MicroRNA-155 as a proinflammatory regulator via SHIP-1 down-regulation in acute gouty arthritis

Introduction

Gout is characterized by episodes of intense joint inflammation in response to intra-articular monosodium urate monohydrate (MSU) crystals. miR-155 is crucial for the proinflammatory activation of human myeloid cells and antigen-driven inflammatory arthritis. The functional role of miR-155 in acute gouty arthritis has not been defined. Therefore, the aim of this study was to examine the role of miR-155 in pathogenesis of acute gouty arthritis.

Methods

Samples from 14 patients with acute gouty arthritis and 10 healthy controls (HCs) were obtained. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were cultured in vitro with MSU crystals, and gene expression (human miR-155 and SHIP-1) were assessed by real-time PCR. THP-1 cells were stimulated by MSU crystals and/or miR-155 transfection and then subjected to Western blot analysis. Levels of human tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β in cell culture supernatants were measured by Luminex. Immunohistochemistry was performed on formalin-fixed gout tissues with anti–SHIP-1 antibody. A C57BL/6 J male mouse model of gout was used to analyze the expressions of miR-155, SHIP-1, and inflammatory cytokines.

Results

The samples from gouty arthritis were highly enriched in miR-155, with levels of expression being higher than those found in PBMC from HC. Treatment of the cells with MSU crystals strongly induced miR-155. In addition, overexpression of miR-155 in the cells decreased levels of SHIP-1 and promoted production of MSU-induced proinflammatory cytokines, such as TNF-α and IL-1β. Consistent with in vitro observations, miR-155 expression was elevated in the mouse model of gout. The production of inflammatory cytokines was markedly increased in MSU crystal induced peritonitis mice.

Conclusions

Overexpression of miR-155 in the gouty SFMC leads to suppress SHIP-1 levels and enhance proinflammatory cytokines.     

P2Y6 receptor signaling pathway mediates inflammatory responses induced by monosodium urate crystals

Gout occurs in individuals with hyperuricemia when monosodium urate (MSU) crystals precipitate in tissues and induce acute inflammation via phagocytic cells such as monocytes. MSU crystals have been demonstrated in skin diseases such as tophaceous gout or psoriasis; however, the importance of MSU crystals in the skin is totally unknown. In this study, we found that MSU crystals, through P2Y(6) receptors, stimulated normal human keratinocytes (NHK) to produce IL-1α, IL-8/CXCL8, and IL-6. P2Y(6) receptor expression increased in MSU-stimulated NHK. Both P2Y(6)-specific antagonist and P2Y(6) antisense oligonucleotides significantly inhibited the production of IL-1α, IL-8/CXCL8, and IL-6 by NHK. Similarly, the P2Y(6)-specific antagonist completely inhibited the MSU-induced production of IL-1β by THP-1 cells, a human monocytic cell line. Remarkably, the P2Y(6)-specific antagonist significantly reduced neutrophil influx in both mouse air pouch and peritonitis models. Thus, these results indicate that the P2Y(6) receptor signaling pathway may be a potential therapeutic target for MSU-associated inflammatory diseases, such as tophaceous gout.     

TRPA1 receptor stimulation by hydrogen peroxide is critical to trigger hyperalgesia and inflammation in a model of acute gout

Acute gout attacks produce severe joint pain and inflammation associated with monosodium urate (MSU) crystals leading to oxidative stress production. The transient potential receptor ankyrin 1 (TRPA1) is expressed by a subpopulation of peptidergic nociceptors and, via its activation by endogenous reactive oxygen species, including hydrogen peroxide (H₂O₂), contributes to pain and neurogenic inflammation. The aim of this study was to investigate the role of TRPA1 in hyperalgesia and inflammation in a model of acute gout attack in rodents. Inflammatory parameters and mechanical hyperalgesia were measured in male Wistar rats and in wild-type (Trpa1^+/+) or TRPA1-deficient (Trpa1^−/−) male mice. Animals received intra-articular (ia, ankle) injection of MSU. The role of TRPA1 was assessed by receptor antagonism, gene deletion or expression, sensory fiber defunctionalization, and calcitonin gene-related peptide (CGRP) release. We found that nociceptor defunctionalization, TRPA1 antagonist treatment (via ia or oral administration), and Trpa1 gene ablation abated hyperalgesia and inflammatory responses (edema, H₂O₂ generation, interleukin-1β release, and neutrophil infiltration) induced by ia MSU injection. In addition, we showed that MSU evoked generation of H₂O₂ in synovial tissue, which stimulated TRPA1 producing CGRP release and plasma protein extravasation. The MSU-elicited responses were also reduced by the H₂O₂-detoxifying enzyme catalase and the reducing agent dithiothreitol. TRPA1 activation by MSU challenge-generated H₂O₂ mediates the entire inflammatory response in an acute gout attack rodent model, thus strengthening the role of the TRPA1 receptor and H₂O₂ production as potential targets for treatment of acute gout attacks.     

How to regulate neutrophils in gout

Most research in gout has concentrated on the proinflammatory mechanisms to explain the inflammation that is generated when leucocytes are in contact with monosodium urate crystals. However, the episodic nature of gout and the absence of inflammation even when crystals are present suggest that there are natural counter-regulatory mechanisms to limit the inflammatory response. Gagné and colleagues showed that myeloid inhibitory C-type lectin, a C-type lectin inhibitory receptor expressed on neutrophils, modulates monosodium urate-induced neutrophil responses in vitro.Neutrophil recruitment and activation play a key role in the acute inflammatory response to monosodium urate (MSU) crystals. In acute gout, our current treatments such as nonsteroidal anti-inflammatory drugs, colchicine or corticosteroids all act on different steps of neutrophil activation. These drugs form part of the first treatment objective in gout – to relieve the painful symptoms of the acute attack – but do not address the second objective, which is to treat the underlying metabolic disorder hyperuricemia. Can neutrophil activation be manipulated or regulated? Are there signals that can be modulated and can this be of clinical relevance?The article by Gagné and colleagues provides evidence for an inhibitory pathway of neutrophil activation that acts through a recently described C-type lectin receptor called the myeloid inhibitory C-type lectin (MICL) [1]. This membrane receptor, also known as CLEC12A, inhibits neutrophil activation when it is engaged. C-type lectin receptors form a large family of proteins that have a common type of carbohydrate-binding domain that mediate cell adhesion and ligand binding in a calcium-dependent manner. Members of the C-type lectin receptors are known to participate in immune regulation, with well-known examples including Dectin-1 (CLE7A), DC-sign (CD209 or CLEC4L) and natural killer cell receptors (Ly49 or KLRA1). The MICL protein is encoded on chromosome 12p13, closely linked to the natural killer gene complex. MICL contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif and is expressed mainly on neutrophils and monocytes. Previous work has shown that the receptor could inhibit cellular activation [2]. The ligands that lead to MICL activation are currently unknown, as there is only a small body of data to show that the receptor interacts with ligands expressed in the bone marrow, thymus and kidney [3].In their studies, Gagné and colleagues showed that MSU crystals as well as a MICL-specific antibody downmodulated MICL expression on neutrophils. Reducing the expression of MICL by transfecting small interfering RNA or by antibody modulation of the receptor led to enhanced production of IL-8 when MSU was added to neutrophils, but no changes in IL-1β secretion were observed. The mechanisms of MICL signaling probably involve tyrosine phosphorylation as well as calcium flux, differing from previous results that showed MICL associated with the phosphatases SHP-1 and SHP-2 [2]. Finally, the addition of colchicine to neutrophils abrogated the negative effect of MSU on MICL expression.These results showed that reduced MICL expression is associated with augmented inflammatory responses from neutrophils, and a higher level of neutrophil MICL expression is associated with a reduced IL-8 production in vitro. As IL-8 is a major neutrophil chemoattractant, this can have important effects on neutrophil recruitment to an inflammatory site in gout. By extrapolation, if MICL expression or signaling could be enhanced or maintained during inflammation, the inhibitory signal may be reinforced and thereby downregulate inflammation. The effect of colchicine in this system is to elevate the expression of MICL, thereby increasing the inhibitory signaling mechanisms that counteract the inflammatory process.A number of caveats need to be mentioned in the interpretation of these results. The data presented were based on in vitro models of inflammation using MSU, and we need to see how this works in vivo before coming to any conclusions, as we have had examples where the in vivo results did not recapitulate the in vitro findings. They convincingly showed that reducing MICL expression on the surface of neutrophils enhanced the proinflammatory signature, but they did not show the converse – that enhanced MICL signaling can further downmodulate inflammation. Furthermore, the ligands that bind and activate MICL are unknown, so we have no idea what is the signal or how to reinforce or manipulate this signaling system. The results presented show that MSU had dual effects on neutrophils – the first is to downregulate MICL expression, and the second is to activate IL-8 production. How are these two mechanisms linked? If MSU acts mainly on the cell membrane internalization of MICL, what is the trigger for the IL-8 secretion? Notwithstanding these uncertainties, the finding that MICL modulates neutrophil activation in gout suggests that there are a number of counter-regulatory mechanisms in operation during an inflammatory process. Identifying these mechanisms may help us to understand the nature of gout as well as open up new therapeutic perspectives.     

Monosodium urate crystals synergize with IFN-gamma to generate macrophage nitric oxide: involvement of extracellular signal-regulated kinase 1/2 and NF-kappa B

Elevated NO production has been detected in patients suffering from various arthropathies; however, its role and regulation during gouty arthritis remain largely unexplored. Monosodium urate (MSU) crystals, the causative agent of gout, have been shown to induce NO generation in vivo and inducible NO synthase (iNOS) expression in human monocytes. The present study was designed to evaluate the ability of MSU crystals to modulate macrophage (M phi) iNOS expression and NO synthesis and to investigate the molecular mechanisms underlying these cellular responses. We found that MSU crystals did not induce NO production in murine J774 M phi. However, a synergistic effect on the level of iNOS expression and NO generation was observed in cells exposed to MSU crystals in combination with IFN-gamma. Characterization of the second messengers involved revealed the requirement of IFN-gamma-mediated Janus kinase 2/STAT1 alpha activation even though MSU crystals did not modulate this signaling cascade by themselves. MSU crystals exerted their up-regulating effect by increasing extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and NF-kappa B nuclear translocation in response to IFN-gamma. The use of specific inhibitors against either NF-kappa B or the ERK1/2 pathway significantly reduced MSU + IFN-gamma-inducible NF-kappa B activity, iNOS expression, and NO production. Altogether, these data indicate that MSU crystals exert a potent synergistic effect on the IFN-gamma-inducible M phi NO generation via ERK1/2- and NF-kappa B-dependent pathways. Understanding the molecular mechanisms through which MSU crystals amplify M phi responses to proinflammatory cytokines such as IFN-gamma will contribute to better define their role in NO regulation during gout, in particular, and inflammation, in general.     

Engagement of CD14 mediates the inflammatory potential of monosodium urate crystals

Phagocyte ingestion of monosodium urate (MSU) crystals can induce proinflammatory responses and trigger acute gouty inflammation. Alternatively, the uptake of MSU crystals by mature macrophages can be noninflammatory and promote resolution of gouty inflammation. Macrophage activation by extracellular MSU crystals involves apparent recognition and ingestion mediated by TLR2 and TLR4, with subsequent intracellular recognition linked to caspase-1 activation and IL-1beta processing driven by the NACHT-LRR-PYD-containing protein-3 inflammasome. In this study, we examined the potential role in gouty inflammation of CD14, a phagocyte-expressed pattern recognition receptor that functionally interacts with both TLR2 and TLR4. MSU crystals, but not latex beads, directly bound recombinant soluble (s) CD14 in vitro. CD14(-/-) bone marrow-derived macrophages (BMDMs) demonstrated unimpaired phagocytosis of MSU crystals but reduced p38 phosphorylation and approximately 90% less IL-1beta and CXCL1 release. Attenuated MSU crystal-induced IL-1beta release in CD14(-/-) BMDMs was mediated by decreased pro-IL-1beta protein expression and additionally by decreased caspase-1 activation and IL-1beta processing consistent with diminished NACHT-LRR-PYD-containing protein-3 inflammasome activation. Coating of MSU crystals with sCD14, but not sTLR2 or sTLR4, restored IL-1beta and CXCL1 production in CD14(-/-) BMDMs in vitro. Gain of function of CD14 directly enhanced TLR4-mediated signaling in response to MSU crystals in transfected Chinese hamster ovary cells in vitro. Last, MSU crystal-induced leukocyte influx at 6 h was reduced by approximately 75%, and local induction of IL-1beta decreased by >80% in CD14(-/-) mouse s.c. air pouches in vivo. We conclude that engagement of CD14 is a central determinant of the inflammatory potential of MSU crystals.     

Animal model of acute gout reproduces the inflammatory and ultrasonographic joint changes of human gout

IntroductionGout is an inflammatory condition induced by the deposition of monosodium urate (MSU) crystals in the joints and soft tissues that can produce acute or chronic arthritis. Several animal models of crystal-induced inflammation have been proposed that involve direct injection of MSU-crystals into different anatomical structures; however, only a few of these models reflect a true diarthrodial joint microenvironment in which an acute gouty attack takes place. The aim of this study was to assess the inflammatory and structural joint changes in a rabbit model of acute gout attack by ultrasound (US), synovial fluid (SF) and histopathological analyses.MethodsUnder US guidance, 42 rabbit knees were randomly injected with a suspension of 50 mg/ml of either MSU or allopurinol synthetic crystals. The control group received intra-articular vehicle of phosphate-buffered saline (PBS). US evaluation, SF and histopathological analyses were performed at days 1, 3, and 7.ResultsA total of 21 rabbit knees were assigned to the control group, 12 to the MSU-crystals group, and 9 to the allopurinol crystals group. By US, the MSU crystals group displayed the double contour sign and bright stippled aggregates in 67% and 75% of joints, respectively. Neither control knees nor allopurinol crystals group displayed these US signs. Power Doppler (PD) signal was moderate to intense in the MSU-crystals group and greater than both the allopurinol crystal and control groups at day 1 (P <0.001) and 3 (P <0.05), with its practical disappearance by day 7. SF leukocyte count was 40,312 ± 6,369 cells/mm³ in the MSU-crystals group, higher than in controls (P = 0.004) and allopurinol crystal group (P = 0.006). At day 7, SF leukocyte count decreased in both MSU and allopurinol crystal groups reaching the non-inflammatory range. Histologically, at day 3 intense synovial polymorphonuclear cells infiltration and MSU aggregates were identified.ConclusionThe rabbit model of MSU crystal-induced acute arthritis efficiently reproduces the inflammatory, US, SF and histopathological changes of the human acute gouty attack.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0550-4) contains supplementary material, which is available to authorized users.     

Diagnostic value of fine needle aspiration cytology in gouty tophi: a report of 7 cases

BACKGROUND The diagnosis of gout can be problematic when the presentation is atypical and serum uric acid is borderline elevated. Demonstration of monosodium urate (MSU) crystals in fine needle aspiration (FNA) smears from nodular masses clinically suspected to be tophi establishes the diagnosis unequivocally. CASES: Of the 7 cases in this study, 4 were suspected clinically to have gouty tophi. Giant cell tumor of tendon sheath, giant cell tumor of bone and metastatic tumor with multicentric involvement of bone were the clinical diagnoses in 1 case each. Serum uric acid levels high enough to be in the diagnostic range for gout were reported in 3 cases, within normal limits in 3 cases and low in 1 chronic alcoholic patient. Bright field microscopy of FNA smears revealed singly scattered or stacks of MSU crystals with variable number of inflammatory cells, with or without foreign body giant cells in 6 cases. In 1 patient, FNA showed stacks of MSU crystals only. Characteristic birefringence of MSU crystals was observed on polarizing microscopy. CONCLUSION: FNA demonstration of MSU crystals on polarizing microscopy can easily establish the nature of the nodules in and around the joints and in soft tissue as gouty tophi and is thus an investigation differentiating this lesion from other masses clinically simulating it.     

Urate Crystal Induced Inflammation and Joint Pain Are Reduced in Transient Receptor Potential Ankyrin 1 Deficient Mice – Potential Role for Transient Receptor Potential Ankyrin 1 in Gout

IntroductionIn gout, monosodium urate (MSU) crystals deposit intra-articularly and cause painful arthritis. In the present study we tested the hypothesis that Transient Receptor Poten-tial Ankyrin 1 (TRPA1), an ion channel mediating nociceptive signals and neurogenic in-flammation, is involved in MSU crystal-induced responses in gout by utilizing three experi-mental murine models.MethodsThe effects of selective pharmacological inhibition (by HC-030031) and genetic depletion of TRPA1 were studied in MSU crystal-induced inflammation and pain by using 1) spontaneous weight-bearing test to assess MSU crystal-induced joint pain, 2) subcutaneous air-pouch model resembling joint inflammation to measure MSU crystal-induced cytokine production and inflammatory cell accumulation, and 3) MSU crystal-induced paw edema to assess acute vascular inflammatory responses and swelling.ResultsIntra-articularly injected MSU crystals provoked spontaneous weight shift off from the affected limb in wild type but not in TRPA1 knock-out mice referring alleviated joint pain in TRPA1 deficient animals. MSU crystal-induced inflammatory cell infiltration and accumulation of cytokines MCP-1, IL-6, IL-1beta, MPO, MIP-1alpha and MIP-2 into subcu-taneous air-pouch (resembling joint cavity) was attenuated in TRPA1 deficient mice and in mice treated with the selective TRPA1 inhibitor HC-030031 as compared to control animals. Further, HC-030031 treated and TRPA1 deficient mice developed tempered inflammatory edema when MSU crystals were injected into the paw.ConclusionsTRPA1 mediates MSU crystal-induced inflammation and pain in experimental models supporting the role of TRPA1 as a potential mediator and a drug target in gout flare.     

Plasma interleukin (IL)-18 (interferon-gamma-inducing factor) and other inflammatory cytokines in patients with gouty arthritis and monosodium urate monohydrate crystal-induced secretion of IL-18

To determine whether levels of interleukin (IL)-18, together with those of IL-1beta, tumor necrosis factor-alpha, IL-6, and IL-8, are elevated in the plasma of patients with gouty arthritis, the plasma concentrations of those cytokines were measured in 31 males with gouty arthritis. Further, CD14+ cells were obtained from human blood and thioglycolate medium-induced peritoneal cells obtained from caspase 1-deficient mice, and then separately cultured in the presence of monosodium urate monohydrate (MSU) crystals. In addition, in an animal in vivo experiment, MSU crystals were injected into subcutaneous air pouches of IL-18-deficient mice. The plasma concentrations of IL-18, IL-6, and IL-8 were elevated in the presence of gouty arthritis in the gout patients. In the in vitro study, the presence of MSU crystals stimulated CD14+ cells (monocytes) to secrete IL-18 and increased the activity of caspase 1 in CD14+ cells, whereas there was no significant effect on IL-18 messenger RNA in CD14+ cells and only a slight induction of IL-18 secretion from thioglycolate medium-induced caspase 1-deficient peritoneal cells. In the in vivo experiment, MSU crystals injected into the air pouch promoted neutrophil accumulation along with an increase in concentrations of keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-1alpha in air-pouch fluids in both IL-18-deficient and wild-type mice. However, there was no increase in the concentration of IL-18 in air-pouch fluids in either mouse strain. Our results suggest that plasma IL-18, IL-6, IL-8, and C-reactive protein (CRP) levels reflect local inflammation associated with gouty arthritis, though IL-18 does not play an important role in neutrophil accumulation. Further, they suggest that MSU crystals accelerate the processing of IL-18 from an inactive to active form via the activation of caspase 1.     

Gout in the spotlight

Understanding how uric acid crystals provoke inflammation is crucial to improving our management of acute gout. It is well known that urate crystals stimulate monocytes and macrophages to elaborate inflammatory cytokines, but the tissue response of the synovium is less well understood. Microarray analysis of mRNA expression by these lining cells may help to delineate the genes that are modulated. Employing a murine air-pouch model, a number of genes expressed by innate immune cells were found to be rapidly upregulated by monosodium urate crystals. These findings provide new research avenues to investigate the physiopathology of gouty inflammation, and may eventually lead to new therapeutic targets in acute gout.     

Development of novel NLRP3-XOD dual inhibitors for the treatment of gout

Gout is a crystalline-related arthropathy caused by the deposition of monosodium urate (MSU). Acute gouty arthritis is the most common first symptom of gout. Studies have shown that NOD-like receptor protein 3 (NLRP3) inflammasome as pattern recognition receptors can be activated by uric acid crystallization, triggering immune inflammation and causing acute gouty arthritis symptoms. Currently, the treatment of gout mainly includes two basic methods: reducing uric acid and alleviating inflammation. In this paper, 22 novel benzoxazole and benzimidazole derivatives were synthesized from deoxybenzoin oxime derivatives. These compounds have good inhibitory effects on NLRP3 and XOD screened by our research group in the early stage. The inhibitory activities of XOD and NLRP3 and their derivatives were also screened. Notably, compound 9b is a multi-targeting inhibitor of NLRP3 and XOD with excellent potency in treating hyperuricemia and acute gouty arthritis.     

Pycnogenol attenuates the inflammatory and nitrosative stress on joint inflammation induced by urate crystals

Acute gouty arthritis results from monosodium urate (MSU) crystal deposition in joint tissues. Deposited MSU crystals induce an acute inflammatory response which leads to damage of joint tissue. Pycnogenol (PYC), an extract from the bark of Pinus maritime, has documented antiinflammatory and antioxidant properties. The present study aimed to investigate whether PYC had protective effects on MSU-induced inflammatory and nitrosative stress in joint tissues both in vitro and in vivo. MSU crystals upregulated cyclooxygenase 2 (COX-2), interleukin 8 (IL-8) and inducible nitric oxide synthase (iNOS) gene expression in human articular chondrocytes, but only COX-2 and IL-8 in synovial fibroblasts. PYC inhibited the up-regulation of COX-2, and IL-8 in both articular chondrocytes and synovial fibroblasts. PYC attenuated MSU crystal induced iNOS gene expression and NO production in chondrocytes. Activation of NF-κB and SAPK/JNK, ERK1/2 and p38 MAP kinases by MSU crystals in articular chondrocytes and synovial fibroblasts in vitro was attenuated by treatment with PYC. The acute inflammatory cell infiltration and increased expression of COX-2 and iNOS in synovial tissue and articular cartilage following intra-articular injection of MSU crystals in a rat model was inhibited by coadministration of PYC. Collectively, this study demonstrates that PYC may be of value in treatment of MSU crystal-induced arthritis through its anti-inflammatory and anti-nitrosative activities.     

Morin,a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators,Intracellular ROS Levels and NF-κB Activation

Our previous studies had reported that morin, a bioflavanoid exhibited potent anti-inflammatory effect against adjuvant-induced arthritic rats. In this current study, we investigated the anti-inflammatory mechanism of morin against monosodium urate crystal (MSU)-induced inflammation in RAW 264.7 macrophage cells, an in vitro model for acute gouty arthritis. For comparison purpose, colchicine was used as a reference drug. We have observed that morin (100–300 μM) treatment significantly suppressed the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1 and VEGF), inflammatory mediators (NO and PEG₂), and lysosomal enzymes (acid phosphatase, β-galactosidase, N-acetyl glucosamindase and cathepsin D) in MSU-crystals stimulated macrophage cells. The mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1), inflammatory enzymes (iNOS and COX-2), and NF-κBp65 was found downregulated in MSU crystal stimulated macrophage cells by morin treatment, however, the mRNA expression of hypoxanthine phospho ribosyl transferse (HPRT) was found to be increased. The flow cytometry analysis revealed that morin treatment decreased intracellular reactive oxygen species levels in MSU crystal stimulated macrophage cells. The western blot analysis clearly showed that morin mainly exerts its anti-inflammatory effects by inhibiting the MSU crystal-induced COX-2 and TNF-α protein expression through the inactivation of NF-κB signaling pathway in RAW 264.7 macrophage cells similar to that of BAY 11–7082 (IκB kinase inhibitor). Our results collectively suggest that morin can be a potential therapeutic agent for inflammatory disorders like acute gouty arthritis.     

TLR2 signaling in chondrocytes drives calcium pyrophosphate dihydrate and monosodium urate crystal-induced nitric oxide generation

Microcrystals of calcium pyrophosphate dihydrate (CPPD) and monosodium urate (MSU) deposited in synovium and articular cartilage initiate joint inflammation and cartilage degradation in large part by binding and directly activating resident cells. TLRs trigger innate host defense responses to infectious pathogens, and the expression of certain TLRs by synovial fibroblasts has revealed the potential for innate immune responses to be triggered by mesenchymally derived resident cells in the joint. In this study we tested the hypothesis that chondrocytes also express TLRs and that one or more TLRs centrally mediate chondrocyte responsiveness to CPPD and MSU crystals in vitro. We detected TLR2 expression in normal articular chondrocytes and up-regulation of TLR2 in osteoarthritic cartilage chondrocytes in situ. We demonstrated that transient transfection of TLR2 signaling-negative regulator Toll-interacting protein or treatment with TLR2-blocking Ab suppressed CPPD and MSU crystal-induced chondrocyte release of NO, an inflammatory mediator that promotes cartilage degeneration. Conversely, gain-of-function of TLR2 in normal chondrocytes via transfection was associated with increased CPPD and MSU crystal-induced NO release. Canonical TLR signaling by parallel pathways involving MyD88, IL-1R-associated kinase 1, TNF receptor-associated factor 6, and IkappaB kinase and Rac1, PI3K, and Akt critically mediated NO release in chondrocytes stimulated by both CPPD and MSU crystals. We conclude that CPPD and MSU crystals critically use TLR2-mediated signaling in chondrocytes to trigger NO generation. Our results indicate the potential for innate immunity at the level of the articular chondrocyte to directly contribute to inflammatory and degenerative tissue reactions associated with both gout and pseudogout.     

Interleukin 1 (IL 1) as a mediator of crystal arthritis. Stimulation of T cell and synovial fibroblast mitogenesis by urate crystal-induced IL 1

We reported before that monosodium urate (MSU) crystals were potent stimulators of endogenous pyrogen (EP) production from human and rabbit mononuclear phagocytes, and proposed that this property of MSU crystals may be important in the pathogenesis of gout. EP activity is now attributed to interleukin 1 (IL 1) peptides but IL 1 is not the only pyrogenic monocyte-derived cytokine, since both interferon-alpha (alpha-IFN) and tumor necrosis factor (TNF) are also pyrogenic in rabbits. Using a T cell comitogenic assay based on a murine helper T cell clone that does not respond to IFN or TNF, we now report the release of IL 1 activity from human blood monocytes and synovial fluid mononuclear cells (MNC), following stimulation with MSU crystals. MSU-induced supernatants with IL 1 activity were neutralized with rabbit antiserum to human IL 1 and also stimulated the growth ([3H]thymidine incorporation) of long-term fibroblast-like cell lines derived from human synovial rheumatoid exudate. Two other crystals associated with articular inflammation were tested: hydroxyapatite was a much less potent stimulus compared with MSU crystals, and calcium pyrophosphate dihydrate did not stimulate IL 1 release from human monocytes or synovial fluid MNC. As a model for the inflammatory consequences of acute and chronic overproduction of IL 1, gout is the only sterile inflammatory disease where the local and systemic pathology is compatible with such overproduction; raised IL 1 levels have been found at the site of inflammation, and a necessary etiologic agent, crystalline urate, has been shown unequivocally to be a direct activator of mononuclear IL 1 release.     

Fine needle aspiration of tophi for crystal identification in problematic cases of gout. A report of two cases

BACKGROUND: The definitive diagnosis of gout is best established by demonstration of monosodium urate (MSU) crystals in the synovial fluid or biopsy. Fine needle aspiration cytology (FNAC) of tophi can play a crucial role in diagnosis. CASES: A 36-year-old chronic alcoholic male developed subcutaneous nodules on both malleoli without a history of arthropathy and with normal serum uric acid levels. FNAC of the nodules demonstrated stacks and sheaves of needle-shaped crystals of MSU. A 50-year-old diabetic male developed multiple nodules on the feet. He gave a past history of painful athropathy. A roentgenogram of the feet was suspicious for gout; however, joint aspiration failed, and the serum uric acid levels were normal. At this juncture FNAC of the feet tophi clinched the diagnosis of gout. In both cases, polarization of needle washings (wet mount) and the fixed, Papanicolaoustained smears showed negatively birefringent, needle-shaped crystals of MSU, thus confirming the diagnosis of gout. CONCLUSION: FNAC of gouty tophi is an easy alternative to synovial biopsy and joint fluid analysis. It is simpler, easier and less painful. As crystals are preserved in stained smears, they can be employed for polarization and confirmation of gout.     

Identification of novel monosodium urate crystal regulated mRNAs by transcript profiling of dissected murine air pouch membranes

Introduction  

The murine air pouch is a bursa-like space that resembles the human synovial membrane. Injection of monosodium urate (MSU) crystals into the pouch elicits an acute inflammatory response similar to human gout. We conducted the present study to identify mRNAs that were highly regulated by MSU crystals in the pouch membrane.     

Neutrophil extracellular trap formation is associated with IL-1β and autophagy-related signaling in gout

Background

Gout is a prevalent inflammatory arthritis affecting 1–2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β.

Methodology/Principal Findings

Synovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra.

Conclusions/Significance

These findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.