Equine neutrophils (PMN) play a critical role in inflammatory processes in horses. The objective of this study was to characterize equine PMN function ex vivo following stimulation with calcium ionophore A23187 (A23187) and phorbol myristate acetate (PMA). These stimulants trigger different branches of the PMN activation process that occurs in vivo. Equine PMN were isolated from the whole blood of six clinically normal geldings using a one-step discontinuous Percoll gradient technique. Neutrophil aggregation, degranulation, and superoxide anion production were evaluated in assay systems which had previously been established to guantitate PMN function. Dose-response curves for A23187 and PMA were derived for the three functions. Results indicate that equine PMN aggregation and superoxide anion production are more responsive to activation by PMA as the maximum change in percent transmittance and maximum nanomoles of superoxide anion produced following PMA stimulation (60.82% and 10.4nmols/10⁶cells, respectively) were greater than those values stimulated by A23187 (41.5% and 5.2nmols/10⁶cells, respectively). However, degranulation was found to be more responsive to A23187 stimulation (maximum percent degranulation= 56.1%) than to PMA stimulation (maximum percent degranulation= 30.7%). Dose-response curves following A23187 and PMA stimulation revealed that superoxide anion production had the lowest threshold concentration among the three functions. Degranulation had the highest threshold concentration among the three functions for both stimulants. Results indicate that equine PMN functions differ in their dependence on second messengers in the activation pathway. These functions also occur in a dose-dependent manner and differ in the threshold concentrations required for their stimulation. / Master of Science

Sphingolipid metabolites play a role in the initiation and perpetuation of inflammatory responses. Since intestinal inflammation is a driving force in the development of colon cancer, in the present study, we investigated the suppression of dextran sodium sulfate (DSS)-induced colitis by dietary sphingomyelin in mice that lack functional peroxisome proliferator-activated receptor γ (PPAR-γ) in intestinal epithelial and immune cells. Dietary spingomyelin decreased colonic inflammation in mice of both genotypes but more efficiently in mice expressing PPAR-γ. Using a real-time polymerase chain reaction array, we detected an up-regulation in genes involved in Th1 (interferon γ) and Th17 (interleukin [IL]-17 and IL-23) responses despite the reduced inflammation. However, the genes involved in Th2 (IL-4, IL-13 and IL-13ra2) and Treg (IL-10rb) anti-inflammatory responses were up-regulated in a PPAR-γ-dependent manner. In order to direct mechanistic studies of how PPAR-γ expression is involved in SM-induced suppression of DSS colitis, we investigated the effect of dietary SM in DSS-treated mice that lack PPAR-γ in the CD4+ T-cells. While the pathogenesis of colitis was independent of PPAR-γ expression in CD4+ T-cells, dietary SM decreased disease activity and colonic inflammation in mice of both genotypes but more efficiently in mice expressing PPAR-γ, indicating both PPAR-γ dependent and independent signaling pathways. In conclusion, in contrast to endogenous sphingolipid metabolites, dietary SM modulated both pro- and anti-inflammatory responses at the early stages of the disease in a partially PPAR-γ dependent manner resulting in a suppression of inflammation that may be critical for the suppression of inflammation-driven colon cancer. / Master of Science

Inflammation is a central feature of various metabolic diseases including obesity and type-II diabetes. For this study, we hypothesized postprandial metabolism following an acute, high fat (HF) meal to be impaired in mice pre-injected with an inflammatory agonist. To this end, C57BL/6J mice were injected with saline or lipopolysaccharide (LPS, 1μg/kgbw) following an overnight fast and gavaged 2hr post-injection with water or a HF meal in liquid form (5kcal; 21.4%SF, 40.8%UF, 27.1%CHO, 10.7%PRO). Blood and muscle samples taken 3hr post-gavage underwent ex vivo analysis. Overall, results demonstrated a metabolic response to a HF meal that was blocked in the presence of LPS. Metabolic flexibility, though unchanged following the HF meal alone, was reduced following the HF meal in the presence of LPS. Additionally, state-4 uncoupled mitochondrial respiration, which was increased following the HF meal, was also reduced following the HF meal in the presence of LPS. Similar near-significant trends were also observed with total palmitate oxidation. Although no independent response to a HF meal or LPS exposure was observed, a unique interaction between treatments significantly diminished ADP dependent, state-3 and maximal respiration. These effects do not appear to be dependent on the production of reactive oxygen species (ROS) since neither the HF meal nor LPS exposure resulted in increased production of ROS. In conclusion, these results demonstrate that acute activation of inflammatory pathways results in alterations in metabolic response to a HF meal in skeletal muscle from mice, although the mechanism underlying these effects is not yet understood. / Master of Science

The ability to sense and respond to external environmental signals is closely regulated by a plurality of cell signaling pathways, thereby maintaining homeostasis. In particular, the inflammatory signaling cascade contributes to cellular homeostasis and regulates responses prompted by external stimuli. Such responses are diverse and range from a variety of processes, including tissue repair, cell fate decisions, and even immune-cell signaling. As with any signaling cascade, strict regulation is required for proper functioning, as abnormalities within the pathway are often associated with pathologic outcomes. A hyperactive inflammatory response within the gastrointestinal tract, for example, contributes to inflammatory bowel disease (IBD), presenting as Crohn’s disease or ulcerative colitis. Furthermore, as a chronic condition, IBD is associated with an increased risk for the development of colitis-associated cancer. In order to resolve inflammation and thus restore homeostasis, negative regulation may be utilized to mediate the activity of inflammatory molecules. The mechanistic action of a specific negative regulator of interest, interleukin receptor associated kinase M (IRAK- M), is explored in detail within the present dissertation. Investigation of IRAK-M in mouse models of colitis, which mimics human IBD, and in mouse models of inflammation-driven tumorigenesis, which models colitis associated cancer, demonstrated that loss of this molecule contributes to host protection. Therefore, IRAK- M may be a suitable target for inhibition in order to advance therapeutic options for human patients afflicted with a GI-related inflammatory disease, such as IBD and colitis associated cancer. Furthermore, an ex vivo method that models the interaction of intestinal epithelial cells with microbes present in the GI tract was optimized and is described in the present dissertation. This method takes advantage of primary intestinal derived organoids, also termed “mini-guts”, which display similar features corresponding to intestinal tissue in vivo. For this reason, the use of “mini-guts” has several advantages, particularly for the enhancement of personalized medicine. The method discussed herein aims to normalize experimental conditions in order to enhance reproducibility, which can further be used to uncover microbial-epithelial interactions that contribute to a pathological state, such as IBD. Finally, this method of intestinal epithelial cell culture was utilized to evaluate the role of a protein, termed NF-κB inducing kinase (NIK), in intestinal epithelial cell growth and proliferation. Ultimately, ex vivo organoid culture can serve as an important model system to study the contribution of NIK in intestinal stem cell renewal, cancer progression, as well as in maintenance of the integrity of the gastrointestinal barrier. / Ph. D. / Inflammation is a tightly regulated physiologic process that is employed by body systems such as the gastrointestinal (GI) tract to handle pathogenic insult, aid in wound healing, and help prevent infections. When abnormal inflammatory responses occur, this can lead to the progression of severe diseases such as ulcerative colitis and Crohn's disease. When inflammation persists in the GI tract, such as in inflammatory bowel disease, this can predispose patients to the development of inflammation- associated colorectal cancer. In order to improve the treatment options for patients afflicted with these maladies, this dissertation is aimed at studying the signaling pathways of the innate immune system that regulate such inflammatory responses. Furthermore, this body of work encompasses a detailed method for isolating and culturing intestinal stem cells, which can be applied in personalized medicine for patients with intestinal diseases. This method was utilized in this dissertation to study genetically modified intestinal stem cells, and can further be used to investigate the interactions of intestinal epithelial cells with pyogenic bacteria that contribute to inflammatory maladies in the GI tract.

Whether innate immune cells may be adapted into potential memory states has becoming an important question in the field of immunity. Although previous conceptual paradigm failed to acknowledge this important question, emerging clinical and basic observations have started to shed intriguing clues to shake the previous dogma regarding innate immunity of being "simple", "raw", "first-line defense with no memory". We have aimed to further address this fundamental issue in this dissertation work, under the close guidance of Dr. Liwu Li. We have chosen to use the model system of Toll-Like-Receptor (TLR) signaling networks within primary monocytes. TLRs play fundamental roles in sensing pathogen-associated molecular patterns (PAMPs) and modulation of innate immunity. Lipopolysaccharide (LPS), an endotoxin found on the cell membrane of gram-negative bacteria, is the ligand of TLR4 and induces a range of inflammatory as well as anti-inflammatory responses. Higher dosages of LPS were known to cause robust yet transient expression of pro-inflammatory mediators. On the other hand, the effects of super-low dose LPS, commonly manifested in humans with adverse health conditions, have been largely ignored in the basic research field. Super-low dose LPS may skew host immune environment into a mild non-resolving pro-inflammatory state, which is a risk factor for inflammatory diseases such as atherosclerosis, compromised wound healing, and elevated risks for sepsis. Our central hypothesize is that monocytes may be adapted by super-low dose LPS into a non-resolving low-grade inflammatory state conducive for the pathogenesis of inflammatory diseases. We have employed both in vitro cell culture system as well as in vivo disease models to test this hypothesis. For the in vitro system, we have cultured primary murine monocytes with increasing signal strength of LPS. Monocyte phenotypes such as the expression of key inflammatory mediators including cytokines, chemokines, and cellular surface markers were studied. Potential molecular and cellular mechanisms were examined. We revealed a novel low-grade inflammatory monocyte phenotype termed ML adapted by super-low dose LPS, mediated through IRF5. For the in vivo system, we have employed both acute and chronic models of inflammation. For the chronic model, we have tested the effects of super-low dose LPS on monocyte polarization in vivo, as well as its contribution to the pathogenesis of atherosclerosis. Furthermore, we have tested the effects of programmed monocytes on wound healing. For the acute model, we have tested the effects of pre-conditioning with super-low dose LPS on the subsequence risks of sepsis elicited by cecal ligation and puncture. We have demonstrated aggravated atherosclerosis, compromised wound healing, and increased sepsis mortality in mice pre-conditioned with super-low dose LPS. Taken together, our findings reveal that monocytes can be differentially programmed into distinct states, depending on the signal strength of LPS. The differential programming and adaptation of monocytes can occur both in vitro and in vivo, and may bear profound pathological consequences. / Ph. D.

Atherosclerosis, the underlying cause of cardiovascular diseases such as coronary artery disease, is driven by inflammation and cell death of leukocytes, particularly macrophages. These processes contribute significantly to plaque destabilization, leading to the development of vulnerable plaques prone to rupture and thrombotic events. Understanding the mechanisms underlying leukocyte inflammation and cell death in atherosclerosis is crucial for identifying novel therapeutic targets to stabilize plaques and reduce the risk of cardiovascular events. Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a key player in atherosclerosis, with its role in lipid metabolism and plaque development being extensively studied. While PCSK9 is predominantly produced in hepatocytes, emerging evidence suggests its expression in leukocytes and macrophages may have distinct effects on atherosclerosis, beyond its role in lipid regulation. In this thesis, the role of leukocyte PCSK9 in atherosclerosis was investigated by restoring normal PCSK9 expression in leukocytes of PCSK9 and ApoE-deficient mice. Contrary to expectations, restoring leukocyte PCSK9 expression did not significantly alter plaque size, macrophage or smooth muscle cell content, pro-inflammatory cytokine expression, or apoptosis within plaques. These findings suggest that leukocyte PCSK9 expression may not significantly impact atherosclerotic plaque development or the local plaque environment in this mouse model. In this thesis, we also explored the role of ApoA1 deficiency and Bim-mediated apoptosis in atherosclerotic plaque development. ApoA1 deficiency increased plaque size, necrotic core size, and apoptosis in macrophages within plaques, potentially through upregulation of Bim protein. Inhibiting Bim in all bone marrow-derived cells and myeloid-specific cells reduced plaque apoptosis, necrotic core sizes, and plaque sizes, highlighting the significance of Bim in atherosclerosis progression, and suggesting a specific role of myeloid cell-derived Bim in plaque stability. These findings provide insights for future PCSK9 research and contribute to our understanding of HDL's protective effects and a potential therapeutic target for apoptotic cell death in atherosclerosis. / Thesis / Doctor of Philosophy (Medical Science)

Chronic, low-grade inflammation is a contributing factor in diseases that impact the health and well-being of horses. Pharmaceutical treatments reduce inflammation, but their use results in negative digestive and kidney disturbances. Polyunsaturated fatty acids (PUFA) play a role in mitigating the inflammatory response and are therefore explored as a dietary approach to attenuate inflammation. γ-Linolenic acid (GLA) is a unique PUFA that when supplemented in the diet can increase the production of anti-inflammatory eicosanoids; however, it is uncommon in the dietary components normally fed to horses. Interest in industrial hemp (Cannabis sativa L.) as a novel source of PUFA stems from the presence of GLA and the potential to reduce inflammation; although, concerns over cannabinoid contamination limit its acceptance. Six Thoroughbred geldings were used in a crossover study with two 63-d periods to measure PUFA metabolism, inflammatory biomarkers, and cannabinoid accumulation in response to hempseed oil (HSO) fed to sedentary horses compared to controls (CON). Treatment diets were offered for the first 35 d of each period and then all horses resumed a uniform feeding rate from d 36 to 63. Serum and synovial fluid PUFA reflected dietary intake. GLA was greater in serum (0.465 vs. 0.046; P < 0.0001) and synovial fluid (0.270 vs. 0; P < 0.0001) in horses fed HSO compared to CON. This contributed to greater dihomo-γ-linolenic acid (DGLA) conversion in serum (0.287 vs. 0.195; P < 0.0001) and synovial fluid (0.348 vs. 0.262; P < 0.04) but not arachidonic acid (AA). Serum GLA returned to baseline concentrations by two weeks post-supplementation, but no treatment x time effect was observed for synovial fluid. HSO did not affect FA in muscle; it is likely the length or quantity of supplementation was inadequate to see changes in muscle PUFA. HSO increased serum interleukin 1β (IL1β; P = 0.01) but there was no treatment by time interaction (P = 0.62). No other inflammatory biomarkers were influenced by treatment. Stride length was not affected by HSO supplementation but was inversely correlated (P ≤ 0.01) with synovial fluid prostaglandin E2 (PGE2; r = -0.56), and positively correlated with serum tumor necrosis factor α (TNFα; r = 0.58), serum IL6 (r = 0.61), and serum IL1β (r = 0.65). Cannabinoids were measured in the HSO supplement, but no cannabinoids were detected in plasma or synovial fluid of horses fed HSO when tested to a 50-ppb limit of detection. These results demonstrate the suitability of HSO as a novel source of PUFA and, more specifically, as a source of GLA without further increasing AA, however, implications for its effect on inflammation require further evaluation. / Doctor of Philosophy / Inflammation contributes to diseases in the horses that reduce their health and well-being. Anti-inflammatory drugs reduce inflammation but are associated with negative health effects including gastric ulcer formation and kidney damage. Diet can influence the inflammatory response and is therefore targeted to moderate inflammation. Specific dietary targets include polyunsaturated fatty acids (PUFA). Hempseed (Cannabis sativa L.) oil (HSO) contains a unique and uncommon dietary PUFA, γ-Linolenic acid (GLA), which can increase the production of anti-inflammatory biomolecules. The goal of this research was to measure PUFA accumulation, specifically GLA, in horses fed HSO for 35 d and then clearance for 28 d post-supplementation. Additionally, we looked at inflammatory markers to determine the effect on inflammation in sedentary horses. Finally, we measured cannabinoids to evaluate if the low level of cannabinoid contamination found in HSO transfers to horse plasma and synovial fluid. To accomplish these goals, we conducted a feeding trial from May 2022 to September 2022 using six Thoroughbred geldings in a cross over study with two 63 d periods. HSO was supplemented the first 35 d of each period and then removed. Serum and synovial fluid PUFA reflected dietary PUFA. Inflammatory biomarkers had a mixed response that could be influenced by additional, unknown factors. The low-level of cannabinoids in the HSO supplement were not detected in plasma or synovial fluid. HSO shows promise as a novel source of PUFA, specifically GLA, without concerns of cannabinoid contaminants.

Keratins are fibrous structural proteins found in human hair that have been used to develop bioactive and biocompatible constructs for a wide variety of tissue engineering and healthcare applications. Their ubiquity, capacity for self-assembly, ease of use and versatility in blended materials, and ability to modulate cell behavior and promote tissue ingrowth have made keratins well-suited for the development of regenerative therapies. In particular, keratins have demonstrated bioactivity in both in-vivo and in-vitro studies, by altering immune and stem cell phenotype and function and promoting an anti-inflammatory/wound healing environment. This work seeks to build on previous research by investigating the ability of low and high molecular weight keratins to augment anti-inflammatory primary macrophage phenotypes and examining the influence of keratin biomaterials on cellular and inflammatory mechanisms in two models of injury and disease. Rodent models of blast induced neurotrauma (BINT) and severe osteoporosis were used to inform the development of 2D and 3D in-vitro models of macrophage/endothelial cell injury and osteogenic differentiation respectively. Keratin biomaterials exhibited some potential to alter macrophage and endothelial cell dynamics following blast, specifically by promoting anti-inflammatory (M2c-like) macrophage polarization and diminishing endothelial cell injury responses (i.e. endothelial glycocalyx shedding). A more clinically relevant model of osteoporosis found that stem cells harvested from older, osteoporotic animals demonstrated limited proliferative and bone differentiation potential compared to healthy cells. However, 3D constructs (especially keratin-based materials) were able to enhance calcification and osteogenic gene expression of diseased cells. These results highlight the complexity of macrophage phenotypic switching and cellular dynamics in these systems. However, keratin-based therapeutics may prove useful for facilitating tissue regeneration and limiting detrimental inflammatory and cellular responses in various models of injury and disease. / Ph. D. / Keratins are proteins found in human hair that have been used for a wide variety of healthcare applications. Their availability, ease of use as coatings, gels, and scaffolds, and their ability to alter cell function have made keratins well-suited for regenerative therapies. In particular, keratins have demonstrated the ability to alter immune and stem cell function by promoting a wound healing environment. This work seeks to investigate the ability of different keratins to enhance wound healing immune cell types and examine the influence of keratin materials on stem and blood vessel cell behavior in two models of injury and disease. Rodent models of blast-wave induced traumatic brain injury (concussion) and severe osteoporosis (bone brittleness) were used to develop cell culture models of immune cell and blood vessel cell injury as well as the conversion of stem cells to bone-building cells respectively. Keratin-based materials exhibited some potential to alter immune and blood vessel cell function following blast injury, specifically by promoting wound healing immune cell transformation and diminishing blood vessel cell injury responses. A more clinically relevant model of osteoporosis found that stem cells harvested from older animals had a more limited ability to divide and transform to bone cells compared to healthy cells. However, 3D gels (especially keratin-based materials)—unlike 2D coatings—were able to enhance calcium deposition and other bone markers in diseased cell cultures. These results highlight the complexity of cell responses in these systems. However, keratin-based therapeutics may prove useful for promoting tissue regeneration and limiting detrimental inflammatory and cellular responses in various models of injury and disease.

Background: Dietary and serum antioxidants and physical activity can effect inflammation, which is associated with breast cancer risk and recurrence. This study investigated the relationship between diet, serum antioxidant capacity, physical activity, and inflammation in breast cancer survivors and individuals without cancer. Methods: Existing demographic, dietary intake, and physical activity data of 78 breast cancer survivors and 30 individuals without cancer from the Day and Night Study conducted at Virginia Commonwealth University were used. Participants were recruited from southern Virginia. Metabolic equivalents were calculated through type, intensity, and duration of physical activity. Dietary antioxidant intake (FRAP) was calculated from Harvard Food Frequency Questionnaire data. Serum samples were analyzed for inflammation (hsCRP,IL-6,IL-1,and TNF alpha) and serum antioxidant capacity (ORAC) at Virginia Tech. Results: Anthropometrics and inflammation were higher, and FRAP and ORAC lower in breast cancer survivors compared to individuals without cancer, although not significant. There was a significant direct relationship between FRAP and ORAC and inverse relationship between FRAP and hsCRP. Breast cancer survivors 6+ years since diagnosis showed significant direct FRAP and IL-1 association, and inverse ORAC and TNF-alpha association. BMI was directly associated with IL-6 and CRP. Inflammation was not associated with METs or weekly activity, although there was an increasing inverse relation between METs, IL-1 and TNF- α with increasing ORAC. Conclusion: There is a significant relationship between dietary antioxidant intake and serum antioxidant capacity and inflammation. Increased body mass index increases inflammation. Diets high in antioxidants and maintaining a healthy weight may help reduce inflammation in breast cancer survivors. / Master of Science / Background: Dietary and serum antioxidants and physical activity can effect inflammation, which is associated with breast cancer risk and recurrence. This study investigates the relationship between diet, serum antioxidant capacity, physical activity, and inflammation in breast cancer survivors and individuals without cancer. Methods: Demographic, dietary intake, and physical activity data of 78 breast cancer survivors and 30 individuals without cancer from the Day and Night Study conducted at Virginia Commonwealth University were used. Participants were recruited in southern Virginia. Metabolic equivalents, a measure of physical activity, were calculated from type, intensity, and duration of physical activity. Dietary antioxidant intake (FRAP) was calculated from Harvard Food Frequency Questionnaire data. Serum samples were analyzed for inflammation (hsCRP, IL-6, IL-1, and TNF-alpha) and serum antioxidant capacity (ORAC) at Virginia Tech. Results: Anthropometrics and inflammation were higher in breast cancer survivors while FRAP and ORAC were lower. Significance existed between dietary antioxidant intake and serum antioxidant capacity as well as dietary antioxidant intake and hsCRP. Higher body mass index was associated with increased inflammation. Breast cancer survivors 6+ years since diagnosis with higher dietary antioxidant intake had lower IL-1, and with serum antioxidant capacity and TNF-alpha. Conclusion: In this population there is a significant relationship between dietary and serum antioxidant capacity, as well as dietary antioxidant capacity and hsCRP. In breast cancer survivors 6+ years since diagnosis there are significant associations in antioxidant capacity and inflammation. This is evidence that dietary antioxidants and maintaining a healthy weight can help reduce inflammation.

Prenatal maternal inflammation (PNMI) is associated with neuropsychiatric outcomes in offspring, including depression and schizophrenia. Despite high levels of comorbidity between these disorders, and overlap in symptomatology, longitudinal examinations of PNMI and its relationship to symptom-level, behavioral outcomes in adulthood have yet to be examined. In both cross-sectional and longitudinal studies conducted within the postnatal period, inflammation is associated with emergence of “sickness behavior,” a cluster of symptoms including dysregulation of appetite, sleep, energy, and motivation. The present studies examined the relationship between maternal levels of specific inflammatory biomarkers during the first and second trimesters of pregnancy, and dimensions of depressive symptomatology, subtypes of anhedonia, and subjective sleep quality/duration, in offspring from the Child Health and Development Studies (CHDS) at mid-life (ages 57-61). The roles of fetal sex and timing of exposure also were explored. First trimester IL-6 was associated with poorer subjective sleep quality in offspring. In sex-stratified analyses, results seem to diverge by sex. Higher first trimester levels of inflammation in mothers pregnant with in male offspring were associated with greater consummatory pleasure and poorer subjective sleep quality. Alternatively, higher second trimester levels of inflammation in mothers pregnant with female offspring were associated with lower anticipatory pleasure, only prior to post hoc correction. These studies expand the developmental scope of examinations of the relationship between PNMI and symptom-level outcomes to mid-life and provide meaningful insight into areas for prevention and intervention across the lifespan. / Psychology