The Anti-Inflammatory and Uric Acid Lowering Effects of Si-Miao-San on Gout.

Background: Si-Miao-San (SMS) is a well-known traditional Chinese medicine. This study aims to evaluate the anti-inflammatory effects of SMS on gouty arthritis and its potential mechanism of action. Methods: The effects and mechanism of SMS were evaluated in monosodium urate (MSU)-treated mice or macrophages. The expression of cytokines and PI3K/Akt was analyzed using real-time PCR and Western blotting analyses. Macrophage polarization was assessed with immunofluorescence assays, real-time PCR, and Western blotting. Mass spectrometry was used to screen the active ingredients of SMS. Results: Pretreatment with SMS ameliorated MSU-induced acute gouty arthritis in mice with increased PI3K/Akt activation and M2 macrophage polarization in the joint tissues. In vitro, SMS treatment significantly inhibited MSU-triggered inflammatory response, increased p-Akt and Arg-1 expression in macrophages, and promoted M2 macrophage polarization. These effects of SMS were inhibited when PI3K/Akt activation was blocked by LY294002 in the macrophages. Moreover, SMS significantly reduced serum uric acid levels in the hyperuricemia mice. Using mass spectrometry, the plant hormones ecdysone and estrone were detected as the potentially effective ingredients of SMS. Conclusion: SMS ameliorated MSU-induced gouty arthritis and inhibited hyperuricemia. The anti-inflammatory mechanism of SMS may exert anti-inflammatory effects by promoting M2 polarization via PI3K/Akt signaling. Ecdysone and estrone might be the potentially effective ingredients of SMS. This research may provide evidence for the application of SMS in the treatment of gout.

Berberine, an isoquinoline alkaloid suppresses TXNIP mediated NLRP3 inflammasome activation in MSU crystal stimulated RAW 264.7 macrophages through the upregulation of Nrf2 transcription factor and alleviates MSU crystal induced inflammation in rats.

The current study was designed to investigate the therapeutic potential of berberine on monosodium urate (MSU) crystal stimulated RAW 264.7 macrophages and in MSU crystal induced rats. Our results indicate that berberine (25, 50 and 75µM) suppressed the levels of pro-inflammatory cytokines (interleukin-1beta (IL-1ß) and tumor necrosis factor alpha (TNFα)) and intracellular reactive oxygen species in MSU crystal stimulated RAW 264.7 macrophages. The mRNA expression levels of IL-1ß, caspase 1, nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3), thioredoxin interacting protein (TXNIP) and kelch-like ECH-associated protein 1 (Keap1) were found downregulated with the upregulation of nuclear factor erythroid-2-related factor 2 (Nrf2) transcription factor and its associated anti-oxidant enzymes: Heme oxygenase I (HO-1), superoxide dismutase (SOD1), glutathione peroxidase (GPx), NADPH quinone oxidoreductase-1 (NQO1) and catalase (CAT) in MSU crystal stimulated RAW 264.7 macrophages upon berberine treatment. Subsequently, western blot analysis revealed that berberine decreased the protein expression of IL-1ß and caspase 1 and increased Nrf2 expression in RAW 264.7 macrophages. Immunofluorescence analysis also explored increased expression of Nrf2 in MSU crystal stimulated RAW 264.7 macrophages by berberine treatment. In addition, the paw edema, pain score, pro-inflammatory cytokines (IL-1ß and TNFα) and articular elastase activity were found significantly reduced in berberine (50mg/kgb·wt) administered MSU crystal-induced rats. Conclusively, our current findings suggest that berberine may represent as a potential candidate for the treatment of gouty arthritis by suppressing inflammatory mediators and activating Nrf2 anti-oxidant pathway.

Uric acid enhances the antitumor immunity of dendritic cell-based vaccine.

Uric acid (UA) released from dying cells has been recognized by the immune system as a danger signal. In response to UA, dendritic cells (DC) in the immune system mature and enhance the T cell response to foreign antigens. It is conceivable that the antitumor immunity of a tumor vaccine could be promoted by the administration of UA. To test this concept, we applied UA as an adjuvant to a DC-based vaccine, and discovered that the administration of UA as an adjuvant significantly enhanced the ability of the tumor lysate-pulsed DC vaccine in delaying the tumor growth. The antitumor activity was achieved with adoptively transferred lymphocytes, and both CD8(+) T cells and NK cells were required to achieve effective immunity. This resulted in an increased accumulation of activated CD8(+) T cells and an elevated production of IFN-γ. Collectively, our study shows that the administration of UA enhances the antitumor activity of tumor lysate-pulsed DC vaccine, thus providing the preclinical rationale for the application of UA in DC-based vaccine strategies.

Effect of chronic khat (Catha edulis, Forsk) use on outcome of Plasmodium berghei ANKA infection in Swiss albino mice.

BACKGROUND: The objective of this study was to explore effects of khat (Catha edulis) on outcome of rodent malaria infection and its anti-plasmodial activities on Plasmodium berghei ANKA (PbA). METHODS: Female Swiss albino mice were orally treated with crude khat (Catha edulis) extracts (100, 200 and 300 mg/kg) on a daily basis for 4 weeks prior to PbA infection. Physical, clinical, hematological, biochemical and histo-pathological features of the mice were assessed. In addition, in vivo anti-plasmodial activities of khat were evaluated. RESULTS: The finding of this study showed that khat use was strongly associated with increment of levels of liver and kidney biomarkers, leucopenia, severe anemia, rise in level of inflammation biomarkers: C-reactive protein (CRP), uric acid (UA), increased monocyte-lymphocyte count ratio (MLCR), manifestation of cerebral malaria symptoms such as ataxia, paralysis and deviation of the head but with no pulmonary edema. Significantly lower level of parasitemia (P<0.05), rectal temperature, but, high level of hemoglobin were observed at the early stage of the PbA infection in khat treated mice than the control. With extension of the treatment period, however, drastic increments were observed in parasite load and rectal temperature although there was reduction in hemoglobin (Hb) level. Moreover, khat showed poor anti-plasmodial activity with <10% parasite suppression activity and lack protection against major malaria symptoms. The significant reduction (P<0.01) of hematological parameters during PbA infection strengthen the notion that hematological parameters could be good predictors of severe malaria complications in human. CONCLUSIONS: In mice model treated with khat prior to infection with the rodent malaria parasite, khat was found to worsen manifestation of most malaria complications. Furthermore, the same plant showed poor in vivo anti-plasmodial activity and protection against major malaria symptoms.

Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa.

Although type 2 immune responses to environmental Ags are thought to play pivotal roles in asthma and allergic airway diseases, the immunological mechanisms that initiate the responses are largely unknown. Many allergens have biologic activities, including enzymatic activities and abilities to engage innate pattern-recognition receptors such as TLR4. In this article, we report that IL-33 and thymic stromal lymphopoietin were produced quickly in the lungs of naive mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and thymic stromal lymphopoietin sensitized naive animals to an innocuous airway Ag OVA, which resulted in production of type 2 cytokines and IgE Ab, and eosinophilic airway inflammation when mice were challenged with the same Ag. Importantly, upon exposure to proteases, uric acid (UA) was rapidly released into the airway lumen, and removal of this endogenous UA by uricase prevented type 2 immune responses. UA promoted secretion of IL-33 by airway epithelial cells in vitro, and administration of UA into the airways of naive animals induced extracellular release of IL-33, followed by both innate and adaptive type 2 immune responses in vivo. Finally, a potent UA synthesis inhibitor, febuxostat, mitigated asthma phenotypes that were caused by repeated exposure to natural airborne allergens. These findings provide mechanistic insights into the development of type 2 immunity to airborne allergens and recognize airway UA as a key player that regulates the process in respiratory mucosa.

PAMPs and DAMPs in allergy exacerbation models.

Sensitization of mice to real-life allergens or harmless antigen with the use of adjuvants will lead to the induction of DAMPs in the immune system. We have shown that the Th2-inducing adjuvant aluminum hydroxide or exposure of the airways to house dust mite leads to the release of DAMPs: uric acid, ATP, and IL-1. Exposure to DAMPs or PAMPs present in allergens or added to harmless allergens, such as the experimental allergen ovalbumin, induces several immune responses, including cellular influx and activation. Cellular influx can be analyzed by flow cytometry. Likewise, cellular activation can be assessed by measuring increased expression and release of chemokines and cytokines. These inflammatory mediators can be analyzed by ELISA or confocal microscopy. Here, we describe the protocols for these assessments and a protocol that takes advantage of bone marrow chimeric mice to further elucidate mechanism.

To forge a solid immune recognition.

Phagocytosis and innate immune responses to solid structures are topics of interest and debate. Alum, monosodium urate, calcium pyrophosphate dehydrate, silica and by extension all solid entities draw varying degrees of attention from phagocytes, such as antigen presenting cells. For some, innocuous soluble metabolites turn into fierce irritants upon crystallization, pointing to divergent signaling mechanisms of a given substance in its soluble and solid states. Over the years, many mechanisms have been proposed, including phagocytic receptors, toll like receptors, and NACHT-LRRs (NLRs), as well as several other protein structure mediated recognition of the solids. Is there a more general mechanism for sensing solids? In this perspective, I present an alternative view on the topic that membrane lipids can engage solid surfaces, and the binding intensity leads to cellular activation. I argue from the stands of evolution and biological necessity, as well as the progression of our understanding of cellular membranes and phagocytosis. The effort is to invite debate of the topic from a less familiar yet equally thrilling viewing angle.

Uric acid-driven Th17 differentiation requires inflammasome-derived IL-1 and IL-18.

Uric acid is released from damaged cells and serves as a danger signal that alerts the immune system to potential threats, even in the absence of microbial infection. Uric acid modulation of innate immune responses has been extensively studied, but the impact of this damage-associated molecular pattern on adaptive responses remains largely unknown. In this study, we report that, in the presence of NF-κB signaling, uric acid crystals were capable of stimulating dendritic cells to promote the release of cytokines associated with Th17 polarization. Accordingly, naive CD4(+) T cells cocultured with uric acid-treated dendritic cells differentiated toward the Th17 lineage. Th17 differentiation required the inflammasome-dependent cytokines IL-1α/ß and IL-18 in both in vitro and in vivo models, and the inflammasome adaptor protein ASC and caspase-1 were essential for Th17 responses. Collectively, our findings indicate a novel role for the danger signal uric acid, in cooperation with NF-κB activation, in driving proinflammatory Th17 differentiation. Our data indicate that sterile inflammation shapes adaptive immunity, in addition to influencing early innate responses.

The role of uric acid as an endogenous danger signal in immunity and inflammation.

Gout is an ancient disease that still plagues us. Its pathogenic culprit, uric acid crystal deposition in tissues, is a strong inflammatory stimulant. In recent years, the mechanisms through which uric acid crystals promote inflammation have been a subject of increasing interest among rheumatologists and immunologists. Uric acid has been identified as an endogenous adjuvant that drives immune responses in the absence of microbial stimulation. Because uric acid is a ubiquitous metabolite that is produced in high quantities upon cellular injury, the ramifications of its effects may be considerable in health and in disease. Uric acid crystals also have been shown to trigger interleukin-1ß-mediated inflammation via activation of the NOD-like receptor protein (NLRP)3 inflammasome, a multimolecular complex whose activation appears to be central to many pathological inflammatory conditions. In this article, we review the possible mechanisms of uric acid-mediated inflammation and offer some historical perspectives on what has been learned about the complex effects of a relatively simple substance.

Recent developments in immune activation by uric acid crystals.

Uric acid crystals, the causative agent of gout, have recently gained widespread attention due to their role as a natural endogenous adjuvant. Uric acid crystals, first sensed extracellularly by membrane lipid alterations, are internalized and subsequently activate the NLRP3 inflammasome. Currently, various aspects of this particular novel pathway are poorly defined. This short review will focus on some recent discoveries regarding this simple crystalline structure and address areas requiring further investigation. The fact that uric acid crystals activate innate host defense mechanisms, triggering robust inflammation and immune activation, may lead to engineering potent adjuvants for future vaccines. Furthermore, the elucidation of uric acid's mechanism of inflammation may lay the foundation for other solid inflammatory structures such as silica, asbestos, and alum.

Human eosinophils recognize endogenous danger signal crystalline uric acid and produce proinflammatory cytokines mediated by autocrine ATP.

Eosinophils are multifunctional leukocytes involved in various inflammatory processes, as well as tissue remodeling and immunoregulation. During inflammation and infection, injured cells and damaged tissues release uric acid and monosodium urate (MSU) crystals as important endogenous danger signals. Uric acid is also implicated in the immunogenic effects of an authentic Th2 adjuvant, aluminum hydroxide. Eosinophils often localize at sites of Th2-type chronic inflammation; therefore, we hypothesized that eosinophils may react to endogenous danger signals. We found that human eosinophils migrate toward soluble uric acid and MSU crystals in a gradient-dependent manner. Eosinophils incubated with MSU crystals, but not those incubated with uric acid solution, produced elevated levels of IL-6 and IL-8/CXCL8. Other cytokines and chemokines, including IL-1beta, IL-10, IL-17, IFN-gamma, CCL2, CCL3, CCL4, TNF-alpha, G-CSF, GM-CSF, fibroblast growth factor, vascular endothelial growth factor, and TGF-beta, were also produced by eosinophils incubated with MSU crystals. Eosinophils exposed to MSU crystals rapidly (i.e., within 1 min of exposure) released ATP into the extracellular milieu. Importantly, this autocrine ATP was necessary for eosinophils to produce cytokines in response to MSU crystals, and P2 nucleotide receptors, in particular P2Y(2), are likely involved in this positive feedback loop. Finally, at higher concentrations, MSU crystals promoted P2R-dependent release of a granule protein (eosinophil-derived neurotoxin) and cell death. Thus, human eosinophils may respond to particulate damage-associated endogenous danger signals. These responses by eosinophils to tissue damage may explain the self-perpetuating nature of chronic inflammation in certain human diseases, such as asthma.

The inflammasomes: guardians of the body.

The innate immune system relies on its capacity to rapidly detect invading pathogenic microbes as foreign and to eliminate them. The discovery of Toll-like receptors (TLRs) provided a class of membrane receptors that sense extracellular microbes and trigger antipathogen signaling cascades. More recently, intracellular microbial sensors have been identified, including NOD-like receptors (NLRs). Some of the NLRs also sense nonmicrobial danger signals and form large cytoplasmic complexes called inflammasomes that link the sensing of microbial products and metabolic stress to the proteolytic activation of the proinflammatory cytokines IL-1beta and IL-18. The NALP3 inflammasome has been associated with several autoinflammatory conditions including gout. Likewise, the NALP3 inflammasome is a crucial element in the adjuvant effect of aluminum and can direct a humoral adaptive immune response. In this review, we discuss the role of NLRs, and in particular the inflammasomes, in the recognition of microbial and danger components and the role they play in health and disease.

Mechanism of action of clinically approved adjuvants.

Aluminum-containing adjuvants continue to be the most widely used adjuvants for human use. In the last year a major breakthrough has been the realization that alum adjuvant triggers an ancient pathway of innate recognition of crystals in monocytes and triggers them to become immunogenic dendritic cells, nature's adjuvant. This recognition can occur directly, via the triggering of the NALP3 inflammasome by alum crystals, or indirectly through release of the endogenous danger signal uric acid. It is also clear now that adjuvants trigger the stromal cells at the site of injection, leading to the necessary chemokines that attract the innate immune cells to the site of injection. How exactly these pathways interact remains to be determined.

A role of IgM antibodies in monosodium urate crystal formation and associated adjuvanticity.

Uric acid is released from injured cells and can act as an adjuvant signal to the immune system. Uric acid crystals invoke strong inflammatory responses in tissues. Although their biological effects are evident and the associated signaling mechanisms are becoming clear, it remains unexplained as to why uric acid precipitates rapidly in vivo, in sharp contrast to the minimal crystallization in vitro. We report in this study that a group of IgM Abs is able to bind to these crystals, which is interesting in light that B cell-deficient mice do not sense the proinflammatory adjuvant effect of uric acid. The titers of these Abs increase upon immunization with uric acid crystals. We have produced large quantities of such mAbs. The purified IgM Abs can significantly facilitate uric acid precipitation to form the inflammatory crystals in vitro. Infusion of these Abs into B cell-deficient mice significantly increases the basal level of inflammation in these recipients and restores the host's ability to sense uric acid's adjuvanticity. Therefore, we have identified a factor in determining uric acid precipitation and possibly its ability to function as an endogenous adjuvant. This finding suggests a new mechanism of the pathogenesis of gouty arthritis and uric acid-induced immune activation.

The endogenous danger signal uric Acid augments contact hypersensitivity responses in mice.

OBJECTIVE: The danger hypothesis proposes that the immune system responds not only to foreign antigens but also to damaged cells or tissues. Recently, uric acid crystals (monosodium urate, MSU) from necrotic cell lysates were identified as a danger signal for dendritic cells (DCs). Our aim was to determine whether MSU modulates immune responses in the skin. METHOD: We analyzed the effect of MSU on trinitrochlorobenzene-induced contact hypersensitivity responses using BALB/c mice administered potassium oxonate, an uricase inhibitor, to prevent MSU degradation. Ear swelling response after elicitation and activation profiles of DCs and T cells in draining lymph nodes after sensitization were assessed. RESULTS: Intradermal administration of MSU augmented the ear swelling response in potassium oxonate-administered mice and enhanced expression of CD86 and CD40 molecules on DCs in the lymph nodes. Activation of DCs was followed by an increase in CD69+ and CD44+ T cells in CD4+ and/or CD8+ subsets in the lymph nodes 4 days after trinitrochlorobenzene sensitization. CONCLUSION: These observations demonstrate that MSU is an endogenous danger signal, which augments the contact hypersensitivity response in mice. MSU released from damaged skin may act as an endogenous adjuvant to augment immune response.

Uric acid, a nucleic acid degradation product, down-regulates dsRNA-triggered arthritis.

Uric acid, the naturally occurring degradation product of purine metabolism, is a danger signal, driving maturation of dendritic cells. It is well known that uric acid crystals display potent proinflammatory properties--the cause of gout--whereas the biological properties of soluble uric acid are less well documented. We have demonstrated previously that nucleic acids of endogenous and exogenous origin display proinflammatory properties. The aim of the present study was to assess the impact of soluble uric acid on in vivo inflammatory responses. Mice were administered with uric acid suspension in saline or saline alone prior to induction of neutrophil-mediated inflammation, delayed-type hypersensitivity, histamin-induced edema (measure of vasodilation capacity), as well as double-stranded (ds)RNA-triggered arthritis. Frequency and severity of arthritis were decreased significantly in mice exposed to dsRNA and simultaneously treated with uric acid as compared with saline-treated controls. Also, granulocyte-mediated inflammatory response and vasodilation capacity were reduced significantly in mice treated with uric acid as compared with their control group. The data suggest that down-regulation of inflammation was mediated by skewing the inflammatory response from the peripheral sites to the peritoneal cavity and down-regulating vasodilatatory capacity and thereby affecting leukocyte migration. In contrast, the T cell-mediated delayed-type hypersensitivity reaction was not affected significantly in mice exposed to uric acid. These findings demonstrate that uric acid displays a potent, distant anti-inflammatory effect in vivo. This property seems to be mediated by down-regulation of neutrophil influx to the site of inflammatory insult.

Membrane repair and immunological danger.

Antigens are able to elicit productive immune responses only when second signals are provided by adjuvant molecules. It is well established that exogenously acquired, pathogen-associated molecular patterns fulfil this adjuvant role when recognized by specific receptors on antigen-presenting cells. Recent evidence points to the existence of another class of adjuvant, which is apparently released from injured cells. Such endogenous adjuvants, referred to as 'danger' signals, could alert the immune system to situations that cause cell damage, but not necessarily those that involve infections. Endogenous adjuvants provide a good explanation for immune responses generated against tumours and autologous tissues, but it has been difficult to explain how a constant activation of the immune system is avoided, considering the frequency at which cells are injured in vivo. Here, we suggest that the efficiency with which cells reseal wounds in their plasma membrane might be an important factor in the balance between tolerance and autoimmunity. Recent observations in synaptotagmin-VII-deficient mice suggest that defective membrane repair could lead to autoimmunity in tissues that are more susceptible to mechanical injury.

Inhibition of monosodium urate monohydrate crystal-induced acute inflammation by retrovirally transfected prostaglandin D synthase.

OBJECTIVE: Hematopoietic prostaglandin D synthase (H-PGDS) is a key enzyme in the production of prostaglandin D and its J series metabolites. We evaluated the antiinflammatory effect of retrovirally transfected H-PGDS in order to investigate the role of H-PGDS in monosodium urate monohydrate (MSU) crystal-induced acute inflammation. METHODS: Expression of endogenous PGDS in a murine air-pouch model of MSU crystal-induced acute inflammation was determined by real-time polymerase chain reaction. H-PGDS complementary DNA (cDNA) was retrovirally transfected into C57BL/6J fibroblasts, and the cells were designated as C57-PGDS cells. Production of prostaglandins by C57-PGDS cells was measured by enzyme immunoassay. The effect of C57-PGDS cells on crystal-induced inflammation was investigated. RESULTS: Injection of the crystals caused a rapid decrease in H-PGDS expression by infiltrating cells and by the soft tissues around the air pouches. In contrast, expression of interleukin-1beta (IL-1beta) and macrophage inflammatory protein 2 (MIP-2) as well as cellular infiltration were significantly increased during the early stage of inflammation. C57-PGDS cells, but not control cells, produced an increased amount of PGD(2) in vitro, but suppressed production of PGE(2). Injection of C57-PGDS cells into air pouches inhibited cellular infiltration and MIP-2 and IL-1beta expression. CONCLUSION: In this murine air-pouch model of MSU crystal-induced inflammation, retrovirally transfected H-PGDS cDNA could reduce cellular infiltration, at least partly by inhibiting MIP-2 and IL-1beta. These findings suggest that gene therapy with H-PGDS may be useful for treating inflammatory diseases.

Prime solutions for cardiopulmonary bypass in neonates: antioxidant capacity of prime based on albumin or fresh frozen plasma.

OBJECTIVE: Oxidative damage and inflammation are believed to play an important role in postoperative complications after cardiopulmonary bypass. During bypass, a prime solution with a high antioxidant capacity may reduce the oxidative damage and inflammation. We investigated total antioxidant capacity and individual scavengers during the preparation of 2 different prime solutions. METHODS: The prime solutions were prepared with either pasteurized human albumin or fresh frozen plasma. The total antioxidant capacity was measured with the total radical antioxidant parameter assay and with the ferric-reducing ability of plasma assay. The individual scavengers vitamin C, sulfhydryl groups, uric acid, and total protein were measured before, during, and after the prime preparation. Malondialdehyde was measured as a parameter for lipid peroxidation. RESULTS: Neither prime solution showed a total radical antioxidant parameter value. The ferric-reducing ability of plasma value of prime solutions was lower than that of undiluted human albumin or fresh frozen plasma. Addition of mannitol did not increase the ferric-reducing ability of plasma value. Vitamin C was only found in the fresh frozen plasma prime. Both prime solutions contained sulfhydryl groups and uric acid in low concentrations. During ultrafiltration, low-molecular-weight antioxidants were lost into the ultrafiltrate. CONCLUSIONS: We showed that prime solutions based on either albumin or fresh frozen plasma had very low antioxidant capacity and that ultrafiltration of the prime solution further lowers this capacity. A prime solution with a low antioxidant capacity may increase oxidative stress in neonates undergoing cardiopulmonary bypass.

Aspiration of the asymptomatic metatarsophalangeal joint in gout patients and hyperuricemic controls.

Asymptomatic metatarsophalangeal joints were aspirated in a group of patients with gout, in 2 control groups with hyperuricemia, and in 1 normouricemic control group. Extracellular urate crystals were present in 70% of gout patients, in 1 of 19 patients with asymptomatic hyperuricemia, and in 2 of 9 patients with renal failure and hyperuricemia but no history of joint disease. Crystals were not found in the 10 normouricemic patients who had other types of arthritis. The presence of crystals in the subjects with gout was not correlated with a history of podagra, duration of gout, presence of tophi, or degree of control of hyperuricemia. Though crystals were found on rare occasions in joint fluid of asymptomatic hyperuricemic subjects, the presence of these crystals in asymptomatic joints was more common in subjects with gout.