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Heritability of plasma neopterin levels in the Old Order Amish
Journal of Neuroimmunology 307 (2017) 37–41
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Heritability of plasma neopterin levels in the Old Order Amish Uttam K. Raheja a,b, Dietmar Fuchs c, Christopher A. Lowry d,e,f,g,h, Sarah H. Stephens i,j, Mary A. Pavlovich i,j, Hira Mohyuddin a, Hassaan Yousufi a, Kathleen A. Ryan i,j, Jeff O'Connell i,j, Lisa A. Brenner d,e,f, Cecile Punzalan k, Andrew J. Hoisington f,l, Gursharon K. Nijjar a, Maureen Groer m, Alan R. Shuldiner i,j, Toni I. Pollin i,j, John W. Stiller a,n, Braxton D. Mitchell i,j,o, Teodor T. Postolache a,d,f,p,⁎ a
Mood and Anxiety Program, University of Maryland School of Medicine, 685 W. Baltimore Street, Suite# 930, Baltimore, MD 21201, USA Child and Adolescent Psychiatry Residency Program, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, 12 Executive Park Drive, Atlanta, GA 30329, USA c Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innrain 80, 6020, Innsbruck, Austria d Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC), Veterans Integrated Service Network (VISN) 19, 1055 Clermont St, Denver, CO 80220, USA e Department of Physical Medicine and Rehabilitation, University of Colorado Anschutz Medical Campus, 13001 E 17th Pl, Aurora, CO 80045, USA f Military and Veteran Microbiome Consortium for Research and Education (MVM-CoRE), 1055 Clermont St, Denver, CO 80220, USA g Department of Integrative Physiology and Center for Neuroscience, University of Colorado Boulder, Boulder, CO 80309, USA h Center for Neuroscience, University of Colorado Anschutz Medical Campus, 13001 E 17th Pl, Aurora, CO 80045, USA i Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland School of Medicine, 685 W. Baltimore Street, Baltimore, MD 21201, USA j Program for Personalized and Genomic Medicine, University of Maryland School of Medicine, 685 W. Baltimore Street, Baltimore, MD 21201, USA k U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition Office of Analytics and Outreach, Division of Public Health Informatics and Analytics, 5001 Campus Drive, College Park, MD 20740, USA l Department of Civil and Environmental Engineering, US Air Force Academy, Colorado Springs, CO 80840, USA m College of Nursing, University of South Florida, 4202 E Fowler Ave, Tampa, FL 33620, USA n Department of Behavioral Health, St. Elizabeth's Hospital, 1100 Alabama Ave SE, Washington, DC 20032, USA o Geriatrics Research and Education Clinical Center, Baltimore Veterans Administration Medical Center, Baltimore, MD 20201, USA p Mental Illness Research, Education and Clinical Center (MIRECC), Veterans Integrated Service Network (VISN) 5, VA Capitol Health Care Network, 209 West Fayette Street, Baltimore, MD 21201, USA b
a r t i c l e
i n f o
Article history: Received 31 October 2016 Received in revised form 7 February 2017 Accepted 21 February 2017 Available online xxxx Keywords: Neopterin Heritability Old Order Amish Biomarkers Psychiatric disorders
a b s t r a c t Background: We examined the heritability of neopterin, a biomarker for cell-mediated immunity and oxidative stress, and potentially for psychiatric disorders, in the Old Order Amish. Methods: Plasma neopterin levels were determined in 2015 Old Order Amish adults. Quantitative genetic procedures were used to estimate heritability of neopterin. Results: Heritability of log-neopterin was estimated at 0.07 after adjusting for age, gender, and household (p = 0.03). The shared household effect was 0.06 (p b 0.02). Conclusions: We found a low heritability of neopterin and small household effect, suggesting that non-household environmental factors are more important determinants of variance of neopterin levels in the Amish. © 2017 Published by Elsevier B.V.
1. Introduction Neopterin, a biomarker for cell-mediated immunity and oxidative stress as a result of immune system activation (Berdowska and Zwirska-Korczala, 2001), is produced by interactions among dendritic cells (Sucher et al., 2013; Wirleitner et al., 2002), T helper-1 lymphocytes, macrophages, and granulocytes by the guanosine triphosphate (GTP) pathway through GTP-cyclohydrolase I (GTP-CH-I) (Fig. 1). Activation of GTP-CH-I in macrophages by interferon-γ and, to a lesser extent, interferon-α, tumor necrosis factor, and endotoxins increases ⁎ Corresponding author at: 685 W. Baltimore Street, Suite #930, Baltimore, MD 21201, USA. E-mail address: [email protected] (T.T. Postolache).
http://dx.doi.org/10.1016/j.jneuroim.2017.02.016 0165-5728/© 2017 Published by Elsevier B.V.
production of neopterin precursors. Neopterin is also produced by microglial cells in the central nervous system (CNS) (Kuehne et al., 2013; Millner et al., 1998). It is detectable in various body fluids, including blood, cerebrospinal fluid (CSF), and urine and is useful clinically as an inflammatory marker (Zuo et al., 2016) in multiple organ systems [e.g., cardiovascular (Fuchs et al., 2009, Grammer et al., 2009, Weiss et al., 1994), respiratory (Fuchs et al., 1984), musculoskeletal (Altindag et al., 1998), nervous (Millner et al., 1998)] and in various neoplasms (Kronberger et al., 1995; Melichar et al., 2014; Murr et al., 1999; Sucher et al., 2010; Yildirim et al., 2008). Chronic, low-grade inflammation is thought to be a risk factor for several psychiatric disorders (Mondelli et al., 2015). Thus, neopterin could become a potentially useful immune/oxidative-stress biomarker for depression (Maes, 2011; Maes et al., 2012; Taymur et al., 2015),
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Fig. 1. Neopterin, an aromatic pteridine, is produced as a result of activation of GTP-cyclohydrolase I by interferon-γ from activated T-lymphocytes, and is a marker of cell-mediated immunity and oxidative stress. Household- and, particularly, non-household-related environmental factors influence pathogen and allergen exposures that enhance T helper type 1 (Th1)-specific immune responses, via pathogen/allergen interactions with dendritic cells (DCs), antigen-presenting cells that drive differentiation of naïve T cells to Th1 cells that secrete interferon-γ. Conversely, exposure to “Old Friends”, including non-pathogenic, immunoregulatory environmental microorganisms, limit inappropriate inflammation through interactions with DCs that drive differentiation of naïve T cells to regulatory T cells (Treg) that secrete anti-inflammatory cytokines, including interleukin 10 (IL-10) and transforming growth factor beta (TGF-β). Together, these environmental factors control Th1 cell secretion of interferon-γ that in turn activates GTP-cyclohydrolase I in macrophages, leading to enzymatic conversion of GTP to neopterin. Genetic factors, not shown, may act at any level of the host immune response to influence neopterin secretion. Abbreviations: DC, dendritic cell; GTP, guanosine-5′-trisphosphate; HOCl, hypochlorite; IL-10, interleukin 10; Th1, T helper type 1 cells; TGF-β, transforming growth factor beta; Treg, regulatory T cells.
postpartum depression (Krause et al., 2014), autism spectrum disorder (Harrison and Pheasant, 1995; Sweeten et al., 2003; Zhao et al., 2015), attention-deficit/hyperactivity disorder (Ceylan et al., 2014), and schizophrenia (Bechter et al., 2010; Chittiprol et al., 2010; Kuehne et al., 2013). Additionally, neopterin has been useful as a biomarker for treatment response and course of multiple sclerosis and HIV/AIDS (Bagnato et al., 2003; Baier-Bitterlich et al., 1996; Fahey et al., 1998; Giovannoni et al., 1997; Hoffmann et al., 2003; Mildvan et al., 2005). The levels of light subunit of neurofilament protein (NF-L) in the CNS, a marker of ongoing axonal injury, correlate with neopterin levels in neuro-asymptomatic HIV patients (Jessen Krut et al., 2014). Neopterin is elevated in acute ischemic stroke (Lin et al., 2012), consistent with its role as biomarker of brain injury. Neopterin production is constitutive to the brain and increases during and is protective against oxidative stress (Ghisoni and Latini, 2015; Ghisoni et al., 2015). Rather than just being a metabolic byproduct with relevance as a biomarker, neopterin may have regulatory roles in inflammation and oxidative stress, enhancing memory and hippocampal long-term potentiation, as shown by intracerebroventricular injection in rodents (Ghisoni et al., 2016). There are indications of allelic variations that impact the enzymatic steps involved in neopterin synthesis. Segawa disease, a hereditary progressive dystonia, results from a GCH1 (GTP-CH-I gene) mutation, and is characterized by low CSF neopterin (Fink et al., 1988). Besides additive genetic effects, household and non-household environmental effects can also contribute to neopterin variance. Household effects refer to shared environmental effects by virtue of living in the same household and having similar environmental exposures and built environment, e.g., the household clustering of Staphylococcus aureus strains in the Old Order Amish (Roghmann et al., 2014). Non-household environmental effects are independent of living in the same household. In line with the “hygiene hypothesis”, research (Stein et al., 2016) has identified environmental factors likely responsible for lower rates of asthma and allergic sensitization in the Old Order Amish as compared to Hutterites. Community infections, allergies, dietary factors, weight, nutrition rich in antioxidants, and high-fat meals can affect cell-mediated immune responses and neopterin levels (Alipour et al., 2007; Brodin et al., 2015; Cheng et al., 2010; Ledochowski et al., 1999; Myles, 2014; Nappo et al., 2002; Strasser et al., 2016; Tilg and Moschen, 2015). There are no studies, to our knowledge, of the heritability of neopterin. In this report, we estimate the heritability of plasma
neopterin levels in the Old Order Amish, a rural population ideal for studying heritability because of their relative lifestyle homogeneity and limited alcohol, tobacco, and substance use disorders that reduce the regular major environmental sources of variation. 2. Methods 2.1. Recruitment The Amish Wellness Study was initiated in 2010 with the purpose of providing wellness screening for cardiometabolic health to members of the Old Order Amish community in Lancaster County, Pennsylvania, USA and performing genetic research related to cardiometabolic health. All participants gave informed consent after a nurse and Amish liaison read the consent to the participant and ascertained understanding. All participants were individuals 18 years of age or older. At the enrollment visit, medical and family histories were obtained and a visit for fasting blood draw was scheduled. Fasting blood draw was obtained by venipuncture either in the mobile clinic, the participant's home, or at the Amish Research Clinic. Whole blood, collected in heparinized tubes, was centrifuged at 5 °C at 3330–3350 rpm for 10 min, and plasma was separated and stored at −80 °C until the time of assay. 2.2. Neopterin Neopterin concentrations were determined using enzyme-linked immunosorbent assay (ELISA) (BRAHMS GmbH, Hennigsdorf, Germany), following the manufacturer's instructions (Groer et al., 2011). Sensitivity of the assay was 2-nmol/L neopterin. 2.3. Statistical analyses Heritability of neopterin was estimated with adjustment for age, gender, and household (Raheja et al., 2013). Modeling the phenotypic covariance between any two individuals in the pedigree as a function of their degree of biological association was used to estimate heritability, defined as the proportion of the total trait variance attributable to the additive effect of genes. Heritability was estimated using the maximum likelihood method with the sequential oligogenic linkage analysis routines (SOLAR) software package (Texas Biomedical Research Institute,
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San Antonio, TX, USA) (Almasy and Blangero, 2010). The household effect represented non-genetic (i.e., environmental) effects shared by individuals living in the same household. Both the genetic and household effects were modeled as random effects in a linear model captured by the kinship matrix (genetic effects) and household matrix (household effects), respectively. The household effect was parameterized by assigning a value of 1 if the relative pair shared the same household and a value of 0 if they did not, as a random effect. 3. Results 3.1. Demographic characteristics The sample (N = 2015) included for this analysis consisted of 850 men and 1165 women. Average age (± S.D.) was 44.0 (± 17.0) years (range 18–90 years). Mean neopterin level was 6.2 (± 3.0) nmol/L with a range of 3.2 to 48.9 nmol/L. The demographic characteristics of the sample are presented in Table 1. 3.2. Heritability of neopterin As neopterin values were not normally distributed, a natural logarithmic transformation was applied. Heritability of log-neopterin was estimated at 0.08 without adjustment for covariates (p = 0.01) and 0.07 when adjusted for age, gender, and household (p = 0.03). The shared household effect was estimated at 0.06 (p b 0.02). 4. Discussion Heritability of immune markers is important to understand their usefulness as potential trait versus state indicators in neuropsychiatric disorders and to estimate immune factors involved in predisposition and symptom perpetuation. Environmental effects may point towards potentially modifiable factors leading to more effective prevention methods. We found a low heritability of neopterin in the Old Order Amish, with genetic factors accounting for only 7% of the neopterin variance. This low heritability may be related to the characteristics of the biomarker or of the studied population. Neopterin levels, a reflection of cellular immunity and oxidative stress, may be more reactive to the environment (e.g., bacterial, viral, parasitic infections; allergens; stress). Alternatively, as a result of particular exposure to allergens, microbes,
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and microbial products in agricultural dust (Stein et al., 2016), nonhousehold-related environmental immune triggers or modulators may override genetic effects. Our study suggests that the majority of variation in neopterin levels in the Amish (up to 87%) is accounted for by non-shared environmental effects, with shared household effects accounting for only 6% of the variance. This result is congruent with that of Brodin and colleagues who evaluated over 200 immune parameters including cells, serum cytokines, and serum proteins and found that most of the variance in these factors was accounted for by non-heritable effects, and that the heritable factors were enmeshed by non-heritable ones in an interconnected network, thereby emphasizing the reactivity of the healthy immune system and greater influence of non-heritable factors (e.g., pathogens) with increasing age (Brodin et al., 2015). It is possible that variation in household factors such as meal preparation and food (e.g. probiotic, prebiotic) may be minimal due to the relative cultural homogeneity of the Amish. Non-household environmental factors such as occupation and school participation might be more important for exposure to pathogens and allergens and contribute to a low household effect of neopterin. One factor contributing to environmental effects on neopterin concentrations may be exposure to environmental organisms that are known to enhance immunoregulation and suppress inappropriate inflammation (Rook et al., 2015). These exposures may occur outdoors, with variability in individual exposures related to occupation, or indoors. The microbiome of the built environment, a potentially important immune modulator (Hoisington et al., 2015; Lowry et al., 2016), is a factor that could be significantly less variable and more similar to the outdoor environment in Amish homes that do not have traditional heating, ventilation, and air conditioning systems, relying instead on opening and closing windows for temperature control and ventilation. Additionally, these homes lack energy efficient building materials present in non-Amish homes that would result in higher air exchange rates, essentially normalizing the indoor and outdoor microbiome. The use of mechanical ventilation systems has been shown to alter the indoor microbiome from the outdoor state when compared to a room with open windows (Kembel et al., 2014; Meadow et al., 2014). Consistent exposure to the outdoor microbiome has been postulated as a method of naturally developing and sustaining immunoregulatory processes (Rook et al., 2014). A recent study has shown indoor dust recovered from the Northern Indiana Amish community was more protective against airway hyper-responsiveness in
Table 1 Demographic and clinical characteristics of the sample.
Gender Age Occupation • Housekeeping, housewife • Farming (dairy, plant, tobacco), farmer's wife, horse keeping, field worker • Shopkeeping, marketing, gardening, waitress, cook, cleaning, livestock auction, caring, cashier • Clerk work, needlework, quilting, teaching, order buyer at hay sales, craft making, municipal waste management, shoe repair • Carpenter, smith, woodwork, mason, mechanic, welder, painting, sawmill, fencing, house framer • Construction, contractor, mobile home builder • Retired • Other Cigarette smoking BMI Medical conditions • Hypertension • Diabetes • Coronary disease Neopterin ⁎ p-Value based on log-transformed variable.
Total
Male
Female
p
2015 44.0 ± 17.0
850 (42.2%) 45.5 ± 16.5
1165 (57.8%) 43.0 ± 17.2
NA 0.001
829/2000 (41.5%) 368/2000 (18.4%) 289/2000 (14.5%)
5/846 (0.6%) 298/846 (35.2%) 95/846 (11.2%)
824/1154 (71.4%) 70/1154 (6.1%) 194/1154 (16.8%)
b0.0001 b0.0001 0.0005
63/2000 (3.2%)
23/846 (2.7%)
40/1154 (3.5%)
0.34
340/2000 (17.0%)
331/846 (39.1%)
9/1154 (0.8%)
b0.0001
25/2000 (1.3%) 26/2000 (1.3%) 60/2000 (3.0%) 98/1982 (4.9%) 26.6 ± 5.0
25/846 (3.0%) 22/846 (2.6%) 47/846 (5.6%) 98/820 (12.0%) 26.0 ± 4.1
0/1154 (0.0%) 4/1154 (0.4%) 13/1154 (1.1%) 0/1162 (0.0%) 27.0 ± 5.5
b0.0001 b0.0001 b0.0001 b0.0001 b0.0001
343/2009 (17.1%) 92/2009 (4.6%) 80/2005 (4.0%) 6.2 ± 3.0
131/849 (15.4%) 34/846 (4.0%) 52/846 (6.2%) 6.1 ± 2.8
212/1160 (18.3%) 58/1163 (5.0%) 28/1159 (2.4%) 6.3 ± 3.1
0.09 0.31 b0.0001 0.15⁎
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mice compared to indoor dust in a South Dakota Hutterite farming community that utilizes Western technology (Stein et al., 2016). Although speculative in nature, this line of evidence is consistent with less variation in the microbiome of the built environment in the Old Order Amish and likely higher exposure to the outdoor or occupation-related microbiome. These ideas could be tested by future research. In conclusion, variance in plasma neopterin levels in the Amish is likely reflective more of non-household environmental effects rather than genetic or household-related environmental factors. Given its low heritability, neopterin may be a particularly appropriate marker for longitudinal studies investigating immune triggers and modulators in both naturalistic and interventional studies. Funding source Research reported in this publication was supported by the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health via the Mid-Atlantic Nutrition Obesity Research Center Pilot NORC grant preliminary/developmental offspring (TTP) of the parent grant P30 DK072488 (BDM). Additional support was provided, in part, by The American Foundation for Suicide Prevention, Distinguished Investigator Award, (TTP), the Rocky Mountain MIRECC for Suicide Prevention, Denver, CO, USA, the Military and Veteran Microbiome Consortium for Research and Education (LAB, CAL, AJH, TTP) and the University of Maryland, College Park, Joint Institute for Food Safety and Applied Nutrition and the U.S. Food and Drug Administration for their support through the cooperative agreement FDU.001418. The findings and conclusions in this study are those of the authors and do not necessarily represent the official positions of the NIH, VA, FDA, or the American Foundation for Suicide Prevention. Presentations The findings of this study were presented at the “Advances in Toxoplasma gondii Research” conference at the FDA White Oak Campus, Silver Springs, MD, USA on February 17, 2016, and at the Society of Biological Psychiatry annual meeting on May 13, 2016, in Atlanta, GA, USA, and at the American Psychiatric Association annual meeting on May 16, 2016, in Atlanta, GA, USA. Conflict of interest The authors declare they have no conflicts of interest related to this study. Acknowledgements We would like to thank the entire personnel of the University of Maryland School of Medicine Amish Research Clinic, Lancaster, PA, USA, in particular the Amish liaisons, including Naomi Esh and Hanna King, and the nurses of the Amish Research Clinic, including Donna Trubiano, Yvonne Rohrer, Theresa Roomet, Mary Morrissey, Nancy Weitzel and Susan Shaub, and finally, to all members of the Old Order Amish community in Lancaster who took part in this study. We are grateful to Philip H. Siebler for preparation of the graphical abstract. We also thank Aline Dagdag, Winny Mwaura, Afshan Qureshi, and Sunghee Flores for their substantial administrative support during this project. References Alipour, A., Elte, J.W., Van Zaanen, H.C., Rietveld, A.P., Cabezas, M.C., 2007. Postprandial inflammation and endothelial dysfuction. Biochem. Soc. Trans. 35, 466–469. Almasy, L., Blangero, J., 2010. Variance component methods for analysis of complex phenotypes. Cold Spring Harb. Protoc. 2010. http://dx.doi.org/10.1101/pdb.top77. Altindag, Z.Z., Sahin, G., Inanici, F., Hascelik, Z., 1998. Urinary neopterin excretion and dihydropteridine reductase activity in rheumatoid arthritis. Rheumatol. Int. 18, 107–111.
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Rook, G.A., Raison, C.L., Lowry, C.A., 2014. Microbial ‘old friends’, immunoregulation and socioeconomic status. Clin. Exp. Immunol. 177, 1–12. Rook, G.A., Lowry, C.A., Raison, C.L., 2015. Hygiene and other early childhood influences on the subsequent function of the immune system. Brain Res. 1617, 47–62. Stein, M.M., Hrusch, C.L., Gozdz, J., Igartua, C., Pivniouk, V., Murray, S.E., Ledford, J.G., Marques Dos Santos, M., Anderson, R.L., Metwali, N., Neilson, J.W., Maier, R.M., Gilbert, J.A., Holbreich, M., Thorne, P.S., Martinez, F.D., Von Mutius, E., Vercelli, D., Ober, C., Sperling, A.I., 2016. Innate immunity and asthma risk in Amish and Hutterite farm children. N. Engl. J. Med. 375, 411–421. Strasser, B., Gostner, J.M., Fuchs, D., 2016. Mood, food, and cognition: role of tryptophan and serotonin. Curr. Opin. Clin. Nutr. Metab. Care 19, 55–61. Sucher, R., Schroecksnadel, K., Weiss, G., Margreiter, R., Fuchs, D., Brandacher, G., 2010. Neopterin, a prognostic marker in human malignancies. Cancer Lett. 287, 13–22. Sucher, R., Kurz, K., Margreiter, R., Fuchs, D., Brandacher, G., 2013. Antiviral activity of interferon-γ involved in impaired immune function in infectious diseases. Pteridines 24, 149–164. Sweeten, T.L., Posey, D.J., Mcdougle, C.J., 2003. High blood monocyte counts and neopterin levels in children with autistic disorder. Am. J. Psychiatry 160, 1691–1693. Taymur, I., Ozdel, K., Ozen, N.E., Gungor, B.B., Atmaca, M., 2015. Urinary neopterine levels in patients with major depressive disorder: alterations after treatment with paroxetine and comparison with healthy controls. Psychiatr. Danub. 27, 25–30. Tilg, H., Moschen, A.R., 2015. Food, immunity, and the microbiome. Gastroenterology 148, 1107–1119. Weiss, G., Willeit, J., Kiechl, S., Fuchs, D., Jarosch, E., Oberhollenzer, F., Reibnegger, G., Tilz, G.P., Gerstenbrand, F., Wachter, H., 1994. Increased concentrations of neopterin in carotid atherosclerosis. Atherosclerosis 106, 263–271. Wirleitner, B., Reider, D., Ebner, S., Bock, G., Widner, B., Jaeger, M., Schennach, H., Romani, N., Fuchs, D., 2002. Monocyte-derived dendritic cells release neopterin. J. Leukoc. Biol. 72, 1148–1153. Yildirim, Y., Gunel, N., Coskun, U., Pasaoglu, H., Aslan, S., Cetin, A., 2008. Serum neopterin levels in patients with breast cancer. Med. Oncol. 25, 403–407. Zhao, H.X., Yin, S.S., Fan, J.G., 2015. High plasma neopterin levels in Chinese children with autism spectrum disorders. Int. J. Dev. Neurosci. 41, 92–97. Zuo, H., Ueland, P.M., Ulvik, A., Eussen, S.J., Vollset, S.E., Nygard, O., Midttun, O., Theofylaktopoulou, D., Meyer, K., Tell, G.S., 2016. Plasma biomarkers of inflammation, the kynurenine pathway, and risks of all-cause, cancer, and cardiovascular disease mortality: the Hordaland health study. Am. J. Epidemiol. 183, 249–258.
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Heritability of plasma neopterin levels in the Old Order Amish Uttam K. Raheja a,b, Dietmar Fuchs c, Christopher A. Lowry d,e,f,g,h, Sarah H. Stephens i,j, Mary A. Pavlovich i,j, Hira Mohyuddin a, Hassaan Yousufi a, Kathleen A. Ryan i,j, Jeff O'Connell i,j, Lisa A. Brenner d,e,f, Cecile Punzalan k, Andrew J. Hoisington f,l, Gursharon K. Nijjar a, Maureen Groer m, Alan R. Shuldiner i,j, Toni I. Pollin i,j, John W. Stiller a,n, Braxton D. Mitchell i,j,o, Teodor T. Postolache a,d,f,p,⁎ a
Mood and Anxiety Program, University of Maryland School of Medicine, 685 W. Baltimore Street, Suite# 930, Baltimore, MD 21201, USA Child and Adolescent Psychiatry Residency Program, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, 12 Executive Park Drive, Atlanta, GA 30329, USA c Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innrain 80, 6020, Innsbruck, Austria d Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC), Veterans Integrated Service Network (VISN) 19, 1055 Clermont St, Denver, CO 80220, USA e Department of Physical Medicine and Rehabilitation, University of Colorado Anschutz Medical Campus, 13001 E 17th Pl, Aurora, CO 80045, USA f Military and Veteran Microbiome Consortium for Research and Education (MVM-CoRE), 1055 Clermont St, Denver, CO 80220, USA g Department of Integrative Physiology and Center for Neuroscience, University of Colorado Boulder, Boulder, CO 80309, USA h Center for Neuroscience, University of Colorado Anschutz Medical Campus, 13001 E 17th Pl, Aurora, CO 80045, USA i Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland School of Medicine, 685 W. Baltimore Street, Baltimore, MD 21201, USA j Program for Personalized and Genomic Medicine, University of Maryland School of Medicine, 685 W. Baltimore Street, Baltimore, MD 21201, USA k U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition Office of Analytics and Outreach, Division of Public Health Informatics and Analytics, 5001 Campus Drive, College Park, MD 20740, USA l Department of Civil and Environmental Engineering, US Air Force Academy, Colorado Springs, CO 80840, USA m College of Nursing, University of South Florida, 4202 E Fowler Ave, Tampa, FL 33620, USA n Department of Behavioral Health, St. Elizabeth's Hospital, 1100 Alabama Ave SE, Washington, DC 20032, USA o Geriatrics Research and Education Clinical Center, Baltimore Veterans Administration Medical Center, Baltimore, MD 20201, USA p Mental Illness Research, Education and Clinical Center (MIRECC), Veterans Integrated Service Network (VISN) 5, VA Capitol Health Care Network, 209 West Fayette Street, Baltimore, MD 21201, USA b
a r t i c l e
i n f o
Article history: Received 31 October 2016 Received in revised form 7 February 2017 Accepted 21 February 2017 Available online xxxx Keywords: Neopterin Heritability Old Order Amish Biomarkers Psychiatric disorders
a b s t r a c t Background: We examined the heritability of neopterin, a biomarker for cell-mediated immunity and oxidative stress, and potentially for psychiatric disorders, in the Old Order Amish. Methods: Plasma neopterin levels were determined in 2015 Old Order Amish adults. Quantitative genetic procedures were used to estimate heritability of neopterin. Results: Heritability of log-neopterin was estimated at 0.07 after adjusting for age, gender, and household (p = 0.03). The shared household effect was 0.06 (p b 0.02). Conclusions: We found a low heritability of neopterin and small household effect, suggesting that non-household environmental factors are more important determinants of variance of neopterin levels in the Amish. © 2017 Published by Elsevier B.V.
1. Introduction Neopterin, a biomarker for cell-mediated immunity and oxidative stress as a result of immune system activation (Berdowska and Zwirska-Korczala, 2001), is produced by interactions among dendritic cells (Sucher et al., 2013; Wirleitner et al., 2002), T helper-1 lymphocytes, macrophages, and granulocytes by the guanosine triphosphate (GTP) pathway through GTP-cyclohydrolase I (GTP-CH-I) (Fig. 1). Activation of GTP-CH-I in macrophages by interferon-γ and, to a lesser extent, interferon-α, tumor necrosis factor, and endotoxins increases ⁎ Corresponding author at: 685 W. Baltimore Street, Suite #930, Baltimore, MD 21201, USA. E-mail address: [email protected] (T.T. Postolache).
http://dx.doi.org/10.1016/j.jneuroim.2017.02.016 0165-5728/© 2017 Published by Elsevier B.V.
production of neopterin precursors. Neopterin is also produced by microglial cells in the central nervous system (CNS) (Kuehne et al., 2013; Millner et al., 1998). It is detectable in various body fluids, including blood, cerebrospinal fluid (CSF), and urine and is useful clinically as an inflammatory marker (Zuo et al., 2016) in multiple organ systems [e.g., cardiovascular (Fuchs et al., 2009, Grammer et al., 2009, Weiss et al., 1994), respiratory (Fuchs et al., 1984), musculoskeletal (Altindag et al., 1998), nervous (Millner et al., 1998)] and in various neoplasms (Kronberger et al., 1995; Melichar et al., 2014; Murr et al., 1999; Sucher et al., 2010; Yildirim et al., 2008). Chronic, low-grade inflammation is thought to be a risk factor for several psychiatric disorders (Mondelli et al., 2015). Thus, neopterin could become a potentially useful immune/oxidative-stress biomarker for depression (Maes, 2011; Maes et al., 2012; Taymur et al., 2015),
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Fig. 1. Neopterin, an aromatic pteridine, is produced as a result of activation of GTP-cyclohydrolase I by interferon-γ from activated T-lymphocytes, and is a marker of cell-mediated immunity and oxidative stress. Household- and, particularly, non-household-related environmental factors influence pathogen and allergen exposures that enhance T helper type 1 (Th1)-specific immune responses, via pathogen/allergen interactions with dendritic cells (DCs), antigen-presenting cells that drive differentiation of naïve T cells to Th1 cells that secrete interferon-γ. Conversely, exposure to “Old Friends”, including non-pathogenic, immunoregulatory environmental microorganisms, limit inappropriate inflammation through interactions with DCs that drive differentiation of naïve T cells to regulatory T cells (Treg) that secrete anti-inflammatory cytokines, including interleukin 10 (IL-10) and transforming growth factor beta (TGF-β). Together, these environmental factors control Th1 cell secretion of interferon-γ that in turn activates GTP-cyclohydrolase I in macrophages, leading to enzymatic conversion of GTP to neopterin. Genetic factors, not shown, may act at any level of the host immune response to influence neopterin secretion. Abbreviations: DC, dendritic cell; GTP, guanosine-5′-trisphosphate; HOCl, hypochlorite; IL-10, interleukin 10; Th1, T helper type 1 cells; TGF-β, transforming growth factor beta; Treg, regulatory T cells.
postpartum depression (Krause et al., 2014), autism spectrum disorder (Harrison and Pheasant, 1995; Sweeten et al., 2003; Zhao et al., 2015), attention-deficit/hyperactivity disorder (Ceylan et al., 2014), and schizophrenia (Bechter et al., 2010; Chittiprol et al., 2010; Kuehne et al., 2013). Additionally, neopterin has been useful as a biomarker for treatment response and course of multiple sclerosis and HIV/AIDS (Bagnato et al., 2003; Baier-Bitterlich et al., 1996; Fahey et al., 1998; Giovannoni et al., 1997; Hoffmann et al., 2003; Mildvan et al., 2005). The levels of light subunit of neurofilament protein (NF-L) in the CNS, a marker of ongoing axonal injury, correlate with neopterin levels in neuro-asymptomatic HIV patients (Jessen Krut et al., 2014). Neopterin is elevated in acute ischemic stroke (Lin et al., 2012), consistent with its role as biomarker of brain injury. Neopterin production is constitutive to the brain and increases during and is protective against oxidative stress (Ghisoni and Latini, 2015; Ghisoni et al., 2015). Rather than just being a metabolic byproduct with relevance as a biomarker, neopterin may have regulatory roles in inflammation and oxidative stress, enhancing memory and hippocampal long-term potentiation, as shown by intracerebroventricular injection in rodents (Ghisoni et al., 2016). There are indications of allelic variations that impact the enzymatic steps involved in neopterin synthesis. Segawa disease, a hereditary progressive dystonia, results from a GCH1 (GTP-CH-I gene) mutation, and is characterized by low CSF neopterin (Fink et al., 1988). Besides additive genetic effects, household and non-household environmental effects can also contribute to neopterin variance. Household effects refer to shared environmental effects by virtue of living in the same household and having similar environmental exposures and built environment, e.g., the household clustering of Staphylococcus aureus strains in the Old Order Amish (Roghmann et al., 2014). Non-household environmental effects are independent of living in the same household. In line with the “hygiene hypothesis”, research (Stein et al., 2016) has identified environmental factors likely responsible for lower rates of asthma and allergic sensitization in the Old Order Amish as compared to Hutterites. Community infections, allergies, dietary factors, weight, nutrition rich in antioxidants, and high-fat meals can affect cell-mediated immune responses and neopterin levels (Alipour et al., 2007; Brodin et al., 2015; Cheng et al., 2010; Ledochowski et al., 1999; Myles, 2014; Nappo et al., 2002; Strasser et al., 2016; Tilg and Moschen, 2015). There are no studies, to our knowledge, of the heritability of neopterin. In this report, we estimate the heritability of plasma
neopterin levels in the Old Order Amish, a rural population ideal for studying heritability because of their relative lifestyle homogeneity and limited alcohol, tobacco, and substance use disorders that reduce the regular major environmental sources of variation. 2. Methods 2.1. Recruitment The Amish Wellness Study was initiated in 2010 with the purpose of providing wellness screening for cardiometabolic health to members of the Old Order Amish community in Lancaster County, Pennsylvania, USA and performing genetic research related to cardiometabolic health. All participants gave informed consent after a nurse and Amish liaison read the consent to the participant and ascertained understanding. All participants were individuals 18 years of age or older. At the enrollment visit, medical and family histories were obtained and a visit for fasting blood draw was scheduled. Fasting blood draw was obtained by venipuncture either in the mobile clinic, the participant's home, or at the Amish Research Clinic. Whole blood, collected in heparinized tubes, was centrifuged at 5 °C at 3330–3350 rpm for 10 min, and plasma was separated and stored at −80 °C until the time of assay. 2.2. Neopterin Neopterin concentrations were determined using enzyme-linked immunosorbent assay (ELISA) (BRAHMS GmbH, Hennigsdorf, Germany), following the manufacturer's instructions (Groer et al., 2011). Sensitivity of the assay was 2-nmol/L neopterin. 2.3. Statistical analyses Heritability of neopterin was estimated with adjustment for age, gender, and household (Raheja et al., 2013). Modeling the phenotypic covariance between any two individuals in the pedigree as a function of their degree of biological association was used to estimate heritability, defined as the proportion of the total trait variance attributable to the additive effect of genes. Heritability was estimated using the maximum likelihood method with the sequential oligogenic linkage analysis routines (SOLAR) software package (Texas Biomedical Research Institute,
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San Antonio, TX, USA) (Almasy and Blangero, 2010). The household effect represented non-genetic (i.e., environmental) effects shared by individuals living in the same household. Both the genetic and household effects were modeled as random effects in a linear model captured by the kinship matrix (genetic effects) and household matrix (household effects), respectively. The household effect was parameterized by assigning a value of 1 if the relative pair shared the same household and a value of 0 if they did not, as a random effect. 3. Results 3.1. Demographic characteristics The sample (N = 2015) included for this analysis consisted of 850 men and 1165 women. Average age (± S.D.) was 44.0 (± 17.0) years (range 18–90 years). Mean neopterin level was 6.2 (± 3.0) nmol/L with a range of 3.2 to 48.9 nmol/L. The demographic characteristics of the sample are presented in Table 1. 3.2. Heritability of neopterin As neopterin values were not normally distributed, a natural logarithmic transformation was applied. Heritability of log-neopterin was estimated at 0.08 without adjustment for covariates (p = 0.01) and 0.07 when adjusted for age, gender, and household (p = 0.03). The shared household effect was estimated at 0.06 (p b 0.02). 4. Discussion Heritability of immune markers is important to understand their usefulness as potential trait versus state indicators in neuropsychiatric disorders and to estimate immune factors involved in predisposition and symptom perpetuation. Environmental effects may point towards potentially modifiable factors leading to more effective prevention methods. We found a low heritability of neopterin in the Old Order Amish, with genetic factors accounting for only 7% of the neopterin variance. This low heritability may be related to the characteristics of the biomarker or of the studied population. Neopterin levels, a reflection of cellular immunity and oxidative stress, may be more reactive to the environment (e.g., bacterial, viral, parasitic infections; allergens; stress). Alternatively, as a result of particular exposure to allergens, microbes,
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and microbial products in agricultural dust (Stein et al., 2016), nonhousehold-related environmental immune triggers or modulators may override genetic effects. Our study suggests that the majority of variation in neopterin levels in the Amish (up to 87%) is accounted for by non-shared environmental effects, with shared household effects accounting for only 6% of the variance. This result is congruent with that of Brodin and colleagues who evaluated over 200 immune parameters including cells, serum cytokines, and serum proteins and found that most of the variance in these factors was accounted for by non-heritable effects, and that the heritable factors were enmeshed by non-heritable ones in an interconnected network, thereby emphasizing the reactivity of the healthy immune system and greater influence of non-heritable factors (e.g., pathogens) with increasing age (Brodin et al., 2015). It is possible that variation in household factors such as meal preparation and food (e.g. probiotic, prebiotic) may be minimal due to the relative cultural homogeneity of the Amish. Non-household environmental factors such as occupation and school participation might be more important for exposure to pathogens and allergens and contribute to a low household effect of neopterin. One factor contributing to environmental effects on neopterin concentrations may be exposure to environmental organisms that are known to enhance immunoregulation and suppress inappropriate inflammation (Rook et al., 2015). These exposures may occur outdoors, with variability in individual exposures related to occupation, or indoors. The microbiome of the built environment, a potentially important immune modulator (Hoisington et al., 2015; Lowry et al., 2016), is a factor that could be significantly less variable and more similar to the outdoor environment in Amish homes that do not have traditional heating, ventilation, and air conditioning systems, relying instead on opening and closing windows for temperature control and ventilation. Additionally, these homes lack energy efficient building materials present in non-Amish homes that would result in higher air exchange rates, essentially normalizing the indoor and outdoor microbiome. The use of mechanical ventilation systems has been shown to alter the indoor microbiome from the outdoor state when compared to a room with open windows (Kembel et al., 2014; Meadow et al., 2014). Consistent exposure to the outdoor microbiome has been postulated as a method of naturally developing and sustaining immunoregulatory processes (Rook et al., 2014). A recent study has shown indoor dust recovered from the Northern Indiana Amish community was more protective against airway hyper-responsiveness in
Table 1 Demographic and clinical characteristics of the sample.
Gender Age Occupation • Housekeeping, housewife • Farming (dairy, plant, tobacco), farmer's wife, horse keeping, field worker • Shopkeeping, marketing, gardening, waitress, cook, cleaning, livestock auction, caring, cashier • Clerk work, needlework, quilting, teaching, order buyer at hay sales, craft making, municipal waste management, shoe repair • Carpenter, smith, woodwork, mason, mechanic, welder, painting, sawmill, fencing, house framer • Construction, contractor, mobile home builder • Retired • Other Cigarette smoking BMI Medical conditions • Hypertension • Diabetes • Coronary disease Neopterin ⁎ p-Value based on log-transformed variable.
Total
Male
Female
p
2015 44.0 ± 17.0
850 (42.2%) 45.5 ± 16.5
1165 (57.8%) 43.0 ± 17.2
NA 0.001
829/2000 (41.5%) 368/2000 (18.4%) 289/2000 (14.5%)
5/846 (0.6%) 298/846 (35.2%) 95/846 (11.2%)
824/1154 (71.4%) 70/1154 (6.1%) 194/1154 (16.8%)
b0.0001 b0.0001 0.0005
63/2000 (3.2%)
23/846 (2.7%)
40/1154 (3.5%)
0.34
340/2000 (17.0%)
331/846 (39.1%)
9/1154 (0.8%)
b0.0001
25/2000 (1.3%) 26/2000 (1.3%) 60/2000 (3.0%) 98/1982 (4.9%) 26.6 ± 5.0
25/846 (3.0%) 22/846 (2.6%) 47/846 (5.6%) 98/820 (12.0%) 26.0 ± 4.1
0/1154 (0.0%) 4/1154 (0.4%) 13/1154 (1.1%) 0/1162 (0.0%) 27.0 ± 5.5
b0.0001 b0.0001 b0.0001 b0.0001 b0.0001
343/2009 (17.1%) 92/2009 (4.6%) 80/2005 (4.0%) 6.2 ± 3.0
131/849 (15.4%) 34/846 (4.0%) 52/846 (6.2%) 6.1 ± 2.8
212/1160 (18.3%) 58/1163 (5.0%) 28/1159 (2.4%) 6.3 ± 3.1
0.09 0.31 b0.0001 0.15⁎
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mice compared to indoor dust in a South Dakota Hutterite farming community that utilizes Western technology (Stein et al., 2016). Although speculative in nature, this line of evidence is consistent with less variation in the microbiome of the built environment in the Old Order Amish and likely higher exposure to the outdoor or occupation-related microbiome. These ideas could be tested by future research. In conclusion, variance in plasma neopterin levels in the Amish is likely reflective more of non-household environmental effects rather than genetic or household-related environmental factors. Given its low heritability, neopterin may be a particularly appropriate marker for longitudinal studies investigating immune triggers and modulators in both naturalistic and interventional studies. Funding source Research reported in this publication was supported by the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health via the Mid-Atlantic Nutrition Obesity Research Center Pilot NORC grant preliminary/developmental offspring (TTP) of the parent grant P30 DK072488 (BDM). Additional support was provided, in part, by The American Foundation for Suicide Prevention, Distinguished Investigator Award, (TTP), the Rocky Mountain MIRECC for Suicide Prevention, Denver, CO, USA, the Military and Veteran Microbiome Consortium for Research and Education (LAB, CAL, AJH, TTP) and the University of Maryland, College Park, Joint Institute for Food Safety and Applied Nutrition and the U.S. Food and Drug Administration for their support through the cooperative agreement FDU.001418. The findings and conclusions in this study are those of the authors and do not necessarily represent the official positions of the NIH, VA, FDA, or the American Foundation for Suicide Prevention. Presentations The findings of this study were presented at the “Advances in Toxoplasma gondii Research” conference at the FDA White Oak Campus, Silver Springs, MD, USA on February 17, 2016, and at the Society of Biological Psychiatry annual meeting on May 13, 2016, in Atlanta, GA, USA, and at the American Psychiatric Association annual meeting on May 16, 2016, in Atlanta, GA, USA. Conflict of interest The authors declare they have no conflicts of interest related to this study. Acknowledgements We would like to thank the entire personnel of the University of Maryland School of Medicine Amish Research Clinic, Lancaster, PA, USA, in particular the Amish liaisons, including Naomi Esh and Hanna King, and the nurses of the Amish Research Clinic, including Donna Trubiano, Yvonne Rohrer, Theresa Roomet, Mary Morrissey, Nancy Weitzel and Susan Shaub, and finally, to all members of the Old Order Amish community in Lancaster who took part in this study. We are grateful to Philip H. Siebler for preparation of the graphical abstract. We also thank Aline Dagdag, Winny Mwaura, Afshan Qureshi, and Sunghee Flores for their substantial administrative support during this project. References Alipour, A., Elte, J.W., Van Zaanen, H.C., Rietveld, A.P., Cabezas, M.C., 2007. Postprandial inflammation and endothelial dysfuction. Biochem. Soc. Trans. 35, 466–469. Almasy, L., Blangero, J., 2010. Variance component methods for analysis of complex phenotypes. Cold Spring Harb. Protoc. 2010. http://dx.doi.org/10.1101/pdb.top77. Altindag, Z.Z., Sahin, G., Inanici, F., Hascelik, Z., 1998. Urinary neopterin excretion and dihydropteridine reductase activity in rheumatoid arthritis. Rheumatol. Int. 18, 107–111.
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