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Enhancing Engulfment of Apoptotic cells: basic biology to Therapy

Periodic Reporting for period 1 - EATing more (Enhancing Engulfment of Apoptotic cells: basic biology to Therapy)

Reporting period: 2018-06-01 to 2020-05-31

The controlled death and turnover of cells in our bodies is critical for healthy physiology, development and resolving inflammatory responses. Many cells can ‘eat’ other cells, and normally dying cells are rapidly removed by phagocytosis. Professional eaters (phagocytes), such as macrophages, have a large capacity for dead cell engulfment. This process, specifically referred to as efferocytosis, is generally anti-inflammatory and contributes to resolution and tissue healing. Problems with dead cell clearance contribute to chronic inflammation and disease: numerous inflammatory diseases are associated with a high burden of uncleared cell corpses, and a reduced capacity for macrophages to perform efferocytosis. For example, atherosclerosis, arthritis, inflammatory bowel disease, chronic respiratory conditions and autoimmunity. This project aimed to address the hypothesis that enhancing dying cell clearance is an effective strategy to treat disease. The rationale for this is twofold: first, by enhancing removal of dying cells, since these uncleared corpses are a form of 'debris' that can perpetuate inflammation. Second, the ‘anti-inflammatory’ effects of efferocytosis could promote healing in specific disease settings.

There is a need for appropriate tools to boost dead cell removal in the body. We (1) aimed to identify compounds, from libraries of biologically active drugs, that were able to increase macrophage capacity for dead cell elimination in vitro. Then, (2) we addressed how these small molecules act at the cellular level to boost efferocytosis by macrophages – and whether they have the same effect on other phagocytes in the body. (3) We aimed to investigate how such molecules could modulate the inflammatory environment, e.g. by the subsequent release of soluble factors by macrophages. The eventual goal (4) is to test the identified small molecule boosters of efferocytosis for beneficial effects in models of inflammatory disease, to potentially offer a new strategy in therapies. Furthermore, our findings will provide insights into the process of dead cell clearance itself.
Based on a drug screen previously performed in the laboratory, I selected 2 primary candidates for further analysis. I identified and confirmed, by a range of experimental approaches, that these small molecules act as boosters of macrophage efferocytosis. These 2 molecules are known to target (inhibit) a common pathway. We confirmed that the pathway in question is important in macrophages for modulating dead cell uptake. Moreover, the transient inhibition of the pathway is important: deleting the gene coding for the responsible protein does not have the same effect. To further understand how this works, we intend to measure the release of soluble factors downstream of this, and related pathways. To reliably measure these soluble factors, I have started a collaboration with experts in the field, and validated the in-house techniques for sample collection.

We successfully optimised the techniques for high throughput imaging of macrophages eating dying cells, and the analysis capacity to screen for molecules that boost efferocytosis. By this approach, we screened 2,560 molecules. From these, we selected 85 molecules for a repeat confirmation screen, and are currently applying our analysis criteria to identify the top candidates.

In the 19 of 24 months of this IF action, 3 out of 4 objectives were largely achieved for top candidate molecules. We demonstrated that macrophage capacity for dead cell elimination can be boosted using small molecules, and we have identified one pathway of interest. These findings have been disseminated by way of multiple lab meetings, a departmental seminar, and a poster presentation at an international congress. One manuscript and one review article are in preparation.

The experimental approaches to realise these objectives are now well-established, which will expedite the evaluation of new candidate molecules from the most recent screen.
Towards our final objective, our pilot experiments to boost dead cell uptake at baseline (in the lungs) were unsuccessful. These data are important, however, as we need to rethink our models to specifically address contexts in which there is a defect in clearance. We also need to confirm the effective pharmaceutical dosing of candidate compounds, and may achieve this by modifying the delivery method. The outcomes of this action have thus informed how we can exploit the results for future study.
We used state-of-the art high-capacity imaging and developed a novel workflow to assess macrophage efferocytosis, and then optimised this for high-throughput screening. Beyond the direct results of the action, in the lab we have also developed the protocols to evaluate transcriptional responses following macrophage phagocytosis of dying cells. By applying state-of-the-art nucleomics approaches we can separate the RNA transcripts deriving from the phagocyte from that of the cargo. These techniques will be used for continuing work related to the project.

The small molecule “boosters” that we describe here will be important tools to exploit in the context of future research, in particular to dissect pathways of processing during dead cell clearance. We intend to address the effect of boosting macrophage efferocytosis in contexts relevant to inflammatory disease, but our findings may have implications in developmental or infectious contexts.
Despite the preemptive termination of the action, we remain intent upon the goal to test the benefit of “boosters” of efferocytosis. To that end, we have refined a new workplan: to assess the effect of compound treatment when there is a high burden of uncleared cell corpses, and to test models in which we can directly monitor efferocytosis upon compound treatment. Importantly, our top candidate molecules are known anti-inflammatory agents. These data could highlight a new mechanism of action for a known drug, and may, in the long term, imply new indications for clinical usage.

Personally, this IF action has had an extremely postitive impact on my professional development: I have enhanced my existing skill set and acquired new competencies. Scientifically, I have developed my technical and analytical approaches, and conceptual engagement and understanding, both with my colleagues, collaborators, and the wider scientific community. I have benefited from the exceptional complementary skills training and career guidance offered by VIB. I have developed new collaborations within and outside of the laboratory, both directly relating to the project objectives and for new research directions.
Important contributions to the community (scientific/public) over the course of the action:
-Attendance at two EMBO workshops – one at which I presented (Crete, Oct 2019)
-Attendance at Society for Leukocyte Biology’s Annual Meeting (+ workshops)(Boston, USA, Nov 2019). Included active participation, assisting to organise events for junior researchers. Membership of the SLB’s Members in Transition and Training group.
-Participation at VIB Biotech Day (public outreach event) Oct. 2018. Communication relating to “What is CRIPSR/Cas9?”
-Ongoing membership of the VIB PostDoc Committee. Co-organisation of two one-day symposia for postdocs of VIB.
-Guided Bachelor Students in a lab project (May 2018). Offered a project for 2020 rotations. Offered a Masters II project to start in late 2020.
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