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

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

Comorbidities increase in-hospital mortality in dengue patients in Brazil

<div><p>Dengue remains an unmet public health burden. We determined risk factors for dengue in-hospital mortality in Brazil. Of 326,380 hospitalised dengue cases in 9-45-year-old individuals, there were 971 deaths. Risk of dying was 11-times higher in the presence of underlying common comorbidities (renal, infectious, pulmonary disease and diabetes), similar to the risk of dying from severe dengue and much higher with the combination. Ensuring access to integrated dengue preventative measures in individuals aged ≥ 9 years including those with comorbidities may help achieve the WHO objective of 50% reduction in mortality and 25% reduction in morbidity due to dengue by 2020.</p></div

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 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 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