Citrobacter rodentium induced liver changes in C57BL/6 mice : animal model of acute inflammatory stress and injury

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011.Each page number preceded by chapter or appendix number. Cataloged from PDF version of thesis.Includes bibliographical references (p. F-150 - F-163).The activation of inflammatory responses, while critical for host defense, contributes to hepatic injury in numerous acute and chronic liver disease states as well as drug-induced liver injury (DILI). The interactions that mediate susceptibility to liver injury and disease, however, are still poorly understood, underscored by the complexity of immune interactions and the diverse cellular composition and functions of the liver. Using Citrobacter rodentium, a well characterized rodent-specific enteric pathogen as a source of extrahepatic inflammatory stress; host liver responses, metabolic dysregulation, and susceptibility to injury in C57BL/6 mice were investigated. For the first time, we show altered liver pathology during the early course of C. rodentium infection, characterized by periportal necrosis indicative of thrombic ischemic injury, correlating with distinct circulating and tissue specific cytokine/chemokine profiles. Using Acetaminophen (APAP), a widely used analgesic and well-characterized hepatotoxin, we evaluated liver responses in isolation and in the context of host inflammation to gain insight into the role of live bacterial infection in altering liver metabolism and susceptibility to DILI. We combined systemic and tissue-specific cytokine/chemokine levels, clinical serum chemistries, and histopathological assessments of hepatic and enteric inflammation and necrosis to measure molecularlevel responses to treatment and their physiological effect. Using principal components analysis (PCA), clustering, partial least squares regression (PLSR), and a combination mutual-information-correlation network, enabled detection and visualization of both linear and nonlinear dependencies between molecules and physiological states across tissues and timepoints. C. rodentium-induced inflammatory stress was finally investigated for its potential in altering drug pharmacokinetics (PK) of substrates varying in their metabolic biotransformation and clearance mechanisms. Infection resulted in increased systemic oral exposure (AUC) of clinically relevant xenobiotics such as verapamil, propranolol, and digoxin. Functionally, these changes were not found dependent on CYP-mediated biotransformation of parent compounds; rather, they appear driven more by proposed gut barrier compromise. In conclusion, gastrointestinal infection with C. rodentium alters systemic and hepatocytes specific responses, not previously appreciated from this enteric pathogen, making it a useful model for studying host-pathogen interactions under acute hepatic inflammatory stress and injury.by Arkadiusz R. Raczynski.Ph.D

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2[superscript −/−] mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2′-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.National Institutes of Health (U.S.) (Grant CA26731)Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant ES002109))Massachusetts Institute of Technology (Merck Fellowship)German Academic Exchange Service (Fellowship

17 -Estradiol and Tamoxifen Prevent Gastric Cancer by Modulating Leukocyte Recruitment and Oncogenic Pathways in Helicobacter Pylori-Infected INS-GAS Male Mice

Helicobacter pylori infection promotes male-predominant gastric adenocarcinoma in humans. Estrogens reduce gastric cancer risk and previous studies demonstrated that prophylactic 17β-estradiol (E2) in INS-GAS mice decreases H. pylori-induced carcinogenesis. We examined the effect of E2 and Tamoxifen, on H. pylori-induced gastric cancer in male and female INS-GAS mice. After confirming robust gastric pathology at 16 weeks post-infection (WPI), mice were implanted with E2, Tamoxifen, both E2 and Tamoxifen, or placebo pellets for 12 weeks. At 28 WPI, gastric histopathology, gene expression and immune cell infiltration were evaluated, and serum inflammatory cytokines measured. After treatment, no gastric cancer was observed in H. pylori-infected males receiving E2 and/or Tamoxifen, while 40% of infected untreated males developed gastric cancer. E2, Tamoxifen and their combination significantly reduced gastric precancerous lesions in infected males compared to infected untreated males (P<0.001, 0.01 and 0.01, respectively). However, Tamoxifen did not alter female pathology regardless of infection status. Differentially expressed genes from males treated with E2 or Tamoxifen (n=363 and n=144, Q<0.05) associated highly with cancer and cellular movement, indicating overlapping pathways in the reduction of gastric lesions. E2 or Tamoxifen deregulated genes associated with metastasis (PLAUR and MMP10) and Wnt inhibition (FZD6 and SFRP2). Compared to controls, E2 decreased gastric mRNA (Q<0.05) and serum levels (P<0.05) of CXCL1, a neutrophil chemokine, leading to decreased neutrophil infiltration (P<0.01). Prevention of H. pylori-induced gastric cancer by E2 and Tamoxifen may be mediated by estrogen signaling and is associated with decreased CXCL1, decreased neutrophil counts and downregulation of oncogenic pathways

Enteric Infection with Citrobacter rodentium Induces Coagulative Liver Necrosis and Hepatic Inflammation Prior to Peak Infection and Colonic Disease

Acute and chronic forms of inflammation are known to affect liver responses and susceptibility to disease and injury. Furthermore, intestinal microbiota has been shown critical in mediating inflammatory host responses in various animal models. Using C. rodentium, a known enteric bacterial pathogen, we examined liver responses to gastrointestinal infection at various stages of disease pathogenesis. For the first time, to our knowledge, we show distinct liver pathology associated with enteric infection with C. rodentium in C57BL/6 mice, characterized by increased inflammation and hepatitis index scores as well as prominent periportal hepatocellular coagulative necrosis indicative of thrombotic ischemic injury in a subset of animals during the early course of C. rodentium pathogenesis. Histologic changes in the liver correlated with serum elevation of liver transaminases, systemic and liver resident cytokines, as well as signal transduction changes prior to peak bacterial colonization and colonic disease. C. rodentium infection in C57BL/6 mice provides a potentially useful model to study acute liver injury and inflammatory stress under conditions of gastrointestinal infection analogous to enteropathogenic E. coli infection in humans.United States. Army Research Office (Institute for Soldier Nanotechnology grant 6915539 (SRT))National Institutes of Health (U.S.) (Grant P01 CA026731)National Institutes of Health (U.S.) (Grant P30 ES02109)National Institutes of Health (U.S.) (Toxicology Training grant ES-070220

<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

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 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

<i>C. rodentium</i> significantly increases Ki-67⁺ labeling index and STAT3 phosphorylation in livers.

<p>(<b>A</b>) Top panels are 200× and lower panels are 400× views of the same frame. Brown-colored pigment indicates positively stained cells for Ki-67. (<b>B</b>) Average number of Ki-67⁺ cells/mm² liver determined in 15 fields (magnification, 400×, 3 fields/lobe) per mouse (<i>n</i> = 3 for 0, 7, and 14 DPI; <i>n</i> = 2 for 3 DPI (NEC); <i>n</i> = 1 for 3 DPI (No NEC)). Data is represented as box-whisker plots, where boxes represent the first to third quartile and a horizontal line indicates the median. Bars represent ranges. (One-way ANOVA with Tukey's multiple comparison test: *** P<0.001). (<b>C</b>) P-STAT3 levels in liver lysates were detected using a Bio-Rad phosphoprotein panel and expressed as a fold change of mean fluorescence intensity (MFI) relative to controls (0 DPI). STAT3 was significantly activated in livers of mice inoculated with <i>C. rodentium</i> at 3 and 7 DPI (Kruskal-Wallis test with Dunn's multiple comparisons test: * P<0.05, ** P<0.01).</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

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