Association of leukocyte mitochondrial DNA copy number with longitudinal C-reactive protein levels and survival in older adults: a cohort study

Chronically high blood levels of C-reactive protein (CRP), an acute-phase protein induced by the systemic effects of proinflammatory cytokines [1], and some proinflammatory cytokines themselves are frequently observed in older individuals, even those who appear healthy [2, 3]. Of note, multiple lines of evidence have indicated that simply having a higher blood level of CRP is associated with greater risks of age-related chronic diseases, chronic health conditions, and multiple adverse health outcomes [2‐6]. As the population ages, the prevalence of these inflammaging-related health problems, which is already sizable, is likely to increase.

However, the systemic chronic inflammation that tends to develop with age remains incompletely understood [2, 3]. The systemic chronic inflammation has been theorized as a key feature of the aging process [3, 7‐9]. Aging is associated with alterations in mitochondria [9]. The mitochondrion has its own genome, which encodes components of the oxidative phosphorylation system and exists in multiple copies [10, 11]. The mitochondrial DNA content of peripheral blood cells, usually measured as the quantity of mitochondrial DNA relative to that of nuclear DNA (mitochondrial DNA copy number [mtDNA_CN]) [9, 11‐14], often decreases with age and is a potential biomarker of mitochondrial function in aging [15‐18]. Studies have demonstrated that the lower the mtDNA_CN of blood cells is, the greater the risk of specific age-related diseases is [12, 19‐21]. These diseases include, but not limited to, coronary heart disease, stroke, kidney function decline, and neurodegenerative disorders. For instance, Ashar et al. [19] showed that one standard deviation lower leukocytes mtDNA_CN was associated with greater risks of incident coronary heart disease and stroke. Tin et al. [20] demonstrated that blood cells mtDNA_CN at the highest quartile was associated with a lower risk of incident chronic kidney disease. These findings suggest that blood cells mtDNA_CN is inversely related to the risk of these diseases. Notably, chronic inflammation is a common feature of many of these diseases, in particular coronary heart disease, ischemic stroke, and kidney function decline [5, 22]. Therefore, an inference from these observations is that a lower blood cell mtDNA_CN may be related to systemic chronic inflammation. In line with this, accumulating experimental evidence suggests that immune cell mitochondria are active players in immunity and inflammation and may play etiological roles in the inflammatory pathogenic process [23, 24].

The blood levels of high-sensitivity CRP (hs-CRP) are primarily determined by its synthesis rate in hepatocytes, which in turn is determined by the levels of circulating proinflammatory cytokines raised by a distal inflammatory pathogenic process [1]. As such, serum hs-CRP is a commonly used clinical laboratory test, and its high level typically indicates an active inflammatory pathogenic process. Most studies on hs-CRP have focused on levels measured at a single time point. Although its blood levels at a single time point carry rich prognostic information [2], more recent studies suggested that its temporal profile may reflect the activity of the corresponding inflammatory pathogenic process in a more timely and proportional manner and that it may additionally provide more detailed information about the activity of the process [25, 26].

The aim of this study was twofold (Additional file 1: Supplementary Fig. 1). The first aim was to prospectively evaluate the relationship of the peripheral blood leukocyte mtDNA_CN with longitudinal hs-CRP levels in a large cohort of older persons without systemic inflammation. The second aim was to assess the relationship between the longitudinal hs-CRP levels and mortality risk. We hypothesized that a lower peripheral blood leukocyte mtDNA_CN would be associated with persistently higher time-varying hs-CRP levels and that these higher time-varying hs-CRP levels would be associated with a higher risk of death after adjusting for the effects of multiple potential confounders.

The characteristics of the study participants at baseline are presented in Table 1. The mean age was 69 years, and 52% of the participants were women. The median leukocyte mtDNA_CN at baseline was 72.00. Of the 3928 participants, 837 died during a median follow-up period of 10.6 years. Participants who died during the follow-up period had a significantly lower leukocyte mtDNA_CN at baseline (68.00) than those who survived (73.00).

In this longitudinal study of community-dwelling older adults, we identified the relationship of peripheral blood leukocyte mtDNA_CN with longitudinal serum hs-CRP levels and the relationship of the longitudinal serum hs-CRP levels with the risk of death. This study was the first to demonstrate that even without accompanying signs of systemic inflammation, a lower peripheral blood leukocyte mtDNA_CN at baseline was associated with persistently higher hs-CRP levels. Moreover, our analysis indicated that these higher longitudinal hs-CRP levels were associated with a higher risk of death. These associations were independent of baseline hs-CRP levels, infectious processes, high-risk cardiovascular inflammation and potential confounders.

Blood levels of hs-CRP are a well-recognized and extensively used biomarker of inflammation [1, 25, 26, 35, 37, 38]. As an acute-phase protein, CRP is rapidly synthesized in hepatocytes, stimulated mainly by circulating cytokine interleukin-6, and released into circulation in response to a range of inflammatory pathologies at distant sites in a dose–response manner [1, 39, 40]. With a constant half-life in the circulation, the level of hs-CRP in an individual’s blood is mainly determined by its rate of synthesis, which in turn is determined by the activity of the underlying cytokines-producing inflammatory pathologies [41]. Therefore, an examination of an individual’s temporal profile of serum hs-CRP by measuring the levels of hs-CRP in their blood serially—rather than at a single time point, as in most studies—offers a unique opportunity to reliably track the activity of the underlying inflammatory pathogenic process [1, 25, 26, 37, 38].

To the best of our knowledge, this study is the first to examine the relationship between the peripheral blood leukocyte mtDNA_CN and the temporal profile of serum hs-CRP. We demonstrated that among older adults free of clinically relevant systemic inflammation, a lower peripheral blood leukocyte mtDNA_CN was associated with a persistently higher level of hs-CRP in the subsequent years. This observation was consistent across the multiple analyses that took into account the potential effects of confounders and intercurrent hs-CRP-raising disease processes. These results suggest that a lower leukocyte mtDNA_CN may be associated with an active inflammatory pathogenic process throughout the years. In general, the observation of an inverse relationship between the peripheral blood leukocyte mtDNA_CN and serum hs-CRP is consistent with the results of other studies, although most analyses have been cross-sectional in nature and thus have precluded the determination of the chronicity of hs-CRP elevation. For instance, in another study, we revealed that the peripheral blood leukocyte mtDNA_CN was negatively correlated with hs-CRP in a cohort of 1990 older adults [42]. In a cohort of 9058 participants without chronic kidney disease [20], Tin et al. noted that those with lower peripheral blood cells mtDNA_CN had a higher serum hs-CRP level. In another cohort of 4812 patients with chronic kidney disease [43], those with a lower whole blood mtDNA_CN were also observed to have a higher hs-CRP.

In this study, by conducting the joint longitudinal and survival analysis, we further demonstrated that a higher level of hs-CRP during follow-up was instantaneously associated with a higher risk of death independent of confounders. Most importantly, this effect of follow-up hs-CRP levels on death risk was independent of baseline hs-CRP levels. On the other hand, the baseline hs-CRP levels were not associated with the death risk after adjusting for the longitudinal hs-CRP levels. Interestingly, after excluding individuals with follow-up hs-CRP levels exceeding thresholds that may indicate an intercurrent infection or high-risk cardiovascular inflammation, the study sample consisted of older adults likely at average or lower risk of diseases (e.g., cardiovascular diseases). Nevertheless, the effects were still observed in this sample of older adults. In sensitivity analysis, we examined whether a different analytic assumption about how the death risk depends on the longitudinal hs-CRP levels might affect the results. Assuming that the risk of death at time t depends on the whole hs-CRP exposure history up to time t, we still observed significant, albeit small, effects of hs-CRP level on the risk of death. The overall results of the survival analysis agree with the argued values of serially determining hs-CRP levels and imply that the longitudinal hs-CRP levels may carry additional information about the near-term health event hazard. Even below clinically relevant thresholds, the chronic low-grade inflammatory process associated with a lower leukocyte mtDNA_CN might uniquely confer an immediately higher mortality risk in older adults, including low-risk individuals. However, we could not exclude the possibility that the chronic low-grade inflammatory process caused by factors other than lower leukocyte mtDNA_CN confers a higher mortality risk.

The findings of this longitudinal study are consistent with recent results of experimental investigations that have suggested a causal role of blood cell mitochondria in the development of pathogenic systemic chronic inflammation. First, emerging evidence from human studies has indicated that a lower blood cells mtDNA_CN may reflect a lower expression of many mitochondrial- and nuclear-encoded genes involved in mitochondrial DNA replication and function in blood cells [13]. Second, as the hub of multiple metabolic and signaling pathways, mitochondria in immune cells play pivotal roles in shaping immune and inflammatory responses [23]. Third, experiments in mice have demonstrated that decreased activity in immune cells' mitochondria metabolic pathways, including mitochondrial respiration, drives systemic chronic inflammation in aging, thereby, the development of age-related diseases [24].

The major strengths of this study include its prospective design with serial measurements of hs-CRP and the use of an advanced analytical method suitable for studying events with a mechanism of significant chronicity (e.g., aging). In addition, this study analyzed prospectively collected data from a well-established cohort with comprehensive baseline information obtained through a standardized procedure. Notably, the measurements of the mtDNA_CN and hs-CRP in this study were centralized and standardized. All these characteristics helped this study minimize the likelihood of observing spurious associations. Nevertheless, our study has some limitations. First, as an observational study, inferences regarding causality and mechanism should be drawn with caution. Second, although we identified potential confounders a priori and, guided by DAGs, attempted to adjust for confounding bias during model building, residual confounding by unmeasured or uncontrolled covariates still could not be excluded. We could not adjust for the potential confounding effects of time-varying covariates on the association between higher longitudinal hs-CRP levels and a higher risk of death. To assess how likely the observed association could be due to uncontrolled confounding, we calculated the E-value in the sensitivity analysis. We found that an uncontrolled covariate would need to be associated with both the time-varying hs-CRP levels and mortality risk with a risk ratio of at least 1.56 (lower limit: 1.42) to explain the observed association fully. Baseline stroke was the covariate most strongly associated with mortality, with an HR of 1.74. After excluding stroke from the model, we still obtained an E-value of 1.56 with a lower confidence limit of 1.42. We reasoned that the observed association was unlikely to be due to uncontrolled confounding. Third, the sample of this observational study consisted of adults aged 55 years and older at study entry. We could not completely exclude the possibility of survival bias and the possibility of underestimating the effects of lower leukocyte mtDNA_CN. Fourth, we excluded those with higher baseline hs-CRP from this study to examine the association of leukocyte mtDNA_CN with hs-CRP trajectory among those who had not yet developed systemic inflammation at the time of mtDNA_CN measurement. As such, our findings may not be generalizable to other populations of older adults who might be more ill. Fifth, further research would be worthwhile to determine the clinical relevance of the sub-threshold hs-CRP levels in older adults. Sixth, we showed that the inflammatory process might have a cumulative effect on the risk of death, as demonstrated by the sensitivity analysis. The clinical relevance of such findings is unclear and is needed to be further determined. Finally, both high and low blood cells mtDNA_CN have been associated with higher risks of a variety of cancers. The roles of inflammation in this context would also be worth investigating.

In conclusion, among older persons without systemic inflammation at baseline, a lower peripheral blood leukocyte mtDNA_CN was associated with persistently higher hs-CRP levels. Moreover, the higher time-varying hs-CRP levels were immediately associated with higher risks of death. Further studies are warranted to clarify the underlying mechanisms.