Find-me and eat-me: understanding how signals from dying cells control and subvert macrophage behaviour

Apoptotic cell death is fundamental in sculpting our bodies as we develop, and in maintaining homeostasis. We now know that clearance of apoptotic cells (efferocytosis) not only disposes of effete cells, but also trains our immune system, helps terminate inflammatory responses and reprograms innate immune cell function. These important roles are conserved through evolution to flies (Weavers et al., Cell 2016). Failures in efferocytosis are associated with chronic inflammatory conditions (e.g. chronic obstructive pulmonary disease, COPD) and damaging autoimmune conditions in humans and impaired immune responses in flies (primary supervisor, manuscript in preparation). Vertebrate macrophages are thought to detect and migrate towards apoptotic cells via the release of diffusible "find-me" signals (e.g. ATP, UTP; Ravichandran, Immunity 2011). This migratory response leads to phagocytosis and subsequent degradation of the apoptotic cell prey. Nonetheless find-me cues remain poorly studied, particularly in vivo. We hypothesise such cues exist in Drosophila and will use this highly genetically-tractable organism, in which apoptosis and macrophage migration can be imaged simultaneously, to dissect genetically how macrophages detect dying cells.
Research aims: 1a. Infer presence and characteristics of find-me cues in vivo. We have developed methods to induce apoptosis and follow fluorescently-labelled macrophage responses to dying cells within Drosophila embryos. Mathematical modeling, based upon quantitative data taken from such movies, will be used to demonstrate the existence of find-me cues and determine their characteristics. This will be achieved through adaptation of a model previously used (Third Supervisor et al., 2014 Cereb. Cortex) to understand directed migration of neuronal growth cones in response to diffusible gradients of morphogens (months 1-12). 1b. Identify novel find-me cues/regulators of their production. Candidate find-me cues identified via parameters gleaned from modelling will be tested in Drosophila embryos using mutants that disrupt their production or detection. In parallel, unbiased genetic screening will uncover novel components of cue production and release by identifying genes necessary for recruitment of Drosophila macrophages to sites of apoptosis induction (months 3-24).
2. Study novel find-me cues in human macrophages.Where homologues exist, we will use siRNA or pharmacological inhibitors to disrupt pathways identified in aim 1 in human macrophages to recreate phenotypes observed in the fly. Microfluidic chambers will be used to image chemotaxis towards apoptotic white blood cells/synthetic find-me cues. We will also study how novel find-me cues interact with other chemokines (e.g cytokines/inflammatory factors) by challenging human macrophages with competing gradients, determining the hierarchy of migratory responses and impact on efferocytosis (months 22-34).3. Translate findings to macrophages from patients with inflammatory disease.
As a proof of principle, we will investigate our findings in the context of chronic inflammation by examining how find-me cues impact migration and efferocytosis in macrophages from patients with COPD with the translational aim of correcting efferocytic defects in this population (months 30-36). In summary, given the broad range of processes and diseases upon which regulation of efferocytosis impacts, a greater understanding of find-me cues and how they influence cell behaviour will bear significant relevance to both medicine and discovery biology.