Role of Interleukin 17 in arthritis chronicity through survival of synoviocytes via regulation of synoviolin expression

Background: The use of TNF inhibitors has been a major progress in the treatment of chronic inflammation. However, not all patients respond. In addition, response will be often lost when treatment is stopped. These clinical aspects indicate that other cytokines might be involved and we focus here on the role of IL-17. In addition, the chronic nature of joint inflammation may contribute to reduced response and enhanced chronicity. Therefore we studied the capacity of IL-17 to regulate synoviolin, an E3 ubiquitin ligase implicated in synovial hyperplasia in human rheumatoid arthritis (RA) FLS and in chronic reactivated streptococcal cell wall (SCW)-induced arthritis.<p></p> Methodology/Principal Findings: Chronic reactivated SCW-induced arthritis was examined in IL-17R deficient and wild-type mice. Synoviolin expression was analysed by real-time RT-PCR, Western Blot or immunostaining in RA FLS and tissue, and p53 assessed by Western Blot. Apoptosis was detected by annexin V/propidium iodide staining, SS DNA apoptosis ELISA kit or TUNEL staining and proliferation by PCNA staining. IL-17 receptor A (IL-17RA), IL-17 receptor C (IL-17-RC) or synoviolin inhibition were achieved by small interfering RNA (siRNA) or neutralizing antibodies. IL-17 induced sustained synoviolin expression in RA FLS. Sodium nitroprusside (SNP)-induced RA FLS apoptosis was associated with reduced synoviolin expression and was rescued by IL-17 treatment with a corresponding increase in synoviolin expression. IL-17RC or IL-17RA RNA interference increased SNP-induced apoptosis, and decreased IL-17-induced synoviolin. IL-17 rescued RA FLS from apoptosis induced by synoviolin knockdown. IL-17 and TNF had additive effects on synoviolin expression and protection against apoptosis induced by synoviolin knowndown. In IL-17R deficient mice, a decrease in arthritis severity was characterized by increased synovial apoptosis, reduced proliferation and a marked reduction in synoviolin expression. A distinct absence of synoviolin expressing germinal centres in IL-17R deficient mice contrasted with synoviolin positive B cells and Th17 cells in synovial germinal centre-like structures.<p></p> Conclusion/Significance: IL-17 induction of synoviolin may contribute at least in part to RA chronicity by prolonging the survival of RA FLS and immune cells in germinal centre reactions. These results extend the role of IL-17 to synovial hyperplasia.<p></p&gt

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles

BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS

Characterisation of tau in the human and rodent enteric nervous system under physiological conditions and in tauopathy

Abstract Tau is normally a highly soluble phosphoprotein found predominantly in neurons. Six different isoforms of tau are expressed in the adult human CNS. Under pathological conditions, phosphorylated tau aggregates are a defining feature of neurodegenerative disorders called tauopathies. Recent findings have suggested a potential role of the gut-brain axis in CNS homeostasis, and therefore we set out to examine the isoform profile and phosphorylation state of tau in the enteric nervous system (ENS) under physiological conditions and in tauopathies. Surgical specimens of human colon from controls, Parkinson’s disease (PD) and progressive supranuclear palsy (PSP) patients were analyzed by Western Blot and immunohistochemistry using a panel of anti-tau antibodies. We found that adult human ENS primarily expresses two tau isoforms, localized in the cell bodies and neuronal processes. We did not observe any difference in the enteric tau isoform profile and phosphorylation state between PSP, PD and control subjects. The htau mouse model of tauopathy also expressed two main isoforms of human tau in the ENS, and there were no apparent differences in ENS tau localization or phosphorylation between wild-type and htau mice. Tau in both human and mouse ENS was found to be phosphorylated but poorly susceptible to dephosphorylation with lambda phosphatase. To investigate ENS tau phosphorylation further, primary cultures from rat enteric neurons, which express four isoforms of tau, were pharmacologically manipulated to show that ENS tau phosphorylation state can be regulated, at least in vitro. Our study is the first to characterize tau in the rodent and human ENS. As a whole, our findings provide a basis to unravel the functions of tau in the ENS and to further investigate the possibility of pathological changes in enteric neuropathies and tauopathies

Role of interleukin 17 in arthritis chronicity through survival of synoviocytes via regulation of synoviolin expression.

BackgroundThe use of TNF inhibitors has been a major progress in the treatment of chronic inflammation. However, not all patients respond. In addition, response will be often lost when treatment is stopped. These clinical aspects indicate that other cytokines might be involved and we focus here on the role of IL-17. In addition, the chronic nature of joint inflammation may contribute to reduced response and enhanced chronicity. Therefore we studied the capacity of IL-17 to regulate synoviolin, an E3 ubiquitin ligase implicated in synovial hyperplasia in human rheumatoid arthritis (RA) FLS and in chronic reactivated streptococcal cell wall (SCW)-induced arthritis.Methodology/principal findingsChronic reactivated SCW-induced arthritis was examined in IL-17R deficient and wild-type mice. Synoviolin expression was analysed by real-time RT-PCR, Western Blot or immunostaining in RA FLS and tissue, and p53 assessed by Western Blot. Apoptosis was detected by annexin V/propidium iodide staining, SS DNA apoptosis ELISA kit or TUNEL staining and proliferation by PCNA staining. IL-17 receptor A (IL-17RA), IL-17 receptor C (IL-17-RC) or synoviolin inhibition were achieved by small interfering RNA (siRNA) or neutralizing antibodies. IL-17 induced sustained synoviolin expression in RA FLS. Sodium nitroprusside (SNP)-induced RA FLS apoptosis was associated with reduced synoviolin expression and was rescued by IL-17 treatment with a corresponding increase in synoviolin expression. IL-17RC or IL-17RA RNA interference increased SNP-induced apoptosis, and decreased IL-17-induced synoviolin. IL-17 rescued RA FLS from apoptosis induced by synoviolin knockdown. IL-17 and TNF had additive effects on synoviolin expression and protection against apoptosis induced by synoviolin knowndown. In IL-17R deficient mice, a decrease in arthritis severity was characterized by increased synovial apoptosis, reduced proliferation and a marked reduction in synoviolin expression. A distinct absence of synoviolin expressing germinal centres in IL-17R deficient mice contrasted with synoviolin positive B cells and Th17 cells in synovial germinal centre-like structures.Conclusion/significanceIL-17 induction of synoviolin may contribute at least in part to RA chronicity by prolonging the survival of RA FLS and immune cells in germinal centre reactions. These results extend the role of IL-17 to synovial hyperplasia

The impact of vedolizumab and ustekinumab on articular extra-intestinal manifestations in inflammatory bowel disease patients : a real-life multicentre cohort study

Background and aims: Extra-intestinal manifestations are frequently reported in inflammatory bowel diseases. However, data comparing the effect of vedolizumab and ustekinumab on articular extra-intestinal manifestations are limited. The aim was to evaluate differences in new onset and evolution of pre-existing joint extra-intestinal manifestations during both treatments. Methods: An international multicentric retrospective study was performed on inflammatory bowel disease patients who started vedolizumab or ustekinumab between May 2010 and December 2020. Extra-intestinal manifestations were assessed at baseline and joint extraintestinal manifestations were evaluated throughout the 2-year follow-up. Arthropathy was defined by joint inflammation (arthritis/sacroiliitis), diagnosed by a rheumatologist, and arthralgia as articular pain without confirmed inflammation. Additionally, skin, ocular and hepatic extra-intestinal manifestations were assessed at baseline. Uni- and multivariate analyses were performed. Results: In total 911 patients (vedolizumab:584; ustekinumab:327) were included. Deterioration of pre-existing arthropathy and rate of new onset arthropathy were not significantly associated with vedolizumab over ustekinumab. Arthropathy was reason to stop treatment in 6 vedolizumab and 2 ustekinumab patients. The odds of developing new arthralgia within 6 months was higher in patients who took vedolizumab compared to ustekinumab (aOR: 2.28 [1.01-5.15], p=0.047). However, this effect was not sustained during the 2-year follow-up (aOR: 1.35 [0.80-2.29], p=0.259). Deterioration of pre-existing arthralgia was comparable between ustekinumab and vedolizumab treated patients. In 2 vedolizumab-treated patients arthralgia was reason to stop treatment. Conclusions: Vedolizumab and ustekinumab can be used safely in patients with articular extra-intestinal manifestations. Only a temporary increased risk for developing arthralgia has been observed under vedolizumab. Keywords: biologicals; inflammatory bowel disease; spondyloarthropathy

Effect of IL-17 on synoviolin expression in RA FLS.

<p>RA FLS were stimulated with IL-17A or IL-17F (50 ng/ml), IL-1 10 ng/ml, TNF 10 ng/ml or LPS 100 ng/ml for 24 h and synoviolin mRNA expression measured by real-time RT-PCR (<b>A, upper panel</b>). The results based on a ratio of synoviolin/β-actin mRNA amplification are presented as the fold induction in synoviolin mRNA expression relative to control samples, n = 3, mean ± SEM. * <i>P</i><0.05 compared to no treatment control. RA FLS were treated with IL-17A or IL-17F (50 ng/ml) for 24 h and synoviolin expression measured by Western Blotting (<b>A, lower panel</b>). The membrane was stripped and reprobed for β-actin and the result is representative of n = 3. RA FLS were treated with IL-17 5-200 ng/ml for 24 h (<b>B, left panel</b>) or IL-17 50 ng/ml over 24 h (<b>B, right panel</b>) and synoviolin expression measured by real-time RT-PCR, n = 3, mean ± SEM, * <i>P</i><0.05. Synoviolin mRNA expression in RA FLS, OA FLS and OA dermal fibroblasts that were untreated (–) or were treated with 50 ng/ml or 10 ng/ml of IL-1 for 24 h <b>(C).</b> * <i>P</i><0.05 versus <b>untreated FLS.</b> RA FLS were preincubated with PD98059 (50μM), SB203580 (5μM) or SP600125 (50μM) for 1 hour, then treated with IL-17 50 ng/ml for 1 hour. Synoviolin expression was measured by real-time PCR, mean ± SEM, n = 3 (<b>D</b>). * p<0.05 compared to IL-17 treatment.</p

Severity of chronic streptococcal cell wall-induced arthritis in wild-type (WT) or IL-17R ^−/− mice A).

<p>(A representative image of haematoxylin and eosin-stained synovial knee joint sections in WT or IL-17R <b>⁻</b>^/<b>−</b> mice at day 42 after 5 repeated injections of streptococcal cell wall (SCW) fragments (magnification ×40). Arthritis severity and inflammatory infiltrate were scored histologically in injected joints of WT or IL-17R <b>⁻</b>^/<b>−</b> mice. Arthritis severity was scored on a scale of 0–2 and synovial infiltrate on a scale of 0–3. Results are expressed as mean ± SEM of 2 separate experiments, n = 10 mice per group per experiment, * <i>P</i><0.05 compared to WT mice. <b>Synoviolin expression is reduced in IL-17R ^−/− mice with chronic SCW-induced arthritis</b> (<b>B</b>). Synoviolin expression in sections of arthritic knee joints from, a wild-type (WT) or IL-17R <b>⁻</b>^/<b>−</b> mouse after 5 repeated injections of SCW fragments (magnification x 40, day 42, n = 10 mice in WT or IL-17R <b>⁻</b>^/<b>−</b> groups from 2 separate experiments). Synoviolin expression was quantified in 10 high power fields, averaged and expressed as the number of synoviolin positive cells/mm², * <i>P</i><0.05 compared to WT mice. <b>Synovial apoptosis in WT or IL-17R ^−/− mice with chronic streptococcal cell wall-induced arthritis</b> (<b>C</b>). A representative image of increased TUNEL staining in IL-17R <b>⁻</b>^/<b>−</b> synovium (magnification ×200.). The number of TUNEL-positive cells was quantified in 10 high power fields, averaged and expressed as the number of TUNEL positive cells/mm², mean ± SEM, * <i>P</i><0.05 compared to WT mice. <b>Synovial PCNA staining in knee joint sections from WT or IL-17R ^−/− mice with chronic streptococcal cell wall-induced arthritis</b> (<b>D</b>). A representative image of PCNA staining in synovial sections (magnification ×200). The number of PCNA-positive cells was quantified in 10 high power fields, averaged and expressed as the number of PCNA positive cells/mm², mean ± SEM, * <i>P</i><0.05 compared to WT mice.</p

Effect of synoviolin knockdown on apoptosis in RA FLS.

<p>RA FLS were nucleofected (amaxa) for 24 h with 0.5 µg of 4 synoviolin siRNA duplexes (S-01 to S-04), synoviolin smartpool siRNA duplex or siCONTROL siRNA (sictl) and synoviolin expression analysed by real-time RT-PCR <b>(A, left panel)</b> or Western Blot <b>(A, right panel).</b> Synoviolin mRNA was normalized to GAPDH and results expressed as the percentage fold reduction compared to sictl treated samples, n = 5, mean ± SEM of duplicate experiments. * <i>P</i><0.05, compared to sictl nucleofected RA FLS. (<b>B</b>), RA FLS were nucleofected as above then treated with SNP 0.1–1 µM overnight and apoptosis measured by the SS DNA apoptosis assay. Results are expressed as fold induction in apoptosis, n = 3 from 3 separate RA donors, mean ± SEM of duplicate experiments. * <i>P</i><0.05, compared to sictl nucleofected RA FLS. (<b>C</b>), RA FLS were nucleofected as above, serum starved overnight then pretreated with 100 ng/ml IL-17A for 2 h then cotreated with SNP 0.1–1 µM overnight and apoptosis measured by SS DNA apoptosis assays expressed as fold induction of apoptosis, n = 3 from 3 separate RA donors, mean ± SEM of duplicate experiments. * <i>P</i><0.05, compared to sictl nucleofected RA FLS. † <i>P</i><0.05 compared to SNP treatment in S-08 nucleofected RA FLS.</p

Effect of IL-17 and TNF on RA FLS synoviolin expression and apoptosis.

<p>RA FLS were stimulated with IL-17A (50 or 100 ng/ml) and/or TNF (1 or 10 ng/ml) for 24 h and synoviolin mRNA expression measured by real-time RT-PCR (<b>A</b>). The results based on a ratio of synoviolin/β-actin mRNA amplification are presented as the fold induction in synoviolin mRNA expression relative to control samples, n = 3, mean ± SEM. * <i>P</i><0.05 compared to no treatment control. † <i>P</i><0.05 compared to TNF treated samples. RA FLS were pretreated with IL-17 100 ng/ml and TNF 10 ng/ml for 2 h then cotreated with SNP 0.5 µM (<b>B, left panel</b>) or 0.1 µM (<b>B, right panel</b>) for 24 h and apoptosis measured by SS DNA apoptosis kit. The results are presented as the fold induction in apoptosis relative to control samples, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * <i>P</i><0.05 compared to SNP treatment in S-08 nucleofected RA FLS.</p

Effect of IL-17 on RA FLS apoptosis.

<p>RA FLS were treated with SNP 0.01-1 µM for 24 h and apoptosis measured by SS DNA apoptosis kit (<b>A</b>). The results are presented as the fold induction in apoptosis relative to control samples, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * <i>P</i><0.05 compared to no treatment control. RA FLS were treated with SNP 0.01–1 µM for 24 h (<b>B, left panel</b>) or 0.1 µM over 24 h (<b>B, right panel</b>) and synoviolin expression measured by real-time RT-PCR. The results are expressed as the ratio of synoviolin/β-actin mRNA amplification, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * <i>P</i><0.05 compared to no treatment control. RA FLS were pretreated with IL-17 100 ng/ml for 2 h then cotreated with SNP 0.1-1 µM for 24 h and apoptosis measured by SS DNA apoptosis kit (<b>C</b>). The results are presented as the fold induction in apoptosis relative to control samples, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * <i>P</i><0.05 compared to no treatment control. RA FLS were treated as above and annexin V analysed. Representative serial fluorescent microscopic pictures of Annexin V positive cells (green), double labelled with Propidium iodide (PI, red) at magnification x 200 (<b>D</b>).</p