Growth effects of N-acylethanolamines on gut bacteria reflect altered bacterial abundances in inflammatory bowel disease

Abstract

Inflammatory bowel diseases (IBD) are associated with alterations in gut microbial abundances and lumenal metabolite concentrations, but the effects of specific metabolites on the gut microbiota in health and disease remain largely unknown. Here, we analysed the influences of metabolites that are differentially abundant in IBD on the growth and physiology of gut bacteria that are also differentially abundant in IBD. We found that N-acylethanolamines (NAEs), a class of endogenously produced signalling lipids elevated in the stool of IBD patients and a T-cell transfer model of colitis, stimulated growth of species over-represented in IBD and inhibited that of species depleted in IBD in vitro. Using metagenomic sequencing, we recapitulated the effects of NAEs in complex microbial communities ex vivo, with Proteobacteria blooming and Bacteroidetes declining in the presence of NAEs. Metatranscriptomic analysis of the same communities identified components of the respiratory chain as important for the metabolism of NAEs, and this was verified using a mutant deficient for respiratory complex I. In this study, we identified NAEs as a class of metabolites that are elevated in IBD and have the potential to shift gut microbiota towards an IBD-like composition.

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Fig. 1: Effects of stool metabolites on bacterial growth in exponential (Vmax) and stationary (max OD600 nm) phases.
Fig. 2: NAEs are elevated in IBD patients and a mouse model of colitis.
Fig. 3: Effects of NAEs on bacterial growth.
Fig. 4: Effects of NAEs on the composition of a complex microbial community.
Fig. 5: Changes in transcriptional activity in complex microbial communities in response to NAE treatment.

Data availability

Metagenomic, metatranscriptomic and transcriptomic data are available in the NCBI Sequence Read Archive as BioProject PRJNA532456. Tables of processed mouse microbial species, monoculture transcriptomic data and chemostat microbial species are available as Supplementary Datasets 4, 7 and 8, respectively. Source data for Figs. 1 and 2 and Extended Data Figs. 3 and 4 are provided with the paper.

Code availability

Custom scripts used to analyse monoculture transcriptomic data are available at https://github.com/broadinstitute/split_merge_pl. The bioBakery tools (KneadData, MetaPhlAn2 and HUMAnN2) used to process meta’omic sequencing data from the two chemostats are available via http://huttenhower.sph.harvard.edu/biobakery as source code and installable packages. Downstream analyses were conducted using custom Python and R scripts. This code (and associated usage notes) is available from the corresponding authors upon request.

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Acknowledgements

We are grateful to A. Vrcic and S. Figueroa-Lazu for preparing the metabolites and to T. Poon for coordinating DNA and RNA sequencing. Broad Technology Labs generated metagenomic libraries, and RNA-Seq libraries were constructed and sequenced at the Broad Institute of MIT and Harvard by the Microbial ‘Omics Core and Genomics Platform, respectively. The Microbial ‘Omics Core also provided guidance on experimental design and conducted preliminary analysis for all RNA-seq data. We are deeply indebted to T. Reimels for helpful discussions, editing the manuscript and figure generation. We thank B. Hall, D. Kenny, D. Plichta, Z. Costliow, G. Jasso, J. Rush and X. Ke for insightful discussions. This work was funded by grants from the National Institutes of Health (NIH) R24DK110499 and U54DK102557 (C.H. and R.J.X.), R01AT009708 (R.J.X.), P01DK094779 and P40OD010995 (RBS), the Crohn’s and Colitis Foundation (R.J.X.) and the Center for Microbiome Informatics and Therapeutics (R.J.X.).

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Contributions

N.F., A.C.T., H.V. and R.J.X. designed the research. N.F. performed bacterial growth and chemostat experiments. E.A.F. and J.B. analysed metagenomic and metatranscriptomic data. J.W.A. performed mass spectrometry analysis. A.O. and R.B.S. provided mouse stool and helped with mass spectrometry data interpretation. J.L.-P. analysed PRISM and iHMP metabolomics data. T.D.A. and A.G. contributed chemostat communities. T.D.A. also provided bacterial isolates. J.A.-P. confirmed the presence of AEA in PRISM and iHMP stool. N.F., H.J.H., J.A.P., C.B.C., C.H., H.V. and R.J.X. supervised the project. R.B.S., C.H. and R.J.X. acquired funding.

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Correspondence to Hera Vlamakis or Ramnik J. Xavier.

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Extended data

Extended Data Fig. 1 Validation of metabolite screen results in dose assays.

Growth curves are shown for three strains using seven metabolites in multiple concentrations. Growth was monitored over time in a volume of 40 µL per well in 384-well plates. The final concentration of DMSO per treated and control well was 0.25%. Growth curves representative of three independent tests are shown and error bars in controls represent the standard deviation of the mean of six technical replicates.

Extended Data Fig. 2 Correlation between absolute and relative NAE abundances in stool from PRISM subjects.

NAEs detected in stool from PRISM Crohn’s disease (CD) patients (n=21) in absolute abundances (ng mg-1) are plotted against their respective relative abundances8. Pearson correlation coefficients (r) are shown. Progenesis QI (nonlinear DYNAMICS) was used for the extraction of non-targeted LC-MS features and TraceFinder (Thermo Fisher Scientific) was used for the manual peak extraction of known metabolites on basis of their mass to charge ratio (m/z) and retention times determined using authentic standards.

Extended Data Fig. 3 Growth effects of NAEs on intestinal bacteria elevated in IBD.

Palmitoylethanolamide (PEA), linoleoyl ethanolamide (LEA), oleoyl ethanolamide (OEA) and arachidonoyl ethanolamide (AEA) were added to growing cells (in the range of 106 to 108 CFU mL-1) in three concentrations (0 µM, 50 µM and 100 µM), and growth was monitored in an absorbance reader in the anaerobic chamber over time. Controls contained 0.4% DMSO. Growth curves representative of two independent experiments are shown and error bars represent the standard deviation of the mean of three technical replicates. Source data

Extended Data Fig. 4 Growth effects of NAEs on intestinal bacteria depleted or invariable in IBD.

Palmitoylethanolamide (PEA), linoleoyl ethanolamide (LEA), oleoyl ethanolamide (OEA) and arachidonoyl ethanolamide (AEA) were added to growing cells (in the range of 106 to 108 CFU mL-1) in three concentrations (0 µM, 50 µM and 100 µM), and growth was monitored in an absorbance reader in the anaerobic chamber over time. Controls contained 0.4% DMSO. Growth curves representative of two independent experiments are shown and error bars represent the standard deviation of the mean of three technical replicates. Source data

Extended Data Fig. 5 Transcriptional responses of Bacteroides fragilis to linoleoyl ethanolamide (LEA) and arachidonoyl ethanolamine (AEA).

a, Differential gene expression between three independent exponential cultures treated for 10 minutes with a sub-inhibitory concentration (25 µM) of LEA or AEA and controls (0.04% DMSO). Differential expression was determined with edgeR, and gene functions were defined using InterPro (EMBL), the NCBI Conserved Domain Database (CDD) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Selected significantly differentially expressed genes (|log2 fold-change (treated/control)|>0.5, FDR<0.05; Benjamini-Hochberg FDR values were derived from p-values calculated using the likelihood-ratio test) are shown in color. b, Genomic environment of the differentially expressed genes using colors that correspond with (a). Genes in white were not significantly differentially expressed. Coordinate maps refer to the genome of strain B. fragilis ATCC 25285. Gene products that have been experimentally shown to be associated with the outer membrane by LC-MS/MS analysis are in bold (Wilson, M. M., Anderson, D. E. & Bernstein, H. D. Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms. PLoS ONE 10, e0117732 (2015)).

Extended Data Fig. 6 Oleic acid, oleoyl ethanolamide and linoleoyl ethanolamide do not enhance growth of a complex I mutant.

Growth of wild-type (WT) E. coli BW25113 and a derivative deficient for complex I (NADH:quinone oxidoreductase, ΔnuoB) in minimal medium (M9 with glucose 4 g L-1 and 1% trace minerals) supplemented with EtOH 0.05%, 3.5 µM, 7 µM and 14 µM of oleic acid (OA), oleoyl ethanolamide (OEA) or linoleoyl ethanolamide (LEA). Growth curves representative of two independent experiments are shown and error bars represent the standard deviation of the mean of three technical replicates.

Extended Data Fig. 7 Effects of NAEs on the composition of a complex microbial community.

a, Taxonomic abundances in chemostat B at the family level. Vertical colored bars represent the relative abundance of bacterial families in samples 1 and 12 hr after addition of DMSO (0.5%), individual NAEs, or a combination of all four NAEs (denoted as NAE-mix). Individual NAEs were added to a final concentration of 500 µM. In combination, the PEA:OEA:LEA:AEA ratio was 125:125:125:125 µM. Heatmaps show log2 fold-changes in b, family- and c, species-level taxonomic abundances between treated samples and DMSO controls (total n=41 with per-treatment n ranging from 6 to 7). Species that shifted with statistical significance in response to treatment are shown (*q<0.20, **q<0.05; FDR q-values derived from nominal two-tailed p-values of the “treatment” coefficient across per-taxon linear regression analyses). Species enriched (red) and depleted (blue) in PRISM CD stool relative to controls (q≤0.1) are indicated. d, Principal coordinate (PCo) analysis on Bray-Curtis dissimilarities between chemostat B (n=21) and PRISM (n=155) metagenomes. Times of exposure to AEA, LEA and DMSO control are indicated.

Extended Data Fig. 8 Changes in transcriptional activity in complex microbial communities in response to NAE treatment.

We used a linear model combining profiles of functional activity from both chemostats over time to identify gene families (KEGG orthologies) that were consistently differentially expressed under NAE treatment relative to DMSO controls (at the community level; total n=96 ranging from n=15 to 17 per treatment). The top 100 such orthologies ranked by mean absolute log-scaled fold change in relative expression are shown. Each of these orthologies was significantly differentially expressed under at least one treatment after correcting for multiple hypothesis testing. Pathway–treatment pairs with open circles had FDR q<0.2; those with closed circles had FDR q<0.05, while an “x” indicates that measurements were insufficient to perform the test (FDR q-values derived from nominal two-tailed p-values of the ‘treatment’ coefficient across per-orthology linear regression analyses). While trends were most significant under AEA treatment, effect sizes trended similarly under LEA and OEA treatment as well.

Extended Data Fig. 9 Changes in transcriptional activity (KEGG modules) in complex microbial communities in response to NAE treatment.

Log2 relative expression is shown for bacterial families contributing to a selection of KEGG modules in samples from chemostats A and B treated with DMSO control, individual NAEs, a combination of all four NAEs (denoted as NAE-mix), or oleic acid (OA). Family-level relative expression values were computed at the species level, averaged over replicates, and then averaged within-family while weighting by species abundance. Unknown (“x”) values represent cases where a function’s DNA and/or RNA abundance were zero for a given stratification (resulting in non-finite log2 relative expression). Col_sort refers to the measure (treatment, time or chemostat) used to order the metadata columns.

Extended Data Fig. 10 Changes in transcriptional activity (KEGG orthologies) in complex microbial communities in response to NAE treatment.

Log2 relative expression is shown for bacterial families contributing to a selection of KEGG orthologies in samples from chemostats A and B treated with DMSO control, individual NAEs, a combination of all four NAEs (denoted as NAE-mix), or oleic acid (OA). Family-level relative expression values were computed at the species level, averaged over replicates, and then averaged within-family while weighting by species abundance. Unknown (“x”) values represent cases where a function’s DNA and/or RNA abundance were zero for a given stratification (resulting in non-finite log2 relative expression). Col_sort refers to the measure (treatment, time or chemostat) used to order the metadata columns.

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Data 1

Metabolites used in this study. Properties of metabolites used in this study, including metabolite class, name, molecular weight, HMDB ID and formula as found in the Human Metabolome Database (http://www.hmdb.ca).

Supplementary Data 2

IBD–metabolite associations. Model results (t-statistics, P values and q values) for metabolite features associated with the two IBD phenotypes (CD and UC) in the PRISM (n = 155) and iHMP (n = 132) cohorts. This list is reproduced from Franzosa et al.8 and Lloyd-Price et al.12 to include the newly validated arachidonoyl ethanolamine.

Supplementary Data 3

Metabolite effects on bacterial growth. Growth rate (Vmax) and maximum cell growth (max OD600 nm) per concentration of metabolite.

Supplementary Data 4

Rag2−/− mouse microbial species relative abundance profiles. Taxonomic composition of the Rag2−/− mouse faecal microbiota determined using MetaPhlAn263. Units are relative abundance (out of 1.0).

Supplementary Data 5

NAE effects on bacterial growth. Growth rate (Vmax) and maximum cell growth (max OD600 nm) per concentration of NAE.

Supplementary Data 6

Microbial species–NAE associations. Spearman correlations between species relative abundances and NAEs that were differentially abundant in IBD samples from the PRISM cohort (n = 155)8.

Supplementary Data 7

Transcriptomic responses of B. fragilis to LEA and AEA. Transcriptomic data of B. fragilis treated with LEA or AEA for 10 min. Differential expression analysis between DMSO- and NAE-treated cultures was conducted with edgeR60.

Supplementary Data 8

Chemostat microbial species relative abundance profiles. Taxonomic composition of the chemostat community before and after treatment with NAEs, determined using MetaPhlAn263.

Supplementary Data 9

Model results in chemostat communities. Outputs of statistical models comparing microbial species relative abundance, microbial family relative abundance, KO relative expression and KEGG module relative expression to NAE treatment (one tab per feature type) in chemostats A (total n = 66 with per-treatment n ranging from 8 to 10) and B (total n = 41 with per-treatment n ranging from 6 to 7). Columns indicate the dependent variable (for example, an individual species), covariate (for example, treatment with LEA), model coefficient, P value and FDR q value.

Supplementary Data 10

KEGG modules and KEGG orthologies in chemostat communities.

Source data

Source Data Fig. 1

Mean growth curves.

Source Data Fig. 2

Metabolite concentrations.

Source Data Extended Data Fig. 3

Growth curves.

Source Data Extended Data Fig. 4

Growth curves.

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Fornelos, N., Franzosa, E.A., Bishai, J. et al. Growth effects of N-acylethanolamines on gut bacteria reflect altered bacterial abundances in inflammatory bowel disease. Nat Microbiol 5, 486–497 (2020). https://doi.org/10.1038/s41564-019-0655-7

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