Background: Ischemic stroke initiates a complex inflammatory cascade that contributes to both tissue repair and secondary injury, driven largely by resident microglia and monocyte-derived cells (MdCs). Microglia rapidly activate while recruited monocytes differentiate into diverse MdC subsets that can either promote recovery or exacerbate damage. The nuclear receptor NR4A1 (Nur77) has been identified as a key regulator of myeloid cell fate and function, but its role in post-stroke neuroinflammation remains unclear. Methods: To delineate the temporal dynamics of microglial and MdC responses following transient middle cerebral artery occlusion (tMCAO), we developed a novel dual-reporter mouse model and innovative tracking methods. This model uses adoptive bone marrow transfer and Cx3cr1- and Tmem119-cre drivers to discriminate between MdCs (Fig. 1A, green) and microglia (Fig. 1A, red) in real-time. Proliferating cells were pulse-labeled with 5-ethynyl-2’-deoxyuridine (EdU), which allows for longitudinal tracking of monocyte recruitment in addition to MdC and microglial expansion. EdU incorporation and NR4A1 protein expression were quantified with flow cytometry and population distributions were quantified using fluorescence microscopy. The effect of NR4A1 in microglia was interrogated using knockout (KO) recipients. Results: The analysis of EdU enrichment shows that MdCs are primarily derived from recently matured monocytes (Fig. 1B). Microglial proliferation was variable but correlated strongly with EdU+ MdC abundance, suggesting coordinated expansion. NR4A1 levels correlated strongly with the expansion of both MdCs and microglia. Deletion of Nr4a1 in microglia led to three times the MdC numbers compared to wild-type controls, supporting the role of NR4A1 in regulating MdC expansion (Fig. 1C). Taken together, these preliminary data suggest that NR4A1 deletion in microglia directly affects MdC recruitment and/or expansion. Conclusions: Our findings reveal a tightly regulated relationship between microglial and MdC expansion following ischemic stroke, with NR4A1 acting as a critical modulator of both cell types. NR4A1 appears to drive early MdC expansion while enhancing microglia expansion at later time points. Future studies will further investigate NR4A1’s cell-type-specific roles and explore its feasibility as a therapeutic target.
Lipfert et al. (Thu,) studied this question.