GPR108 works as an activator but limits TLRs-triggered NF-κB and IFNβ response.

Abstract

(A) Two Gpr108-inducible clones 1–1 and 2–2, derived from Gpr108-/- iBMDM cells were stimulated with Dox (1μg/ml) for 48 hours. The expressions of Gpr108 were determined by RT-qPCR with actin gene as an internal control. (B) Two Gpr108-inducible clones 1–1 and 2–2 were transduced with NF-κB reporter containing luciferase. Cells were pretreated with or without Dox for 48 hours and then the luciferase activities were measured after the stimulation with LPS (100 ng/ml) and immiquimod (50 μg/ml) for 18 hours. (C) Gene expressions of IL-1β, IL-6, TNFα and IFNβ in DOX treated or untreated cells (clone1-1) (48 hours) were analyzed after exposure to LPS (100ng/ml) and immiquimod (50 μg/ml) for 1.5 hours. Actin was used as an internal control. (D) HEK293 cells were transfected with NF-κB-luciferase reporter and Flag-vector or Flag-TLRs, together with GFP or GPR108 plasmids. The NF-κB luciferase activities (fold increase) were analyzed after 48 hours transfection. (E) HEK293/TLR3, TLR4 and TLR7 stable cell lines were transfected with NF-κB-luciferase or IFNβ-luciferase reporter, together with control vector or Gpr108. Cells were treated with their corresponding agonists Poly (I:C) (TLR3), LPS (TLR4) and R484 (TLR7) at different doses for 18 hours and were analyzed for luciferase activities (fold increase). (F) Schematic diagram of the Gpr108 mutants: m1, an N-terminal domain deletion mutant with a leader peptide (green box); m2, loop3 replacement (AVPFQ (red line) was replaced with GGGGS (green line)); m3, a C-terminal domain deletion (purple line). (G) HEK293 cells were transfected with NF-κB-luciferase reporter, together with HA-vector, Gpr108 or Gpr108 mutants. The luciferase activities (fold increase) were analyzed after 48 hours transfection. Data were shown as mean±SD.*p < 0.05, **p < 0.01, ***p<0.001. All the experiments were repeated for at least three times.</p