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 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

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

International audienceBackground: Calcium pyrophosphate (CPP) microcrystal deposition is associated with wide clinical phenotypes, including acute and chronic arthritis, that are interleukin 1b (IL-1b)-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-CPPTb) 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-1b, IL-6 and IL-8 production by ELISA; and mitogen-activated protein kinase (MAPK) activation by immunoblot analysis. NF-kB 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-1b and CXCL1 in the exudate. The role of NF-kB was determined by a pharmacological approach, both in vivo and in vitro.Results: In vitro, IL-1b production induced by m- and t-CPPD and m-CPPTb crystals was NLRP3 inflammasome dependent. m-CPPD crystals were the most inflammatory by inducing a faster and higher production and gene expression of IL-1b, IL-6, and IL-8 than t-CPPD, m-CPPTb and MSU crystals. The a-CPP phase did not show an inflammatory property. Accordingly, m-CPPD crystals led to stronger activation of NF-kB, p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) MAPKs. Inhibition of NF-kB completely abrogated IL-1b and IL-8 synthesis and secretion induced by all CPP crystals. Also, inhibition of JNK and ERK1/2 MAPKs decreased both IL-1b secretion and NF-kB activation induced by CPP crystals. In vivo, IL-1b and CXCL1 production and neutrophil infiltration induced by m-CPPD crystals were greatly decreased by NF-kB inhibitor treatment.Conclusion: Our results suggest that the inflammatory potential of different CPP crystals relies on their ability to activate the MAPK-dependent NF-kB pathway. Studies are ongoing to investigate the underlying mechanisms

PHYTOBS dataset - French National Service of Observation for Phytoplankton in coastal waters

The PHYTOBS dataset includes long-term time series on marine microphytoplankton, since 1987, along the whole French metropolitan coast. Microphytoplankton data cover microscopic taxonomic identifications and counts. The whole dataset is available, it includes 25 sampling locations.PHYTOBS network studies microphytoplankton diversity in the hydrological context along French coasts under gradients of anthropogenic pressures. PHYTOBS network allows to analyse the responses of phytoplankton communities to environmental changes, to assess the quality of the coastal environment through indicators, to define ecological niches, to detect variations in bloom phenology, and to support any scientific question by providing data.The PHYTOBS network provides the scientific community and stakeholders with validated and qualified data, in order to improve knowledge regarding biomass, abundance and composition of marine microphytoplankton in coastal and lagoon waters in their hydrological context.PHYTOBS originates of two networks. The historical REPHY (French Observation and Monitoring program for Phytoplankton and Hydrology in coastal waters) supported by Ifremer since 1984 and the SOMLIT (Service d'observation en milieu littoral) supported by INSU-CNRS since 1995. The monitoring has started in 1987 on some sites and later in others.Hydrological data are provided by REPHY or SOMLIT network as a function of site locations