Rapid exposure of macrophages to drugs resolves four classes of effects on the leading edge sensory pseudopod: Non-perturbing, adaptive, disruptive, and activating.

Thomas C Buckles,Joseph J Falke,Danijel Djukovic,Brian P Ziemba,Rossana C.N Melo

doi:10.1371/journal.pone.0233012

Abstract

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.

Highlights

Leukocytes, including macrophages and neutrophils, possess a sophisticated chemosensory system that controls cellular migration up primary attractant gradients to sites of infection, injury, or tumor formation during the innate immune response [1,2,3,4,5,6,7,8]
The chemosensory pathway that directs leukocyte migration up primary and secondary attractant gradients is localized on the broad sensory pseudopod at the leading edge of the polarized cell, where pathway components assemble on the cytoplasmic leaflet of the plasma membrane
The leading edge pseudopod possesses an intrinsic adaptation mechanism that damps out fluctuations in the chemical environment as required for movement in an attractant gradient [42, 69, 70]

Summary

Introduction

Leukocytes, including macrophages and neutrophils, possess a sophisticated chemosensory system that controls cellular migration up primary attractant gradients to sites of infection, injury, or tumor formation during the innate immune response [1,2,3,4,5,6,7,8]. The chemosensory pathway that directs leukocyte migration up primary and secondary attractant gradients is localized on the broad sensory pseudopod at the leading edge of the polarized cell, where pathway components assemble on the cytoplasmic leaflet of the plasma membrane (reviewed in [3,4,5, 9, 15,16,17]). The leukocyte pathway possesses a positive feedback loop that is able to maintain the stability and ruffling activity of the leading edge pseudopod even in the absence of an attractant gradient, enabling the pseudopod to rapidly detect and direct migration up a newly appearing gradient [3, 4, 16, 18,19,20,21,22,23,24,25,26]. Inhibition of any loop component yields inactivation of other loop components, decreased PI3K activity and PIP3 levels, and contraction of the pseudopod

Methods

Results

Conclusion