TLR-6 SNP P249S is associated with healthy aging in nonsmoking Eastern European Caucasians - A cohort study
© Hamann et al. 2016
Received: 3 February 2016
Accepted: 11 March 2016
Published: 17 March 2016
To investigate mechanisms that determine healthy aging is of major interest in the modern world marked by longer life expectancies. In addition to lifestyle and environmental factors genetic factors also play an important role in aging phenotypes. The aged immune system is characterized by a chronic micro-inflammation, known as inflamm-aging, that is suspected to trigger the onset of age-related diseases such as cardiovascular disease, Alzheimer’s disease, cancer, and Diabetes Mellitus Type 2 (DMT2). We have recently shown that a Toll-like receptor 6 variant (P249S) is associated with susceptibility to cardiovascular disease and speculated that this variant may also be associated with healthy aging in general by decreasing the process of inflamm-aging.
Analyzing the PolSenior cohort we show here that nonsmoking S allele carriers are significantly protected from age-related diseases (P = 0.008, OR: 0.654). This association depends not only on the association with cardiovascular diseases (P = 0.018, OR: 0.483) for homozygous S allele carriers, but is also driven by a protection from Diabetes Mellitus type 2 (P = 0.010, OR: 0.486) for S allele carriers. In addition we detect a trend but no significant association of this allele with inflamm-aging in terms of baseline IL-6 levels.
We confirm our previous finding of the TLR-6 249S variant to be protective regarding cardiovascular diseases. Furthermore, we present first evidence of TLR-6 249S being involved in DMT2 susceptibility and may be in general associated with healthy aging possibly by reducing the process of inflamm-aging.
The process of successful aging and longevity is complex and far from being understood. Lifestyle, environment, and a limited aging-related impairment of the immune system (termed immunosenescence) are among the most important factors contributing to the healthy aging phenotype. The aged adaptive immune system is characterized by a decreased effectiveness due to decreased T- and B-cell responses and accumulation of functionally impaired memory lymphocytes . Furthermore, the aged adaptive immune system is characterized by a dysregulation of T-cell subtypes, with a relative increase of Th2 lymphocytes resulting in increased levels of Th2 cytokines . The aged innate immune system is characterized by reduced functions of neutrophils, macrophages, NK cells, and dendritic cells . In addition, an important feature of the aged immune system is a chronic low grade inflammatory state, also known as micro-inflammation or inflamm-aging, which has been suspected to trigger aging-associated diseases such as cardiovascular disease (CVD), diabetes mellitus type 2 (DMT 2), metabolic syndrome, and neurodegeneration . The mechanisms inducing the process of inflamm-aging are multifactorial and not completely understood. One reason may be the dysregulation of T lymphocyte subtypes. An enhanced translocation of gut bacteria as well as an enhanced DNA-damage in the elderly, which both trigger the innate pro-inflammatory immune response, are also discussed [5–7]. Furthermore, epigenetic changes in innate immune cells are discussed to be involved in inflamm-aging .
However, numerous genetic factors have also been proposed to influence aging. Several studies show that healthy aging and particularly longevity are highly heritable. For example, siblings of centenarians also have an enhanced probability for a longer healthy life [9, 10]. Genetic variations associated with the onset of age-related diseases such as atherosclerosis resulting in CVD, DMT 2, Alzheimer’s Disease, and cancer may play a pivotal role in determining successful aging . Among aging-associated diseases, CVD plays a crucial role regarding mortality in the middle-aged population of the Western world [12, 13]. Recently, we have shown that the Toll-like receptor (TLR)-6 SNP P249S is associated with the risk for CVD with the homozygous S/S variant protecting from CVD. We also speculated whether this SNP may influence successful aging in general .
It has been shown that aging is associated with the dysregulation of expression and signal transduction of a variety of innate immune receptors, among them TLRs . TLRs are important receptors of the innate immune system recognizing “pathogen associated molecular patterns (PAMPs)” followed by the activation of the innate immune response . Although the process of inflammation is fundamental for survival in terms of combating infections or coping with damaging agents, it is also an important cause of many aging-associated diseases such as atherosclerosis, DMT 2, cognitive decline, and cancer, since these diseases are all associated with chronic inflammation . The inflammatory response is driven by a variety of signaling pathways, however, the transcription factor NFκB has been considered to be the master regulator of inflammation , and is also a central part of TLR signaling .
Since inflamm-aging seems to be one of the reasons for the development of aging-related diseases, we postulated previously that variations within the TLR-system that decrease the inflammatory response may lead to a decreased process of inflamm-aging and may be, therefore, beneficial in terms of healthy aging. First evidence for this hypothesis was given by the finding that such variations are overrepresented in healthy elderly subjects in comparison to healthy younger subjects . Following this discovery, we here investigated a cohort of elderly Caucasians and show that in nonsmoking elderly subjects the TLR-6 SNP P249S is associated with healthy aging. Analysis of certain aging related diseases revealed a significant protective effect on CVD and DMT 2.
A sub-group of 1544 participants of the PolSenior program, the first ones for whom the complete medical records (including, among others, data on cardio-vascular and respiratory diseases, cancer, diabetes, stroke and cognitive impairment) and DNA samples were available at the beginning of current study, was analyzed. PolSenior was a multicenter, interdisciplinary project, designed to assess health and socio-economic status of the Polish Caucasians aged ≥65 years. Details of the PolSenior recruitment are described elsewhere . Project participants completed a detailed questionnaire regarding their medical, social, and economic past and current health status, underwent an examination including elements of comprehensive geriatric assessment, and donated blood for biochemical and genetic analyses. Steady state IL-6 level were measured using ELISA method (R&D System, Minneapolis, MN, USA, sensitivity 0.04 pg/mL). Blood pressure was measured three times during the first and second visit using validated, automatic blood pressure measuring device (A&D UA 787, A&D Company Limited, Tokyo, Japan) in a seated position. The study subject was diagnosed as suffering from hypertension if his/her average blood pressure from these measurements was ≥ 140/90 mmHg, or the disease has been previously diagnosed and the patient was taking hypotensive drugs over the past two weeks. The study was approved by the Bioethics Commission of the Medical University of Silesia in Katowice. All participants gave a written, informed consent for participation in the study. All investigations were carried out in accordance with the ethical guidelines of the 1975 Declaration of Helsinki. Subjects with MMSE < 24, determined according to the Polish version of the Mini-Mental State Examination test, were classified as subjects with cognitive impairment. According to Akbaraly et al. we set IL-6 levels ≤1.0 ng/L as low, 1.1–2.0 ng/L as intermediate, and >2 ng/L as high . Low IL-6 levels were used as reference in logistic regression analysis.
Genomic DNA was prepared by standard procedures from whole blood. Genotyping for TLR-6 SNP P249S (rs5743810) was performed by melting curve analysis as described previously . Melting curve analysis was carried out employing primers gaaagactctgaccaggcat (forward), ctagtttattcgctatccaagtg (reverse), and FRET-hybridization probes: accagaggtccaaccttactgaa-FL and LC-red 640-ttaccctcaaccacatagaaacgacttgga resulting in melting points of 61, and 52 °C for the wild-type and the mutated allele, respectively.
Binary logistic regression analyses, Mann–Whitney U-test, Kruskal-Wallis, and T-test have been performed employing the IBM SPSS Statistics software package (version 20.0, IBM, Munich, Germany).
Association of common risk factors with age related diseases
Baseline characteristic of the study subjects
Mean age (SD)
Mean BMI (SD)
Smoking: never/past/current (%)
849 (55.0)/534 (34.6)/152 (9.8)
Hypertension yes/no (%)
Baseline IL-6 (ng/L)
Lung disease (%)
DMT 2 (%)
Cognitive impairment (%)
TLR-6 PP/PS/SS (%)
615 (39.8)/718 (46.5)/211 (13.7)
Association of common risk factors with aging-related diseases in the PolSenior group. Univariate analysis of common risk factors and aging related diseases
75.7 (6.6)/77.3 (6.3)
OR (95 % CI)
Association of common risk factors with aging-related diseases in the PolSenior group. Multivariate analysis of common risk factors and aging-related diseases
OR (95 % CI)
The TLR-6 variant 249S/S is significantly associated with protection from aging-related diseases in non-smoking subjects
TLR-6 P249S is associated with healthy aging in non-smokers
OR (95 % CI)
Combined aging related diseases
P/S + S/S
Combined aging related diseases
P/S + S/S
TLR-6 P249S is associated with CVD and DMT 2 in non-smokers
OR (95 % CI)
P/S + S/S
P/S + S/S
P/S + S/S
P/S + S/S
P/S + S/S
Combined aging related diseases, CVD excluded
P/S + S/S
No association of TLR-6 6 P249S variation with baseline IL-6 levels
TLR-6 P249S is not associated with baseline levels of IL-6
IL-6 (ng/L, mean, SD)
Aging related diseases
We have recently shown that TLR-6 P249S is associated with coronary artery disease, one of the most important aging-related diseases, and have speculated that this genetic variation may be also associated with healthy aging in general by decreasing the process of inflamm-aging due to decreased sensitivity of the innate immune response [14, 19]. Healthy, or successful aging is affected by a variety of factors including environmental factors, life style, micro-inflammation, and genetic factors that determine the occurrence, onset and course of aging-related diseases. Since most age-related diseases are associated with chronic inflammation , a sensitive immune system may be an example of antagonistic pleiotropy with beneficial effects at early age but adverse effects in later life since evolution optimizes for fitness, not for longevity . By analyzing a middle-aged population-based cohort, exhibiting the typical distribution of aging-related diseases, we here confirmed our previous finding of TLR-6 P249S being associated with CVD and showed that this SNP is associated with healthy aging in non-smoking subjects of the PolSenior cohort. This positive association with healthy aging remains significant after exclusion of subjects suffering from CVD indicating a more general effect, not only restricted to CVD. Furthermore we present first evidence of this SNP to be also involved in DMT-2 and the process of inflamm-aging in non-smoking subjects.
We have currently no conclusive explanation why this effect could not be shown in smoking subjects. Although our study does not show smoking to be one of the strongest risk factors for aging-related diseases in individuals 65 years old and older, the effect of TLR-6 variants on healthy aging may nevertheless be masked by smoking. Such modifying effect of smoking has recently been reported for several genetic associations in age-related macular degeneration which are largely restricted to non-smokers . One possible explanation could be that in our cohort smoking is associated with both, lower ager and lower BMI which in turn are associated with the absence of aging-related diseases. The association of TLR-6 P249S with DMT 2 has not been shown before. However, since the number of non-smoking subjects with DMT 2 was rather low in our cohort (n = 79), and TLR-6 has not been found by GWAs for DMT 2, these findings have to be confirmed in larger studies .
Analyzing the whole cohort for common risk factors by multivariate regression analysis showed, as expected, increased age, BMI, and inflamm-aging to be strongly associated with the occurrence of aging-related diseases. Since we have previously speculated that less functional TLR variants decreasing the sensitivity of the innate immune system may dampen the process of inflamm-aging, we analyzed the association of TLR-6 P249S with baseline IL-6 levels. Although the S allele at position 249 of TLR-6 has been shown to decrease TLR-6 signaling , we could not find a significant effect on inflamm-aging. However, we could show a trend towards decreased IL-6 levels in S allele carriers. The failure of finding significant changes may potentially be explained by the fact that other pro-inflammatory cytokines such as TNF-α and IL-1β, the serum-levels of which were not available to us, may be more involved in the process of inflamm-aging as compared to IL-6. Recent data show that inflamm-aging is not simply the chronic increase of pro-inflammatory markers such as IL-6 and CRP. Inflamm-aging seem to be much more complex and is characterized by the simultaneous change in both pro- and anti- inflammatory markers . Furthermore, repeated measurements of IL-6 levels, which were not available to us in this cohort are known to be more accurate in determining inflamm-aging . Finally, the SNPs investigated by us may lead to changes in cytokines in certain tissues, which cannot be monitored by measuring serum levels.
On the other hand, comparing healthy subjects with diseased subjects, a strong correlation of increased IL-6 baseline levels and aging-related diseases was shown. This may be evidence for the fact that inflamm-aging is driven mainly by a dysregulated T lymphocyte subpopulation  and chronic stimulation of the innate immune system via TLRs does not play the dominant role.
Since the limitation of our study is relatively small sample number, especially in disease subgroups, our results require further investigation by independent studies.
In conclusion we confirm here our previous results of less functional TLR-6 variant 249S to protect from CVD. In addition we show here that this variant protects also from DMT-2 and is in general associated with healthy aging in the PolSenior cohort. Furthermore, we could show a trend for a decreased process of inflamm-aging by the TLR-6 249S allele as measured by IL-6 levels. However, the effect of TLR variants on the process of inflamm-aging needs to be confirmed by further studies.
Financial support was provided by Charité - Universitätsmedizin Berlin (grant 2007-486), the Berliner Krebsgesellschaft e.V. (all to R.R.S), and by the Polish Ministry of Science and Higher Education grant PBZ-MEiN-9/2/2006 – K143/P01/2007/1 (to M.P.K., M.S. and M.M.). We thank Inga Wyroslak for outstanding technical assistance.
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