Khadija Tahiri

Oestrogens inhibit interleukin 1β‐mediated nitric oxide synthase expression in articular chondrocytes through nuclear factor‐κB impairment

Objectives: To investigate the presence and functionality of oestrogen receptor α (ERα) in interleukin (IL)1β-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17β oestradiol (E2) effects on IL1β-induced inducible nitric oxide synthase (iNOS) expression. Methods: The presence and functionality of ERα were investigated by immunocytochemistry and transient expression of an E2-responsive reporter construct. iNOS expression and production were determined by transient expression of a chimeric iNOS promoter–luciferase construct and protein immunoblotting. Nitric oxide (NO) production was determined by the Griess reaction. DNA-binding activities of nuclear factor-κB (NF-κB) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)–ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry. Results: ERα was identified in the nucleus of chondrocytes. ERα efficiently transactivated a transiently expressed E2-responsive construct. On IL1β treatment, ERα partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1β-induced NO production (−34%, −31% and −36%, respectively). E2 decreased IL1β-induced iNOS protein expression (−40%). Transient expression of an iNOS promoter construct strongly suggested that iNOS expression was inhibited at the transcriptional level, and EMSA-ELISA assays showed that E2 reduced (−60%) the IL1β-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1β was also strongly decreased by E2. Conclusions: Our data support a reciprocal antagonism between oestrogens and IL1β, ultimately resulting in the decrease of cytokine-dependent NO production through transcriptional inhibition of iNOS expression. This effect was associated with selective inhibition of p65 DNA binding and nuclear translocation.

The Val158Met COMT polymorphism is a modifier of the age at onset in Parkinson's disease with a sexual dimorphism

The catechol-O-methyltranferase (COMT) is one of the main enzymes that metabolise dopamine in the brain. The Val158Met polymorphism in the COMT gene (rs4680) causes a trimodal distribution of high (Val/Val), intermediate (Val/Met) and low (Met/Met) enzyme activity. We tested whether the Val158Met polymorphism is a modifier of the age at onset (AAO) in Parkinsons disease (PD). The rs4680 was genotyped in a total of 16 609 subjects from five independent cohorts of European and North American origin (5886 patients with PD and 10 723 healthy controls). The multivariate analysis for comparing PD and control groups was based on a stepwise logistic regression, with gender, age and cohort origin included in the initial model. The multivariate analysis of the AAO was a mixed linear model, with COMT genotype and gender considered as fixed effects and cohort and cohort-gender interaction as random effects. COMT genotype was coded as a quantitative variable, assuming a codominant genetic effect. The distribution of the COMT polymorphism was not significantly different in patients and controls (p=0.22). The Val allele had a significant effect on the AAO with a younger AAO in patients with the Val/Val (57.1±13.9, p=0.03) than the Val/Met (57.4±13.9) and the Met/Met genotypes (58.3±13.5). The difference was greater in men (1.9 years between Val/Val and Met/Met, p=0.007) than in women (0.2 years, p=0.81). Thus, the Val158Met COMT polymorphism is not associated with PD in the Caucasian population but acts as a modifier of the AAO in PD with a sexual dimorphism: the Val allele is associated with a younger AAO in men with idiopathic PD.

The autophagy/lysosome pathway is impaired in SCA7 patients and SCA7 knock-in mice

There is still no treatment for polyglutamine disorders, but clearance of mutant proteins might represent a potential therapeutic strategy. Autophagy, the major pathway for organelle and protein turnover, has been implicated in these diseases. To determine whether the autophagy/lysosome system contributes to the pathogenesis of spinocerebellar ataxia type 7 (SCA7), caused by expansion of a polyglutamine tract in the ataxin-7 protein, we looked for biochemical, histological and transcriptomic abnormalities in components of the autophagy/lysosome pathway in a knock-in mouse model of the disease, postmortem brain and peripheral blood mononuclear cells (PBMC) from patients. In the mouse model, mutant ataxin-7 accumulated in inclusions immunoreactive for the autophagy-associated proteins mTOR, beclin-1, p62 and ubiquitin. Atypical accumulations of the autophagosome/lysosome markers LC3, LAMP-1, LAMP2 and cathepsin-D were also found in the cerebellum of the SCA7 knock-in mice. In patients, abnormal accumulations of autophagy markers were detected in the cerebellum and cerebral cortex of patients, but not in the striatum that is spared in SCA7, suggesting that autophagy might be impaired by the selective accumulation of mutant ataxin-7. In vitro studies demonstrated that the autophagic flux was impaired in cells overexpressing full-length mutant ataxin-7. Interestingly, the expression of the early autophagy-associated gene ATG12 was increased in PBMC from SCA7 patients in correlation with disease severity. These results provide evidence that the autophagy/lysosome pathway is impaired in neurons undergoing degeneration in SCA7. Autophagy/lysosome-associated molecules might, therefore, be useful markers for monitoring the effects of potential therapeutic approaches using modulators of autophagy in SCA7 and other autophagy/lysosome-associated neurodegenerative disorders.

Natural chondroitin sulphates increase aggregation of proteoglycan complexes and decrease adamts-5 expression in interleukin 1β-treated chondrocytes

Objective: To assess the effect of natural chondroitin sulphate (CS) on the ability of neosynthesised sulphated proteoglycans (PGs) to aggregate in cultured chondrocytes treated with interleukin (IL)1β. Methods: Primary cultured rabbit articular chondrocytes were treated or not with IL1β alone or with concentrations of CS for 20 h. Neosynthesised PGs were labelled by incorporation of [35SO4]-sulphate and analysed by chromatography on Sepharose 2B columns. Gelatinolytic activity was measured by zymography, and matrix metalloproteinase (MMP)1 mRNA level in chondrocytes underwent real-time PCR. Expression of ADAMTS (for “a disintegrin and metalloproteinase with thrombospondin motifs”) -4 and -5 was analysed by real-time PCR and western blotting. Results: The production of [35SO4]-labelled PGs was significantly increased with 10 μg/ml CS in the cellular pool rather than in the incubation medium. The addition of CS to IL1β-treated cells inhibited in part the disaggregation of sulphated PGs induced by IL1β. This inhibitory effect of CS is associated with a significant decrease in ADAMTS-5 expression at the mRNA and protein levels. No effect of CS was observed on IL1β-induced gelatinolytic activity, MMP1 mRNA expression or ADAMTS-4 expression. Conclusion: CS increases the production of functional sulphated PGs in the direct environment of chondrocytes in vitro. This beneficial effect of CS in IL1β-treated cells is associated with decreased expression of ADAMTS-5.

Hes1, a new target for interleukin 1β in chondrocytes

Objectives To investigate the effects of interleukin 1β (IL1β) treatment on the Notch1/Hes1 pathway in chondrocytes in vitro. Methods Mouse articular chondrocytes in primary culture were challenged with IL1β, alone or combined with Notch1 and IL1β pathway inhibitors. Notch1 and Hes1 expressions were investigated by immunocytochemistry, western blot and real-time quantitative (q)PCR. IL1β-responsive genes were assessed by real-time qPCR and a specific siRNA against Hes1 was used to identify Hes1 target genes. Results Notch1 labelling remained nuclear and stable in intensity irrespective of treatment, suggesting a steady state activation of this pathway in our model. IL1β transiently increased Hes1 mRNA (2.5-fold) and protein expression in treated versus naive chondrocytes. Hes1 mRNA level then decreased below control and its cyclic pattern of expression was lost. This was associated with nuclear translocation of the cytoplasmic Hes1 protein. IL1β induced increase in Hes1 mRNA was transcriptional, occurred through nuclear factor (NF)κB activation and appeared to be associated with downregulation by its own protein. Hes1 induction was insensitive to the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), which suggested its independence from novel Notch1 activation. Hes1 expression was efficiently silenced by a specific siRNA. This experiment revealed that Hes1 did not mediate IL1β-induced downregulation of Sox9, type II collagen and aggrecan transcription but mediated IL1β induction of matrix metalloproteinase (MMP)13 and ADAM metallopeptidase with thrombospondin type 1 motif, 5 (ADAMTS5). The Hes1-related repressor Hey1 was expressed at a very low level and was not inducible by IL1β. Conclusion Hes1 is a novel IL1β target gene in chondrocytes which influences a discrete subset of genes linked to cartilage matrix remodelling and/or degradation.

Transcriptome Analysis of Peripheral Blood in Chronic Inflammatory Demyelinating Polyradiculoneuropathy Patients Identifies TNFR1 and TLR Pathways in the IVIg Response

AbstractWe have studied the response to intravenous immunoglobulins (IVIg) by a transcriptomic approach in 11 chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients (CIDP duration = 6 [0.83–6.5] years). RNA was extracted from cells in whole blood collected before and 3 weeks after IVIg treatment, and hybridized on Illumina chips. After RNA quality controls, gene expression was analyzed using statistical tests fitted for microarrays (R software, limma package), and a pathway analysis was performed using DAVID software. We identified 52 genes with expression that varied significantly after IVIg (fold change [FC] > 1.2, P < 0.001, false discovery rate [FDR] <0.05). Among these 52 genes, 7 were related to immunity, 3 were related to the tumor necrosis factor (TNF)-&agr; receptor 1 (TNFR1) pathway (inhibitor of caspase-activated DNase (ICAD): FC = 1.8, P = 1.7E-7, FDR = 0.004; p21 protein-activated kinase 2 [PAK2]: FC = 1.66, P = 2.6E-5, FDR = 0.03; TNF-&agr;-induced protein 8-like protein 1 [TNFAIP8L1]: P = 1.00E-05, FDR = 0.026), and 2 were related to Toll-like receptors (TLRs), especially TLRs 7 and 9, and were implicated in autoimmunity. These genes were UNC93B1 (FC = 1.6, P = 2E-5, FDR = 0.03), which transports TLRs 7 and 9 to the endolysosomes, and RNF216 (FC = 1.5, P = 1E-05, FDR = 0.03), which promotes TLR 9 degradation. Pathway analysis showed that the TNFR1 pathway was significantly lessened by IVIg (enrichment score = 24, Fischer exact test = 0.003). TNF-&agr; gene expression was higher in responder patients than in nonresponders; however, it decreased after IVIg in responders (P = 0.04), but remained stable in nonresponders. Our data suggest the actions of IVIg on the TNFR1 pathway and an original mechanism involving innate immunity through TLRs in CIDP pathophysiology and the response to IVIg. We conclude that responder patients have stronger inflammatory activity that is lessened by IVIg.