Mechanisms of autoinflammation and therapy targets in Gout

Contains fulltext : 178237.pdf (publisher's version ) (Open Access)Radboud University, 15 november 2017Promotores : Joosten, L.A.B., Netea, M.G.289 p

Innate immune memory: Implications for host responses to damage-associated molecular patterns

Cells of the innate immune system build immunological memory via epigenetic reprogramming after stimulations with microbial ligands. This functional readjustment allows for enhanced nonspecific inflammatory responses upon secondary challenges, a process termed "trained immunity." The epigenomic blueprint of trained monocytes has been recently reported, which revealed several important immunologic and metabolic mechanisms that underlie these changes. Interestingly, similar long-term reprogramming of cytokine production has also been described to be induced by endogenous damage-associated molecular patterns (DAMPs). Here, we present an overview of the novel data showing that endogenous alarm signals associated with tissue damage and sterile inflammation can induce trained immunity through epigenetic regulation of transcriptional programs. We describe new and old evidence of persistent effects of DAMPs in driving inflammation and enforce the concept that the influence of tissue-derived signals is critical in adjusting the magnitude and type of immune response built by the host. The better characterization of trained immunity for the persistence of inflammation induced by DAMPs would provide new possibilities for intervention in aging and autoinflammatory disorders

New gout test: enhanced ex vivo cytokine production from PBMCS in common gout patients and a gout patient with Kearns-Sayre syndrome

Item does not contain fulltextMonosodium urate (MSU) monohydrate crystals synergize with various toll-like receptor (TLR) ligands to induce interleukin-(IL)-1beta production. Data are shown from a young male with mitochondriopathy in Kearns-Sayre syndrome (KSS) who developed gout and underwent urate-lowering therapy (ULT) versus a group of common gout patients. Peripheral blood mononuclear cells (PBMCs) are exposed in vitro to MSU crystals in the presence/absence of TLR2 ligands palmitic acid (C16:0) or palmitoyl-3-cysteine (Pam3Cys); proinflammatory cytokine production (IL-1beta, IL-6, IL-8) is assessed by specific ELISA's. MSU crystals alone failed to induce IL-1beta, IL-6, or IL-8 in both the KSS patient and gout controls. A strong synergy between MSU crystals and C16:0 or Pam3Cys for induction of IL-1 beta/IL-6 is found in gout patients, but in gout with KSS, we found even more response than in control gout patients. Pam3Cys exposure reveals an enhanced response in cells originating from the KSS patient, indicating a high producer phenotype in response to TLR2 stimulation. During ULT, serum urate levels dropped in the KSS case. The hyperresponse of TLR2 may be secondary to the high serum urate concentration of 0.92 mmol/l that was initially found in circulation in vivo. Within a 6-month period, the serum urate concentration dropped, and the in vitro stimulation tests improved but did not fully normalize yet. The ex vivo cytokine production in gout patients is promising a novel gout test; PBMCs' responses in the mitochondriopathic gout patient is enhanced when compared with common gout patients, indicating a supersensitive gout patient profile. The non-inflammatory presentation in the KSS case with bulky gout is due to less inflammatory MSU crystals, i.e., specific crystal stereochemical/conformational properties. For developing gout attacks, the serum urate level and specific crystal properties both are of importance. Key Messages 1. Ex vivo cell tests are promising to serve as a novel gout lab test for screening purposes. 2. Ex vivo cellular responses are reduced following intraarticular glucocorticoid injection and/or urate-lowering therapy. 3. Crystal conformation properties play a role in the inflammatory in vivo and ex vivo response in gout. 4. A young male with Kearns-Sayre syndrome is described with less pronounced inflammatory PMBC responses to his own MSU crystals which explains the advanced stage of urate accumulation in this individual

Enhanced interleukin-1beta production of PBMCs from patients with gout after stimulation with Toll-like receptor-2 ligands and urate crystals

Contains fulltext : 107754.pdf (publisher's version ) (Open Access)ABSTRACT: INTRODUCTION: Monosodium urate monohydrate (MSU) crystals synergize with various toll-like receptor (TLR) ligands to induce cytokine production via activation of the NOD-like receptor (NLR) family, pyrin domain-containing 3 (NLPR3) inflammasome. This has been demonstrated in vitro using human cell lines or monocytes of healthy volunteers. In the present study, we have investigated the effect of MSU crystals and of their combination with TLR ligands in peripheral blood mononuclear cells (PBMC) of patients with gout. METHODS: PBMCs from 18 patients with primary gout and 12 healthy donors were exposed to MSU crystals in the presence or absence of saturated fatty acid C18:0 (free fatty acid, TLR2 ligand), palmitoyl-3-cystein (Pam3Cys, TLR1/2 ligand) and fibroblast stimulating factor-1 (FSL-1, TLR 2/6 ligand). Production of IL-1beta, IL-6, IL-8, IL-17 and tumor necrosis factor alpha (TNFalpha) was determined by ELISA. mRNA transcripts of IL-1beta were measured by real-time PCR. RESULTS: MSU crystals alone failed to induce IL-1beta, IL-6 or TNFalpha in both patients and control groups, but a stronger synergy between MSU/Pam3Cys and MSU/C18:0 for the induction of IL-1beta was found in patients with gout compared to healthy controls. IL-6, but not IL-8, followed the kinetics of IL-1beta. No production of the neutrophil-recruiting IL-17 was detectable after stimulation of the patients' PBMCs with MSU in both the presence or absence of TLR ligands. No change of gene transcripts of IL-1beta after stimulation with MSU and Pam3Cys or with MSU and C18:0 was found. A positive correlation was found between synergy in IL-1beta production from PBMCs of patients between C18:0 and MSU crystals, as well as the annual number of attacks of acute gouty arthritis (rs: +0.649, P: 0.022). CONCLUSIONS: The synergy between MSU crystals and TLR-2 ligands is more prominent in patients with gout than in controls. This is likely mediated by the enhanced maturation of pro-IL-1beta into IL-1beta

TLR1 polymorphisms in Europeans and spontaneous pregnancy loss.

Toll-like receptors (TLRs) are critical components of the pathogen recognition by the host innate immune system. Recently it has been shown that TLR1 is under evolutionary pressure in Europeans. This involves the positive selection of the nonsynonymous TLR1 1805G variant in Europeans, although this is associated with poor TLR1 response and unfavorable prognosis in various infections. In terms of natural selection, differential fertility is another mechanism, independent of infection susceptibility, that may explain the polymorphism pattern observed for TLR1. To test this hypothesis, we assessed the correlation of two TLR1 SNPs (T1805G and G239C) with spontaneous pregnancy loss in a case-control study that included 132 spontaneous pregnancy loss patients and 142 control volunteers. Similar allele frequencies of T1805G were observed between cases and controls, but GG genotype tended to be associated with pregnancy loss (OR 1.91; 95%CI 1.03, 3.53). No differences were observed for the TLR1 G239C SNP. Our findings showed slight differences in the distribution of T1805G variants in women with pregnancy loss, but these were not indicative of a protective effect of the TLR1 1805G allele for this fertility disorder. Although our hypothesis was not proven, potential effects of TLR1 polymorphisms on pregnancy outcome have been suggested, and future studies in larger cohorts are warranted

Soluble uric acid primes TLR-induced proinflammatory cytokine production by human primary cells via inhibition of IL-1Ra

OBJECTIVES: The study of the proinflammatory role of uric acid has focused on the effects of its crystals of monosodium urate (MSU). However, little is known whether uric acid itself can directly have proinflammatory effects. In this study, we investigate the priming effects of uric acid exposure on the cytokine production of primary human cells upon stimulation with gout-related stimuli. METHODS: Peripheral blood mononuclear cells (PBMCs) were harvested from patients with gout and healthy volunteers. Cells were pretreated with or without uric acid in soluble form for 24 h and then stimulated for 24 h with toll-like receptor (TLR)2 or TLR4 ligands in the presence or absence of MSU crystals. Cytokine production was measured by ELISA; mRNA levels were assessed using qPCR. RESULTS: The production of interleukin (IL)-1beta and IL-6 was higher in patients compared with controls and this correlated with serum urate levels. Proinflammatory cytokine production was significantly potentiated when cells from healthy subjects were pretreated with uric acid. Surprisingly, this was associated with a significant downregulation of the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra). This effect was specific to stimulation by uric acid and was exerted at the level of gene transcription. Epigenetic reprogramming at the level of histone methylation by uric acid was involved in this effect. CONCLUSIONS: In this study we demonstrate a mechanism through which high concentrations of uric acid (up to 50 mg/dL) influence inflammatory responses by facilitating IL-1beta production in PBMCs. We show that a mechanism for the amplification of IL-1beta consists in the downregulation of IL-1Ra and that this effect could be exerted via epigenetic mechanisms such as histone methylation. Hyperuricaemia causes a shift in the IL-1beta/IL-1Ra balance produced by PBMCs after exposure to MSU crystals and TLR-mediated stimuli, and this phenomenon is likely to reinforce the enhanced state of chronic inflammation

Suppression of monosodium urate crystal-induced cytokine production by butyrate is mediated by the inhibition of class I histone deacetylases

OBJECTIVES: Acute gouty arthritis is caused by endogenously formed monosodium urate (MSU) crystals, which are potent activators of the NLRP3 inflammasome. However, to induce the release of active interleukin (IL)-1beta, an additional stimulus is needed. Saturated long-chain free fatty acids (FFAs) can provide such a signal and stimulate transcription of pro-IL-1beta. In contrast, the short-chain fatty acid butyrate possesses anti-inflammatory effects. One of the mechanisms involved is inhibition of histone deacetylases (HDACs). Here, we explored the effects of butyrate on MSU+FFA-induced cytokine production and its inhibition of specific HDACs. METHODS: Freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with MSU and palmitic acid (C16.0) in the presence or absence of butyrate or a synthetic HDAC inhibitor. Cytokine responses were measured with ELISA and quantitative PCR. HDAC activity was measured with fluorimetric assays. RESULTS: Butyrate decreased C16.0+MSU-induced production of IL-1beta, IL-6, IL-8 and IL-1beta mRNA in PBMCs from healthy donors. Similar results were obtained in PBMCs isolated from patients with gout. Butyrate specifically inhibited class I HDACs. The HDAC inhibitor, panobinostat and the potent HDAC inhibitor, ITF-B, also decreased ex vivo C16.0+MSU-induced IL-1beta production. CONCLUSIONS: In agreement with the reported low inhibitory potency of butyrate, a high concentration was needed for cytokine suppression, whereas synthetic HDAC inhibitors showed potent anti-inflammatory effects at nanomolar concentrations. These novel HDAC inhibitors could be effective in the treatment of acute gout. Moreover, the use of specific HDAC inhibitors could even improve the efficacy and reduce any potential adverse effects

Uric acid priming in human monocytes is driven by the AKT-PRAS40 autophagy pathway

Contains fulltext : 174695.pdf (publisher's version ) (Closed access)Metabolic triggers are important inducers of the inflammatory processes in gout. Whereas the high serum urate levels observed in patients with gout predispose them to the formation of monosodium urate (MSU) crystals, soluble urate also primes for inflammatory signals in cells responding to gout-related stimuli, but also in other common metabolic diseases. In this study, we investigated the mechanisms through which uric acid selectively lowers human blood monocyte production of the natural inhibitor IL-1 receptor antagonist (IL-1Ra) and shifts production toward the highly inflammatory IL-1beta. Monocytes from healthy volunteers were first primed with uric acid for 24 h and then subjected to stimulation with lipopolysaccharide (LPS) in the presence or absence of MSU. Transcriptomic analysis revealed broad inflammatory pathways associated with uric acid priming, with NF-kappaB and mammalian target of rapamycin (mTOR) signaling strongly increased. Functional validation did not identify NF-kappaB or AMP-activated protein kinase phosphorylation, but uric acid priming induced phosphorylation of AKT and proline-rich AKT substrate 40 kDa (PRAS 40), which in turn activated mTOR. Subsequently, Western blot for the autophagic structure LC3-I and LC3-II (microtubule-associated protein 1A/1B-light chain 3) fractions, as well as fluorescence microscopy of LC3-GFP-overexpressing HeLa cells, revealed lower autophagic activity in cells exposed to uric acid compared with control conditions. Interestingly, reactive oxygen species production was diminished by uric acid priming. Thus, the Akt-PRAS40 pathway is activated by uric acid, which inhibits autophagy and recapitulates the uric acid-induced proinflammatory cytokine phenotype

Alpha-1-anti-trypsin-Fc fusion protein ameliorates gouty arthritis by reducing release and extracellular processing of IL-1beta and by the induction of endogenous IL-1Ra

OBJECTIVES: In the present study, we generated a new protein, recombinant human alpha-1-anti-trypsin (AAT)-IgG1 Fc fusion protein (AAT-Fc), and evaluated its properties to suppress inflammation and interleukin (IL)-1beta in a mouse model of gouty arthritis. METHODS: A combination of monosodium urate (MSU) crystals and the fatty acid C16.0 (MSU/C16.0) was injected intra-articularly into the knee to induce gouty arthritis. Joint swelling, synovial cytokine production and histopathology were determined after 4 h. AAT-Fc was evaluated for inhibition of MSU/C16.0-induced IL-1beta release from human blood monocytes and for inhibition of extracellular IL-1beta precursor processing. RESULTS: AAT-Fc markedly suppressed MSU/C16.0-induced joint inflammation by 85-91% (p<0.001). Ex vivo production of IL-1beta and IL-6 from cultured synovia were similarly reduced (63% and 65%, respectively). The efficacy of 2.0 mg/kg AAT-Fc in reducing inflammation was comparable to 80 mg/kg of plasma-derived AAT. Injection of AAT-Fc into mice increased circulating levels of endogenous IL-1 receptor antagonist by fourfold. We also observed that joint swelling was reduced by 80%, cellular infiltration by 95% and synovial production of IL-1beta by 60% in transgenic mice expressing low levels of human AAT. In vitro, AAT-Fc reduced MSU/C16.0-induced release of IL-1beta from human blood monocytes and inhibited proteinase-3-mediated extracellular processing of the IL-1beta precursor into active IL-1beta. CONCLUSIONS: A single low dose of AAT-Fc is highly effective in reducing joint inflammation in this model of acute gouty arthritis. Considering the long-term safety of plasma-derived AAT use in humans, subcutaneous AAT-Fc emerges as a promising therapy for gout attacks

Crohn's disease-associated ATG16L1 polymorphism modulates pro-inflammatory cytokine responses selectively upon activation of NOD2

Contains fulltext : 96772.pdf (publisher's version ) (Closed access)OBJECTIVE: Autophagy has recently been shown to modulate the production of pro-inflammatory cytokine production and to contribute to antigen processing and presentation through the major histocompatibility complex. Genetic variation in the autophagy gene ATG16L1 has been recently implicated in Crohn's disease pathogenesis. The mechanisms underlying this association are not yet known, although experimental models suggest an inhibitory effect of autophagy on interleukin 1beta (IL-1beta) responses. Here, the effect of ATG16L1 genetic variation on cytokine responses has been assessed in humans. DESIGN AND SETTING: Peripheral blood mononuclear cells from healthy individuals and patients with Crohn's disease with different ATG16L1 genotypes were stimulated with ligands for Toll-like receptor 2 (TLR2), TLR4 and nucleotide-binding oligomerisation domain 2 (NOD2), with or without the autophagy inhibitor 3-methyladenine. Induction of cytokine production and related factors were measured at the mRNA and protein level. Furthermore, protein levels of ATG16L1 were assessed by western blot. RESULTS: The present study demonstrates that cells isolated from individuals bearing the ATG16L1 Thr300Ala risk variant, which is shown to affect ATG16L1 protein expression upon NOD2 stimulation, display increased production of the pro-inflammatory cytokines IL-1beta and IL-6, specifically after stimulation with NOD2 ligands. In contrast, no differences were found when cells were stimulated with TLR2 or TLR4 agonists. These findings were confirmed in two independent cohorts of volunteers and in a group of patients with Crohn's disease. The increased production could be ascribed to increased mRNA expression, while processing of pro-IL-1beta by caspase-1 activation was not affected. The effect of the ATG16L1 polymorphism was abrogated when autophagy was blocked. CONCLUSIONS: The present study is the first to link the ATG16L1 polymorphism with an excessive production of IL-1beta and IL-6 in humans, which may explain the effects of this polymorphism on the inflammatory process in Crohn's disease