As the principal antigen-presenting cells to T cells, dendritic cells (DCs) have a key role in the balance of immunity and autoimmunity. They are essential in two major, converse roles - eliciting T cell immune responses to pathogenic material, and maintaining peripheral tolerance to self-tissue by inhibiting self-reactive T cells. These functions involve the processing of pathogenic or self antigens and subsequent presentation of antigenic peptides on MHC to antigen-specific T cells. DC recognition of conserved pathogenic markers induces a mature phenotype that governs immunogenic presentation to T cells and, consequently, the adaptive immune response. In contrast, DC recognition of self tissue suppresses maturation, instead inducing a tolerogenic phenotype that induces self antigen-specific T cell to die, become anergised, or converted to T regulatory cells. Apoptotic cells are the major source of self-antigen for the maintenance of peripheral tolerance, and their defective clearance by DCs is implicated in autoimmunity. Apoptotic cells are thought to actively suppress maturation of DCs and inhibit the possible immune responses promoted by proinflammatory mediators released from necrotic cells. However, the immune function of apoptotic cells and their relative influence over necrotic cells are highly contested, partially due to the complex nature of immunogenicity arising from the sourcing and generation of apoptotic cells. In this investigation, various methods of inducing apoptosis and necrosis are evaluated. Definitive methods of inducing well-characterised cell death are then employed to compare the effects of apoptotic and necrotic cells on dendritic cells and in vitro and in vivo immune responses. Reported here are in vitro findings that support previous reports of the anti-inflammatory response of DCs to apoptotic cells, and the inflammatory response of DCs to necrotic cells. The previously-reported inhibitory effect of apoptotic cells on LPS-induced secretion of Th1 cytokines is supported here, but the inhibitory effect of apoptotic cells on LPS-induced upregulation of co-stimulatory molecules is contested. Novel findings describe the upregulation of DC expression of co-inhibitory molecules induced by both apoptotic cells and necrotic cells. Apoptotic cells, but not necrotic cells, had a suppressive effect on CpG-induced upregulation of co-stimulatory molecules and pro-inflammatory cytokines. Apoptotic cells suppressed the capacity of untreated and CpG-treated, but not LPS-treated, DCs to elicit IFNγ production by T cells. Apoptotic cells, but not necrotic cells, induced regulatory T cells and partially restored their CpG-suppressed induction. Finally, apoptotic cell-modulation of DCs inhibited the induction of autoimmunity in a novel modification of an in vivo model of diabetes. Interestingly, novel evidence for the possibility of necrotic cell-induced tolerance by means of direct T cell killing is addressed.

Fibroblast growth factor 2 (FGF2) is a multifunctional protein translated as CUG-initiated, high molecular weight (hi FGF2) or AUG-initiated, low molecular weight (lo FGF2) isoforms with potentially distinct functions. Previous work showed that overexpression of hi- but not lo FGF2 elicited chromatin compaction resulting in cell death, by an intracrine route. A series of studies were undertaken aimed at extending our understanding of the intracrine action of Hi FGF2. Major findings are as follows: a. Hi FGF2 overexpression induces apoptotic cell death, as indicated by increased TUNEL staining, and mitochondrial participation (cytochrome c release to cytosol, rescue of the hi FGF2 phenotype by the anti-apoptotic protein Bcl-2. b. Increased expression of pro-survival signals/proteins that are known to upregulate Bcl-2, such as nuclear Akt; the PIM-1 kinase; and the heat shock protein hsp70, also rescued the hi FGF2-induced phenotype. c. The hi-FGF2 effect was associated with sustained, intracrine, activation of ERK, and was blocked by ERK inhibitors. d. FGF2 isoform specific affinity chromatography followed by mass spectroscopy identified several proteins as potentially interacting with hi FGF2; of these, the p68 RNA helicase and the hsp70 were further confirmed as interacting partners, by co-immunoprecipitation. e. Increased nuclear co-localization, and possibly interaction, between hi FGF2 and overexpressed hsp70 correlated with rescue from hi FGF2 induced cell death. f. Factors associated with cardiac pathology (isoproterenol, angiotensin II, endothelin I) also upregulated endogenous hi FGF2 in cardiac cells in culture. Adriamycin-induced cardiotoxicity in the rat, known to be linked to increased incidence of apoptosis, was also associated with increased endogenous hi FGF2. g. Hi FGF2 is expressed in the human heart (atria) and localizes in both cytosol and nuclei, suggesting a participation in human heart physiology and pathophysiology. Work presented here is consistent with the notion that endogenous hi FGF2 up-regulation may play a role in promoting cell death during prolonged tissue stress and dysfunction. It follows that processes related to hi FGF2 upregulation, hi FGF2-nuclear protein interactions and mechanisms of hi FGF2 induced cell death, represent potential therapeutic targets for modulating cell death.

Fibroblast growth factor 2 (FGF2) is a multifunctional protein translated as CUG-initiated, high molecular weight (hi FGF2) or AUG-initiated, low molecular weight (lo FGF2) isoforms with potentially distinct functions. Previous work showed that overexpression of hi- but not lo FGF2 elicited chromatin compaction resulting in cell death, by an intracrine route. A series of studies were undertaken aimed at extending our understanding of the intracrine action of Hi FGF2. Major findings are as follows: a. Hi FGF2 overexpression induces apoptotic cell death, as indicated by increased TUNEL staining, and mitochondrial participation (cytochrome c release to cytosol, rescue of the hi FGF2 phenotype by the anti-apoptotic protein Bcl-2. b. Increased expression of pro-survival signals/proteins that are known to upregulate Bcl-2, such as nuclear Akt; the PIM-1 kinase; and the heat shock protein hsp70, also rescued the hi FGF2-induced phenotype. c. The hi-FGF2 effect was associated with sustained, intracrine, activation of ERK, and was blocked by ERK inhibitors. d. FGF2 isoform specific affinity chromatography followed by mass spectroscopy identified several proteins as potentially interacting with hi FGF2; of these, the p68 RNA helicase and the hsp70 were further confirmed as interacting partners, by co-immunoprecipitation. e. Increased nuclear co-localization, and possibly interaction, between hi FGF2 and overexpressed hsp70 correlated with rescue from hi FGF2 induced cell death. f. Factors associated with cardiac pathology (isoproterenol, angiotensin II, endothelin I) also upregulated endogenous hi FGF2 in cardiac cells in culture. Adriamycin-induced cardiotoxicity in the rat, known to be linked to increased incidence of apoptosis, was also associated with increased endogenous hi FGF2. g. Hi FGF2 is expressed in the human heart (atria) and localizes in both cytosol and nuclei, suggesting a participation in human heart physiology and pathophysiology. Work presented here is consistent with the notion that endogenous hi FGF2 up-regulation may play a role in promoting cell death during prolonged tissue stress and dysfunction. It follows that processes related to hi FGF2 upregulation, hi FGF2-nuclear protein interactions and mechanisms of hi FGF2 induced cell death, represent potential therapeutic targets for modulating cell death.

L'efferocytose est un phénomène physiologique par lequel des millions de cellules apoptotiques sont efficacement éliminées par phagocytose sans provoquer une réaction inflammatoire. L'efficacité de ce processus nécessite une interaction rapide entre la cellule apoptotique et son phagocyte afin d'éviter l'entrée de la première dans une phase de nécrose secondaire. Cette interaction implique des motifs de la surface des 2 cellules qui peuvent interagir directement ou aussi indirectement via des molécules de pontage. Ce dernier rôle est associé à C1q, premier composant du système du complément. En effet, C1q favorise l'élimination des cellules apoptotiques et aussi la réponse tolérogène en interagissant avec des ligands présents de part et d'autre de la synapse efferocytaire. La calréticuline (CRT) de surface fait partie de ces ligands. Initialement connue comme co-récepteur, avec CD91, de la queue collagène de C1q à la surface du phagocyte, la CRT est aujourd'hui décrite comme un signal « eat-me » de la surface des cellules apoptotiques où elle peut aussi interagir avec les têtes globulaires de C1q. Des études récentes ont soulevé le potentiel immunogénique de la CRT, notamment au cours de la mort des cellules cancéreuses. Ainsi, la CRT de surface joue un rôle important dans l'efferocytose même si l'on ne sait pas exactement comment cette protéine chaperonne résidente du réticulum endoplasmique est exposée à la membrane plasmique. Dans un premier temps, j'ai démontré que la CRT augmente à la surface des cellules Jurkat rapidement après l'induction de l'apoptose, à un stade où la phosphatidylsérine, marqueur emblématique de l'apoptose, n'est pas encore détectée. D'une manière intéressante, C1q est capable d'interagir directement avec la CRT exposée à la surface des cellules à ce stade « pré-apoptotique », et de favoriser significativement leur phagocytose par les macrophages THP1. Dans un deuxième temps, j'ai mis en évidence la présence de la CRT dans le milieu extracellulaire et montré qu'elle varie avec l'évolution de l'apoptose. De plus, la CRT soluble est capable d'induire la migration des macrophages THP1, d'augmenter l'expression à leur surface de CD14, récepteur impliqué dans l'efferocytose, et de stimuler la macropinocytose, un processus utilisé par les phagocytes lors de la phagocytose des cellules apoptotiques. Cela suggère que la CRT extracellulaire peut moduler la biologie du phagocyte. Enfin, la CRT exogène peut se lier à la surface des macrophages et peut être ainsi une source externe de la CRT retrouvée à la surface des phagocytes. / Efferocytosis is a physiological phenomenon whereby millions of apoptotic cells are efficiently removed by phagocytosis without inducing an inflammatory response. The efficiency of this process requires rapid interaction between the apoptotic cell and the phagocyte in order to prevent the entry of the dying cell in a secondary necrosis phase. This interaction involves patterns of the surface of the 2 cells that can interact directly or indirectly via bridging molecules. The latter role is associated to C1q, the first component of the complement system. Indeed, C1q promotes the removal of apoptotic cells and the tolerogenic response by interacting with ligands present on either side of the efferocytic synapse. Surface exposed calreticulin (CRT) is one of these ligands. Initially known as the co-receptor, with CD91, to the collagenous tail of C1q on the phagocyte surface, CRT is now described as an “eat-me" signal of the apoptotic cell surface where it can also interact with the globular heads of C1q. Recent data have revealed the immunogenic potential of CRT, especially in the case of cancer cell death. Thus, surface exposed CRT plays an important role in efferocytosis even if it is not fully understood how this reticulum endoplasmic resident protein gets to the plasma membrane. I firstly demonstrated that CRT increases rapidly on the surface of Jurkat cells after the induction of apoptosis, at a stage where phosphatidylserine, emblematic marker of apoptosis, is not yet detected. Interestingly, C1q is capable of interacting directly with this “pre-apoptotic” cell surface exposed CRT, and promotes significantly the uptake of Jurkat cells by THP1 macrophages at this stage. Secondly, I demonstrated the presence of CRT in the extracellular medium whose content depends on the evolution of apoptosis. Furthermore, soluble CRT induces the migration of THP1 macrophages, increases their surface expression of CD14, a receptor involved in efferocytosis, and stimulates macropinocytosis, a process used by phagocytes during phagocytosis of apoptotic cells. These results suggest that the extracellular CRT can modulate the biology of the phagocyte. Finally, exogenous CRT binds to the surface of macrophages and can therefore be an external source of the CRT found on the phagocyte surface.

Cell death is a fundamental aspect of development and homeostasis; its dysregulation is commonly associated with disease. Historically, apoptosis has been the most heavily studied type of cell death, but there are many other non-apoptotic forms of cell death. The Drosophila ovary provides a powerful in vivo model to study non-apoptotic cell death. Each egg chamber in the ovary contains 15 nurse cells that support an oocyte throughout development, and at the end of oogenesis the nurse cells are surrounded by stretch follicle cells and undergo non-apoptotic cell death. The work in this dissertation investigated the role of stretch follicle cells in nurse cell death. Genetic ablation of the stretch follicle cells revealed that they are required for multiple nurse cell death events including the transport of cytoplasm to the oocyte and DNA fragmentation. We found that phagocytic machinery is required in the stretch follicle cells for the acidification and elimination of nurse cells, suggesting nurse cells die by phagoptosis. Furthermore, live imaging and a transgenic engulfment detector demonstrated that nurse cells are not engulfed piece-wise despite the requirement of phagocytosis machinery, but are instead surrounded and acidified extracellularly. To determine the mechanism driving nurse cell acidification, we performed a targeted RNAi screen against lysosome-associated genes. Using tissue-specific RNAi, we demonstrated that the V-ATPase proton pump is required in the stretch follicle cells for nurse cell acidification. GFP fusion proteins and antibody staining revealed that V-ATPases become enriched and localize to the stretch follicle cell plasma membranes to acidify the nurse cells that they surround. Following acidification, the stretch follicle cells were found to release cathepsins, lysosomal proteases, to break down and degrade the nurse cell. To uncover novel pro-death proteins that mediate signaling between the stretch follicle cells and nurse cells, we utilized proximity-dependent protein labeling and identified proteins enriched in the stretch follicle cells. Altogether this work uncovers a new role for lysosomal machinery acting at the plasma membrane of stretch follicle cells to drive nurse cell death, and identifies pro-death proteins in the stretch follicle cells that promote nurse cell death.

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.

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