An overlooked subset of Cx3cr1wt/wt microglia in the Cx3cr1CreER-Eyfp/wt mouse has a repopulation advantage over Cx3cr1CreER-Eyfp/wt microglia following microglial depletion
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ABSTRACT:
PROVIDER: PRJNA775631 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16599306.fastq.gz | Fastqsanger.gz | |||
| SRR16599307.fastq.gz | Fastqsanger.gz | |||
| SRR16599308.fastq.gz | Fastqsanger.gz | |||
| SRR16599309.fastq.gz | Fastqsanger.gz | |||
| SRR16599310.fastq.gz | Fastqsanger.gz |
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Project description:Cx3cr1CreER-Eyfp/wt mice contain a subset of microglia lacking Cre and EYFP expression. These microglial escape Cre-mediated recombination and gain a repopulation advantage following Cre-driven DTA-mediated microglial depletion.
2022-02-01 | GSE186700 | GEO
Project description:BackgroundFluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cellular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1CreER-Eyfp/wt mouse strain for studies of microglia.MethodsImmunohistochemistry, Flow Cytometry, RNA sequencing and whole-genome sequencing were used to identify the subpopulation of microglia in Cx3cr1CreER-Eyfp/wt mouse brains. Genetically mediated microglia depletion using Cx3cr1CreER-Eyfp/wtRosa26DTA/wt mice and CSF1 receptor inhibitor PLX3397 were used to deplete microglia. Primary microglia proliferation and migration assay were used for in vitro studies.ResultsWe unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1 expression than Cx3cr1CreER-Eyfp/wtCre+Eyfp+ microglia in Cx3cr1CreER-Eyfp/wt mouse brains, thus termed Cx3cr1highCre-Eyfp- microglia. This subpopulation constituted less than 1% of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1highCre-Eyfp- microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further demonstrated that the Cx3cr1highCre-Eyfp- microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1highCre-Eyfp- microglia are Cx3cr1wt/wtCre-Eyfp-. Finally, we demonstrated that CX3CL1-CX3CR1 signaling regulates microglial repopulation both in vivo and in vitro.ConclusionsOur results raise a cautionary note regarding the use of Cx3cr1CreER-Eyfp/wt mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies.
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Project description:Microglia are crucial for CNS homeostasis and involved in a wide range of neurodegenerative and neuroinflammatory diseases. Systemic inflammation and infections can contribute to neurodegeneration later in life by affecting microglia. Like other innate immune cells, microglia can develop innate immune memory in response to a challenge, altering their response to future stimuli. Innate immune memory can ameliorate or worsen CNS pathology, but its persistence is unclear. Recently, several methods have been developed to stimulate microglia turnover. Here, we investigated whether colony-stimulating factor 1 (CSF1R)-dependent microglia depletion followed by repopulation reversed microglial tolerance to systemic inflammation in mice. Repopulated microglia displayed a reduced expression of homeostatic genes and genes related to mitochondrial respiration and TCA cycle metabolism and an increased expression of immune effector and activation genes. Nonetheless, the blunted inflammatory gene expression induced by the LPS-preconditioning was retained after a depletion-repopulation cycle. Our study highlights the persistence of epigenetic changes underlying microglial tolerance and indicates potential implications of microglia depletion and repopulation therapies on microglia functions.
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Project description:Although most tissue macrophages are embryonically derived it is evident that circulating monocytes can compete for virtually any macrophage niche. Monocytes can thus become long-lived replacements of tissue macrophages that are indistinguishable from their embryonic counterparts, but the factors regulating this process are incompletely understood. To study niche competition in the CNS we depleted microglia with >95% efficiency using CX3CR1CreER/+R26DTA/+ mice and monitored long-term repopulation. The microglial niche was repopulated within weeks by a combination of local microglia proliferation giving rise to CX3CR1+F4/80lowClec12a– microglia, as well as infiltration of CX3CR1+F4/80hiClec12a+ macrophages. Adoptive transfer experiments demonstrated that peripherally-derived macrophages arose directly from Ly6Chi monocytes without contribution from hematopoietic progenitors and was independent of BBB breakdown. After repopulation we sorted microglia and monocyte-derived macrophages and performed transcriptional, epigenetic (DNA methylation) and ex vivo functional profiling. This revealed that Ly6Chi monocytes upregulated microglia gene expression and adopted microglia DNA methylation signatures. However, in contrast to proliferating microglia, which rapidly regained their homeostatic gene signature, monocyte-derived macrophages retained a distinct gene signature associated with antigen presentation, interferon signaling and chemotaxis. This translated into functional changes in monocyte-derived macrophages, as demonstrated by differences in surface marker expression, phagocytosis and cytokine production. Our results demonstrate that monocytes are imprinted by the CNS microenvironment but remain transcriptionally, epigenetically and functionally distinct. This may have implications for neuroinflammatory disease states and direct design of novel therapies.
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Project description:In this study, we aimed to deplete the aging microglia and investigate the impact of the newly-born microglia in Alzheimer's pathology using the 3xTg mouse model. Specifically, >24 mo male 3xTg mice were given chow containing 1200mg/kg PLX5622 (AIN-76A-D1001i, Research Diests, NJ, USA) for 2 weeks to deplete their microglia. Control diet with the same base formula were given to control group and to experimental groups during a 4-week repopulation phase immediately following PLX treatment. At the end of the repopulation, we pooled sorted, viable microglia (CD45int CD11b) from 3-5 mice/group and ran single-cell RNAseq analysis to determine the similarities and differences between the aged and repopulated cells in the context of Alzheimer's Disease. We also included non-transgenic controls as a separate group.
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