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 ... -  《-》

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 ... -  《-》

Resident macrophages initiating and driving inflammation in a monosodium urate monohydrate crystal-induced murine peritoneal model of acute gout.

Martin WJ ，Walton M ，Harper J 《ARTHRITIS AND RHEUMATISM》

The effect of diet-induced obesity on the inflammatory phenotype of non-adipose-resident macrophages in an in vivo model of gout.

Gout is strongly associated with obesity. The aim of this study was to determine if obesity altered the inflammatory phenotype of non-adipose tissue-resident macrophages in response to the gout-causing agent monosodium urate (MSU) crystals. C57BL/6J mice were fed a high-fat diet for 12 weeks. Resident peritoneal macrophages were stimulated ex vivo with MSU crystals (200 µg/ml for 18 h) and the supernatants were collected. Mice were challenged with MSU crystals in vivo (3 mg, intraperitonal) and the peritoneal lavage fluid was collected (8 and 16 h). Cytokine and chemokine levels were analysed by multiplex bead array and peritoneal cell populations were analysed by flow cytometry. Peritoneal macrophages from obese mice produced elevated background levels of IL-6, monocyte chemoattractant protein 1 (MCP-1) and keratinocyte-derived cytokine (KC) that decreased following MSU crystal stimulation ex vivo. MSU-induced IL-1β production was higher for macrophages from obese mice compared with controls. High background levels of IL-6, MCP-1, KC and GM-CSF, but not IL-1β, were measured in the peritoneal fluid of unchallenged obese mice. MSU crystal challenge in vivo raised IL-1β levels equally in both control and obese mice, whereas elevated background levels of IL-6, MCP-1, KC and GM-CSF levels dropped in obese mice. There was a consistent trend towards lower numbers of naive peritoneal resident macrophages and MSU-recruited monocytes and neutrophils in obese mice. Obesity induces a background pro-inflammatory environment orchestrated by non-adipose tissue-resident macrophages. However, this may not automatically translate into exacerbation of MSU crystal-induced inflammation in gout.

Shaw OM ，Pool B ，Dalbeth N ，Harper JL ... -  《-》

Octacalcium phosphate crystals induce inflammation in vivo through interleukin-1 but independent of the NLRP3 inflammasome in mice.

To determine the mechanisms involved in inflammatory responses to octacalcium phosphate (OCP) crystals in vivo. OCP crystal-induced inflammation was monitored using a peritoneal model of inflammation in mice with different deficiencies affecting interleukin-1 (IL-1) secretion (IL-1α(-/-) , IL-1β(-/-) , ASC(-/-) , and NLRP3(-/-) mice) or in mice pretreated with IL-1 inhibitors (anakinra [recombinant IL-1 receptor antagonist] and anti-IL-1β). The production of IL-1α, IL-1β, and myeloid-related protein 8 (MRP-8)-MRP-14 complex was determined by enzyme-linked immunosorbent assay. Peritoneal neutrophil recruitment and cell viability were determined by flow cytometry. Depletion of mast cells or resident macrophages was performed by pretreatment with compound 48/80 or clodronate liposomes, respectively. OCP crystals induced peritoneal inflammation, as demonstrated by neutrophil recruitment and up-modulation of IL-1α, IL-1β, and MRP-8-MRP-14 complex, to levels comparable with those induced by monosodium urate monohydrate crystals. This OCP crystal-induced inflammation was both IL-1α- and IL-1β-dependent, as shown by the inhibitory effects of anakinra and anti-IL-1β antibody treatment. Accordingly, OCP crystal stimulation resulted in milder inflammation in IL-1α(-/-) and IL-1β(-/-) mice. Interestingly, ASC(-/-) and NLRP3(-/-) mice did not show any alteration in their inflammation status in response to OCP crystals. Depletion of the resident macrophage population resulted in a significant decrease in crystal-induced neutrophil infiltration and proinflammatory cytokine production in vivo, whereas mast cell depletion had no effect. Finally, OCP crystals induced apoptosis/necrosis of peritoneal cells in vivo. These data indicate that macrophages, rather than mast cells, are important for initiating and driving OCP crystal-induced inflammation. Additionally, OCP crystals induce IL-1-dependent peritoneal inflammation without requiring the NLRP3 inflammasome.

Narayan S ，Pazar B ，Ea HK ，Kolly L ，Bagnoud N ，Chobaz V ，Lioté F ，Vogl T ，Holzinger D ，Kai-Lik So A ，Busso N ... -  《-》