Understanding how neutrophils self-organise their migration to sites of inflammation in vivo

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Poplimont, Hugo Clément Geoffrey  ORCID logo  https://orcid.org/0000-0002-2319-363X

Neutrophils are major effectors of acute inflammation and microbial defence. Their infiltration and migration in injured tissue are critical for the inflammatory response. They are often observed navigating in a highly co-ordinated and directed manner leading to their aggregation at the target site of infection. This self-organised cell gathering is referred to as swarming. It is known that neutrophil swarming is driven by autocrine attractant production, notably of the lipid leukotriene B4 (LTB4). The decision to release attractants at the single-cell level is important and impacts the magnitude of the entire immune response. However, the precise mechanisms triggering this decision remain unclear. In this study, I employed in vivo imaging of zebrafish larvae to reveal the molecular processes that trigger the release of LTB4 and initiation of swarms. I developed a 2-Photon laser wound assay to elicit and visualise neutrophil swarming. A major limitation in previous studies of neutrophil swarms was the lack of tools to understand cell signalling dynamics during the response. To overcome this, I generated a new biosensor to probe for calcium levels in neutrophils as it correlates with the production of chemoattractants. Using this new tool, I revealed that neutrophils clustering at the target inflammatory site are experiencing sustained high calcium elevation. Using a new probe to follow the production dynamics of LTB4, I demonstrated that the rise of intracellular calcium promotes the biosynthesis of this key attractant. I further demonstrated that these calcium fluxes are triggered upon contact with necrotic tissue. This prompted me to interrogate the damage molecules driving these calcium signals in neutrophils. I discovered that the calcium fluxes were mediated by ATP that binds gated ion channels (P2X1) leading to rapid intracellular calcium uptake. Surprisingly, I found that live neutrophils can also trigger this calcium flux in other neutrophils upon mutual contact. Using chemical and genetic inhibition, I found that connexin-43 (Cx43) hemichannels, through their ability to release ATP, enable amplification of the calcium signal leading to chemoattractant production and subsequent neutrophil recruitment. I concluded that activation of LTB4 synthesis is a group decision reached via Cx43-dependent communication in pioneer clustering neutrophils. As Cx43 inhibition significantly reduced neutrophil aggregation at the target site, I investigated if this inhibition could have consequences for wound defence. For this, I developed a wound colonisation assay with Pseudomonas aeruginosa. Using this approach, I showed that Cx43 was crucial for the protection of wounds from opportunistic bacteria. Finally, I designed and generated an optogenetic tool to manipulate LTB4 biosynthesis dynamics in vivo. I demonstrated the effectiveness of this tool in immortalised cells and zebrafish larvae. The unique features of this tool make it very useful for a wide range of research applications on signalling dynamics. In conclusion, I have shown that by reinforcing damage signalling, Cx43 channels coordinate attractant biosynthesis in pioneer neutrophils. This generates an effective chemoattractant gradient source and promotes targeted aggregation and defence. This study, therefore, reveals a new mechanistic principle of collective behaviour that could be exploited in future pathological research.

Sarris, Milka
Neutrophil, Swarming, Inflammation, Infection, Optogenetic, Zebrafish, Microscopy, Live imaging, LTB4, Connexin, Calcium
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Wellcome trust four-year programme