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Investigation of the role and mechanism of action of NOD2-mediated isoform selective PI3K signalling in gut immunity and inflammation

Periodic Reporting for period 1 - Signalling-in-NODs (Investigation of the role and mechanism of action of NOD2-mediated isoform selective PI3K signalling in gut immunity and inflammation)

Reporting period: 2017-05-01 to 2019-04-30

Inflammatory bowel diseases (IBD), which include Crohn’s disease and ulcerative colitis, is a complex chronic inflammatory condition mainly affecting western societies. Although there has been progress in keeping inflammation down in chronic IBD, each therapy works only in a percentage of patents with variable outcomes. Therefore, there is an unmet need for new therapeutic strategies, which can only be achieved through a better understanding of the molecular mechanisms of host immunity.
Phosphoinositide 3-kinases (PI3Ks) are an evolutionarily conserved family of enzymes that generate lipid second messengers and couple to pattern recognition receptors (PRRs) mediating immune responses. Nucleotide oligomerisation domain (NOD)2 is a PRR that detects muramyl dipeptide (MDP) from the bacterial peptidoglycan wall and is the first susceptibility gene identified for Crohn's disease. PI3K inhibitors are important drug targets in cancer and inflammation and are in clinical trials, and yet a major side effect of these therapies is the clinical manifestation of colitis.
The host lab previously discovered that a single PI3K isoform, p110δ controls TLR-mediated signalling and impacts gut immunity and inflammation. Specifically, genetic inactivation of the kinase activity of p110δ PI3K in mice results in gut inflammation and colitis. Therefore, we proposed to unravel key control mechanisms by which p110δ conveys host protective functions through PRRs and in special NOD2 pathway and maintains gut immunity and tolerance. The broad aims were to (1) Determine the cell-intrinsic role and mechanism of action of p110δ PI3K orchestrating NOD2 and other PRRs signalling pathways in dendritic cells to induce antimicrobial functions. (2) Discover the role of p110δ PI3K in gut immunity using p110δ PI3K kinase inactivated mice alongside with pharmacological targeting strategies in vivo.
PI3K isoform p110δ role was explored in ubiquitous or conditional PI3K-gene-targeted mice and with PI3K-selective inhibitors in models of gut inflammation in vivo and PRR-associated events in bone marrow-derived dendritic cells (DCs) in vitro.
The in vivo inactivation of p110δ rendered dysbiosis and a hyper-inflammatory state revealed by an exaggerated mucosal Th1/Th17 cytokine profile with increase in pro-inflammatory cytokines (IL-1β, IL-18 and IL-12p70) but significantly reduced anti-inflammatory cytokines levels (IL-10) in response to an infection.
At a molecular level, PRR-mediated activation of DCs defective in p110δ activity coordinated a defective production of intra-phagosomal reactive oxygen species (ROS) by NOX-2 enzyme, increasing caspase-1 mediated inflammasome activation, and defective activation of the effector non-canonical autophagy via defective coupling of RAC2-NOX2. Lack of p110δ PI3K activity also revealed inefficacy in promoting regulatory T cells proliferation in favour to INFγ-producing cells in response to microbial ligands, preventing Treg-dependent immune tolerance and favouring inflammation facilitating the development of colitis.
The results of the proposed research have led to the identification of a new role and mechanism of action of a lipid kinase molecule PI3K, in a p110δ isoform selective manner in 1. DC-intrinsic NOD2-dependent activation of autophagy and dampening of inflammasome activity, 2. DC-intrinsic anti-inflammatory role of NOD2 signalling in vitro and in vivo, 3. New NOD2-driven regulatory pathways to induce DC anti-inflammatory properties to protect mammalian organism from colitis, 4. New immunotherapeutic angle to generate anti-/pro-inflammatory DCs in vitro and in vivo.
The results were presented in National and International Scientific events such as Autophagy UK Network Meeting, UCL Partners III Symposium, Toll Editing Innate Immunity, London Inflammation Network and Autophagy in inflammation. They were also presented at a local level in the Biochemical Pharmacology Department meetings and William Harvey Research Institute seminars.
This research proposal will impact positively the scientific knowledge on the class I PI3K isoforms, particularly p110δ in gut immunity and will have important effects on the human health and therapies where PI3K-mediated regulation of innate immune signals could be therapeutically targeted.
It will be especially useful in immunotherapy where temporal inhibition of peripheral regulatory T cell activities would be beneficial such as in cancer and chronic viral infections. We also expect these results to have a great impact on other diseases in which patients develop an inflammatory bowel disease similar to Crohn's disease such as granulomatous disease (CGD). There are clinical and pathological resemblances between this two pathologies, supporting the possibility of mechanistic similarities in their pathogenesis. Our results provide evidence of temporary temper of those pathways regulated by p110δ activity could contribute to the amelioration of the symptoms. It could have a benefit in terms of the potential to develop personalized, preventive or therapeutic strategies that could lead to potential savings in terms of healthcare, use of healthcare resources and quality of life for those patients affected.
This research project has also had an important impact in my career and has allowed me to progress beyond the ‘state-of-art’. I was given the opportunity to work in the prestigious William Harvey Research Institute, as the largest pharmacological research institute in the UK and meet internationally excellent scientists from 45 countries and develop new networks both within the host institution and beyond. I developed important collaborations with experts on different fields related to the project. I benefited from a great range of transferable skills workshops in scientific writing, leadership training, research management and creative thinking to support my professional development. I also benefited from technical and scientific skills training (flow cytometry, confocal microscopy) and accessed outstanding equipment to conduct my work successfully. I was encouraged to present my research and discuss problems encountered during regular group and institutional meetings, but also to participate in social events and public engagement organized by the Institute such as Barts and QMUL science festival and Pint of Science festival to bring science closer to the general public and patients. I also had the opportunity of supervising 2 Master students and I participated in problem-based learning activities with alumni of the Queen Mary University as my first contact with the academic part of the science. The greatest achievement for my career is the article that is in preparation and will be submitted to Nature Immunology in July 2019. I have two more reviews on gut immunity, tolerance, and antigen presentation that are also in preparation and I expect to publish them in the near future.
Pathways associated with susceptibility genes for IBD and CGD