Epigenetic Adaptation Drives Monocyte Differentiation into Microglia-Like Cells Upon Engraftment into the Central Nervous System

Reviewer #1 (Public review):

Microglia are mononuclear phagocytes in the CNS and play essential roles in physiology and pathology. In some conditions, circulating monocytes may infiltrate in the CNS and differentiated into microglia or microglia-like cells. However, the specific mechanism is large unknown. In this study, the authors explored the epigenetic regulation of this process. The quality of this study will be significantly improved if a few questions are addressed.

(1) The capacity of circulating myeloid cell-derived microglia are controversial. In this study, the authors utilized CX3CR1-GFP/CCR2-DsRed (hetero) mice as a lineage tracing line. However, this animal line is not an appropriate approach for this purpose. For example, when the CX3CR1-GFP/CCR2-DsRed as the undifferentiated donor cell, they are GFP+ and DsRed+. When the cell fate has been changed to microglia, they will change into GFP+ and DsRed- cells. However, this process is mediated with busulfan and artificially introduced bone marrow cells in the circulating cell, which is not existed in physiological and pathological conditions. These artifacts will potentially bring in artifacts and confound the conclusion, as the classical wrong text book knowledge of the bone marrow derived microglia theory and subsequently corrected by Fabio Rossi lab1,2. This is the most risk for drawing this conclusion. The top evidence is from the parabiosis animal model. Therefore, A parabiosis study before making this conclusion, combining a CX3CR1-GFP (hetero) mouse with a WT mouse without busulfan conditioning and looking at whether there are GFP+ microglia in the GFP- WT mouse brain. If there are no GFP+ microglia, the author should clarify this is not a physiological or pathological condition, but a defined artificial host condition, as previously study did3.

(2) In some conditions, peripheral myeloid cells can infiltrate and replace the brain microglia4,5. Discuss it would be helpful to better understand the mechanism of microglia replacement.

References:

(1) Ajami, B., Bennett, J.L., Krieger, C., Tetzlaff, W., and Rossi, F.M. (2007). Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nature neuroscience 10, 1538-1543. 10.1038/nn2014.

(2) Ajami, B., Bennett, J.L., Krieger, C., McNagny, K.M., and Rossi, F.M.V. (2011). Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nature neuroscience 14, 1142-1149. http://www.nature.com/neuro/journal/v14/n9/abs/nn.2887.html#supplementary-information.

(3) Mildner, A., Schmidt, H., Nitsche, M., Merkler, D., Hanisch, U.K., Mack, M., Heikenwalder, M., Bruck, W., Priller, J., and Prinz, M. (2007). Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nature neuroscience 10, 1544-1553. 10.1038/nn2015.

(4) Wu, J., Wang, Y., Li, X., Ouyang, P., Cai, Y., He, Y., Zhang, M., Luan, X., Jin, Y., Wang, J., et al. (2025). Microglia replacement halts the progression of microgliopathy in mice and humans. Science 389, eadr1015. 10.1126/science.adr1015.

(5) Xu, Z., Rao, Y., Huang, Y., Zhou, T., Feng, R., Xiong, S., Yuan, T.F., Qin, S., Lu, Y., Zhou, X., et al. (2020). Efficient strategies for microglia replacement in the central nervous system. Cell reports 32, 108041. 10.1016/j.celrep.2020.108041.

Reviewer #2 (Public review):

Mouse fate mapping studies have established that the bulk of microglia derives from cells that seed the brain early during development. However, monocytes were also shown to give rise to parenchymal CNS macrophages and thus are potential candidates for microglia replacement therapy. Whether monocyte-derived cells adopt bona fide microglia identities has remained under debate. The study of Liu et al addresses this important outstanding question, focusing on the retina.

Specifically, the authors investigate monocyte-derived macrophages that arise upon challenges in the murine retina using scRNAseq and ATACseq analyses, combined with flow cytometry and histology. They complement this approach with an analysis of BM chimeras and analyses of the latter. The authors conclude that monocyte-derived cells acquire markers that have originally been proposed to be microglia-specific, including P2ry12, Tmem119, and Fcrls.

In 2018, four comprehensive independent studies reported the analyses of monocyte-derived CNS macrophages (PMID 30451869, 30523248, 29643186, 29861285). Following transcriptome and epigenome analyses, these teams came to the collective conclusion that HSC-derived cells remain distinct from microglia. Using advanced fate mapping and better isolation and profiling tools, a more recent study, however, concluded that, if given sufficient time of CNS residence, most monocyte-derived macrophages can, at the transcriptome level, become essentially identical to microglia (PMID 40279248, https://www.biorxiv.org/content/10.1101/2023.11.16.567402v1).

Given this controversy, the study of Paschalis and colleagues, which focuses largely on retinal monocyte-derived cells, could have been a valuable resource and complement for clarification. Indeed, interestingly, their data suggest that microglia adaptation of monocyte-derived macrophages might be faster in the retina than in the CNS. However, for the reasons outlined below, the study falls in its present form short of providing significant new insight and is a missed opportunity.

Comments:

The major shortcoming of the study is that the authors decided to focus on a very limited number of genes to make their case, rather than performing a more informative, unbiased, and detailed global analysis. In contrast to what the authors state, much of the microglia community is, I believe, aware of experimental limitations and the problem with markers. Showing gain of microglia marker expression on monocyte-derived cells, or loss of monocyte markers, such as Ly6C, is not novel.

This is highlighted Fig. 3F. No one argues today that monocyte-derived tissue macrophages differ from blood monocytes (although the authors repeatedly emphasize this as novelty). However, the heatmap shows that the engrafted cells clearly differ from naïve and injured microglia. What are these genes, their associated pathways ?

Also, how about expression of the Sall1 gene that encodes a repressor that is considered important to maintain microglia identity (PMID37322178, 27776109). Somewhat surprisingly, Sall1 was recently also shown to be expressed by monocyte-derived CNS macrophages (PMID 40279248). It would be valuable information if the authors can corroborate this finding.

The authors state in their discussion that monocyte-derived macrophages seem 'hardwired for inflammatory responses'. While this is an interesting suggestion, the NFkB motif enrichment is insufficient and should be complemented with a target list. Again, it would be important to be aware of heterogeneity.

A critical factor when analyzing CNS macrophages is the exclusion of perivascular CNS border-associated cells, which also holds for the retina (see PMID 38596358). This should be addressed. Can the authors discriminate BAM from microglia in their scRNAseq data set, for instance, by their CD206 expression or other markers ? BAM have been shown to display distinct transcriptomes and even as a contamination could introduce significant bias.

Even for the genes the authors focus on, it is hard to understand from the way the authors present the data what fraction of cells are positive. This would be critical information since there could be some heterogeneity. Flowcytometry analysis, including double staining for P2ry12, Tmem119, and Fcrls to see correlations, would here be valuable.

The authors state in their title that 'epigenetic adaptation drives monocyte differentiation'. However, since all gene expression is governed by the epigenome, this is trivial. I would argue that to gain meaningful insight and justify such a statement, it would require an in-depth global comparative analysis of the chromatin status of yolk sac microglia and monocyte-derived CNS macrophages, including CUT&RUN analysis for specific histone marks and methylation patterns.

Please cite and discuss PMID 30451869, 30523248, 29643186, 29861285, and in particular the more recent highly relevant study PMID 40279248.