Introduction

Geriatric patients are often affected by low-grade, chronic systemic inflammatory state. This process referred to as “inflamm-aging” was suggested to participate to the decline of physiological functions and increased vulnerability encountered in older patients [1]. Indeed, inflammatory markers, such as serum interleukin (IL)-6, C-reactive protein (CRP) or TNF-alpha are powerful predictors of morbidity and mortality in very old humans [2–5]. Furthermore, several reports indicate an association between inflammatory markers and geriatric conditions such as frailty, functional decline or depression [6–8].

Because of the overlap between geriatric syndromes and comorbidities, it is extremely difficult from the current literature to identify which clinical parameters are associated with increased inflammatory markers. Cardiovascular (CV) diseases are among the most prevalent comorbidities in geriatric patients [9]. CV diseases and related risk factors were proposed to influence the inflammatory state [10]. Other factors such as chronic cytomegalovirus (CMV) infections could also contribute to the association between geriatric conditions and low-grade inflammation [11,12]. The aim of this study was to assess the contribution of these underlying conditions in the association between inflammatory markers and common geriatric conditions (such as comorbidities, functional dependence, cognitive disorders, depression and malnutrition). For this purpose, we recruited old subjects in the general population or hospitalized for chronic medical conditions. We performed a comprehensive geriatric assessment (CGA) to identify comorbidities and common geriatric conditions. In this cross-sectional study, we also included younger subjects (with or without CV diseases) to assess the contribution of ongoing chronic co-morbidities in low-grade inflammation independently of age or geriatric conditions.

We focused our analysis on plasmatic IL-6, a classical systemic inflammatory marker. In order to more specifically address the functional status of blood innate immune cells, we also monitored cytokine production upon ex vivo stimulation with Alum. This vaccine adjuvant is a classical activator of the NLRP3-dependent inflammasome [13]. On its own, it does not directly support transcriptional activation of pro-inflammatory genes [13]. However, if cells are in a pre-activated state that leads to accumulation of pro-IL1β (e.g. in response to Toll-like receptor (TLR) ligands), alum will induce the secretion of mature IL-1β and downstream IL-6 production. Hence, cytokine production in these conditions will indirectly reflect the global activation status of blood innate immune cells.

Materials and Methods

Results

Occurrence of cardiovascular diseases is independently associated with high circulating IL-6 levels

We analyzed basal plasma IL-6 levels rather than spontaneous secretion by isolated PBMC or monocytes as it also reflects the contribution of other potential sources such as adipocytes, muscle or endothelial cells.

Using univariate analyses, we first attempted to identify relevant associations between clinical and biological parameters on one hand and basal IL-6 levels on the other. We found a significant association for monocytes (R²=0.8; p= 0.01) and WBC counts (R²=0.12; p=0.001), age (R²= 0.05; p=0.03), CV risk factors (R²=0.08; p=0.01) and diseases (R²=0.14; p=0.0007), type 2 diabetes (R²=0.13; p=0.013) and a trend for hypertension (R²=0.08; p=0.06).

To assess the relative contribution of these variables, we next performed multivariate analysis (summarized in Table 5) including age, gender, cardiovascular risk factors and diseases, WBC and monocytes counts, creatinin clearance and CMV status. Age, CV diseases and WBC counts appeared to be associated with basal IL-6 levels. IL-6 values were then normalized to WBC counts and we performed the same multivariate analysis. Once again, age and CV diseases were found to be significantly associated with basal IL-6 levels/WBC ratio.

N=100	R2 ; F value	Standardized Coefficient	p value
Log10 IL-61	R2=0.31; F=10.93
WBC		0.363	<0.001
CV diseases		0.319	0.002
Age		0.255	0.0013
Log10 IL-6/WBC ratio 2	R2=0.31; F=10.93
CV diseases		0.35	0.001
Age		0.232	0.03

Table 5. Multivariate analyses for basal IL-6 levels in the whole study group.

1adjusted for age, gender, CV risk factors and diseases, WBC and monocytes counts, creatinin clearance, CMV status.

2adjusted for age, gender, CV risk factors and diseases, creatinin clearance, CMV status.

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Secondly, we assessed the association between geriatric conditions and low-grade inflammation. Geriatric scales are only validated in aged individuals. Hence, to assess the association between inflammatory state and geriatric conditions, we restricted our analyses to subjects >75 years of age. We compared plasma IL-6 levels between older individuals with ISAR>1 and ISAR≤1 point. IL-6 levels were significantly increased in this second group (Figure 1). To take into account possible confounding variables, we performed univariate analyses. No geriatric conditions (MMSE, CIRS-G and grip strength) were significantly correlated with this inflammatory parameter but there was a trend for Katz, MNA and GDS. As for the entire study group, we observed a significant association between CV diseases (R²=0.1; p=0.04), type 2 diabetes (R²=0.13; p=0.02) and plasma IL-6 as well as a trend for CV risk factors (R²=0.08; p=0.07). We performed multivariate analyses with age, CV risk factors, gender and geriatric scales. As geriatric scales influence each other, we analyzed separately MNA, GDS or MMSE scores in multivariate analysis. No geriatric score appeared as a significant predictive factor (cfr table 6).

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Figure 1. Inflammatory markers in individuals with ISAR

≤1 and >1 point.

(A) Plasmatic IL-6 levels were analyzed by ELISA. (B-C) Whole blood samples were incubated with alum (500µg/ml) for 18h. Cell-free supernatants were collected and analyzed for IL-6 and IL-1β levels by ELISA. ISAR>1 point, n=27; ISAR≤1 point, n=25. For statistical analysis, we used the Mann-Whitney rank sum test *p<0.05; **p<0.01.

https://doi.org/10.1371/journal.pone.0081911.g001

N=52	R2 ; F value	Standardized Coefficient	p value
Log10 IL-61	R2=0.13; F=6.5
Age		0.03	0.014
Log10 IL-62	R2=0.14; F=7
Age		0.03	0.011
Log10 IL-63	R2=0.13; F=6.5
Age		0.03	0.014
Log10 IL-6/WBC ratio4	R2=0.15; F=7.7
Age		0.04	0.008
Log10 IL-6/WBC ratio5	R2=0.16; F=8.1
Age		0.04	0.007
Log10 IL-6/WBC ratio6	R2=0.15; F=7.7
Age		0.04	0.008

Table 6. Multivariate analyses for basal IL-6 levels in subjects

> 75 years.

1adjusted for Katz, age, gender, CV risk factors and WBC counts.

2adjusted for MNA, age, gender, CV risk factors and WBC counts.

3adjusted for GDS, age, gender, CV risk factors and WBC counts.

4adjusted for Katz, age, gender, CV risk factors and diseases.

5adjusted for MNA, age, gender, CV risk factors and diseases.

6adjusted for GDS, age, gender, CV risk factors and diseases.

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Discussion

Previous studies on inflammation in older individuals and geriatric patients yielded conflicting conclusions. The main difficulty in the interpretation of the results comes from the heterogeneity of the population and differences in the classification, definitions and inclusion/exclusion criteria used. A summary of such studies is shown in Table S1. We included main study parameters (size of the group, age groups, inclusion criteria, clinical data, design and statistical methods used) and their conclusions. Increased inflammatory markers are generally associated with age, CV comorbidities and geriatric conditions. However, when confounding factors are taken into account, the relative contribution of these parameters is less clear. Herein, we took particular care to characterize the clinical parameters of the enrolled subjects. As CV disorders are associated with low-grade inflammation and as healthy old individuals might present underlying CV disorders without any symptoms, we included younger subjects (with or without CV diseases) to assess the contribution of ongoing chronic co-morbidities in low-grade inflammation independently of age or geriatric conditions. Indeed, in enrolled younger subjects (mean age: 49 years), proportion of CV diseases or risk factors (hypertension: 38%, type 2 diabetes: 10%, hypercholesterolemia: 63% and ongoing CV diseases: 27%) was comparable to that observed in older individuals. We observed that the strongest predictive factors for increased IL-6 plasmatic levels were the history of CV diseases or risk factors and age. A previous study indicated that IL-6 levels were associated with age but not with CV risk factors (assessed by BMI, blood pressure or lipidemia) in a 20-84 year old healthy population. However, the authors did not report the occurrence of CV diseases in their study population [7,28]. Forsey et al. also observed an increase of IL-6 with age and poor health status (using SENIEUR, OCTO and NONA criteria) but they did not characterize their group for ongoing comorbidities [29]. Hence, as suggested previously [10], our findings support the notion that part of age-associated inflammatory state could be linked to accumulation of CV risk factors and morbidities in the geriatric population. In another study, high inflammatory markers (in particular basal IL-6) were found to be predictive for the development of CV events within a 3-year period in older well-functioning individuals [30]. In people without known CV diseases, CRP level is also an important predictive factor for CV events [31]. Furthermore, two large mendelian randomized studies showed that polymorphisms of the IL-6R are associated with reduced CRP levels and the risk of coronary artery diseases. However, both studies showed increased concentrations of IL-6 levels that seem in contradiction with a causal role of IL-6. This paradox might be resolved by the fact that both IL-6R polymorphisms seem to be associated with downregulation of IL-6R signaling and that increased IL-6 levels could be a result of a feedback loop to increase IL-6 signaling [32,33]. A meta-analysis of prospective studies involving white populations confirmed that polymorphisms of the IL-6 gene were also associated with the development of coronary artery diseases [34].

We observed that IL-1β and IL-6 production by blood cells in response to alum was also strongly associated with CV diseases and risk factors. This result suggests that in these patients, circulating innate immune cells could be in a pre-activated state. Indeed, CV risk factors such as hypercholesterolemia, high saturated fat diet, obesity and hyperglycemia are known to promote inflammation through different pathways such as the endoplasmic reticulum (ER) stress, the inflammasomes and the TLRs [3,35,36]. ER stress and TLR pathways both lead to transcriptional activation of inflammatory genes such as pro-IL1β that could account for the association between high IL-1β and IL-6 production in response to alum and CV diseases that we observed. However, the design of our study does not allow us to establish a causal links between CV diseases and low-grade inflammation. Coronary artery diseases, high BMI and atherosclerosis are associated with an increase in circulating inflammatory monocytes (CD14^dimCD16⁺). Furthermore, the proportion of intermediate monocytes (CD14⁺⁺CD16⁺) is also associated with increased CV risk factors and is predictive of CV events and poor outcome [37,38]. In comparison to classical monocytes (CD14⁺⁺CD16), this subset displays an activated phenotype, reduced telomere length and produces high amounts of proinflammatory cytokines [39,40]. It would therefore be important to determine the responsiveness of these cells to alum in the context of CV diseases.

Along this line, chronic CMV infection was also associated with modulation of monocyte functions [41] that could account for the association between CMV status and IL-6 levels upon alum that we observed. Chronic infection with CMV is seen as a major factor that could influence exhaustion of memory T lymphocytes in older individuals. CMV seropositivity is part of the “immune risk phenotype” (IRP) and is associated with increased all-cause mortality [42]. It has been suggested that immune response to persistent CMV infection could also participate to the establishment or maintenance of inflamm-aging processes [2,42]. Several studies previously reported an association between IL-6 levels and CMV status. In patients with coronary artery diseases, Blanckenberg et al. observed that CMV titers were correlated to IL-6 levels and together could predict cardiac mortality [43]. In community-dwelling post-menopausal women, mean IL-6 levels were found to be higher in CMV+ subjects but significance was lost after adjustment for confounding factors [11,44]. Interestingly, in the “Hertfordshire Ageing study” group, increase in plasma IL-6 levels upon aging was similar in seronegative, seropositive volunteers and volunteers who became seropositive for CMV, arguing against a central role of CMV infection in this process [45]. We identified CMV status as an independent predictor of IL-6 production upon alum stimulation but not for other inflammatory parameters, suggesting that CMV could directly or indirectly influence the capacity of the immune cells to produce this cytokine. It would be important to define the potential mechanistic links responsible for these observations.

In our study, geriatric patients with ISAR>1 point display increased basal and alum-induced IL-6 production compared to old individuals with ISAR<1 point. However, we did not find any significant association between geriatric conditions (assessed by Katz, MNA, GDS, CIRS-G, MMSE, Grip strength) and low-grade inflammation. Studies that have identified association between frailty and high IL-6 levels generally used criteria developed by Fried et al. (slow gait speed, low physical activity, unintentional weight loss, self-reported exhaustion and muscle weakness) [46]. Initial studies indicated an association between elevated CRP levels and frailty after exclusion of CV diseases and diabetes [47]. However, association between increased IL-6 and frailty was less clear in other studies. For example, in the “Longitudinal Aging Study Amsterdam” (LASA), IL-6 was not associated with frailty parameters [48]. While IL-6 could be predictive for frailty in a cohort of women aged 70 to 79 [11], increased IL-6 levels were not found to be predictive for the later development of frailty in the “Hertfordshire Ageing study” (HAS, 10 year follow-up, age between 65-70 years) [49]. The use of NSAID and corticoids, the heterogeneity of the population and follow-up bias because of mortality and various frailty criteria could partly explain these discrepancies.

Dependency is a direct expression of frailty. In institutionalized subjects older than 65 years old selected for cerebral vascular diseases, a decline in activities of daily living (Katz ADL and Barthel index) was associated with high IL-6 levels [8]. Gonzalo-Calvo et al. showed the same results in institutionalized volunteers [8,50]. However, variability in interpretation of Katz score hampers interpretation and comparison of these studies with the present one [51].

Muscle strength and walking speed are major phenotypic criteria of frailty. Several studies used this parameter to assess the link between frailty and inflammation. Indeed, Schaap et al. observed that higher IL-6 and CRP levels were associated with decreased muscle strength in a cohort of independent individuals over 55 years after adjustment for confounders such as age, depressive symptoms, chronic diseases and cognitive troubles [52]. Similar conclusions were also previously reached by Ferruci et al. in community-dwelling women (above 65) [53]. In a cohort of healthy subjects between 20 to 72 year old, Blain and colleagues observed a similar association between IL-6 levels and walking performance but not with muscle strength [54]. Interestingly, Bautmans et al. showed that while better grip strength and fatigue resistance were associated with higher IL-6 levels in well-functioning subjects, this was not the case for nursing home patients and hospitalized patients [26,55,56]. Taken together, these data indicate that association between inflammation and grip strength is highly dependent on the population under study and might reflect variations in important confounding factors. In particular, CV diseases and underlying pathological conditions (such as atherosclerosis) are likely to participate to the development of frailty and reduced multi-organ physiological reserve in old age. Hence, CV diseases, frailty and geriatric conditions are probably deeply intertwined.

Cognitive and psychological factors are other important aspects of the geriatric patient. As these factors also participate to the general health status of the patients and could impact their immunological profile, we used a CGA to characterize our patients [57,58]. Some studies found an association between depressive or stress symptoms and IL-6 levels [6,7,59]. Meta-analysis confirmed the association between IL-6, CRP and IL-1β levels and depression. However, it was not significant anymore once age or medication intake were taken into account [60]. In the context of this study, for ethical reasons and accuracy of the other geriatric scales, we did not recruit patients presenting with strong cognitive impairment. The potential association of these clinical parameters and inflammation is therefore probably underestimated. Several studies have shown an association with cognitive decline and IL-6 [61,62] but most studies observed an association with vascular dementia but not Alzheimer’s dementia [63,64], suggesting that underlying CV conditions could be responsible for such association. However, other studies that included patients with low MMSE scores did not detect an association for TNFα and IL-6 levels [65–67].

One limit of our study is the small size of the group given its heterogeneity. We performed numerous comparisons that increase α error of the statistical analyses. As we recruited patients that were hospitalized, acute medical conditions could bias inflammatory markers. To reduce this potential effect, data were collected just before discharge to home. Although conclusions were similar when the whole group was analyzed (not shown), we limited evaluation of geriatric scales to subjects above 75 years of age, as these clinical parameters might not reflect the same underlying conditions in younger patients. As we included younger and older individuals with chronic diseases, we cannot fully assume that the impact of these diseases on physiological and immune functions will be the same in both age groups. Transversal studies do not take into account the evolution of clinical and immunological parameters. We did not specifically look at periodontal diseases known to be associated with chronic inflammation [68].

Despite these limitations, we identified a clear association between CV diseases and inflammatory parameters suggesting that among clinical parameters, these factors have the largest impact on the development of low-grade inflammation observed in the geriatric population.

Supporting Information

Acknowledgments

The authors would like to thank Arnaud Marchant, Muriel Nguyen, Julie Callenaere, Yves Delmarcelle, Thierry Honorez, Julie Hardy, Zohra Bourkhich, the geriatric team of Erasme hospital, Pierre and Brigitte Compté, Vincent Geenen, Henri Martens and all volunteers.