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1.
Proc Natl Acad Sci U S A ; 118(28)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260399

RESUMO

Forward genetic studies use meiotic mapping to adduce evidence that a particular mutation, normally induced by a germline mutagen, is causative of a particular phenotype. Particularly in small pedigrees, cosegregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the identification of mutations causing immune phenotypes in mice by creating Candidate Explorer (CE), a machine-learning software program that integrates 67 features of genetic mapping data into a single numeric score, mathematically convertible to the probability of verification of any putative mutation-phenotype association. At this time, CE has evaluated putative mutation-phenotype associations arising from screening damaging mutations in ∼55% of mouse genes for effects on flow cytometry measurements of immune cells in the blood. CE has therefore identified more than half of genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.


Assuntos
Mutação em Linhagem Germinativa/genética , Leucócitos/metabolismo , Aprendizado de Máquina , Meiose/genética , Algoritmos , Animais , Automação , Feminino , Citometria de Fluxo , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Probabilidade , Reprodutibilidade dos Testes , Software
2.
Proc Natl Acad Sci U S A ; 116(23): 11380-11389, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31097594

RESUMO

LPS-responsive beige-like anchor (LRBA) protein deficiency in humans causes immune dysregulation resulting in autoimmunity, inflammatory bowel disease (IBD), hypogammaglobulinemia, regulatory T (Treg) cell defects, and B cell functional defects, but the cellular and molecular mechanisms responsible are incompletely understood. In an ongoing forward genetic screen for N-ethyl-N-nitrosourea (ENU)-induced mutations that increase susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice, we identified two nonsense mutations in Lrba Although Treg cells have been a main focus in LRBA research to date, we found that dendritic cells (DCs) contribute significantly to DSS-induced intestinal inflammation in LRBA-deficient mice. Lrba-/- DCs exhibited excessive IRF3/7- and PI3K/mTORC1-dependent signaling and type I IFN production in response to the stimulation of the Toll-like receptors (TLRs) 3, TLR7, and TLR9. Substantial reductions in cytokine expression and sensitivity to DSS in LRBA-deficient mice were caused by knockout of Unc93b1, a chaperone necessary for trafficking of TLR3, TLR7, and TLR9 to endosomes. Our data support a function for LRBA in limiting endosomal TLR signaling and consequent intestinal inflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Colite/metabolismo , Endossomos/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T Reguladores/metabolismo , Animais , Autoimunidade/fisiologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Colite/induzido quimicamente , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Sulfato de Dextrana/farmacologia , Feminino , Inflamação/metabolismo , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos
3.
Proc Natl Acad Sci U S A ; 115(49): E11523-E11531, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30442666

RESUMO

The SMCR8-WDR41-C9ORF72 complex is a regulator of autophagy and lysosomal function. Autoimmunity and inflammatory disease have been ascribed to loss-of-function mutations of Smcr8 or C9orf72 in mice. In humans, autoimmunity has been reported to precede amyotrophic lateral sclerosis caused by mutations of C9ORF72 However, the cellular and molecular mechanisms underlying autoimmunity and inflammation caused by C9ORF72 or SMCR8 deficiencies remain unknown. Here, we show that splenomegaly, lymphadenopathy, and activated circulating T cells observed in Smcr8-/- mice were rescued by triple knockout of the endosomal Toll-like receptors (TLRs) TLR3, TLR7, and TLR9. Myeloid cells from Smcr8-/- mice produced excessive inflammatory cytokines in response to endocytosed TLR3, TLR7, or TLR9 ligands administered in the growth medium and in response to TLR2 or TLR4 ligands internalized by phagocytosis. These defects likely stem from prolonged TLR signaling caused by accumulation of LysoTracker-positive vesicles and by delayed phagosome maturation, both of which were observed in Smcr8-/- macrophages. Smcr8-/- mice also showed elevated susceptibility to dextran sodium sulfate-induced colitis, which was not associated with increased TLR3, TLR7, or TLR9 signaling. Deficiency of WDR41 phenocopied loss of SMCR8. Our findings provide evidence that excessive endosomal TLR signaling resulting from prolonged ligand-receptor contact causes inflammatory disease in SMCR8-deficient mice.


Assuntos
Proteína C9orf72/metabolismo , Proteínas de Transporte/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Receptores Toll-Like/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Proteínas Relacionadas à Autofagia , Proteína C9orf72/genética , Proteínas de Transporte/genética , Colite/induzido quimicamente , Sulfato de Dextrana , Regulação da Expressão Gênica , Hematopoese/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Mutação , Transdução de Sinais/imunologia , Receptores Toll-Like/genética
4.
Proc Natl Acad Sci U S A ; 114(7): E1273-E1281, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28137860

RESUMO

Creatine, a nitrogenous organic acid, replenishes cytoplasmic ATP at the expense of mitochondrial ATP via the phosphocreatine shuttle. Creatine levels are maintained by diet and endogenous synthesis from arginine and glycine. Glycine amidinotransferase (GATM) catalyzes the rate-limiting step of creatine biosynthesis: the transfer of an amidino group from arginine to glycine to form ornithine and guanidinoacetate. We screened 36,530 third-generation germline mutant mice derived from N-ethyl-N-nitrosourea-mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). Among 27 colitis susceptibility phenotypes identified and mapped, one was strongly correlated with a missense mutation in Gatm in a recessive model of inheritance, and causation was confirmed by CRISPR/Cas9 gene targeting. Supplementation of homozygous Gatm mutants with exogenous creatine ameliorated the colitis phenotype. CRISPR/Cas9-targeted (Gatmc/c ) mice displayed a normal peripheral immune response and immune cell homeostasis. However, the intestinal epithelium of the Gatmc/c mice displayed increased cell death and decreased proliferation during DSS treatment. In addition, Gatmc/c colonocytes showed increased metabolic stress in response to DSS with higher levels of phospho-AMPK and lower levels of phosphorylation of mammalian target of rapamycin (phospho-mTOR). These findings establish an in vivo requirement for rapid replenishment of cytoplasmic ATP within colonic epithelial cells in the maintenance of the mucosal barrier after injury.


Assuntos
Colite/prevenção & controle , Creatina/farmacologia , Homeostase/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Amidinotransferases/genética , Amidinotransferases/metabolismo , Animais , Sistemas CRISPR-Cas , Colite/genética , Colite/metabolismo , Colo/efeitos dos fármacos , Colo/metabolismo , Creatina/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Mucosa Intestinal/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Substâncias Protetoras/metabolismo , Substâncias Protetoras/farmacologia
5.
Proc Natl Acad Sci U S A ; 112(5): E440-9, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25605905

RESUMO

With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.


Assuntos
Mutação Puntual , Alelos , Animais , Feminino , Genes Letais , Ligação Genética , Masculino , Camundongos , Linhagem , Fenótipo , Locos de Características Quantitativas
6.
Nat Commun ; 14(1): 3652, 2023 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-37339972

RESUMO

A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. Using a forward genetic screen, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Trans-Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells, leading to a decrease in antimicrobial peptides and more penetrable mucus layer. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficient colonocytes have a common deficiency of several critical glycosylation enzymes. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo.


Assuntos
Mucosa Intestinal , Intestinos , Proteínas de Membrana , Animais , Camundongos , Microbioma Gastrointestinal , Glicosilação , Células Caliciformes/metabolismo , Complexo de Golgi/metabolismo , Homeostase , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestinos/metabolismo , Proteínas de Membrana/metabolismo , Muco , Celulas de Paneth/metabolismo
7.
J Invest Dermatol ; 143(7): 1138-1146.e12, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36708947

RESUMO

Morphea is characterized by initial inflammation followed by fibrosis of the skin and soft tissue. Despite its substantial morbidity, the pathogenesis of morphea is poorly studied. Previous work showed that CXCR3 ligands CXCL9 and CXCL10 are highly upregulated in the sera and lesional skin of patients with morphea. We found that an early inflammatory subcutaneous bleomycin mouse model of dermal fibrosis mirrors the clinical, histological, and immune dysregulation observed in human morphea. We used this model to examine the role of the CXCR3 chemokine axis in the pathogenesis of cutaneous fibrosis. Using the REX3 (Reporting the Expression of CXCR3 ligands) mice, we characterized which cells produce CXCR3 ligands over time. We found that fibroblasts contribute the bulk of CXCL9-RFP and CXCL10-BFP by percentage, whereas macrophages produce high amounts on a per-cell basis. To determine whether these chemokines are mechanistically involved in pathogenesis, we treated Cxcl9-, Cxcl10-, or Cxcr3-deficient mice with bleomycin and found that fibrosis is dependent on CXCL9 and CXCR3. Addition of recombinant CXCL9 but not CXCL10 to cultured mouse fibroblasts induced Col1a1 mRNA expression, indicating that the chemokine itself contributes to fibrosis. Taken together, our studies provide evidence that CXCL9 and its receptor CXCR3 are functionally required for inflammatory fibrosis.


Assuntos
Dermatite , Esclerodermia Localizada , Humanos , Animais , Camundongos , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Regulação para Cima , Ligantes , Quimiocina CXCL9/genética , Quimiocina CXCL9/metabolismo , Fibrose , Inflamação , Fibroblastos/metabolismo , Bleomicina/toxicidade , Receptores CXCR3/genética , Receptores CXCR3/metabolismo
8.
J Exp Med ; 217(4)2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-31985756

RESUMO

In a forward genetic screen of N-ethyl-N-nitrosourea (ENU)-induced mutant mice for aberrant immune function, we identified mice with a syndromic disorder marked by growth retardation, diabetes, premature death, and severe lymphoid and myeloid hypoplasia together with diminished T cell-independent (TI) antibody responses. The causative mutation was in Pdia6, an essential gene encoding protein disulfide isomerase A6 (PDIA6), an oxidoreductase that functions in nascent protein folding in the endoplasmic reticulum. The immune deficiency caused by the Pdia6 mutation was, with the exception of a residual T cell developmental defect, completely rescued in irradiated wild-type recipients of PDIA6-deficient bone marrow cells, both in the absence or presence of competition. The viable hypomorphic allele uncovered in these studies reveals an essential role for PDIA6 in hematopoiesis, but one extrinsic to cells of the hematopoietic lineage. We show evidence that this role is in the proper folding of Wnt3a, BAFF, IL-7, and perhaps other factors produced by the extra-hematopoietic compartment that contribute to the development and lineage commitment of hematopoietic cells.


Assuntos
Linfócitos/imunologia , Células Mieloides/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Animais , Fator Ativador de Células B/imunologia , Linhagem Celular , Feminino , Células HEK293 , Hematopoese/imunologia , Humanos , Interleucina-7/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Proteína Wnt3A/imunologia
9.
Sci Immunol ; 5(43)2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980486

RESUMO

T follicular helper cells (TFH) participate in germinal center (GC) development and are necessary for B cell production of high-affinity, isotype-switched antibodies. In a forward genetic screen, we identified a missense mutation in Prkd2, encoding the serine/threonine kinase protein kinase D2, which caused elevated titers of immunoglobulin E (IgE) in the serum. Subsequent analysis of serum antibodies in mice with a targeted null mutation of Prkd2 demonstrated polyclonal hypergammaglobulinemia of IgE, IgG1, and IgA isotypes, which was exacerbated by the T cell-dependent humoral response to immunization. GC formation and GC B cells were increased in Prkd2-/- spleens. These effects were the result of excessive cell-autonomous TFH development caused by unrestricted Bcl6 nuclear translocation in Prkd2-/- CD4+ T cells. Prkd2 directly binds to Bcl6, and Prkd2-dependent phosphorylation of Bcl6 is necessary to constrain Bcl6 to the cytoplasm, thereby limiting TFH development. In response to immunization, Bcl6 repressed Prkd2 expression in CD4+ T cells, thereby committing them to TFH development. Thus, Prkd2 and Bcl6 form a mutually inhibitory positive feedback loop that controls the stable transition from naïve CD4+ T cells to TFH during the adaptive immune response.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas Quinases/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Animais , Linfócitos B/imunologia , Transplante de Medula Óssea , Diferenciação Celular , Feminino , Centro Germinativo/imunologia , Células HEK293 , Humanos , Imunoglobulinas/sangue , Imunoterapia Adotiva , Masculino , Camundongos Transgênicos , Mutação , Proteína Quinase D2 , Proteínas Quinases/genética
10.
J Invest Dermatol ; 139(9): 1848-1853.e1, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31445571

RESUMO

The primary goals of modern genetics are to identify disease-causing mutations and to define the functions of genes in biological processes. Two complementary approaches, reverse and forward genetics, can be used to achieve this goal. Reverse genetics is a gene-driven approach that comprises specific gene targeting followed by phenotypic assessment. Conversely, forward genetics is a phenotype-driven approach that involves the phenotypic screening of organisms with randomly induced mutations followed by subsequent identification of the causative mutations (i.e., those responsible for phenotype). In this article, we focus on how forward genetics in mice can be used to explore dermatologic disease. We outline mouse mutagenesis with the chemical N-ethyl-N-nitrosourea and the strategy used to instantaneously identify mutations that are causative of specific phenotypes. Furthermore, we summarize the types of phenotypic screens that can be performed to explore various aspects of dermatologic disease.


Assuntos
Testes Genéticos/métodos , Projetos de Pesquisa , Dermatopatias/genética , Animais , Cruzamento/métodos , Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Mutagênese/efeitos dos fármacos , Mutagênicos/toxicidade , Mutação/efeitos dos fármacos , Fenótipo , Transdução de Sinais/genética , Pele/patologia , Dermatopatias/diagnóstico , Dermatopatias/patologia
11.
Science ; 364(6440)2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31073040

RESUMO

Precise control of Wnt signaling is necessary for immune system development. In this study, we detected severely impaired development of all lymphoid lineages in mice, resulting from an N-ethyl-N-nitrosourea-induced mutation in the limb region 1-like gene (Lmbr1l), which encodes a membrane-spanning protein with no previously described function in immunity. The interaction of LMBR1L with glycoprotein 78 (GP78) and ubiquitin-associated domain-containing protein 2 (UBAC2) attenuated Wnt signaling in lymphocytes by preventing the maturation of FZD6 and LRP6 through ubiquitination within the endoplasmic reticulum and by stabilizing "destruction complex" proteins. LMBR1L-deficient T cells exhibited hallmarks of Wnt/ß-catenin activation and underwent apoptotic cell death in response to proliferative stimuli. LMBR1L has an essential function during lymphopoiesis and lymphoid activation, acting as a negative regulator of the Wnt/ß-catenin pathway.


Assuntos
Linfopoese/genética , Receptores de Superfície Celular/fisiologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptores de Superfície Celular/genética
12.
Dis Model Mech ; 11(12)2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30563851

RESUMO

Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) is a member of the basic helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) transcription factor family. ARNT2 heterodimerizes with several members of the family, including single-minded homolog-1 (SIM1) and neuronal PAS domain protein 4 (NPAS4), primarily in neurons of the central nervous system. We screened 64,424 third-generation germline mutant mice derived from N-ethyl-N-nitrosourea (ENU)-mutagenized great-grandsires for weight abnormalities. Among 17 elevated body weight phenotypes identified and mapped, one strongly correlated with an induced missense mutation in Arnt2 using a semidominant model of inheritance. Causation was confirmed by CRISPR/Cas9 gene targeting to recapitulate the original ENU allele, specifying Arg74Cys (R74C). The CRISPR/Cas9-targeted (Arnt2R74C/R74C) mice demonstrated hyperphagia and increased adiposity as well as hepatic steatosis and abnormalities in glucose homeostasis. The mutant ARNT2 protein showed decreased transcriptional activity when coexpressed with SIM1. These findings establish a requirement for ARNT2-dependent genes in the maintenance of the homeostatic feeding response, necessary for prevention of obesity and obesity-related diseases.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diabetes Mellitus/genética , Fígado Gorduroso/genética , Predisposição Genética para Doença , Hiperfagia/genética , Mutação/genética , Obesidade/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Peso Corporal , Etilnitrosoureia , Feminino , Glucose/metabolismo , Células HEK293 , Homeostase , Homozigoto , Humanos , Hipotálamo/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Transcrição Gênica
13.
Dis Model Mech ; 11(9)2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30279225

RESUMO

Myosin ID (MYO1D) is a member of the class I myosin family. We screened 48,649 third generation (G3) germline mutant mice derived from N-ethyl-N-nitrosourea-mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). We found and validated mutations in Myo1d as a cause of increased susceptibility to DSS-induced colitis. MYO1D is produced in the intestinal epithelium, and the colitis phenotype is dependent on the nonhematopoietic compartment of the mouse. Moreover, MYO1D appears to couple cytoskeletal elements to lipid in an ATP-dependent manner. These findings demonstrate that MYO1D is needed to maintain epithelial integrity and protect against DSS-induced colitis.


Assuntos
Colite/metabolismo , Colite/prevenção & controle , Lipídeos de Membrana/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Diferenciação Celular , Colite/genética , Colite/patologia , Sulfato de Dextrana , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Genes Recessivos , Hematopoese , Mucosa Intestinal/patologia , Intestinos/patologia , Camundongos Endogâmicos C57BL , Mutação/genética , Fenótipo , Reprodutibilidade dos Testes
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