The clinical course and eventual outcome, or prognosis, of complex diseases varies enormously between affected individuals. This variability critically determines the impact a disease has on a patient's life but is very poorly understood. Here, we exploit existing genome-wide association study data to gain insight into the role of genetics in prognosis. We identify a noncoding polymorphism in FOXO3A (rs12212067: T > G) at which the minor (G) allele, despite not being associated with disease susceptibility, is associated with a milder course of Crohn's disease and rheumatoid arthritis and with increased risk of severe malaria. Minor allele carriage is shown to limit inflammatory responses in monocytes via a FOXO3-driven pathway, which through TGFβ1 reduces production of proinflammatory cytokines, including TNFα, and increases production of anti-inflammatory cytokines, including IL-10. Thus, we uncover a shared genetic contribution to prognosis in distinct diseases that operates via a FOXO3-driven pathway modulating inflammatory responses.
Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.
Genotype at rs12212067 Drives Allele-Specific Expression of
FOXO3A and Modulates Inflammatory Cytokine Production in Monocytes (A) Allele-specific expression assay showing the ratio of rs12212067 alleles in genomic DNA (gDNA) and complementary DNA (cDNA; synthesized from pre-mRNA) from peripheral monocytes of heterozygous individuals. Ninety-six colonies were genotyped per individual per condition (either unstimulated [unstim.] or stimulated with 100 ng/ml LPS [stim.]); n = 6. (B) TNFα and IL-10 production by PBMC from minor (G) and major (T) allele homozygotes at rs1221267 following 24 hr stimulation with LPS; n = 15 per group, assayed in triplicate. (C) Intracellular staining of TNFα and IL-10 production in LPS-stimulated (100 ng/ml) CD3 + T cells, CD19 + B cells and CD14 + monocytes. Isotype control shown as shaded histogram. Results representative of 20 experiments. (D) TNFα and IL-10 production by purified monocytes from minor and major allele homozygotes following 24 hr stimulation with LPS (100 ng/ml); n = 11 per group, assayed in triplicate. 20 of these individuals had provided samples used in B. (E) TNFα and IL-10 production by PBMC from minor and major allele homozygotes following 24 hr stimulation with other TLR ligands; n = 11 per group (same individuals’ samples as D), assayed in triplicate. (F) IL-1β, IL-6, and IL-8 production by purified monocytes from minor and major allele homozygotes following 24 hr stimulation with LPS (same samples as in D), n = 11 per group. Data are represented as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S2, S3, S4 and Tables S4 and S5.
Genotype at rs12212067 Influences the Nuclear Recovery of FOXO3 during Cellular Activation (A) The subcellular localization of FOXO3 in monocytes during LPS stimulation (representative images). FOXO3 (red) and nucleus (DAPI, blue). (B) The ratio of nuclear to cytoplasmic FOXO3 over time (mean immunofluorescence intensity) in LPS-stimulated monocytes from minor (G) and major (T) allele homozygotes at rs12212067 (100 ng/ml, n = 10). The dotted line indicates transition from higher signal in nucleus (above) to cytoplasm (below). (C–D) Production of TNFα (C) and IL-10 (D) by monocytes analyzed in (B). Cytokines were quantified in triplicate by ELISA. Data are represented as mean ± SEM,
∗p < 0.05, ∗∗p < 0.01. See also Figure S5.
A TGFβ1-Dependent Mechanism Is Responsible for the Effect of rs12212067 on Monocyte Cytokine Production (A) TGFβ1 production by PBMC (n = 20 per group) and monocytes (n = 13 per group) from minor (G) and major (T) allele homozygotes at rs1221267 following 24 hr stimulation with LPS (100 ng/ml), assayed in triplicate. (B) TNFα and IL-10 production by PBMC from minor and major allele homozygotes following 24 hr stimulation with LPS (100 ng/ml) in the presence of a TGFβ1-neutralizing antibody or isotype control; n = 10 per group, assayed in triplicate. (C) Production of TGFβ1 by monocytes in the time-course experiment shown in Figure 2, measured in triplicate by ELISA. (D) The effect of TGFβ1 or a TGFβ1-neutralizing antibody upon TNFα and IL-10 production by LPS-stimulated monocytes, assessed using flow cytometry (mean fluorescence intensity, MFI). Data are log
2 transformed following normalization, Wilcoxon signed-rank test; n = 6. Data are represented as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, NS, nonsignificant.
FOXO3 Regulates TGFβ1 Production in Stimulated Monocytes (A) Jaspar sequence logo of the FOXO3-binding site (
http://jaspar.genereg.net). (B) ChIP-qPCR analysis of the TGFβ1 promoter following FOXO3 immunoprecipitation. Enrichment for each region is shown relative to input (5 ng) and normalized against the results of a negative control immunoprecipitate (polyclonal IgG). Validated positive and negative control genomic regions were used. Controls shown are HBB (negative) and a region upstream of MSTN (positive). n = 3. (C) Luciferase reporter assay showing effect of silencing FOXO3A on transcription from the TGFβ1 promoter in two monocyte cell lines (U937 and MONO-MAC6). No transfection (Nil) and scrambled controls (Scr) are shown. Data representive of three experiments, each with seven biological replicates. (D) Apoptosis in monocytes from minor (G) and major (T) allele homozygotes at rs12212067 following 24 hr stimulation with LPS (100 ng/ml); assessed by flow cytometry (7-AAD +, Annexin V +), n = 12 per group. (E) The change in the concentration of mono- and oligonucleosomes in the cytoplasm (apoptosis) and supernatant (necrosis) of monocytes following LPS stimulation (24 hr, 100 ng/ml), assessed in triplicate by ELISA. Dotted lines link paired samples; Wilcoxon signed-rank test, n = 8 per group. Data are represented as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, NS, nonsignificant. See also Figure S6 and Table S6.
Foxo3a Activity Predisposes to More Severe Colitis in a Mouse Model (A–F) Colitis was induced in littermate Foxo3a , −/− Foxo3a , and +/− Foxo3 mice using 2% DSS (7 d). Results representative of two experiments each with 5 mice per group, Mann Whitney test unless indicated. Data are represented as mean ± SEM, +/+ ∗p < 0.05, ∗∗p < 0.01. (A) Light microscopy of H&E stained colonic sections from Foxo3a and −/− Foxo3 mice. Colons removed on day 12. (B) Histological assessment of colitis severity, scored with a standard tool (Dieleman et al., 1998). (C) Percentage change in total body weight (measured daily). Two-way ANOVA. (D) Colon weight (day 12). (E) Colon length (day 12). (F) Quantitative PCR of TNFα and IL-10 mRNA extracted from paraffin-embedded colonic sections. Normalization to beta-actin (left) and CD11b (right). See also Figure S7. +/+
Cochran-Armitage Plot of Candidate Gene Association Analysis, Related to Table 1 Cochran Armitage plot of –log(10) p values against chromosomal position for the candidate gene association analysis results (primary cohort only—668 aggressive CD, 389 indolent CD). Each point represents a SNP. 1134 SNPs within 81 genes in the IL-2 and IL-7 signaling pathways were included in the analysis. Manhattan plot generated using Haploview (Barrett et al., 2005).
Linkage Disequilibrium around rs12212067 Is Limited to
FOXO3A, Related to Figure 1 Regional LD plot—adapted from a plot generated using SNP Annotation and Proxy software (Johnson et al., 2008)—demonstrating linkage disequilibrium around rs12212067. Each SNP is represented by a diamond and its position is plotted against its r 2 value with rs12212067. Those with r 2 ≥ 0.5 are filled blue. Rs12212067 is represented by the red diamond. The dotted lines indicate a region of LD where r 2 ≥ 0.5. The horizontal arrowed lines below represent the genes in this region, with the arrows indicating the direction of transcription and the exons in FOXO3A shown as red bars. The purple line indicates the recombination rate.
Genotype at rs12212067 Correlates with TNFα and IL-10 Production in Stimulated Monocytes, Related to Figure 1 The amount of TNFα and IL-10 produced by monocytes following LPS stimulation (6 hr
) was quantified by intracellular flow cytometry. The increase in mean fluorescence intensity (compared with autologous unstimulated monocytes) is shown—stratified by genotype (GG, minor allele homozygotes; TT, major allele homozygotes). n = 20, data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.
Genotype at rs12212067 Does Not Alter Monocyte Production of GMCSF, IL-12p70, IL-2, IL-4, or IL-5, Related to Figure 1 GMCSF, IL-12p70, IL-2, IL-4 and IL-5 production by purified monocytes from minor and major allele homozygotes following 24 hr’ stimulation with LPS (same samples as used in Figures 1D and 1F), n = 22. None of the comparisons were significantly different (all p ≥ 0.05). Data are represented as mean ± SEM.
Inhibition of Protein Synthesis Retards the Recovery of Nuclear FOXO3 in Stimulated Monocytes, Related to Figure 2 The effect of cycloheximide (CHX, 5 μg/ml) upon the ratio of nuclear:cytoplasmic FOXO3 in stimulated monocytes was assessed by immunofluorescence. Quantification of immunofluorescence was performed by an independent observer blinded to the experimental details. The dotted line indicates the transition from higher signal in nucleus (above) to higher signal in cytoplasm (below). All live cells in 4 high-powered fields assessed at each time point. Data are represented as mean ± SEM,
∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Apoptotic Cells Modulate Inflammatory Responses in a TGFβ1-Dependent Manner, Related to Figure 4 The effect of coincubating autologous apoptotic neutrophils or autologous nonapoptotic neutrophils (PMN) upon TNFα and IL-10 production by LPS-stimulated monocytes, assessed using flow cytometry. Data are log
2 transformed following normalization, Wilcoxon signed-rank test; n = 6, data represented as mean ± SEM, ∗p < 0.05.
Foxo3a Activity Leads to Increased IL-6 Expression in the Colon in a Mouse Model of Colitis, Related to Figure 5 Colitis was induced in littermate Foxo3a , −/− Foxo3a and +/− Foxo3 mice using 2% DSS in drinking water for 7 days. Mice were killed on day 12 and colons were removed and paraffin-embedded. Quantitative PCR of IL-6 mRNA extracted from paraffin-embedded sections. Representative data are shown for one of two experiments each with 5 mice per group, Mann Whitney test. Data are represented as mean ± SEM, +/+ ∗p < 0.05.
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Research Support, Non-U.S. Gov't
Arthritis, Rheumatoid / genetics*
Arthritis, Rheumatoid / physiopathology
Cell Nucleus / metabolism
Crohn Disease / genetics*
Crohn Disease / physiopathology
Extracellular Matrix Proteins / immunology
Forkhead Transcription Factors / genetics*
Forkhead Transcription Factors / metabolism
Malaria, Falciparum / genetics*
Malaria, Falciparum / physiopathology
Polymorphism, Single Nucleotide*
Transforming Growth Factor beta / immunology
Extracellular Matrix Proteins
Forkhead Transcription Factors
Transforming Growth Factor beta
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