Serum Metabolomics in a <i>Helicobacter hepaticus</i> Mouse Model of Inflammatory Bowel Disease Reveal Important Changes in the Microbiome, Serum Peptides, and Intermediary Metabolism

Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder of the bowel. The etiology remains unknown, but IBD is immune-driven and multiple factors including genetic, environmental, and microbiological components play a role. Recombinase-activating gene-2-deficient (Rag2^–/–) mice infected with <i>Helicobacter hepaticus</i> (<i>H. hepaticus</i>) have been developed as an animal model to imitate naturally occurring inflammatory events and associated key features of chronic inflammatory responses in humans. In this study, we have combined mass spectrometry-based metabolomics and peptidomics to analyze serum samples of Rag2^–/– mice infected with <i>H. hepaticus</i>. Metabolomics profiling revealed that <i>H. hepaticus</i> infection dramatically changed numerous metabolite pathways, including tryptophan metabolism, glycerophospholipids, methionine-homocysteine cycle, citrate cycle, fatty acid metabolism and purine metabolism, with the majority of metabolites being down-regulated. In particular, there were notable effects of gut microflora on the blood metabolites in infected animals. In addition, the peptidomics approach identified a number of peptides, originating from proteins, including fibrinogen, complement C4, and alpha-2-macroglobulin, with diverse biological functions with potentially important implications for the progress of IBD. In summary, the strategy of integrating a relevant animal model and sensitive mass spectrometry-based profiling may offer a new perspective to explore biomarkers and provide mechanistic insights into IBD

Glycan receptor distribution for human-adapted influenza A virus HA.

<p>Recombinant SC18 (H1) HA and Alb58 (H2) HA expressed in insect cells were used to stain ferret cranial, caudal and lung hilar regions at a concentration of 20 µg/ml. <b>A</b>, SC18 HA stained only the submucosal glands in all the three regions of ferret respiratory tract. This is in contrast to human trachea where goblet cells (marked as *) were stained by SC18 HA. This restricted binding pattern of SC18 HA can be attributed to its stringent binding specificity to long α2–6 linked (6′SLNLN) glycans. <b>B</b>, Alb58 HA stained the submucosal glands, the underlying mucosa and some goblet cells (in the caudal region) similar to SNA-I. This staining pattern is similar to that in human trachea wherein all the goblet cells (marked as *), submucosal glands and the glycocalyx are stained with Alb58HA. The significant goblet cell staining of Alb58 HA of human trachea as compared to ferret respiratory tract is in accordance with predominant expression of O-linked α2–6 sialic acid in human tracheal goblet cells as compared to that in ferret respiratory tract (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027517#pone-0027517-g003" target="_blank"><b>Figure 3</b></a>). The nuclei were stained with PI (<i>red</i>). The apical surface is marked with a <i>white</i> arrow.</p

α2–3 linked glycan distribution in ferret respiratory tract.

<p>MAL-II lectin (<i>green</i>) was used to stain ferret cranial, caudal and lung hilar regions. As seen from the images, MAL-II stained the submucosal glands, the underlying mucosa and some goblet cells (marked as *) in the caudal region. There was no staining of the lung hilar region indicating an absence of α2–3 glycans. The nuclei were stained with PI (<i>red</i>). The apical surface is marked with a <i>white</i> arrow.</p

Co-staining of recombinant Alb58 (H2) HA with Jacalin.

<p>Ferret cranial, caudal and lung hilar regions were co-stained with 10 µg/ml of Jacalin (FITC labeled) and 20 µg/ml of recombinant Alb58 HA. The co-staining is indicated by a <i>yellow</i> staining pattern. As seen from the images, co-staining was predominantly seen in the submucosal glands of ferret trachea and lung hilar region. Some co-staining was also seen in the goblet cells of trachea (marked as *) and lung hilar region. This staining pattern is in contrast to human trachea wherein co-staining is predominantly seen in goblet cells (marked as *). The apical surface is marked with a <i>white</i> arrow.</p

Visual scoring of lectin staining of various cell types in tissue sections used in this study.

<p>− No staining + Moderate staining +/− Only few cells stained (<10%) ++ Extensive staining.</p

PLS-DA and OPLS component contributions for discrimination (R² Y) and variance (Q²) of necrosis at 3 DPI.

<p>Model results are indicated based on using serum or tissue targets alone as well as combined models using the top variables from each independent model.</p

<i>C. rodentium</i>-induced colonic effects in C57BL/6 mice.

<p>(<b>A</b>) Mock inoculated animals at 0 DPI with normal colonic architecture where epithelial integrity and goblet cells appear intact. (<b>B</b>) Colon at 3 DPI showing epithelial defects at the top of the crypt. (<b>C</b>) Colon at 14 DPI demonstrating hyperplastic crypts and depletion of goblet cells. (<b>D</b>) <i>C. rodentium</i> induced statistically significant histological changes as early as 7 DPI (inflammation, edema, epithelial defects) in colonic sections and found to be most dramatic at 14 DPI. Crypt atrophy and minimal dysplastic changes were only noticeable at 14 DPI. Changes in inflammation, edema, epithelial defects, and hyperplasia as early as 3 DPI were noted, but failed to reach statistical significance (Kruskal-Wallis non-parametric test with Dunn's multiple comparison test: <b>*</b> P<0.05, <b>**</b> P<0.01, <b>***</b> P<0.001). Symbols indicate individual animals and lines indicate group means.</p

Liver-specific OPLS analysis of <i>C. rodentium</i> infected animals at 3 DPI.

<p>OPLS analysis of <i>C. rodentium</i> infected animals at 3 DPI using serum cytokines and chemistries (X) for prediction of ALT levels (Y). (<b>A</b>) Mice segregated well in the predictive component (principal component 1) with an R²X (1) = 0.35, indicating this component captured ∼35% of the variance present in the X variables. (<b>B</b>) The predictive weight and covariation of serum targets (<b>X variables, </b><b>black triangles</b>) in relation to serum ALT (<b>Y, </b><b>red square</b>). (<b>C</b>) Observed vs predicted plot for ALT resulted in a R² = 0.9892 indicating a highly predictive model based on hepatic cytokines. (<b>D</b>) Table representing the VIPs >1 for the predictive component for serum ALT.</p

Serum-specific PLS-DA analysis of <i>C. rodentium</i> infected C57BL/6 mice at 3 DPI.

<p>(<b>A</b>) Partial least squares discriminate analysis (PLS-DA) showing separation of mice with and without necrosis using the first two principal components. (<b>B</b>) Cytokine covariation based on class discrimination using cytokine targets as independent variables (<b>X, black triangles</b>) and pathological states (presence of absence of necrotic lesions) as the dependent dummy variables (<b>Y, red squares</b>). (<b>C</b>) Variables in projection (VIPs) for principal components 1 and 2, where targets with values >1 have positive influence in discriminating between classes. Table represents the serum-specific VIPs and their respective scores that best discriminate necrotic from non-necrotic mice at 3 DPI.</p

<i>C. rodentium</i>-induced necrosis and histological liver changes in C57BL/6 mice.

<p>(<b>A</b>) Control livers appeared normal with minimal observable histology. (<b>B</b>) At 3 DPI the appearance of a focus of lobular injury and inflammation indicating a pro-inflammatory state (indicated by <b>#</b>). (<b>C</b>) Multifocal venous thrombi and associated periportal hepatocellular coagulative necrosis (indicated by <b>*</b>) was observed at 3 DPI suggestive of thrombotic ischemic injury. (<b>D</b>) Higher magnification (400×) view showing necrotic hepatocytes with eosinophilic cytoplasm, appearance of pyknotic or absence of hepatic nuclei, and loss of normal cellular architecture. (<b>E</b>) <i>C. rodentium</i> induced histological changes as early as 3 DPI in liver sections, statistically significant at 7 DPI (portal, lobular, interface inflammation, # lobes with >5 inflammatory foci, and hepatitis index score), with moderate improvement by 14 DPI. (<b>F</b>) The degree of necrosis determined by pathological assessment as well as serum ALT measurements. (<b>G</b>) The pattern of necrosis was assessed as centrilobular, midzonal, or periportal in distribution. (Kruskal-Wallis with Dunn's post test compared to controls: <b>*</b> P<0.05, <b>**</b> P<0.01, <b>***</b> P<0.001). Symbols indicate individual animals and lines indicate group means.</p