title: Determining Cellular and Molecular Mechanisms Behind Glial Cell Phagocytosis
Creators: name: Nazareth, Lynn
Publisher: name: Griffith University
Year: 2021
Subjects: subject: Phagocytosis
subject: Cell eating
subject: Olfactory ensheathing cells
subject: Schwann cells

description: Phagocytosis (“cell eating”) is an immunobiological process required for maintenance of systemic homoeostasis under normal physiological conditions (during development and adulthood) and in various pathologies. Phagocytosis is a receptor-mediated event, wherein a phagocytic cell recognizes, engulfs and degrades specific targets that need to be eliminated. The targets can be either “self-targets”, such as dead or damaged cells, or “non-self-targets”, such as microorganisms. In the nervous system, the “first responding” phagocytes are usually the supporting glial cells. Based on the location they are present in, glial cells are classified as either CNS or PNS glia. The key phagocytic glia in the CNS are astrocytes and microglia, and in the PNS, Schwann cells (SCs). Some peripheral nerves, however, have other glial types which mediate this function, such as olfactory ensheathing cells (OECs) in the olfactory nerve. Efficient phagocytosis is essential for regeneration after nervous system injury, but after CNS injury, glial phagocytosis is often inefficient. In contrast, after PNS injury, glia rapidly phagocytose and clear the cellular and myelin debris resulting from the injury. Due to their ability to support nerve growth, particularly via physical support and secretion of growth/guidance factors, while simultaneously performing phagocytosis; transplantation of SCs and OECs have promising potential to treat CNS injuries. However, phagocytosis is a highly specialized function and the key molecular and cellular components in PNS glial phagocytosis are largely unknown. If these could be characterized, new drug targets may be revealed that can further promote glial-mediated neural regeneration (but without causing an excessive inflammatory response). The site of a CNS injury is a complex environment, with cell death occurring via different mechanisms. These include distinct types of necrosis and apoptosis, and glia may respond differently to these distinct “self-targets”. Hence, in this Thesis, I investigated key cellular and molecular mechanisms involved in OEC- and SC-mediated phagocytosis of cells undergoing various forms of death. I discovered that OECs and SCs are indeed competent phagocytes that can recognize, internalize and degrade a range of “self-targets”. Both cell types expressed a number of phagocytic receptors, including phosphatidylserine (PS) recognition receptors, pathogen recognition receptors (PRRs), scavenger receptors, Fcγ receptors (FcγRs) and complement receptors (CRs). OECs and SCs both rapidly recognised and engulfed various cellular targets (within 2 h). Recognition of targets occurred mainly via PS displayed on the dying cell surface, with potential involvement of PRRs. The family of small Rho GTPases (Rac, Cdc42 and Rho) were also important for target engulfment. However, while engulfment was rapid, breakdown was relatively slow, particularly when the targets were necrotic bodies and myelin debris (especially when compared to professional phagocytes, i.e., macrophages). Engulfment of apoptotic targets resulted in anti-inflammatory cytokine production, however, necrotic target uptake led to a proinflammatory response. Overall, OECs phagocytosed larger amounts of targets over time, as well as processed targets faster, than SCs. During the process of phagocytosis, OECs also produced less pro-inflammatory, but more immunomodulatory, factors than SCs. Thus, OECs were more efficient in phagocytosing “self-targets” than SCs, accompanied by a more favourable immune response, suggesting that OECs may be better transplantation candidates than SCs. Two peripheral nerves, the olfactory nerve and the trigeminal nerve (intranasal branches) extend between the nasal cavity and the brain. These nerves are populated by OECs and SCs, respectively. These nerves have been shown to function as a pathway by which certain microbes can enter the brain, leading to CNS infection. The nasal mucosa, and associated nasal-associated lymphoid tissue (NALT) constitute a strong physical and immunological barrier against microorganisms, and those that do manage to penetrate the mucosa are considered to be phagocytosed by OECs and SCs in the nerves. However, microbes that can infect the CNS via these two peripheral nerves have been shown to evade phagocytic destruction and instead infect PNS glia. In this Thesis, I also investigated how OECs and SCs respond to bacterium thought to infect the CNS via nerves - Chlamydia muridarum. I chose this bacterium as it is commonly used to model C. pneumoniae infections in mice. C. pneumoniae CNS infection has been suggested to contribute to the development of late-onset dementia, thus being clinically relevant. I found that C. muridarum, which replicates in intracellular inclusion bodies, infected both OECs and SCs, however, the glia were not as susceptible to infection and intracellular bacterial growth as non-immune cells. Both OECs and SCs mounted a significant immune response to bacterial challenge, with OECs producing the strongest response. Despite this, C. muridarum could manipulate various intracellular and phagocytic machinery pathways to survive inside the glia, including pathways involving small Rho GTPases (Rac, Cdc42 and Rho) and PI3K/Akt. C. muridarum also suppressed lysosomal recruitment by “hijacking” Ras-like small GTPases (Rabs) responsible for intracellular trafficking and host nutrient scavenging. Thus, C. muridarum could escape phagocytosis (degradation) and grow inside glia. This is potentially a key reason by which the bacteria may disseminate through peripheral nerves, leading to CNS infection. The findings presented in this Thesis (including resultant publications), increases our understanding of how PNS glia remove dying and damaged “self”, including key cellular and molecular mechanisms involved in OEC and SC-mediated phagocytosis. The current study also, by characterizing how the glia responded to C. muridarum, explored the crucial dichotomy between phagocytosis vs infection. Internalization of bacteria into a cell can lead to either or both. In the case of OECs and SCs, C. muridarum challenge led to infection but also an immune response, restricted bacterial growth and likely also killing of a proportion of bacteria. This understanding may provide us with tools/drug targets for manipulation of various aspects of the PNS glia-mediated phagocytic processes. This could involve improved clearance of cellular debris without adverse inflammatory events post-transplantation into a nervous system injury site. Tweaking certain aspects of the phagocytic pathway may also prevent infections by microbes that can use the nose-to-brain pathway to infect the CNS, without using antibiotics (thus, not contributing to antimicrobial resistance). Finally, this thesis has also given us some interesting insights into differences between the two types of PNS glia. OECs and SCs, were considered to be quite similar in the past and both are deemed as good transplantation candidates. Overall, OECs were found to be more efficient phagocytes and equipped with additional molecular components of phagocytic pathways than SCs. OECs also produced a more favourable immune response than SCs in response to damaged “self”. In contrast, OECs mounted a stronger bactericidal immune response to C. muridarum than SCs, suggesting that OECs exhibit better antimicrobial protection mechanisms than SCs.