CLD is a debilitating pro-inflammatory ‘scarring’ condition that often results in the development of age-associated co-morbidities (especially physical frailty) leading to reduced quality of life and ultimately increased mortality (4, 5). Raised systemic inflammation is recognised as a key driver of the ageing phenotype (8), which increases the risk of multiple life limiting diseases (39). Here, we investigated whether CLD increases the rate of biological ageing in skeletal muscle and in the immune system, and interrogated these biological systems for known hallmark mechanisms of ageing that could explain the increased incidence of sarcopenia (29) and reduced immunity (40) in this patient population. Our findings provide the first evidence of increased biological ageing in patients with CLD, across these two biological systems, utilising epigenetic and immune phenotype-based measures. Clinically, the identification of a divergence of biological age from chronological age, or the presence of a negative ageing trajectory, may highlight CLD patients at greatest risk of disease progression, allowing early therapeutic intervention, including medicines that directly modulate ageing processes (41).
Evidence of increased skeletal muscle ageing in patients with CLD
It has previously been reported that patients with CLD display hallmarks of ageing, including reduced telomere length in liver tissue, hepatocytes and leukocytes, with such telomere attrition being positively associated with mortality risk and hepatic fibrosis (42, 43). In line with this, we observed greater epigenetic age acceleration in the skeletal muscle tissue of CLD patients, suggesting epigenetic muscle ageing may be a contributing factor to the development of muscle dysfunction, reported in up to 70% of patients with CLD (44). Ageing is also associated with a chronic elevation of circulating pro-inflammatory cytokines and a reduction of anti-inflammatory cytokines, a process referred to as ‘inflammageing’ (45). GDF-15 has recently gained recognition as a key age-associated cytokine, with elevated serum concentrations positively correlated with all-cause mortality (46, 47), multimorbidity (48), frailty (49, 50) and sarcopenia (51–53), while recent work by Kim-Muller et al demonstrated that GDF-15 neutralisation restored both muscle mass and function in mice (54). In line with these data and consistent with other reports in CLD (55, 56), we observed significantly greater circulating levels of GDF-15 in CLD patients, which were positively associated with muscle epigenetic age acceleration, further supporting an aged phenotype of the skeletal muscle in CLD patients.
Although the mechanisms that drive age-related epigenetic changes are not fully understood, elevation of circulating factors including proinflammatory cytokines such as TNFα (57), IL-6 (58) and IL-12 (59) reputedly play a role in modulating epigenetic modifications of DNA (60). Similarly, increased adiposity, which is also strongly associated with chronic low-grade inflammation (61), has been reported to drive epigenetic age acceleration in other tissues, including the liver (62). Therefore, it is possible that alterations in circulating factors, such as increased levels of proinflammatory cytokines (63) or ammonia (64) following primary liver dysfunction may drive epigenetic changes in secondary tissues such as skeletal muscle, negatively impacting their ageing trajectory. However, it will be important to elucidate key factors that drive epigenetic ageing, and those that become elevated secondary to age associated changes in cellular function.
In addition to modifications of the muscle epigenome, it is also possible that the epigenetic muscle ageing we observed here could be attributed to differential epigenome content in CLD patients, reflecting changes in the muscle tissue composition. Indeed, we have recently reported evidence of significantly greater intramuscular adipose tissue infiltration within the quadricep muscle of CLD patients (29), while a shift in fibre-type composition such as a loss of type II fibres typically observed with age, may also have impacted the epigenetic content of our CLD cohort (65).
Along with greater muscle epigenetic age, the skeletal muscle tissue of CLD patients was enriched for the novel SenMayo gene set (36), suggesting a greater presence of senescent cells within the muscle tissue of CLD patients, another hallmark of ageing. Accumulation of senescent cells is associated with tissue dysfunction, partly mediated by the pro-inflammatory senescence associated secretory phenotype (66) and can further compound tissue dysfunction through induction of senescence in neighbouring cells (66). Despite the terminally differentiated nature of muscle fibres, increased senescent cell burden has recently been reported in skeletal muscle of older individuals, likely driven by a p21 mediated mechanism (3, 13, 67). Importantly, increased muscle senescence has been shown to negatively correlate with markers of muscle function and to impair muscle regeneration and promote fibrosis in mice (3, 68). Conversely, targeted removal of senescent cells with senolytic compounds improved grip strength and increased muscle satellite cell proliferation in mice (3, 68). Therefore, senolytic based therapeutic interventions may provide a novel and feasible strategy to maintain or improve muscle function in patients with CLD, with similar approaches recently being shown to improve physical function in other chronic inflammatory disease states, specifically idiopathic pulmonary fibrosis (19).
Patients with CLD display greater ageing of the immune system.
We also observed a significant increase in indicators of ageing in the immune cells of patients with CLD, utilising the IMM-AGE score (37, 69), which agrees with previous data demonstrating immune cell telomere attrition in CLD (42, 43, 70). Interestingly, unlike epigenetic age in either muscle tissue or PBMCs, IMM-AGE correlated well with measures of skeletal muscle mass and may therefore also be a more clinically relevant model for predicting declines in skeletal muscle mass and function. Additionally, it is well established that immune cells infiltrate skeletal muscle and contribute to regulation of muscle biology (71). Therefore, increased numbers of senescent immune cells may have contributed to the raised muscle SenMayo score observed here, as well as contributing to localised inflammation within the muscle itself, impairing function. Interestingly, such immune system ageing may also play an important role in driving intramuscular adipose tissue accumulation, as a positive association between immune system ageing and muscle IMAT was still observed when only considering lean individuals. Collectively, these findings may help to explain the high prevalence of sarcopenia in CLD patients.
In addition to IMM-AGE, we also observed greater epigenetic ageing of PBMCs in CLD patients. Epigenetic age acceleration was particularly evident with the PACE of ageing clock, which indicates the ageing trajectory, rather than predicting chronological age and so could more accurately reflect disease progression (28). Additionally, PBMC epigenetic ageing positively correlated with IMM-AGE, demonstrating consistent biological age acceleration when utilising different methods. Therefore, PMBC epigenetic clocks, may provide useful clinical biomarker tools to identify CLD patients at the greatest risk of mortality (69).
We also identified a significant positive correlation between the epigenetic ageing of the immune system and skeletal muscle tissue epigenetic age acceleration, indicating that CLD is associated with epigenetic ageing across different biological systems. In the only other study of this kind, Sillanpää et al similarly reported a correlation of epigenetic age across muscle tissue and blood samples obtained from matched individuals (72). Translationally, PBMC epigenetic clocks have the advantage of greater capability for high throughput analysis in comparison to the immune cell phenotyping necessary for IMM-AGE calculation, or obtaining muscle biopsy samples for measures of muscle ageing.