In vitro and in vivo studies confirm the role of regulatory volume decrease

Experimental in vitro and in vivo studies confirmed the important role of the so-called regulatory volume decrease (RVD), an endogenous defence mechanism that allows cells to return to their initial volume after cellular swelling. Moreover, LRRC8A seems to be the key protein required for this mechanism.

Acute arthritis induced by monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals depends on IL-1β activated by the NLRP3 inflammasome. Different stimuli, such as hypo-osmolarity and ATP, can trigger the NLRP3 inflammasome. Secondary to water influx, there is cellular swelling. As an endogenous defence mechanism, cells set up the RVD, allowing cells to return to their initial volume. LRRC8A is the obligatory key protein required to form active VRAC channels, responsible for water efflux and cell volume reduction.

In their study, Dr Twinu Wilson Chirayath (Paris Cité University, France) and his team tried to evaluate the role of the LRRC8/ VRAC channel in cell volume regulation and MSU- and CPP- crystals-induced release of IL-1β by stimulating primed monocytes with synthetic sterile MSU and CPP [1]. Cytokine production was then quantified by ELISA, and cell volume variations were visualised by live video recording. Moreover, the role of the LRRC8/VRAC channel was evaluated with the help of the pharmacological inhibitor DCPIB or by silencing the LRRC8A subunit (shLRRC8A RNA) in these cells.

In addition, the researchers injected MSU and CPP crystals into air pouches created subcutaneously (mimicking synovial cavity) in wildtype mice and conditional LRRC8A knock-out mice. Supernatants and pouch lavages were used to measure cytokine production by ELISA.

Both in cells treated with the pharmacological inhibitor and in cells where LRRC8A expression was silenced, MSU- and CPP-induced NF-Κb activation was markedly reduced. This was accompanied by a significantly reduced IL-1β production compared with wildtype cells (P<0.001). MSU and CPP crystals exposure induced a biphasic cell volume change characterised by a significant increase followed by a decrease induced by a RVD-like phenomenon. Cells treated with the pharmacological inhibitor or cells with silenced LRRC8A subunits did not show the biphasic cell volume changes, supporting that LRRC8A is required to form active VRAC channels.

The in vivo results confirmed the effects seen in the in vitro experiment: Inflammation induced by MSU and CPP crystals assessed in lavage fluid and by conventional histology was lower in LRRC8A knock-out mice as compared with wildtype mice both in terms of IL-1β production and cell infiltrate.

The authors concluded that MSU- and CPP-crystal-induced inflammation are involved in IL-1β release, and cell volume variation can be regulated by the LRRC8/VRAC channel. These findings add further complexity to crystal-induced inflammation and inflammasome activation.

1. Chirayath TW, et al. Monosodium urate and calcium pyrophosphate crystal-induced inflammation relies on cell volume regulation and LRRRC8/VRAC channel OP0095, EULAR 2023, 31 May–3 June, Milan, Italy.

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Posted on 31 July, 202328 March, 2024