CD11b integrin receptor as a regulator of microglial function and a target for non-viral gene delivery

Smolny, M 2013, CD11b integrin receptor as a regulator of microglial function and a target for non-viral gene delivery, Doctor of Philosophy (PhD), Medical Sciences, RMIT University.


Document type: Thesis
Collection: Theses

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Title CD11b integrin receptor as a regulator of microglial function and a target for non-viral gene delivery
Author(s) Smolny, M
Year 2013
Abstract Microglia are the principal immune cells of the central nervous system and as such are crucial for maintaining integrity of the neuronal network in health and disease. Microglia become activated when they sense changes in their extracellular environment. Acute microglial activation is important to mediate and resolve neuroinflammation. However, sustained activation of microglia is observed in chronic neuropathologies such as Alzheimer’s Disease. It still remains controversial whether chronically activated microglial cells are neuroprotective or neurotoxic. Thus, a better understanding of the mechanisms of microglial activation is required.

One aim of this project was to investigate the role of integrin receptor CD11b/CD18 (complement receptor 3, CR3) in microglial activation. This receptor has a prominent role in the immune response of closely related peripheral immune cells, but has been inadequately studied in microglia. Another aim was to develop non-viral vehicles (‘immunogenes’) based on monoclonal antibodies in order to study microglial function by targeted gene transfer which has not been achieved so far.

Part 1 of this project used the monoclonal α-CD11b antibody OX42 as tool to investigate CD11b function in vitro. Immunofluorescence studies identified a role of microglial CD11b in mediating several aspects of microglial activation that depend on the CD11b binding site and/or other co-receptors. Soluble, intact OX42 antibody induced partial microglial activation (macropore formation), while particulate zymosan, a compound stimulating the CD11b lectin-binding site, triggered a vigorous release of reactive oxygen species (ROS). Further, beads coated with intact OX42 antibody triggered a phagocytosis-induced respiratory burst. This strong inflammatory response most likely also involved Fcγ-receptors (FcγR), because beads coated with OX42-F(ab’)2 antibody fragments that lack FcγR binding sites did not stimulate the respiratory burst despite being phagocytosed by microglia via CD11b/CR3. These data suggested that intact OX42 antibody is useful for targeted non-viral gene transfer into microglia. Thus, an immunogene was synthesized in part 2 of this project, using the targeting ligand OX42 antibody conjugated to polyethyleneglycol (PEG)-engrafted polyethyleneimine (PEI). The OX42-immunogene transfected only a few microglia. Microglial cells that had taken up the OX42-immunogene displayed mainly non-specific auto-fluorescence apparently associated with intracellular degradation of the immunogene in vitro and in vivo. Further experiments were conducted to elucidate the barriers to non-viral gene transfer into microglia. The results revealed that aggregated OX42-immunogene triggered a strong immune response (ROS production and CD11b exocytosis) that likely involved FcγRs and caused intracellular destruction of the OX42-immunogene. Endosomal escape was also found to be limited in microglia. This project identified a differential role of CD11b/CR3 in mediating the inflammatory response of microglia. CD11b/CR3 can mediate microglial activation in absence or presence of the respiratory burst. The results of this work suggest that CD11b/CR3 could play an important role in the switch of the microglial phenotype from protective to toxic and contribute to neurotoxic ROS production in neuropathologies. Nevertheless, CD11b may be a good target for antibody-mediated gene transfer into microglia if FcγR-stimulation is avoided. Thus, targeted gene transfer may be accomplished with a second generation OX42-F(ab’)2-immunogene that lacks binding sites for FcγRs.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Medical Sciences
Keyword(s) Microglia
integrin
CD11b
CR3
complement receptor
neuroinflammation
phagocytosis
reactive oxygen species
respiratory burst
OX42
trafficking
gene transfer
immunogene
PEI
polyethyleneimine
non-viral vehicle
transfection
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Created: Mon, 16 Dec 2013, 10:06:42 EST by Brett Fenton
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