Dataset Information

An overlooked subset of Cx3cr1^wt/wt microglia in the Cx3cr1^{CreER-Eyfp/wt} mouse has a repopulation advantage over Cx3cr1^{CreER-Eyfp/wt} microglia following microglial depletion.

ABSTRACT:

Background

Fluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cellular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1^{CreER-Eyfp/wt} mouse strain for studies of microglia.

Methods

Immunohistochemistry, Flow Cytometry, RNA sequencing and whole-genome sequencing were used to identify the subpopulation of microglia in Cx3cr1^{CreER-Eyfp/wt} mouse brains. Genetically mediated microglia depletion using Cx3cr1^{CreER-Eyfp/wt}Rosa26^DTA/wt mice and CSF1 receptor inhibitor PLX3397 were used to deplete microglia. Primary microglia proliferation and migration assay were used for in vitro studies.

Results

We unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1 expression than Cx3cr1^{CreER-Eyfp/wt}Cre⁺Eyfp⁺ microglia in Cx3cr1^{CreER-Eyfp/wt} mouse brains, thus termed Cx3cr1^highCre^-Eyfp^- microglia. This subpopulation constituted less than 1% of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1^highCre^-Eyfp^- microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further demonstrated that the Cx3cr1^highCre^-Eyfp^- microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1^highCre^-Eyfp^- microglia are Cx3cr1^wt/wtCre^-Eyfp^-. Finally, we demonstrated that CX3CL1-CX3CR1 signaling regulates microglial repopulation both in vivo and in vitro.

Conclusions

Our results raise a cautionary note regarding the use of Cx3cr1^{CreER-Eyfp/wt} mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies.

SUBMITTER: Zhou K

PROVIDER: S-EPMC8783445 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Diabetic retinopathy, a microvascular disease characterized by irreparable vascular damage, neurodegeneration and neuroinflammation, is a leading complication of diabetes mellitus. There is no cure for DR, and medical interventions marginally slow the progression of disease. Microglia-mediated inflammation in the diabetic retina is regulated via CX3CR1-FKN signaling, where FKN serves as a calming signal for microglial activation in several neuroinflammatory models. Polymorphic variants of CX3CR1, hCX3CR1I249/M280 , found in 25% of the human population, result in a receptor with lower binding affinity for FKN. Furthermore, disrupted CX3CR1-FKN signaling in CX3CR1-KO and FKN-KO mice leads to exacerbated microglial activation, robust neuronal cell loss and substantial vascular damage in the diabetic retina. Thus, studies to characterize the effects of hCX3CR1I249/M280 -expression in microglia-mediated inflammation in the diseased retina are relevant to identify mechanisms by which microglia contribute to disease progression. Our results show that hCX3CR1I249/M280 mice are significantly more susceptible to microgliosis and production of Cxcl10 and TNFα under acute inflammatory conditions. Inflammation is exacerbated under diabetic conditions and coincides with robust neuronal loss in comparison to CX3CR1-WT mice. Therefore, to further investigate the role of hCX3CR1I249/M280 -expression in microglial responses, we pharmacologically depleted microglia using PLX-5622, a CSF-1R antagonist. PLX-5622 treatment led to a robust (~70%) reduction in Iba1+ microglia in all non-diabetic and diabetic mice. CSF-1R antagonism in diabetic CX3CR1-WT prevented TUJ1+ axonal loss, angiogenesis and fibrinogen deposition. In contrast, PLX-5622 microglia depletion in CX3CR1-KO and hCX3CR1I249/M280 mice did not alleviate TUJ1+ axonal loss or angiogenesis. Interestingly, PLX-5622 treatment reduced fibrinogen deposition in CX3CR1-KO mice but not in hCX3CR1I249/M280 mice, suggesting that hCX3CR1I249/M280 expressing microglia influences vascular pathology differently compared to CX3CR1-KO microglia. Currently CX3CR1-KO mice are the most commonly used strain to investigate CX3CR1-FKN signaling effects on microglia-mediated inflammation and the results in this study indicate that hCX3CR1I249/M280 receptor variants may serve as a complementary model to study dysregulated CX3CR1-FKN signaling. In summary, the protective effects of microglia depletion is CX3CR1-dependent as microglia depletion in CX3CR1-KO and hCX3CR1I249/M280 mice did not alleviate retinal degeneration nor microglial morphological activation as observed in CX3CR1-WT mice.

Dataset Information

An overlooked subset of Cx3cr1^wt/wt microglia in the Cx3cr1^{CreER-Eyfp/wt} mouse has a repopulation advantage over Cx3cr1^{CreER-Eyfp/wt} microglia following microglial depletion.

Background

Methods

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

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.

Background

Methods

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

An overlooked subset of Cx3cr1^wt/wt microglia in the Cx3cr1^{CreER-Eyfp/wt} mouse has a repopulation advantage over Cx3cr1^{CreER-Eyfp/wt} microglia following microglial depletion.