IL-6 can singlehandedly drive many features of frailty in mice

Abstract

Frailty is a geriatric syndrome characterized by age-related declines in function and reserve resulting in increased vulnerability to stressors. The most consistent laboratory finding in frail subjects is elevation of serum IL-6, but it is unclear whether IL-6 is a causal driver of frailty. Here, we characterize a new mouse model of inducible IL-6 expression (IL-6^TET-ON/+ mice) following administration of doxycycline (Dox) in food. In this model, IL-6 induction was Dox dose-dependent. The Dox dose that increased IL-6 levels to those observed in frail old mice directly led to an increase in frailty index, decrease in grip strength, and disrupted muscle mitochondrial homeostasis. Littermate mice lacking the knock-in construct failed to exhibit frailty after Dox feeding. Both naturally old mice and young Dox-induced IL-6^TET-ON/+ mice exhibited increased IL-6 levels in sera and spleen homogenates but not in other tissues. Moreover, Dox-induced IL-6^TET-ON/+ mice exhibited selective elevation in IL-6 but not in other cytokines. Finally, bone marrow chimera and splenectomy experiments demonstrated that non-hematopoietic cells are the key source of IL-6 in our model. We conclude that elevated IL-6 serum levels directly drive age-related frailty, possibly via mitochondrial mechanisms.

Keywords: Frailty; IL-6; Mouse; Transgenic models.

Fig. 1

Increased IL-6 and frailty with age. WT C57BL/6 male and female mice of varying ages (A, 6 mo) were bled retro-orbitally and their a serum IL-6 levels were measured by ELISA. b frailty was assessed by frailty index (FI) consisting of 31 health-related variables. c IL-6 serum levels were correlated with frailty index in individual mice (R²=0.7131). Scheme of generation of inducible IL-6 mice. d Mice with Flexible Accelerated STOP TetO knocked in the IL-6 gene (F.A.S.T. IL-6 mice) were crossed to ROSA-rtTA (reverse tetracycline-controlled transactivator) mice resulting in generation of IL-6^TET-ON/+ mice. In the presence of doxycycline, rtTA binds the tetO sequence allowing for IL-6 transcription. e Adult IL-6^TET-ON/+ mice fed 30 ppm Dox for 7 months exhibited serum IL-6 to levels seen in 28-month-old mice. f Switching mice on and off Dox diet resulted in on/off pattern of increase and decrease in serum IL-6 levels. a–c data pooled from two independent experiments (total N=32). e–f data representative of two independent experiments with N=5 per group. *p<0.05; **p<0.01, ***p<0.001; ****p<0.0001

Fig. 2

IL-6 overexpression results in increased frailty. IL-6^TET-ON/+ mice on Dox diet exhibited a an increase in frailty index scores after six weeks, b reduced grip strength after 3 weeks, and c after 7 months induction, 10 mo old mice had frailty scores equivalent to much older mice. d gastrocnemius, but not soleus muscle mass was reduced. e Relative expression of PGC1α and NDUFB8 normalized to β-actin were increased in the gastrocnemius muscle and f spleen. Data representative of 2 experiments, with n=5 mice/expt. *p<0.05; **p<0.01; ***p<0.001; ****p< 0.0001

Fig. 3

IL-6 is increased in spleen homogenates. a IL-6 levels were measured in spleen homogenates of adult (4 mo), old (26-28mo), and IL-6^TET-ON/+ mice fed 30 ppm dox diet for two months. b IL-6 levels of the same mice were measured in fat and c muscle homogenates. d Multiplex cytokine analysis of splenic homogenates from 4 mo WT mice (top 5 rows), 28 mo WT mice (middle 5 rows), and 30 ppm Dox-induced IL-6^TET-ON/+ mice (bottom 5 rows). e ELISA measures of soluble IL-6R demonstrate significant increase in serum of Dox-induced (30 ppm) mice. n=5/group, representative of 2 experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001

Fig. 4

Neutrophils are the main producers of IL-6 in spleens of aged mice. a Flow cytometric plot shows Ly6G (A81) positive cells have the highest intracellular expression (MFI) of IL-6 in A WT mice which is reduced in IL-6^TET-ON/+ mice. b Most IL-6+ cells were expressing Ly6G (A81) in adult and old WT mice but to a lesser extent in IL-6^TET-ON/+ mice. c Absolute counts of neutrophils were increased in spleens of old (26-28 mo) mice and 30 ppm induced mice. d Mixed bone marrow chimeras were able to induce IL-6 when IL-6^TET-ON/+ cells were of non-hematopoietic origin. e Splenectomized IL-6^TET-ON/+ mice induced IL-6 to same levels as non-splenectomized mice in response to Dox food (30 PPM). f Same mice exhibited decreased induction of serum IL-6 in response to LPS stimulation (5 μg/mouse, 6 h, intraperitoneal injection). b, c Data pooled from two experiments with N=5/group. d Data from one experiment with N=8/group. e, f data representative of two experiments with N=3-5 mice per group. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.