Epigenetic Adaptation Drives Monocyte Differentiation into Microglia-Like Cells Upon Engraftment into the Central Nervous System
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The authors investigated the epigenetic mechanisms regulating the differentiation of circulating monocytes that infiltrate the CNS and adopt microglia-like characteristics. The work is useful to the field, as the contribution of circulating myeloid cell-derived microglia remains controversial. However, the evidence presented is inadequate as the analyses are based on a very limited set of genes, which does not sufficiently support the authors' central claims.
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Abstract
The identification of specific markers to distinguish resident microglia from infiltrating monocytes has been a long-standing challenge in neuroscience. Recently, proteins such as P2RY12, TMEM119, and FCRLS have been proposed as microglia-specific and are now widely used to define microglial populations in health and disease. The specificity of these markers was predicated on the assumption that circulating monocytes retain their distinct signatures after entering the central nervous system (CNS). Here, we challenge this paradigm. Using a combination of bone marrow chimeras, single-cell RNA sequencing, ATAC-seq, flow cytometry, and immunohistochemistry, we demonstrate that monocytes engrafting into the CNS acquire de novo expression of these established microglia markers. This phenotypic conversion is driven by profound epigenetic reprogramming, characterized by dynamic changes in chromatin accessibility at key gene loci, including P2ry12, Tmem119, and Aif1 (Iba1), and a shift in transcription factor binding motifs toward a microglial profile. We show this process occurs in the retina following injury and, remarkably, under physiological conditions in the brain and spinal cord, where blood-derived monocytes progressively contribute to the resident myeloid pool. Furthermore, engrafted monocytes downregulate canonical monocyte markers (Ly6C, CD45), eventually becoming indistinguishable from embryonic microglia based on conventional phenotyping. Our findings reveal that infiltrating monocytes undergo extensive epigenetic and transcriptional remodeling to adopt a microglia-like fate, challenging the specificity of current markers and necessitating a re-evaluation of the distinct roles of these two cell populations in CNS pathology.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17779506.
This preprint investigates the adaptations that brain-infiltrating peripheral monocytes undergo to phenotypically resemble resident microglia in the central nervous system (CNS). Using single-cell RNA sequencing, ATACseq, flow cytometry, immunohistochemistry, and qPCR in a busulfan-conditioned bone marrow chimera mouse model, the authors report that once infiltrated, monocytes progressively upregulate canonical "microglia-specific" markers such as P2RY12, TMEM119, FCRLS, and IBA1. Moreover, they undergo extensive chromatin remodeling at microglial gene loci, accompanied by enrichment of transcription factor motifs associated with microglial identity. The authors ultimately conclude that …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17779506.
This preprint investigates the adaptations that brain-infiltrating peripheral monocytes undergo to phenotypically resemble resident microglia in the central nervous system (CNS). Using single-cell RNA sequencing, ATACseq, flow cytometry, immunohistochemistry, and qPCR in a busulfan-conditioned bone marrow chimera mouse model, the authors report that once infiltrated, monocytes progressively upregulate canonical "microglia-specific" markers such as P2RY12, TMEM119, FCRLS, and IBA1. Moreover, they undergo extensive chromatin remodeling at microglial gene loci, accompanied by enrichment of transcription factor motifs associated with microglial identity. The authors ultimately conclude that infiltrating monocytes acquire transcriptional and epigenetic features that make them indistinguishable from embryonic microglia by conventional phenotyping, while maintaining potentially distinct inflammatory potential. This study is highly relevant to ongoing efforts to rigorously define both microglial identity and the roles of infiltrating monocytes in the CNS, particularly at a time when interest in their contribution to neuroinflammatory and neurodegenerative processes is rapidly expanding.
Strengths: The study is technically sophisticated, integrating complementary molecular, cellular, and epigenetic approaches that together provide a comprehensive view of monocyte-to-microglia reprogramming. The dataset provides valuable multiomic insight into the dynamic plasticity of CNS myeloid populations and meaningfully contributes to a field faced with the limitations of canonical markers.
Weaknesses: Some interpretative claims—particularly regarding the extent of "indistinguishability by conventional phenotyping" and the generalization of these findings to physiological contexts—would benefit either from additional supporting data or from language that more explicitly acknowledges the limitations of the experimental model. Clearer figure labeling and methodological detail would also improve transparency and strengthen alignment between data and conclusions.
Major Points
1. The evidence presented shows that infiltrating monocytes acquire many microglia-like molecular and epigenetic features, but the conclusion that they become fully "indistinguishable" from embryonic microglia by conventional phenotyping requires additional evidence. The current data suggest substantial convergence toward a microglial profile, yet also indicate retained specificity such as residual separation between day 0 and day 7 clusters (Fig. 1A), partial overlap rather than merging of CD45 expression (Fig. 1E), lower P2RY12 and TMEM119 levels in engrafted cells compared to resident microglia (Figs. 2–3, S2), persistence of CCR2⁺Ly6C⁺ subsets (Fig. 5), and distinct transcription factor motif enrichment (Fig. 6). Together, these examples suggest that infiltrating monocytes adopt many canonical microglial features while maintaining discrete molecular signatures, which influences how the results can be interpreted. It would be helpful for the authors to comment on this possibility and clarify whether they view these differences as biologically meaningful or within the expected range of phenotypic convergence. Quantifying the fraction of marker-positive cells across time points and assessing functional microglial behaviors (e.g., cytokine release) would further clarify the extent of convergence.
2. The authors suggest that monocyte infiltration and reprogramming occur under physiological conditions. However, the study design relies on busulfan-mediated myeloablation to label infiltrating monocytes, an approach that alters hematopoiesis, cytokine signaling, and blood–brain barrier integrity, potentially facilitating both monocyte entry and reprogramming. Without controls in unconditioned or lineage-traced animals, it remains uncertain whether the observed engraftment represents a physiological process or an artifact of conditioning. The authors should clarify the limitations of this approach and suggest follow-up studies using complementary models such as genetic lineage tracing in order to determine whether similar reprogramming occurs under physiological conditions.
3. ATAC-seq results clearly demonstrate increased accessibility at microglial gene loci (e.g., P2ry12, Tmem119, Ms4a7, C1qa) among infiltrating monocytes. However, without direct manipulation of key transcriptional regulators (e.g., PU.1, IRF8, MITF), the observed motif enrichment indicates only a correlation between the loci accessibility during monocyte reprogramming and transcription factor sequences associated with microglial identity. Without demonstrating TF binding or necessity, motif enrichment alone cannot establish mechanistic causation. Integrating ATAC-seq and scRNA-seq data, such as by linking accessibility peaks to differentially expressed genes or motif expression correlations, would connect chromatin remodeling to transcriptional outcomes more convincingly, and incorporating CUT&RUN, ChIP-seq, or perturbation experiments would further strengthen mechanistic inference. At minimum, the authors' claim that chromatin remodeling "drives" phenotypic convergence should be rephrased to emphasize association, and the discussion could be further strengthened by offering alternative interpretations such as chromatin remodeling functioning as a permissive or downstream adaptation to microenvironmental cues.
4. The discussion effectively highlights the implications of marker ambiguity. However, while the authors persuasively challenge the field's reliance on canonical markers, they stop short of leveraging their own data to propose distinguishing criteria or mechanistic frameworks for future studies. For example, sustained enrichment of MITF and NF-κB1 motifs and persistent accessibility of inflammatory gene loci could represent actionable distinguishing features. Emphasizing these differences would shift the discussion from identifying what existing approaches get wrong to demonstrating what the field can do right.
Minor Points
1. The manuscript alternates between "monocyte," "macrophage," "microglia-like," and "monocyte-derived microglia" when describing the same population. Consistent nomenclature would prevent confusion between lineage and phenotype.
2. Exact n values, statistical tests, and effect sizes are not consistently reported in figure legends. Including these for each panel would improve transparency and reproducibility.
3. In Figure 1, the caption does not define what the t-SNE plots in panels C–D represent or how they relate to the UMAP in 1A. Clarifying whether these reflect transcriptional clusters, marker expression, or sample time points would improve interpretability.
4. In Figure 4, motif analysis compares naïve microglia to engrafted macrophages but omits injury conditioned microglia as a control, making it difficult to separate monocyte-specific injury responses from those of the broader myeloid population. Including injury-conditioned microglia in the comparison, or explicitly clarifying why such a control was not incorporated, would help contextualize the motif differences.
5. In Figure 6, the caption could briefly clarify that this panel summarizes preceding findings rather than presenting new quantitative data, to ensure readers interpret it as a schematic overview.
6. The limitations section could be expanded beyond busulfan conditioning to address restricted sample sizes, potential phenotypic reversal at later timepoints, lack of functional validation, and the conflation of injury-related and physiological contexts.
Competing interests
The authors declare that they have no competing interests.
Use of Artificial Intelligence (AI)
The authors declare that they did not use generative AI to come up with new ideas for their review.
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eLife Assessment
The authors investigated the epigenetic mechanisms regulating the differentiation of circulating monocytes that infiltrate the CNS and adopt microglia-like characteristics. The work is useful to the field, as the contribution of circulating myeloid cell-derived microglia remains controversial. However, the evidence presented is inadequate as the analyses are based on a very limited set of genes, which does not sufficiently support the authors' central claims.
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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 …
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.
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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 …
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.
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