Figure 1. Prolonged stress impairs iNKT cells’ capacity to trigger IL-4 and IFN-γ production and potentiates an abnormal inflammatory response to glycolipid Ags.

(A) WT B6 mice were restrained for 12 h. Control animals remained undisturbed but were deprived of food and water. Mice subsequently received αGC, αCGC, or vehicle (Veh) i.p., or a combination of IL-12 and IL-18 i.v.

(B and C) At the indicated time points post-αGC injection, serum IL-4 (B) and IFN-γ (C) were quantified by ELISA (n = 10 per group).

(D and E) Two hours after αGC injection, IL-4⁺ and IFN-γ⁺ cell frequencies among hepatic (D) and splenic (E) TCRβ⁺PBS-57-loaded CD1d tetramer⁺ iNKT cells were determined by flow cytometry.

(F) Two, 12, and 24 h after αGC (or Veh) administration, serum cytokine levels were measured via multiplex assays, and average values (n = 3 per cohort) were used to generate a heatmap.

(G) Separate cohorts (n = 4) were injected with αCGC or Veh, and blood IL-4 and IFN-γ levels were measured by ELISA.

(H) Parallel cohorts received IL-12 and IL-18 and were sacrificed 1 h later for their livers and spleens, in which IFN-γ⁺ iNKT cell percentages were determined.

Each symbol in (D), (E), and (H) represents an individual mouse. Error bars represent SEM. *, **, ***, and **** denote differences with p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively, by two-way ANOVA with Dunnett’s correction (B, C, and G) or unpaired Student’s t tests (D, E, and H).