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1.
Nature ; 577(7788): 103-108, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31827281

RESUMO

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.

3.
Front Immunol ; 10: 1433, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354696

RESUMO

Signal transducer and activator of transcription (STAT1)1 gain of function (GOF) pathogenic variants have been associated with increased levels of phosphorylated STAT1 and STAT1-dependent cellular responses. Delayed dephosphorylation was proposed as the underlying mechanism leading to the characteristically raised pSTAT1 levels. We examined the levels of STAT1 protein and message as well as rates of STAT1 phosphorylation, dephosphorylation, and degradation associated with STAT1 GOF pathogenic variants. Fresh peripheral blood mononuclear cells (PBMC) from 14 STAT1 GOF patients carrying 10 different pathogenic variants in the coiled-coil, DNA binding, and SH2 domains and healthy donors were used to study STAT1 levels and phosphorylation (pSTAT1) following IFNγ and IFNα stimulation. STAT1 protein levels were measured by flow cytometry and immunoblot. STAT1 mRNA levels were measured using quantitative reverse transcription PCR. STAT1 protein degradation was studied using cycloheximide. Patient IFNγ and IFNα induced peak pSTAT1 was higher than in healthy controls. The velocity of pSTAT1 dephosphorylation after treatment of IFNγ stimulated CD14+ monocytes with the Janus Kinase (JAK)-inhibitor ruxolitinib was significantly faster in patient cells. STAT1 protein levels in patient CD14+ monocytes and CD3+ T cells were higher than in healthy donors. There was a strong and positive correlation between CD14+ STAT1 protein levels and peak pSTAT1 levels. Patient fresh PBMC STAT1 mRNA levels were increased at rest and after 16 h of incubation. STAT1 protein degradation was similar in patient and healthy volunteer cells. Patient IFNγ receptors 1 and 2 and JAK2 levels were normal. One patient in our cohort was treated with the oral JAK inhibitor ruxolitinib. Treatment was associated with normalization of both STAT1 protein and peak pSTAT1 levels. After JAK inhibitor treatment was stopped the patient's CD14+ monocyte STAT1 protein and peak phosphorylation levels increased proportionally. These findings suggest that patients with STAT1 GOF mutations have higher levels of total STAT1 protein, leading to high levels of pSTAT1 after stimulation, despite rapid STAT1 dephosphorylation and normal degradation.

4.
J Exp Med ; 216(9): 1986-1998, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31235509

RESUMO

IL-6 excess is central to the pathogenesis of multiple inflammatory conditions and is targeted in clinical practice by immunotherapy that blocks the IL-6 receptor encoded by IL6R We describe two patients with homozygous mutations in IL6R who presented with recurrent infections, abnormal acute-phase responses, elevated IgE, eczema, and eosinophilia. This study identifies a novel primary immunodeficiency, clarifying the contribution of IL-6 to the phenotype of patients with mutations in IL6ST, STAT3, and ZNF341, genes encoding different components of the IL-6 signaling pathway, and alerts us to the potential toxicity of drugs targeting the IL-6R.

5.
Front Pediatr ; 7: 55, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024866

RESUMO

In the era of newborn screening (NBS) for severe combined immunodeficiency (SCID) and the possibility of gene therapy (GT), it is important to link SCID phenotype to the underlying genetic disease. In western countries, X-linked interleukin 2 receptor gamma chain (IL2RG) and adenosine deaminase (ADA) deficiency SCID are two of the most common types of SCID and can be treated by GT. As a challenge, both IL2RG and ADA genes are highly polymorphic and a gene-based diagnosis may be difficult if the variant is of unknown significance or if it is located in non-coding areas of the genes that are not routinely evaluated with exon-based genetic testing (e.g., introns, promoters, and the 5'and 3' untranslated regions). Therefore, it is important to extend evaluation to non-coding areas of a SCID gene if the exon-based sequencing is inconclusive and there is strong suspicion that a variant in that gene is the cause for disease. Functional studies are often required in these cases to confirm a pathogenic variant. We present here two unique examples of X-linked SCID with variable immune phenotypes, where IL2R gamma chain expression was detected and no pathogenic variant was identified on initial genetic testing. Pathogenic IL2RG variants were subsequently confirmed by functional assay of gamma chain signaling and maternal X-inactivation studies. We propose that such tests can facilitate confirmation of suspected cases of X-linked SCID in newborns when initial genetic testing is inconclusive. Early identification of pathogenic IL2RG variants is especially important to ensure eligibility for gene therapy.

7.
Front Immunol ; 10: 479, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30936877

RESUMO

Background: HOIP is the catalytic subunit of the linear ubiquitination chain assembly complex (LUBAC) that is essential for NF-κB signaling and thus proper innate and adaptive immunity. To date only one patient with HOIP deficiency has been reported with clinical characteristics that include autoinflammation, immunodeficiency, amylopectinosis, and systemic lymphangiectasia. Case: We sought to identify a genetic cause of a disease for an 8 year-old girl who presented with early-onset immune deficiency and autoinflammation. Methods: Targeted next generation sequencing of 352 immune-related genes was performed. Functional studies included transcriptome analysis, cytokine profiling, and protein analysis in patients' primary cells. Results: We identified biallelic variants in close proximity to splice sites (c.1197G>C and c.1737+3A>G) in the RNF31 gene. RNA extracted from patient cells showed alternatively spliced transcripts not present in control cells. Protein expression of HOIP and LUBAC was reduced in primary cells as shown by western blotting. Patient-derived fibroblasts demonstrated attenuated IL-6 production, while PBMCs showed higher TNF production after stimulation with proinflammatory cytokines. RNA sequencing of whole blood RNA and PBMCs demonstrated a marked transcriptome wide change including differential expression of type I interferon regulated genes. Conclusion: We report the second case of HOIP deficiency with novel compound heterozygous mutations in RNF31 and distinct clinical and molecular features. Our results expand on the clinical spectrum of HOIP deficiency and molecular signatures associated with LUBAC deficiency.

9.
J Clin Invest ; 128(7): 3071-3087, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29889099

RESUMO

Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.


Assuntos
Mutação em Linhagem Germinativa , Fator de Transcrição Ikaros/genética , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Mutação com Perda de Função , Adolescente , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos , Linfócitos B/imunologia , Criança , Pré-Escolar , Feminino , Genes Dominantes , Heterozigoto , Humanos , Fator de Transcrição Ikaros/química , Fator de Transcrição Ikaros/imunologia , Lactente , Masculino , Células Mieloides/imunologia , Linhagem , Fenótipo , Domínios Proteicos/genética , Homologia de Sequência de Aminoácidos , Linfócitos T/imunologia , Adulto Jovem
10.
Nat Commun ; 9(1): 1239, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29588478

RESUMO

Ikaros family zinc finger 1 (IKZF1) is a haematopoietic transcription factor required for mammalian B-cell development. IKZF1 deficiency also reduces plasmacytoid dendritic cell (pDC) numbers in mice, but its effects on human DC development are unknown. Here we show that heterozygous mutation of IKZF1 in human decreases pDC numbers and expands conventional DC1 (cDC1). Lenalidomide, a drug that induces proteosomal degradation of IKZF1, also decreases pDC numbers in vivo, and reduces the ratio of pDC/cDC1 differentiated from progenitor cells in vitro in a dose-dependent manner. In addition, non-classical monocytes are reduced by IKZF1 deficiency in vivo. DC and monocytes from patients with IKZF1 deficiency or lenalidomide-treated cultures secrete less IFN-α, TNF and IL-12. These results indicate that human DC development and function are regulated by IKZF1, providing further insights into the consequences of IKZF1 mutation on immune function and the mechanism of immunomodulation by lenalidomide.


Assuntos
Células Dendríticas/fisiologia , Fator de Transcrição Ikaros/fisiologia , Haploinsuficiência , Hematopoese , Humanos , Interferon-alfa/metabolismo , Interleucina-12/metabolismo , Lenalidomida
12.
Open Forum Infect Dis ; 4(4): ofx202, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29226168

RESUMO

Heterozygous STAT1 gain-of-function (GOF) mutations are associated with chronic mucocutaneous candidiasis and a broad spectrum of infectious, inflammatory, and vascular manifestations. We describe therapeutic failures with the Janus Kinase (JAK) inhibitor ruxolitinib in 2 STAT1 GOF patients with severe invasive or cutaneous fungal infections.

13.
Blood ; 130(13): 1553-1564, 2017 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-28778864

RESUMO

NF-κB signaling through its NFKB1-dependent canonical and NFKB2-dependent noncanonical pathways plays distinctive roles in a diverse range of immune processes. Recently, mutations in these 2 genes have been associated with common variable immunodeficiency (CVID). While studying patients with genetically uncharacterized primary immunodeficiencies, we detected 2 novel nonsense gain-of-function (GOF) NFKB2 mutations (E418X and R635X) in 3 patients from 2 families, and a novel missense change (S866R) in another patient. Their immunophenotype was assessed by flow cytometry and protein expression; activation of canonical and noncanonical pathways was examined in peripheral blood mononuclear cells and transfected HEK293T cells through immunoblotting, immunohistochemistry, luciferase activity, real-time polymerase chain reaction, and multiplex assays. The S866R change disrupted a C-terminal NF-κΒ2 critical site affecting protein phosphorylation and nuclear translocation, resulting in CVID with adrenocorticotropic hormone deficiency, growth hormone deficiency, and mild ectodermal dysplasia as previously described. In contrast, the nonsense mutations E418X and R635X observed in 3 patients led to constitutive nuclear localization and activation of both canonical and noncanonical NF-κΒ pathways, resulting in a combined immunodeficiency (CID) without endocrine or ectodermal manifestations. These changes were also found in 2 asymptomatic relatives. Thus, these novel NFKB2 GOF mutations produce a nonfully penetrant CID phenotype through a different pathophysiologic mechanism than previously described for mutations in NFKB2.


Assuntos
Códon sem Sentido , Subunidade p52 de NF-kappa B/genética , Imunodeficiência Combinada Severa/genética , Insuficiência Adrenal/genética , Imunodeficiência de Variável Comum/genética , Displasia Ectodérmica/genética , Hormônio do Crescimento/deficiência , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Fenótipo
14.
Sci Rep ; 7(1): 3702, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28623346

RESUMO

Syndromic primary immunodeficiencies are rare genetic disorders that affect both the immune system and other organ systems. More often, the immune defect is not the major clinical problem and is sometimes only recognized after a diagnosis has been made based on extra-immunological abnormalities. Here, we report two sibling pairs with syndromic primary immunodeficiencies that exceptionally presented with a phenotype resembling early-onset common variable immunodeficiency, while extra-immunological characteristics were not apparent at that time. Additional features not typically associated with common variable immunodeficiency were diagnosed only later, including skeletal and organ anomalies and mild facial dysmorphism. Whole exome sequencing revealed KMT2A-associated Wiedemann-Steiner syndrome in one sibling pair and their mother. In the other sibling pair, targeted testing of the known disease gene for Roifman syndrome (RNU4ATAC) provided a definite diagnosis. With this study, we underline the importance of an early-stage and thorough genetic assessment in paediatric patients with a common variable immunodeficiency phenotype, to establish a conclusive diagnosis and guide patient management. In addition, this study extends the mutational and immunophenotypical spectrum of Wiedemann-Steiner and Roifman syndromes and highlights potential directions for future pathophysiological research.


Assuntos
Agamaglobulinemia/diagnóstico , Agamaglobulinemia/imunologia , Cardiomiopatias/diagnóstico , Cardiomiopatias/imunologia , Imunodeficiência de Variável Comum/diagnóstico , Imunodeficiência de Variável Comum/imunologia , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/imunologia , Retardo Mental Ligado ao Cromossomo X/diagnóstico , Retardo Mental Ligado ao Cromossomo X/imunologia , Osteocondrodisplasias/diagnóstico , Osteocondrodisplasias/imunologia , Doenças Retinianas/diagnóstico , Doenças Retinianas/imunologia , Idade de Início , Biomarcadores , Citogenética , Diagnóstico Diferencial , Estudo de Associação Genômica Ampla , Humanos , Imunofenotipagem , Recém-Nascido , Masculino , Linhagem , Fenótipo , RNA Nuclear Pequeno/genética , Análise de Sequência de DNA , Irmãos
16.
Proc Natl Acad Sci U S A ; 113(36): 10127-32, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27559085

RESUMO

Systemic autoinflammatory diseases are caused by mutations in genes that function in innate immunity. Here, we report an autoinflammatory disease caused by loss-of-function mutations in OTULIN (FAM105B), encoding a deubiquitinase with linear linkage specificity. We identified two missense and one frameshift mutations in one Pakistani and two Turkish families with four affected patients. Patients presented with neonatal-onset fever, neutrophilic dermatitis/panniculitis, and failure to thrive, but without obvious primary immunodeficiency. HEK293 cells transfected with mutated OTULIN had decreased enzyme activity relative to cells transfected with WT OTULIN, and showed a substantial defect in the linear deubiquitination of target molecules. Stimulated patients' fibroblasts and peripheral blood mononuclear cells showed evidence for increased signaling in the canonical NF-κB pathway and accumulated linear ubiquitin aggregates. Levels of proinflammatory cytokines were significantly increased in the supernatants of stimulated primary cells and serum samples. This discovery adds to the emerging spectrum of human diseases caused by defects in the ubiquitin pathway and suggests a role for targeted cytokine therapies.


Assuntos
Alelos , Endopeptidases/genética , Fibroblastos/patologia , Doenças Hereditárias Autoinflamatórias/genética , Leucócitos Mononucleares/patologia , Mutação , Idade de Início , Criança , Pré-Escolar , Consanguinidade , Citocinas/genética , Citocinas/imunologia , Dermatite/fisiopatologia , Endopeptidases/deficiência , Endopeptidases/imunologia , Insuficiência de Crescimento/fisiopatologia , Feminino , Febre/fisiopatologia , Fibroblastos/enzimologia , Fibroblastos/imunologia , Regulação da Expressão Gênica , Células HEK293 , Doenças Hereditárias Autoinflamatórias/diagnóstico , Doenças Hereditárias Autoinflamatórias/enzimologia , Doenças Hereditárias Autoinflamatórias/patologia , Humanos , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/imunologia , Masculino , NF-kappa B/genética , NF-kappa B/imunologia , Paniculite/fisiopatologia , Linhagem , Transdução de Sinais , Ubiquitina/genética , Ubiquitina/imunologia
17.
PLoS Pathog ; 11(12): e1005293, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26679537

RESUMO

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/imunologia , Candidíase/imunologia , Infecções do Sistema Nervoso Central/imunologia , Síndromes de Imunodeficiência/imunologia , Infiltração de Neutrófilos/imunologia , Animais , Western Blotting , Proteínas Adaptadoras de Sinalização CARD/deficiência , Feminino , Citometria de Fluxo , Humanos , Síndromes de Imunodeficiência/microbiologia , Camundongos , Camundongos Knockout
19.
J Clin Immunol ; 35(4): 348-55, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25814141

RESUMO

Caspase-8 deficiency (CED) was originally described in 2002 in two pediatric patients presenting with clinical manifestations resembling autoimmune lymphoproliferative syndrome (ALPS) accompanied by infections, and T, B and NK cell defects. Since then, no new CED patients were published. Here we report two adult siblings (Pt1 and Pt2) presenting in their late thirties with pulmonary hypertension leading to lung transplant (Pt1), and a complex neurological disease leading to multiple cranial nerves palsies (Pt2) as their main manifestations. A thorough clinical and immunological evaluation was performed at the Primary Immunodeficiency Clinic at NIH, followed by whole exome sequencing. The patients had multiorgan lymphocytic infiltration and granulomas, as well as clinical signs of immune deficiency/ immune dysregulation. Both siblings carried homozygous mutations in CASP8, c.1096C > T, p.248R > W. This was the same mutation described on the previously published CED patients, to whom these new patients were likely distantly related. We report two new CED patients presenting during adulthood with life-threatening end-organ lymphocyte infiltrates affecting the lungs, liver, spleen, bone marrow and central nervous system. This phenotype broadens the clinical spectrum of manifestations associated with this disease and warrants the search of CASP8 mutations in other cohorts of patients.


Assuntos
Síndrome Linfoproliferativa Autoimune/diagnóstico , Síndrome Linfoproliferativa Autoimune/genética , Caspase 8/genética , Granuloma/patologia , Mutação , Irmãos , Adulto , Síndrome Linfoproliferativa Autoimune/complicações , Biópsia , Citocinas/biossíntese , Exoma , Feminino , Hepatomegalia/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Imunofenotipagem , Pulmão/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Esplenomegalia/diagnóstico , Tomografia Computadorizada por Raios X
20.
Science ; 345(6204): 1623-1627, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25213377

RESUMO

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory receptor found on immune cells. The consequences of mutations in CTLA4 in humans are unknown. We identified germline heterozygous mutations in CTLA4 in subjects with severe immune dysregulation from four unrelated families. Whereas Ctla4 heterozygous mice have no obvious phenotype, human CTLA4 haploinsufficiency caused dysregulation of FoxP3(+) regulatory T (Treg) cells, hyperactivation of effector T cells, and lymphocytic infiltration of target organs. Patients also exhibited progressive loss of circulating B cells, associated with an increase of predominantly autoreactive CD21(lo) B cells and accumulation of B cells in nonlymphoid organs. Inherited human CTLA4 haploinsufficiency demonstrates a critical quantitative role for CTLA-4 in governing T and B lymphocyte homeostasis.


Assuntos
Antígeno CTLA-4/genética , Mutação em Linhagem Germinativa , Haploinsuficiência , Doenças do Sistema Imunitário/genética , Imunidade/genética , Adulto , Animais , Linfócitos B/imunologia , Feminino , Fatores de Transcrição Forkhead/imunologia , Humanos , Masculino , Camundongos , Camundongos Mutantes , Linhagem , Linfócitos T Reguladores/imunologia , Adulto Jovem
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