RESUMO
In infantile nephropathic cystinosis, variants of the CTNS gene cause accumulation of cystine in lysosomes, causing progressive damage to most organs. Patients usually present before 1 year of age with signs of renal Fanconi syndrome. Cysteamine therapy allows cystine clearance from lysosomes and delays kidney damage but does not prevent progression to end-stage kidney disease, suggesting that pathways unrelated to cystine accumulation are also involved. Among these, impaired autophagy, altered endolysosomal trafficking, and increased apoptosis have emerged in recent years as potential targets for new therapies. We previously showed that luteolin, a flavonoid compound, improves these abnormal pathways in cystinotic cells and in zebrafish models of the disease. Herein, we have investigated if prolonged luteolin treatment ameliorates kidney damage in a murine model of cystinosis. To this end, we have treated Ctns-/- mice from 2 to 8 months with 150â¯mg/kg/day of luteolin. No significant side effects were observed. Compared to untreated animals, analyses of kidney cortex samples obtained after sacrifice showed that luteolin decreased p62/SQSTM1 levels (p <0.001), improved the number, size, and distribution of LAMP1-positive structures (p <0.02), and decreased tissue expression of cleaved caspase 3 (p <0.001). However, we did not observe improvements in renal Fanconi syndrome and kidney inflammation. Kidney function remained normal during the time of the study. These results indicate that luteolin has positive effects on the apoptosis and endo-lysosomal defects of cystinotic proximal tubular cells. However, these beneficial effects did not translate into improvement of renal Fanconi syndrome.
RESUMO
Cystinosis is a rare autosomal recessive disorder caused by mutations in the CTNS gene that encodes cystinosin, a ubiquitous lysosomal cystine/H+ antiporter. The hallmark of the disease is progressive accumulation of cystine and cystine crystals in virtually all tissues. At the kidney level, human cystinosis is characterized by the development of renal Fanconi syndrome and progressive glomerular and interstitial damage leading to end-stage kidney disease in the second or third decade of life. The exact molecular mechanisms involved in the pathogenesis of renal disease in cystinosis are incompletely elucidated. We have previously shown upregulation of NLRP2 in human cystinotic proximal tubular epithelial cells and its role in promoting inflammatory and profibrotic responses. Herein, we have investigated the role of NLRP2 in vivo using a mouse model of cystinosis in which we have confirmed upregulation of Nlrp2 in the renal parenchyma. Our studies show that double knock out Ctns-/- Nlrp2-/- animals exhibit delayed development of Fanconi syndrome and kidney tissue damage. Specifically, we observed at 4-6 months of age that animals had less glucosuria and calciuria and markedly preserved renal tissue, as assessed by significantly lower levels of inflammatory cell infiltration, tubular atrophy, and interstitial fibrosis. Also, the mRNA expression of some inflammatory mediators (Cxcl1 and Saa1) and the rate of apoptosis were significantly decreased in 4-6-month old kidneys harvested from Ctns-/- Nlrp2-/- mice compared to those obtained from Ctns-/-mice. At 12-14 months of age, renal histological was markedly altered in both genetic models, although double KO animals had lower degree of polyuria and low molecular weight proteinuria and decreased mRNA expression levels of Il6 and Mcp1. Altogether, these data indicate that Nlrp2 is a potential pharmacological target for delaying progression of kidney disease in cystinosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Cistinose , Nefropatias , Animais , Cistina/metabolismo , Cistinose/genética , Cistinose/metabolismo , Cistinose/patologia , Rim/patologia , Nefropatias/patologia , RNA Mensageiro , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Modelos Animais de Doenças , CamundongosRESUMO
TLR7 recognizes pathogen-derived single-stranded RNA (ssRNA), a function integral to the innate immune response to viral infection. Notably, TLR7 can also recognize self-derived ssRNA, with gain-of-function mutations in human TLR7 recently identified to cause both early-onset systemic lupus erythematosus (SLE) and neuromyelitis optica. Here, we describe two novel mutations in TLR7, F507S and L528I. While the L528I substitution arose de novo, the F507S mutation was present in three individuals from the same family, including a severely affected male, notably given that the TLR7 gene is situated on the X chromosome and that all other cases so far described have been female. The observation of mutations at residues 507 and 528 of TLR7 indicates the importance of the TLR7 dimerization interface in maintaining immune homeostasis, where we predict that altered homo-dimerization enhances TLR7 signaling. Finally, while mutations in TLR7 can result in SLE-like disease, our data suggest a broader phenotypic spectrum associated with TLR7 gain-of-function, including significant neurological involvement.
Assuntos
Mutação com Ganho de Função , Lúpus Eritematoso Sistêmico , Feminino , Masculino , Humanos , Receptor 7 Toll-Like , Mutação , Dimerização , RNARESUMO
Haploinsufficiency of A20 (HA20) is an inflammatory disease caused by mutations in the TNFAIP3 gene classically presenting with Behcet's-like disease. A20 acts as an inhibitor of inflammation through its effect on NF-kB pathway. Here we describe four consanguineous patients (three sisters and their mother) with a predominantly autoimmune phenotype, including thyroiditis, type I diabetes, hemolytic anemia and chronic polyarthritis. All patients had recurrent oral ulcers, with only 1 patient presenting also recurrent fever episodes, as a classical autoinflammatory feature. Next generation sequencing identified a novel heterozygous frameshift mutation (p.His577Alafs*95) that causes a premature stop codon in the zinc finger domain of A20, leading to a putative haploinsufficiency of the protein. Functional analyses confirmed the pathogenicity of the mutation. The variant was associated with decreased levels of A20 in blood cells. Accordingly, ex-vivo lipopolysaccharide (LPS)-stimulated patients' peripheral blood mononuclear cells (PBMCs) showed higher levels of p65 NF-kB phosphorylation, as well as increased production of the proinflammatory cytokines IL-1ß, IL-6 and TNF-α. Moreover, in agreement with recent observations, demonstrating a role for A20 in inhibiting STAT1 and IFNγ pathways, markedly higher circulating levels of the two IFNγ-inducible chemokines CXCL9 and CXCL10 were detected in all patients. Supporting the findings of a hyperactivation of IFNγ signaling pathway in HA20 patients, patients' monocytes showed higher levels of STAT1 without stimulation, as well as higher phosphorylated (active) STAT1 levels following IFNγ stimulation. In conclusion, our study show that in the clinical spectrum of HA20 autoimmune features may predominate over autoinflammatory features and demonstrate, from a molecular point of view, the involvement of A20 in modulating not only the NF-kB, but also the IFNγ pathway.
Assuntos
Doenças Autoimunes/diagnóstico , Doenças Autoimunes/etiologia , Autoimunidade/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Alelos , Família , Genótipo , Humanos , FenótipoRESUMO
Objective: To investigate the activation of the IFNγ signaling pathway in monocytes of patients with secondary hemophagocytic lymphohistiocytosis (sHLH)/macrophage activation syndrome (MAS) and to evaluate whether levels of phosphorylated STAT1 represent a biomarker for the identification of patients at early stages of the disease. Methods: Fresh whole blood samples from pediatric patients with active sHLH/MAS, not receiving (n=10) and receiving glucocorticoids (n=14) at time of sampling, were prospectively collected. As disease control groups, patients with active systemic juvenile idiopathic arthritis (sJIA) without MAS, patients with sHLH/MAS in remission and patients with other rheumatic diseases were also sampled. Whole blood cells were left unstimulated or stimulated with increasing concentrations of IFNγ for 10 minutes and the intracellular Tyrosine (701)-phosphorylated STAT1 (pSTAT1) levels were evaluated in monocytes by flow cytometry. Results: Monocytes from untreated sHLH/MAS patients showed significantly higher basal levels of pSTAT1 compared to those observed in monocytes from glucocorticoid-treated sHLH/MAS patients and from all the other disease controls. In addition, a significant increase in responsiveness to IFNγ, as assessed by increased levels of pSTAT1 following ex vivo stimulation, was observed in monocytes from untreated sHLH/MAS patients. pSTAT1 levels in monocytes distinguished patients with sHLH/MAS not treated with glucocorticoids from patients with active sJIA or with other rheumatic diseases [AUC, 0.93; 95% confidence interval 0.85-1.00, p<0.001]. Statistically significant correlations between IFNG mRNA levels in whole blood cells, circulating IFNγ levels and pSTAT1 levels in sHLH/MAS monocytes were found. Conclusion: Our data demonstrating higher basal levels of pSTAT1 as well as a hyperreactivity to IFNγ stimulation in monocytes from patients with sHLH/MAS point to perturbations in the activation of downstream IFNγ signaling pathway as a contributor to the hyperinflammation occurring in these patients. Finally, the observation that glucocorticoids affect pSTAT1 levels in vivo, makes it difficult to consider the measurement of pSTAT1 levels as a biomarker to identify patients at early stages of sHLH/MAS in clinical practice.