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.

Approximately 50% of all malignant pleural mesotheliomas (MPM) are deficient in argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine biosynthesis, and are sensitive to arginine deprivation. This discovery in MPM has been translated into the clinic using the arginine depletor pegylated arginine deiminase (ADI-PEG20), which showed a halving in the risk of disease progression in a randomised phase II study. However, unstudied to date, stromal resistance to ADI-PEG20 may reduce its efficacy. Here, I studied the effect of macrophages, abundant in mesothelioma, on the tumour cytotoxicity of ADI-PEG20. A distinct pro-inflammatory cytokine gene expression signature involved in macrophage recruitment and activation was identified and validated in ADI-PEG20-treated ASS1 negative MPM cell lines. In vivo induction of pro-inflammatory cytokines was also seen in ADI-PEG20-treated patient plasma. Notably, in vitro co-culture experiments demonstrated a significant increase in ASS1 negative MPM cell viability upon co-culture with macrophages in the presence of ADI-PEG20. This was accompanied by a significant increase in ASS1 expression in co-cultured macrophages, with a corresponding increase in argininosuccinate lyase (ASL) expression in co-cultured tumour cells and a doubling in levels of the arginine precursor, argininosuccinate, in cell supernatant. The addition of argininosuccinate to tumour cell media rescued ASS1 negative MPM cells from ADI-PEG20 cytotoxicity, while the macrophage-mediated resistance to ADI-PEG20 was abrogated following ASL knockdown in MPM cells. Finally, xenograft studies demonstrated a significant reduction in tumour volume in mice treated with ADI-PEG20 in combination with macrophage depletion, compared with ADI-PEG20 alone. Collectively, the data indicate that as a result of metabolic 'cross-talk' between macrophages and ASS1 negative MPM cells, macrophages mediate MPM resistance to ADI-PEG20 via the provision of argininosuccinate. My studies provide a rationale for combining ADI-PEG20 with an inhibitor of macrophage recruitment in the treatment of ASS1-deficient mesothelioma.

Released in response to cellular activation, microvesicles are a major vector mechanism for the delivery of protein, nucleic acid and bioactive lipid payloads in local tissues and plasma. Large numbers of microvesicles (including those from neutrophils) are found within inflammatory sites, such as the rheumatoid synovium. Human neutrophil microvesicles promote tissue protection, and in some cases repair, by affecting function and phenotype of other inflammatory cells. Of these, tissue macrophages are central to the recovery of homeostasis after an inflammatory insult. The data herein indicate that microvesicles released by activated neutrophils impede lipopolysaccharide and interferon gamma-induced \M1-like" polarisation of macrophages via phosphatidylserine (PtdSer) exposure, and induce annexin A1-dependent release of transforming growth factor beta (TGFb). Macrophages treated with these vesicles stimulate the production of cartilage matrix from chondrocytes, and are unable to induce an inflammatory phenotype in fi broblasts. The efficacy of these vesicles is reproduced in two in vivo models of acute inflammation, zymosan-induced peritonits and K/BxN serum-transfer arthritis. Finally, the possibility of using both autologous, and cell-line-derived microvesicles as pharmacodynamic tools is explored. Microvesicles generated from neutrophils from patients with rheumatoid arthritis are found to be protective, and can outcompete the pro-inflammatory effects of both platelet microvesicles, and those isolated from synovial fluid of patients with rheumatoid arthritis. By building on the observation that anxA1 on microvesicles stimulates TGFb release in macrophages, a cell line was transfected to release anxA1+ microvesicles, and their e ects compared to those of their wild type counterparts.

O Transtorno Bipolar (TB) é uma doença psiquiátrica grave, altamente incapacitante que está associada com diversas comorbidades médicas e altas taxas de suicídio. Embora sua fisiopatologia não esteja completamente elucidada, inúmeros estudos têm mostrado alterações no sistema imune de indivíduos com TB. A resposta crônica destes indivíduos ao estresse parece gerar um aumento da inflamação sistêmica bem como da neuroinflamação. A micróglia ativada devido aos estímulos inflamatórios contínuos deve ocasionar diferentes prejuízos tanto bioquímicos quanto funcionas. Os macrófagos, primeira linha de defesa, são células de característica plástica de extrema importância do sistema imune e podem ser estimulados a polarizar para diferentes formas com liberação de fatores pró e antiinflamatórios, estimulando ou mantendo a homeostase no ambiente agredido de alguma forma. Desta forma, nosso trabalho buscou investigar a resposta fenotípica dos macrófagos contra o meio ambiente pró-inflamatório sistêmico observado no plasma de pacientes bipolares eutímicos, maníacos e depressivos em comparação aos controles. A amostra incluiu 5 controles saudáveis, 8 pacientes bipolares remetidos, 5 pacientes maníacos e 5 pacientes depressivos. As citocinas e quimiocinas de RNAm em células U937 tratadas com plasma mostraram um padrão de expressão diferente relativo entre controles saudáveis e pacientes com TB. As citoquinas inflamatórias tais como IL-1β e TNF-α, em pacientes bipolares maníacos e depressivos demonstram maiores quantidades de IL-1β mRNA do que os pacientes eutímicos e pacientes depressivos induziram maiores quantidades de RNAm de TNF-α do que os pacientes eutímicos em células U937. Já a expressão das quimiocinas CXCL9 e CXCL10 no plasma de pacientes com TB depressivos, demostraram ser de menor expressão significativa no grupo de pacientes maníacos quando comparados a controles e pacientes bipolares eutímicos. Nossos resultados sugerem que as citocinas periféticas devem modular a polarização M1 ou M2 de macrófagos no TB. / Bipolar Disorder (BD) is a severe and highly incapacitating psychiatric disorder which is associated with the presence of medical comorbidities. The progression of BD is related to an important cognitive deficit and also to biological and clinical manifestations that lead to treatment resistance and worse prognosis. Immune disturbances have been widely observed and investigated in BD patients. Chronic inflammatory responses induce neuroinflammation, mainly by pro-inflammatory microglial activation, and result in biochemical and functional impairment. Macrophages are the first line of defense of the immune system and exhibit cell plasticity. As well, microglia represents the resident macrophage of the central nervous system been responsible for its protection. Both cells can be stimulated to polarize into two different phenotypes, mainly pro- and anti-inflammatory, maintaining the homeostasis under physiologic and pathologic conditions. Therefore, we aimed to investigate macrophages phenotypical response when submitted to BD patients plasma in different episodes, which is considered a pro-inflammatory environment, and healthy controls plasma. Subjects included healthy controls (n=5), remitted BD patients (n=8), manic patients (n=5) and depressive patients (n=5). The mRNA expression of chemokynes and cytokines from U937 cells treated with BD patients plasma were different from those submitted to healthy controls plasma. Higher mRNA expression of IL-1β was observed in those cells submitted to manic and depressive BD patients plasma when compared to euthymic patients. Also, depressive BD patients plasma induced higher expression of TNF-α compared to euthymic patients. However, chemokynes expression, such as CXCL9 and CXCL10, were reduced in depressive BD patients. However, chemokynes expression, such as CXCL9 and CXCL10, were reduced in depressive BD patients. Inflammatory cytokines such as IL-1β and TNF-α in bipolar manic and depressive patients demonstrate higher amounts of IL-1β mRNA that euthymic patients and depressive patients induced higher amounts of TNF-α mRNA levels than the patients in euthymic U937. Since the expression of CXCL9 and CXCL10 chemokines in plasma from patients with depressive TB, proved less significant expression in the group of manic patients when compared to controls and euthymic bipolar patients.

Thesis. 1976. M.S.--Massachusetts Institute of Technology. Dept. of Nutrition and Food Science. / Microfiche copy available in Archives and Science. / Vita. / Bibliography: leaves 69-78. / M.S.

Human rhinovirus (HRV) is responsible for the majority of common cold infections and asthma exacerbations. HRV predominantly replicates in the epithelial cells of the upper airway, where common cold symptoms are produced. However, HRV also enters the lower airway, encountering the epithelial cells and alveolar macrophages thought to produce inflammatory responses during HRV-induced asthma exacerbations. Notably, alveolar macrophages release inflammatory mediators such as MCP1/CCL2 and RANTES/CCL5 in response to HRV despite the fact that limited if any HRV replication occurs in these cells. The present study seeks to address the mechanism by which alveolar macrophages are susceptible but not permissive to HRV replication and to identify the step in the HRV replication cycle that restricts HRV to abortive replication in macrophages. Evidence presented herein demonstrates that major-group (ICAM-1 tropic) HRV replicate with limited success in cell line-derived macrophages, whereas minor-group (LDLR tropic) HRV do not replicate in these monocyte-lineage cells. In contrast, neither major- nor minor-group HRV replicate in primary human PBMC-derived macrophages. Capsid swap experiments demonstrated that difference in replicative capacity between major- and minor-group HRV is mediated at the level of permissiveness rather than susceptibility. RNA- Seq gene expression studies identified candidate host genes that may act to regulate HRV replication. These RNA-Seq studies also revealed positive- and negative-sense HRV RNA genomes in monocyte-lineage cells, suggesting that abortive HRV replication takes place within them. Overexpressing interferon-stimulated genes (ISGs) implicated in restricting the replication of poliovirus did not affect the accumulation of HRV RNA. Further study will continue to investigate the differences between major- and minor-group HRV responsible for differential replication success in cell-line derived macrophages and characterize the point(s) in the HRV replication cycle at which replication is blocked in primary macrophages. The ultimate goals of these studies are to reveal vulnerabilities in the HRV replication cycle and to identify host factors whose expression might be pharmacologically altered to attenuate HRV infection, thereby providing novel treatment options for controlling the common cold and HRV-induced asthma exacerbations.

The intestine is constantly exposed to a wide variety of dietary antigens, commensal bacteria and pathogens, toward which it has evolved complex immune responses to protect the host. The intestinal immune system relies on innate immune cells, such as mononuclear phagocytes (MNPs), that include dendritic cells (DCs), monocytes (Mo) and macrophages (Mfs), to sense and respond to luminal and mucosal challenges. MNPs are essential players as they instruct adaptive immune cells, in particular T cells, to discriminate between innocuous and harmful antigens. Generation of different CD4 T cell responses to commensal and pathogenic bacteria is crucial for maintaining a healthy gut environment, but the associated cellular mechanisms are poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by epithelium-associated commensal segmented filamentous bacteria (SFB). Several reports suggest that the cytokine environment induced by gut bacteria is sufficient to drive LP Th17 cell differentiation. In this context, intestinal DCs are proposed to facilitate the conversion of naïve CD4 T cells to Th17 cells within gut-draining lymph nodes. Whether such mechanisms control commensal-mediated Th17 cell differentiation has not been examined. In this work, I explore the mechanisms of induction of Th17 cells by SFB, with a particular focus on the role of antigen-presenting cells in this process. Initiation of CD4 T cell responses requires both major histocompatibility II (MHCII)-mediated antigen presentation and cytokine stimulation, which can be provided by the same or different subsets of intestinal MNPs. To test the requirement for either function in the induction of Th17 cells by SFB, we analyzed the role of SFB-induced cytokine environment in driving Th17 cell differentiation of non-SFB transgenic CD4 T cells. We find that although the cytokine environment is important, it is not sufficient to promote Th17 cell differentiation of activated CD4 T cells. In fact, we show that MHCII-dependent antigen presentation of SFB antigens by intestinal MNPs is crucial for Th17 cell induction. Expression of MHCII on CD11c+ cells was necessary and sufficient for SFB-induced Th17 cell differentiation. We also show that most SFB-induced Th17 cells respond to SFB antigens, which stressed that they carry T cell receptors that recognize SFB moieties. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. Our results outline the complex role of MNPs in the regulation of intestinal Th17 cell homeostasis, and we investigated the contribution of individual subsets to SFB-specific Th17 cell differentiation. Although the role of DCs in initiating T cell responses is well appreciated, how Mfs contribute to the generation of CD4 T cell responses to intestinal microbes is unclear. To this end, I examined the role of mucosal DCs and Mfs in Th17 induction by SFB in vivo. Employing DC and Mf subset-specific depletion and gain-of-function mouse models, I show that Mfs, and not conventional CD103+ DCs, are essential for generation of SFB-specific Th17 responses. Thus, Mfs drive mucosal T cell responses to certain commensal bacteria.

The regulated differentiation and polarisation of macrophages are essential for successful wound healing process. During wound repair, macrophages are involved in the early inflammatory process of healing, as well in later regenerative phases by producing cytokines and growth factors relevant for each stage. Their plasticity made macrophages able to change their phenotype from M1 inflammatory during the inflammatory phase of healing to M2 reparative during regenerative phases of healing. Diabetes affects the ability of macrophages to mature from the bone marrow and on their ability to polarise to different phenotypic subsets. Whereas the non-diabetic macrophages can mature normally to M2 macrophages during mid-stages of healing, diabetic wound continues o display immature proinflammatory macrophages resulting in mixed M1/M2 macrophages in the wound that remain until late stages of healing. We previously showed that sustained expression of Hoxa3 reduced the-the excessive number of leukocytes recruited to the wound, suggesting an anti-inflammatory effect of Hoxa3 upon all leukocytes population. Hoxa3 protein transduction also promoted the differentiation of HSC/P into pro-angiogenic Gr1+CD11b+ myeloid cells. Here we showed that Hoxa3 promoted the differentiation of macrophages and upregulated the transcriptional machinery controlling macrophage differentiation, in THP-1 monocytes and primary macrophages from non-diabetic and diabetic mice. Using qRT-PCR and protein analysis of bone marrow derived macrophages from diabetic mice, we showed that Hoxa3 upregulated the master regulator of macrophages differentiation, Pu.1 transcriptionally and post- transcriptionally and that Hoxa3 protein interacted with Pu.1 protein in vitro and in vivo within macrophages proposing a mechanism of their regulation. Hoxa3 also inhibited proinflammatory markers in classically activated macrophages and augmented pro-healing markers in alternatively activated macrophages. Investigating the IL-4/Stat6 pathway of M2 macrophage activation revealed that Hoxa3 upregulated Stat6 and increased Stat6 phosphorylation, a novel effect of Hoxa3 on the signaling pathway of alternative macrophage activation. In vivo analysis of Hoxa3's effect on wound derived macrophages in diabetic mice, confirmed that Hoxa3 promoted the generation of pro-healing macrophages and showed reduced Nos2+ (M1) cells and increased Arg1+ (M2) cells suggesting that Hoxa3 can rescue the phenotype of diabetic macrophages in the wound. Altogether, this work has delineated the specific role of Hoxa3 in rescuing maturation and phenotype of diabetic macrophages thereby providing a better understanding of the therapeutic role of this transcription factor for myeloid cells dysregulation in diabetes.

A common feature of glomerular disease is a protein leak into the urine. Proteinuria occurs in kidney disease and is an important risk factor for cardiovascular disease (CVD). ET‐1 is a potent vasoconstrictor/pressor peptide that is up‐regulated in CVD and many forms of inflammatory renal diseases. The actions of ET‐1 are mediated via two G‐protein coupled receptors, the ETAR which serves primarily in the pro‐hypertensive actions of ET‐1 and is often considered as the main pathological receptor subtype, with the ETBR serving to clear circulating ET‐1. Antagonism of one or both of receptors has been shown to be of clinical benefit in the treatment of hypertension. This research demonstrated a beneficial effect of selective ETAR antagonism using Sitaxsentan in a rat model of GN. ETAR blockade reduced blood pressure and importantly reduced glomerular inflammation as assessed by glomerular macrophage (Mϕ) infiltration. Further, we aimed to demonstrate that Mϕ, key mediators of inflammation are activated by ET‐1 to adopt a pro‐inflammatoy phenotype. However, early studies demonstrated that ET‐1 does not activate Mϕ as hypothesised. Mϕ were more phagocytic, and ET‐1 was chemokinetic for macrophages, an ETBR medicated event. ET‐1 was also removed by Mϕ, suggesting a potential regulatory role of Mϕ in the ET system. This phenomenon led to inclusion of additional in vivo studies to investigate the role of Mϕ in the regulation of ET‐1 and its pressor effects. These effects were investigated in a murine model of Mϕ ablation using CD11b‐DTR mice. These experiments determined in vivo that Mϕ ablation augments pressor responses to ET‐1, suggesting that Mϕ are required to regulate ET‐1. In vitro, Mϕ remove ET‐1 by several mechanisms involving proteolytic degradation of the peptide and ETBR mediated clearance, demonstrating a potential mechanism for the in vivo observation. Furthermore, proteinuria is believed to be due to damage or effacement of specialized visceral glomerular epithelial cells or podocytes. We identified in vitro that the ETAR mediates ET‐1 induced human podocyte cell effacement by actin cytoskeleton aberrations and slit‐diaphragm protein down-regulation, ET‐1 and pro‐inflammatory cytokine production. This thesis provides evidence to support our initial hypotheses that selective ETAR antagonism ameliorates proteinuric renal disease via its effects on podocytes and macrophages. Continued studies both in vitro and in vivo will strengthen the body of evidence to promote the therapeutic use of ETR antagonists in inflammatory renal disease.

Injuries to connective tissues such as ligaments and tendons are common, and rather than healing, repair typically results in fibrosis, or the formation of mechanically inferior and disorganized scar tissue. This fibrotic repair response is due in part to inflammation, during which the injury site is invaded by a number of cell types, including macrophages, neighboring fibroblasts, and homed stem cells or progenitor cells. Activation of macrophages is believed to be modulated by communications with fibroblasts and stem cells, prompting either a pro-fibrotic or a pro-regenerative response. Beyond changes to the cellular microenvironment, fibrosis also results in changes to the organization and mechanical properties of the matrix microenvironment. For healthy fibrous connective tissues, the matrix is comprised of aligned collagen fibers, while scar tissue is disorganized and exhibits weaker mechanical properties than healthy tissue. To date, the nature of the cell-cell and cell-matrix interactions and their relevance in tissue healing or repair remain understudied. To better understand the cellular and matrix-based cues that direct scar formation versus tissue regeneration, and using anterior cruciate ligament (ACL) injuries as a model, Aim 1 of this thesis tests the hypothesis that in vitro models of cellular communications between fibroblasts, macrophages, and mesenchymal stem cells (MSC) can be used to determine the effects of cellular interactions on macrophage activation and fibrosis. In Aim 2, the contribution of matrix-based cues (alignment and mechanical properties) to the inflammatory and fibrotic response, as well as their modulation of cellular interactions, were examined. Findings from these two aims reveal that 1) communications between native tissue fibroblasts and macrophages drive inflammation and fibrosis, while stem cells modulate the repair process through a combination of trophic signaling and immunomodulatory roles, and 2) matrix alignment and mechanical properties exert combined regulation on cell response during inflammation. From a clinical application perspective, stem cells delivered in conjunction with an engineered matrix that provides the critical cues for driving stem cell immunomodulation and trophic signaling will be essential for promoting tissue regeneration and minimizing fibrosis. In particular, an aligned matrix with an elastic modulus similar to that of developing connective tissue may serve to further minimize inflammation and scar formation, and activate stem cell-guided regeneration of mechanically functional connective tissue.