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

Abstract

Background: Calcium pyrophosphate (CPP) microcrystal deposition is associated with wide clinical phenotypes, including acute and chronic arthritis, that are interleukin 1β (IL-1β)-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-CPPTβ) 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-1β, 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-1β 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-1β production induced by m- and t-CPPD and m-CPPTβ crystals was NLRP3 inflammasome dependent. m-CPPD crystals were the most inflammatory by inducing a faster and higher production and gene expression of IL-1β, IL-6, and IL-8 than t-CPPD, m-CPPTβ 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-1β and IL-8 synthesis and secretion induced by all CPP crystals. Also, inhibition of JNK and ERK1/2 MAPKs decreased both IL-1β secretion and NF-κB activation induced by CPP crystals. In vivo, IL-1β 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.

Submicron Crystals of the Parkinson’s Disease Substantia nigra: Calcium Oxalate, Titanium Dioxide and Iron Oxide

“…particles. Both are phagocytized, [25,[35][36][37][38][39] the TiO2 also by microglia [20,40]. In mice, activation of microglial NLRP3 inflammasome by misfolded α-synuclein causes the death of proximal dopaminergic neurons.…”

“…Both COD and TiO2 are phagocytized and NLRP3 inflammasome activating. [20,24,25,38,[41][42][43][44][45][46][47] If they activate also the microglial NLRP3 inflammasome, as is likely, proximal dopaminergic neurons would die. Because the number of microglia approaches the number of neuronal cells of the brain [65] each dopaminergic cell has on the average more than one proximal microglial cell.…”

“…It is a long-known cytotoxic component of urinary tract and kidney calculi, [30][31][32] associated also with retinopathy. [33,34] Both COD and TiO2 are phagocytized, [25,[35][36][37][38][39] TiO2 also by microglia. [40] Both activate the NLRP3 inflammasome.…”

“…[40] Both activate the NLRP3 inflammasome. [24,25,38,[41][42][43][44][45][46][47] Crystalline COD is associated with human lesions [31,33,34,[48][49][50] and has been reported in the human brain, [51] particularly when patients are lethally poisoned by the antifreeze ethylene glycol. [52][53][54] Ethylene glycol is metabolized to oxalic acid [55,56] and is precipitated in the brain as calcium oxalate [57].…”

<p>Calcium Pyrophosphate And Monosodium Urate Activate The NLRP3 Inflammasome Within Bladder Urothelium Via Reactive Oxygen Species And TXNIP</p>

“…In similar studies of osteoarthritis, both CPPD and MSU promote inflammation mediated by this inflammasome. 13 In their role as DAMPs, these two components are thought to potentiate NLRP3 inflammasome activation by stimulating intracellular reactive oxygen species (ROS) production. Under normal conditions, TXNIP, a structural co-factor of the assembled inflammasome, is bound to the cellular antioxidant thioredoxin.…”