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Additional file 2: Appendix 1–3. Appendix 1. Significantly functions categories in Gene Ontology (GO) enrichment analysis in discovery set. Appendix 2. Significant Differentially Methylated Regions (DMRs) with annotated genes by hump-hunting method in the discovery group. Appendix 3. Significant Differentially Methylated Regions (DMRs) with annotated genes by Probe Lasso method in the discovery group.

Figure S2. Immunofluorescence staining results. (A) CCRL2 and ChemR23 knockdown efficiency in 18.5-day-old fetal mice and 7-day-old offspring from diabetic dams and macrophages isolated from the peritoneal cavity of mice. (B) IOD/area of chemerin- and CCRL2-positive cells in forebrain tissues of 18.5-day-old fetal mice and 7-day-old offspring from controls, chemerin-induced diabetic dams, and chemerin-induced diabetic dams with ChemR23 knockdown mice. (C) IOD/area of F4/80- and MAP2-positive cells. (D) IOD/area of chemerin- and F4/80-positive cells. (E) IOD/area of β-III-tubulin level in coronal cortical sections at E18.5 and NeuN level in Olfactory bulb and dentate gyrus of 8-week-old offspring from controls, chemerin-induced diabetic dams, chemerin-induced diabetic dams with ChemR23 knockdown and chemerin-induced diabetic dams with CCRL2 knockdown mice. * chemerin-induced diabetic dams vs. controls (E18.5d); #chemerin-induced diabetic dams with ChemR23 knockdown/CCRL2 knockdown vs. chemerin-induced diabetic dams (E18.5d); ^GDM group vs. controls (B7.0d); $chemerin-induced diabetic dams with ChemR23 knockdown/CCRL2 knockdown vs. chemerin-induced diabetic dams (B7.0d). * and #, P < 0.05; **, ##, ^^ and $$, P < 0.01. (PDF 5692 kb)

Figure S3. FACS sorting for macrophages and effect of chemerin on migration of macrophages. (A) Macrophages, microglia, and other cell fractions were sorted by FACS from a pool of mononuclear cells isolated from brain tissues of 18.5-day-old fetal mice. CD45 high, CD11b high, F4/80 high, and Gr-1 low indicate the macrophage fraction; CD45 intermediate and CD11b intermediate indicate the microglial fraction; and CD11b negative and Gr-1 high indicates other cell fractions. (B) Levels of ChemR23 were detected by western blotting in a pool of macrophages isolated from the peritoneal cavity of normal mice. Macrophages were stimulated with 0, 1, 10, 100, or 1000 nM chemerin for 30 min. The histogram represents the gray values of bands normalized to GAPDH. (C) The proportion of migrated macrophages was measured by Transwell assay. CXCL8 treatment was defined as the positive control. Data are presented as mean with 95% CI. * chemerin treatment vs. control. **P < 0.01. (PDF 4414 kb)

Figure S1. Effects of chemerin on cortex, mature neurons and the concentration of chemerin in brain tissue. (A) Integrated optical density per unit area (IOD/area) of β-III-tubulin level in coronal cortical sections at E18.5 and NeuN level in Olfactory bulb and dentate gyrus of 8-week-old offspring from controls and chemerin-induced diabetic group. (B) The concentration of chemerin measured by ELISA kit in brain tissue of E18.5 and 7-day-old offspring from chemerin-induced diabetic dams. *chemerin or chemerin+CCRL2-knockdown vs control; #chemerin+CCRL2-knockdown vs chemerin. #, P < 0.05; ** and ##, P < 0.01. (PDF 1005 kb)

Robustness analysis with respect to other epigenetic biomarkers of aging in the WHI. A-C Results for the Horvath method when 47 out of 353 CpGs were removed from the epigenetic clock (because they are in the vicinity of a SNP). Since none of the remaining clock CpGs are near a SNP, the resulting age acceleration is not trivially related to race/ethnicity. A DNA methylation age versus chronological age. B Ethnicity versus age acceleration (defined as residual resulting from regressing DNAm age on chronological age). C Intrinsic epigenetic age acceleration versus ethnicity. D, E Alternative epigenetic biomarker of aging based on 589 age-related CpGs from Teschendorff [13]. The biomarker was defined using the following steps. First, the DNA methylation levels of each CpGs were standardized (to mean zero and variance 1). Second, a weighted average was formed by multiplying each CpG by the T test statistic from the chronological age relationship based on the table from the original reference. Third, the weighted average was regressed on chronological age to arrive at a residual. The resulting residual is referred to as extrinsic measure of age acceleration since it was not adjusted for blood cell counts. Fourth, the resulting measure was regressed on estimated blood cell counts (analogous to those used for IEAA) in order to arrive an intrinsic measure of age acceleration. F, G Epigenetic measures of age acceleration using the Hannum method 71 CpGs [19]. D, F Results for intrinsic measures, i.e. measures of age acceleration that adjust both for blood cell counts and chronological age. E, G reports extrinsic measures, i.e. no adjustment for imputed blood cell counts. Each bar plot depicts 1 standard error and reports the results from a Kruskalâ Wallis test. (PDF 55 kb)

Educational level versus age acceleration in the WHI. Each row relates educational level (x-axis) to three respective measures of epigenetic age acceleration: (A-D) Age Accel., (E-H) IEAA, and (I-L) EEAA. The columns correspond to different groups of women from the WHI. The first, second, third, and fourth columns report findings for (A, E, I) all women, (B, F, J) Caucasians, (C, G, K) African Americans, and (D, H, L) Hispanics, respectively. Each bar plot reports the mean values, 1 standard error, and the p value from a non-parametric group comparison test (Kruskalâ Wallis). Education was assessed using the form â Demographics and Study Membership.â We find that education predicts future EEAA. (PDF 6 kb)

Epigenetic age acceleration in Hispanics versus country of residence in the WHI. Each column corresponds to different measure of age acceleration: (A, D) age acceleration residual, (B, E) IEAA (C, F) EEAA. (A-C, first row) results for “country of birth” (x-axis). (D-F, second row) results for “country of residence” at age 35 years, which was defined by combining two variables country of birth and “living in the US at age 35.” The left-most bar corresponds to Hispanic women who were born outside the US and lived outside the US at age 35 years, the middle bar corresponds to Hispanic women who were born outside the US but lived already in the US at the age of 35 years; the right-most bar reports results for women who were born in the US and lived in the US at age 35 years. Incidentally, all of these postmenopausal Hispanic women lived in the US at the age of the blood draw. As a caveat, we mention the relatively small group sizes (small gray numbers underneath the bars). (PDF 3 kb)

Lymphoblastoid cell lines from Han Chinese, Caucasians, and African Americans. A Gray line corresponds to a natural spline regression through Caucasian samples. Age acceleration was defined as residual with respect to this line. B Marginally significant evidence that African Americanâ s are younger than other ethnic groups. (PDF 33 kb)

Demographic and physiologic characteristics of women from the WHI. Case-control status refers to CHD. Two designs were used to select samples: case/control and case-cohort. (DOC 48 kb)

Type II diabetes status versus age acceleration in the Bogalusa study. Each row relates type II diabetes status (x-axis) to three respective measures of epigenetic age acceleration: (A-C) Age Accel., (D-F) IEAA, and (G-I) EEAA. The columns correspond to different groups. The first, second, and third columns report findings for (A, D, G) all participants, (B, E, H) Caucasians, (C, F, I) African Americans, respectively. Each bar plot reports the mean values, 1 standard error, and the p value from a non-parametric group comparison test (Kruskalâ Wallis). Type 2 diabetes status was defined as fasting glucoseâ >â =126Â mg/dl or taking diabetes medication. (PDF 3 kb)

Hypertension status versus age acceleration in the Bogalusa study. Each row relates hypertension status (x-axis) to three respective measures of epigenetic age acceleration: (A-C) Age Accel., (D-F) IEAA, and (G-I) EEAA. The columns correspond to different groups. The first, second, and third columns report findings for (A, D, G) all participants, (B, E, H) Caucasians, (C, F, I) African Americans, respectively. Each bar plot reports the mean values, 1 standard error, and the p value from a non-parametric group comparison test (Kruskalâ Wallis). Hypertension status was defined as meeting any of the three conditions: (1) blood pressureâ >â =140/90; (2) taking medication; or (3) having been diagnosed as having hypertension. (PDF 4 kb)

Educational level versus age acceleration in the Bogalusa study. Each row relates educational level (x-axis) to three respective measures of epigenetic age acceleration: (A-C) Age Accel., (D-F) IEAA, and (G-I) EEAA. The columns correspond to different groups. The first, second, and third columns report findings for (A, D, G) all participants, (B, E, H) Caucasians, (C, F, I) African Americans, respectively. Each bar plot reports the mean values, 1 standard error, and the p value from a non-parametric group comparison test (Kruskal–Wallis). Education was grouped as follows: group 1 = grades 1–7; group 2 = grades 8–9; group 3 = grades 10–12; group 4 = vocational/tech training; group 5 = college; group 6 = postgraduate. (PDF 5 kb)

Hypertension status versus age acceleration in the Bogalusa study. Each row relates hypertension status (x-axis) to three respective measures of epigenetic age acceleration: (A-C) Age Accel., (D-F) IEAA, and (G-I) EEAA. The columns correspond to different groups. The first, second, and third columns report findings for (A, D, G) all participants, (B, E, H) Caucasians, (C, F, I) African Americans, respectively. Each bar plot reports the mean values, 1 standard error, and the p value from a non-parametric group comparison test (Kruskalâ Wallis). Hypertension status was defined as meeting any of the three conditions: (1) blood pressureâ >â =140/90; (2) taking medication; or (3) having been diagnosed as having hypertension. (PDF 4 kb)

Accuracy of imputed blood cell counts. Here we used an independent dataset, which was not used to develop estimators of blood cell counts based on DNA methylation data, to evaluate the accuracy of the imputed blood cell counts. For each participant, both flow cytometric measures and Illumina Inf450 data were available from 96 participants as described in [88]. A-G The scatter plots depict the predicted abundance of blood cell count (based on DNA methylation levels) versus the corresponding observed flow cytometric measurement (y-axis). Each panel reports a robust correlation coefficient (biweight midcorrelation) and a corresponding p value. The Houseman method was used to impute (A) CD8+ T cells, (B) CD4+ T, (C) B cells. The epigenetic clock software was used for imputing (D) naïve CD8+ T cells, (E) naïve CD4 + T cells, (F) plasma blasts, and (G) exhausted CD8+ T cells. H, I Another flow cytometric dataset was used to test for ethnic differences in naïve CD4+ T cells. The y-axis shows the log transformed flow cytometric measurement of naïve CD4+ T cells (adjusted for age). Specifically, the y-axis reports the residual resulting from regressing log(naïve CD4+ T cell abundance) on chronological age. H Findings for HIV– participants (198 Caucasians versus 34 Hispanics). I Findings for HIV+ participants (101 Caucasians, 58 Hispanics). Stouffer’s meta-analysis across the two strata (HIV+ and HIV– strata) shows that Hispanics have significantly fewer naïve CD4+ T cells (Stouffer’s p = 0.030, Stouffer’s Z = (1.75 + 1.31)/sqrt(2)). (PDF 130 kb)

Educational level versus age acceleration in the Bogalusa study. Each row relates educational level (x-axis) to three respective measures of epigenetic age acceleration: (A-C) Age Accel., (D-F) IEAA, and (G-I) EEAA. The columns correspond to different groups. The first, second, and third columns report findings for (A, D, G) all participants, (B, E, H) Caucasians, (C, F, I) African Americans, respectively. Each bar plot reports the mean values, 1 standard error, and the p value from a non-parametric group comparison test (Kruskal–Wallis). Education was grouped as follows: group 1 = grades 1–7; group 2 = grades 8–9; group 3 = grades 10–12; group 4 = vocational/tech training; group 5 = college; group 6 = postgraduate. (PDF 5 kb)