Muir Clare F, Reyes-Aldasoro Constantino Carlos, Prajsnar Tomasz K, Michno Bartosz J, Cholewa-Waclaw Justyna, Ho Yin X, Bernut Audrey, Loynes Catherine A, Elworthy Stone, Bowden Kieran A, Cadby Ashley J, Prince Lynne R, King Jason S, Ellett Felix, Condliffe Alison M, Renshaw Stephen A
Bateson Centre, School of Medicine and Population Health, University of Sheffield, Sheffield S10 2TN, UK.
Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh EH25 9RG, UK.
Dis Model Mech. 2025 Sep 1;18(9). doi: 10.1242/dmm.052042. Epub 2025 Aug 29.
Class I PI3 kinases (PI3Ks) coordinate the delivery of microbicidal effectors to the phagosome by forming phosphatidylinositol (3,4,5)-trisphosphate (PIP3). However, the dynamics of PIP3 in neutrophils during a live bacterial tissue infection are unknown. We therefore developed an in vivo, live zebrafish infection model that enables visualisation of dynamic changes in Class 1 PI3K signalling in neutrophil phagosomes in real time. We identified that, on ∼12% of neutrophil phagosomes, PHAkt-eGFP, a reporter for Class 1 PI3K signalling, repeatedly fades and re-recruits in pulsatile bursts. This phenomenon occurred on phagosomes containing live and dead bacteria as well as beads, and was dependent on the activity of the Class 1 PI3K isoform PI3Kγ. Detailed imaging suggested that pulsing phagosomes represent neutrophils transiently re-opening and re-closing phagosomes, a conclusion supported by observations that a subset of phagosomes in human neutrophils rapidly accumulate dye from the extracellular space. Therefore, we propose that some neutrophil phagosomes remain unsealed and are consequently able to exchange contents with the extracellular environment, with implications for phagosome fate and communication with surrounding cells.
I类磷脂酰肌醇-3激酶（PI3Ks）通过形成磷脂酰肌醇（3,4,5）-三磷酸酯（PIP3）来协调将杀菌效应物递送至吞噬体。然而，在活细菌组织感染期间中性粒细胞中PIP3的动态变化尚不清楚。因此，我们开发了一种体内活斑马鱼感染模型，该模型能够实时可视化中性粒细胞吞噬体中I类PI3K信号传导的动态变化。我们发现，在约12%的中性粒细胞吞噬体上，I类PI3K信号的报告基因PHAkt-eGFP会以脉冲爆发的形式反复消失并重新募集。这种现象发生在含有活细菌、死细菌以及珠子的吞噬体上，并且依赖于I类PI3K亚型PI3Kγ的活性。详细的成像表明，脉冲吞噬体代表中性粒细胞暂时重新打开和重新关闭吞噬体，这一结论得到了以下观察结果的支持：人类中性粒细胞中的一部分吞噬体迅速从细胞外空间积累染料。因此，我们提出一些中性粒细胞吞噬体保持未封闭状态，因此能够与细胞外环境交换内容物，这对吞噬体命运以及与周围细胞的通讯具有影响。
