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.
Highlights
Calcium pyrophosphate dihydrate (CPPD) microcrystal deposition disease affects more than 17.5% of people over 75 years old [1, 2]
On optical microscopy of THP1 cells cultured with CPP phases, m-CPPD crystals exhibited a needle-shaped structure and were the smallest and were more dispersed in cell culture than the others, whereas t-CPPD crystals had a rectangular shape, m-CPPTβ crystals were platelet-like and formed aggregates and the amorphous CPP (a-CPP) phase corresponded to large shapeless particles (Figure 1B)
interleukin 1β (IL-1β) and IL-8 production induced by m-CPPD and t-CPPD were both substantially decreased by JNK and extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinases (MAPKs) inhibitors (Figure 8C) but were not modulated by the p38 MAPK inhibitor (Figure 8D)
Summary
Calcium pyrophosphate dihydrate (CPPD) microcrystal deposition disease affects more than 17.5% of people over 75 years old [1, 2]. Monoclinic and triclinic CPPD phases (m-CPPD and t-CPPD, respectively) are the two types of CPPD crystals identified in synovial fluids at the time of acute arthritis, but the existence of precursor phases such as m-CPP tetrahydrate beta (m-CPPTβ) and amorphous CPP (a-CPP) have been suggested in vitro [3, 4]. Smaller crystals are more active than larger ones: the crystal surface plays a major role in determining cellular response and m-CPPD may be more active than t-CPPD [6,7,8,9,10] In line with these preclinical results, acute inflammatory synovial fluids contain a greater proportion of m-CPPD crystals and low inflammatory samples contain a greater proportion of t-CPPD crystals [7]. 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
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