The Contribution Of Toll Like Receptors To The Pathogenesis Of Gout

BackgroundToll-like receptors (TLRs) are major pattern recognition receptors (PRRs) of the innate immune system. It can detect pathogen-associated molecular patterns (PAMP), trigger intracellular signaling cascades, ultimately culminate in the expression of a variety of proinflammatory molecules, which is a prerequisite for the subsequent activation and shaping of adaptive immunity. Thus they serve as a bridge to long-term adaptive immune responses. Recent studies suggest that monosodium urate (MSU) crystals may act as a kind of danger-associated molecular patterns (DAMPs) which convey similar messages and elicit similar responses as PAMPs, and may be involved in the pathogenesis of gout. Findings from existing studies are mostly based on animal models or cell lines except that very few studies directly choose patients with gout as their objects to investigate the association between TLRs and the inflammatory mechanisms of gout.ObjectivePatients with gout in acute stage or non-acute stage were chosen as objects of our study to investigate the association between TLRs and the inflammatory mechanisms and its clinical significance in gout, to investigate the role of TLR4 in inflammatory reaction and its signal transduction in gout, to suggest the possibility of blocking the abnormal inflammatory reaction of gout at a cellular level, and to find potential therapeutic targets of gout. MethodsThe study consists of two parts1. The expression of Toll-like receptor 2 and Toll-like receptor 4 mRNA in peripheral blood mononuclear cells from the patients with primary gout. Expression changes of TLRs in PBMC from patients with acute gout (32cases) or non-acute gout (20cases) were involved in this study. The expression of TLR2 and TLR4 mRNA in PBMCs from patients with gout was determined by RQ-PCR. ELISA and western blot were used to analyze the level of IL-1βin plasma and expression of NF-κB p65 protein in the nucleoprotein of PBMC. The concentration of uric acid in plasma was measured by automatic biochemistry analyzer. All results were adopted to compare with healthy controls(32cases)and correlation between TLR4 mRNA and the plasma uric acid , IL-1βlevel of the gout patients were measured.2. The study of the NF-κB p65 activation and IL-1βlevel through TLR4 signaling pathway after stimulation with LPS. Blood from 8 patients with acute gout and 8 healthy volunteers was aliquoted into 2.5 ml samples and received the following: (1) Treated with NS as blank control. (2) Treated with 10μg/ml LPS for 1 hour. (3) After pretreated with finity purified anti-human TLR4 (5μg/ml to 20μg/ml) for 2 hours, 10μg/ml LPS was added into the whole blood and incubated for 1 hour. After the above treatments , PBMCs for lysates were prepared from the whole blood by Ficoll density gradient centrifugation, the detection of transcript factor p65 was followed by TransAM NF-κB p65 Kit. 200μl whole blood after the above treatments were transfered into 96-well plates and incubated for 4 hours,plasma for IL-1βdeterminations was separated and examined by ELISA.Results1. Patients with acute gout showed higher TLR4 mRNA and NF-κB p65 protein level in their PBMC nucleus and higher IL-1βlevel, higher uric acid concentration in their plasma than patients with non-acute gout and healthy controls(p<0.01). Significant differences were also observed between the Non-acute gout patients and healthy controls(p<0.05).2. No significant difference of TLR2 mRNA level was observed between the Acute patients and non-acute gout patients or healthy controls(p>0.05). 3. The expression level of TLR4 mRNA had significant positive correlationwith the plasma uric acid and IL-1βlevel of the acute and non-acutegout patients. So does the expression level of TLR4 mRNA and IL-1βlevel in healthy controls. But the level of plasma uric acid was not significantly correlated with the IL-1βlevel or the expression level of TLR4 mRNA in healthy controls.4. LPS can significantly increase TLR4 mRNA in gout patients and healthy controls (p<0.001).5. LPS can significantly increase PBMC NF-κB p65 activation and plasma IL-1βlevel in gout patients and healthy controls (p<0.001); Gout patients represent higher IL-1βlevel increase after the treatment of LPS(p<0.001),but no significant difference was observed between each group in the increasing extent of NF-κB p65 activation(p=0.185).6. NF-κB p65activation and IL-1βlevel induced by LPS were greatly inhibited by pretreatment of anti-TLR4 in a dose-dependent manner (p<0.01). Pretreatment of 30μg/ml anti-TLR4 can inhibit the NF-κB p65 activation induced by LPS to its untreated condition, but can not inhibit IL-1βlevel induced by LPS to its untreated condition.Conclusion 1. Gout patients showed higher expression of TLR4 mRNA and plasma IL-1βlevel, higher NF-κB p65 protein in nucleus of their PBMC. TLR4 mRNA and plasma IL-1βlevel have positive correlation with UA in gout patients which indicates TLR4-NF-κB p65-IL-1βsignal transduction participates in the inflammatory mechanisms of gout. The inflammation appears as the increasing level of inflammatory factors such as IL-1βin plasma. Uric acid may be one of the agents which can activate this passway.2. LPS can promote gout patients and healthy controls express more TLR4 mRNA, stronger NF-κB p65 activation and higher plasma IL-1βlevel. The increasing extent of IL-1βin gout patients is higher than that of the controls. That activation can be inhibited by anti-TLR4 in a dose-dependent manner. The dose of anti-TLR4 which completely inhibited NF-κB p65 activation induced by LPS could not fully inhibit the increased IL-1βlevel induced by LPS. This phenomenon is more obvious in gout patients, which indicates LPS-TLR4-NF-κB p65-IL-1βsignal transduction may be involved in the inflammatory mechanisms of gout. Besides TLRs, there are other signaling pathways involved in the pathogenesis of gout.