Fecal microbiome and metabolome differ in healthy and food-allergic twins

Abstract

BACKGROUNDThere has been a striking generational increase in the prevalence of food allergies. We have proposed that this increase can be explained, in part, by alterations in the commensal microbiome.METHODSTo identify bacterial signatures and metabolic pathways that may influence the expression of this disease, we collected fecal samples from a unique, well-controlled cohort of twins concordant or discordant for food allergy. Samples were analyzed by integrating 16S rRNA gene amplicon sequencing and liquid chromatography-tandem mass spectrometry metabolite profiling.RESULTSA bacterial signature of 64 operational taxonomic units (OTUs) distinguished healthy from allergic twins; the OTUs enriched in the healthy twins were largely taxa from the Clostridia class. We detected significant enrichment in distinct metabolite pathways in each group. The enrichment of diacylglycerol in healthy twins is of particular interest for its potential as a readily measurable fecal biomarker of health. In addition, an integrated microbial-metabolomic analysis identified a significant association between healthy twins and Phascolarctobacterium faecium and Ruminococcus bromii, suggesting new possibilities for the development of live microbiome-modulating biotherapeutics.CONCLUSIONTwin pairs exhibited significant differences in their fecal microbiomes and metabolomes through adulthood, suggesting that the gut microbiota may play a protective role in patients with food allergies beyond the infant stage.TRIAL REGISTRATIONParticipants in this study were recruited as part of an observational study (ClinicalTrials.gov NCT01613885) at multiple sites from 2014 to 2018.FUNDINGThis work was supported by the Sunshine Charitable Foundation; the Moss Family Foundation; the National Institute of Allergy and Infectious Diseases (NIAID) (R56AI134923 and R01AI 140134); the Sean N. Parker Center for Allergy and Asthma Research; the National Heart, Lung, and Blood Institute (R01 HL 118612); the Orsak family; the Kepner family; and the Stanford Institute for Immunity, Transplant and Infection.

Keywords: Allergy; Immunology.

Conflict of interest statement

Conflict of interest: KCN reports grants from Allergenis and Ukko Pharma and research sponsorship by Novartis, Sanofi, Astellas, and Nestle as well as personal fees from Regeneron, Astrazeneca, Immuneworks, and Cour Pharmaceuticals. KCN is involved in clinical trials at Regeneron, Genentech, AImmune Therapeutics, DBV Technologies, AnaptysBio, Adare Pharmaceuticals, and Stallergenes-Greer. KCN is a data and safety monitoring board member at Novartis and NHLBI; is the cofounder of BeforeBrands, Alladapt, ForTra, and Iggenix; and is the director of FARE and World Health Organization Center of Excellence. CRN is the president and cofounder of ClostraBio Inc. CRN, KCN, RB, and LAH are inventors on a provisional US patent (63/122,833) filed on December 8, 2020.

Figures

Figure 1. Flow diagram of study design and participating patients.

Figure 2. Relative abundance of microbial composition of healthy and allergic twins does not differ at the family level.

(A) Relative abundance of taxonomy at the family level. Sample IDs are shown on the x axis (n = 34). Discordant twins (12 pairs, n = 24), for which one member was healthy and the other member was allergic; concordant twins (5 pairs, n = 10), for which both members were allergic. Of 36 total samples in the twin cohort, 1 sample (S5077) failed sequencing and yielded 0 reads, hence the corresponding twin pair (no. 13) was excluded from 16S analysis. (B and C) Correlation of OTU abundance between members from each twin pair, with the comparison between concordant and discordant twin pairs shown in B and the comparison between dizygotic and monozygotic twins shown in C. Each dot denotes 1 twin pair (17 pairs shown). (D and E) Shannon α diversity index between healthy and allergic groups, with all samples are shown in D (n = 34) and only discordant twins shown in E (n = 24). Each dot denotes 1 sample. In B–E, the bounds of the boxes represent the 25th and 75th percentiles, the horizontal centers line indicate the medians, and the whiskers extend to data points within a maximum of 1.5 times the IQR. Two-tailed Wilcoxon’s rank-sum test was used in B–D, and two-tailed Wilcoxon’s signed-rank test was used in E.

Figure 3. Healthy twins exhibit a fecal microbial profile distinct from allergic siblings.

Relative abundance heatmap of the 64 OTUs identified to be differentially abundant between healthy (n = 12) and allergic (n = 22) twins. Of these 64 OTUs, 62 were more abundant in the healthy group (healthy-abundant OTUs), and 2 were more abundant in the allergic group (allergic-abundant OTU). OTU IDs are shown on the row in the format of “OTU_ID|Family,” and those annotated with the Clostridia class (Lachnospiraceae, Ruminococcaceae, unclassified Clostridiales) are highlighted in pink. Sample IDs are shown on the column, with annotation bars above the heatmap indicating concordant/discordant twin members, sex, and zygosity. A binary presence/absence heatmap of the 64 OTUs is shown in Supplemental Figure 3. Of 36 samples total in the twin cohort, 1 sample (S5077) failed sequencing and yielded 0 reads; therefore, the corresponding twin pair (no. 13) was excluded from 16S analysis. DS-FDR was used on all samples (P < 0.05) and 2-tailed Wilcoxon’s signed-rank test was used on discordant twin pairs (P < 0.10), respectively. Unadjusted P value thresholds were used to filter for OTUs of interest. After BH-FDR correction, no OTUs passed the FDR cutoff of 0.10 threshold, potentially due to small sample size.

Figure 4. Healthy and allergic twins exhibit within-twin pair differences in microbial composition.

(A) Bubble plot showing the per–twin pair abundance differences of the 64 OTUs shown in Figure 3 between the healthy and allergic groups. The size of each circle corresponds to the relative abundance of an OTU. Samples were arranged as discordant twins (12 pairs, n = 24), where one member is healthy and the other member is allergic; concordant twins (5 pairs, n = 10), where both members are allergic. (B) The aggregated OTU abundance score was significantly higher in healthy (n = 12) relative to allergic twins (n = 22). The score was calculated using the 64 differentially abundant OTUs from A. The score for discordant twin pairs only is shown in Supplemental Figure 4. Each dot denotes 1 sample. The bounds of the boxes represent the 25th and 75th percentiles, the horizontal center lines indicate the medians, and the whiskers extend to data points within a maximum of 1.5 times the IQR. In A, DS-FDR was used on all samples (P < 0.05) and 2-tailed Wilcoxon’s signed-rank test was used on discordant twin pairs (P < 0.10), respectively. Unadjusted P value thresholds were used to filter for OTUs of interest. After BH-FDR correction, no OTUs passed the FDR cutoff of 0.10 threshold, potentially due to small sample size. In B, 2-tailed Wilcoxon’s rank-sum test was used on all samples.

Figure 5. Healthy and allergic twins exhibit differential enrichment in fecal metabolic pathways.

(A) Of 36 samples, 33 metabolites were more abundant in the healthy (n = 13) group relative to the allergic (n = 23) group. Metabolites are shown on the row in the format of “COMP_ID|Biochemical_Name|Super_Pathway|Sub_Pathway.” Sample IDs are shown on the column, with annotation bars above the heatmap indicating concordant/discordant twin members, sex, and zygosity. (B) Of 36 samples, 64 metabolites were more abundant in the allergic group (n = 23) relative to the healthy (n = 13) group. Same annotations as in A. In A and B, 2-tailed Welch’s 2-sample t test was used on all samples (P < 0.10) and unadjusted P value thresholds were used to filter for individual metabolites of interest. After FDR correction, no individual metabolites passed the FDR cutoff of 0.10 threshold, potentially due to small sample sizes.

Figure 6. Distinct metabolic pathways are enriched in healthy and allergic twins.

(A) Metabolites more abundant in the healthy group (from Figure 5A) or in the allergic group (from Figure 5B) were enriched in different subpathways shown. Relative enrichment fold change is shown on the x axis, and the name of subpathway is shown on the y axis. P value and FDR-adjusted P value of each subpathway enrichment are shown next to each horizontal bar. (B and C) Representative examples of metabolites in the enriched subpathways in the healthy or allergic group. (B) The linoleoyl-linolenoyl-glycerol (18:2/18:3) [1]* (subpathway: Diacylglycerol) was higher in healthy (n = 13) compared with allergic (n = 23) twin members. (C) The secoisolariciresinol (subpathway: Food Component/Plant) was higher in allergic twin pairs (n = 23) compared with healthy twin pairs (n = 13). Supplemental Figure 10 shows the result of discordant twin pairs only that correspond to metabolites shown in B and C. In B and C, units shown on the y axis represent the normalized raw area counts of UPLC-MS/MS peaks, rescaled to set the median equal to 1.00 for each biochemical (see Methods). Each dot denotes 1 sample. The bounds of the boxes represent the 25th and 75th percentiles, the horizontal center lines indicate the medians, and the whiskers extend to data points within a maximum of 1.5 times the IQR. In A, the hypergeometric test was used to compute the P values of relative enrichment of metabolite subpathways and filtered by FDR-adjusted P < 0.10. Pathways consisting of at least 2 significant metabolites were included in the statistical test. After BH-FDR multiple-testing correction DAG remained as the most significantly enriched subpathway in metabolites more abundant in healthy twins (FDR-adjusted P < 0.00001). In B and C, 2-tailed Welch’s 2-sample t test was used on all samples.

Figure 7. The OTUs differentially abundant between healthy and allergic groups are correlated with different sets of metabolites and pathways.

Of 64 OTUs from Figure 3, 4 OTUs showed a strong correlation with the 97 metabolites from Figure 5, A and B. The filtering of OTUs is illustrated in the analytical workflow (Supplemental Figure 1). Metabolites are shown on the row in the format of “COMP_ID|Biochemical_Name|Super_Pathway|Sub_Pathway,” and OTU IDs are shown on the column in the format of “OTU_ID|Family.” Three OTUs that match to bacteria species at >99% identity are bolded. On the heatmap, between each OTU and each metabolite, a positive correlation is shown in red, and a negative correlation is shown in blue. OTUs were divided into 4 clusters based on same height on the dendrogram shown on the column using R function cut.tree. Similarly, metabolites were divided into 5 groups based on same height on the dendrogram shown on the row. Annotation to metabolite groups 1–5 was added based on the distribution of Spearman’s correlation coefficient ρ among the healthy-abundant OTU clusters 1–3 consisting of 21 OTUs (Supplemental Figure 12). Cluster 4 only contains 1 OTU from allergic-abundant bacteria and, hence, was not used for metabolite group annotation. Spearman’s correlation was used.

Figure 8. Two bacterial species correlated with pathways that were differentially abundant between healthy and allergic twins.

(A) Distribution of pathways in group 1 and 2 metabolites from Figure 7. Top: Superpathways in each group. The fraction of metabolites from each superpathway on the y axis was calculated by the number of metabolites that belong to this pathway divided by the total number of metabolites in a group. Bottom: Number of metabolites that belong to each subpathway; (left) group 1, (right) group 2. SAM, S-adenosylmethionine. (B) OTU 556835 (family Acidaminococcaceae) is significantly more abundant in the healthy group compared with the allergic group by 16S sequencing. This OTU was annotated as Phascolarctobacterium faecium at the species level. (C) Quantitative PCR (qPCR) validates the abundance differences between healthy and allergic groups using P. faecium–specific primers. (D) OTU188079 (family Ruminococcaceae) is significantly more abundant in the healthy group compared with the allergic group by 16S sequencing. This OTU was annotated as Ruminococcus bromii at the species level. (E) qPCR validates the abundance differences between healthy and allergic groups using R. bromii–specific primers. Units shown on the y axis in C and E represent 2–Ct normalized to total 16S rRNA copies per gram of fecal material and multiplied by a constant (1 × 1022) to bring all values above 1 (see Methods). In B–E, n = 30 samples (15 twin pairs) with DNA available for qPCR validation are shown (10 healthy, 20 allergic). Each dot denotes 1 sample. The bounds of the boxes represent the 25th and 75th percentiles, the horizontal center lines indicate the medians, and the whiskers extend to data points within a maximum of 1.5 times the IQR. DS-FDR was used in B and D. In C and E, qPCR data were log10 transformed, and 2-tailed Wilcoxon’s rank-sum test was used.