consistent with this, bone marrow chimera studies show that aberrant Pkhd1 must be expressed in the target tissue (cholangiocytes) and the immune system (bone marrow). Mutations of Pkhd1 produce biliary abnormalities in mice but have not been previously associated with autoimmunity. In this study, we eliminate clinical biliary disease by backcrossing this Pkhd1 mutation onto the C57BL/6 genetic background; thus, the NOD genetic background (which promotes autoimmunity) is essential for disease. We propose that loss of functional Pkhd1 on the NOD background produces early bile duct abnormalities, initiating a break in tolerance that leads to autoimmune cholangitis in NOD.Abd3 congenic mice. This model is important for understanding loss of tolerance to cholangiocytes and is relevant to the pathogenesis of several human cholangiopathies.
Asunto(s)
Enfermedades Autoinmunes/genética , Colangitis/genética , Diabetes Mellitus/genética , Cirrosis Hepática Biliar/genética , Mutación/genética , Receptores de Superficie Celular/genética , Animales , Quimera , Modelos Animales de Enfermedad , Antecedentes Genéticos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Secuencias Repetidas Terminales/genéticaRESUMEN
Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and Treg function, making Treg-specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rßγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D)2. The reduced affinity of IgG-(IL-2N88D)2 for the IL-2Rßγ receptor resulted in a Treg-selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D)2 induced sustained preferential activation of Tregs accompanied by a corresponding 10-14-fold increase in CD4+ and CD8+ CD25+FOXP3+ Tregs; conditions that had no effect on CD4+ or CD8+ memory effector T cells. The expanded cynomolgus Tregs had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D)2 increased Tregs while wild-type IgG-IL-2 increased NK cells in addition to Tregs. The expanded human Tregs had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and Treg-selective IL-2 that activates and expands functional Tregsin vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures.
Asunto(s)
Enfermedades Autoinmunes/terapia , Inmunoglobulina G/inmunología , Inmunoterapia/métodos , Interleucina-2/inmunología , Linfotoxina-alfa/inmunología , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Sitios de Unión , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Proliferación Celular , Metilación de ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Epigénesis Genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/química , Inmunoglobulina G/genética , Interleucina-2/administración & dosificación , Interleucina-2/química , Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2/inmunología , Activación de Linfocitos/efectos de los fármacos , Linfotoxina-alfa/administración & dosificación , Linfotoxina-alfa/química , Linfotoxina-alfa/genética , Macaca fascicularis , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Transducción de Señal , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
By congenic strain mapping using autoimmune NOD.C57BL/6J congenic mice, we demonstrated previously that the type 1 diabetes (T1D) protection associated with the insulin-dependent diabetes (Idd)10 locus on chromosome 3, originally identified by linkage analysis, was in fact due to three closely linked Idd loci: Idd10, Idd18.1, and Idd18.3. In this study, we define two additional Idd loci--Idd18.2 and Idd18.4--within the boundaries of this cluster of disease-associated genes. Idd18.2 is 1.31 Mb and contains 18 genes, including Ptpn22, which encodes a phosphatase that negatively regulates T and B cell signaling. The human ortholog of Ptpn22, PTPN22, is associated with numerous autoimmune diseases, including T1D. We, therefore, assessed Ptpn22 as a candidate for Idd18.2; resequencing of the NOD Ptpn22 allele revealed 183 single nucleotide polymorphisms with the C57BL/6J (B6) allele--6 exonic and 177 intronic. Functional studies showed higher expression of full-length Ptpn22 RNA and protein, and decreased TCR signaling in congenic strains with B6-derived Idd18.2 susceptibility alleles. The 953-kb Idd18.4 locus contains eight genes, including the candidate Cd2. The CD2 pathway is associated with the human autoimmune disease, multiple sclerosis, and mice with NOD-derived susceptibility alleles at Idd18.4 have lower CD2 expression on B cells. Furthermore, we observed that susceptibility alleles at Idd18.2 can mask the protection provided by Idd10/Cd101 or Idd18.1/Vav3 and Idd18.3. In summary, we describe two new T1D loci, Idd18.2 and Idd18.4, candidate genes within each region, and demonstrate the complex nature of genetic interactions underlying the development of T1D in the NOD mouse model.
Asunto(s)
Antígenos CD2/genética , Cromosomas de los Mamíferos/genética , Diabetes Mellitus Tipo 1/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Alelos , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Antígenos CD2/inmunología , Cromosomas de los Mamíferos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Regulación de la Expresión Génica/inmunología , Sitios Genéticos/inmunología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Datos de Secuencia Molecular , Proteína Tirosina Fosfatasa no Receptora Tipo 22/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately twofold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we show that engineered haplodeficiency of Il2 gene expression not only reduces T cell IL-2 production by twofold but also mimics the autoimmune dysregulatory effects of the naturally occurring susceptibility alleles of Il2. Reduced IL-2 production achieved by either genetic mechanism correlates with reduced function of CD4(+) CD25(+) regulatory T cells, which are critical for maintaining immune homeostasis.
Asunto(s)
Autoinmunidad/genética , Diabetes Mellitus Tipo 1/inmunología , Interleucina-2/genética , Linfocitos T Reguladores/inmunología , Alelos , Animales , Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Homeostasis/inmunología , Interleucina-2/biosíntesis , Interleucina-2/inmunología , Ratones , Ratones Congénicos , Ratones Endogámicos NOD , Linfocitos T Reguladores/metabolismo , Transcripción GenéticaRESUMEN
Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.
Asunto(s)
Interleucina-2/metabolismo , Transducción de Señal , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Biomarcadores/metabolismo , Antígeno CTLA-4/metabolismo , Relación Dosis-Respuesta a Droga , Eosinofilia/inducido químicamente , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Interleucina-2/análogos & derivados , Interleucina-2/farmacología , Subunidad alfa del Receptor de Interleucina-2/deficiencia , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Recuento de Linfocitos , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , Fenotipo , Fosforilación/efectos de los fármacos , Unión Proteica , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/farmacología , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
In the NOD mouse model of type 1 diabetes, insulin-dependent diabetes (Idd) loci control the development of insulitis and diabetes. Independently, protective alleles of Idd3/Il2 or Idd5 are able to partially protect congenic NOD mice from insulitis and diabetes, and to partially tolerize islet-specific CD8(+) T cells. However, when the two regions are combined, mice are almost completely protected, strongly suggesting the existence of genetic interactions between the two loci. Idd5 contains at least three protective subregions/causative gene candidates, Idd5.1/Ctla4, Idd5.2/Slc11a1, and Idd5.3/Acadl, yet it is unknown which of them interacts with Idd3/Il2. Through the use of a series of novel congenic strains containing the Idd3/Il2 region and different combinations of Idd5 subregion(s), we defined these genetic interactions. The combination of Idd3/Il2 and Idd5.3/Acadl was able to provide nearly complete protection from type 1 diabetes, but all three Idd5 subregions were required to protect from insulitis and fully restore self-tolerance. By backcrossing a Slc11a1 knockout allele onto the NOD genetic background, we have demonstrated that Slc11a1 is responsible for the diabetes protection resulting from Idd5.2. We also used Slc11a1 knockout-SCID and Idd5.2-SCID mice to show that both loss-of-function alleles provide protection from insulitis when expressed on the SCID host alone. These results lend further support to the hypothesis that Slc11a1 is Idd5.2.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Epistasis Genética , Sitios de Carácter Cuantitativo , Alelos , Animales , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Femenino , Predisposición Genética a la Enfermedad , Glucosa-6-Fosfatasa/inmunología , Tolerancia Inmunológica/genética , Islotes Pancreáticos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas/inmunologíaRESUMEN
Type 1 diabetes is an autoimmune disease influenced by multiple genetic loci. Although more than 20 insulin-dependent diabetes (Idd) loci have been implicated in the nonobese diabetic (NOD) mouse model, few causal gene variants have been identified. Here we show that RNA interference (RNAi) can be used to probe candidate genes in this disease model. Slc11a1 encodes a phagosomal ion transporter, Nramp1, that affects resistance to intracellular pathogens and influences antigen presentation. This gene is the strongest candidate among the 42 genes in the Idd5.2 region; a naturally occurring mutation in the protective Idd5.2 haplotype results in loss of function of the Nramp1 protein. Using lentiviral transgenesis, we generated NOD mice in which Slc11a1 is silenced by RNAi. Silencing reduced the frequency of type 1 diabetes, mimicking the protective Idd5.2 region. Our results demonstrate a role for Slc11a1 in modifying susceptibility to type 1 diabetes and illustrate that RNAi can be used to study causal genes in a mammalian model organism.
Asunto(s)
Proteínas de Transporte de Catión/genética , Diabetes Mellitus Tipo 1/genética , Predisposición Genética a la Enfermedad , Interferencia de ARN , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos NODRESUMEN
Nonobese diabetic (NOD) mice congenic for C57BL/10 (B10)-derived genes in the Idd9 region of chromosome 4 are highly protected from type 1 diabetes (T1D). Idd9 has been divided into three protective subregions (Idd9.1, 9.2, and 9.3), each of which partially prevents disease. In this study we have fine-mapped the Idd9.1 and Idd9.2 regions, revealing further genetic complexity with at least two additional subregions contributing to protection from T1D. Using the NOD sequence from bacterial artificial chromosome clones of the Idd9.1 and Idd9.2 regions as well as whole-genome sequence data recently made available, sequence polymorphisms within the regions highlight a high degree of polymorphism between the NOD and B10 strains in the Idd9 regions. Among numerous candidate genes are several with immunological importance. The Idd9.1 region has been separated into Idd9.1 and Idd9.4, with Lck remaining a candidate gene within Idd9.1. One of the Idd9.2 regions contains the candidate genes Masp2 (encoding mannan-binding lectin serine peptidase 2) and Mtor (encoding mammalian target of rapamycin). From mRNA expression analyses, we have also identified several other differentially expressed candidate genes within the Idd9.1 and Idd9.2 regions. These findings highlight that multiple, relatively small genetic effects combine and interact to produce significant changes in immune tolerance and diabetes onset.
Asunto(s)
Mapeo Cromosómico , Diabetes Mellitus Tipo 1/genética , Animales , Cromosomas de los Mamíferos , Susceptibilidad a Enfermedades , Femenino , Expresión Génica , Estudios de Asociación Genética , Haplotipos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
We previously described the NOD.c3c4 mouse, which is protected from type 1 diabetes (T1D) because of protective alleles at multiple insulin-dependent diabetes (Idd) genes, but develops autoimmune biliary disease (ABD) resembling primary biliary cirrhosis (PBC). In this paper, we characterize the NOD.ABD strain, which is genetically related to the NOD.c3c4 strain but develops both ABD and T1D. Histologically, NOD.ABD biliary disease is indistinguishable from that in NOD.c3c4 mice. The frequency of effector memory (CD44(+)CD62L(-)) and central memory (CD44(+)CD62L(+)) CD8 T cells is significantly increased in the intrahepatic lymphocyte fraction of NOD.ABD mice, and NOD.ABD CD8 T cells produce more IFN-γ and TNF-α, compared with controls. NOD.ABD splenocytes can transfer ABD and T1D to NOD.c3c4 scid mice, but only T1D to NOD scid mice, suggesting that the genetic origin of the target organ and/or its innate immune cells is critical to disease pathogenesis. The disease transfer model, importantly, shows that biliary duct damage (characteristic of PBC) and inflammation precede biliary epithelial cell proliferation. Unlike T1D where both CD4 and CD8 T cells are required for disease transfer, purified NOD.ABD CD8 T cells can transfer liver inflammation into NOD.c3c4 scid recipients, and disease transfer is ameliorated by cotransferring T regulatory cells. Unlike NOD.c3c4 mice, NOD.ABD mice do not develop anti-nuclear or anti-Smith autoantibodies; however, NOD.ABD mice do develop the antipyruvate dehydrogenase Abs typical of human PBC. The NOD.ABD strain is a model of immune dysregulation affecting two organ systems, most likely by mechanisms that do not completely coincide.
Asunto(s)
Conductos Biliares/inmunología , Conductos Biliares/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Cirrosis Hepática Biliar/inmunología , Cirrosis Hepática Biliar/patología , Traslado Adoptivo , Animales , Cruzamientos Genéticos , Diabetes Mellitus Tipo 1/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Células K562 , Cirrosis Hepática Biliar/genética , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Especificidad de Órganos/genética , Especificidad de Órganos/inmunologíaRESUMEN
Environmental and genetic factors define the susceptibility of an individual to autoimmune disease. Although common genetic pathways affect general immunological tolerance mechanisms in autoimmunity, the effects of such genes could vary under distinct immune challenges within different tissues. In this study, we demonstrate this by observing that autoimmune type 1 diabetes-protective haplotypes at the insulin-dependent diabetes susceptibility region 10 (Idd10) introgressed from chromosome 3 of C57BL/6 (B6) and A/J mice onto the NOD background increase the severity of autoimmune primary biliary cirrhosis induced by infection with Novosphingobium aromaticivorans, a ubiquitous alphaproteobacterium, when compared with mice having the NOD and NOD.CAST Idd10 type 1 diabetes-susceptible haplotypes. Substantially increased liver pathology in mice having the B6 and A/J Idd10 haplotypes correlates with reduced expression of CD101 on dendritic cells, macrophages, and granulocytes following infection, delayed clearance of N. aromaticivorans, and the promotion of overzealous IFN-γ- and IL-17-dominated T cell responses essential for the adoptive transfer of liver lesions. CD101-knockout mice generated on the B6 background also exhibit substantially more severe N. aromaticivorans-induced liver disease correlating with increased IFN-γ and IL-17 responses compared with wild-type mice. These data strongly support the hypothesis that allelic variation of the Cd101 gene, located in the Idd10 region, alters the severity of liver autoimmunity induced by N. aromaticivorans.
Asunto(s)
Antígenos CD/genética , Predisposición Genética a la Enfermedad/genética , Infecciones por Bacterias Gramnegativas/inmunología , Hepatitis Autoinmune/inmunología , Cirrosis Hepática Biliar/inmunología , Sphingomonadaceae/inmunología , Animales , Antígenos CD/inmunología , Femenino , Infecciones por Bacterias Gramnegativas/genética , Infecciones por Bacterias Gramnegativas/patología , Hepatitis Autoinmune/genética , Hepatitis Autoinmune/microbiología , Cirrosis Hepática Biliar/genética , Cirrosis Hepática Biliar/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Transgénicos , Índice de Severidad de la EnfermedadRESUMEN
We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 mice accounts for the protection from type 1 diabetes (T1D) provided by the insulin-dependent diabetes susceptibility region 10 (Idd10), a <1 Mb region on mouse chromosome 3. In this study, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including Foxp3(+) regulatory CD4(+) T cells, CD11c(+) dendritic cells, and Gr1(+) myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Because CD101 has been associated with regulatory T and Ag presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.
Asunto(s)
Antígenos CD/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Animales , Antígenos CD/biosíntesis , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Células CHO , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Genotipo , Haplotipos , Ratones , Ratones Congénicos , Ratones Endogámicos A , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Datos de Secuencia MolecularRESUMEN
Primary biliary cirrhosis (PBC) is an autoimmune disease with a strong genetic component characterized by biliary ductular inflammation with eventual liver cirrhosis. The serologic hallmark of PBC is antimitochondrial antibodies that react with the pyruvate dehydrogenase complex, targeting the inner lipoyl domain of the E2 subunit (anti-PDC-E2). Herein we demonstrate that NOD.c3c4 mice congenically derived from the nonobese diabetic strain develop an autoimmune biliary disease (ABD) that models human PBC. NOD.c3c4 (at 9-10 wk, before significant biliary pathology) develop antibodies to PDC-E2 that are specific for the inner lipoyl domain. Affected areas of biliary epithelium are infiltrated with CD3+, CD4+, and CD8+ T cells, and treatment of NOD.c3c4 mice with monoclonal antibody to CD3 protects from ABD. Furthermore, NOD.c3c4-scid mice develop disease after adoptive transfer of splenocytes or CD4+ T cells, demonstrating a central role for T cells in pathogenesis. Histological analysis reveals destructive cholangitis, granuloma formation, and eosinophilic infiltration as seen in PBC, although, unlike PBC, the extrahepatic biliary ducts are also affected. Using a congenic mapping approach, we define the first ABD (Abd) locus, Abd1. These results identify the NOD.c3c4 mouse as the first spontaneous mouse model of PBC.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/inmunología , Cirrosis Hepática Biliar/inmunología , Proteínas Mitocondriales/inmunología , Traslado Adoptivo , Animales , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Colangitis/genética , Colangitis/inmunología , Colangitis/patología , Mapeo Cromosómico , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/genética , Modelos Animales de Enfermedad , Granuloma/genética , Granuloma/inmunología , Granuloma/patología , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Cirrosis Hepática Biliar/genética , Cirrosis Hepática Biliar/patología , Cirrosis Hepática Experimental/genética , Cirrosis Hepática Experimental/inmunología , Cirrosis Hepática Experimental/patología , Ratones , Ratones Congénicos , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Proteínas Mitocondriales/genética , Estructura Terciaria de Proteína/genética , Sitios de Carácter Cuantitativo/genética , Sitios de Carácter Cuantitativo/inmunologíaRESUMEN
SAR studies of the substitution effect on the central phenyl ring of the biphenyl scaffold were carried out using anacetrapib (9a) as the benchmark. The results revealed that the new analogs with substitutions to replace trifluoromethyl (9a) had a significant impact on CETP inhibition in vitro. In fact, analogs with some small groups were as potent or more potent than the CF(3) derivative for CETP inhibition. Five of these new analogs raised HDL-C significantly (>20mg/dL). None of them however was better than anacetrapib in vivo. The synthesis and biological evaluation of these CETP inhibitors are described.
Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Oxazolidinonas/farmacología , Animales , Química Farmacéutica/métodos , HDL-Colesterol/metabolismo , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Transgénicos , Modelos Químicos , Relación Estructura-ActividadRESUMEN
We have used the public sequencing and annotation of the mouse genome to delimit the previously resolved type 1 diabetes (T1D) insulin-dependent diabetes (Idd)18 interval to a region on chromosome 3 that includes the immunologically relevant candidate gene, Vav3. To test the candidacy of Vav3, we developed a novel congenic strain that enabled the resolution of Idd18 to a 604-kb interval, designated Idd18.1, which contains only two annotated genes: the complete sequence of Vav3 and the last exon of the gene encoding NETRIN G1, Ntng1. Targeted sequencing of Idd18.1 in the NOD mouse strain revealed that allelic variation between NOD and C57BL/6J (B6) occurs in noncoding regions with 138 single nucleotide polymorphisms concentrated in the introns between exons 20 and 27 and immediately after the 3' untranslated region. We observed differential expression of VAV3 RNA transcripts in thymocytes when comparing congenic mouse strains with B6 or NOD alleles at Idd18.1. The T1D protection associated with B6 alleles of Idd18.1/Vav3 requires the presence of B6 protective alleles at Idd3, which are correlated with increased IL-2 production and regulatory T cell function. In the absence of B6 protective alleles at Idd3, we detected a second T1D protective B6 locus, Idd18.3, which is closely linked to, but distinct from, Idd18.1. Therefore, genetic mapping, sequencing, and gene expression evidence indicate that alteration of VAV3 expression is an etiological factor in the development of autoimmune beta-cell destruction in NOD mice. This study also demonstrates that a congenic strain mapping approach can isolate closely linked susceptibility genes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Sitios Genéticos/inmunología , Predisposición Genética a la Enfermedad , Insulina/fisiología , Ratones Congénicos , Mapeo Físico de Cromosoma/métodos , Proteínas Proto-Oncogénicas c-vav/genética , Alelos , Animales , Cruzamientos Genéticos , Diabetes Mellitus Tipo 1/patología , Modelos Animales de Enfermedad , Exones/genética , Femenino , Regulación de la Expresión Génica/inmunología , Insulina/genética , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Proteínas del Tejido Nervioso/genética , Netrinas , Proteínas Proto-Oncogénicas c-vav/biosíntesisRESUMEN
Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoEâ»/â» and low density lipoprotein receptor (LDLr)â»/â» mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETPâº/â» hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETPâº/â» mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.
Asunto(s)
Apolipoproteínas B/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/genética , LDL-Colesterol/sangre , Modelos Animales de Enfermedad , Receptores de LDL/genética , Animales , Apolipoproteínas B/sangre , Apolipoproteínas E/sangre , Apolipoproteínas E/genética , Aterosclerosis/patología , Línea Celular Tumoral , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Efecto Fundador , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hemicigoto , Humanos , Metabolismo de los Lípidos/genética , Liposomas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nanopartículas/administración & dosificación , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Receptores de LDL/metabolismo , Triglicéridos/sangreRESUMEN
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.
Asunto(s)
LDL-Colesterol/sangre , Fragmentos Fab de Inmunoglobulinas/farmacología , Receptores de LDL/química , Serina Endopeptidasas/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Fluoroinmunoensayo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Macaca mulatta , Masculino , Ratones , Ratones Transgénicos , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores de LDL/metabolismo , Serina Endopeptidasas/químicaRESUMEN
We describe structure-activity studies leading to the discovery of 2-arylbenzoxazole 3, the first in a series to raise serum high-density lipoprotein cholesterol levels in transgenic mice. Replacement of the 4-piperidinyloxy moiety with piperazinyl provided a more synthetically tractable lead, which upon optimization resulted in compound 4, an excellent inhibitor of cholesteryl ester transfer protein function with good pharmacokinetic properties and in vivo efficacy.
Asunto(s)
Acetanilidas/química , Benzoxazoles/química , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , HDL-Colesterol/sangre , Acetanilidas/síntesis química , Acetanilidas/farmacocinética , Animales , Benzoxazoles/síntesis química , Benzoxazoles/farmacocinética , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Ratones , Ratones Transgénicos , Relación Estructura-ActividadRESUMEN
Idd5.1 regulates T1D susceptibility in nonobese diabetic (NOD) mice and has two notable candidate genes, Ctla4 and Icos. Reduced expression of one of the four CTLA-4 isoforms, ligand-independent CTLA-4 (liCTLA-4), which inhibits in vitro T cell activation and cytokine production similarly to full-length CTLA-4 (flCTLA-4), has been hypothesized to increase type 1 diabetes (T1D) susceptibility. However, further support of this hypothesis is required since the Idd5.1 haplotypes of the diabetes-susceptible NOD and the resistant B10 strains differ throughout Ctla4 and Icos. Using haplotype analysis and the generation of novel Idd5.1-congenic strains that differ at the disease-associated Ctla4 exon 2 single-nucleotide polymorphism, we demonstrate that increased expression of liCTLA-4 correlates with reduced T1D susceptibility. To directly assess the ability of liCTLA-4 to modulate T1D, we generated liCTLA-4-transgenic NOD mice and compared their diabetes susceptibility to nontransgenic littermates. NOD liCTLA-4-transgenic mice were protected from T1D to the same extent as NOD.B10 Idd5.1-congenic mice, demonstrating that increased liCTLA-4 expression alone can account for disease protection. To further investigate the in vivo function of liCTLA-4, specifically whether liCTLA-4 can functionally replace flCTLA-4 in vivo, we expressed the liCTLA-4 transgene in CTLA-4(-/-) B6 mice. CTLA-4(-/-) mice expressing liCTLA-4 accumulated fewer activated effector/memory CD4(+) T cells than CTLA-4(-/-) mice and the transgenic mice were partially rescued from the multiorgan inflammation and early lethality caused by the disruption of Ctla4. These results suggest that liCTLA-4 can partially replace some functions of flCTLA-4 in vivo and that this isoform evolved to reinforce the function of flCTLA-4.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/inmunología , Diabetes Mellitus Tipo 1/genética , Linfocitos T/inmunología , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Autoinmunidad/inmunología , Antígeno CTLA-4 , Diabetes Mellitus Tipo 1/inmunología , Predisposición Genética a la Enfermedad , Haplotipos/genética , Haplotipos/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-17/biosíntesis , Interleucina-17/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Transgénicos , Polimorfismo de Nucleótido Simple/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Linfocitos T/metabolismoRESUMEN
Human genome wide association studies (GWAS) have recently identified at least four new, non-MHC-linked candidate genes or gene regions causing type one diabetes (T1D), highlighting the need for functional models to investigate how susceptibility alleles at multiple common genes interact to mediate disease. Progress in localizing genes in congenic strains of the nonobese diabetic (NOD) mouse has allowed the reproducible testing of gene functions and gene-gene interactions that can be reflected biologically as intrapathway interactions, for example, IL-2 and its receptor CD25, pathway-pathway interactions such as two signaling pathways within a cell, or cell-cell interactions. Recent studies have identified likely causal genes in two congenic intervals associated with T1D, Idd3, and Idd5, and have documented the occurrence of gene-gene interactions, including "genetic masking", involving the genes encoding the critical immune molecules IL-2 and CTLA-4. The demonstration of gene-gene interactions in congenic mouse models of T1D has major implications for the understanding of human T1D since such biological interactions are highly likely to exist for human T1D genes. Although it is difficult to detect most gene-gene interactions in a population in which susceptibility and protective alleles at many loci are randomly segregating, their existence as revealed in congenic mice reinforces the hypothesis that T1D alleles can have strong biological effects and that such genes highlight pathways to consider as targets for immune intervention.