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

Kai Zhou; Jinming Han; Harald Lund; Nageswara Rao Boggavarapu; Volker M Lauschke; Shinobu Goto; Huaitao Cheng; Yuyu Wang; Asuka Tachi; Cuicui Xie; Keying Zhu; Ying Sun; Ahmed M Osman; Dong Liang; Wei Han; Kristina Gemzell-Danielsson; Christer Betsholtz; Xing-Mei Zhang; Changlian Zhu; Martin Enge; Bertrand Joseph; Robert A Harris; Klas Blomgren

doi:10.1186/s12974-022-02381-6

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

J Neuroinflammation. 2022 Jan 21;19(1):20. doi: 10.1186/s12974-022-02381-6.

Authors

Kai Zhou^{1

2}, Jinming Han^{3

4}, Harald Lund^{3

5}, Nageswara Rao Boggavarapu⁶, Volker M Lauschke^{5

7

8}, Shinobu Goto^{6

9}, Huaitao Cheng¹⁰, Yuyu Wang⁶, Asuka Tachi^{6

11}, Cuicui Xie⁶, Keying Zhu³, Ying Sun¹², Ahmed M Osman⁶, Dong Liang⁶, Wei Han⁶, Kristina Gemzell-Danielsson^{6

13}, Christer Betsholtz^{12

14}, Xing-Mei Zhang³, Changlian Zhu^{15

16}, Martin Enge¹⁰, Bertrand Joseph¹⁷, Robert A Harris³, Klas Blomgren^{18

19

20}

Affiliations

¹ Henan Neurodevelopment Engineering Research Center for Children, Children's Hospital Affiliated to Zhengzhou University, Zhengzhou, China. kai.zhou@ki.se.
² Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden. kai.zhou@ki.se.
³ Applied Immunology and Immunotherapy, Department of Clinical Neuroscience, Karolinska Institutet, Center for Molecular Medicine, Karolinska University Hospital, Stockholm, Sweden.
⁴ Neuroimmunology Center, Department of Neurology, Xuanwu Hospital, Capital Medical University, National Center for Neurological Disorders, Beijing, China.
⁵ Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
⁶ Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden.
⁷ Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
⁸ University of Tuebingen, Tuebingen, Germany.
⁹ Department of Obstetrics and Gynecology, Nagoya City University Hospital, Nagoya, Japan.
¹⁰ Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden.
¹¹ Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
¹² Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
¹³ WHO-Centre, Karolinska University Hospital, Stockholm, Sweden.
¹⁴ Department of Medicine Huddinge, Karolinska Institutet, Campus Flemingsberg, Huddinge, Sweden.
¹⁵ Centre for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden.
¹⁶ Henan Key Laboratory of Child Brain Injury and Henan Pediatric Clinical Research Center, The Third Affiliated Hospital and Institute of Neuroscience, Zhengzhou University, Zhengzhou, China.
¹⁷ Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
¹⁸ Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden. klas.blomgren@ki.se.
¹⁹ Henan Key Laboratory of Child Brain Injury and Henan Pediatric Clinical Research Center, The Third Affiliated Hospital and Institute of Neuroscience, Zhengzhou University, Zhengzhou, China. klas.blomgren@ki.se.
²⁰ Pediatric Oncology, Karolinska University Hospital, Stockholm, Sweden. klas.blomgren@ki.se.

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.

Keywords: Cre; Cx3cr1; Diphtheria toxin subunit A (DTA); GFP; Homologous recombination; Loss of heterozygosity (LOH); Microglia; Microglial depletion; Microglial repopulation; YFP.

MeSH terms

Animals
CX3C Chemokine Receptor 1 / genetics
CX3C Chemokine Receptor 1 / metabolism
Mice
Mice, Transgenic
Microglia* / metabolism
Signal Transduction*

Substances

CX3C Chemokine Receptor 1
Cx3cr1 protein, mouse

Abstract

MeSH terms

Substances

Grants and funding