The emergence of the eukaryotic cell was one of the key events that paved the path to the vast variety of complex life visible all around us. A pivotal event in the process of eukaryogenesis was an endosymbiosis of two prokaryotic cells which in time resulted in the mitochondria. Phagocytosis, which is the process in which  larger cells envelope and digest smaller cells is one of the feasible explanations for how one cell came to be within another. It is common in eukaryotes, however, no contemporary examples of prokaryotes practicing phagocytosis exist. But phagocytosis had to evolve at some point, so how did it happen? This thesis gives an attempt to determine what conditions facilitate the evolution of phagocytosis. In this endeavour, we constructed chemostatic models for two different population structures where secondary consumers are subject to an invasion by a mutant with predatory capabilities. The different population structures are denoted as "competition" and "cross-feeding". By using random parameter sampling we show that the predator has a higher likelihood to invade in the cross-feeding environment. We also showed using adaptive dynamics that a mutant that is able to invade and replace its predecessor favours evolving towards a higher predation strategy in the cross-feeding case whereas in competition  such behaviour is less clear and a more balanced strategy seems preferable.

Trained immunity and immune tolerance have been identified as long-term response patterns of the innate immune system. The causes of these opposing reactions remain elusive. Here, we report about differential inflammatory responses of microglial cells derived from neonatal mouse brain to increasing doses of the endotoxin LPS. Prolonged priming with ultra-low LPS doses provokes trained immunity, i.e., increased production of pro-inflammatory mediators in comparison to the unprimed control. In contrast, priming with high doses of LPS induces immune tolerance, implying decreased production of inflammatory mediators and pronounced release of anti-inflammatory cytokines. Investigation of the signaling processes and cell functions involved in these memory-like immune responses reveals the essential role of phosphoinositide 3-kinase γ (PI3Kγ), one of the phosphoinositide 3-kinase species highly expressed in innate immune cells. Together, our data suggest profound influence of preceding contacts with pathogens on the immune response of microglia. The impact of these interactions—trained immunity or immune tolerance—appears to be shaped by pathogen dose.

Human polymorphonuclear leukocytes (PMN) are a first line of defense against fungal infections. PMN express numerous pattern recognition receptors (PRR) that facilitate identification of invading microorganisms and ultimately promote resolution of disease. Dectin-1 (β-glucan receptor) is a PRR expressed on several cell types and has been studied on monocytes and macrophages. However, the role played by dectin-1 in the recognition and killing of fungi by PMN is unknown. We investigated the ability of dectin-1 to mediate human PMN phagocytosis and fungicidal activity. Dectin-1 was expressed on the surface of PMN from all subjects tested (n=29) and in an intracellular compartment that co-sedimented with azurophilic granules in Percoll density gradients. Soluble β-glucan and mAb GE2 (anti-dectin-1) inhibited binding and phagocytosis of zymosan by human PMN (e.g., ingestionwas inhibited 40.1% by 3O min, p<0.001), and blocked reactive oxygen species production. Notably, soluble β-glucan and GE2 inhibited phagocytosis and killing of Candida albicans by PMN (inhibition of killing was 54.8% for β-glucan and 36.2% for GE2, p<0.01). Our results reveal a mechanism whereby PMN dectin-1 plays a key role in the recognition and killing of fungal pathogens by the innate immune system.

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Cell clearance is critical for the resolution of inflammation. Defects in cell clearance can result in pathologies associated with chronic inflammation. Cells are cleared by phagocytes. These phagocytes can be either professional phagocytes whose main function is to engulf, or non-professional phagocytes, which have other tissue-resident functions but can engulf when needed. While the molecular biology of cell clearance has been heavily studied, the differences between mechanisms of engulfment by non-professional and professional phagocytes are not yet known. The study of cell clearance by non-professional phagocytes is important due to the presence of these cells in all tissues of the human body. The Drosophila ovary is a genetically tractable, in vivo model system to study engulfment by non-professional phagocytes. In response to protein deprivation, apoptosis is induced in the germline and the surrounding follicle cells engulf the dying corpses. The back and forth signaling that occurs between the dying germline and engulfing epithelial follicle cells is critical for efficient death and clearance. The germline must display eat-me signals for phagocyte recognition. The interaction of the eat-me signal and the engulfment receptor allows follicle cells to activate downstream signaling for internalization and corpse degradation. Interestingly, engulfment receptors have an additional role in driving germline death progression. The research in this dissertation focuses on the signals that drive the reciprocal signaling between non-professional phagocytes and dying cells. The results of this study demonstrate that Draper, an engulfment receptor, induces germline cell death by activating a Shark/JNK/NADPH oxidase signaling axis that is dependent on a YxxL motif. The results from this work indicate that the apoptotic machinery is activated and nurse cell nuclei are degraded in response to Draper overexpression, but Draper-induced cell death occurs independent of effector caspases, indicating that Draper induces cell death via other death pathways. An unbiased, high throughput approach to identify novel death-inducing receptors and ligands is also described. This study describes the reciprocal signaling mechanisms between engulfing and dying cells and opens up new avenues for targeting engulfment-mediated cell death. / 2021-11-08T00:00:00Z

Engulfment of dead cells by epithelial cells is crucial for the health of an organism. Defective engulfment can lead to serious conditions such as retinitis pigmentosa and asthma. In the Drosophila melanogaster ovary, protein starvation induces apoptotic cell death of germline cells, which are then engulfed by adjacent epithelial follicle cells. The follicle cells synchronously enlarge approximately four-to-five fold as they engulf the dying germline, suggesting that significant changes are required. However, the molecular changes needed to drive enlargement and engulfment by epithelial cells are still poorly understood. In this dissertation, I determined the role of integrins in engulfment, the interactions between the core engulfment machinery and the corpse processing pathway, and the roles of GTPases in engulfment by epithelial cells. First, I found that the integrin heterodimer, αPS3/βPS, becomes apically enriched and is required in the epithelial follicle cells for engulfment. αPS3/βPS is trafficked in a polarized fashion using much of the same machinery as migrating cells, suggesting similarities between engulfing and migrating cells. The canonical corpse processing pathway has been well-characterized, however, little is known about how the core engulfment machinery interacts with the corpse processing pathway. I found that the phagocytic receptor Draper is present on the phagocytic cup and early phagosomes, whereas integrins are maintained on the cell surface. Engulfment mutants fell into three distinct categories based on their specific effects on internalization and phagosome maturation, suggesting that components of the core engulfment machinery are required for distinct steps of corpse processing. Last, I investigated the roles of the Rho family GTPases during engulfment. Strikingly, Rac2 becomes induced during engulfment whereas Rac1 does not change its expression pattern. Both Rac1 and Rac2 are required for engulfment, however, Rac1 has defects in both enlargement and vesicle uptake whereas Rac2 only has defects in enlargement. Furthermore, I found that Rac1 and Rac2 may have differential effects on the Jun kinase signaling pathway, which suggests complexity in the regulatory network controlling engulfment in epithelial cells. Together, this work has provided a greater understanding of the molecular changes required within epithelial cells for proper engulfment. / 2016-12-01T00:00:00Z

Early postnatal period is characterised by generally immature phenotype of the newborn's immune system. The maturation of the immune system including setting appropriate regulatory responses is occurring during this period and encountering pioneering bacteria colonizing neonate plays an important role. In the early days after birth, the immune system of a newborn is very limited, and the adaptive part is mostly represented by antibodies transferred from the mother by cord blood (CB) in the womb and then by colostrum and mother's milk after labour. Therefore, innate immunity plays a key role in defence (against pathogens) in neonates and is largely represented by neutrophils. This study aims to better understand neutrophil biology and phenotype in umbilical CB, compared to neutrophils from peripheral blood (PB) of mothers and healthy non pregnant women (referred to as HC). The assessment of neutrophil phenotype based on surface markers was performed using flow cytometry. Expression of genes linked to antimicrobial function was measured using quantitative PCR. Functional properties of neutrophils, metabolic activity during activation and phagocytosis, and suppressive properties were assessed using the SeaHorse machine and flow cytometry, respectively. Here we confirm the presence of immature CD16low...

Natural IgM producing phagocytic B cells (NIMPABs) are a rare population of immune cells that can produce antibodies to broadly target and eliminate cancer cells via phosphatidylcholine (PtC)-specific phagocytosis. A novel, dual-labeled lipid-shelled superparamagnetic iron oxide nanoparticle (SPION)-based (SLNP) system was developed to trigger specific phagocytotic behavior in and subsequent enrichment of NIMPABs for a potential immunotherapy. Here we propose the design of an in vitro model to assess cell-SLNP interactions with J774A.1 monocyte cell line and the optimization of SLNP formulation. First, we developed and examined the morphology, size, concentration, purification, sterilization, and storage conditions of oleylamine-coated SPIONs and SLNPs using various microscopy methods, spectroscopy, dynamic light scattering, and zeta potential. Our data confirmed SPIONs are magnetic and 7-8 nm in diameter. SLNPs containing SPIONs retained the magnetic property, and are typically measured between 100-120 nm in diameter, and had a positive zeta potential. Fluorescence labeling of the SLNPs did not affect their properties. Second, we examined cytotoxicity and phagocytosis of SLNPs with J774A.1 cells. Our preliminary data showed that significant percentage of phagocytosis can be observed as early as 2 hours. However, longer than 2 hour incubation resulted in significant cytotoxic effects. The source of SLNP cytotoxicity was examined with transmission electron microscopy and characterization techniques, identifying high un-encapsulated free oleylamine-coated SPION content in SLNP samples contributing to a positive zeta potential for these samples. Simplified SLNPs with oleic acid-coated SPIONs were synthesized and resulting examined particles had a negative zeta potential, reduced free SPION content and improved SPION incorporation into SLNP cores. Based on these findings, SLNP criteria, characterization techniques, and cell assays were revised to establish a rigorous, standardized workflow essential for determining the optimal SLNP formulation. Future work must continue to modify SLNP formulation with information obtained from all characterization techniques and cellular assays outlined in the in vitro model. Once optimized, selective SLNP-mediated isolation of NIMPAB cells can be validated ex vivo with murine peritoneal cavity washout cells and then human peripheral blood samples. / 2024-08-26T00:00:00Z

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