Doctor of Philosophy in Experimental Medicine (PhD) 
The role of SHIP1 in the inhibition of macrophage activation
Gowling WLG (Canada) LLP
Interleukin-10 (IL10) is best studied for its anti-inflammatory action on immune cells including macrophages, but it also exerts littler known effects on non-immune cells such as prostate cancer (PCa) epithelial cells. In this thesis I examined the contribution of an IL10-induced protein called EP4 on IL10 inhibition of macrophage activation, as well as the ability of IL10 to stimulate expression of proteins in PCa cells which are associated with tumour progression. Macrophages sense molecules on pathogens such as lipopolysaccharide (LPS) and respond by producing inflammatory cytokines including TNFα and other mediators which coordinate the host defense against the pathogen. IL10 is produced as part of the resolution (anti-inflammatory) phase of inflammation. IL10 receptor (IL10R) signalling in macrophages involves the STAT3 transcription factor and the SHIP1 inositol phosphatase which work together to support the expression of genes involved in the anti-inflammatory response. I report here that IL10 upregulates the expression of prostaglandin E2 (PGE2) receptor EP4. The EP4 receptor has previously been shown to mediate PGE2’s anti-inflammatory action. Knockdown of EP4 impaired IL10’s ability to inhibit LPS-induced signalling events and TNFα production. I propose that part of the IL10 anti-inflammatory effect on macrophages may thus be mediated through LPS-induced, autocrine PGE2, binding to IL10-induced EP4. The direct effect of IL10 on epithelial cancer cells has not been well studied. Serum levels of IL10 increase in PCa patients as their tumours progress, and this is thought to contribute to the suppressed immune system observed in these patients. PCa patients are treated with androgen antagonists such as enzalutamide (ENZ) to inhibit androgen receptor dependent PCa cell growth. Development of ENZ resistance is associated with tumour differentiation to a more aggressive neuroendocrine phenotype. I found that IL10 like ENZ, induce markers of neuroendocrine differentiation and inhibit androgen receptor reporter activity in PCa cells. Both also upregulated the levels of PDL1 (CD274) which would promote tumour survival in vivo through its interaction with the immune cell inhibitory receptor PD1 to suppress an anti-tumour immunity. These findings suggest that IL10’s direct action on PCa cells contributes to PCa progression independent of IL10’s suppression of host immune cells.
Inflammation is a protective mechanism against infection, but it must be appropriately regulated to prevent pathological consequences including inflammatory diseases. Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that inhibits the activation of many immune cell types, including the macrophage, to prevent exaggerated immune responses. Understanding the signalling pathway downstream of IL-10 and IL-10 receptor (IL-10R) is essential in developing therapeutics to treat inflammatory diseases. Canonically, IL-10R signalling is described as solely depending on the activation of the transcription factor STAT3 and the expression of STAT3-response genes. However, our laboratory has previously shown that IL-10 also activates the inositol 5'-phosphatase SHIP1 to mediate its anti-inflammatory responses, such as inhibiting the expression of pro-inflammatory cytokines. Moreover, we have now found that IL-10 is able to inhibit expression of miRNA-155 (miR-155) in macrophages activated by the bacterial product lipopolysaccharide (LPS). This inhibition by IL-10 requires both STAT3 and SHIP1, and occurs at the maturation step, but not at the transcription step, of miR-155. Consistent with SHIP1’s involvement in IL-10 function, a previously described SHIP1 activator, AQX-MN100, mimics IL-10 and inhibits LPS-induced miR-155. Next, we investigated the roles of STAT3 and SHIP1 in IL-10 regulation of global gene expression in activated macrophages. Among the genes identified as IL-10 regulated, we have found different subsets of genes potentially to be SHIP1-regulated, STAT3-regulated, and SHIP1-STAT3 regulated. Considering the importance of SHIP1 activation in IL-10 function, SHIP1 activators provide an alternate way to control inflammation. We have previously shown that SHIP1 activators bind to SHIP1’s C2 domain and regulate SHIP1 enzymatic activity allosterically. Using x-ray crystallography, we obtained the crystal structure of SHIP1’s phosphatase and C2 domains, the minimal region necessary for allosteric activation by SHIP1 activators. Analysis of the crystal structure revealed differences between SHIP1 and related phosphatases. Biochemical and biophysical methods have been employed to identify amino acid residues potentially interacting with SHIP1 activators. Together, this work strengthens the model that STAT3 and SHIP1 work together to mediate the anti-inflammatory function of IL-10. We also described the structure of SHIP1, providing the first step in rational drug design to generate better small molecule SHIP1 activators.
Interleukin-10 (IL-10) is an anti-inflammatory cytokine essential for maintaining immune homeostasis. One of its major targets is the macrophage where it inhibits production of pro-inflammatory cytokines, chemokines and other soluble mediators. However, the intracellular signaling mechanisms by which IL-10 achieves macrophage deactivation remain under intense investigation. Our studies suggest that in addition to canonical STAT3 signaling, IL-10 mediates its early phase anti-inflammatory response through SHIP1 in a STAT3-independent manner. Upon macrophage activation by bacterial lipopolysaccharide, the phosphoinositide 3′ kinase (PI-3 kinase) pathway is activated to produce cytokines such as tumor necrosis factor α (TNFα). SHIP1 is a negative regulator of the PI-3 kinase pathway and its activation downstream of the IL-10 receptor suppresses PI-3 kinase-initiated signals that trigger transcriptional elongation of TNFα and other pro-inflammatory related genes. We next investigated whether SHIP1 activation could mimic the anti-inflammatory actions of IL-10. We screened for small-molecule activators of SHIP1 and isolated the meroterpenoid compound Pelorol. Pelorol and its derivatives specifically enhanced SHIP1’s phosphatase activity and thus suppressed inflammation in macrophage cultures, and in murine models of endotoxic shock, acute anaphylaxis, and inflammatory bowel disease. Closer examination of SHIP1’s enzyme kinetics indicated that SHIP1 is subject to allosteric activation by its product phosphatidylinositol-3,4-bisphosphate (PI-3,4-P₂). We subsequently identified a previously unrecognized C2 domain residing C-terminal of SHIP1’s phosphatase domain which is required for its allosteric activation and is the binding site for both PI-3,4-P₂ and the small-molecule SHIP1 agonists. Bioinformatic and structural analyses also revealed another previously unappreciated domain located N-terminal of SHIP1’s catalytic domain. Using NMR spectroscopy, we characterized this domain as having pleckstrin homology (PH) domain-like topology. We demonstrate that SHIP1’s PH-related (PH-R) domain participates in recruiting SHIP1 to the plasma membrane upon cell stimulation via direct interactions with phosphatidylinositol-3,4,5-trisphosphate. The PH-R domain is essential for SHIP1 inhibition of FcR-dependent phagocytosis and represents another target to which to develop modulators of SHIP1 function. Together, this work suggests that IL-10 activation of SHIP1 is important in its inhibition of macrophage activation, and that mimicking IL-10 with small-molecule SHIP1 agonists could be an effective and viable approach to treating various inflammatory and autoimmune conditions.
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.
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.
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.
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.