Department of Biochemistry, and Molecular Biophysics Program, University of Colorado Boulder, Boulder, Colorado, United States America.
PLoS One. 2020 May 29;15(5):e0233012. doi: 10.1371/journal.pone.0233012. eCollection 2020.
Leukocyte migration is controlled by a membrane-based chemosensory pathway on the leading edge pseudopod that guides cell movement up attractant gradients during the innate immune and inflammatory responses. This study employed single cell and population imaging to investigate drug-induced perturbations of leading edge pseudopod morphology in cultured, polarized RAW macrophages. The drugs tested included representative therapeutics (acetylsalicylic acid, diclofenac, ibuprofen, acetaminophen) as well as control drugs (PDGF, Gö6976, wortmannin). Notably, slow addition of any of the four therapeutics to cultured macrophages, mimicking the slowly increasing plasma concentration reported for standard oral dosage in patients, yielded no detectable change in pseudopod morphology. This finding is consistent with the well established clinical safety of these drugs. However, rapid drug addition to cultured macrophages revealed four distinct classes of effects on the leading edge pseudopod: (i) non-perturbing drug exposures yielded no detectable change in pseudopod morphology (acetylsalicylic acid, diclofenac); (ii) adaptive exposures yielded temporary collapse of the extended pseudopod and its signature PI(3,4,5)P3 lipid signal followed by slow recovery of extended pseudopod morphology (ibuprofen, acetaminophen); (iii) disruptive exposures yielded long-term pseudopod collapse (Gö6976, wortmannin); and (iv) activating exposures yielded pseudopod expansion (PDGF). The novel observation of adaptive exposures leads us to hypothesize that rapid addition of an adaptive drug overwhelms an intrinsic or extrinsic adaptation system yielding temporary collapse followed by adaptive recovery, while slow addition enables gradual adaptation to counteract the drug perturbation in real time. Overall, the results illustrate an approach that may help identify therapeutic drugs that temporarily inhibit the leading edge pseudopod during extreme inflammation events, and toxic drugs that yield long term inhibition of the pseudopod with negative consequences for innate immunity. Future studies are needed to elucidate the mechanisms of drug-induced pseudopod collapse, as well as the mechanisms of adaptation and recovery following some inhibitory drug exposures.
白细胞迁移受到前沿伪足上基于膜的化学感觉通路的控制，该通路在先天免疫和炎症反应期间引导细胞沿吸引剂梯度运动。本研究采用单细胞和群体成像技术，研究了培养的极化 RAW 巨噬细胞中前沿伪足形态的药物诱导扰动。测试的药物包括代表性的治疗药物（乙酰水杨酸、双氯芬酸、布洛芬、对乙酰氨基酚）和对照药物（PDGF、Gö6976、wortmannin）。值得注意的是，将四种治疗药物中的任何一种缓慢添加到培养的巨噬细胞中，模拟患者标准口服剂量报告的血浆浓度缓慢增加，不会导致伪足形态发生可检测的变化。这一发现与这些药物的良好临床安全性一致。然而，快速向培养的巨噬细胞中添加药物，会对前沿伪足产生四种不同的影响：（i）非干扰性药物暴露不会导致伪足形态发生可检测的变化（乙酰水杨酸、双氯芬酸）；（ii）适应性暴露会导致延伸伪足暂时坍塌，其标志性的 PI(3,4,5)P3 脂质信号随后缓慢恢复延伸伪足形态（布洛芬、对乙酰氨基酚）；（iii）破坏性暴露会导致长期伪足坍塌（Gö6976、wortmannin）；（iv）激活性暴露会导致伪足扩张（PDGF）。适应性暴露的新观察结果使我们假设，快速添加适应性药物会使内在或外在的适应系统不堪重负，导致暂时坍塌，然后适应性恢复，而缓慢添加则使系统能够实时逐渐适应，以抵消药物扰动。总体而言，这些结果说明了一种方法，这种方法可能有助于识别在极端炎症事件中暂时抑制前沿伪足的治疗药物，以及长期抑制伪足并对先天免疫产生负面影响的有毒药物。未来的研究需要阐明药物诱导伪足坍塌的机制，以及一些抑制性药物暴露后的适应和恢复机制。
