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Institute of Cancer, Barts & The London School of Medicine, Queen Mary, University of London (QMUL)
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Abstract bachelorproef 2016-2017: The role and function of p110 delta PI3K isoform in gut Immunity and inflammation
Inflammatory bowel disease (IBD) is a complex, immunologically-mediated disease that comprises Crohn’s disease (CD) and ulcerative colitis (UC), and constitutes a major health burden in developed countries. Phosphoinositide 3-kinases (PI3K) are important drug targets in cancer and inflammation, and PI3K inhibitors are in clinical trials for these diseases. Nucleotide oligomerisation domain (NOD)2 is a pattern recognition receptor that recognises the bacterial peptidoglycan component muramyl dipeptide (MDP) and regulates innate immune defences at mucosal sites. NOD2 is the first susceptibility gene identified for CD, but the molecular mechanism regulating NOD2-mediated gut homeostasis is not well understood. Recently Dr. Aksoy’s group discovered that mice carrying an inactivating mutation in the kinase domain of p100δ isoform of PI3K spontaneously develop colitis in the presence of intestinal, opportunistic pathogens.
PI3K isoform p110δ is a lipid kinase that is highly expressed in leukocytes. The hypothesis is that the loss of p110δ PI3K enzymatic activity results in colitis in vivo and therefore may couple to NOD2 pathway to maintain gut immunity and health.
This study had two aims. The first aim is to delineate the impact of genetic or pharmacological loss of p110δ PI3K in NOD2-mediated gut inflammation/colitis in vivo. To achieve these results, in vivo studies were carried out using p110δ knock-in (KI) mice and wild type (WT) mice, which were treated with p110δ inhibitor. Using the real-time PCR technique, the expression of a number of bacterial species were examined. The gut microbiota following treatment with the inhibitor was carried out and the macroscopic analysis of the mouse colons were performed. In the in vivo study, using p110δ inhibitor, it was found that inhibition of p110δ predisposes mice to dextran sodium sulfate induced colitis, marked by shortened colons similar to the results observed by genetic inactivation of p110δ. The RT-PCR data from KI and WT drug-treated mice showed differences in the expression of several bacteria species in comparison to the WT control counterparts.
The second aim was to investigate the role and mechanism of action of p110δ PI3K in NOD2 signalling mediated by muramyl dipeptide (MDP) in cell-based system in phagocytes. The bone marrow-derived dendritic cells (BMDCs) from WT mice and KI mice were generated in vitro and then examined for their responses to MDP using western blotting. The data showed that MDP-NOD2 pathway induces activation of PI3K-Akt phosphorylation, which was abolished in p110δ PI3K deficient BMDCs. On the contrary, the stress pathway p38 activation was increased from p110δ KI DCs.
The data obtained in vitro and in vivo demonstrated that p110δ PI3K controls NOD2-mediated immune responses to bacterial peptide MDP and dampens inflammation. In vivo inactivation of p110δ PI3K predisposes mice to colitis due to uncontrolled growth of intestinal pathobionts and increased inflammation.
Samenvatting eindwerk 2012-2013: Isoform selective roles of p110β class I PI3K in microcrystal uptake and inflammasome activation in phagocytes
Gout is a painful auto-inflammatory disease, which is caused by the deposition of monosodium urate (MSU) crystals in the joints. The mechanisms involved in the uptake of MSU crystals are not well understood but reported to involve professional phagocytes, mainly dendritic cells and macrophages. The initial stages of micro-crystal uptake involve active probing, particle capture and macropinocytosis/phagocytosis processes, some or all of whichmight be regulated by phophatidylinositol 3-kinases (PI3Ks). The crystal uptake leads to inflammasome activation, which induces auto-inflammation processes.
The main aim of this study is to determine the role of p110β, a class I PI3K, in the uptake of micro-crystals and thus inflammasome activation in phagocytes. In the first part of this study, the activation of caspase-1 auto-processing (or cleavage) and Akt phosphorylation is investigated using western blot analysis. Caspase-1 is an important molecule in the inflammasome pathway, while Akt is a well-known PI3K downstream target. The p110β deficient phagocytes showed decreasedcaspase-1 and Akt activation after stimulation with distinct micro-crystals. These lower levels of inflammasome activation can lead to lower levels of inflammatory cytokines and thus to lower levels of inflammation. To verify this, the levels of interleukin (IL)-1β and TNF are measured with enzyme-linked immunosorbent assays. Both IL-1β and TNF are cytokines and lead directly to the actual inflammation. The p110β deficient phagocytes show selective decrease of IL-1β but not of TNF after stimulation with micro-crystals. These findings suggest that p110β deficient phagocytes display less inflammatory phenotype after micro-crystal stimulation than their normal counterparts. The second part of this study focuses on the mechanisms that can control inflammasome activity in phagocytes. Since the p110β isoform can contribute to micro-crystal uptake through cytoskeletal arrangements and the drug of choice for gout treatment, colchicin, is a microtubule disrupting agent, the actin and tubulin structures and dynamics are investigated and analyzed. This is done using confocal microscopy and flow cytometry analysis in phagocytes from wild-type and p110β deficient mice. The data shows differences between actin and, at a lesser level, tubulin structures in phagocytes who lack p110β. In addition, the p110β deficient phagocytes appear to have longer uropods, but no significant differences in their filopodia numbers. When stimulated with the micro-crystals, the p110β deficient phagocytes are able to capture and bind, but unable to uptake the particles. This uptake disability appears to be caused by differences in actin and tubulin dynamics, with a lower depolymerization rate of actin. This lower depolymerization can result in the actin accumulating at the base of the phagocytic cups, antagonizing the internalization of the micro-crystals.
Future prospects include the investigation into more detailed mechanism of action of class I PI3K, p110b and that might be involved in micro-crystal induced autoinflammation.
Samenvatting eindwerk 2010-2011:The study of the role of class I and III phospoinositide 3-kinases isoforms in phagocytic cells
Each year 7.6 million people worldwide die from cancer. For centuries, researchers search for the ultimate cure. To find an effective treatment, the investigation of all possible pathways, leading to cancer promotion is necessary.
Phosphoinositide-3‑kinases (PI3Ks) are lipid kinases that are one of the most frequently deregulated pathways in cancer and other disease context. PI3Ks have roles in the early intracellular signal transduction pathways and affect many biological functions such as survival, growth and migration. This signaling is quite complex because of the existence of eight PI3K isoforms, which are divided into class I, class II and class III PI3Ks. PI3K signaling has been implicated in metabolic control, immunity, angiogenesis and cardiovascular homeostasis, which makes them, attractive targets for therapeutic intervention.
A better understanding of
Each year 7.6 million people worldwide die from cancer. For centuries, researchers search for the ultimate cure. To find an effective treatment, the investigation of all possible pathways, leading to cancer promotion is necessary.
Phosphoinositide-3‑kinases (PI3Ks) are lipid kinases that are one of the most frequently deregulated pathways in cancer and other disease context. PI3Ks have roles in the early intracellular signal transduction pathways and affect many biological functions such as survival, growth and migration. This signaling is quite complex because of the existence of eight PI3K isoforms, which are divided into class I, class II and class III PI3Ks. PI3K signaling has been implicated in metabolic control, immunity, angiogenesis and cardiovascular homeostasis, which makes them, attractive targets for therapeutic intervention.
A better understanding of how the different PI3K isoforms are regulated and how they affect the downstream signaling can uncover their roles in pathology and reveal in which disease contexts their blockade could be beneficial. The general aim of this work is to uncover the isoform selective functions of class I PI3Ks, particularly of p110δ in phagocyte immune responses in particular lipopolysaccharide (LPS)-mediated Toll-like receptors 4 (TLR4) pathway.
The first part of the study, involves the characterization of the bone marrow derived macrophages that carry an inactivating mutation in p110 δ class I PI3K isoform. Microscopy and FACS analysis of the F-/G- actin levels give more information about morphologic changes which p110d inactivation could induce in these cell types. The analysis shows that the p110 δ macrophages have prolonged tails, minor or none formation of lamellipodia and smaller ruffles around the plasma membrane. The FACS analysis of the actin levels demonstrate that p110δ plays an important role in the actin polymerization and thus cytoskeleton rearrangements.
In the following assays, the examination of the role of PI3K isoforms in endocytosis and phagocytosis is carried out by selective or general inhibition of this pathway. The total (pan) PI3K inhibitors have the most radical effect on the uptake of transferrin particles. This proves that most of the PI3K classes are involved in clathrin-dependent endocytosis. Selective PI3Kδ and PI3Kγ inhibitors have the most significant effect amongst the isoform selective drugs. This is an indication that p110δ and p110γ are more involved than the other isoforms, since they are also highly expressed in leukocytes.
The second part of this thesis focuses on the PI3K-mediated regulation of the protein expression of molecules, which are important for transmitting signaling downstream of the Toll-like receptor 4, the receptor for lipopolysaccharide (LPS). MyD88-adapter like (MAL) is an important adapter protein which transmits early LPS signals from the cell surface localized TLR4 since MAL adheres at the plasma membrane through binding to phosphoinositide (4,5) bisphosphate (PIP2). Wiskott-aldrich syndrome protein (WASP) is an important protein involved in actin polymerization which also binds to PIP2. The pharmaceutical inhibition of certain PI3K isoforms gives information on how PI3K regulates LPS-induced degradation of PIP2-binding proteins and that it is possible to interfere with the degradation by targeting PI3K. What can be important in inflammatory diseases. The western blotting assays also show that phospholipase C (PLC)γ, calpain- and cytoskeleton inhibitors of actin polymerization have the considerate effect to repress MAL degradation. The positive control in the WASP assays did not degrade as expected, so it is difficult to subscribe a role for any PI3K isoform in this phenomenon.
The last part of this thesis, involves the assessment of the localization of the phospholipids in bone marrow derived dendritic cells, with the focus on phosphatidylinositol-3-phospate (PI3P). Localization of PI3P through confocal microscopy gives the information that PI3P is found in the cytosol. Inhibition of the p110δ PI3K pathway does not affect the amount of PI3P.
Future prospects is to reveal further downstream protein effectors of p110δ isoform of PI3K and translating to drugs development. The long-term aim is to delineate the roles of all PI3K isoforms in biology and signalling, and to uncover their potential utility as drug targets. how the different PI3K isoforms are regulated and how they affect the downstream signaling can uncover their roles in pathology and reveal in which disease contexts their blockade could be beneficial. The general aim of this work is to uncover the isoform selective functions of class I PI3Ks, particularly of p110δ in phagocyte immune responses in particular lipopolysaccharide (LPS)-mediated Toll-like receptors 4 (TLR4) pathway.
The first part of the study, involves the characterization of the bone marrow derived macrophages that carry an inactivating mutation in p110 δ class I PI3K isoform. Microscopy and FACS analysis of the F-/G- actin levels give more information about morphologic changes which p110d inactivation could induce in these cell types. The analysis shows that the p110 δ macrophages have prolonged tails, minor or none formation of lamellipodia and smaller ruffles around the plasma membrane. The FACS analysis of the actin levels demonstrate that p110δ plays an important role in the actin polymerization and thus cytoskeleton rearrangements.
In the following assays, the examination of the role of PI3K isoforms in endocytosis and phagocytosis is carried out by selective or general inhibition of this pathway. The total (pan) PI3K inhibitors have the most radical effect on the uptake of transferrin particles. This proves that most of the PI3K classes are involved in clathrin-dependent endocytosis. Selective PI3Kδ and PI3Kγ inhibitors have the most significant effect amongst the isoform selective drugs. This is an indication that p110δ and p110γ are more involved than the other isoforms, since they are also highly expressed in leukocytes.
The second part of this thesis focuses on the PI3K-mediated regulation of the protein expression of molecules, which are important for transmitting signaling downstream of the Toll-like receptor 4, the receptor for lipopolysaccharide (LPS). MyD88-adapter like (MAL) is an important adapter protein which transmits early LPS signals from the cell surface localized TLR4 since MAL adheres at the plasma membrane through binding to phosphoinositide (4,5) bisphosphate (PIP2). Wiskott-aldrich syndrome protein (WASP) is an important protein involved in actin polymerization which also binds to PIP2. The pharmaceutical inhibition of certain PI3K isoforms gives information on how PI3K regulates LPS-induced degradation of PIP2-binding proteins and that it is possible to interfere with the degradation by targeting PI3K. What can be important in inflammatory diseases. The western blotting assays also show that phospholipase C (PLC)γ, calpain- and cytoskeleton inhibitors of actin polymerization have the considerate effect to repress MAL degradation. The positive control in the WASP assays did not degrade as expected, so it is difficult to subscribe a role for any PI3K isoform in this phenomenon.
The last part of this thesis, involves the assessment of the localization of the phospholipids in bone marrow derived dendritic cells, with the focus on phosphatidylinositol-3-phospate (PI3P). Localization of PI3P through confocal microscopy gives the information that PI3P is found in the cytosol. Inhibition of the p110δ PI3K pathway does not affect the amount of PI3P.
Future prospects is to reveal further downstream protein effectors of p110δ isoform of PI3K and translating to drugs development. The long-term aim is to delineate the roles of all PI3K isoforms in biology and signalling, and to uncover their potential utility as drug targets.
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Tags: biotechnology human biology |
Address
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4 Newark St, Whitechapel
London E1 2AT
United Kingdom |
Contacts
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Traineeship supervisor
Prof. Bart Vanhaesebroeck
+44 207 882 8269 bart.vanh@qmul.ac.uk |
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Traineeship supervisor
Dr. Ezra Aksoy
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