Preprint Club
A cross-institutional Journal Club Initiative

Keywords

● Monocytes

● Mitochondrial Reactive Oxygen Species

● Multiple Sclerosis

Main Findings

In this preprint, the authors investigate the source of reactive oxygen species (ROS) that determine the severity of multiple sclerosis (MS) in mice. Tissue damage due to ROS production is a key feature in MS. However, the major contributing cells responsible for this damage remain poorly classified. Using multiple human and murine high resolution sequencing data sets, the authors generate an integrated map of brain macrophages and identify an enrichment of a ROS gene expression signature. Confirming these results in the murine data sets the authors reveal and confirm that Monocyte-derived Cells (MdC) rather than Microglia express highest levels of ROS. Using a combination of inducible Cre lines, the investigators provide data indicating that ROS production in EAE, a murine experimental model for MS, is independent of the NADPH Oxidase 2-associated protein gp91phox. Identifying that MdC show elevated expression of the mitochondrial complex I (a central part of the electron transport chain) inspired the authors to explore the role of reverse electron transport in generating mitochondrial ROS (mtROS). Using a transgene encoding allowing the conditional overexpression of mitochondrial-tagged Catalase (mCAT), the authors next demonstrate that CCR2-expressing MdC, overexpressing cells mCAT show significantly lower levels of mtROS productions. In line with these observations, the authors report a decrease in infiltrating immune cells (monocytes and T cells), a decrease in demyelination and a significant amelioration of EAE pathology. The authors conclude that MdC-derived mtROS rather then microglia contribute to ROS-mediated tissue damage in EAE/MS.

Limitations

● While the source and pathway contributing to ROS are nicely dissected, the fate of MdC or monocytes under the describe circumstances (mCAT overexpression and brain inflammation) remain unclear. One wonders if mCAT overexpression impacts inflammatory cytokine production and the availability of DAMPs in the brain, or whether mCAT overexpression alters myeloid cell migration, or survival.

● A more detailed spatial characterization of myeloid cells in active lesions and the lesion edges would be a fantastic addition to the preprint’s last figure. Why are there fewer MdC/inflammatory cells? Pairing such an analysis with an assessment of neuronal cell death, the characterization of T cells and their regional distribution would be very valuable information.

● An additional aspect that could improve the authors’ report is an answer to the question of how inflammatory cytokines contribute to mtROS production in EAE. Integrating the role of IFNg or GM-CSF into the activity of complex I would be of great value particularly in light of recently published data https://doi.org/10.1038/s41586-024-07167-9 . A role for RORgt+ T cells could be considered here as well.

● Another point that arises from the authors data is the question on what pathway or mechanism facilitates RET in MdC but not in microglia?

● While the use of the reported Cre lines is a widely-accepted and elegant strategy, it remains unclear to what extent, or if at all, infiltrating monocytes contribute to the pool of “microglia-like cells” under conditions of EAE. A clearer definition of MdCs’ contribution to the population of “microglia/microglia-like” cells would strengthen the authors’ claim and help to define whether a transition of MdC to cells with features and phenotypes of microglia could be considered as path towards ROS production by microglia/microglia-like cells. As a re-occurring issue in the field, the authors’ report and experimental set up has the potential to provide clarifying answers to this issue.

Significance/Novelty

The authors elegantly demonstrate that RET and mtROS produced by CCR2-expressing cells is a key contributor to tissue damage in EAE. The use of the mCAT transgene makes this work particularly interesting. The authors experimental set up strongly suggests that Monocyte-derived Cells are the major producers of tissue-damaging ROS through the use of mitochondrial RET. This pathway and its contribution to the disease are novel and a great advancement to the field that now allows the integration of reports on mitochondrial dysfunction and MS into a testable, experimental setting.

This work nicely demonstrates the use of an elegant in vivo system that facilitates the exploration of an underappreciated pathway implicated in inflammation-driven tissue damage. The provided results inspire the use of such tools for many additional inflammatory conditions to dissect the contribution of monocyte-derived cells and tissue-resident macrophages to the severity of chronic and acutely inflammatory diseases.

Credit

Reviewed by Arthur Mortha (University of Toronto, Department of Immunology) as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interest in relation to their involvement in the review.