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Gut Microbes Volume 15, 2023 - Issue 1
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Research Paper

Diet high in linoleic acid dysregulates the intestinal endocannabinoid system and increases susceptibility to colitis in Mice

Poonamjot Deola Department of Molecular, Cell and Systems Biology, University of California, Riverside, CA, USA;b Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USACorrespondence[email protected]
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Paul Rueggerb Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USAView further author information
,
Geoffrey D. Loganb Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USAView further author information
,
Ali Shawkic Division of Biomedical Sciences, University of California, Riverside, CA, USAView further author information
,
Jiang Lic Division of Biomedical Sciences, University of California, Riverside, CA, USAView further author information
,
Jonathan D. Mitchellb Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USAView further author information
,
Jacqueline Yua Department of Molecular, Cell and Systems Biology, University of California, Riverside, CA, USAView further author information
,
Varadh Piamthaib Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USAView further author information
,
Sarah H. Radia Department of Molecular, Cell and Systems Biology, University of California, Riverside, CA, USAView further author information
,
Sana Hasnaina Department of Molecular, Cell and Systems Biology, University of California, Riverside, CA, USAView further author information
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Kamil Borkowskid West Coast Metabolomics Center, Genome and Biological Sciences Facility, University of California Davis, Davis, CA, USAView further author information
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John W. Newmand West Coast Metabolomics Center, Genome and Biological Sciences Facility, University of California Davis, Davis, CA, USA;e United States Department of Agriculture, Agricultural Research Service, Western Human Nutrition Research Center, Davis, CA, USAView further author information
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Declan F. McColec Division of Biomedical Sciences, University of California, Riverside, CA, USAView further author information
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Meera G. Nairc Division of Biomedical Sciences, University of California, Riverside, CA, USAView further author information
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Ansel Hsiaob Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USAView further author information
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James Bornemanb Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USACorrespondence[email protected]
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Frances M. Sladeka Department of Molecular, Cell and Systems Biology, University of California, Riverside, CA, USACorrespondence[email protected]
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Article: 2229945 | Received 08 Feb 2023, Accepted 21 Jun 2023, Published online: 03 Jul 2023

Figures & data

Figure 1. Experimental design.

Notes: (a) In vivo experiments using four different models (C57BL6 adult males) and the indicated treatments with high fat diets (SO+f, soybean oil plus fiber; SO, soybean oil; Plenish, low LA soybean oil and olive oil), DSS, and/or gavage with linoleic acid (LA). (b) In vitro experiments with the indicated bacterial strains and treatment of mammalian cells (CaCO2, J774) or growth condition. The assays used in each model are indicated as well as the figures presenting the results.
Figure 1. Experimental design.

Figure 2. A diet high in LA increases susceptibility to colitis in IL10−/− mice. IL10−/− mice fed SO+f or Viv diet for 10 weeks.

Notes: (s) Average weekly body weights and at harvest; the body weight at harvest is a few days after the last data point in the weekly body weight graph. (b) hemoccult assay. (c) colon length and crypt length. Scale bar is 400 microns. Colon sections are shown in Supplementary Figure 1. * vs Viv. T-test, P < .05 N = 5–12 per group.
Figure 2. A diet high in LA increases susceptibility to colitis in IL10−/− mice. IL10−/− mice fed SO+f or Viv diet for 10 weeks.

Figure 3. A diet high in LA increases colitis susceptibility and decreases barrier function in WT and α1HMZ mice.

Notes: (a) WT mice on Viv chow or SO+f diet for 15 weeks were treated with 2.5% DSS in their drinking water for 6 d: % weight loss of DSS-treated mice, body weight at harvest; the body weight at harvest is a few days after the last data point in the weekly body weight graph. * vs Viv P < .05, T-test N = 3–4 per group (b) Schematic of the mouse Hnf4a gene showing the two promoters (P1 and P2) (top), and the exon-swap (1D to 1A) that drives the expression of only P1-HNF4α in α1HMZ mice. α1HMZ mice on Viv chow or SO+f diet for 15 weeks were treated with 2.5% DSS for 6 d. (c) % body weight loss, weight at harvest, * vs α1HMZ Viv P < .05, T-test N = 3–4 per group. (d) Representative colonic histology. Big arrow, immune infiltrate; small arrow, loss of crypt structure; line, thickening of muscularis in SO+f. Scale bar is 400 microns. (e) Colon length and crypt length (at least 10 crypts were measured per mouse) Additional sections are shown in Supplementary Figures S2 and 3. a vs untreated WT, b vs untreated α1HMZ Viv, c vs untreated α1HMZ SO+f. One-way ANOVA, Tukey’s post-hoc. N = 5–12 per group.
Figure 3. A diet high in LA increases colitis susceptibility and decreases barrier function in WT and α1HMZ mice.

Figure 4. A diet high in LA increases immune dysfunction and decreases barrier function in WT and α1HMZ mice.

Notes: Immune cell analysis (Eos, eosinophils; Mono, monocytes; Neutro, neutrophils) in DSS-treated WT (a) or α1HMZ (b) mice (see Supplementary Figure S2D and S3D for data from untreated mice); * vs WT or α1 HMZ Viv P < .05, T-test was performed between Viv and SO+f for each cell type, N = 3–4 per group. (c) Epithelial barrier permeability in WT and α1HMZ mice fed either Viv or SO+f diets for 12 weeks or SO+f for 8 weeks, followed by 4 weeks of Viv (reversed) (one outlier removed from WT reversed group). * vs Viv, ** vs Viv and Reversed. P < .05, one-way ANOVA, Sidak’s post hoc comparison. N = 6–22 per group. (d) HNF4α immunoblots of whole cell extracts (WCE, 30 µg) from distal colon of WT mice fed Viv chow or SO+f diet for 12 weeks or SO+f for 8 weeks followed by Viv chow for 4 weeks (reversed). Each lane contains WCE from a different mouse. P1 control-nuclear extract from HCT116 cells expressing P1-HNF4α; P2 control-nuclear extract from α7HMZ mouse (see Supplementary Figure S4 for entire blots for WT mice and P1 blot for α1HMZ mice). (e) Quantification of the P1-and P2-HNF4α immunoblot signals (shown in ) normalized to total protein, as determined by actin staining of the same blot. * P < .05, one-way ANOVA, Tukey’s post hoc comparison. N = 3–4 per group.
Figure 4. A diet high in LA increases immune dysfunction and decreases barrier function in WT and α1HMZ mice.

Figure 5. A diet high in LA increases the abundance of SO-mAIEC in WT mouse intestines.

Notes: (a) Bacterial species plots of intestinal epithelial cells (IECs) from the small and large intestines (N = 4–5) of mice fed Viv chow or an SO diet, with no added fiber. (b) Correlations between indicated mouse metadata and log10 of SO-mAIEC relative abundance in the IECs of mice fed Viv or SO+f diets. Spearman’s correlation coefficient (r) (for the body weight and adipose tissue) and Pearson correlation coefficient (for colon length) (r) goodness of fit or R2 values for linear regression and P value of the correlations (P) are indicated on the graphs. (c) Phenotypic characterization of the E. coli isolate enriched by SO (SO-mAIEC), compared with the human AIEC LF82 and the nonpathogenic E. coli K12. Assessments were made for bacterial adherence to Caco-2BBe cells, intracellular invasion of CaCo-2BBe cells, and replication in J774A.1 murine macrophages as indicated. * P < .05, one-way ANOVA, Tukey’s post hoc comparison. N = 12 across four experiments.
Figure 5. A diet high in LA increases the abundance of SO-mAIEC in WT mouse intestines.

Figure 6. Soybean oil increases oxylipins and decreases endocannabinoid system metabolites in SO-mAIEC cultured in vitro.

Notes: (a) Experimental workflow and Principal Components Analysis (PCA) of oxylipin and endocannabinoid/NAE metabolites in SO-mAIEC grown with or without SO in the media. Absolute levels of fatty acids (b), oxylipin metabolites (c), endocannabinoid system metabolites (d) and (e) prostaglandins measured in SO-mAIEC grown in vitro in the presence or absence of SO in the media. *P < .05, T-test. N = 6 per group. (f) Growth curves for SO-mAIEC and E. coli K-12 grown in Minimum Essential Medium (M9) with LA, ethanol, or glucose as the carbon source. *P < .05, T-test. N = 3 replicates per culture. See Supplementary Figure S6.
Figure 6. Soybean oil increases oxylipins and decreases endocannabinoid system metabolites in SO-mAIEC cultured in vitro.

Figure 7. A diet high in LA decreases barrier function and alters the metabolome in the intestines of conventionally raised and germ-free mice.

Notes: (a) Epithelial barrier permeability as measured by FITC-Dextran in the blood in GF mice fed either Viv chow or SO+f diet for 12 weeks. T-test. N = 3–5 per group. See Supplementary Figure S6D for comparison of Conv and GF FITC. (b) PCA of oxylipin and endocannabinoid/NAE metabolites in IECs isolated from Conv and GF mice fed either Viv chow or SO+f diet for 8 weeks. N = 6 per group. (c) Schematic and Venn analysis of metabolomics data in B. Absolute levels of fatty acids (d), graph showing ratio of product-substrate for metabolism of LA to AA (omega-6) and ALA to EPA (omega-3) (e) and absolute levels of endocannabinoids/NAEs (f) measured in IECs from Conv and GF mice fed either Viv chow or SO+f diets for 8 weeks. a vs Conv Viv, b vs Conv SO+f, c vs GF Viv P < .05, P = .07 for AA Conv Viv vs GF Viv, One-way ANOVA (Holm-Sidak posthoc). N = 6 per group. See Supplementary Figures S7 and S8.
Figure 7. A diet high in LA decreases barrier function and alters the metabolome in the intestines of conventionally raised and germ-free mice.

Figure 8. Impact of LA on gut microbiome.

Notes: WT mice on Viv chow, SO or PL diets for 12 weeks were treated with 2.5% DSS for 6 d, followed by 3 d recovery: % body weight loss (a) and colonic histology (b) of DSS-treated mice. Big arrow, immune infiltrate; small arrow, loss of crypt structure; Scale bar is 400 microns. Additional sections are shown in Supplementary Figure 9. (c) Disease activity index (DAI) after 6 weeks on an indicated diet. See Supplementary Table 2 for details on DAI scores. * vs Viv, a vs untreated WT Viv, b vs untreated WT SO+f. T-test, P < .05 N = 5–12 per group.
Figure 8. Impact of LA on gut microbiome.

Figure 9. Proposed model by which a soybean oil-based HFD increases susceptibility to colitis.

Notes: (a) Impact of a diet high in soybean oil on the intestinal epithelium showing specific effects on host (left) and microbial cells (right) which ultimately leads to disruption of barrier function and IBD. (b) Simplified metabolic pathway of linoleic (LA) and alpha-linolenic acid (ALA). (c) Disrupted balance between ALA- and LA-derived anti- and pro-inflammatory metabolites caused by a diet high in soybean oil, which is tipped even further by the pathobiont SO-mAIEC. See Discussion for additional details.
Figure 9. Proposed model by which a soybean oil-based HFD increases susceptibility to colitis.
Supplemental material

Supplemental Material

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Data availability statement

The bacterial rRNA ITS sequences have been deposited in the National Center for Biotechnology Information (NCBI)’s Sequence Read Archive (SRA) under the SRA BioProject Accession PRJNA622821 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA622821/). The metabolomics data supporting the findings of this study are available within the article and the supplementary materials.

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