Fig 2: GRM1 Mutations Affect Receptor Activity and Can Be Pharmacologically Modulated In Vitro(A) Overview of the modified luciferase reporter assay used to assess mGluR1 activity. The GRM1-Tango construct, into which mutations were introduced, consists of a FLAG-tagged GRM1 sequence followed by the Tango element, i.e., a V2 tail capable of recruiting β-arrestin, a cleavage site for the tobacco etch virus (TEV) protease, and a tetracycline-controlled transactivator (tTA). GRM1 mutations are indicated by black stars. Signaling via mGluR1 results in a conformational change in the V2 tail and recruitment of β-arrestin, followed by TEV protease-mediated cleavage and release of tTA, which translocates to the nucleus and activates transcription of the luciferase reporter gene, resulting in a quantifiable output of mGluR1 activity in the form of luminescence (figure adapted from Kroeze et al.15).(B) Structure of the mGluR1 endogenous ligand, L-glutamate, and the inhibitors used in this study: competitive inhibitor MCPG, inverse agonist BAY36-7620, and the FDA-approved negative allosteric modulator Nitazoxanide.(C) Relative activity of mGluR1 mutants. HTLA cells, stably expressing a β-arrestin/TEV protease complex and a tTA-dependent luciferase reporter gene, were seeded at 70,000 cells/well onto poly-L-lysine-coated 96-well plates in DMEM without L-glutamine (Life Technologies), containing penicillin, streptomycin, hygromycin B, and puromycin. After 24 hr, cells were transiently transfected with the four GRM1-Tango constructs (WT, p.Tyr262Cys, p.Tyr792Cys, and p.Gly1056Argfs∗49) and incubated for a further 24 hr. Cells were then treated overnight with 500 μM (RS)-MPCG (Tocris), 10 μM BAY36-7620 (Tocris), or 10 μM Nitazoxanide (Sigma-Aldrich), diluted in assay buffer (20 mM HBSS, 1× HEPES [pH 7.4], both Life Technologies), before cell lysis in Bright-Glo solution (Promega) and luminescence reading. Data were analyzed statistically in GraphPad Prism using a two-way analysis of variance (ANOVA), followed by Bonferroni’s multiple comparison post hoc test. Significance was defined as p < 0.05 and is shown here relative to mGluR1 wild-type (WT) in the untreated condition and relative to the corresponding untreated sample for all other treatment conditions, unless otherwise indicated. Data shown are mean ± SEM from one experiment, representative of results recorded in four biological replicates, each consisting of three technical replicates per construct per condition. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 3: AIM2 deficiency inhibits arsenic-induced cleavage of pro-casepase-1 and secretion of IL-1β and IL-18 in the skin of AIM2 KO mice. (A) AIM2 deficiency was confirmed by protein gel blot (upper panel) and arsenic-induced cleavage of pro-casepase-1 was inhibited in the skin of AIM2 KO mice. (B) Arsenic-induced secretion of IL-1β and IL-18, but not IL-1α, in the skin of wildtype mice was inhibited in AIM2 KO mice. The experiments were repeated three times. Data are represented as mean ± SEM of three experiments. *p < 0.05 vs. control.

Fig 4: PKR mutation inhibits arsenic-induced activation of AIM2 inflammasome and secretion of IL-1β and IL-18, but not IL-1α, in the skin of C-PKR−/− mice. The C-PKR−/− mice and controlled C57BL/6J mice were treated with 0.25 µM sodium arsenite in drinking water for 8 weeks. The skin tissues were collected. (A) The protein levels of AIM2, cleaved caspase-1, IL-1α, IL-1β and IL-18 were determined by Western-blot and quantified by densitometry. (B) The secretion levels of IL-1α, IL-1β and IL-18 were determined by ELISA assay. The experiments were repeated three times. Data are represented as mean ± SEM of three experiments. *p < 0.05.

Fig 5: Effect of rHuIL-12 or Placebo on EPO, IL-18 and CXCL10 levels in the Phase 1b expansion study. EPO, IL-18, and CXCL10 were measured at the indicated time points using validated assays. The mean changes from baseline after treatment with rHuIL-12 (12 μg) or placebo are shown as follows: A) EPO; B) IL-18; C) CXCL10.