Obesity is associated with a state of chronic inflammation, which is believed to contribute to insulin resistance. We previously identified interferon regulatory factor 3 (IRF3) as an anti-adipogenic transcription factor with high expression in adipocytes. Because IRF3 is known to drive expression of pro-inflammatory genes in immune cells, we hypothesized that it may also promote inflammation and insulin resistance in adipocytes. Consistent with our expectations, we found that the expression of inflammatory genes in adipocytes was induced by IRF3 overexpression, while knockdown of IRF3 had the opposite effect. Despite this effect on local adipocyte gene expression, we found that \(Irf3^{-/-}\) mice did not show evidence of altered systemic inflammation. Nonetheless, \(Irf3^{-/-}\) mice did display altered metabolism relative to their wild type (WT) littermates. For example, high fat diet (HFD) fed \(Irf3^{-/-}\) mice exhibited increased lean mass and decreased fat mass compared to WT, accompanied by increased food intake and energy expenditure. Further investigation showed that the white adipose tissue (WAT) of \(Irf3^{-/-}\) mice had increased expression of brown adipocyte selective genes compared to WT, and the inguinal WAT of the \(Irf3^{-/-}\) mouse contain multilocular adipocytes that resemble brown adipocytes. These data suggest that IRF3 affects energy homeostasis by regulating the development of brown adipocyte-like cells in WAT. Additionally, \(Irf3^{-/-}\) mice are significantly more insulin sensitive and glucose tolerant compared to WT when kept on HFD. Consistent with in vivo observations, IRF3 knockdown in 3T3-L1 adipocytes resulted in enhanced insulin-stimulated glucose uptake and lipogenesis, while overexpression of constitutively active IRF3 had the opposite effect. Several IRF3 target genes in adipocytes were identified using transcriptional profiling. Interestingly, the expression level of Slc2a4 (encoding the Glut4 protein) was inversely correlated with that of IRF3 in both WAT and cultured adipocytes. Analysis of the Slc2a4 proximal promoter identified a putative IRF3 binding site upstream of the transcription start site, and luciferase assay in 3T3-L1 adipocytes showed that IRF3 negatively regulates Slc2a4 expression via this site. Taken together, these data indicate that IRF3 plays a role in whole body glucose homeostasis by repressing thermogenic gene expression as well as the expression of adipose Glut4.

Allergens are complex mixes of proteins and other compounds that have innate signaling capacity leading to Th2 inflammation. Understanding the role of each of these signals is essential to determining what separates allergens from innocuous proteins. Here, we examine two models for Th2 inflammation: infection with the helminth Trichinella spiralis and footpad immunization with papain, a cysteine protease structurally similar to proteases found in many common allergens including grass pollen and dust mites and helminth-secreted proteases secreted. Together, these studies highlight previously unappreciated effector roles of accessory cells during Th2 inflammation.

Age-related macular degeneration (AMD) is a leading cause of visual impairment and blindness, affecting over 30 million people worldwide. It is characterized by the appearance of insoluble deposits known as drusen in the outer retina, between the retinal pigment epithelium (RPE) and Bruch's membrane. Drusen are heterogeneously composed of many compounds, including cholesterol, amyloid-β, and complement proteins. AMD also involves the accumulation of pigments collectively termed lipofuscin in RPE lysosomes. The underlying causes of AMD are unknown, but studies have implicated inflammatory processes in its pathogenesis.

Obesity is an established risk and progression factor for many types of cancer, including pancreatic and colon cancer, and is characterized by abnormal metabolic hormone production and a chronic low-grade state of inflammation. However, the links between obesity, hormones, inflammation and tumorigenesis in colon and pancreatic tissue are poorly understood. Calorie restriction (CR), an anti-obesity dietary regimen with potent anticancer effects, reduces serum metabolic hormones and protumorigenic cytokines. Insulin-like growth factor (IGF)-1 is a metabolic hormone that activates NF-[kappa]B, a key regulator of inflammation. NF-[kappa]B is a transcription factor that mediates transcription of many cancer- and inflammation-related genes and is upregulated in both colon and pancreatic cancer. We hypothesized that CR inhibits colon and pancreatic tumor cell growth through modulation of hormone-stimulated NF-[kappa]B activation and protumorigenic gene expression. To test this hypothesis, we used CR and ad libitum feeding to generate a lean and overweight (control) phenotype, respectively; in C57BL/6 mice transplanted with MC38 colon cancer cells or Panc 02 pancreatic cancer cells, and analyzed the effect of diet on circulating hormone levels, markers of inflammation, and tumor growth. We also investigated the in vitro effects of IGF-1 on NF-[kappa]B activation and downstream protumorigenic gene expression in MC38 and Panc 02 cells. CR, relative to control diet, reduced body weight, circulating IGF-1 levels, and transplanted MC38 and Panc 02 tumor growth, as well as protumorigenic gene expression in the MC38 and Panc 02 tumor microenvironment. IGF-1 increased cell viability, NF-[kappa]B nuclear translocation and DNA binding, transcriptional activation, and downstream gene expression of inflammation and other protumorigenic genes in MC38 colon cancer cells and Panc 02 pancreatic cancer cells in vitro. Knockdown studies of NF-[kappa]B in Panc 02 cells using si-RNA established that the IGF-1-induced increase in protumorigenic gene expression is mediated, at least partially, through an NF-[kappa]B-dependent mechanism. In conclusion, these findings in models of pancreatic and colon cancer help clarify the links between obesity, IGF-1, NF-[kappa]B-mediated inflammation, and cancer. This work provides the underpinnings for several new molecular targets and strategies to test in model systems and translational studies for preventing or controlling obesity-related cancer. / text

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein, is involved in neutrophil recruitment into peritoneum. Because mechanisms of excessive migration of activated neutrophils into inflamed lungs, credited with tissue damage, are not fully understood, we explored the hitherto unknown expression and role of LSP1 in neutrophil migration in a mouse model of acute lung inflammation. We induced acute lung inflammation through intranasal E. coli lipopolysacharide (LPS) (80μg) in wild-type 129/SvJ (WT) and LSP1 deficient (LSP1-/-) mice. WT (n=10) and LSP1-/- (n=11) mice showed significant neutrophilia and more neutrophils in bronchoalveolar lavage (BAL) at 9 hour post-LPS challenge compared to respective saline-treated controls (WT=7; LSP1-/-=10). LPS treatment induced more BAL neutrophils (P<0.001), myeloperoxidase concentrations and Gr-1+ neutrophils in lung tissues in WT mice compared to LSP1-/- mice. Lung myeloperoxidase and Gr-1+ (P<0.05) were higher in LPS-treated WT compared to the LSP1-/- mice. Lung tissue and BAL fluid KC, MCP-1, MIP-1α and MIP-1β concentration and vascular permeability were not different between LPS-treated WT and LSP1-/- mice but TNF-α concentration was higher in LPS-treated WT mice. Hematoxylin and eosin staining showed more septal congestion in LPS-treated WT mice compared to LSP1-/- mice. LSP1 expression was increased in lungs from LPS-treated mice compared to saline control. The autopsied lungs from septic humans, compared to their respective controls, showed increased expression of LSP1. These data show that LSP1 expression is modulated in acute lung inflammation and that LSP1 deficiency reduces neutrophil migration into acute lung inflammation.

Atherosclerotic cardiovascular diseases (CVD) are the leading cause of death and often develop due to obesity-induced complications including hyperlipidemia, elevated blood pressure (BP), inflammation, and oxidative stress. Epidemiological, animal model, and cell culture studies indicate that citrus, and grapefruit specifically, exert cardiovascular health benefits, likely due to the high flavonoid content in citrus fruits. Grapefruit and/or isolated grapefruit flavonoids elicit cardiovascular benefits via improvements in lipid metabolism and endothelial reactivity, and by antioxidant and anti-inflammatory actions. The aim of this work was to determine the role of six-week daily consumption of grapefruit on weight, lipid, and BP control as well as inflammatory and oxidative stress markers in overweight/obese adults. Further, we sought to evaluate the acute, postprandial effects of grapefruit consumption on metabolic, inflammatory, and oxidative stress markers in response to a high fat, high calorie (HFHC) double meal challenge. Participants were randomized to either a grapefruit group (n=42) in which they consumed 1.5 grapefruit/day for six weeks or to a control condition (n=32). Ten participants who completed the feeding trial also participated in the postprandial study. On two test days participants consumed a HFHC meal for breakfast and again for lunch. A ruby red grapefruit was consumed with breakfast on the first test day. Blood samples were collected at baseline and for the subsequent eight hours on each day. In the feeding trial, grapefruit consumption resulted in reductions in waist circumference (p<0.001), systolic BP (p=0.03), total cholesterol (p=0.001), and LDL-cholesterol (p=0.021) compared to baseline values. F2-isoprostanes and hsCRP values were nonsignificantly lower in the grapefruit vs. control arm following the intervention (p=0.063 and p=0.073, respectively). In the postprandial evaluation, insulin concentrations were significantly higher 30 minutes (p=0.007) and 2 hours (p=0.025) post HFHC + grapefruit meal consumption vs. HFHC alone. HFHC + grapefruit intake resulted in lower IL-6 concentrations after two hours (p=0.017) and lower F2-isoprostanes after 5 hours (p=0.0125). These findings suggest that regular grapefruit consumption may reduce CVD risk by targeting many of the risk factors and pathogenic factors involved in endothelial dysfunction. However, this dietary change alone is unlikely to result in significant CVD risk reduction.

Background: Clinical and epidemiologic studies have investigated the effects of diet, physical activity, and inflammation on the risk of colorectal adenoma occurrence and recurrence. Inflammation has been proposed as a mechanism of action for the development of colorectal adenoma and cancer. Research indicates that fruit phenolic exposure may attenuate the inflammatory response and some data suggest that berries are effective in mitigating this process. Inflammatory cytokines such as interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor alpha (TNF-α) are of particular interest due to their role in adenoma development. Epidemiological investigations have studied the association between bioactive fruit phenolic compounds and colorectal neoplasia; however, epidemiological data for the association between consumption of berries, which contain high concentrations of these compounds, and colorectal adenoma recurrence are limited. In addition to a potential role of phenolics in reducing inflammation, physical activity has also been proposed as a mitigator of this process. Numerous studies have investigated the association between physical activity and colorectal neoplasia, yet data on sedentary behavior and colorectal adenoma recurrence are limited. This dissertation was designed to further elucidate the role of fruit phenolics and sedentary behavior on colorectal adenoma recurrence and to specifically highlight the potential role of black raspberries in mitigating the postprandial inflammatory response among overweight and obese individuals. Methods: Ten overweight or obese males (BMI>25 kg/m²), ages 55-72, participated in an open-label, randomized, 14-day, pilot crossover study. Subjects consumed a high- fat, high- calorie (HFHC) meal, with (Group 1) or without (Group 2) a 5 day regimen of 45 g of black raspberry powder in the form of a slurry. The study included a two-day washout period before Group 1 and Group 2 were crossed over. The two-day washout period was based on a pharmacokinetic study conducted with black raspberry powder (1); peak plasma concentrations of ellagic acid and anthocyanin metabolites peaked at 1 to 2 hours following consumption of 45 grams of black raspberry powder and by 12 hours, plasma concentrations of these metabolites were almost fully washed out, with plasma concentrations returning to near baseline levels. Blood samples were obtained prior to consumption of the HFHC breakfast and at 1, 2, 4, 8, and 12 hours afterwards, during two 14-hour clinic visits. The primary study outcomes were changes in areas under the curves (AUCs) of serum biomarkers of TNF-α, CRP, and IL-6. A secondary pooled analysis was conducted among participants from two randomized, double blind, placebo-controlled Phase III clinical trials to investigate the association between berry consumption and colorectal adenoma recurrence, and the association between sedentary behavior and colorectal adenoma recurrence. Analyses included 2,502 subjects who had completed the baseline Arizona Food Frequency Questionnaire to ascertain berry consumption history in the past year and 1,730 men and women who had completed the baseline Arizona Activity Frequency Questionnaire to ascertain sedentary behavior. All subjects had a follow-up colonoscopy during the trial. Logistic regression modeling was employed to estimate the effect of sedentary behavior or berry consumption on colorectal adenoma recurrence. Results: The mean AUC of serum IL-6 was significantly lower (p=0.03) with black raspberry (BRB) feeding (45.5±36.3 pg/mL; mean±SD), compared to high fat, high calorie meal alone (56.7±50.0 pg/mL). No statistically significant differences were observed in the mean AUC of serum TNF-α or CRP. In the pooled analysis, no significant associations were observed between berry consumption and adenoma recurrence in the pooled population or when stratified by sex. In the evaluation of association between sedentary behavior and adenoma recurrence, subjects in the second, third, and fourth quartiles of sedentary behavior experienced higher odds of adenoma recurrence; however, the difference was only statistically significant for the third quartile. Sex-stratified analyses revealed that in men, sedentary activity was statistically significantly associated with 45% higher odds of adenoma recurrence. Compared to the lowest quartile of sedentary activity, the ORs (95% CIs) for the second, third, and fourth quartiles among men were 1.31 (0.93, 1.84), 1.47 (1.04, 2.09), and 1.45 (1.02, 2.06) respectively (P trend=0.03). In contrast, no association with sedentary activity was observed in women. Conclusion: Polyphenol exposure in the form of a black raspberry slurry significantly decreased post-prandial IL-6 in a clinical trial among ten older overweight and obese men. These findings suggest short-term attenuation of an inflammatory maker may not translate to decreased adenoma recurrence, however, long term randomized clinical trials with black raspberries are needed to evaluate this further. However, in an epidemiological analysis, consumption of up to 1 cup per week of whole berries was not associated with lower odds for adenoma recurrence among a pooled population of participants in the Wheat Bran Fiber and Ursodeoxycholic Acid Phase III clinical trials. While the epidemiological results indicated that berry consumption are not associated with the development of early colorectal neoplasia, the effects on later stages of carcinogenesis are unknown. Higher levels of berry consumption may be required in order to reach a cancer inhibitory effect. Finally, results of the physical activity study suggest that sedentary behavior is associated with a higher risk of adenoma recurrence among men, providing evidence of detrimental effects of a sedentary lifestyle early in the carcinogenesis pathway. Efforts to further evaluate these findings in other cohorts or in an intervention trial should be considered.

A novel cell culture system was designed to serve as a model of neuroinflammation. Neurons, astrocytes, and microglia derived from embryonic and perinatal rat cortical tissue were combined in a three-dimensional hydrogel utilizing a method that facilitated cell maturation and viability. Chemical challenge of the cultures with a broad pro-inflammatory stimulus resulted in the production of inflammatory cytokines and other associated molecules commensurate with the response observed in vivo and in other in vitro systems. It was hypothesized that mechanical deformation of the multitypic neural cell cultures would produce a similar response and thus validate the system as an in vitro model of traumatic brain injury-induced neuroinflammation. Mechanical injury delivered using custom-manufactured culture chambers and injury devices successfully imparted a moderate level of cell death to the cultures. It was determined that a mechanically-induced inflammatory response required chemical stimulation prior to the injury. The research presented here describes the generation and characterization of a novel in vitro culture system and its implementation in experiments designed to model secondary injury mechanisms associated with injury-induced neuroinflammation. The findings of these studies, applications of the culture system, and future research avenues are discussed.

The mechanisms responsible for the enhanced inflammatory response in type 2 diabetes (T2DM) and its contribution to the severe ischemia/reperfusion (I/R) injury observed in the T2DM heart are unclear. I/R is associated with an acute inflammatory response recognized by reactive oxidant production, complement activation, and leukocyte-endothelial cell adhesion, among others. Complement activation plays an important role in the inflammatory response and is involved in the manifestation of I/R injury in the non-diabetic heart, and is a potent chemoattractant for circulating neutrophils (PMNs). The purpose of this dissertation research was to test the hypothesis that the complement system, predominantly the lectin pathway, is a significant contributor to the excessive response of the Zucker Diabetic Fatty (ZDF), a rat model of T2DM, to myocardial I/R injury. Following 30min of coronary artery occlusion and 120min of reperfusion we measured C3 deposition, PMN accumulation, PMN CD11b expression, and ICAM-1 expression. We found significantly more C3 deposition, PMN accumulation, ICAM-1 and PMN CD11b expression in diabetic samples compared to non-diabetic samples. To elucidate a role for complement system activation, we treated animals with FUT-175, a broad complement inhibitor. In vivo, FUT-175 treatment significantly decreased complement deposition (66%), PMN accumulation (59%), and infarct size (55%) compared to untreated animals in both non-diabetic Sprague-Dawley and diabetic ZDF rats. To specifically examine the role of the lectin pathway, we selectively inhibited rat MBL-A prior to myocardial I/R in ZDF rats. Anti-MBL treatment significantly decreased infarct size, C3 deposition and PMN accumulation in the ZDF post-ischemic left ventricle (LV). Genomic analysis revealed that gene expression of the pro-inflammatory cytokines IL-6 and IL-1α was enhanced in the ZDF heart following reperfusion, and quantitative RT-PCR results confirmed IL-6 upregulation. We found significantly increased complement C5a receptor (CD88) expression on diabetic neutrophils prior to ischemia, suggesting that diabetic PMNs are "primed" to respond to complement activation. Taken together, these results provide evidence that 1) the ZDF rat is a good model for chronic inflammation in the setting of T2DM, 2) lectin pathway activation plays a significant role in the inflammatory response to I/R injury in the ZDF heart, and 3) anti-complement therapy may be particularly cardio-protective in T2DM.

Correct dosing in pharmacotherapeutics is based on the idea that too much of a drug will cause toxicity, while too little will result in failure to elicit the desired response. A major factor in the ability of a patient to handle any dose of a drug is the capacity to metabolize and eliminate that drug from the body. For the vast majority of drugs, the liver plays a key role in determining the rate at which drugs are eliminated. First, drugs must be taken up across the cell membrane into hepatocytes by uptake transporters. Once inside the hepatocyte, biotransformation enzymes metabolize and conjugate the drug to a more water-soluble compound, the distribution of which is more easily controlled. These water-soluble metabolites are then transported out of the hepatocyte by additional drug transporters either into bile for elimination, or back into the blood.More than 2 million severe adverse drug reactions occur in the US each year and often result from interindividual variation in the ability to metabolize and eliminate drugs. This number does not include medical errors, but rather circumstances where an individual is unable to handle the standard dose of the correctly prescribed drug. Although genetics plays an important role, the greatest source of variation comes from other environmental factors such as disease states. Nonalcoholic fatty liver disease (NAFLD) is a chronic condition that comprises a spectrum of histopathologies that range from simple steatosis to the more severe steatohepatitis. Specifically, nonalcoholic steatohepatitis (NASH) has become one of the leading causes for liver transplantation in the United States, and thus clearly become a considerable burden to the U.S. healthcare system.It is not known whether the capacity of the liver to metabolize and excrete drugs is altered in patients with NASH. Because the liver plays such a critical role in drug metabolism and disposition, any disease state that disrupts or modifies these functions will alter the fate of a given drug within the body. It is therefore very likely that the ability of the liver to metabolize and excrete clinically relevant drugs is compromised in NASH patients.