Thesis (Ph. D.)--Ohio State University, 20043. / Title from first page of PDF file. Document formatted into pages; contains xiv, 115 p.; also includes graphics. Includes abstract and vita. Advisor: Clay B. Marsh, Dept.of Pathology. Includes bibliographical references (p. 102-115).

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

In this thesis I examined the role of circulating monocytes and lung macrophages in the pathogenesis of the early fibrotic, progressive fibrotic and resolution phases of pulmonary fibrosis. Pulmonary fibrosis with destruction of lung architecture and consequent respiratory failure and death represents a massive worldwide health burden. Although idiopathic pulmonary fibrosis (IPF) is the archetypal and most common cause of lung fibrosis, numerous respiratory diseases can progress to pulmonary fibrosis, and this usually signifies a worse prognosis. Importantly, the incidence and prevalence of IPF continue to rise and it remains one of the few respiratory conditions for which there are no effective therapies. The mechanisms resulting in pulmonary fibrosis are controversial. Early work in the 1980s and 1990s suggested that lung macrophages were important. However, at the turn of the 21st century there was a shift to a belief that pulmonary fibrosis resulted from aberrant wound healing as a consequence of repetitive epithelial injury from an as yet unknown cause. However, with the ever expanding knowledge of the importance of macrophages in other fibrotic conditions such as the kidney and liver, the potential importance of macrophages in pulmonary fibrosis has become more pertinent. Using an in vivo depletional strategy in several murine models of lung fibrosis, in conjunction with human studies, I sought to characterise the role of circulating monocytes and lung macrophages in the pathogenesis of pulmonary fibrosis. I have established that circulating monocytes and lung macrophages are not critical for the development of early lung fibrosis. In contrast, circulating monocytes and lung macrophages are important during the progressive fibrotic phase of lung fibrosis. Furthermore, my data suggest that the pro-fibrotic alternatively activated macrophages may be the sub-class of macrophages that mediate this fibrogenic effect. In addition and in contrast, I have established that lung macrophages are required for the resolution of fibrosis. This finding is in keeping with important work performed in the field of liver fibrosis. There is an ever increasing literature examining the role of matrix metalloproteinases (MMPs) during tissue fibrosis and repair. My work has suggested that during lung fibrosis there may be compartmental specific functions of MMPs that regulate lung fibrogenesis, although more work is required before this exciting finding can be properly defined.

Résumé Par leur capacité de moduler la réponse immune, les rayons ultraviolets (UV) ont trouvé des applications dans le traitement de diverses maladies immunes. Leurs mécanismes d’action sont encore incomplètement définis. L’un d’entre eux comporte l’induction de cytokines immunosuppressives et antiinflammatoires. Ce processus peut être provoqué par la phagocytose de corps apoptotiques, l’apoptose constituant une des lésions cellulaires élémentaires provoquée par les UV. Le but de notre travail a été de préciser les cytokines impliquées dans la réponse aux UV, de définir certains mécanismes de leur production et de la potentialiser par des agents pharmacologiques. Notre étude a comporté deux parties: (1) l’une in vivo chez des malades souffrant de GVH chronique résistante aux traitements conventionnels et traités par photochémothérapie extracorporelle, procédure dans laquelle les leucocytes du malades, prélevés par leucaphérèse puis traités par un psoralène et par UVA lui sont finalement réinjectés; (2) l’autre in vitro où des PBMC de volontaires sains ont été irradiés avec 10J/m2 de rayons UVC qui ne nécessitent pas de photosensibilisation par psoralène. Deux cytokines, l’IL-10 et l’IL-1Ra ont été évaluées par RT-PCR dans un système de coculture autologue entre PBMC et PBL rendus apoptotiques par irradiation. L’évolution du processus apoptotique déclenché par les UV a été mesurée par cytomètrie de flux. Celui-ci concernait essentiellement les lymphocytes, les monocytes/macrophages révélant une résistance relative à l’apoptose, il était progressif, culminant entre la 24ème et la 48ème heures. Lors des cocultures entre PBMC et PBL irradiés, un accroissement très significatif du nombre de copies d’ARNm, concernant les deux cytokines étudiées, l’IL-10 et l’IL-1Ra était observé. L’induction d’IL-1Ra était dépendante de l’IL-10. Une préactivation par du LPS était nécessaire pour la révélation du phénomène. Ensuite, nous avons évalué l’implication sur la synthèse de cytokines du processus de phagocytose de lymphocytes rendus apoptotiques par irradiation UV et divers moyens pharmacologiques pour la potentialiser. La préincubation du matériel irradié pendant une nuit (16h) à 37° dans le but d’accroître la proportion de cellules en voie d’apoptose avant mise en contact avec les PBMC a permis d’obtenir un accroissement très marqué sans nécessiter de LPS, portant essentiellement sur la production d’IL-1Ra tant sur l’ARNm que la protéine secrétée; l’induction d’IL-10 était cette fois négligeable. L’implication de la phagocytose dans le processus a été démontrée par deux agents bloquants (a) l’anticorps monoclonal anti-CD36 (corécepteur avec l’intégrine V3 de la thrombospondine) activant la production d’IL-1Ra et mimant par ce fait le processus phagocytaire et (b) la cytochalasine E la bloquant. Nous avons testé diverses substances pharmacologiques dont l’action activatrice de l’IL-1Ra est connue, en l’occurrence les immunoglobulines G à usage IV (IgIV) et le GM-CSF. L’adjonction d’IgIV (1mg/ml) ou GM-CSF (10 ng/ml) une heure après le début de la coculture exerce sur la sécrétion d’IL-1Ra un effet additif avec les UV. Selon la concentration utilisée, les IgIV peuvent agir par deux mécanismes. Outre l’effet d’activation macrophagique lié au récepteur Fc, nous avons démontré à haute concentration un mécanisme nouveau, du à la présence dans les IgIV d’anticorps naturels antiFas induisant l’apoptose des lymphocytes. Une incubation de 16h des lymphocytes avec 25 mg/ml d’IgIV avant mise en culture provoque outre une apoptose importante une augmentation significative de l’IL-1Ra. Dans ce cas, le processus est indépendant du fragment Fc, la fraction F(ab’)2 gardant la capacité d’induire l’apoptose et de provoquer la production d’IL-1Ra. En conclusion, nous avons mis en évidence un mécanisme nouveau d’induction d’IL-1Ra, non décrit auparavant et défini diverses modalités qui pourraient accroître sa production: - L’incubation de 16h du matériel irradié permet d’orienter le système en accroissant la production de l’IL-1Ra sans que la production de l’IL-10 soit modifiée et sans nécessiter de LPS. Nous attribuons cet effet à l’accroissement du processus apoptotique qui en résulte. - Nous avons potentialisé la production d’IL-1Ra par deux agents pharmacologiques, le GM-CSF et les IgIV. Les mécanismes d’action des IgIV dépendent de la concentration utilisée. 1. Aux concentrations de l’ordre de 1mg/ml, les IgIV exercent, avec les UV un effet additif sur l’induction d’IL-1Ra par une action dépendant du fragment Fc. 2. Aux concentrations élevées de 25mg/ml, un effet apoptotique attribuable à l’action d’anticorps anti-Fas agonistes est observé. Une préincubation de 16h de lymphocytes avec cette concentration d’ IgIV avant mise en culture avec les PBMC autologues provoque outre l’apoptose importante des lymphocytes un accroissement significatif de la production d’IL-1Ra. Le processus est indépendant du fragment Fc, la fraction F(ab’)2 gardant la capacité d’induire l’apoptose et la production d’IL-1Ra.

Thèse de doctorat : Biologie Moléculaire. ARN : Biogenèse et structure / fonction : Nancy 1 : 2008. / Titre provenant de l'écran-titre.

Thesis (M.Med.Sc.) - University of Queensland, 2003. / Includes bibliography.

Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains x, 182 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Introduction Chikungunya virus (CHIKV) is an alphavirus in the family Togaviridae. Since 2005 the virus has caused a major epidemic of disease in humans, ranging from Central Africa, South-East Asia, Caribbean and more recently the Americas. The virus is spread by mosquitoes, most notably Aedes aegypti and Ae. albopictus. CHIKV causes an acute disease in humans, which is characterised by a rapid onset of high fever, rash, myalgia and arthralgia. The symptoms typically resolve within a week. Remarkably, up to a third of patients who recover from acute chikungunya develop chronic arthritis/arthralgia, which may last for months or years and has a large negative impact on the quality of life. The mechanism by which this occurs is not yet fully understood. CHIKV can infect human monocytes, and macrophages positive for CHIKV antigen have been observed in joint tissue from patients recovered from acute CHIKV infection but with chronic arthritis. Furthermore, it has been demonstrated that macrophages can be infected with CHIKV in vitro by a mechanism involving apoptotic debris from CHIKV-infected cells. Hypothesis and aims Infection of monocytes and macrophages with CHIKV contributes to clinical disease and virus persistence in vivo. The aim of this project was to investigate the mechanism by which alphaviruses infect macrophages in vitro, and to generate a CHIKV which is unable to replicate in monocytes and macrophages in vitro, and to study its pathogenicity in vivo. Materials and methods HeLa cells were infected with Semliki Forest virus (SFV), an alphavirus closely related to CHIKV, or SFV replicon particles (SFV VRP). Following cell death, whole cell supernatant or clarified cell supernatant from SFV- and SFV VRP-infected cells was passaged onto human monocyte-derived macrophages (MDMs). These cells were observed microscopically for expression of the fluorescent marker encoded by the SFV. Virus and VRP-infected apoptotic debris were inspected for the presence of alphavirus replication complexes by electron microscopy. Subsequently, a recognition element (RE) for a haematopoietic-specific miRNA (miR-142-3P) was incorporated into the genome of SFV (proof-of-concept) and CHIKV to investigate if blocking virus replication in cells of the mononuclear phagocyte system altered virus kinetics in vitro. The replication of the modified viruses was investigated in macrophage/monocyte cell lines Thp-1 and IC-21, and in HEK 293 cells modified to express miR-142-3P under the control of an inducible tetracycline promoter. Modified viruses were tested in animal models of disease (mouse for SFV and non-human primate for CHIKV) to investigate the pathogenicity of these viruses in vivo. Results The presence of apoptotic debris from SFV-infected cells was required to infect MDMs with SFV. The presence or absence of infectious virus particles in the apoptotic debris did not affect the infection rate. Intact alphavirus replication complexes were found within the apoptotic debris. MiR-142-3P RE was successfully incorporated into the genome of both SFV and CHIKV. RE-virus replication in all cells expressing miR-142-3P was reduced by 90-99% when compared to control viruses. RE-virus replication was not affected in cells which did not express miR- 142-3P. In interferon-α/β receptor knockout mice, RE-SFV generated viraemia comparable to the control virus, but could not infect efficiently the population of macrophages resident in the marginal zone of the spleen. RE-CHIKV was found to be genetically stable in vitro following multiple passages on BHK-21 cells in the absence of a selective pressure from miR-142-3P. RE-CHIKV was inoculated into two cynomolgus macaques. The data from this experiment are not yet available. Conclusion SFV was shown to infect MDM via apoptotic debris containing intact alphavirus replication complexes, which were the most likely infectious agent. SFV and CHIKV unable to replicate in haematopoietic cells in vitro were successfully engineered. The pathogenicity of modified SFV and CHIKV was investigated in vivo.

Effective treatment of the acute systemic inflammatory response associated with sepsis is lacking, but likely will require new ways to rebalance dysregulated immune responses. One challenge is that human sepsis often is diagnosed too late to reduce the hyperinflammation of early sepsis. Another is that the sequential response to sepsis inflammation rapidly generates an adaptive and immunosuppressive state, which by epigenetic imprint may last for months or years. Emerging data support that the immunosuppressive phase of sepsis can both directly reprogram gene expression of circulating and tissue cells, and disrupt development and differentiation of myeloid precursor cells into competent immunocytes. We recently reported that adoptive transfer of bone marrow CD34+ cells into mice after sepsis induction by cecal ligation and puncture significantly improves late-sepsis survival by enhancing bacterial clearance through improved neutrophil and macrophage phagocytosis. That study, however, did not examine whether CD34+ transfer can modify noninfectious acute systemic inflammatory responses. Here, we report that CD34+ cell transfer mice that have survived late sepsis also resist lethal lipopolysaccharide (LPS)-induced inflammatory shock (88% lived vs 0% of naive mice). The CD34+ cell-recipient survivor mice administered LPS had globally reduced levels of circulating inflammatory mediators compared with naive mice, but their peritoneal and bone marrow-derived macrophages (BMDMs), unlike those from naïve mice, remained LPS responsive ex vivo. We further found that CD34+ cell transfer into LPS-challenged naïve mice had diminished immunosuppression, as assessed by ex vivo responses of peritoneal and BMDMs to LPS challenge. We conclude that CD34+ cell adoptive transfer rebalances dysregulated immune responses associated with sepsis and endotoxin shock.

We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal β-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the β-glucan receptor (βGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional βGR isoforms - βGR-A and βGR-B - were expressed by these cell populations in peripheral blood; however, only βGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other β-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major β-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.