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UBC Theses and Dissertations

Role of cellular autophagy ("self-eating") in the activity of novel, site-specific immunomodulators Kwok, Martin


Chronic immune activation and inflammation are etiologically associated with the pathology of inflammatory bowel disorders, certain cancers, and persisting infections (eg. HIV)¹⁻³. Recently, a series of novel, microbe-derived, Site-Specific Immunomodulators (SSIs; Qu Biologics) have been described to repurpose the immune response and exert therapeutic effects at specific sites of pathology⁴⁻⁷. The mechanism for these effects are poorly understood, but may involve a phenomenon called innate immune memory⁴⁻⁷. Importantly, innate immune memory is distinct from classic adaptive immune memory, as it enhances immune responses against subsequent exposures to different, rather than the same pathogen⁸'⁹. Cells of the monocyte/macrophage (M/Ms) lineage are key innate immune memory cells¹⁰. Interestingly, it has been established that M/Ms exert many of their effects through autophagy (“self eating”), a critical regulator of cellular homeostasis, immune defense and inflammation¹¹⁻¹³. Therefore, it was hypothesized that autophagy is induced by and is required for the biologic effects of SSIs. U937 monocytic and HT-29 colonic epithelial cell lines were selected as in vitro models. First, a novel flow cytometry-based autophagy detection approach (CYTO-ID®) was validated and used to establish that SSIs may indeed act through their capacity to mobilize the autophagy pathway. Subsequently, the downstream effects of SSI-induced autophagy were investigated by analyzing the expression of cytokines, IL-1β and IL-18, both of which are reputed to be modulated by the autophagy pathway¹⁴. However, neither cytokine was detectable by the ELISA approach used. Finally, experiments to shed light on the molecular mechanisms by which SSIs induce autophagy were undertaken. Knowing that SSIs are derived from specific bacteria⁴⁻⁷, I examined whether SSI-induced autophagy was mediated by the cell’s bacterial lipopolysaccharide Pattern Recognition Receptors (PRRs) (Toll-like receptors (TLR)-2 and TLR4). Since TLR2/TLR4 signaling proceeds via the Myeloid differentiation factor 88 (MyD88) adaptor protein, a MyD88 antagonist was used to establish that SSIs induce autophagy through their bacterial components, and the MyD88 signaling pathway, specifically. Finally, the thesis provides a basis for further investigations into the role of autophagy in SSI-induced therapeutic effects in vivo using the Lewis Lung Carcinoma, spontaneous colitis (Muc2-/-), and other mouse models of disease.

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