NLRP3 inflammasome-mediated neutrophil recruitment and hypernociception depend on leukotriene B(4) in a murine model of gout.

Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)-derived leukotriene B(4) (LTB(4) ) in driving tissue inflammation and hypernociception in a murine model of gout. Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1β (IL-1β), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB(4) activity, cytokine (IL-1β, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively. Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophil-dependent hypernociception. MSU crystal-induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1β/MyD88-dependent manner. LTB(4) was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1-dependent IL-1β production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB(4) after MSU crystal injection, and LTB(4) was relevant in the MSU crystal-induced maturation of IL-1β. Mechanistically, LTB(4) drove MSU crystal-induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome. These results reveal the role of the NLRP3 inflammasome in mediating MSU crystal-induced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB(4) in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.

Amaral FA ，Costa VV ，Tavares LD ，Sachs D ，Coelho FM ，Fagundes CT ，Soriani FM ，Silveira TN ，Cunha LD ，Zamboni DS ，Quesniaux V ，Peres RS ，Cunha TM ，Cunha FQ ，Ryffel B ，Souza DG ，Teixeira MM ... -  《-》

A Role for Gut Microbiota and the Metabolite-Sensing Receptor GPR43 in a Murine Model of Gout.

Host-microbial interactions are central in health and disease. Monosodium urate monohydrate (MSU) crystals cause gout by activating the NLRP3 inflammasome, leading to interleukin-1β (IL-1β) production and neutrophil recruitment. This study was undertaken to investigate the relevance of gut microbiota, acetate, and the metabolite-sensing receptor GPR43 in regulating inflammation in a murine model of gout. Gout was induced by the injection of MSU crystals into the knee joints of mice. Macrophages from the various animals were stimulated to determine inflammasome activation and production of reactive oxygen species (ROS). Injection of MSU crystals caused joint inflammation, as seen by neutrophil influx, hypernociception, and production of IL-1β and CXCL1. These parameters were greatly decreased in germ-free mice, mice treated with antibiotics, and GPR-43-deficient mice. Recolonization or administration of acetate to germ-free mice restored inflammation in response to injection of MSU crystals. In vitro, macrophages produced ROS and assembled the inflammasome when stimulated with MSU. Macrophages from germ-free animals produced little ROS, and there was little inflammasome assembly. Similar results were observed in macrophages from GPR-43-deficient mice. Treatment of germ-free mice with acetate restored in vitro responsiveness of macrophages to MSU crystals. In the absence of microbiota, there is decreased production of short-chain fatty acids that are necessary for adequate inflammasome assembly and IL-1β production in a manner that is at least partially dependent on GPR43. These results clearly show that the commensal microbiota shapes the host's ability to respond to an inflammasome-dependent acute inflammatory stimulus outside the gut.

Vieira AT ，Macia L ，Galvão I ，Martins FS ，Canesso MC ，Amaral FA ，Garcia CC ，Maslowski KM ，De Leon E ，Shim D ，Nicoli JR ，Harper JL ，Teixeira MM ，Mackay CR ... -  《-》

Decoy Receptor 3 Inhibits Monosodium Urate-Induced NLRP3 Inflammasome Activation via Reduction of Reactive Oxygen Species Production and Lysosomal Rupture.

Gout is a common inflammatory arthritis caused by the deposition of monosodium urate (MSU) crystals in the joints. This activates the macrophages into a proinflammatory state by inducing NLRP3-dependent interleukin-1β (IL-1β) secretion, resulting in neutrophil recruitment. Soluble decoy receptor 3 (DcR3) is an immune modulator and can exert biological functions via decoy and non-decoy actions. Previously, we showed that DcR3 suppresses lipopolysaccharides (LPS)- and virus-induced inflammatory responses in the macrophages and promotes the macrophages into the M2 phenotype. In this study, we clarified the actions of DcR3 and its non-decoy action motif heparin sulfate proteoglycan (HSPG) binding domain (HBD) in the MSU crystal-induced NLRP3 inflammasome activation in the macrophages and in mice. In bone marrow-derived macrophages, THP-1 and U937 cells, we found that the MSU crystal-induced secretion of IL-1β and activation of NLRP3 were suppressed by both DcR3.Fc and HBD.Fc. The suppression of the MSU-induced NLRP3 inflammasome activation is accompanied by the inhibition of lysosomal rupture, mitochondrial production of the reactive oxygen species (ROS), expression of cathepsins, and activity of cathepsin B, without affecting the crystal uptake and the expression of NLRP3 or pro-IL-1β. In the air pouch mice model of gout, MSU induced less amounts of IL-1β and chemokines secretion, an increased M2/M1 macrophage ratio, and a reduction of neutrophil recruitment in DcR3-transgenic mice, which expresses DcR3 in myeloid cells. Similarly, the mice intravenously treated with DcR3.Fc or HBD.Fc displayed less inflammation response. These findings indicate that HBD of DcR3 can reduce MSU crystal-induced NLRP3 inflammasome activation via modulation of mitochondrial and lysosomal functions. Therefore, we, for the first time, demonstrate a new therapeutic potential of DcR3 for the treatment of gout.

Pan YG ，Huang MT ，Sekar P ，Huang DY ，Lin WW ，Hsieh SL ... -  《Frontiers in Immunology》

Phosphoinositide-3 kinase gamma regulates caspase-1 activation and leukocyte recruitment in acute murine gout.

This study investigates the participation of PI3Kγ in the development of joint inflammation and dysfunction in an experimental model of acute gout in mice. Acute gout was induced by injection of monosodium urate (MSU) crystals into the tibiofemoral joint of mice. The involvement of PI3Kγ was evaluated using a selective inhibitor and mice deficient for PI3Kγ (PI3Kγ-/- ) or with loss of kinase activity. Neutrophils recovered from the inflamed joint were quantified and stained for phosphorylated Akt (pAkt) and production of reactive oxygen species (ROS). The adherence of leukocytes to the joint microvasculature was assessed by intravital microscopy and cleaved caspase-1 by Western blot. Injection of MSU crystals induced massive accumulation of neutrophils expressing phosphorylated Akt. In the absence of PI3Kγ, there was reduction of pAkt expression, chemokine production, and neutrophil recruitment. Genetic or pharmacological inhibition of PI3Kγ reduced the adherence of leukocytes to the joint microvasculature, even in joints with established inflammation. Neutrophils from PI3Kγ-/- mice produced less ROS than wild-type neutrophils. There was decreased joint damage and dysfunction in the absence of PI3Kγ. In addition, in the absence of PI3Kγ activity, there was reduction of cleaved caspase-1 and IL-1β production in synovial tissue after injection of MSU crystals and leukotriene B4 . Our studies suggest that PI3Kγ is crucial for MSU crystal-induced acute joint inflammation. It is necessary for regulating caspase-1 activation and for mediating neutrophil migration and activation. Drugs that impair PI3Kγ function may be useful to control acute gout inflammation.

Tavares LD ，Galvão I ，Costa VV ，Batista NV ，Rossi LCR ，Brito CB ，Reis AC ，Queiroz-Junior CM ，Braga AD ，Coelho FM ，Dias AC ，Zamboni DS ，Pinho V ，Teixeira MM ，Amaral FA ，Souza DG ... -  《-》

Brief report: Granulocyte-macrophage colony-stimulating factor drives monosodium urate monohydrate crystal-induced inflammatory macrophage differentiation and NLRP3 inflammasome up-regulation in an in vivo mouse model.

To determine the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of inflammatory macrophages in an in vivo model of monosodium urate monohydrate (MSU) crystal-induced inflammation. C57BL/6J mice were treated with either clodronate liposomes to deplete peritoneal macrophages or GM-CSF antibody and were then challenged by intraperitoneal injection of MSU crystals. Peritoneal lavage fluid was collected, and cellular infiltration was determined by flow cytometry. Purified resident and MSU crystal-recruited monocyte/macrophages were stimulated ex vivo with MSU crystals. The interleukin-1β (IL-1β) levels in lavage fluids and ex vivo assay supernatants were measured. GM-CSF-derived and macrophage colony-stimulating factor (M-CSF)-derived macrophages were generated in vitro from bone marrow cells. Protein expression of IL-1β, caspase 1, NLRP3, and ASC by in vitro- and in vivo-generated monocyte/macrophages was analyzed by Western blotting. Depletion of resident macrophages lowered MSU crystal-induced IL-1β and GM-CSF levels in vivo as well as IL-1β production by MSU crystal-recruited monocytes stimulated ex vivo. GM-CSF neutralization in vivo decreased MSU crystal-induced IL-1β levels and neutrophil infiltration. MSU crystal-recruited monocyte/macrophages from GM-CSF-neutralized mice expressed lower levels of the macrophage marker CD115 and produced less IL-1β following ex vivo stimulation. These monocytes exhibited decreased expression of NLRP3, pro/active IL-1β, and pro/active caspase 1. In vitro-derived GM-CSF-differentiated macrophages expressed higher levels of NLRP3, pro/active IL-1β, and pro/active caspase 1 compared to M-CSF-differentiated macrophages. GM-CSF plays a key role in the differentiation of MSU crystal-recruited monocytes into proinflammatory macrophages. GM-CSF production may therefore contribute to the exacerbation of inflammation in gout.

Shaw OM ，Steiger S ，Liu X ，Hamilton JA ，Harper JL ... -  《-》