Engrafted peripheral CX3CR1+ cells remain reactive despite their morphometric quiescence. Engrafted peripheral CX3CR1+ cells remain reactive despite their.

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Engrafted peripheral CX3CR1+ cells remain reactive despite their morphometric quiescence. Engrafted peripheral CX3CR1+ cells remain reactive despite their morphometric quiescence. (A–D) Flow cytometric analysis of retinal cells from CX3CR1+/EGFP mice. (A) In naive mice, CX3CR1+ cells are predominantly CD45lo (96%), and only a small percentage is CD45hi (4%). CX3CR1+ CD45lo cells do not express MHC-II; however, 2.8% of CX3CR1+ CD45hi cells have baseline MHC-II expression. (B) Twenty weeks after ocular injury, 94% percent of the CX3CR1+ cells are CD45lo, 20% of which express MHC-II. Likewise, 3% of the CX3CR1+ cells are CD45hi, 72% of which express MHC-II. (C) Flow cytometry for the differentiation of yolk-sac–derived retinal microglia (YS-μGlia 16w) from peripherally engrafted bone-marrow CX3CR1+ monocytes (MB μG 16w) 16 wk after ocular injury using RFP-conjugated CX3CR1 antibody against bone-marrow–transferred CX3CR1+/EGFP retinal cells. Both cell populations are strongly expressed. (D) Flow cytometry and cell sorting of splenic CX3CR1+ cells (control), retinal yolk-sac–derived microglia, and retinal peripherally engrafted monocytes 16 wk after ocular injury for subsequent analysis of IL-1β and TNF-α expression with qPCR. (E) Compared with splenic CX3CR1+ cells, both yolk-sac–derived and peripheral microglia had elevated TNF-α and IL-1β gene expression. However, peripherally engrafted monocytes had significantly higher expression than yolk-sac–derived microglia 16 wk after injury. *P < 0.05; ****P < 0.0001; Tukey’s method for multiple comparisons. (F and G) Twenty weeks after the injury, peripheral CX3CR1+ monocytes appear to frequently interact with and engulf β3 tubulin+ neuroretinal tissue (F), despite their otherwise ramified and quiescent morphology (G). (H and I) In contrast, in naive eyes, although ramified CX3CR1+ monocytes come into contact with β3 tubulin+ neuronal tissue, they do not internalize it. (Scale bars: F–I, Upper, 50 μm; F–I, Lower, 20 μm.) All experiments were performed in triplicate. Eleftherios I. Paschalis et al. PNAS doi:10.1073/pnas.1807123115 ©2018 by National Academy of Sciences