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.
