Figure 4.

mTOR or TRAF6 activation is only part of the mechanism by which NLRC3 regulates metabolic pathways in tolerant macrophages

(A and B) (A) Schematic of treatments (upper), and immunoblot to detect phosphorylated mTOR, S6K, and 4EBP1 in tolerant or naive RAW 264.7 macrophages transduced with shNLRC3, ovNLRC3, or null vector (lower), and (B) in PMs with or without LPS in vitro challenge after CLP operation (n = 4). (C and I) Seahorse XF^e 24 monitored ECAR in tolerant RAW 264.7 with stable NLRC3 deficiency in the presence of mTOR inhibitor INK-128 (C) or NF-κB inhibitor BAY11-7082 (I) or vehicle after LPS restimulation; and in naive macrophages with stable NLRC3 deficiency in the presence of INK-128 (C) or NF-κB inhibitor BAY11-7082 (I) or vehicle with no LPS stimulation (n = 5). (D, J, and K) ELISA for lactate production, TNF-α, IL-6, and IL-1β in tolerant BMDMs of NLRC3^ΔMac (LysM-Cre⁺ NLRC3^fl/fl) mice in the presence of INK-128 (D) or NF-κB inhibitor BAY11-7082 (J and K) or vehicle after LPS restimulation; and in naive macrophages of NLRC3^ΔMac in the presence of INK-128 (D) or NF-κB inhibitor BAY11-7082 (J and K) or vehicle with no LPS stimulation (n = 5). (E) Immunoprecipitation of IκB and p65 and of TRAF6, IκB, and p65 from the tolerant or naive RAW 264.7 macrophages transduced with null or shNLRC3 or ovNLRC3 vector after 90-min LPS restimulation, followed by immunoblot to detect K63-linked ubiquitination (K63-Ub) of TRAF6. (F) Immunoblot of IκB, and p65 in PMs with or without LPS in vitro challenge after CLP operation (n = 4). (G) RAW 264.7 macrophages with stable NLRC3 deficiency were transfected with shTRAF6 or shNS (control) vector; Seahorse XF^e 24 monitored ECAR in these tolerant cells following LPS stimulation or naive cells with no LPS stimulation (n = 5). (H) ELISA for lactate production in tolerant BMDMs of NLRC3^ΔMac mice with shTRAF6 or shNS (control) vector after restimulation (n = 5), and in naive BMDMs of NLRC3^ΔMac mice with shTRAF6 or shNS vector in the absence of LPS stimulation. Circles represent individual mice. Graphs show the mean ± SE of experimental replicates and are representative of at least three independent experiments. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; NS, not significant (two-way ANOVA or Student’s t test). See also Figures S14 and S15.