Origin and Function of Stress-Induced IL-6 in Murine Models

Abstract

Acute psychological stress has long been known to decrease host fitness to inflammation in a wide variety of diseases, but how this occurs is incompletely understood. Using mouse models, we show that interleukin-6 (IL-6) is the dominant cytokine inducible upon acute stress alone. Stress-inducible IL-6 is produced from brown adipocytes in a beta-3-adrenergic-receptor-dependent fashion. During stress, endocrine IL-6 is the required instructive signal for mediating hyperglycemia through hepatic gluconeogenesis, which is necessary for anticipating and fueling "fight or flight" responses. This adaptation comes at the cost of enhancing mortality to a subsequent inflammatory challenge. These findings provide a mechanistic understanding of the ontogeny and adaptive purpose of IL-6 as a bona fide stress hormone coordinating systemic immunometabolic reprogramming. This brain-brown fat-liver axis might provide new insights into brown adipose tissue as a stress-responsive endocrine organ and mechanistic insight into targeting this axis in the treatment of inflammatory and neuropsychiatric diseases.

Keywords: IL-6; acute stress; beta-adrenergic receptors; brown adipose tissue; gluconeogenesis; immunometabolism; inflammation; neuroendocrine-immune axis; neuroimmunology; tolerance.

Figure 1.. Acute stress induces endocrine IL6.

(A) Plasma IL6 levels in mice after exposure to one of the indicated stress challenges (n=5 per group, representative of 2 experiments). These experiments were performed by H.Q. NT, no treatment. TR, tube restraint. (B) Fold change of the indicated inflammatory cytokines and chemokines four hours after a single retro-orbital bleed or tube restraint (TR). Results are presented as the ratio of cytokine or chemokine levels from stressed subjects compared with no stress controls (n=5 per group). Experiments were performed by R. D. (C) Plasma IL6 levels following repetitive bleeding. Repetitive bleeding was applied to the same mice (n=5) every four hours, while circadian controls were non-stressed mice bled once at the indicated ZT times (n=5 for each time point). Experiments were performed by A.W. (D) Plasma levels of noradrenaline, corticosterone, and IL6 post bleeding at indicated time point (n=5 per time point, representative of 2 experiments). * p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001 See also Figure S1 and S7.

Figure 2.. Stress-inducible IL6 is produced by brown adipocytes.

(A) Plasma levels of IL6 post retro-orbital bleeding from conventionally housed mice (SPF), germ free mice (GF), or mice deficient in key signaling pathways necessary for pathogen detection and response (n=8 for SPF, n=3 for GF, n=3 for Tlr2/4^−/−, n=4 for Myd88/Trif^−/−). (B) Plasma level of IL6 from chimeric mice exposed to bleeding. WT→Il6 KO: Il6 knockout (KO) mice transplanted with bone marrow (BM) cells from wildtype (WT) mice; Il6 KO→WT: WT mice transplanted with BM cells from Il6 KO mice (n=5 per group). (C) Transcriptional analysis of Il6 in tissues from stressed and control mice (n=5 per group, representative of 2 experiments). FB, forebrain. MB, midbrain. HB, hindbrain. BAT, brown adipose tissue. eWAT, epididymal white adipose tissue. rWAT, retroperitoneal white adipose tissue. iWAT, inguinal white adipose tissue. NT, no treatment. TR, tube restraint. (D) Representative images of immunohistochemical staining for IL6 in brown adipose tissue harvested from bled mice or from controls (NT). IL6-positive staining is brown. (E) Plasma level of IL6 post bleeding from mice with surgical removal of brown adipose tissue (BATectomy) or sham surgery controls (sham) (n=3 for sham, n=4 for BATectomy) (F) Transcriptional analysis of Il6 in stromal vascular fraction (SVF) of brown adipose tissue (BAT) from stressed mice (n=3 for NT, n=4 for Bleed or TR group, representative of 2 experiments). Results are presented as fold increase relative to non-stressed controls (NT). TR, tube restraint. (G) Plasma level of IL6 post bleeding from mice with Il6 genetically deleted in brown adipocytes (Il6f/f^ΔUCP1) compared with littermate controls (Il6f/f). NT, no treatment. * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001 See also Figure S1.

Figure 3.. ADRB3 mediates brown adipocyte-derived IL6 in response to acute stress.

(A) Circulating IL6 levels (n=7 per group) and (B) mRNA expression of Il6 post bleeding in Ucp1 KO or WT mice (n=7 for WT, n=11 for WT + bleed, n=7 for Ucp1 KO, n=10 for Ucp1 KO + bleed). NT, no treatment. BAT, brown adipose tissue. eWAT, epididymal white adipose tissue. (C) Plasma IL6 levels in mice bled or restrained after anesthesia induced by ketamine/xylazine or isoflurane (n=5 per group, representative of 2 experiments). NT, no treatment. TR, tube restraint. (D) Circulating IL6 post bleeding from mice with chemical denervation of brown adipose tissue via 6-hydroxydopamine (6-OHDA) administration (n=3 per group). (E) Circulating IL6 levels and (F) mRNA expression of Il6 two hours post injection of beta-adrenergic receptor agonists (n=5 per group). ADRB1/2: isoproterenol, ADRB1 and ADRB2 agonist. ADRB3: CL316,243, ADRB3 agonist. FB, forebrain. Adrenal, Adrenal gland. BAT, brown adipose tissue. (G) Plasma level of IL6 post bleeding at indicated time points from mice pre-treated with the ADRB3 antagonist SR59203A (n=5 per group). (H) Plasma level of IL6 post bleeding in Adrb1/2 KO or Adrb3 KO mice (n=5 per group). NT, no treatment. (I) Plasma level of IL6 post bleeding from mice with conditional UCP1-mediated Adrb3 deletion (Adrb3f/f^ΔUCP1) or littermate controls (Adrb3f/f) (n=5 per group). NT, no treatment. * p<0.05, **p<0.01, *** p<0.001, **** p<0.0001 See also Figure S1 and S7.

Figure 4.. IL6 is necessary for promoting stress-hyperglycemia.

(A) Energy expenditure post bleeding from mice pretreated with IL6Ra antibody or isotype control (n=4 per group, representative of 2 experiments). Statistic represents the area under the curve between groups. (B) Blood glucose levels post bleeding from mice pretreated with IL6Ra inhibitor or isotype control (n=10 per group). CBG: Capillary blood glucose (C) Endogenous glucose production (EGP) post bleeding in mice pretreated with IL6Ra inhibitor or isotype control (n=8 for isotype, n=9 for IL6Ra mAb). (D) Pyruvate tolerance test (PTT) performed four hours post bleeding in mice pretreated with IL6Ra antibody or isotype control (n=5 per group, representative of 2 experiments). AUC, area under curve. (E) PTT performed four hours post bleeding in mice with conditional UCP1-mediated deletion of Adrb3 (Adrb3f/f^ΔUCP1) or (F) Il6 (Il6f/f^ΔUCP1) compared respectively with their littermate controls. (n=8 for Adrb3f/f, n=5 for Adrb3f/f^ΔUCP1, n=6 for Il6f/f or Il6f/f^ΔUCP1). AUC, area under curve. (G) Gluconeogenesis-associated gene expression in the liver harvested three hours post bleeding (n=4 for NT, n=5 for Bleed). Pck1: phosphoenolpyruvate carboxykinase 1. G6pc: glucose-6-phosphatase catalytic subunit. Pcx: pyruvate carboxylase. Fbp1: fructose-1,6-bisphosphatase 1. Gck: glucokinase. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 See also Figure S2 and S3.

Figure 5.. IL6 mediates stress hyperglycemia through hepatocyte reprogramming.

(A) G6pc mRNA in the liver and kidney of Il6 KO or WT mice four hours post bleeding (n=5 per group). (B) Il6ra mRNA in the indicated tissues from stressed mice (n=5 per group, representative of 2 experiments)). FB, forebrain. MB, midbrain. HB, hindbrain. BAT, brown adipose tissue. eWAT, epididymal white adipose tissue. rWAT, retroperitoneal white adipose tissue. iWAT, inguinal white adipose tissue. NT, no treatment. TR, tube restraint. (C) Rates of glucose production and (D) of gluconeogenesis from pyruvate in the liver or kidney of mice treated with IL6Ra inhibitor or isotype control (n=8 for isotype, n=9 for IL6Ra mAb). (E) PTT four hours post bleeding or (F) tube restraint in mice with hepatocyte-specific deletion of Il6ra (Il6raf/f^ΔAlb) compared with littermate controls (Il6raf/f) (n=5 per group). AUC, area under the curve. (G) Time in the dark using the light/dark box paradigm analyzed four hours post bleeding (bleed) from mice with hepatic deletion of Il6ra (Il6raf/f^ΔAlb) compared with littermate controls (Il6raf/f) (n=15 for Il6raf/f, n=9 for Il6raf/f^ΔAlb, representative of 3 experiments). * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001 See also Figure S4.

Figure 6.. ADRB3-dependent IL6 from BAT potentiates lethal endotoxemia secondary to acute stress

(A) Survival rate of LPS-induced endotoxemia in mice pre-exposed to the indicated stressors (n=10 per group). NT, no treatment. TR, tube restraint. (B) Survival of endotoxemic animals pretreated with vehicle (n=8), ADRB1 and ADRB2 agonist (ADRB1/2, n=7) or ADRB3 agonist (ADRB3, n=9). (C) Survival of endotoxemic animals pretreated with stress dose of IL6 (n=8 per group). (D) Survival of endotoxemic animals pretreated with the indicated interventions. n=9 for tube restraint (TR), n=10 for TR with ketamine, n=8 for IL6, n=9 for IL6 with ketamine. (E) Survival rate of endotoxemic mice pre-exposed to restraint with ADRB3 antagonist or vehicle injection. TR, tube restraint. (n=10 per group). (F) Lethality in endotoxemia from mice pre-exposed to restraint with brown adipose tissue specific deletion of Il6 (Il6f/f^ΔUCP1) compared with littermate controls (Il6f/f) (n=10 per group). (G) Circulating creatinine (Cr) levels 0 or 24 hours post LPS injection from mice pre-exposed to bleeding or restraint stress (n=5 per group). NT, no treatment. TR, tube restraint. (H) Circulating creatinine (Cr) levels 0 or 24 hours post LPS injection from mice pre-exposed to restraint stress with brown adipose tissue specific deletion of Il6 (Il6f/f^ΔUCP1) compared with littermate controls (Il6f/f) (n=5 per group). TR, tube restraint. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 See also Figure S5 and S6.

Figure 7.. Model

(A) Acute stresses engage a brain-BAT-liver axis to promote metabolic adaptation in order to support fight or flight physiology (B) Acute stress, unlike fasting or other negative energy states, requires IL6, in contrast to hormones like glucagon or growth hormone (GH), to induce gluconeogenesis for anticipatory, (impending increase in glucose demand), as opposed to responsive adaptation.