Background/Purpose: Monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals are responsible for interleukin (IL)-1β dependent acute arthritis. The release of mature IL-1β is dependent on the NLRP3 inflammasome, which can be activated by other sterile stimuli such as hypo-osmolarity and ATP. During extracellular hypotonic stress, there is cellular swelling secondary to water influx. The cell sets up a defence mechanism, called regulatory volume decrease (RVD), allowing it to return to its initial volume. RVD depends on the anion channel VRAC, a hetero-hexamer composed of members of the LRRC8 family. LRRC8A is the obligatory key protein required to form active VRAC channel. Activation of LRRC8/VRAC channel results in an efflux of anions (mainly chloride) and osmolytes leading to water efflux and cell volume reduction. We evaluate the role of the LRRC8/VRAC channel in cell volume regulation and IL-1β release induced by MSU and CPP crystals.

Methods: In-vitro, primed THP-1 monocytes were stimulated by synthetic sterile MSU and CPP and cytokine production was quantified by ELISA. Cell volume variations were visualized by live video recording and cell surface was measured using ImageJ software. The role of the LRRC8/VRAC channel was evaluated using a pharmacological inhibitor DCPIB or by silencing the LRRC8A subunit (shLRRC8A RNA) in these cells. In vivo, MSU and CPP crystals were injected into air pouches created subcutaneously (mimicking synovial cavity) in wild-type mice and conditional LRRC8A Knock-out mice in the macrophage lineage (Cxcr3Cre_Lrrc8aflox/flox). Supernatants and pouch lavages were used to measure cytokine production by ELISA.

Results: MSU-and CPP-induced NF-κB activation was reduced in WT THP-1 cells treated with DCPIB and in THP-1 cells where LRRC8A expression was silenced. Similarly, IL-1β production induced by MSU and CPP crystals was substantially decreased in WT THP-1 treated with DCPIB (MSU 5200 vs 1080 pg/ml; CPP 11500 vs 4980, p< 0.0001) and in shLRRC8A THP-1 cells compared to crystal-treated WT cells. MSU and CPP crystals exposure induced a biphasic cell volume change characterised by a significant increase followed by a RVD-like phenomenon. These cell volume changes were abolished in the presence of DCPIB and not observed in shLRRC8A THP-1 cells. In vivo, inflammation induced by MSU and CPP crystals assessed in lavage fluid and conventional histology was lower in Cxcr3Cre_Lrrc8aflox/flox mice as compared with wild-type mice in terms of IL-1β production and cell infiltrate.

Conclusion: These results suggested that MSU and CPP crystal-induced inflammation involves cell volume variation regulated by VRAC/LRRC8 channel.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/monosodium-urate-and-calcium-pyrophosphate-crystal-induced-inflammation-relies-on-cell-volume-regulation-and-lrrc8-vrac-channel-activation/