Metabolic perturbations and cellular stress underpin susceptibility to symptomatic live-attenuated yellow fever infection

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Abstract

Flaviviral infections result in a wide spectrum of clinical outcomes, ranging from asymptomatic infection to severe disease. Although the correlates of severe disease have been explored1,2,3,4, the pathophysiology that differentiates symptomatic from asymptomatic infection remains undefined. To understand the molecular underpinnings of symptomatic infection, the blood transcriptomic and metabolomic profiles of individuals were examined before and after inoculation with the live yellow fever viral vaccine (YF17D). It was found that individuals with adaptive endoplasmic reticulum (ER) stress and reduced tricarboxylic acid cycle activity at baseline showed increased susceptibility to symptomatic outcome. YF17D infection in these individuals induced maladaptive ER stress, triggering downstream proinflammatory responses that correlated with symptomatic outcome. The findings of the present study thus suggest that the ER stress response and immunometabolism underpin symptomatic yellow fever and possibly even other flaviviral infections. Modulating either ER stress or metabolism could be exploited for prophylaxis against symptomatic flaviviral infection outcome.

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Fig. 1: Increased baseline expression of ER stress response, sumoylation and cell cycle genes was associated with symptomatic viral infection.
Fig. 2: Reduced TCA cycle genes and intermediates were associated with symptomatic viral infections.
Fig. 3: Induction of oxidative stress after viral infection was associated with symptomatic outcome.
Fig. 4: Increased maladaptive ER stress in subjects with symptomatic outcome correlated with baseline ER stress and sumoylation gene expression.

Data availability

Trial 1 and trial 2 raw baseline gene expression data and the processed Z-scores have been deposited in ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession numbers E-MTAB-7928 and E-MTAB-7931, respectively. The CE-MS/MS metabolite expression data was deposited in the National Institute of Health’s Metabolomics Workbench under accession number ST001176.

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Acknowledgements

We thank all the participants, research coordinators and nurses at the SingHealth Investigational Medicine Unit for their time and assistance in the conduct of the clinical trials. We also thank the anonymous reviewers for their constructive comments. This work is supported by J.G.-H.L.’s Clinician-Scientist Award (NMRC/CSA-INV/0013/2016) and E.E.O.’s Senior Clinician-Scientist Award (NMRC/CSA/060/2014) and Centre Grant (NMRC/CG/M003/2017), all from the National Medicine Research Council of Singapore. We also thank the Tanoto Foundation for their generous support for ViREMiCS.

Author information

K.R.C., J.G.-H.L. and E.E.O. conceptualized and designed the study. J.G.-H.L. and L.W. enrolled the subjects of the trials. K.R.C., E.S.G., C.Y.Y. and S.L.-X.Z. performed the transcriptomic analyses. C.L. and Y.H.L. performed the lipid and cytokine profiling. K.R.C., Y.H.L. and A.B. analyzed the lipid and cytokine data. E.O.Z. and K.R.C. performed the CE-MS/MS analysis. K.R.C. and J.Z.H.L. performed the metabolism assays. K.R.C., Y.H.L., J.G.-H.L. and E.E.O. analyzed the data. K.R.C., J.G.-H.L. and E.E.O. wrote the manuscript.

Correspondence to Kuan Rong Chan or Jenny Guek-Hong Low or Eng Eong Ooi.

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The authors declare no competing interests.

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Peer review information: Alison Farrell is the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Symptomatic subjects display higher baseline transcript expression of genes related to ER stress response, sumoylation and cell cycle.

a, Volcano plot displaying 34,667 genes detected by microarray in subjects from trial 1. Red dots represent increased baseline expression of genes (2,055 DEGs) in symptomatic (n = 19) relative to asymptomatic subjects (n = 8) (unpaired, two-sided, Student’s t-test, P < 0.05 corrected for multiple testing using Bonferroni’s correction). Blue dots indicated reduced baseline expression of genes (1,963 DEGs) in symptomatic relative to asymptomatic subjects. Data are represented as −log10(P value) versus log2(transformed baseline gene expression of symptomatic relative to asymptomatic subjects). b, Pathway analysis of genes that were more abundantly expressed in symptomatic (n = 19) relative to asymptomatic (n = 8) subjects at baseline (unpaired, two-sided, Student’s t-test, P < 0.05 corrected for multiple testing using Bonferroni’s correction). DEGs were analyzed against the Reactome database, and the significance of each pathway was determined by Enrichr. The dotted line indicates P = 0.05. ce, Z-score heatmap showing the baseline expression of genes belonging to the ER stress response, sumoylation and cell cycle in subjects with (n = 19) and without (n = 8) symptoms. Only genes that were significantly different (unpaired, two-sided, Student’s t-test, P < 0.05 corrected for multiple testing using Bonferroni’s correction) between symptomatic and asymptomatic subjects were displayed. Red indicated increased expression whereas blue indicated reduced expression. The genes displayed from c,d were subjected to validation in trial 2. All data are based on biologically independent samples. Source data

Extended Data Fig. 2 Increased baseline abundance of UPR and sumoylation genes in symptomatic subjects are expressed in B cells.

a,b, Volcano plot displaying baseline expression of all 95 and 170 genes from categories ‘R-HSA-381119 UPR’ and ‘R-HSA-2990836 sumoylation’, respectively, in symptomatic (n = 19) relative to asymptomatic subjects (n = 8). Red dots represent increased baseline expression in symptomatic subjects whereas blue dots represent reduced baseline expression in symptomatic subjects (unpaired, two-sided, Student’s t-test, P < 0.05 corrected for multiple testing using Bonferroni’s correction). Data represented as −log10(P value) versus log2(transformed baseline gene expression of symptomatic relative to asymptomatic subjects). c,d, ROC curves of genes in the UPR and sumoylation pathway as indicators of symptomaticity (symptomatic n = 19, asymptomatic n = 8). Calculated sensitivity (y axis) is plotted against 1 − specificity formula (x axis). AUC, P value (two-sided) and 95% CI are displayed. ROC statistics generated using the Wilson/Brown method. e, BTMs that were positively associated with the baseline transcriptional profiles of symptomatic subjects (symptomatic n = 19 versus asymptomatic n = 8). GSEA of the DEGs was used to identify important associations. The top three pathways have FDR < 0.025, where the FDR value is adjusted for gene set size and multiple hypotheses testing. Normalized enrichment scores computed by GSEA with nominal P values < 0.05 (two-sided test) are shown. f, Top module that was notably over-represented in symptomatic subjects at baseline. On the x axis, enrichment score; on the y axis, gene rank in symptomatic versus asymptomatic subjects at baseline. The nominal P values and FDR values were all <0.001, and were directly reflected in GSEA analysis. The FDR value is adjusted for gene set size and multiple hypotheses testing. g,h, White cell and lymphocyte counts at baseline between the symptomatic (n = 43) and asymptomatic (n = 24) subjects. The unpaired, two-sided, Student’s t-test was used for comparison. Center points indicate the median and error bars the s.d. All data are based on biologically independent samples.

Extended Data Fig. 3 Symptomatic subjects display higher baseline abundance of ER stress and sumoylation genes.

a, Volcano plot displaying baseline expression of genes related to ER stress (red) and sumoylation (orange) in symptomatic versus asymptomatic subjects from trial 2 (n = 35), validated using nCounter. The genes subjected for validation were based on trial 1. Genes used for validation were as shown in Extended Data Fig. 1c,d. Data are represented as −log10(P value) versus log2(transformed baseline gene expression of symptomatic relative to asymptomatic subjects). An unpaired, two-sided, Student’s t-test was used for comparison. b, Pearson’s correlation (r) of Z-scores (genes TPYSL2 and UBA2) with number of symptoms based on subjects from trial 1 (n = 27) and trial 2 (n = 35). Red squares indicated Z-scores of subjects from trial 1 whereas purple squares indicated subjects from trial 2. The P values indicate the significance of the slope (two-sided test). No adjustments were made because the subset of genes for validation was selected based on trial 1 results. All data are based on biologically independent samples.

Extended Data Fig. 4 Symptomatic subjects display lower expression of genes related to the TCA cycle.

a, Pathway analysis of genes that were less abundantly expressed in symptomatic (n = 19) relative to asymptomatic (n = 8) subjects at baseline (unpaired, two-sided, Student’s t-test, P <0.05 corrected for multiple testing using Bonferroni’s correction). DEGs were analyzed against the Reactome database, and the significance of each pathway was determined by Enrichr. The dotted line indicates P = 0.05. b, Volcano plot displaying baseline expression of all TCA cycle genes (n = 22) in category R-HSA-71403. Blue dots represented downregulated baseline expression in symptomatic subjects (unpaired, two-sided, Student’s t-test, P < 0.05 corrected for multiple testing using Bonferroni’s correction). Data represented as −log10(P value) versus log2(transformed baseline gene expression of symptomatic relative to asymptomatic subjects). c, ROC curves of genes in the TCA cycle pathway as indicators of symptomaticity. Calculated sensitivity (y axis) is plotted against 1 − specificity formula (x axis). AUC, P value (two-sided) and 95% CI were displayed. ROC statistics generated using the Wilson/Brown method. d, Z-score heatmap showing baseline expression of metabolic genes in subjects with (n = 19) and without (n = 8) symptoms or adverse events after yellow fever vaccination. Genes with significant differences (P  < 0.05) between symptomatic and asymptomatic subjects were shown. Red indicates enrichment whereas blue indicates reduced expression. e, Heatmap showing the normalized baseline expression for individual genes belonging to the category ‘R-HSA-2046104 Alpha-linoleic acid metabolism’ in symptomatic (Symp) and asymptomatic (Asymp) subjects in trial 1 and trial 2 subjects. f, Number of genes (# Genes) represented in the various pathways by the nCounter Vantage RNA Cancer Metabolism Panel. g, Volcano plot displaying baseline expression of genes related to the TCA cycle in symptomatic versus asymptomatic subjects from trial 2 (n = 35), validated using nCounter. Blue dots represent downregulated baseline expression in symptomatic subjects (P value < 0.05). An unpaired, two-sided, Student’s t-test was used for comparison. No adjustments were made. h, The top three enriched downregulated Reactome pathways from trial 2, using genes represented in the customized nCounter Metabolism Panel. DEGs with reduced baseline expression in symptomatic subjects (unpaired, two-sided, Student’s t-test, P < 0.05) were analyzed against the Reactome database, and the significance of each pathway was determined by Enrichr. All data are based on biologically independent samples.

Extended Data Fig. 5 Baseline levels of TCA cycle intermediates were lower in symptomatic subjects, but not oxylipin metabolites and glucose.

a, BTMs that were negatively associated with the baseline transcriptional profiles of symptomatic subjects (symptomatic n = 19, asymptomatic n = 8). GSEA of the DEGs was used to identify the important associations (unpaired, two-sided, Student’s t-test, P < 0.05 corrected for multiple testing using Bonferroni’s correction). Normalized enrichment scores computed by GSEA for the important modules (nominal P value < 0.05, FDR < 0.25) are shown. b, Top module that was significantly under-represented in symptomatic subjects at baseline (symptomatic n = 19, asymptomatic n = 8). On the x axis, enrichment score; on the y axis, gene rank in symptomatic versus asymptomatic subjects at baseline. Nominal P value (two-sided) and FDR values were displayed, where the FDR value was adjusted for gene set size and multiple hypotheses testing. Box and whisker plots of oxylipin (c) and oxylipin (d) derivative concentrations at baseline in symptomatic (n = 17) and asymptomatic (n = 12) subjects. Lines represent medians, boxes represent 25–75% intervals and whiskers represent 5–95% intervals. e, Volcano plot showing the relative expression of metabolites at baseline in symptomatic (n = 7) versus asymptomatic (n = 6) subjects, detected by CE-MS/MS. Indicated in blue are TCA cycle intermediates that were notably downregulated in symptomatic subjects. Data represented as −log10(P value) versus log2(transformed baseline gene expression of symptomatic relative to asymptomatic subjects). An unpaired, two-sided, Student’s t-test with Bonferroni’s correction was used for analysis. f, Baseline glucose concentrations between symptomatic (n = 12) and asymptomatic (n = 17) subjects. Center points indicate the mean and error bars the s.d. Comparisons were based on a two-tailed, Student’s t-test. All data are based on biologically independent samples.

Extended Data Fig. 6 Medium-chain acylcarnitine levels positively correlate with baseline TCA cycle metabolism.

ac, Pearson’s correlation (r) of log2(transformed values of fold changes) (day 1 versus day 0) of 2-octenoylcarnitine with log2(transformed baseline) (day 0) concentrations of malate, isocitrate and citrate. Red circles indicate symptomatic subjects (n = 7) whereas blue circles indicate asymptomatic subjects (n = 6). The P values indicate the significance of the slope, which assesses whether the correlation coefficient of the slope differs from 0 (two-sided). All data are based on biologically independent samples.

Extended Data Fig. 7 Baseline adaptive ER stress and sumoylation expression levels are associated with susceptibility to maladaptive ER stress.

a,b, Pearson’s correlation (r) of log2(transformed values of fold changes) (day 1 versus day 0) of TXNIP with either baseline expression levels of adaptive ER stress or sumoylation genes, using trial 2 subjects. TXNIP expression levels are determined by qPCR, normalized with the control gene (TBP). Composite baseline Z-scores are based on the genes shown in Fig. 1c,d, quantified by NanoString technology. Red circles indicate symptomatic subjects (n = 10) and blue circles asymptomatic subjects (n = 22). P values indicate the significance of the slope, which assesses whether the correlation coefficient of the slope differs from 0 (two-sided). All data are based on biologically independent samples.

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