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Inflammation is fundamentally a beneficial response leading to removal of the offending factor, and resolution, an active regulated process that is essential to maintain tissue integrity and function. Previous work in our department showed a role for COX-2 derived PGD2 and its cyclopentenone metabolite 15d-PGJ2 during resolution in a pleurisy model. I investigated mice lacking hPGD2S and therefore absent PGD2 and 15d-PGJ2 by inducing peritonitis with zymosan. The resolution of peritonitis in hPGD2S mice was delayed compared to wild type C57black VI mice. PGD2 via its action on the DP1 receptor controls the balance of pro- versus antiinflammatory cytokines that regulate leukocyte influx as well as monocyte-derived macrophage efflux from the inflamed peritoneal cavity to draining lymph nodes leading to resolution. Previous data that laid doubt to the presence of 15d-PGJ2 in in vivo models and questioned its role in resolution of innate inflammation. By measurement of peritoneal exudates with LC-MSMS, there is definitive proof that 15d-PGJ2 is synthesised during mammalian inflammatory responses. When the cellular profile was examined by flow cytometry, a biphasic response of lymphocytes was observed with the disappearance during acute inflammation with activation of the DP-1 receptor. The profile of lymphocytes that reappear during resolution differed from the naïve state and comprised of B1, NK, gamma/delta T, CD4+/CD25+ and B2 cells. The lymphocytes do not appear to have a role in resolution as lymphocyte deficient RAG2-/- and wild types resolve uniformly. However repopulating lymphocytes are critical for modulating responses to superinfection as observed by the exaggerated peritonitis/death of RAG2-/- mice when exposed to group B streptococcus following zymosan. In gp91phox-/- mice, an experimental model of CGD, where the peritonitis failed to resolve, the adaptive transfer of resolution phase lymphocytes into the peritoneum, was protective against superinfection. Furthermore in wild type mice when the anti-inflammatory adenosine and downstream cAMP were measured in peritonitis, there was a biphasic response. In gp91phox-/- mice, both adenosine and cAMP were significantly lower at onset and again at resolution. Adenosine, signalling 15 through its A2A receptor and therefore elevating cAMP is not only anti-inflammatory, but also importantly, it does not impair pro-resolution pathways as antagonism of the A2A receptor worsens acute inflammation and prolongs resolution. In summary, resolution is an active regulated process that requires the input COX-2 derived PGD2 as well as anti-inflammatory A2A receptor activation. To further refine this model it was shown that repopulating lymphocytes are essential to prevent super-infection and persistence of inflammation.

The circadian clock drives ~24hr rhythms in a variety of processes, from gene expression through to behaviour, facilitating anticipation of daily changes in the external environment and temporal separation of internal processes. This pacemaker is a critical regulator of immune function and many inflammatory diseases show time-of-day variation in symptom severity. Disruption of the pacemaker by manipulation of the daily cycle of light and dark exposure (experimental 'jet lag') is known to exacerbate inflammatory responses to innate immune challenge, and recent evidence has highlighted immuno-modulatory roles for components of the molecular oscillator in peripheral tissues. The adrenal-derived glucocorticoid hormones are potent anti-inflammatory molecules and are capable of modulating circadian oscillations in peripheral tissues. This, along with their rhythmic secretion profile, makes them key candidates as mediators of circadian regulation of inflammatory signalling. Utilising adrenalectomy, timed glucocorticoid administration, hormone clamp and genetic targeting of the glucocorticoid receptor in mice, I present evidence for an interaction between glucocorticoid signalling and the circadian pacemaker in regulating the pulmonary inflammatory response to lipopolysaccharide (LPS) challenge. The neutrophilic response to aerosolised LPS exhibits a clear time-of-day effect in vivo, which is lost after disruption of endogenous glucocorticoid production via adrenalectomy. However, replacement of a rhythmic circulating glucocorticoid concentration with a flat daily average using a subcutaneous hormone clamp does not disrupt the inflammatory rhythm. Finally, a novel mouse strain was produced with disrupted expression of the glucocorticoid receptor (GR) in bronchial epithelial cells (Ccsp-GR-/-). These cells are critical regulators of circadian rhythmicity in the lung and drive rhythmic neutrophil influx in response to LPS stimulation through production of the chemokine CXCL5. Loss of GR in the bronchial epithelium was associated with a loss of rhythmic neutrophil influx after challenge, but anti-inflammatory sensitivity to the synthetic glucocorticoid dexamethasone remained. Collectively, these data show that appropriate temporal modulation of pulmonary inflammation requires functional glucocorticoid signalling, although the ligand itself does not need to oscillate. The retention of anti-inflammatory dexamethasone sensitivity suggests a role for cross-talk between the bronchial epithelium and additional cell populations, consistent with recent evidence for immuno-suppressive macrophage-epithelium communication in the lung. These are the first studies to dissect the mechanistic links between clocks, glucocorticoids and immunological responses in a target tissue.

Menstruation is triggered by a fall in circulating progesterone (P4), and to a lesser extent, oestradiol (E2) concentrations, and characterised by classical inflammatory features in the endometrium: breakdown of the basal lamina, tissue oedema and an influx of migratory leucocytes. During and following menstruation, endometrial inflammation is resolved and the endometrium is repaired. The successful resolution of acute inflammation in other tissues involves apoptosis and the phagocytic clearance of apoptotic cells. Human endometrial tissues were collected with informed patient consent and local research ethics committee approval. C57Bl/6 mice underwent an induced menstruation protocol (via sequential E2 and P4 exposure followed by P4 withdrawal), both with and without experimental inhibition of apoptosis (using the pan-caspase inhibitor, Q-VD-OPh). Coordinated apoptosis and neutrophil recruitment were hypothesised to be components of the menstrual event and to precede menstrual shedding in the human endometrium. Immunoreactivity histoscoring for cleaved caspase-3 (CC3) revealed extensive apoptosis in the normal human endometrium early in the ‘perimenstrual’ period, and careful stereological delineation of neutrophil (elastase+) recruitment showed a significant influx coincident with menstrual tissue breakdown. Apoptosis and neutrophil recruitment were hypothesised to follow similar courses in the endometria of mice undergoing an induced menstruation protocol, recapitulating human menstrual events. Immunoreactivity histoscoring for CC3 and stereological investigation into neutrophil (Ly6G+) recruitment in mouse endometrial tissues revealed almost identical extents and timings of apoptosis and neutrophil recruitment in women. Whole genome array evidence of differential apoptosis-related gene transcription in the endometria of women with heavy menstrual bleeding (HMB) compared to those of women with normal menstrual bleeding (NMB) led to the hypothesis that apoptosis may be dysregulated in women with HMB and that perhaps this may delay timely repair of the endometrium and lead to prolonged bleeding in consequence. Candidate differentially-regulated gene transcripts identified by the whole genome array were validated by means of RT-qPCR, although immunoreactivity histoscoring for CC3 did not reveal any differences in apoptosis or its localisation between women with NMB and HMB at the menstrual cycle time-points examined. Building on evidence of apoptotic transcriptional dysregulation in the endometria of women with HMB, it was hypothesised that experimental inhibition of apoptosis (via Q-VD-OPh) in a mouse model of induced menstruation could delay endometrial repair and delay resolution of endometrial inflammation. Some evidence of delayed early repair was obtained, alongside the discoveries of delayed inflammatory gene transcription and increased decidual proliferation (BrdU+) in apoptosis-inhibited mice. Apoptosis precedes the classical inflammatory features of menstruation in the human and mouse endometrium, with inhibition of apoptosis in the latter altering repair and the inflammatory micro-environment. An apoptosis-inhibited mouse model of menstruation may therefore represent a viable model for the further study of heavy menstrual bleeding.

Excessive tissue-damaging inflammation can exacerbate acute brain injury, and non-resolving inflammation is implicated in chronic neurodegeneration. Understanding the mechanisms that resolve deleterious inflammation in the brain is imperative to develop new therapeutic strategies. However current knowledge is limited, partly due to a lack of tractable models. Studies in extra-cerebral tissues have shown that myeloid cells are central to the inflammatory response. The aim of this thesis was to develop a model of self-limiting acute brain inflammation that is optimised to address mechanisms controlling resolution. The model was used to define the temporal profile of myeloid cell accumulation in the brain and establish the precise identities, origin and functional contribution of cell subsets in the resolution of the inflammatory response in the brain. Cerebral inflammation was induced by stereotaxic injection of inflammatory stimuli (LPS, HMGB1, MSU); LPS produced a robust inflammatory response and neutrophil influx and loss defined clear phases of initiation and resolution. Cellular changes (e.g. glial activation, endothelial activation and leukocyte influx) in response to LPS were characteristic of acute inflammation. Bone marrow chimaeric (Csf1r-EGFPC57Bl/6J) and monocyte reporter (Ccr2+/RFP) mice were used to distinguish between infiltrating macrophages and resident microglia. Analysis over 28 d showed the temporal profile of myeloid cells during brain inflammation, and monocyte accumulation contributed to expansion of the total mononuclear phagocyte population. Ccr2RFP/RFP knock-in mice showed that monocyte recruitment and resolution were independent of CCR2, and selective depletion of Ly6Clo monocytes with an anti- CSF1R antibody did not affect macrophage recruitment. Monocyte depletion using clodronate failed to deplete the Ly6Cint population and monocytes were still recruited into the brain. Together these results suggest multiple monocyte subsets could be involved in the inflammatory response in the brain. These data show that myeloid cell subsets of distinct origins accumulate in the inflamed brain. This work establishes a model system to identify endogenous mechanisms of resolution in cerebral inflammation and provides a platform to test CNS-targeted pro-resolution agents.

Some mounted ill. Bibliography: leaves 197-226.

The S100s comprise a group of Ca2+-binding proteins of the EF-hand superfamily with varied functions. Within this family, three inflammatory-related proteins - S100A8, S100A9 and S100A12 - form a subcluster known as the 'calgranulins'. S100A12 levels are elevated in sera from patients with inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. S100A12 is constitutively expressed in neutrophils and induced in monocytes by LPS and TNFα, and in macrophages by IL-6. S100A12 is a potent monocyte and mast cell chemoattractant and its potentiation of mast cell activation by IgE cross-linking indicates an important role in allergic inflammation. Importantly, mast cell-dependent activation of acute inflammatory responses and monocyte recruitment is provoked by S100A12 administration in vivo. S100A12 may also influence adhesion molecule expression on endothelial cells, stimulate IL 1β and TNFinduced in monocytes production in BV 2 microglial cells, and stimulate IL 2 secretion by T lymphocytes via ligation of the receptor for advanced glycation end-products (RAGE). To date, the only extracellular receptor characterised for S100A12 is RAGE, although additional/alternate receptors are indicated. In particular, recent studies indicate that chemotaxis and mast cell activation by S100A12 are likely mediated by other receptors. The studies presented here investigated some extracellular functions of S100A12, factors influencing these functions and suggest mechanisms that may be involved. In addition to Ca2+, S100A12 binds Zn2+. Chapter 3 explores the relevance Zn2+ binding to S100A12 structure and function. Zn2+ induced formation of complexes, principally hexamers, and this was not influenced by Ca2+. S100A12 inhibited the gelatinolytic activities of matrix metalloproteinase (MMP)-2 and 9 by chelating Zn2+ from their active sites. MMPs are important in processes leading to plaque rupture. An antibody that specifically recognised Zn2+-induced complexes was generated and immunohistochemical studies demonstrated S100A12, the hexameric complex, and MMP 2 and 9 co-localisation in human atheroma. These results suggest that hexameric S100A12 may form in vivo and may implicate S100A12 in regulating plaque rupture by inhibiting MMP activity. Interestingly S100A12 synergised with LPS to induce MMP 3 and 13 expression in vitamin D3-differentiated THP 1 macrophages (THP 1 macs). S100A12 regulation of MMP expression and activity indicates that it may be involved in a self-regulatory loop, which depends on relative levels of Zn2+ and on other stimuli (eg LPS) in the inflammatory milieu. Chapter 4 describes the development of tools and methods for assessing interactions of S100A12 with cell surface receptors. To assay surface binding, an alkaline phosphatase fusion protein, a biotinylated hinge peptide and biotinylated recombinant S100A12 were generated; only S100A12 b proved useful. Surface binding of S100A12 was detected on several monocytoid/macrophage and mast cells using flow cytometry and immunocytochemistry. Some cells contained intracytoplasmic granular structures that were S100A12-positive. Unexpectedly, a subpopulation of cells in murine bone marrow-derived mast cell cultures that expressed low levels of c-kit, a marker of mature mast cells, bound high levels of S100A12. These may represent haematopoietic stem cells, which express low levels of c kit, and S100A12-mediated functional changes of these cells is worthy of characterisation. Unlike interactions of S100A8/A9 with endothelial cells, pre-incubation of S100A12 with Zn2+ or heparin had no effect on surface binding to THP 1 macs, indicating that Zn2+-induced structural changes were unlikely to alter receptor interactions. Heparan sulfate moieties are unlikely to mediate surface binding of S100A12 even though S100A12 bound heparin with relatively high affinity. Chapter 5 focussed on mechanisms involved in some S100A12 extracellular functions. Based on experiments studying effects of bovine S100A12 on BV-2 murine microglial cells, S100A12 is proposed to induce pro-inflammatory cytokine in monocytes via RAGE. Human peripheral blood mononuclear cells or human THP 1 macs activated with S100A12 did not increase cytokine induction at the mRNA or protein levels, indicating that the 'S100/RAGE pro-inflammatory axis' theory should be re-evaluated. In an attempt to provide insights into a novel receptor, mechanisms involved in S100A12-provoked THP 1 chemotaxis were investigated. This activity was sensitive to pertussis toxin, but not to an ERK1/2 pathway inhibitor, suggesting involvement of a G protein-coupled receptor. Although some RAGE ligands also bind and activate Toll-like receptors (TLRs) antibodies to TLR2 and TLR4 did not block S100A12 binding to THP 1 macs. Affinity enrichment and separation of proteins by SDS PAGE and peptide mapping by mass spectrometry identified the α and γ subunits of F1 ATP synthase, implicating ATP synthase as a putative receptor. Although primarily mitochondrial, this complex is expressed on the surface of several cell types and was confirmed on THP 1 cells and mast cells by flow cytometry. By modulating surface F1 ATP synthase activity, and thereby extracellular ATP/ADP concentrations, S100A12 may mediate its pro-inflammatory functions through G-protein coupled purinergic receptors. This work has generated new directions for studying mechanisms by which S100A12 influences monocyte/macrophage and mast cell functions that are relevant to important inflammatory diseases, such as atherosclerosis and allergic inflammation.

Graduation date: 1997

Thesis (M.S.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Mary P. Miles. Includes bibliographical references (leaves 62-66).

C-reactive protein (CRP) is associated with an increased risk of cardiovascular disease (CVD), peripheral vascular disease, diabetes, and stroke. In addition to traditional risk factors of CVD, some studies have shown that depression and anger independently predict CRP, but other studies have found null results, and few, if any, studies have considered possible roles of physical activity and diet. The purpose of this study was to investigate the ability of certain psychosocial variables to predict CRP controlling for traditional CVD risk factors. Cross-sectional data for 300 healthy women who participated in the Stockholm Female Coronary Risk Study were analyzed. Regression analyses were performed to determine whether anger, depression, social support, marital stress, and self-esteem were associated with CRP levels while controlling for relevant covariates. Analyses investigated possible mediating effects of certain aspects of diet and physical activity and whether body composition (measured by waist circumference) and fasting glucose moderates the relationship between psychosocial variables and CRP. We found that anger symptoms were negatively associated with CRP and anger discussion was positively associated with CRP controlling for several biological variables. Diet and physical activity did not explain the relationship between these anger variables and CRP. Social support in the forms of social attachment and social integration were positively associated with CRP among women with a larger waist circumference and higher fasting glucose, respectively. Marital stress was positively related to CRP among women with a larger waist circumference. Among women with a smaller waist circumference, marital stress was negatively related to CRP and social integration was positively related to CRP. These findings suggest that having a large waist in addition to less social support and more marital stress is disadvantageous with regard to CRP. Furthermore, it is possible that being quite thin may not necessarily be advantageous with regard to inflammation.