Naive T cells are positioned at the origin of adaptive immune responses. The activation of naive T cells is usually viewed from the perspective of IL-2 production and entry into the cell cycle. This activation is antigen specific and MHC restricted via TCR ligation/CD3 signaling but also demands the simultaneous ligation of and signaling via CD28. Naive T cell TCR ligation without appropriate co-stimulus produces an anergic state, in which the naive T cell fails to produce IL-2 or expand clonally. It is implied that cells that might present self-destructive antigens would be incapable of delivering required costimulus thus avoiding initiation of inappropriate immune responses. However, CD45RBhi expressing THP cells express high levels of CD40L that is sustained following extended periods of TCR/CD3 stimulation. CD40L is the major T cell molecule involved in contact-dependent signaling of both B-cell and macrophage effector functions. This suggests that naive CD4+ T cells are capable of participating in and contributing to on-going immune responses following signaling via TCR/CD3 alone. This dissertation represents efforts to analyze the contact signaling capability of naive CD4+ T cells and their ability to trigger macrophage cytocidal/tumoricidal functions. The data generated by this research demonstrate that: Naive T cell purification by endothelial panning was superior to the standard method of CD44 panning for studies on T cell mediated macrophage activation. The activation requirements for contact signaling of macrophages by naive T cells are less stringent than the requirements for activation of naive T cell proliferation. Viable naive THP and antigen presenting splenic macrophages are capable of delivering reciprocal activating signals. Viable naive THP responding to presented antigen were able to trigger IFNg-primed macrophages to produce nitric oxide by both CD40L-dependent and CD40L-independent signaling pathways.

Atherosclerosis, characterized by the build-up of cholesterol, immune cells and cellular debris within arterial walls, is accelerated following myocardial infarction by poorly understood mechanisms. Ubiquitin, a small, well-studied intracellular protein involved in protein turnover via the proteasome pathway, has recently been shown to exert extracellular effects on cardiac myocytes, in vitro, and in mice undergoing myocardial remodeling. This study investigates the potential role of extracellular ubiquitin in atherosclerosis by determining its effects on two critical atherosclerotic processes: the migration of vascular smooth muscles cells and the uptake of modified LDL by monocyte/macrophages in foam cell formation. In the presence of ubiquitin, smooth muscle cell migration was accelerated and foam cell formation was enhanced, suggesting that ubiquitin has an active role in atherosclerosis.

Significant advances in the efficacy of cancer therapy have been accompanied by an escalation of side effects that result from therapy-induced injury to normal tissues. Patients with high grade cancer or metastasis are often treated with chemotherapy, 50% of which are associated with reactive oxygen species generation and cellular oxidative stress. Heart is the normal tissue most susceptible to chemotherapy-induced oxidative stress and heart disease is the most common leading cause of death in cancer survivors. However, early and sensitive biomarkers to identify heart disease are still lacking. Extracellular vesicles (EVs) are released from cells during oxidative stress and send oxidized proteins into the circulation as a compensatory mechanism that prevents cellular proteotoxicity. Thus, the protein contents of EVs released during the pre-degeneration stage reveal that oxidative stress is occurring early in the damaged tissue. Using a mouse model of doxorubicin (DOX)-induced cardiac injury, we demonstrated that EVs can be used as an early diagnostic tool for tissue injury as they are oxidatively modified with 4-hydroxynonenal and contain tissue specific proteins—glycogen phosphorylase brain/heart, muscle, and liver isoforms—that indicate their origins. These biomarkers increased early, before the changes of conventional biomarkers occurred. EVs also mediate intercellular communication by transferring bioactive molecules between cells. In the cell culture system, EVs play an important role in oxidative stress response by inducing macrophage polarization. EVs from cardiomyocytes promoted both proinflammatory (M1) and anti-inflammatory (M2) macrophage polarization evidenced by higher pro- and anti-inflammatory cytokines and nitric oxide generation, as well as mitochondrial oxidative phosphorylation suppression and glycolysis enhancement. In contrast, EVs from the hepatocytes supported anti-inflammatory macrophage (M2) by enhancing oxidative phosphorylation and anti-oxidant proteins. DOX promoted the immunostimulatory effects of cardiomyocyte EVs but not hepatocyte EVs. The differential functions of EVs on macrophage phenotype switching are due to their different effects on Thioredoxin 1 redox state, which regulates activities of redox sensitive transcription factors NFκB and Nrf-2. Our findings shed light on the role of EVs as a redox active mediator of immune response during chemotherapy.

ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.

Dans l'industrie, les poudres de carbure de silicium (SiC) sont élaborées principalement par le procédé Acheson. Durant ce procédé multi-étapes, les poudres subissent diverses opérations pouvant être à l'origine de la dissémination de poussières inhalables dans l'environnement de travail. Jusqu'à présent, la toxicité des poudres de SiC n'a été que très peu étudiée avec des résultats contradictoires soulignant l'intérêt d'évaluer l'activité biologique de poudres de SiC par une approche pluridisciplinaire. Dans une première partie, l'activité biologique de poudres de SiC produites industriellement par le procédé Acheson a été évaluée. Par suite, des modifications de la surface de ces particules par le biais de traitements thermiques oxydants ont permis de mettre en évidence l'influence de la surface des particules dans la réponse cellulaire. Pour finir et compléter l'étude, nous avons étendu le sujet au cas des nanoparticules de SiC, synthétisées par pyrolyse laser et voie sol-gel.<br />Pour évaluer l'activité biologique des particules de SiC, des tests In Vitro sur des macrophages de culture (RAW 264.7) ont été réalisés en étudiant différents domaines de la réponse cellulaire (état d'inflammation, mort cellulaire, stress oxydant) en relation avec les propriétés physico-chimiques des particules (taille, surface, morphologie, structure cristallographique, chimie, état de surface, activité radicalaire).<br />Les résultats, illustrés suivant un modèle vecteur, soulignent que les particules industrielles de SiC sont principalement caractérisées par une activité modérée de l'état inflammatoire, aucun effet cytotoxique et un impact significatif sur le stress oxydant. Des différences en fonction de la taille des particules ont été observées ainsi qu'une forte capacité des particules à générer directement des radicaux libres (HO•, COO•-). Après modification de la surface des particules par traitement thermique oxydant, la réponse cellulaire se caractérise par une forte augmentation de l'état d'inflammation et de la cytotoxicité. Enfin, un stress oxydant significatif est observé avec les nanoparticules de SiC, bien supérieur à celui observé avec les particules industrielles de SiC.

<p>All aspects of cell life are regulated by signal transduction mechanisms. This thesis describes the regulatory roles of a few key signal transduction molecules involved in three major biological responses. The studied pathways include platelet derived growth factor (PDGF)-BB induced transformation of murine fibroblasts, interferon (IFN)-γ stimulated monocyte activation and all-trans retinoic acid (ATRA) induced myeloid differentiation. </p><p>We found that intact phosphoinositide 3OH-kinase (PI3K) activity is essential in the signaling pathway that leads to the morphological alterations and migration pattern characteristic of PDGF-BB transformed NIH/sis and NIH/COL1A1 fibroblasts. Furthermore, our data indicated that the small Rho-GTPase, Rac1 is the predominant mediator of these signals downstream of PI3K.</p><p>The study of the IFN-γ induced activation of monocytic U-937 cells showed that upregulation of the high affinity receptor for IgG (FcγRI) is dependent on the coordination of several regulatory events: the PKR-mediated serine 727 phosphorylation of Stat1, the expression of the hematopoietic lineage specific transcription factor PU.I, and the activation of the NFκB pathway.</p><p>ATRA-induced differentiation and cell cycle arrest are impaired in U-937 sublines expressing phosphorylation deficient Stat1 (Stat1Y701F and Stat1S727A). The findings in paper III indicated that the expression pattern of the myeloid specific transcription factors Stat2, ICSBP and c/EBPε was altered in the sublines and that intact Stat1 activation is critical for maintaining the balance of the transcriptional network during ATRA induced terminal differentiation.</p><p>Finally, ATRA-induced differentiation and growth arrest were blocked by treatment with the IKKα/β inhibitor BMS345541 or by ectopic expression of the NFκB super repressor IκBα (S32A/S36A). The fact that IκB(AA) sublines differentiated normally in response to vitamin D3, showed that NFκB inhibition specifically affected ATRA induced responses. Notably we suggest that the activity of the NFκB pathway may interfere with the differentiation process via a direct effect on the RAR/RXR mediated transcription.</p>

Adipose tissue (AT) expansion and remodeling that maintains healthy function relies on stromal preadipocytes capable of differentiating into new adipocytes (adipogenesis). During chronic positive energy balance, a relative deficit in adipogenesis, from either a decrease in preadipocyte number or their capacity to differentiate, leads to excessive adipocyte hypertrophy and AT dysfunction. AT contains macrophages whose number and activation state is dynamically regulated with changes in AT mass. This study aims to investigate the effect of macrophage-secreted factors on preadipocyte survival. To assess the effect of macrophage-secreted factors on preadipocytes, murine 3T3-L1 preadipocytes or human primary preadipocytes were incubated with macrophage-conditioned medium (MacCM), prepared from either murine (J774A.1, RAW264.7, bone marrow-derived) or human (THP-1, monocyte-derived) macrophage models, respectively. MacCM inhibited preadipocyte apoptosis and activated pro-survival signaling in both preadipocyte models. Inhibition of PDGFR, Akt, or ERK1/2 reduced the pro-survival effect of MacCM in 3T3-L1 preadipocytes. Inhibition of reactive oxygen species (ROS) generation, or enhancement of ROS clearance, reduced MacCM-dependent 3T3-L1 preadipocyte survival. Whereas anti-inflammatory activated macrophages retained the ability to prevent preadipocyte apoptosis, pro-inflammatory activated macrophages did not. TNF-α immunoneutralization restored the survival activity of pro-inflammatory MacCM on 3T3-L1 preadipocytes. These studies reveal a novel pro-survival effect of MacCM on preadipocytes, and identify signaling molecules (PDGF, Akt, ERK1/2, and ROS) that underlie this action. Macrophage activation was found to regulate the pro-survival activity of MacCM. These in vitro cell culture studies are consistent with a model in which the extent of preadipocyte apoptosis in vivo may determine preadipocyte number and the ability of AT to expand while maintaining healthy function during chronic positive energy balance.

The innate immune protein surfactant protein (SP-) D is a carbohydrate binding protein that was originally isolated from mucosal lung tissues. Recently, studies show that SP-D binds to antibodies, including immunoglobulin M (IgM), which interacts with late apoptotic cells. Here we focus on the interaction between SP-D and IgM as they pertain to late apoptotic cell clearance. We hypothesized that the three-way interaction between IgM, SP-D and late apoptotic cells is functionally applicable to clearing late apoptotic cells from the lungs, thereby reducing lung inflammation. We show that SP-D binds to IgM and that IgM binds to the late apoptotic subclass of dying cells. We demonstrate that IgM and SP-D can both bind to late apoptotic cells in mutually distinct regions while also displaying some regional overlap. We show evidence that during LPS-induced lung inflammation both IgM and SP-D levels are elevated and this corresponds to an augmentation of apoptotic cell clearance. We illustrate that the protein interaction of IgM and SP-D is functionally relevant to apoptotic cell clearance in the lungs by showing that late apoptotic cells coated in IgM and/or SP-D are cleared more efficiently than control cells, by alveolar macrophages in vivo. Our ex vivo studies further show that these cells internalize apoptotic cells by engulfing very small particles released from the dying cells. We then showed that IgM preferentially directs the engulfment of small particles (~1 μm) by macrophages, in an apparent size-specific antibody-dependent particle clearance function. Our data reveals a novel relationship amongst IgM, SP-D, apoptotic cells, and alveolar macrophages that contributes to our understanding of apoptotic cell clearance, which may be used in the future to generate strategies addressing apoptotic cell accumulation or clearance deficiency in disease.

The innate immune protein surfactant protein (SP-) D is a carbohydrate binding protein that was originally isolated from mucosal lung tissues. Recently, studies show that SP-D binds to antibodies, including immunoglobulin M (IgM), which interacts with late apoptotic cells. Here we focus on the interaction between SP-D and IgM as they pertain to late apoptotic cell clearance. We hypothesized that the three-way interaction between IgM, SP-D and late apoptotic cells is functionally applicable to clearing late apoptotic cells from the lungs, thereby reducing lung inflammation. We show that SP-D binds to IgM and that IgM binds to the late apoptotic subclass of dying cells. We demonstrate that IgM and SP-D can both bind to late apoptotic cells in mutually distinct regions while also displaying some regional overlap. We show evidence that during LPS-induced lung inflammation both IgM and SP-D levels are elevated and this corresponds to an augmentation of apoptotic cell clearance. We illustrate that the protein interaction of IgM and SP-D is functionally relevant to apoptotic cell clearance in the lungs by showing that late apoptotic cells coated in IgM and/or SP-D are cleared more efficiently than control cells, by alveolar macrophages in vivo. Our ex vivo studies further show that these cells internalize apoptotic cells by engulfing very small particles released from the dying cells. We then showed that IgM preferentially directs the engulfment of small particles (~1 μm) by macrophages, in an apparent size-specific antibody-dependent particle clearance function. Our data reveals a novel relationship amongst IgM, SP-D, apoptotic cells, and alveolar macrophages that contributes to our understanding of apoptotic cell clearance, which may be used in the future to generate strategies addressing apoptotic cell accumulation or clearance deficiency in disease.

All aspects of cell life are regulated by signal transduction mechanisms. This thesis describes the regulatory roles of a few key signal transduction molecules involved in three major biological responses. The studied pathways include platelet derived growth factor (PDGF)-BB induced transformation of murine fibroblasts, interferon (IFN)-γ stimulated monocyte activation and all-trans retinoic acid (ATRA) induced myeloid differentiation. We found that intact phosphoinositide 3OH-kinase (PI3K) activity is essential in the signaling pathway that leads to the morphological alterations and migration pattern characteristic of PDGF-BB transformed NIH/sis and NIH/COL1A1 fibroblasts. Furthermore, our data indicated that the small Rho-GTPase, Rac1 is the predominant mediator of these signals downstream of PI3K. The study of the IFN-γ induced activation of monocytic U-937 cells showed that upregulation of the high affinity receptor for IgG (FcγRI) is dependent on the coordination of several regulatory events: the PKR-mediated serine 727 phosphorylation of Stat1, the expression of the hematopoietic lineage specific transcription factor PU.I, and the activation of the NFκB pathway. ATRA-induced differentiation and cell cycle arrest are impaired in U-937 sublines expressing phosphorylation deficient Stat1 (Stat1Y701F and Stat1S727A). The findings in paper III indicated that the expression pattern of the myeloid specific transcription factors Stat2, ICSBP and c/EBPε was altered in the sublines and that intact Stat1 activation is critical for maintaining the balance of the transcriptional network during ATRA induced terminal differentiation. Finally, ATRA-induced differentiation and growth arrest were blocked by treatment with the IKKα/β inhibitor BMS345541 or by ectopic expression of the NFκB super repressor IκBα (S32A/S36A). The fact that IκB(AA) sublines differentiated normally in response to vitamin D3, showed that NFκB inhibition specifically affected ATRA induced responses. Notably we suggest that the activity of the NFκB pathway may interfere with the differentiation process via a direct effect on the RAR/RXR mediated transcription.