Gout and pseudo-gout-related crystals promote GLUT1-mediated glycolysis that governs NLRP3 and interleukin-1ß activation on macrophages.

OBJECTIVE: Macrophage activation by monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals mediates an interleukin (IL)-1ß-dependent inflammation during gout and pseudo-gout flare, respectively. Since metabolic reprogramming of macrophages goes along with inflammatory responses dependently on stimuli and tissue environment, we aimed to decipher the role of glycolysis and oxidative phosphorylation in the IL-1ß-induced microcrystal response. METHODS: Briefly, an in vitro study (metabolomics and real-time extracellular flux analysis) on MSU and CPP crystal-stimulated macrophages was performed to demonstrate the metabolic phenotype of macrophages. Then, the role of aerobic glycolysis in IL-1ß production was evaluated, as well in vitro as in vivo using 18F-fluorodeoxyglucose positron emission tomography imaging and glucose uptake assay, and molecular approach of glucose transporter 1 (GLUT1) inhibition. RESULTS: We observed that MSU and CPP crystals led to a metabolic rewiring toward the aerobic glycolysis pathway explained by an increase in GLUT1 plasma membrane expression and glucose uptake on macrophages. Also, neutrophils isolated from human synovial fluid during gout flare expressed GLUT1 at their plasma membrane more frequently than neutrophils isolated from bloodstream. Both glucose deprivation and treatment with either 2-deoxyglucose or GLUT1 inhibitor suppressed crystal-induced NLRP3 activation and IL-1ß production, and microcrystal inflammation in vivo. CONCLUSION: In conclusion, we demonstrated that GLUT1-mediated glucose uptake is instrumental during the inflammatory IL-1ß response induced by MSU and CPP crystals. These findings open new therapeutic paths to modulate crystal-related inflammation.

Pseudogout-associated inflammatory calcium pyrophosphate dihydrate microcrystals induce formation of neutrophil extracellular traps.

Pseudogout is an autoinflammatory condition triggered by calcium pyrophosphate dehydrate (CPPD) crystal deposition in the joints. The innate immune system is irritated by and responds to the presence of the crystals with an inflammatory response. The synovial fluid contains activated inflammatory macrophages and neutrophil granulocytes. Several details of crystal-induced macrophage activation were recently uncovered, but very little is known about interactions of CPPD crystals with neutrophils. In this study, we show that human neutrophils engulf CPPD crystals and form large amounts of neutrophil extracellular traps (NETs) in vitro. Released extracellular DNA binds myeloperoxidase and citrullinated histone H4. CPPD crystal-stimulated neutrophils and their nuclear DNA undergo morphological changes characteristic for NET formation. The ERK/MEK signaling pathway, heat shock protein 90, PI3K, and an intact cytoskeleton are required for CPPD-induced NET formation. Blocking crystal-activated respiratory burst has, however, no effect on NETs. Human neutrophils release IL-1ß and IL-8 in response to CPPD crystals, and blocking CXCR2, the main IL-8R, diminishes NET formation. Proinflammatory cytokines, TNF-α, GM-CSF, and IL-1ß, increase NET release by the crystals. Enhanced bacterial killing by CPPD-induced NETs demonstrates their ability to cause cellular damage. Our work documents and provides details about extracellular trap release in human neutrophils activated by CPPD microcrystals. We suggest that crystal-triggered NET formation can be a novel contributor to inflammatory conditions observed in CPPD crystal-driven synovitis.

Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout.

To date, medical diagnosis of gout and pseudogout has been performed by observing the crystals in the joint fluid of patients under a polarized microscope. Conventional diagnostic methods using a polarized microscope have disadvantages, such as time-consuming analysis, a high false negative rate, and difficulty in distinguishing gout with monosodium urate (MSU) crystals and pseudogout with calcium pyrophosphate dihydrate (CPPD) crystals in synovial fluids. In this study, a chromogenic assay for the diagnosis of gout and pseudogout, without the requirement of a polarized microscope and trained experts, was proposed using Fv antibodies with specific binding activities to MSU and CPPD crystals. The IgG VH chain Fv library with randomized complementarity-determining region 3 (CDR3) region was expressed on the outer membrane of Escherichia coli using autodisplay technology. The target Fv antibodies with binding activity to MSU and CPPD crystals were screened from the autodisplayed Fv library on the E. coli outer membrane, and five clones were selected. On the basis of the binding properties of the screened Fv antibodies, peptides with the selected clone of amino acid sequences of the CDR3 region (15 residues) were chemically synthesized. The binding properties of the synthetic peptides with amino acid sequences of CDR3 regions from the selected clones were analyzed using fluorescence imaging and flow cytometry, and the affinity constants (Kd) of each peptide for binding to MSU and CPPD crystals were calculated by fitting based on the isotherm model. A chromogenic assay configuration for gout and pseudogout was developed using synthetic peptides. In this chromogenic assay, synthetic peptides labeled with biotin and streptavidin-horseradish peroxidase (HRP) complex were used, and crystal detection was possible using a chromogenic reaction between HRP and a chromogenic substrate (TMB). Finally, gout and pseudogout were diagnosed by detecting MSU and CPPD crystals in the synovial fluid in the concentration range of 0-300 µg/mL.

Inflammatory Potential of Four Different Phases of Calcium Pyrophosphate Relies on NF-κB Activation and MAPK Pathways.

Background: Calcium pyrophosphate (CPP) microcrystal deposition is associated with wide clinical phenotypes, including acute and chronic arthritis, that are interleukin 1ß (IL-1ß)-driven. Two CPP microcrystals, namely monoclinic and triclinic CPP dihydrates (m- and t-CPPD), have been identified in human tissues in different proportions according to clinical features. m-CPP tetrahydrate beta (m-CPPTß) and amorphous CPP (a-CPP) phases are considered as m- and t-CPPD crystal precursors in vitro. Objectives: We aimed to decipher the inflammatory properties of the three crystalline phases and one amorphous CPP phase and the intracellular pathways involved. Methods: The four synthesized CPP phases and monosodium urate crystals (MSU, as a control) were used in vitro to stimulate the human monocytic leukemia THP-1 cell line or bone marrow-derived macrophages (BMDM) isolated from WT or NLRP3 KO mice. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR; IL-1ß, IL-6 and IL-8 production by ELISA; and mitogen-activated protein kinase (MAPK) activation by immunoblot analysis. NF-κB activation was determined in THP-1 cells containing a reporter plasmid. In vivo, the inflammatory potential of CPP phases was assessed with the murine air pouch model via cell analysis and production of IL-1ß and CXCL1 in the exudate. The role of NF-κB was determined by a pharmacological approach, both in vivo and in vitro. Results:In vitro, IL-1ß production induced by m- and t-CPPD and m-CPPTß crystals was NLRP3 inflammasome dependent. m-CPPD crystals were the most inflammatory by inducing a faster and higher production and gene expression of IL-1ß, IL-6, and IL-8 than t-CPPD, m-CPPTß and MSU crystals. The a-CPP phase did not show an inflammatory property. Accordingly, m-CPPD crystals led to stronger activation of NF-κB, p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) MAPKs. Inhibition of NF-κB completely abrogated IL-1ß and IL-8 synthesis and secretion induced by all CPP crystals. Also, inhibition of JNK and ERK1/2 MAPKs decreased both IL-1ß secretion and NF-κB activation induced by CPP crystals. In vivo, IL-1ß and CXCL1 production and neutrophil infiltration induced by m-CPPD crystals were greatly decreased by NF-κB inhibitor treatment. Conclusion: Our results suggest that the inflammatory potential of different CPP crystals relies on their ability to activate the MAPK-dependent NF-κB pathway. Studies are ongoing to investigate the underlying mechanisms.

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.

How can calcium pyrophosphate crystals induce inflammation in hypophosphatasia or chronic inflammatory joint diseases?

Hypophosphatasia (HP) is a rare inborn error of bone and mineral metabolism characterized by a defect in the tissue non-specific alkaline phosphatase (TNSALP) gene. Calcium pyrophosphate dihydrate (CPPD) crystals are known to accumulate as substrates of TNSALP in tissues and joints of patients with HP. In CPPD-induced arthritis these crystals are known to induce an inflammatory response. HP patients do suffer from pain in their lower extremities. However, it is not clear whether CPPD crystals contribute to these musculoskeletal complaints in HP. As long as there is no curative treatment of HP, symptomatic treatment in order to improve clinical features, especially with regard to pain and physical activity, is of major interest to the patients. Knowledge of the mechanisms underlying crystal-induced cell activation, however, is limited. Here we describe recent advances in elucidating the signal transduction pathways activated by CPPD crystals as endogenous "danger signals". Recent investigations provided evidence that Toll/interleukin-1 receptor (TIR) domain containing receptors including Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R), as well as the triggering receptor expressed on myeloid cells 1 (TREM-1) and the NACHT-leucin rich repeat and pyrin-domain-containing protein (NALP3) containing inflammasome are essentially involved in acute CPPD crystal-induced inflammation. These receptors are considered in part as components of the innate immune system. Further studies are needed to improve our understanding of the pathophysiological mechanisms leading to inflammation and tissue destruction associated with deposition of microcrystals. They might support the development of new therapeutic strategies for crystal-induced inflammation. Eventually, patients with HP might as well profit from such strategies addressing these metabolic disorders secondary to the gene defect.

Inflammatory responses to intradermal crystals in healthy volunteers and patients with rheumatic diseases.

The inflammatory response to intradermal injections of urate, pyrophosphate and hydroxyapatite crystals in human forearm skin is described. Patients with rheumatoid arthritis responded normally to urate crystals, and patients with osteoarthritis or pyrophosphate arthropathy responded normally to hydroxyapatite and pyrophosphate crystals respectively. These results suggest that variation in host response to crystals cannot explain the different patterns of crystal-induced disease seen in man. The model, however, is recommended as a safe, simple ethical and reproducible test of inflammation in human subjects.

Phagocytosis of sodium urate and calcium pyrophosphate crystals by leukocytes of gouty, pseudogouty and health donors.

The initial rates of oxygen consumption and of glucose oxidation via the hexose monophosphate shunt were measured during the phagocytosis of sodium urate and calcium pyrophosphate microcrystals. By applying the kinetics of an enzyme-catalyzed reaction, the affinity of the polymorphonuclear leukocytes for crystals, Km, and the maximum rate, Vmax, were determined. When the comparisons are made between the two kinds of crystals, the differences in Vmax are always significant (p less than 0.02). The lesser phlogistic properties of pyrophosphate crystals stem probably in part from different crystal-protein interactions. The influence of the donor is less marked. Part of the weakly depressed phagocytic activity observed with pseudogouty PMN may be attributed to antiinflammatory drugs.

Superoxide anion production and phagocytosis of crystals by cultured endothelial cells.

OBJECTIVE: To show that cultured human umbilical vein endothelial cells (HUVEC) are capable of phagocytizing inflammation-causing crystals and of generating superoxide anion (SOA) during phagocytosis. METHODS: The superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT) dye was used as a measure of SOA production. Phagocytosis was quantified by light microscopy and confirmed by transmission electron microscopy. Cytochrome C was also studied but was found to undergo spontaneous reduction by monosodium urate (MSU) without cells. RESULTS: Crystals of MSU, calcium oxalate, hydroxyapatite, and calcium pyrophosphate dihydrate (CPPD) were phagocytized and, except for the CPPD crystals, induced NBT reduction. Cholesterol and cholesterol monohydrate were neither phagocytized nor did they induce NBT reduction. CONCLUSIONS: Endothelial cells may be a significant source of oxygen radicals in crystal-associated and other arthritides.