ESM promotes proliferation of intestinal cell and inhibits their LPS-induced expression of major inflammatory cytokines

To determine the role of ESM in intestinal inflammation, we first addressed its impact on LPS-stimulated Caco-2 cells. After a 24-hr ESM treatment, the cell viability was significantly increased both with and without the LPS stimulation (Fig. 1a). The interleukin (IL)-6 concentration in the medium was significantly lower in LPS + ESM-treated cells compared to LPS-stimulated cells alone (Fig. 1b). The real-time reverse transcription-polymerase chain reaction (RT-PCR) results indicated the following in LPS-treated cells: the expression of genes for IL-1β (a vital mediator of the inflammatory response) were significantly down-regulated by the supplementation with ESM; the same supplementation up-regulated the cell proliferation-related factors connective tissue growth factor (CTGF) and platelet-derived growth factor alpha (PDGFA), and the potent vasoconstrictor peptide endothelin 1 (EDN1) (Fig. 1c).

Figure 1: Effects of ESM on LPS-stimulated Caco-2 cells. (a) Cell viability, (b) IL-6 concentration in medium, (c) gene expression in cells. Results are mean ± SE in each group (n = 3). Data with different letters (a,b,c) are significantly different at P < 0.05 by Dunnett’s test. Full size image

ESM-supplemented mice displayed ameliorated severity of DSS-induced colitis

Since cell proliferation is associated with intestinal epithelial restitution in injured tissue, we next investigated whether ESM promotes intestinal epithelial restitution and ameliorates the intestinal injury in DSS-induced colitis. Only nonsignificant differences in the total food and water intake before DSS-drinking were observed among the three groups, and in the DSS intake (water intake after DSS administration) between the DSS and D-ESM8 groups (Suppl. Table S3).

The mice that received DSS alone showed significant body weight loss (Fig. 2a) and diarrhea (Suppl. Fig. S2), resulting in significantly severe disease activity index (DAI) values (Fig. 2b) as well as shortened colon lengths (Fig. 2c), reduced weight of the gastrocnemius muscle (Fig. 2d), increased plasma IL-6 levels (Fig. 2e) and augmented colonic MPO activity (P = 0.12, Fig. 2f). The D-ESM8 mice revealed low susceptibility to these pathological conditions of IBD, resulting in a significant amelioration of DAI from day 7. In addition, a significant loss of crypts and inflammatory cell infiltration into the mucosa were observed in the H&E-stained colon sections of the DSS group, whereas ESM supplementation revealed clear re-epithelialization of ulcerated areas (Fig. 2g).

Figure 2: General characteristics of the study groups. (a) body weight, (b) DAI, (c) length of colon, (d) weight of the gastrocnemius muscle, (e) plasma IL-6 levels, (f) MPO activity in colon, and (g) H&E staining of colon. The rectangle encircles the part of deprived colon crypts and arrows are pointing to the infiltrated inflammatory cells. CON, control mice; DSS, mice administered only DSS; D-ESM8, mice administered DSS and ESM (80 g kg−1). Results are mean ± SE in each group (n = 8). Data with different letters (a,b,c) are significantly different at P < 0.05 by Dunnett’s test. Full size image

Variations in the colonic gene expression profile

To obtain further insight into the modulating effect of ESM during intestinal inflammation, we performed a DNA microarray analysis to unravel the biological pathways involved. The most highly ranked functional clusters and categories for genes that are altered in the colon in accord with the Ingenuity Pathway Analysis (IPA, http://www.ingenuity.com/, date accessed: Jan. 2014) are those involved in the functions of immune cells and epithelial restitution (Suppl. Table S4). In each immune cell function-related category, inflammatory cytokines and chemokines were highlighted, such as Il1β, Il6, and members of the chemokine (C-X-C motif) ligand (CXCL) family and the chemokine (C-C motif) ligand (CCL) family. All of these up-regulated genes (Il1β, Il6, Cxcl9, Cxcl13, Ccl9 and Ccl11) in the DSS group were suppressed in the D-ESM8 group (Fig. 3a). Exposure of DSS-treated mice to ESM also down-regulated the gene expression levels of the matrix metalloproteinase (MMP) family (Mmp7 and Mmp9) and up-regulated the gene expression of tissue inhibitors of metalloproteinase (Timp2 and Timp3), which are involved in the breakdown of the extracellular matrix and tissue remodeling in normal physiological processes and disease progression.

Figure 3 Colonic mRNA expressions of genes related to (a) inflammation as evaluated by RT-PCR, (b) nutritional immunity by RT-PCR, and (c) intestinal cell proliferation and restitution as shown by microarray data. The numbers in the upper left spaces are P-values indicating the gene proportion mapping to a function or pathway analyzed via an IPA. V-shape, cytokine/growth factor; spiral, enzyme; dumbbell, transcription regulator; Y-shape, transmembrane receptor; cupule, transporter; three-leaf, kinase; double circle, complex; circle, other; gear, function. CON, control mice; DSS, mice administered only DSS; D-ESM8, mice administered DSS and ESM (80 g kg−1). Results are mean ± SE in each group (n = 8). Data with different letters (a,b,c) are significantly different at P < 0.05 by Dunnett’s test. Full size image

In addition, the expression levels of the genes coding for antimicrobial peptides and microbial growth related genes (lipocalin-2, Lcn2; regenerating islet-derived 3 gamma, Reg3γ; S100 calcium binding protein, S100a8, S100a9 and formyl peptide receptor 1, Fpr1), as well as an oxidative activity-associated gene nitric oxide synthase 2 (Nos2) were also lower in the D-ESM8 group compared to the DSS group (Fig. 3b).

Consistent with the ESM’s regulatory action in Caco-2 cells, the ESM diet attenuated the transcription levels of critical intestinal epithelial proliferation- and restitution-related genes, which are involved in the migration of cells, homing, organization of the cytoskeleton, proliferation of cells, and necrosis (Fig. 3c, Suppl. Fig. S3).

LPS responses in ESM-supplemented mice

Since ulcerative colitis is associated with an increased bacterial translocation to the liver and an elevated LPS level is linked to increased intestinal permeability9, we examined the LPS level in the liver of DSS-treated mice. This level tended to be suppressed in the D-ESM8 group compared to the DSS group (Fig. 4a). ESM also decreased the hepatic gene expression of biomolecules involved in LPS responses (Fig. 4b), including LPS binding protein (Lbp), Cd14 and toll-like receptor (Tlr4) (Fig. 4c) and the chemokines Ccl6, Cxcl11 and Cxcl13 (Fig. 4d). The metagenomic analysis revealed that the relative abundance of one of the LPS-producing pathogenic bacteria, the Enterobacteriaceae family member Escherichia coli (E. coli) was increased in the DSS groups, whereas ESM restored the E. coli abundance to near-normal levels (Fig. 4e).

Figure 4: LPS responses in liver and producing bacteria. (a) LPS levels in the liver. (b) The LPS response-related IPA pathway. Red and green indicate up- and down-regulation, with the more intense color indicating a greater change. (c) Hepatic mRNA expressions of genes related to the LPS response and (d) inflammation. (e) E. coli abundance in the cecum content by metagenome analysis and RT-PCR. (f) Correlations between E. coli, LPS levels and DAI. CON, control mice; DSS, mice administered only DSS; D-ESM8, mice administered DSS and ESM (80 g kg−1). Results are mean ± SE in each group (n = 8). Data with different letters (a,b,c) are significantly different at P < 0.05 by Dunnett’s test. Full size image

In addition, there was a remarkably high correlation (r = 0.90) between the results of the real-time RT-PCR and the metagenome data of E. coli relative abundance (Fig. 4f). Similarly, the positive correlations between the abundance of E. coli and the LPS concentration (r = 0.63), as well as between the abundance of E. coli and the inflammation score DAI (r = 0.53) (Fig. 4f) indicate that the suppressive effect of ESM on colitis may be associated with its negative impact on the abundance of E. coli which subsequently suppressed inflammation and tissue injury.

Reduced Th (T helper) 17 number in ESM-supplemented mice

Given that mesenteric lymph nodes (MLNs) function as the primary gut-associated inductive site where naïve T cells first encounter enteric antigens and are activated as disease-producing colitogenic Th1 and/or Th17 cells10,11, we isolated MLNs and evaluated the number of Th17 cells by flow cytometry. The results revealed that the DSS administration induced significant CD4+ IL-17A frequency, which was significantly attenuated by ESM (Fig. 5a). Corroborating the flow cytometry analysis results, the gene expression findings showed a significantly lower colonic expression of Il17a in the D-ESM8 mice compared to that of the DSS mice (Fig. 5b), and there was also a tendency (P = 0.09) for low levels of IL-6 in the media of 48 h-incubated MLNs (Fig. 5c). The level of segmented filamentous bacteria (SFB) (Fig. 5d), which promoted the differentiation of Th17 cells, was significantly higher in the DSS group compared to that of the control mice, and this high SFB abundance was reversed in the D-ESM8 group. These findings suggest the possible ability of ESM to regulate the function of SFB and the differentiation of Th17 cells.

Figure 5: The number of Th17 cells and related bacteria. (a) The Th17 number in MLNs shown by the flow cytometric analysis. (b) The Il17a expression in the colon. (c) The IL-6 levels in the medium of 48 h-incubated MLNs. (d) SFB abundance in cecum contents. CON, control mice; DSS, mice administered only DSS; D-ESM8, mice administered DSS and ESM (80 g kg−1). Results are mean ± SE in each group (n = 8). Data with different letters (a,b,c) are significantly different at P < 0.05 by Dunnett’s test. Full size image

The multifaceted integrated omics analysis revealed ameliorated energy metabolism by ESM supplementation

Hepatobiliary manifestations are frequently observed in IBD patients, because of the close anatomic link between the gastrointestinal tract and the hepatobiliary system, and due to the mesenteric venous drainage ascending via the portal vein into the liver9. This prompted us to conduct an omics analysis to elucidate the relationship between colitis and various hepatic functions. The results of this analysis demonstrated that the top hepatic protein functional categories of the differentially changed proteins were those involved in the urea cycle, gluconeogenesis, glycolysis, the tricarboxylic acid (TCA) cycle, ketogenesis, and fatty acid β-oxidation (Suppl. Table S6). These findings also correspond to the transcriptome profile (Fig. 6); that is, the expressions of these pathway related genes were suppressed in the DSS mice compared to the control mice, whereas the supplementation with ESM elevated these expressions.

Figure 6: The energy metabolism-related genes, proteins and metabolites in the liver revealed by the omics analysis. (a) TCA cycle, (b) urea cycle, (c) glycolysis pathway. The numbers in () are fold changes of the gene(g) expression, protein(p) and metabolite(m) levels, respectively, where red indicates an increase and green indicates a decrease in the two comparisons (DSS vs. CON and D-ESM8 vs. DSS). (d) Correlation diagram of each pathway. The colorful arrows represent promoted or increased (red) and suppressed or decreased (green) in the two comparisons. Full size image

Distinct improvements of hepatic intermediates were observed in the TCA cycle (citrate, cis-aconitate, isocitrate) (Fig. 6a, Suppl. Table S8) and glycolysis (glucose-1-phosphate, G-1-P; fructose-1,6-bisphosphate, F-1,6-P) (Fig. 6b) in the DSS mice exposed to ESM. The plasma intermediates of the TCA cycle (citrate, cis-aconitate, isocitrate, 2-oxoglutarate, succinate, fumarate, malate, hydroxyproline and Trp) also indicated that the ESM supplementation improved the weakened glycolysis and the TCA cycle caused by DSS, which is common in inflammatory environments (Suppl. Table S7). Collectively, the expression of energy metabolism-related genes and the levels of proteins and metabolites in glycolysis, the TCA cycle and the urea cycle (Fig. 6a–c) were elevated in the D-ESM8 mice compared to the DSS mice (Fig. 6d).

We also observed changed expressions of proteins involved in mitochondrial dysfunction from functional categories of the proteomic analysis (Suppl. Table S6), including ATP synthase (Atp5c1 and Atp5a1) and NADH hydrolytic enzymes (Ndufab1, Ndufb5, Ndufab10, Ndufv1, Ndufs2, and Ndufs6), which are enzymes involved in the electron transport chain and oxidative phosphorylation (Suppl. Fig. S4). All of these enzymes showed lower levels in the DSS group compared to the CON group, and the levels were preserved in the D-ESM8 group.

ESM improved microbiota dysbiosis and community structure

As the pathogenesis of IBD results from a pathogenic immune response against the gut microbiota12, we next investigated whether the suppressive effects of ESM on DSS-induced colitis also resulted from the alleviation of dysbiosis, by analyzing microbiota functional profiles using a metagenome analysis.

The analysis at the phylum level revealed that the relative abundance of the three most prevalent bacterial phyla was significantly different among the three experimental groups. Bacteroidetes decreased from 67.0% in CON to 34.6% in DSS and recovered to 55.2% in ESM; Firmicutes, from 26.5% to 13.7% and to 18.6%; Proteobacteria, from 0.9% to 40% and to 8% (Fig. 7a), respectively.

Figure 7: Effects of ESM on microbiota dysbiosis and structure as revealed by the metagenome analysis. (a) Phylum level of microbiota profiling (%). (b) Rarefaction curves. (c) Unweighted UniFrac-based principal coordinate analysis. (d) Weighted UniFrac-based principal coordinate analysis. (e) Abundance of IBD-related bacteria. (f) SCFA-producing bacteria. Group names are as in the earlier figures. Results are mean ± SE in each group (n = 8). Data with different letters (a,b,c) are significantly different at P < 0.05 by Dunnett’s test. Full size image

We then assessed two classifications of bacterial diversity termed α and β. According to the α-diversity (rarefaction plot) analysis using Observed species and the Shannon index, the DSS mice exhibited the lowest species richness among the three groups (Fig. 7b), which is expected since a lower richness of bacterial species has been associated with disease states, including IBD in humans and DSS colitis in mice13. The higher richness of the ESM group matched the beneficial effects of ESM on colitis.

We also analyzed the β-diversity — which reveals distinctive views of community structure — by using two phylogenetic distance metrics, i.e., weighted and unweighted Unifrac distances, which measure the presence and absence of abundant lineages and particular bacteria taxa, respectively. The percent variations by the unweighted metric of the CON, DSS and D-ESM8 groups were 21.10%, 7.27% and 13.13%, respectively (Fig. 7c), which indicates that rare bacteria altered the community structure of these groups.

The clustering of the CON, DSS and D-ESM8 groups by the weighted UniFrac distances showed 62.36%, 3.23% and 20.38% variations, respectively indicating that bacterial communities were more evident (Fig. 7d). Regardless of the rare and abundant bacteria, these data suggest that microbial communities are differentially improved by ESM, indicating that the changes in microbiome diversity may be related to the anti-inflammatory effects of ESM.

Increased Enterobacteriaceae, which is a marker of intestinal inflammation and oxidative stress in human IBD and murine colitis, was detected significantly more frequently in the inflammatory DSS groups (Proteobacteria phylum, reaching 39% of the total bacteria) compared to the CON group (Suppl. Table S9). Conversely, Enterobacteriaceae were detected significantly less frequently in the D-ESM8 group (approx. 6%). Similarly, the DSS administration increased the numbers of Enterococcaceae, which were counteracted to near-normal levels by ESM supplementation (Fig. 7e).

In addition, considering that the metabolites of gut microbiota such as short-chain fatty acids (SCFAs) are the primary energy source for colonocytes and directly influence host gene expression, we further focused on the presence of SCFA-producing microbiota (Fig. 7f). The DSS group had a reduced abundance of the SCFA producers Ruminococcaceae (Firmicutes phylum, comprising approx. 10% of the total bacteria) and Porphyromonadaceae (Bacteroidetes phylum) known to be decreased in IBD, all of which were restored by ESM.