Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Bacteremia involving oral bacteria and the systemic inflammatory responses are mechanisms that could causally link oral and systemic diseases. Objective: To use an experimental gingivitis model (EGM) in 2 clinical studies to 1) examine the systemic inflammatory responses to dental plaque, and assess racial differences in these responses; 2) determine whether dental plaque accumulation causes bacteremia and subsequent systemic responses following toothbrushing. Additionally, a laboratory study was conducted to examine the interaction between circulating human neutrophils and Fusobacterium nucleatum. Methods: For both clinical studies, healthy adults, aged 18-31 years, were recruited. In the first study, black and white, males and females participated in a 21-day EGM; in the second study, white adults participated in a 7-day EGM. In both studies, subjects visited the clinic weekly for: 1) measurement of the plaque index (PI) and gingival index (GI); 2) collection of peripheral blood samples to evaluate systemic markers of inflammation. In the second study, to analyze bacteremic episodes during the experimental phase, peripheral blood samples were collected at baseline and at 0.5, 5, and 30 minutes post-toothbrushing. In the laboratory study, interactions between F. nucleatum and circulating neutrophils were examined using a luminol-enhanced chemiluminescence assay. Results: During the experimental phases of both clinical studies, PI and GI increased (p<0.05) with a correlation between PI and GI ≥0.79. In the first study, dental plaque accumulation resulted in a systemic response that manifested as changes (p<0.05) in the level of inflammatory markers, hematologic factors, markers of lipid metabolism, and markers of metabolic change. This systemic response differed between individuals of different gender and race. In the second study, bacteremic episodes and changes in hematologic factors were observed post-toothbrushing during the experimental phase. Activation of neutrophils with F. nucleatum, in the laboratory study, increased the levels of neutrophil chemiluminescence (p<0.05). Conclusions: Overall, the findings of these investigations may shed light on the mechanistic pathways by which oral infection may impose risk for systemic diseases and provide some evidence to support a possible causal association between oral and systemic diseases. The clinical significance of this in systemic inflammatory diseases requires further investigation.

Tissue homeostasis is necessary for optimal organ functioning. The onset of tissue trauma compromises the homeostatic environment resulting in widespread cell death with the likelihood of exposure to invading micro-organisms. Early stage elimination of microbes and immunomodulation is co-ordinated by leukocytes of the innate immune system of which neutrophils and macrophages play a pivotal role. Leukocyte-released pro-inflammatory factors are vital in the containment of infection but bring with it a degree of collateral tissue destruction. Thus cascading stages during inflammation must be tightly regulated to bring about timely tissue regeneration and regained homeostasis. However, chronic inflammatory diseases e.g. rheumatoid arthritis highlights the existence of defective regulation at numerous stages during this transition, often leading to debilitating disease progression. Recently published findings by our research group identified the anti-inflammatory properties of α-defensin - an anti-microbial peptide released from dying human neutrophils - on stimulated macrophages. Thus the main objective of my research was to gain an understanding into the molecular actions of α-defensins which inhibit the macrophage inflammatory potential. Strong evidence supported the propensity of α-defensins to inhibit both intracellular and secreted protein synthesis, as assessed by de novo 35S-radiolabeled Methionine incorporation. Inhibition was not attributed to endoplasmic reticulum stress events, a common diagnosis in the regulation of global translation. Supporting evidence using cell-free systems identified a fundamental block in translation with the inclusion of α-defensin. Biochemical studies linked the ability of α-defensin to bind non-specifically to oligonucleotide sequences. This binding potential was also demonstrated on ribosomal RNA (rRNA), impeding its migration through electrophoretic gels. Immunocytochemical assays proposed an emerging suggestion of α-defensins in macrophages concentrated in close proximity to ribosomes around the perinuclear region. Evidence of suggested defensin/ribosome accumulation after 24hrs after treatment were attempted but to date remained unconfirmed. Attempts to determine the fate of these proposed accumulations were inconclusive, assessed by autophagy assays and ribosome semiquantitation. This thesis describes for the first time an enhanced understanding into the intracellular inhibitory mechanisms of α-defensins on macrophages and possibly other cell types. Understanding the molecular impact of α-defensins provide key insights into this novel inflammatory regulator, with the potential to be utilized in future immunotherapies.

Endometrial adenocarcinoma is the most common gynaecological malignancy in Western countries, affecting mainly post-menopausal women with a frequency of 15-20 per 100 000 women per year. Over-expression of the cyclooxygenase (COX) enzymes and prostaglandin receptors has been demonstrated in endometrial adenocarcinoma as well as other gynaecological pathologies. Increased expression of the prostaglandin F2α (PGF2α) receptor (FP) has been previously demonstrated in endometrial adenocarcinoma. A role for the FP receptor in the promotion of endometrial adenocarcinoma has been shown, with evidence for elevated PGF2α-FP signalling up-regulating angiogenic and tumourigenic genes, and increasing proliferation and migration of neoplastic epithelial cells. This thesis examines signalling pathways regulated by and interacting with the FP receptor that influence chemokine expression and subsequent effects in endometrial adenocarcinoma. To investigate PGF2α-FP interactions in endometrial adenocarcinoma, an endometrial epithelial cell line of adenocarcinoma origin (Ishikawa cells) stably transfected with the FP receptor to levels seen in cancer was used (FPS cells). An antibody array identified the chemokine C-X-C motif Ligand 1 (CXCL1) as a target gene regulated by PGF2α-FP signalling in this cell line. Expression of CXCL1 and its receptor, CXCR2, were elevated in cancer tissue as compared to normal endometrium and localised to glandular epithelium, endothelium and stroma. The induction of CXCL1 expression in FPS cells and endometrial adenocarcinoma explants was determined to be by a signalling pathway involving Gq, the epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK). The infiltration of immune cells into endometrial adenocarcinoma as compared to normal endometrium was then investigated. Increased neutrophils were present in endometrial adenocarcinoma compared with normal endometrium, and the expression of CXCR2 was colocalised to neutrophils. In vitro chemotaxis assays demonstrated that conditioned media from PGF2α-treated FPS cells stimulated human neutrophil chemotaxis which could be abolished by CXCL1 protein immunoneutralisation from the conditioned media or antagonism of CXCR2 on neutrophils. Moreover, xenograft tumours in nude mice arising from inoculation with FPS cells had higher neutrophil infiltration compared to tumours arising from wild-type cells or following treatment of mice bearing FPS tumours with CXCL1-neutralising antibody. Therefore, the up-regulation of CXCL1 by PGF2α promoted neutrophil chemotaxis into endometrial adenocarcinoma. The expression of a further chemokine, CC motif Ligand 20 (CCL20) was determined to be regulated by PGF2α -FP signalling in endometrial adenocarcinoma, and expression of CCL20 and its receptor CCR6 was elevated in endometrial adenocarcinoma. The induction of CCL20 by PGF2α -FP signalling in FPS cells was dependent on the signalling molecules Gq, EGFR, ERK, calcineurin and nuclear factor of activated T-cells (NFAT). The treatment of endometrial epithelial cells with recombinant CCL20 caused a significant increase in proliferation. Finally interactions between the signalling pathway of another pro-inflammatory lipid, lysophosphatidic acid (LPA), and FP receptor signalling in endometrial adenocarcinoma were examined. LPA increased expression of the FP receptor and the FP target genes previously discussed in this thesis, CXCL1 and CCL20, in FPS cells. Expression of the LPA receptors (LPAR) 1, 2 and 3 was localised in endometrial tissue, and LPAR2 and 3 were found to be elevated in endometrial adenocarcinoma compared with normal endometrium, suggesting amplification of the PGF2α -FP signalling pathway by LPA was possible. Collectively, these data demonstrate that inflammatory cytokine signalling pathways regulated by PGF2α-FP activation can promote immune cell infiltration and proliferation of endometrial adenocarcinoma, and that interaction of LPA and PGF2α-FP signalling in endometrial adenocarcinoma may exacerbate the disease.

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1, encoded by Hsd11b1) is an enzyme that predominantly converts inactive glucocorticoids (cortisone in human and most mammals, 11dehydro-corticosterone in mice and rats) into their active forms (cortisol and corticosterone, respectively). Thus 11β-HSD1 amplifies intracellular levels of glucocorticoids. Studies in globally 11β-HSD1 deficient mice have revealed changes in glucocorticoid-regulated physiological and pathological processes, including metabolism, aging, arthritis and angiogenesis. The function of macrophages, which play an important role in inflammation, is also altered. For example, 11β-HSD1 deficiency in macrophages causes a delay in their acquisition of phagocytic capacity. To dissect the role of macrophage 11β-HSD1 in angiogenesis, arthritis and obesity, both in vitro macrophage stimulation and in vivo functional assays in macrophage-specific 11β-HSD1 knockout mice, were conducted. Thioglycollate-elicited peritoneal macrophages from globally 11β-HSD1 deficient and control C57BL/6 mice were used for in vitro studies. In M1/M2 macrophage polarisation experiments, 11β-HSD1 deficient macrophages showed increased expression of mRNAs encoding pro-inflammatory factors upon lipopolysaccharide and interferon-ϒ treatment and decreased expression of pro-resolution genes with interleukin-4 stimulation. However, at cytokine or protein levels, there was little difference between the genotypes except for decrease IL12 p40 levels in 11β-HSD1 deficient macrophages. Hypoxic stress failed to show differences between genotypes in hypoxia-regulated gene expression. These data do not support a strong role for macrophage 11β-HSD1 in inflammation regulation, nor in response to hypoxia, at least when measured in vitro. The discrepancy between transcriptional and translational responses is currently unexplained, but may reflect altered posttranscriptional activity. To investigate the role of macrophage 11β-HSD1 in vivo, macrophage-specific Hsd11b1 knockout mice, LysM-Cre Hsd11b1 flox/flox (MKO) mice and Hsd11b1flox/flox littermate controls were generated. In MKO mice, 11β-HSD1 protein levels and enzyme activity were reduced by >80% in resident peritoneal macrophages. However, 11β-HSD1 protein and enzyme activity levels were unchanged or only modestly reduced in thioglycocollate-elicited peritoneal neutrophils, monocytes/macrophages, or in bone marrow-derived macrophages, despite >80% decrease in Hsd11b1 mRNA levels in these cells. A relatively long half-life of 11β-HSD1 protein compared to that of circulating myeloid cells may underlie this mismatch between transcriptional and translational expression. Furthermore, following 12 days of inflammatory arthritis induced by K/BxN serum transfer, the reduction in 11β-HSD1 protein levels in circulating neutrophils of MKO mice is consistently around 50%, which corroborates the above explanation. MKO mice and littermate controls were subjected to inflammatory models which may involve resident macrophages. First, to address the role of 11β-HSD1 in macrophages in angiogenesis, sponge implants were inserted subcutaneously into the flanks of adult male mice and harvested after 21 days. Chalkley counting on hematoxylin and eosin stained sponge sections showed significantly increased angiogenesis in MKO mice (scores: 5.2±1.0 versus 4.3±0.7; p<0.05, n=9-11). Cdh5 expression (encoding VE-cadherin, a marker of endothelial cells) was higher in sponges from MKO mice (relative expression: 1.5±0.9 versus 0.8±0.6; p<0.05), as was Il1b (encoding IL-1 beta, a marker of inflammation, relative expression: 6.5±6.4 versus 1.5±0.9; p<0.05). Vegfa mRNA (encoding vascular endothelial growth factor alpha) was unchanged, with a trend for higher Angpt1 (encoding angiopoietin 1, p=0.09) expression levels in the MKO group. These results suggest that lack of 11β- HSD1 in resident macrophages increases their pro-angiogenic activity, independently of VEGF-. The K/BxN serum transfer model of arthritis was used to investigate the role of macrophage 11β-HSD1 in arthritis. Adult male MKO and control mice received a single i.p. injection of 125μl K/BxN serum per mouse, followed by 21 days of clinical scoring to assess joint inflammation. The onset of inflammation (d1-8) was similar between MKO and control mice, but MKO mice exhibited greater clinical inflammation scores in the resolution phase of arthritis (d13-21; area-under-the-curve: 86.6±14.7 versus 60.1±13.4; p<0.005), indistinguishable from globally 11β-HSD1- deficient mice. Hematoxylin and eosin staining revealed pronounced fibroplasia predominantly in the supporting mesenchyme associated with the tenosynovium, with new bone and blood vessel formation. These results suggest that macrophage 11β-HSD1 deficiency is fully accountable for the worse arthritis resolution phenotype in the globally 11β-HSD1 deficient mice, but not the earlier onset of inflammation with global 11β-HSD1 deficiency. Macrophage activation states are closely linked with adipose insulin sensitivity. Globally 11β-HSD1 deficient mice are protected from high fat diet induced insulin resistance and adipose tissue hypoxia and fibrosis. To study the effect of macrophage 11β-HSD1 deficiency on insulin sensitivity, adult male MKO and control mice were given a 14 week high fat diet, which typically causes insulin resistance in control but not globally 11β-HSD1 KO mice. The level of fibrosis in subcutaneous adipose tissues was reduced as indicated by quantification of picrosirius red staining of collagen, though GTT data so far does not support protection from insulin resistance in MKO mice. In summary, in vitro macrophage polarisation experiments do not support a strong role of 11β-HSD in M1/M2 macrophage polarisations or response to hypoxia. However, MKO mice reveal, for the first time, an important in vivo role of macrophage 11β-HSD1 to promote angiogenesis and facilitate resolution of K/BxN serum transfer induced arthritis. Modulation of fibrosis is context dependent. Reduced adipose fibrosis may be one of the mechanisms that improve insulin sensitivity. Meanwhile, these findings suggest caution regarding the potential side effects of 11β-HSD1 inhibitors in treating metabolic disease in patients with inflammation-related co-morbidities, such as rheumatoid arthritis.

Chronic in ammation and variations in blood flow have been implicated in the pathogenesis of cardiovascular disease. It is also possible that inflammatory and rheological processes are involved in the development of mild cognitive impairment and dementia, either through their association with vascular disease or via some other, more direct effect on the brain. Evidence is increasing for a causal relationship between Alzheimer's disease and inflammation, possibly related to inflammatory activation of microglia. Inflammatory processes may also be involved in the pathogenesis of cerebral small vessel disease, which in turn has been linked to cognitive impairment and dementia. There is also evidence showing that rheological factors affect cerebral blood flow. However, despite these findings, the associations between inflammatory and rheological markers and cognitive ability have not been extensively studied in large groups of ageing people. The primary aim of this thesis was to test for associations between late-life levels of inflammatory and rheological markers (C-reactive protein (CRP), fibrinogen, tumor necrosis factor (TNF)-α, interleukin (IL)-6, plasma viscosity, and haematocrit) and cognitive ability. A genetic analysis was then performed to model single nucleotide polymorphisms (SNPs) in the genes encoding the markers against cognition in an attempt to determine the weight of evidence for a causal inflammation-cognition association. Four studies were used to test these aims with the majority of the analysis being performed on the Aspirin for Asymptomatic Atherosclerosis (AAA) Trial (n = 3,350), and the Edinburgh Type 2 Diabetes Study (ET2DS) (n = 1,066). The Edinburgh Artery Study (n = 534), and the 1936 Lothian Birth Cohort (n = 1,091), were used as replication cohorts for the genetic analysis. All cohorts comprised community-dwelling, elderly citizens (aged around 70 years) living in central Scotland. With the exception of the ET2DS, all data used were for secondary analyses. Cognitive ability was assessed in all studies using comprehensive batteries of neuropsychological tests that included a measure of crystallised intelligence in the form of a vocabulary test. As performance on such tests varies little across a lifespan, adjusting for these scores in the late-life models enabled the determination of estimated lifetime cognitive change. In the case of the 1936 Lothian Birth Cohort an actual age-11 IQ measure was available in addition to the cognitive follow-up scores recorded at age-70. Linear regression showed small but significant associations between CRP, fibrinogen, and plasma viscosity, and cognition and estimated lifetime cognitive decline in the AAA Trial. Similar results were observed in the ET2DS for CRP, IL-6, and TNF-α. These associations tended to be of a magnitude whereby the markers explained 1% of the variance of the cognitive test scores. The cognitive domains most consistently associated with the markers were processing speed, and a data derived general intelligence factor. A novel genetic analysis was then undertaken to model SNPs against cognitive ability and decline. Most of the results generated were null findings. However, strongly significant associations were found between the rs2227412 fibrinogen beta gene SNP and the cognitive test scores in the ET2DS. Furthermore, the genotype associated with the lowest cognitive scores was also related to higher levels of plasma fibrinogen. Whilst replication of the association between the fibrinogen SNPs and cognition was not found across all cohorts, these results still indicate a potentially causal role for this haemostatic/inflammatory marker. To date, the majority of inflammation-cognition associations have focussed on the acute-phase protein CRP. The main outcomes from this thesis suggest that its close correlate, fibrinogen, is an equally, if not more important factor in the complex process of cognitive ageing.

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Multiple lines of experimental evidence demonstrate that chronic alcoholconsumption causes mitochondrial injury, as well as acinar cell oxidative and metabolicstress. Alcoholics are more susceptible to acute and chronic pancreatitis. Despite alcoholrelatedacinar cell injury, apoptosis, or programmed cell death, appears to be reduced inacinar cells rather than increased, as is seen in the liver.This work describes the possible mechanisms through which alcohol affects theacinar cell apoptosis pathways in the rat pancreas. Two apoptotic pathways wereinvestigated: (1) receptor-mediated apoptosis via Fas/FasL and caspase-8, and (2)mitochondrial-mediated apoptosis via Bcl-2/Bax and caspase-9. Both pathways canactivate the final apoptosis executer caspase-3. Using the Lieber-DeCarli alcohol/controldiet, rats fed alcohol for 14 weeks had a significant decrease of key mediators of theFas/Fas ligand receptor-mediated pathway, while the mitochondria-associated apoptoticpathway is inappropriately deactivated. In addition, this study describes the mRNAexpression of inflammatory cytokines, such as IL-1??, IL-6, TNF??, IL-18 and TGF??,which are reported to influence inflammation and apoptosis. The anti-inflammatoryeffects of alcohol were confirmed with decreased expression of regulatory cytokinesincluding IL-1??, IL-18, TGF?? and IL-6 in alcohol-fed rats.Alcohol appears to block apoptosis in the pancreas through multiple mechanisms.Activity of the Fas/Fas ligand receptor-mediated pathway appears to be suppressed at thelevel of caspase-8, with further inhibition by down-regulation of caspase-3. Despiteknown acinar cell stress and mitochondria injury, the mitochondria-mediated apoptoticpathway was not activated. This data suggest that alcohol consumption suppresses theremoval of mitochondria injured acinar cells, promoting apoptosis resistance, and mayincrease the susceptibility to pancreatitis. The increased susceptibility to pancreatic injurywas further investigated by using lipopolysaccharide (LPS). Alcohol exacerbates LPSinducedpancreatoxicity by enhanced pancreatic apoptosis. The attenuation of apoptosisby ethanol increased the threshold of apoptosis in response to LPS and acceleratesapoptosis. Here it is hypothesized that alcoholics are more susceptible to endotoxinmediatedacute pancreatitis and the response is more severe than in non-alcoholics.

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