Probiotic bacteria produce conjugated linoleic acid locally in the gut that targets macrophage PPAR γ to suppress colitis

Abstract

Background: Inflammatory bowel disease (IBD) therapies are modestly successful and associated with significant side effects. Thus, the investigation of novel approaches to prevent colitis is important. Probiotic bacteria can produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anti-inflammatory effects. This study aimed to investigate the cellular and molecular mechanisms underlying the anti-inflammatory efficacy of probiotic bacteria using a mouse model of colitis.

Methodology/principal findings: The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in a mouse model of DSS colitis. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen, blood and colonic lamina propria cells were phenotypically and functionally characterized. Fecal samples and colonic contents were collected to determine the effect of VSL#3 and CLA on gut microbial diversity and CLA production. CLA and VSL#3 treatment ameliorated colitis and decreased colonic bacterial diversity, a finding that correlated with decreased gut pathology. Colonic CLA concentrations were increased in response to probiotic bacterial treatment, but without systemic distribution in blood. VSL#3 and CLA decreased macrophage accumulation in the MLN of mice with DSS colitis. The loss of PPAR γ in myeloid cells abrogated the protective effect of probiotic bacteria and CLA in mice with DSS colitis.

Conclusions/significance: Probiotic bacteria modulate gut microbial diversity and favor local production of CLA in the colon that targets myeloid cell PPAR γ to suppress colitis.

Figure 1. VSL#3 and conjugated linoleic acid (CLA) ameliorate disease activity.

(A), colonic gross pathology (B), leukocyte infiltration (C), mucosal thickening (D), and down-modulate colonic expression of tumor necrosis factor (TNF-α, E) and monocyte chemoattractant protein 1 (MCP-1, F) in mice with DSS colitis. Data are represented as mean ± standard error. Statistically significant differences (P<0.05) when compared to control (CONT) mice are depicted with an asterisk.

Figure 2. VSL#3 and conjugated linoleic acid (CLA) modulate immune cell subsets in mesenteric lymph nodes from wild type mice were immunophenotyped to identify CCR2+ (A) and TNF-α+ (B) macrophage subsets by flow cytometry.

Data are represented as mean ± standard error. Statistically significant differences (P<0.05) when compared to control (CONT) mice are depicted with an asterisk.

Figure 3. VSL#3 and conjugated linoleic acid (CLA) ameliorate colonic histopathology of PPAR γ-expressing but not macrophage-specific PPAR γ-null mice.

Representative photomicrographs of colons from PPAR γ-expressing (PPAR γ flfl Cre−, top panels A–C) and macrophage-specific PPAR γ null (PPAR γ flfl Cre+, bottom panels D–F) control untreated (A&D), CLA-treated (B&E), and VSL#3-treated (C&F) mice. The colonic sections were excised on day 7 of DSS challenge, stored in formalin, sectioned and stained with hematoxylin and eosin staining. Panels B and C, corresponding to PPAR γ-expressing mice treated with CLA and VSL#3, show improved colitis. The beneficial effects of CLA and VSL#3 are abrogated in colons from mice conditionally deficient in macrophage PPAR γ (E&F). Arrows indicate leukocytic infiltration, asterisks indicate edema, filled arrowheads indicate crypt hyperplasia, open arrowheads indicate epithelial erosion and ulceration, and MT denotes mucosa thickenning. Original magnification at 100×.

Figure 4. VSL#3 and conjugated linoleic acid (CLA) regulate colonic lamina propria lymphocytes (LPL) and colonic gene expression.

Expression of toll-like receptor-4 (TLR-4) was assessed by quantitative real time RT-PCR (A). LP macrophages were immunophenotyped to identify their production of MCP-1 by intracellular staining and flow cytometry (B). Values are means ± SEM, n = 10. Statistically significant differences (P<0.05) when compared to Cre−, Cre+, or both control (CONT) mice are depicted with an asterisk, two asterisks, or the # symbol, respectively.

Figure 5. VSL#3 and conjugated linoleic acid (CLA) alter the composition of the colonic microbial community.

Terminal restriction fragment (TRF) length polymorphism reveals that VSL#3 and CLA treatment alters luminal microbial community composition (A). Ordination plot by multidimensional scaling of luminal microbial communities, with circle size depicting histology score (B). Shannon diversity and Margalef richness (mean +/− SEM); groups were compared by ANOVA plus Tukey's test (C). SIMPER analysis; percent contribution of TRFs (top 80%) that contribute most to similarity within each group (D). Ordination plot in panel A, with circle size depicting standardized abundance of TRF H116.

Figure 6. VSL#3 and conjugated linoleic acid (CLA) regulate plasma and colonic CLA concentrations.

Cis-9, trans-11 CLA concentrations were measured in plasma (A) and colonic contents (B) by gas chromatography. Values are means ± SEM, n = 8. Statistically significant differences (P<0.05) when compared to control (CONT) mice are depicted with an asterisk.

Figure 7. Proposed model for a mechanism of action underlying the protective effects of VSL#3 probiotic bacteria in mouse models of gut inflammation and cancer.

Colonization with VSL#3 probiotic bacteria modulate gut microbial diversity and favor local production of conjugated linoleic acid (CLA) in the colon that targets myeloid cell peroxisome proliferator-activated receptor γ (PPAR γ) to suppress colitis.