Platelets induce endoplasmic reticulum stress in macrophages in vitro

Abstract

Background: Endoplasmic reticulum (ER) stress is a key feature of lipid-laden macrophages and contributes to the development of atherosclerotic plaques. Blood platelets are known to interact with macrophages and fine-tune effector functions such as inflammasome activation and phagocytosis. However, the effect of platelets on ER stress induction is unknown.

Objectives: The objective of this study is to elucidate the potential of platelets in regulating ER stress in macrophages in vitro.

Methods: Bone marrow-derived macrophages and RAW 264.7 cells were incubated with isolated murine platelets, and ER stress and inflammation markers were determined by reverse transcription-quantitative polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. ER morphology was investigated by electron microscopy. Cell viability, lipid accumulation, and activation were measured by flow cytometry. To gain mechanistic insights, coincubation experiments were performed with platelet decoys/releasates as well as lipopolysaccharide, blocking antibodies, and TLR4 inhibitors.

Results: Coincubation of platelets and macrophages led to elevated levels of ER stress markers (BIP, IRE1α, CHOP, and XBP1 splicing) in murine and human macrophages, which led to a pronounced enlargement of the ER. Macrophage ER stress was accompanied by increased release of proinflammatory cytokines and intracellular lipid accumulation, but not cell death. Platelet decoys, but not platelet releasates or lysate from other cells, phenocopied the effect of platelets. Blocking TLR4 inhibited inflammatory activation of macrophages but did not affect ER stress induction by platelet coincubation.

Conclusion: To our knowledge, this study is the first to demonstrate that platelets induce ER stress and unfolded protein response in macrophages by heat-sensitive membrane proteins, independent of inflammatory activation of macrophages.

Keywords: ER stress; atherosclerosis; blood platelets; macrophages; unfolded protein response.