TLR1/2 and 5 induce elevated cytokine levels from rheumatoid arthritis monocytes independent of ACPA or RF autoantibody status

Objective RA is an autoimmune inflammatory joint disease. Both RF and ACPA are associated with more progressive disease and higher levels of systemic inflammation. Monocyte activation of toll-like receptors (TLRs) by endogenous ligands is a potential source of increased production of systemic cytokines. RA monocytes have elevated TLRs, some of which are associated with the disease activity score using 28 joints (DAS28). The aim of this study was to measure TLR-induced cytokine production from monocytes, stratified by autoantibody status, to assess if their capacity to induce cytokines is related to autoantibody status or DAS28. Methods Peripheral blood monocytes isolated from RA patients and healthy controls were stimulated with TLR1/2, TLR2/6, TLR4, TLR5, TLR7, TLR8 and TLR9 ligands for 18 h before measuring IL-6, TNFα and IL-10. Serum was used to confirm the autoantibody status. Cytokine levels were compared with RF, ACPA and DAS28. Results RA monocytes demonstrated significantly increased IL-6 and TNFα upon TLR1/2 stimulation and IL-6 and IL-10 upon TLR5 activation. TLR7 and TLR9 activation did not induce cytokines and no significant differences were observed between RA and healthy control monocytes upon TLR2/6, TLR4 or TLR8 activation. When stratified by ACPA or RF status there were no correlations between autoantibody status and elevated cytokine levels. However, TLR1/2-induced IL-6 did correlate with DAS28. Conclusions Elevated TLR-induced cytokines in RA monocytes were not related to ACPA or RF status. However, TLR1/2-induced IL-6 was associated with disease activity

Contribution of toll-like receptors and the NLRP3 inflammasome in rheumatoid arthritis pathophysiology

Rheumatoid arthritis (RA) is a progressive autoimmune disease that is characterized by inflammation of the synovial joints leading to cartilage and bone damage. The pathogenesis is sustained by the production of pro-inflammatory cytokines including tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6, which can be targeted therapeutically to alleviate disease severity. Several innate immune receptors are suggested to contribute to the chronic inflammation in RA, through the production of pro-inflammatory factors in response to endogenous danger signals. Much research has focused on toll-like receptors and more recently the nucleotide-binding domain and leucine-rich repeat pyrin containing protein-3 (NLRP3) inflammasome, which is required for the processing and release of IL-1β. This review summarizes the current understanding of the potential involvement of these receptors in the initiation and maintenance of inflammation and tissue damage in RA and experimental arthritis models

Expression of sterile-α and armadillo motif in rheumatoid arthritis monocytes correlates with TLR2 induced IL-1β and disease activity

Objective Cartilage and bone damage in rheumatoid arthritis (RA) are associated with elevated IL-1β. The effects of IL-1β can be reduced by biological therapies that target IL-1β or TNFα. However, the mechanisms responsible for increased IL-1β and the effect of anti-TNFα have not been fully elucidated. Recently, sterile-α and armadillo motif-containing protein (SARM) was identified as a negative regulator of toll-like receptor (TLR) induced IL-1β secretion through an interaction with the inflammasome. This study set out to investigate SARM during TLR induced IL-1β secretion in RA peripheral blood monocytes and in patients commencing anti-TNFα treatment. Methods Monocytes were isolated from RA patients and healthy controls; disease activity was measured by DAS28. IL-1β secretion was measured by ELISA following TLR1/2, TLR4 and TLR7/8 stimulation. The mRNA expression of SARM, IL-1β and the components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome were measured by quantitative PCR. SARM protein expression was measured by western blotting. Results TLR1/2 activation induced elevated IL-1β in RA monocytes compared with heathy controls (p= 0.0009), which negatively correlated with SARM expression (p = 0.0086). Lower SARM expression also correlated with higher disease activity (p = 0.0246). Additionally, patients responding to anti-TNFα treatment demonstrated a rapid upregulation of SARM, which was not observed in non-responders. Conclusion Together, these data highlight a potential contribution from SARM to RA pathophysiology where decreased SARM may lead to elevated IL-1β associated with RA pathogenesis. Furthermore, the data additionally present a potential mechanism by which TNFα blockade can modify IL-1β secretion

The role of SIRT1 in the pathogenesis of Spondyloarthritides

HintergrundSpondyloarthritiden(SpA) sind eine Gruppe von chronisch entz\ufcndlichen,rheumatischen Erkrankungen, die durch Entz\ufcndungen der Wirbels\ue4ule, den peripheren Gelenken, den Enthesen, und auch durch axiale Skelettsch\ue4den und Knochenneubildung gekennzeichnet sind. Der Interleukin(IL)-23/T-Helfer(TH)17 Signalweg ist von gro fer Bedeutung in der Krankheitsentstehung von Spondyloarthritis(SpA). Es konnte gezeigt werden, dass die Protein/Histon Deacetylase Sirtuin1(silent mating type information regulation 2 homologSIRT1) als epigenetischer Regulator wirkt und die Entstehung von proinflammatorischen TH17 Zellen, durch die Deacetylierung des Zelllinienspezifischen Transkriptionsfaktors,erh\uf6ht. Die Deletion von SIRT1 in T Zellen und auch die chemische Inhibierung von SIRT1 in vivo sch\ufctzt M\ue4use vor experimenteller autoimmun Encephalomyelitis(EAE),eine TH17 Zellvermittelte Erkrankung. Das Ziel dieser Arbeit war die Untersuchung der Rolle von SIRT1 in der Pathogenese von axialer SpA.Material und MethodenMononukle\ue4re Zellen des peripheren Blutes(PBMCs) wurden aus dem Blut von SpA Patienten und gesunden Kontrollen(HC) isoliert. Anschlie fend wurden naive CD4+T Zellen mittels magnetischer Zellseparation(MACS) isoliert und f\ufcr 11 Tage unter polarisierenden Bedingungen mit SIRT1 Inhibitor oder Aktivator zu TH0 Zellen, regulatorische T Zellen(Treg) und TH17 Zellen differenziert. Die H\ue4ufigkeit der polarisierten Zellen wurde durch die Bestimmung der Expression von Zelllinienspezifischen Cytokinen, Oberfl\ue4chenmarkern und Transkriptionsfaktoren mittels Durchflusszytometrie(FACS) ermittelt. In naiven CD4+T Zellen wurden durch FACS SIRT1 Protein Spiegel bestimmt, mittels real-time PCR(qPCR) wurde SIRT1 Genexpression ermittelt und die SIRT1 Aktivit\ue4t wurde durch einen fluorometrischen SIRT1 Aktivit\ue4tstest gemessen.ErgebnisseIn vitro konnte kein Unterschied zwischen SpA und HC in der Differenzierung von naiven CD4+T Zellen zu TH17 Zellen oder Tregs festgestellt werden. Es wurde eine negative Korrelation zwischen TH17 polarisierten Zellen und Treg polarisierten Zellen in SpA Patienten detektiert[corrcoeff=-0,567,p=0,009]. Ferner zeigte die Krankheitsaktivit\ue4t eine positive Korrelation mit TH17 polarisierten Zellen[corrcoeff=0,549,p=0,023] und eine negative Korrelation mit Treg polarisierten Zellen[corrcoeff=-0,489,p=0,064].Die SIRT1 Genexpression[SpA:8,95(SD=1,52)vs. HC:9,65(SD=2,39),p=0,437] und die SIRT1 Aktivit\ue4t[SpA:15976(SD= 8423)vs. HC:12936(SD=9775),p=0,395] in naiven CD4+T Zellen waren \ue4hnlich zwischen SpA Patienten und HCs. Jedoch zeigte sich, dass der SIRT1 Protein Spiegel in naiven CD4+ T Zellen [SpA:1293 (SD=447) vs. HC:984 (SD=410),p=0,077] und CD4+ Ged\ue4chtniszellen [SpA:1528(SD=527) vs. HC: 1097 (SD=510), p=0,035]. in SpA Patienten erh\uf6ht war im Gegensatz zu HC. Die Behandlung der TH17 Differenzierungskultur mit dem SIRT1 Inhibitor Ex527 (Ex) senkte die Anzahl an TH17 polarisierten Zellen in SpA Patienten [TH17: 6,28% (2,89-11,80) vs. TH17 Ex: 3,42 (1,79-8,21), p0,001] und HCs [TH17: 6,53% (4,27-11,10) vs. TH17 Ex: 3,48% (2,45-9,73), p=0,012]. Die Aktivierung vonSIRT1 w\ue4hrend der Kultivierungszeit hatte einen f\uf6rdernden Effekt auf die TH17 Differenzierung in SpA Patienten [TH17: 6,28% (2,89-11,80) vs. TH17 Cay: 9,55% (4,17-16,20), p0,001].SchlussfolgerungIn dieser Studie konnte in vitro eine f\uf6rdernde Rolle von SIRT1 auf die Differenzierung von naiven CD4+ T Zellen zu TH17 Zellen festgestellt werden. Die erh\uf6hten SIRT1 Protein Spiegel in naiven CD4+ T Zellen und CD4+ Ged\ue4chtniszellen weisen m\uf6glicherweise auf einen epigenetischen Einfluss von SIRT1 auf die Entstehung von proinflammatorischen TH17 Zellen in SpA Patienten hin. Weitere Untersuchungen werden demzufolge n\uf6tig sein, damit krankheitsspezifische Auswirkungen von SIRT1 in vivo gekl\ue4rt werden k\uf6nnen und um herzufinden, ob dieser epigenetischer Regulator gezielt in der Therapie von SpA Patienten eingesetzt werden kann.BackgroundSpondyloarthritides represent a group of chronic inflammatory rheumatic diseases characterized by inflammation of the spine and peripheral Joint oligoarthritis, enthesitis,as well as axial skeletal damage and new bone formation. The interleukin (IL)-23/Thelper (TH)17 pathway is of great relevance in the pathogenesis of Spondyloarthritis (SpA). The protein/histone deacetylase Sirtuin 1 (silent mating type Information regulation 2 homolog SIRT1) acts as epigenetic regulator and has been shown to enhance the development of proinflammatory TH17 cells by deacetylating the linagespecific transcription factor. In vivo, deletion of SIRT1 in T cells as well as chemical inhibition of SIRT1 protected mice from experimental autoimmune encephalomyelitis (EAE), a TH17 cell-mediated disease. The aim of this thesis is investigating the role of SIRT1 in the pathogenesis of axial SpA.Material and MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from the blood of SpA patients and healthy controls (HC). Na\uefve CD4+ T cells were isolated via magnetic assisted cell sorting (MACS) and cultured under TH0-, regulatory T cells (Treg)- and TH17 cell polarizing conditions for 11 days and treated with SIRT1 inhibitor or activator. Frequency of polarized cells was assessed by determining the expression of cell-linage specific cytokines, surface markers and transcription factors via fluorescence assisted cell sorting (FACS). In na\uefve CD4+ T cells, SIRT1 protein levels were evaluated via FACS, SIRT1 gene expression levels were assessed using real-time PCR (qPCR), and SIRT1 activity was measured with a fluorometric SIRT1 activity assay kit.ResultsIn vitro, the differentiation capacity from na\uefve CD4+ T cells to TH17 cells or Tregs was comparable between SpA and HCs. SpA patients exhibited a negative correlation between TH17 polarized cells and Treg polarized cells [corrcoeff = -0.567, p =0.009]. Also, disease activity showed a positive correlation with TH17 polarized cells [corrcoeff=0.549, p=0.023], and a negative correlation with Treg polarized cells [corrcoeff=-0.489, p=0.064].SIRT1 gene expression level [SpA: 8.95 (SD=1.52) vs. HC: 9.65 (SD=2.39), p=0.437] and SIRT1 activity [SpA: 15976 (SD= 8423) vs. HC: 12936 (SD=9775), p=0.395] in na\uefve CD4+ T cells were similar between SpA and HC. However, SIRT1 protein Levels were higher in SpA patients as compared to HC in na\uefve CD4+ T cells [SpA: 1293 (SD=447) vs. HC: 984 (SD=410), p=0.077] as well as in CD4+ memory T cells [SpA:1528 (SD=527) vs. HC: 1097 (SD=510), p=0.035]. Treatment of the TH17 differentiation culture with SIRT1 inhibitor Ex527 (Ex) decreased the level of TH17 polarized cells significantly in both, SpA [TH17: 6.28% (2.89-11.80) vs. TH17 Ex: 3.42 (1.79-8.21), p0.001] and HC [TH17: 6.53% (4.27-11.10) vs. TH17 Ex: 3.48% (2.45-9.73), p=0.012]. Activating SIRT1 during cultivation showed an enhancing effect on TH17 cell differentiation in SpA patients [TH17: 6.28% (2.89-11.80) vs. TH17 Cay: 9.55% (4.17-16.20), p0.001].ConclusionThis study reveals a promoting effect of SIRT1 on the differentiation of na\uefve CD4+ T cells to TH17 cells in vitro. Moreover, enhanced SIRT1 protein levels in na\uefve CD4+ T cells as well as in CD4+ memory T cells in SpA patients might indicate an epigenetic influence of SIRT1 on the development of proinflammatory TH17 cells in SpA patients. Further investigations are required to examine disease specific effects of SIRT1 in vivo, to clarify if this epigenetic regulator might constitute a target for future therapy of SpA.submitted by Sarah UnterbergerZusammenfassungen in Deutsch und EnglischMasterarbeit Karl-Franzens-Universit\ue4t Graz 2018 785

Multiple TLRs elicit alternative NLRP3 inflammasome activation in primary human monocytes independent of RIPK1 kinase activity

The canonical NOD-like receptor family pyrin domain containing 3 (NLRP3) pathway involves a priming step to induce pro-IL-1β followed by a secondary signal such as K+ efflux to activate inflammasome formation. This then leads to the maturation of IL-1β and the formation of gasdermin D (GSDMD) pores that initiate pyroptosis and mediate IL-1β release. In contrast, primary human monocytes also engage an alternative pathway in response to toll-like receptor (TLR) 4 activation, without the need for a secondary signal. Data from a monocyte-like cell line suggest that the alternative pathway functions via the TLR adaptor protein TIR-domain-containing adapter-inducing interferon-β (TRIF), receptor-interacting protein kinase 1 (RIPK1), FAS-associated death domain (FADD) and caspase-8 upstream of NLRP3 activation, but in the absence of K+ efflux or pyroptosis. Usage of the alternative pathway by other members of the TLR family that induce IL-1β but do not signal through TRIF, has yet to be explored in primary human monocytes. Furthermore, the mechanism by which IL-1β is released from monocytes remains unclear. Therefore, this study investigated if the alternative NLRP3 inflammasome pathway is initiated following activation of TLRs other than TLR4, and if GSDMD was necessary for the release of IL-1β. Monocytes were stimulated with ligands that activate TLR1/2, TLR2/6, TLR4 and TLR7 and/or TLR8 (using a dual ligand). Similar to TLR4, all of the TLRs investigated induced IL-1β release in a NLRP3 and caspase-1 dependent manner, indicating that TRIF may not be an essential upstream component of the alternative pathway. Furthermore, inhibition of RIPK1 kinase activity had no effect on IL-1β release. Although IL-1β was released independently of K+ efflux and pyroptosis, it was significantly reduced by an inhibitor of GSDMD. Therefore, it is feasible that low level GSDMD pore formation may facilitate the release of IL-1β from the cell, but not be present in sufficient quantities to initiate pyroptosis. Together these data suggest that the alternative pathway operates independently of RIPK1 kinase activity, downstream of diverse TLRs including TLR4 in primary human monocytes and supports the potential for IL-1β release via GSDMD pores alongside other unconventional secretory pathways.</p

The Effect of Elevated Protein Intake on DNA Damage in Older People: Comparative Secondary Analysis of Two Randomized Controlled Trials

A high protein intake at old age is important for muscle protein synthesis, however, this could also trigger protein oxidation with the potential risk for DNA damage. The aim of this study was to investigate whether an increased protein intake at recommended level or well above would affect DNA damage or change levels of reduced (GSH) and oxidised glutathione (GSSG) in community-dwelling elderly subjects. These analyses were performed in two randomized intervention studies, in Austria and in New Zealand. In both randomized control trials, the mean protein intake was increased with whole foods, in the New Zealand study (n = 29 males, 74.2 ± 3.6 years) to 1.7 g/kg body weight/d (10 weeks intervention; p < 0.001)) in the Austrian study (n = 119 males and females, 72.9 ± 4.8 years) to 1.54 g/kg body weight/d (6 weeks intervention; p < 0.001)). In both studies, single and double strand breaks and as formamidopyrimidine—DNA glycosylase-sensitive sites were investigated in peripheral blood mononuclear cells or whole blood. Further, resistance to H2O2 induced DNA damage, GSH, GSSG and CRP were measured. Increased dietary protein intake did not impact on DNA damage markers and GSH/GSSG levels. A seasonal-based time effect (p < 0.05), which led to a decrease in DNA damage and GSH was observed in the Austrian study. Therefore, increasing the protein intake to more than 20% of the total energy intake in community-dwelling seniors in Austria and New Zealand did not increase measures of DNA damage, change glutathione status or elevate plasma CRP.Education, Faculty ofNon UBCKinesiology, School ofReviewedFacultyResearcherOthe

Identification of Dlk1-Dio3 Imprinted Gene Cluster Noncoding RNAs as Novel Candidate Biomarkers for Liver Tumor Promotion

The molecular events during non-genotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital-mediated liver tumor promotion in vivo. Molecular profiling (mRNA, miRNA, DNA methylation & proteins) of mouse liver during 13 weeks of phenobarbital treatment revealed progressive increases in hepatic expression of long non-coding RNAs and microRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. Phenobarbital-induction of the Dlk1-Dio3 cluster non-coding RNA Meg3 was localised to glutamine synthetase positive hypertrophic perivenous hepatocytes suggesting a role for β-catenin signaling in the dysregulation of Dlk1-Dio3 non-coding RNAs. The carcinogenic relevance of Dlk1-Dio3 locus non-coding RNA induction was further supported by in vivo genetic dependence on Constitutive Androstane Receptor (CAR) and β-catenin pathways. Our data identify Dlk1-Dio3 non-coding RNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds