Defective Mitophagy Impairs Response to Inflammatory Activation of Macrophage-Like Cells
Authors: Orekhov Alexander Nikolaevich , Zhuravlev Alexander Dmitrievich , Vinokurov Andrey Yurievich , Nikiforov Nikita Gennadievich , Omelchenko Andrey Vladimirovich , Sukhorukov Vasily Nikolaevich , Sinyov Vasily Vladimirovich , Sobenin Igor Alexandrovich 
Published: March 2024

Background and Aims:

The role of mitophagy in atherosclerosis has been extensively
studied during the last few years. It was shown that mitophagy is involved in the regulation of
macrophages, which are important players as immune cells in atherosclerosis development. In this
study, we investigated the relationship between mitophagy and response to inflammatory stimulation of
macrophage-like cells. Six cybrid cell lines with normal mitophagy, that is, increasing in response to stimulation,
and 7 lines with defective mitophagy not responding to stimulation were obtained. The objective
of the study was to compare the nature of the inflammatory response in normal and defective mitophagy
in order to elucidate the role of mitophagy defects in inflammation.

Methods:

We used cytoplasmic hybrids (cybrids) as cellular models, created using mitochondrial DNA
from different atherosclerosis patients. Mitophagy was stimulated by carbonyl cyanide m-chlorophenyl
hydrazone (CCCP) and assessed as the degree of colocalization of mitochondria with lysosomes using
confocal microscopy. Western blotting methods were used for the determination of proteins involved in
the exact mechanism of mitophagy. Experiments with stimulation of mitophagy show a high correlation
between these two approaches (microscopy and blotting). The pro-inflammatory response of cybrids
was stimulated with bacterial lipopolysaccharide (LPS). The extent of the inflammatory response was assessed
by the secretion of cytokines CCL2, IL8, IL6, IL1β, and TNF measured by ELISA.

Results:

Basal level of secretion of cytokines CCL2, IL8 and TNF was 1.5-2 times higher in cultures of
cybrids with defective mitophagy compared to cells with normal mitophagy. This suggests a persistently
elevated inflammatory response in cells with defective mitophagy, even in the absence of an inflammatory
stimulus. Such cells in the tissue will constantly recruit other immune cells, which is characteristic
of macrophages derived from monocytes circulating in the blood of patients with atherosclerosis. We
observed significant differences in the degree and type of response to inflammatory activation in cybrids
with defective mitophagy. These differences were not so much quantitative as they were dramatically
qualitative. Compared with cells with normal mitophagy, in cells with defective mitophagy, the relative
(to basal) secretion of IL8, IL6 and IL1b increased after the second LPS activation. This indicates
a possible lack of tolerance to inflammatory activation in cells with defective mitophagy, since typically,
re-activation reveals a smaller pro-inflammatory cytokine response, allowing the inflammatory process
to resolve. In cells with normal mitophagy, exactly this normal (tolerant) inflammatory reaction
was observed.

Conclusion:

Data on the involvement of mitophagy, including defective mitophagy, in disturbances of
the inflammatory response in sepsis, viral infections, autoimmune diseases and other pathologies have
previously been reported. In this work, we studied the role of defective mitophagy in non-infectious
chronic inflammatory diseases using the example of atherosclerosis. We showed a dramatic disruption
of the inflammatory response associated with defective mitophagy. Compared with cybrids with normal
mitophagy, in cybrids with defective mitophagy, the secretion of all studied cytokines changed significantly
both quantitatively and qualitatively. In particular, the secretion of 3 of 5 cytokines demonstrated
an intolerant inflammatory response manifested by increased secretion after repeated inflammatory stimulation.
Such an intolerant reaction likely indicates a significant disruption of the pro-inflammatory response
of macrophages, which can contribute to the chronification of inflammation. Elucidating the
mechanisms of chronification of inflammation is extremely important for the search for fundamentally
new pharmacological targets and the development of drugs for the prevention and treatment of chronic
inflammatory diseases, including atherosclerosis and diseases characteristic of inflammation. Such diseases
account for up to 80% of morbidity and mortality.