NLRP3-dependent in vivo inflammatory responses are elevated in a new mouse avatar of the autoinflammatory disease mevalonate kinase deficiency Skinner OP(a), Munoz MA(a), Fletcher E(a), Jurczyluk J(a), Kristianto E(b), Hodson M(b), Xiao Y(a), Brink R(c), Zahra D(c), Simon A(d), Rogers MJ(a) (a) Healthy Ageing Theme, Garvan Institute of Medical Research, Sydney, Australia (b) Innovation Centre, Victor Chang Cardiac Research Institute, Sydney, Australia (c) Immunity & Inflammation Theme, Garvan Institute of Medical Research, Sydney, Australia (d) Radboud University Medical Center, Laboratory of Experimental Internal Medicine, Nijmegen, The Netherlands Mevalonate kinase deficiency (MKD) is an autoinflammatory disease caused by recessive, pathogenic mutations in MVK encoding an enzyme in the mevalonate pathway. Mutations in MVK cause a characteristic build-up of the enzyme substrate, mevalonic acid, in plasma and urine and prevent protein prenylation (a downstream outcome of the mevalonate pathway) in PBMCs. Defective protein prenylation in MKD enhances inflammasome activation and IL-1β release from PBMCs. However the type of inflammasome involved is controversial, with evidence from cell culture models of NLRP3- and pyrin-dependent mechanisms. To better understand how defective prenylation causes inflammation in MKD, we created the first mouse avatar of this disease. Using CRISPR/Cas9 gene editing we generated mice compound heterozygous for a point mutation in Mvk (Val377Ile, the commonest pathogenic variant in humans) and a 91bp deletion in exon 11. Analysis of PBMCs, bone marrow and splenocytes from Mvk V377I/d91 mice revealed a clear defect in protein prenylation that closely resembled the defect in PBMCs from patients compound heterozygous for pathogenic MVK variants. Levels of mevalonic acid (detected by LC/MS) were also elevated in plasma and bone marrow cells from Mvk V377I/d91 mice. This new mouse model therefore recapitulates the biochemical features of MKD in humans. Under steady state conditions, adult Mvk V377I/d91 mice did not show any difference in the level of B cells, T cells, dendritic cells, neutrophils or monocytes in peripheral blood compared to wildtype or Mvk +/V377I control mice. However, acute in vivo treatment with i.p. LPS caused a significant increase in the level of inflammatory serum cytokines and chemokines (IL-1b, IL-18, IL-6, G-CSF, IL- 12, MIP1a, CCL2 and CCL5) in Mvk V377I/d91 mice compared to controls. These same inflammatory mediators were also higher in serum from a MKD patient compared to healthy volunteers. In Mvk V377I/d91 mice, i.p. administration of a single dose of the NLRP3 inhibitor MCC950 (50mg/kg) 1 hour prior to LPS treatment completely abolished the LPS-induced increase in serum IL-18 and IL-1b. Consistent with enhanced NLRP3-dependent inflammasome activity in vivo, PBMCs from Mvk V377I/d91 mice also released more IL-1b in vitro than PBMCs from control mice after LPS/nigericin stimulation (abolished by MCC950), and macrophages from Mvk V377I/d91 mice did not show any difference in pyrin-dependent IL-1b release after LPS/TcdA stimulation compared to controls. These studies, using the first genetic model of MKD, provide clear evidence of enhanced NLRP3-dependent inflammasome activity as a likely factor in the inflammatory disease phenotype of MKD.