Interleukin-6 Drives Mitochondrial Dysregulation and Accelerates Physical Decline: Insights From an Inducible Humanized IL-6 Knock-In Mouse Model.

Chronic activation of inflammatory pathways (CI) and mitochondrial dysfunction are independently linked to age-related functional decline and early mortality. Interleukin 6 (IL-6) is among the most consistently elevated chronic activation of inflammatory pathways markers, but whether IL-6 plays a causative role in this mitochondrial dysfunction and physical deterioration remains unclear. To characterize the role of IL-6 in age-related mitochondrial dysregulation and physical decline, we have developed an inducible human IL-6 (hIL-6) knock-in mouse (TetO-hIL-6mitoQC) that also contains a mitochondrial-quality control reporter. Six weeks of hIL-6 induction resulted in upregulation of proinflammatory markers, cell proliferation and metabolic pathways, and dysregulated energy utilization. Decreased grip strength, increased falls off the treadmill, and increased frailty index were also observed. Further characterization of skeletal muscles postinduction revealed an increase in mitophagy, downregulation of mitochondrial biogenesis genes, and an overall decrease in total mitochondrial numbers. This study highlights the contribution of IL-6 to mitochondrial dysregulation and supports a causal role of hIL-6 in physical decline and frailty.

Metabolomics-Based Identification of Metabolic Dysfunction in Frailty.

Dysregulation of energy producing metabolic pathways has been observed in older adults with frailty. In this study, we used liquid chromatography-mass spectrometry technology to identify aging- and frailty-related differences in metabolites involved in glycolysis, the tricarboxylic (TCA) cycle, and other energy metabolism-related pathways in the serum of a cohort of community-dwelling adults aged 20-97 (n = 146). We also examined the relationship between serum levels of metabolites and functional measures, physical frailty, and risk status for adverse health outcomes. We observed elevated levels of TCA cycle and glycolytic intermediates in frail subjects; however, the differences in the levels of ATP and other energy metabolites between young, nonfrail, and frail adults were not significant. Instead, we found that serum levels of neurotransmitters N-acetyl-aspartyl-glutamate, glutamate, and γ-aminobutyric acid were significantly elevated in older adults with frailty. These elevations of glycolytic and TCA cycle intermediates, and neurotransmitters may be part of the biological signature of frailty.

Serum Concentrations of Losartan Metabolites Correlate With Improved Physical Function in a Pilot Study of Prefrail Older Adults.

Losartan is an oral antihypertensive agent that is rapidly metabolized to EXP3174 (angiotensin-subtype-1-receptor blocker) and EXP3179 (peroxisome proliferator-activated receptor gamma [PPARγ] agonist), which was shown in animal studies to reduce inflammation, enhance mitochondrial energetics, and improve muscle repair and physical performance. We conducted an exploratory pilot study evaluating losartan treatment in prefrail older adults (age 70-90 years, N = 25). Participants were randomized to control (placebo) or treatment (daily oral losartan beginning at 25 mg per day and increasing every 8 weeks) for a total of 6 months. Fatigue, hyperkalemia, and hypotension were the most observed side effects of losartan treatment. Participants in the losartan group had an estimated 89% lower odds of frailty (95% confidence interval [CI]: 18% to 99% lower odds, p = .03), with a 0.3-point lower frailty score than the placebo group (95% CI: 0.01-0.5 lower odds, p = .04). Frailty score was also negatively associated with serum losartan and EXP3179 concentrations. For every one standard deviation increase in EXP3179 (ie, 0.0011 ng/µL, based on sample values above detection limit) and EXP3174 (ie, 0.27 ng/µL, based on sample values above detection limit), there was a 0.0035 N (95% CI: 0.0019-0.0051, p < .001) and a 0.0027 N (95% CI: 0.00054-0.0043, p = .007) increase in average knee strength, respectively.

Targeted Deletion of Interleukin-6 in a Mouse Model of Chronic Inflammation Demonstrates Opposing Roles in Aging: Benefit and Harm.

Chronic inflammation (CI) in older adults is associated with reduced health span and life span. Interleukin-6 (IL-6) is one CI marker that is strongly associated with adverse health outcomes and mortality in aging. We have previously characterized a mouse model of frailty and chronic inflammatory pathway activation (IL-10tm/tm, IL-10 KO) that demonstrates the upregulation of numerous proinflammatory cytokines, including IL-6. We sought to identify a more specific role for IL-6 within the context of CI and aging and developed a mouse with targeted deletion of both IL-10 and IL-6 (IL-10tm/tm/IL-6tm/tm, DKO). Phenotypic characteristics, cytokine measurements, cardiac myocardial oxygen consumption, physical function, and survival were measured in DKO mice and compared to age- and gender-matched IL-10 KO and wild-type mice. Our findings demonstrate that selective knockdown of IL-6 in a frail mouse with CI resulted in the reversal of some of the CI-associated changes. We observed increased protective mitochondrial-associated lipid metabolites, decreased cardiac oxaloacetic acid, improved myocardial oxidative metabolism, and better short-term functional performance in DKO mice. However, the DKO mice also demonstrated higher mortality. This work shows the pleiotropic effects of IL-6 on aging and frailty.

Plasma levels of corticosterone, tumor necrosis factor receptor 1 and interleukin 6 are influenced by age, sex and chronic inflammation in mice treated with acute temperature stress.

Resiliency is the ability to respond to, adapt to and recover from stressors. Deterioration of resiliency in older adults has been hypothesized to be regulated by age-related changes in stress response systems, including the Hypothalamic Pituitary Adrenal (HPA) axis and the innate immune system response. Although age-related chronic inflammation is strongly related to lack of resiliency, the impact of chronic inflammation on acute stress response is unclear. Here we describe the impact of a five-hour exposure to cold temperature acute stressor, on immune and corticosterone response using older and younger IL-10tm/tm mice, a mouse model with chronic inflammatory pathway activation, and age and gender matched C57/Bl6 background control (WT) mice. Overall, mice exposed to 4 °C for 5 h had significantly higher plasma corticosterone levels compared to those that remained at room temperature (25 °C), with the exception of the WT females. Cold stressed mice had lower plasma tumor necrosis factor receptor 1 (TNFR1) levels with varying significance, in all ages and phenotypes, with the exception of the old female WT mice. In contrast, the effects of cold stress on pro-inflammatory cytokine interleukin 6 (IL-6) levels were inconsistent and not significant, with the exception of the female IL-10tm/tm mice. In conclusion, these findings demonstrate that sex, age and chronic inflammatory pathway activation all influence corticosterone secretion and inflammatory processes in the face of acute cold stress.

Frailty transitions, inflammation, and mortality among persons aging with HIV infection and injection drug use.

OBJECTIVE: Frailty is a critical aging-related syndrome marked by diminished physiologic reserve and heightened vulnerability to stress, predictive of major adverse clinical outcomes in HIV-infected and uninfected adults. Frailty is a dynamic state, yet little data exist on predictors and consequences of frailty transitions. DESIGN/METHODS: Frailty was assessed semiannually among HIV-infected and uninfected persons with prior injection drug use using the five Fried phenotype domains. An inflammatory index score was constructed from IL-6 and soluble TNF-α receptor-1 data. Markov transition models assessed determinants of frailty transitions. Cox proportional hazards models estimated mortality risk. RESULTS: Among 1353 AIDS Linked to the IntraVenous Experience participants with 9559 frailty transition assessments, 33% were HIV-infected. Younger age, higher education, employment, reduced comorbidity, HIV virologic suppression, elevated CD4 nadir (>500 cells/µl) and absence of a prior AIDS diagnosis were significantly associated with both reduced frailty progression and greater frailty recovery. Each SD decrease in inflammatory index score was associated with decreased frailty progression [odds ratio 0.78; 95% confidence interval (CI), 0.65, 0.92] and increased frailty recovery (odds ratio 1.29; 95% CI, 1.08, 1.53). Being frail at one of two consecutive visits was associated with increased mortality, compared with maintenance of a nonfrail state. Being frail at both of two consecutive visits demonstrated the highest mortality risk (hazard ratio 3.23; 95% CI, 2.1, 4.96). CONCLUSION: Sustained, and to a lesser degree, intermittent frail states are associated with increased mortality. HIV virologic suppression with earlier antiretroviral therapy, reduced comorbidity, and reduced inflammation may prevent frailty progression and promote frailty recovery, consequently improving survival for persons aging with HIV and persons with prior injection drug use.

Kynurenines link chronic inflammation to functional decline and physical frailty.

Chronic inflammation is associated with physical frailty and functional decline in older adults; however, the molecular mechanisms of this linkage are not understood. A mouse model of chronic inflammation showed reduced motor function and partial denervation at the neuromuscular junction. Metabolomic profiling of these mice and further validation in frail human subjects showed significant dysregulation in the tryptophan degradation pathway, including decreased tryptophan and serotonin, and increased levels of some neurotoxic kynurenines. In humans, kynurenine strongly correlated with age, frailty status, TNF-αR1 and IL-6, weaker grip strength, and slower walking speed. To study the effects of elevated neurotoxic kynurenines on motor neuronal cell viability and axonal degeneration, we used motor neuronal cells treated with 3-hydroxykynurenine and quinolinic acid and observed neurite degeneration in a dose-dependent manner and potentiation of toxicity between 3-hydroxykynurenine and quinolinic acid. These results suggest that kynurenines mediate neuromuscular dysfunction associated with chronic inflammation and aging.

Upregulated ex vivo expression of stress-responsive inflammatory pathway genes by LPS-challenged CD14(+) monocytes in frail older adults.

Frailty has been increasingly recognized as an important clinical syndrome in old age. The frailty syndrome is characterized by chronic inflammation, decreased functional and physiologic reserve, and increased vulnerability to stressors, leading to disability and mortality. However, molecular mechanisms that contribute to inflammation activation and regulation in frail older adults have not been investigated. To begin to address this, we conducted a pathway-specific gene array analysis of 367 inflammatory pathway genes by lipopolysaccharide (LPS)-challenged CD14(+) monocytes from 32 community-dwelling frail and age-, race-, and sex-paired nonfrail older adults (mean age 83 years, range 72-94). The results showed that ex vivo LPS-challenge induced average 2.0-fold or higher upregulated expression of 116 genes in frail participants and 85 genes in paired nonfrail controls. In addition, frail participants had 2-fold or higher upregulation in LPS-induced expression of 7 stress-responsive genes than nonfrail controls with validation by quantitative real time RT-PCR. These findings suggest upregulated expression of specific stress-responsive genes in monocyte-mediated inflammatory pathway in the syndrome of frailty with potential mechanistic and interventional implications.

Upregulated monocytic expression of CXC chemokine ligand 10 (CXCL-10) and its relationship with serum interleukin-6 levels in the syndrome of frailty.

Frailty is an important geriatric syndrome that predicts disability and mortality. Substantial evidence suggests inflammation marked by elevated IL-6 levels as a key pathophysiologic factor that contributes to frailty. CXCL-10, a potent pro-inflammatory chemokine, has increased levels with age and is implicated in several inflammatory conditions. To better understand molecular mechanisms of inflammation activation in frailty, we evaluated monocytic expression of CXCL-10 and other inflammatory pathway genes by pathway-specific gene array analysis and quantitative RT-PCR. Frailty status was determined by the validated criteria. Sixteen pairs of community-dwelling frail and age-, race-, and sex-matched non-frail participants (mean age 83 years, range 72-94) completed the study. Here we report that frail participants had higher CXCL-10 expression levels than matched non-frail controls (1.05+/-0.88 versus 0.53+/-0.39, p=0.04). CXCL-10 expression correlated with IL-6 levels only in frail participants (Spearman correlation coefficient r=0.52, p=0.03). Furthermore, frailty-associated CXCL-10 upregulation was highly correlated with IL-6 elevation, both measured by frail-over-non-frail ratios (r=0.93, p<0.0001). These findings suggest upregulated monocytic expression of CXCL-10 as an important molecular mechanism that contributes to inflammation activation in frail older adults. Therapeutic implications include potential development of CXCL-10-based interventional strategies for the prevention and treatment of frailty in older adults.

The physical and biological characterization of a frail mouse model.

BACKGROUND: The development of animal models that approximate human frailty is necessary to facilitate etiologic and treatment-focused frailty research. The genetically altered IL-10(tm/tm) mouse does not express the antiinflammatory cytokine interleukin 10 (IL-10) and is, like frail humans, more susceptible to inflammatory pathway activation. We hypothesized that with increasing age, IL-10(tm/tm) mice would develop physical and biological characteristics similar to those of human frailty as compared to C57BL/6J control mice. METHODS: Strength, activity, serum IL-6, and skeletal muscle gene expression were compared between age-matched and gender-matched IL-10(tm/tm) mice on C57BL/6J background and C57BL/6J control mice using a longitudinal design for physical characteristics and cross-sectional design for biological characteristics. RESULTS: Strength levels declined significantly faster in IL-10(tm/tm) compared to control mice with increasing age. Serum IL-6 levels were significantly higher in older compared to younger IL-10(tm/tm) mice and were significantly higher in older IL-10(tm/tm) compared to age- and gender-matched C57BL/6J control mice. One hundred twenty-five genes, many related to mitochondrial biology and apoptosis, were differentially expressed in skeletal muscle between 50-week-old IL-10(tm/tm) and 50-week-old C57BL/6J mice. No expression differences between IL-10(tm/tm) age groups were identified by quantitative polymerase chain reaction. CONCLUSION: These physical and biological findings suggest that the IL-10(tm/tm) mouse develops inflammation and strength decline consistent with human frailty at an earlier age compared to C57BL/6J control type mice. This finding provides rationale for the further development and utilization of the IL-10(tm/tm) mouse to study the biological basis of frailty.

Impaired mitochondrial degradation by autophagy in the skeletal muscle of the aged female interleukin 10 null mouse.

Mitochondrial dysfunction, chronic inflammation and muscle aging are closely linked. Mitochondrial clearance is a process to dampen inflammation and is a critical pre-requisite to mitobiogenesis. The combined effect of aging and chronic inflammation on mitochondrial degradation by autophagy is understudied. In interleukin 10 null mouse (IL-10(tm/tm)), a rodent model of chronic inflammation, we studied the effects of aging and inflammation on mitochondrial clearance. We show that aging in IL-10(tm/tm) is associated with reduced skeletal muscle mitochondrial death signaling and altered formation of autophagosomes, compared to age-matched C57BL/6 controls. Moreover, skeletal muscles of old IL-10(tm/tm) mice have the highest levels of damaged mitochondria with disrupted mitochondrial ultrastructure and autophagosomes compared to all other groups. These observations highlight the interface between chronic inflammation and aging on altered mitochondrial biology in skeletal muscles.

Rapamycin increases grip strength and attenuates age-related decline in maximal running distance in old low capacity runner rats.

Rapamycin is known to extend lifespan. We conducted a randomized placebo-controlled study of enteric rapamycin-treatment to evaluate its effect on physical function in old low capacity runner (LCR) rats, a rat model selected from diverse genetic background for low intrinsic aerobic exercise capacity without genomic manipulation and characterized by increased complex disease risks and aging phenotypes. The study was performed in 12 male and 16 female LCR rats aged 16-22 months at baseline. The treatment group was fed with rapamycin-containing diet pellets at approximately 2.24mg/kg body weight per day and the placebo group with the same diet without rapamycin for six months. Observation was extended for additional 2 months. Physical function measurements include grip strength measured as maximum tensile force using a rat grip strength meter and maximum running distance (MRD) using rat physical treadmill test. The results showed that rapamycin improved grip strength by 13% (p=.036) and 60% (p=.001) from its baseline in female and male rats, respectively. Rapamycin attenuated MRD decline by 66% (p=.001) and 46% (p=.319) in females and males, respectively. These findings provide initial evidence for beneficial effect of rapamycin on physical functioning in an aging rat model of high disease risks with significant implication in humans.

Skeletal muscle ATP kinetics are impaired in frail mice.

The interleukin-10 knockout mouse (IL10(tm/tm)) has been proposed as a model for human frailty, a geriatric syndrome characterized by skeletal muscle (SM) weakness, because it develops an age-related decline in SM strength compared to control (C57BL/6J) mice. Compromised energy metabolism and energy deprivation appear to play a central role in muscle weakness in metabolic myopathies and muscular dystrophies. Nonetheless, it is not known whether SM energy metabolism is altered in frailty. A combination of in vivo (31)P nuclear magnetic resonance experiments and biochemical assays was used to measure high-energy phosphate concentrations, the rate of ATP synthesis via creatine kinase (CK), the primary energy reserve reaction in SM, as well as the unidirectional rates of ATP synthesis from inorganic phosphate (Pi) in hind limb SM of 92-week-old control (n = 7) and IL10(tm/tm) (n = 6) mice. SM Phosphocreatine (20.2 ± 2.3 vs. 16.8 ± 2.3 µmol/g, control vs. IL10(tm/tm), p < 0.05), ATP flux via CK (5.0 ± 0.9 vs. 3.1 ± 1.1 µmol/g/s, p < 0.01), ATP synthesis from inorganic phosphate (Pi → ATP) (0.58 ± 0.3 vs. 0.26 ± 0.2 µmol/g/s, p < 0.05) and the free energy released from ATP hydrolysis (∆G ∼ATP) were significantly lower and [Pi] (2.8 ± 1.0 vs. 5.3 ± 2.0 µmol/g, control vs. IL10(tm/tm), p < 0.05) markedly higher in IL10(tm/tm) than in control mice. These observations demonstrate that, despite normal in vitro metabolic enzyme activities, in vivo SM ATP kinetics, high-energy phosphate levels and energy release from ATP hydrolysis are reduced and inorganic phosphate is elevated in a murine model of frailty. These observations do not prove, but are consistent with the premise, that energetic abnormalities may contribute metabolically to SM weakness in this geriatric syndrome.

Losartan improves measures of activity, inflammation, and oxidative stress in older mice.

Sarcopenia is an age-related decline in skeletal muscle mass and function that is multifactorial in etiology. Age-related changes in the renin-angiotensin system (RAS), increased oxidative stress, and chronic inflammation likely all contribute to its development. Losartan, an angiotensin II type I receptor blocker (ARB) decreases RAS activity and likely influences oxidative stress and inflammation. Given this, we hypothesized that losartan would improve activity levels and parameters related to inflammation and oxidative stress in older mice. We sought to test this hypothesis by comparing functional and molecular parameters between 18-month-old C57BL/6 mice treated with 50-70 mg/kg/day of losartan over a 4 month-period and age- and gender-matched mice receiving placebo. Losartan treatment significantly improved several activity measurements during treatment period compared to placebo controlled group, including increased time on treadmill, traveling activity, standing activity, and decreased grid contacts (p-values<0.05, 0.001, 0.01; and 0.04 respectively). Grip strength did not improve in treatment group relative to control group over time. Serum IL-6 level in the treated group was significantly lower than that in the control group at the end of treatment (30.3±12.9 vs. 173.0±59.5pg/ml, p<0.04), and mRNA expression of antioxidant enzymes catalase (3.9±0.9 vs. 1.0±0.4) and glutathione peroxidase (4.7±1.1 vs. 1.0±0.4) was significantly higher (p-value: 0.02, and 0.03 respectively) in quadriceps muscle after 4 months of treatment in treated and control groups. These results support the hypothesis that chronic losartan treatment improves skeletal muscle related activity measures in older mice, and that it is associated with more favorable relevant biological profiles in the treatment group. Additional studies are needed to 1) further quantify this functional improvement, 2) further identify mechanisms that influence this improvement, and 3) provide additional rationale for translating these findings into older adults.

Simple biologically informed inflammatory index of two serum cytokines predicts 10 year all-cause mortality in older adults.

BACKGROUND: Individual measurements of inflammation have been utilized to assess adverse outcomes risk in older adults with varying degrees of success. This study was designed to identify biologically informed, aggregate measures of inflammation for optimal risk assessment and to inform further biological study of inflammatory pathways. METHODS: In total, 15 nuclear factor-kappa B-mediated pathway markers of inflammation were first measured in baseline serum samples of 1,155 older participants in the InCHIANTI population. Of these, C-reactive protein, interleukin-1-receptor antagonist, interleukin-6, interleukin-18, and soluble tumor necrosis factor-α receptor-1 were independent predictors of 5-year mortality. These five inflammatory markers were measured in baseline serum samples of 5,600 Cardiovascular Health Study participants. A weighted summary score, the first principal component summary score, and an inflammation index score were developed from these five log-transformed inflammatory markers, and their prediction of 10-year all-cause mortality was evaluated in Cardiovascular Health Study and then validated in InCHIANTI. RESULTS: The inflammation index score that included interleukin-6 and soluble tumor necrosis factor-α receptor-1 was the best predictor of 10-year all-cause mortality in Cardiovascular Health Study, after adjusting for age, sex, education, race, smoking, and body mass index (hazards ratio = 1.62; 95% CI: 1.54, 1.70) compared with all other single and combined measures. The inflammation index score was also the best predictor of mortality in the InCHIANTI validation study (hazards ratio 1.33; 95% CI: 1.17-1.52). Stratification by sex and CVD status further strengthened the association of inflammation index score with mortality. CONCLUSION: A simple additive index of serum interleukin-6 and soluble tumor necrosis factor-α receptor-1 best captures the effect of chronic inflammation on mortality in older adults among the 15 biomarkers measured.

Inflammation and mortality in a frail mouse model.

Mice homozygous for targeted deletion of the interleukin 10 gene (Il-10) have been partially characterized as a model for human frailty. These mice have increased serum interleukin (IL)-6 in midlife, skeletal muscle weakness, and an altered skeletal muscle gene expression profile compared to age and sex-matched C57BL/6 (B6) control mice. In order to further characterize for use as a frailty model, we evaluated the evolution of inflammatory pathway activation, endocrine change, and mortality in these mice. Serum was collected in groups of age- and sex-matched B6.129P2-Il10(tm1Cgn)/J (IL-10(tm/tm)) mice and B6 control mice at age 12, 24, 48, 72, and 90 weeks. Cytokines including IL-6, interleukin 1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), chemokine (C-X-C motif) ligand 1 (KC), IL-12, and IL-10 were measured using electro-chemiluminescent multiplex immunoassay and insulin-like growth factor 1 (IGF-1) was measured using solid-phase enzyme-linked immunosorbent assay. A separate longitudinal cohort was monitored from age 35 weeks to approximately 100 weeks. Survival was evaluated by Kaplan-Meier survival estimates and detailed necropsy information was gathered in a subset of mice that died or were sacrificed. In IL-10(tm/tm) mice compared to B6 controls, serum IL-6, IL-1ß, TNF-α, IFN-γ, KC levels were significantly elevated across the age groups, serum mean IGF-1 levels were higher in the 48-week-old groups, and overall mortality rate was significantly higher. The quadratic relationship between IGF-1 and age was significantly different between the two strains of mice. Serum IL-6 was positively associated with IGF-1 but the effect was significantly larger in IL-10(tm/tm) mice. These findings provide additional rationale for the use of the IL-10(tm/tm) mouse as a model for frailty and for low-grade inflammatory pathway activation.

Decreased cell proliferation and altered cytokine production in frail older adults.

BACKGROUND AND AIMS: Frailty is a geriatric syndrome that predicts increased morbidity and mortality. In order to investigate specific immune system modulations that may contribute to frailty, eleven age- and sex-matched pairs of community-dwelling frail and non-frail older adults were identified. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated and PBMC proliferation and production of IL-6, tumor necrosis factor (TNF)-alpha, and IL-10 in the presence and absence of lipopolysaccharide (LPS) were examined. RESULTS: We found that frail subjects had a significantly lower LPS-induced PBMC proliferation ratio compared with non-frail subjects (2.1+/-0.9 vs 3.11+/-1.9, p<0.03). In addition, frail subjects had higher IL-6 production by PBMC at 48 hours after LPS stimulation (35678+/-15637 vs 25178+/-6342 pg/mL, p<0.03). No significant differences were observed in TNF-alpha, and IL-10 production between groups. CONCLUSIONS: These results suggest that, compared with non-frail controls, frail older adults have both decreased LPS-induced proliferation and increased IL-6 production by PBMC.