Genetic and environmental determinants of population variation in interleukin-6, its soluble receptor and C-reactive protein: Insights from identical and fraternal twins
Introduction
The pleiotropic cytokine, interleukin-6 (IL-6), is produced by many cells and tissues, and plays a major role in normal physiology and inflammatory responses. It has been widely employed in research on population health and aging because circulating levels of IL-6 tend to rise in old age, with obesity, and following stressful life events (Fried et al., 1998, Friedman et al., 2005, Kiecolt-Glaser et al., 2003). It has also been associated with chronic stress and vulnerability to depression (Bob et al., 2010, Lutgendorf et al., 1999, Miller et al., 2002). Although blood levels of IL-6 are often quantified in isolation, its biological actions are determined by two distinct membrane bound glycoproteins expressed on the surface of target cells: (1) a classical transmembrane IL-6 receptor (mIL-6r), and (2) a signal-transducing non-ligand binding subunit, gp130, which is activated by a complex formed by IL-6 and the soluble form of the IL-6 receptor (sIL-6r) (Kallen, 2002, Peters et al., 1998). Further, the sIL-6r has also been shown to moderate central actions of IL-6 within the brain (Schöbitz et al., 1995). Therefore, to more completely understand variation in IL-6 synthesis and responses across individuals, it is important to also quantify the soluble receptor, which was done in the following study.
IL-6 and sIL-6r are coded by different genes and controlled by distinct mechanisms of expression (Crichton et al., 1996, Jones et al., 2001, Lust et al., 1992). In addition, sIL-6r may be produced either by alternative mRNA splicing or by proteolytic cleavage and shedding from the surface of cells (Jones et al., 2001, Müllberg et al., 1993). Thus, it is of significance to determine the extent and similarity of the genetic constraints on IL-6 and its soluble receptor. It is known that the magnitude of the IL-6 response in inflammatory conditions can be affected by different single nucleotide polymorphisms (SNPs) associated with the IL-6 gene (Bruunsgaard et al., 2004, Sen et al., 2011, Walston et al., 2007). Accordingly, an examination of IL-6 responses to a strong inflammatory stimulus yielded high heritability estimates (de Craen et al., 2005). However, this general conclusion is often, and inappropriately, overgeneralized to all aspects of IL-6 synthesis and release. In the absence of inflammatory stimuli, SNPs have a weak or no association with baseline levels of IL-6 in the blood stream (Bagli et al., 2003, Bennermo et al., 2004, Brull et al., 2001, Burzotta et al., 2001, Herbert et al., 2006, Lieb et al., 2004, Nauck et al., 2002, Sen et al., 2011, Shah et al., 2013, Van Oijen et al., 2006, Wernstedt et al., 2004). On the other hand, SNPs affecting sIL-6r production appear to be more consistently associated with its levels in systemic circulation (Galicia et al., 2004, Rafiq et al., 2007, Sasayama et al., 2012, Van Dongen et al., 2014).
Serum IL-6 and sIL-6r are both influenced by body adiposity, although IL-6 appears to be affected to a greater extent than the soluble receptor (Crichton et al., 1996, Mehra et al., 2006, Mohamed-Ali et al., 1999, Pini et al., 2012). Accordingly, sIL-6r can serve as an independent biomarker for certain pathological conditions. It does not always react to the same physiological stimuli as IL-6; it is not always responsive to changes in IL-6 levels, and it does not play a role in all IL-6 pathways (Hurst et al., 2001, Kallen, 2002, Lamas et al., 2013, Mehra et al., 2006, Montero-Julian, 2001, Peters et al., 1998). Adipocytes, as well as the macrophages embedded in fat tissue, are known to be a major source of the IL-6 found in blood, especially in the non-inflammatory, healthy state (Coppack, 2007, Fried et al., 1998, Khaodhiar et al., 2004, Suganami and Ogawa, 2010, Weisberg et al., 2003, Wisse, 2004). The adipokine leptin also stimulates the release of IL-6 from leukocytes and macrophages (Agrawal et al., 2011, Behrendt et al., 2010, Kredel et al., 2013). Although these biological pathways often interact in a bidirectional and reciprocal manner, there is considerable evidence to suggest that adiposity exerts a far greater influence on inflammatory physiology (Miller, 2003, Welsh et al., 2010). Given the strong association between obesity and IL-6, it is likely that heritable factors influencing weight gain would also have a parallel effect on IL-6, a hypothesized linkage specifically tested in our analyses.
Likewise, the acute phase reactant, C-reactive protein (CRP) has been consistently associated with adiposity and inflammatory conditions, including cardiovascular disease (Carroll et al., 2009, Gupta et al., 2012, Saijo et al., 2004). Obesity can increase production of CRP by the liver as well as in adipose tissue (Anty et al., 2006, Calabro et al., 2005). Although previous genetic and heritability studies have demonstrated direct and independent effects on CRP levels(De Maat et al., 2004, Dehghan et al., 2011, Pankow et al., 2001, Wörns et al., 2006), there is evidence suggesting a causal relationship between the genetics of obesity and circulating CRP levels. SNPs associated with body mass index (BMI) can influence blood levels of CRP, while the converse has not been demonstrated (Holmes et al., 2014, Welsh et al., 2010). Thus, to further probe the unique nature and strength of the association between adiposity and IL-6, we also considered the relationship between adiposity and CRP. It is known that both CRP and IL-6 are involved in inflammatory response pathways, but CRP production appears to be more readily stimulated by IL-6, even within adipose tissue (Anty et al., 2006, Calabro et al., 2005, Heinrich et al., 1990, Volanakis, 2001). In addition, circulating levels of CRP and IL-6 are regulated independently by different genes (Shah et al., 2013). By analyzing these associations in identical and fraternal twins, we were able to directly compare and contrast the relative influence of adiposity on both CRP and IL-6, and to consider the reciprocal relationship between CRP and IL-6.
Previous twin studies have determined that adiposity, measured as BMI, is highly heritable (Hjelmborg et al., 2008, Schousboe et al., 2003, Segal et al., 2008). Associations between a genetic score, consisting of 14 SNPs related to BMI, and multiple cardiovascular and inflammatory traits including both IL-6 and CRP, were recently assessed (Holmes et al., 2014). However, it is still not known whether the heritable influence of obesity on IL-6 and its soluble receptor are coordinated, and whether CRP is affected in a similarly linked manner. We probed these relationships by comparing IL-6, sIL-6r and CRP levels in identical and fraternal adult twins, who also varied in adiposity and anthropometric concordance.
Twin studies take advantage of the different degree of genetic relatedness between monozygotic (MZ) and dizygotic (DZ) twins to estimate the relative contribution of genetic and environmental effects contributing to the phenotypic variance of a trait, as well as to the covariance between traits. Typical twin studies rely on the assumption that MZ twins share 100% and DZ twins share 50% of their genes, while both types of twins, regardless of zygosity, share a common rearing environment (Hall, 2003). In addition to the shared environment, it is also possible to discern effects of the non-shared environment, reflecting individual experiences not shared in common. Greater phenotypic similarity for MZ twins than found in DZ twins would be indicative of higher heritable contributions. On the other hand, when MZ and DZ twins present a similar phenotype, more variance attributable to life style and common environmental processes is assumed. Likewise, when monozygotic twins are discordant, it is usually attributed to unshared environmental influences (Boomsma et al., 2002, Christensen et al., 2001, Duffy et al., 1998, Hjelmborg et al., 2008).
We utilized bivariate ACE heritability models based on structural equation modeling (SEM) to estimate the proportion of IL-6, sIL-6r and CRP variation accounted for by genetic, shared and unshared environmental factors, as well as the heritable influences shared with adiposity. Our a priori hypothesis was that obesity would have a larger effect on IL-6 than on sIL-6r. Secondarily, the a posteriori hypothesis was tested: obesity would exert a, heritable influence on CRP, but one that was only moderately associated with the genetics of IL-6. We also compared IL-6 and sIL-6r intra-class correlations (ICCs) between co-twins in order to confirm the greater similarity between MZ co-twins than between DZ co-twins, indicative of heritable influences. To further test this hypothesis, we compared the ICC for MZ co-twins with that of genetically unrelated control participants matched to each MZ twin case. Assuming that environmental and lifestyle factors, BMI in particular, play a greater role in accounting for IL-6, we predicted that ICCs between the actual MZ co-twins would be no higher than for control adults who were closely matched on age, gender, BMI and a socio-economic index (SEI). By matching control subjects to the twin cases on these attributes, we simulated 4 of the major environmental and host factors known to influence IL-6 (Ershler and Keller, 2000, Friedman et al., 2005, Hjelmborg et al., 2008, Johnson and Krueger, 2005, O’Connor et al., 2007, Wisse, 2004). Hence, we could infer whether BMI and demographics (i.e., age, gender and SEI) accounted for the concordance between matched controls and co-twins. Given the likelihood of strong genetic constraints on sIL-6r, we anticipated small or negligible ICCs for sIL-6r with the unrelated, matched controls.
These analyses were possible because the recruitment strategy of a large survey of health and aging in the United States, Midlife Development in the United States (MIDUS), which included an over-sampling of twin siblings. It provided the unique opportunity to determine the heritability of circulating IL-6, sIL-6r and CRP, as well as the heritable contribution of obesity.
Section snippets
Participants
Participants were drawn from the MIDUS II Biomarker project, 2004–2009, a continuation of an earlier MIDUS 1 survey supported by the MacArthur Foundation in 1995–96. In addition to a representative probability sample, MIDUS 1 recruited a national sample of twin pairs, from which the current cohort was selected. Between 2003 and 2005, blood specimens were obtained, enabling the determination of cytokines and other biomarkers for each twin pair (Love et al., 2010). The twin sample was comprised
Twin sample
The MIDUS Biomarker project has been shown previously to be comparable to the larger MIDUS participants for most socio-demographic, health status, and health behavior indicators, but not race (Love et al., 2010). The latter was not an issue for the current analyses because the 2 African-American twin pairs were excluded. Table 1 presents the socio-demographic and clinical information, and mean cytokine values for MZ and DZ twins and the unrelated controls who were matched with the cases, as well
Discussion
By taking advantage of the fact that monozygotic twins are genetically more similar than dizygotic twins, our analyses demonstrated that the genetic constraints on IL-6 and its soluble receptor are clearly distinct. In addition, the association between BMI and IL-6 was more evident than for sIL-6r. This differential relationship with adiposity affected the heritability estimates. By fitting bivariate ACE models, we calculated variance and covariance components, parsing out genetic and
Conflict of interest
Amaral, W.Z. has no conflict of interest to declare.
Krueger, R.F. does not have any conflicts of interest.
Ryff, C.D. does not have any conflicts of interest.
Coe, C.L. does not have any conflicts of interest.
Acknowledgments
This research was supported by a grant from the National Institute on Aging (P01 AG020166). The original MIDUS study was supported by the MacArthur Foundation Research Network on Successful Midlife Development. Specimen collection was facilitated by the General Clinical Research Centers program (M01-RR023942 [Georgetown], M01-RR00865 [UCLA]), and at UW from the Clinical and Translational Science Award (CTSA) program of the National Center for Research Resources (1UL1RR025011). The contributions
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