Alice Mui

The host defence against pathogen infection or tissue damage includes activation of immune cells such as macrophages, leading to a process called inflammation, which needs to be appropriately regulated to avoid the development of inflammatory diseases. The anti-inflammatory cytokine Interleukin 10 (IL10) plays a central role in inhibiting macrophage activation. The stimulation of macrophages with the bacterial cell wall component lipopolysaccharide (LPS) results in the production of inflammatory cytokines, mediators and increased expression of intracellular signaling molecules such as the microRNA (miRNA) miR-155. IL10 signaling inhibits the production of pro-inflammatory cytokines and miR-155, in part by downregulation of miR-155. Our previous work showed that IL10 inhibits the maturation of the precursor of miR-155 (pre-miR-155) to a mature miR-155. This thesis aimed to characterize the mechanism by which IL10 signaling interferes with miR-155 maturation by identifying proteins that interact with pre-miR-155. Our studies showed CELF2 to be a pre-miR-155 interacting protein. A study of CELF2 deficient cells suggests that this protein contributes to the expression of mature miR-155 and IL10 inhibition of miR-155 and inflammatory cytokine production.

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Introduction: Emotional lability (EL), the uncontrollable and unmotivated expression of emotion, is a rare and distressing symptom of brainstem compression from extra-axial posterior fossa tumours. In published case reports, EL is alleviated by surgical resection of the tumour. The primary goal of this study was to radiographically compare the degree of compression from mass lesions onto brainstem structures between EL cases and matched controls. The secondary goal was to compare changes in patient reported quality of life (QOL) pre- and post-operatively.Methods: In a retrospective review of patients treated for posterior fossa tumours with brainstem compression between 2002 and 2018 at Vancouver General Hospital, 11 patients with confirmed EL were identified. Each case was matched to 3 controls. Pre-operative axial T2-weighted FLAIR MRI scans were reviewed, and a lateral brainstem compression scale was used to characterize the degree of mass effect at the level of the medulla, pons, and midbrain. Compression was compared between EL patients and control patients. Clinical variables were also compared. Patients routinely complete the Short form-36 (SF36v1) general health survey at clinic appointments, these surveys were retrospectively obtained from patient charts. Pre- versus post-operative changes in survey scores were compared between EL patients and controls. Results: EL symptoms ceased post-operatively for all EL patients. EL tumours exert greater compression onto the pons (EL mean compression score=2.91, control mean compression score=1.91, one-tailed p=0.03). EL patients more commonly have cerebellar findings (p=0.003) pre-operatively. Patients with EL-causing tumours experienced greater improvement post-operatively in “Health Change” (two-tailed p=0.05), which was maintained over time. Control patients experienced greater improvement in the “Role Limitations Due to Physical Health” SF36v1 sub-score (two-tailed p=0.03), which was not maintained over time.Conclusion: This is the largest case series to-date investigating adult extra-axial posterior fossa tumours that cause EL. Findings suggest that compression onto the pons inhibits control over involuntary, stereotyped expression of emotion This adds to evidence that EL may be attributed to deafferentation of the cerebellum from cortical and limbic structures through the basis pontis, leading to impaired modulation of emotional response. The QOL results augment benefits of obtaining EL-alleviating surgery.

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Background: Inflammation is a major immune response that protects the host from infections. Upon pathogen encounter, immune cells are activated and produce pro-inflammatory mediators. Although inflammation is a critical step in both innate and acquired immune response, excessive inflammation can damage tissues and lead to inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Interleukin 10 (IL10) is a key anti-inflammatory cytokine that deactivates various immune cells including macrophages. IL10 signaling has classically been described to include ligand stimulated phosphorylation of the IL10 receptor (IL10R), recruitment of the signal transducer and activator of transcription 3 (STAT3) and the subsequent phosphorylation, dimerization and translocation of STAT3 to the nucleus, where it stimulates transcription of anti-inflammatory genes. Our studies have shown that in addition to the canonical STAT3 signaling, IL10 mediates its anti-inflammatory effect through SHIP1 inositol phosphatase. We hypothesized that IL10 signaling leads to the recruitment and activation of a SHIP1-STAT3 complex that then mediates the anti-inflammatory effects of IL10.Our SHIP1 pulldown studies have revealed that SHIP1 and STAT3 form a complex in response to IL10. In addition, we observed the AMP-activated protein kinase (AMPK), which has been previously reported to play a role in IL10 signaling, to be part of this complex. The pro-inflammatory counterpart of IL10, IL6 is unable to initiate the formation of such complex although both IL10 and IL6 induce tyrosine phosphorylation of STAT3. In addition to IL10, our small molecule SHIP1 agonist, AQX-151, was also found to induce the formation of the SHIP1-STAT3-AMPK complex. Our experiments in STAT3 knock-out bone marrow derived macrophages suggested that STAT3 mediates the association of SHIP1 and AMPK. We also mutated a number of phospho-Tyr residues in SHIP1 and assessed IL10 signaling. Tyr190 was identified to play an important role in both complex assembly and cellular response to IL10 (evaluated by inhibition of TNFα secretion).These studies showed for the first time the importance of SHIP1, STAT3 and AMPK complex in IL10 signaling. Understanding the mechanisms by which IL10 down regulates immune cell function will give insight into development of novel therapeutics that mimic the beneficial effects of IL10.

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Inflammation is an important step in the body’s defense against pathogen infection. However, it must be tightly regulated and appropriately terminated to prevent pathological consequences. Interleukin-10 (IL10) is one of the body’s most important anti-inflammatory cytokine that can inhibit many molecular events necessary for promoting inflammation including production of pro-inflammatory cytokines such as Tumor Necrosis Factor α (TNFα). Our laboratory has recently shown that SH2-domain containing Inositol 5ʹ phosphatase (SHIP1) is involved in IL10 signaling in macrophages, and although the mechanism of how this occurs is not well studied, our laboratory have obtained data suggesting SHIP1 mediates IL10 signalling through its phosphatase activity or interaction with other signalling proteins. SHIP2 is the only other known homologue of SHIP1 with approximately 38% amino acid sequence identity, yet they possess several similar functions including mediating FcγIIB signaling and phagocytosis. Because of their similarities and SHIP1’s involvement in IL10 signaling, we sought to investigate whether SHIP2 is also involved in inhibiting inflammatory response in macrophage by knocking it out using CRISPR/Cas9-mediated genome editing. Overall, we were unable to determine whether SHIP2 plays a role in macrophage anti-inflammatory response due to the large variation in cell sensitivity to IL10 and we also observed that transduction of macrophages with CRISPR/Cas9 virus alters the cellular response to IL10 which confounded our investigation of SHIP2 function.

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The inflammatory response is an important physiological mechanism for hosts’ defense against pathogens and post-assault tissue repair. However, excessive inflammation leads to tissue damage and pathology. Therefore, inflammation is tightly regulated by anti-inflammatory factors such as interleukin-10 (IL-10) in order to maintain homeostasis. IL-10 inhibits macrophages’ activation by suppressing macrophages’ antigen presenting ability and production of pro-inflammatory cytokines such as tumor necrosis factor α (TNFα). The signal transducer and activator of transcription 3 (STAT3) pathway has been regarded as the only downstream pathway of IL-10 for decades. However, our lab has previously shown that IL-10’s early anti-inflammatory action uses the lipid phosphatase SH2 domain containing inositol 5´ phosphatase (SHIP1) but not STAT3. Previous results in our lab suggested that IL-10 activates SHIP1 to inhibit the phosphoinositide 3-kinase (PI3K) pathway; and this accounts for IL-10’s early anti-inflammatory effects. Since the previous results were mostly obtained using cell lines, we sought to verify the results in the more physiological relevant mouse primary cells. We first investigated whether IL-10 activates SHIP1 by assessing the physical interaction of IL-10 receptor (IL-10R) and SHIP1, the localization of SHIP1, and the phosphorylation state of SHIP1 upon IL-10 stimulation. We could not observe any effect of IL-10 on altering the activation state of SHIP1. We next investigated IL-10’s effect on the activation of Akt, a downstream molecule of PI3K, in lipopolysaccharide (LPS) activated macrophages. We demonstrated that IL-10 inhibited phosphorylation of Akt in macrophages from C57BL/6 mice but not macrophages from Balb/C mice. Lastly, we investigated the roles of SHIP1 and STAT3 in IL-10’s inhibition of TNFα protein. We found that the TNFα production profile in SHIP1-/- and STAT3-/- cells were extremely similar. Closer examination showed that SHIP1messenger RNA (mRNA) expression was significantly reduced in STAT3 knock out (-/-) macrophages. Although this work failed to demonstrate some of the observations obtained in cell lines, it shows the significance of genetic background of the cells used in experiments. It also suggests that STAT3-/- macrophages’ unresponsiveness to IL-10 may due to the lower SHIP1 level in these cells, indicating a potentially important role of SHIP1 in IL-10’s anti-inflammatory properties.

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Inflammation is a physiological process required for defense against pathogens and the repair of damaged tissues. However, excessive or improper inflammation can be detrimental and results in a number of diseases such as rheumatoid arthritis and inflammatory bowel disease. To prevent the negative effects of inflammation, the inflammatory response is tightly regulated by the anti-inflammatory cytokine interleukin-10 (IL-10). The main target of IL-10 are activated macrophages whose exposure to IL-10 results in the anti-inflammatory response (AIR) characterized by depressed antigen presentation and the inhibited secretion of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα). To induce the AIR in macrophages, IL-10 binds to cell surface receptors which activate the transcription factor STAT3 leading to the transcription of gene products responsible for carrying out the AIR. However, we hypothesized that IL-10 also uses a STAT3-independent mechanism to induce the AIR. Here we demonstrate that IL-10 utilizes the lipid phosphatase SHIP1 to inhibit TNFα production in activated macrophages. SHIP1 is responsible for dissociating TNFα mRNA from polysomes leading to inhibited translation of TNFα mRNA during the AIR. This effect of SHIP1 occurs early in the AIR and helps to immediately halt TNFα production. We also demonstrate that the tyrosine kinase Btk, a reported positive regulator of TNFα production in macrophages, is also utilized by IL-10 in the early AIR to inhibit TNFα production. However, Btk is not required for IL-10 to dissociate TNFα mRNA from polysomes suggesting that Btk and SHIP1 are involved in distinct IL-10 signalling pathways. Finally we show that TIA-1, a RNA binding protein that silences TNFα mRNA translation is not involved in the IL-10 AIR. These results clearly demonstrate the existence of non-STAT3 signalling pathways in the IL-10 AIR and suggest that SHIP1 and Btk activators could be used as potential therapeutics in the treatment of inflammatory disorders.

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