Fig 2: CX3CL1/CX3CR1 signal mediates hypoxia-induced-M1 type microglia. Primary microglia were treated with 1% O2 for 20 h followed by the co-treatment with 50 ng/ml CX3CL1 for 4 h. (A–D) Western blot detection of CD16, CD206, and IL-1β levels in microglia after hypoxia and CX3CL1 treatment (A), and the grayscale values of each band were counted (B–D), n = 3. (E–H) Primary microglia generated from wild-type (WT) or CX3CR1-deficient (KO) mice were treated with 1% O2 for 24 h. The cells were lysed to determine CD16, CD206, and IL-1β levels by Western blot, n = 3. (I–L) Primary KO microglia were treated with 1% O2 for 20 h followed by the co-treatment of 50 ng/ml CX3CL1 for 4 h. Cells were lysed to detect CD16, CD206, and IL-1β levels by Western blot, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, and n.s. indicates no statistical difference.

Fig 3: Treadmill training inhibits osteoclast formation by suppressing CX3CL1 secretion: (A, B) Cytokine microarray analysis technique to detect cytokine secretion levels in the tendon-bone insertion; (C) The ZDOCK score values and best pose interaction of CX3CL1 and CX3CR1. (D, E). PCR analysis of gene expression levels of CX3CL1 and CX3CR1 in the tendon insertion. (F, G) Osteoclast induction of primary monocyte-macrophages with the addition of CX3CL1 recombinant protein or CX3CR1 antagonist protein (AZD8797) and TRAP staining of culture results and counting of positive cells. Data are shown as the mean ± SEM and n = 6 per group per experiment. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test with ****P < 0.0001.

Fig 4: Hypobaric hypoxia (HH) exposure induces CX3CL1/CX3CR1 signal activation and M1-type polarization of hippocampal microglia. (A) Immunofluorescence labeling of microglia/macrophage marker Iba1 in CA1, Scale bar = 100 μm. (B–G) Statistics of panel (A), including the number of Iba1+ cells [(B), n = 5], circularity [(C), n = 118, brain sections from 5 mice of each group were stained, and 23–24 cells of each mouse were quantified], soma area [(D), n = 61 or 68, brain sections from 5 mice of each group were stained, and 12–13 cells of each mouse were quantified], MI [(E), MI = soma area/arborization area, n = 62 or 64, brain sections from 5 mice of each group were stained, and 13–14 cells in CA1 region of each mouse were quantified] and number of branches [(F,G), n = 45, brain sections from 5 mice of each group were stained, and 9 cells of each mouse were quantified]. (H) Immunofluorescence labeling of microglia/macrophage marker Iba1 in CA1 region of CX3CR1-GFP mice, Scale bar = 100 μm. (I) Statistics of the ratio of Iba1+GFP+ cells in GFP + cells in panel (H), n = 4. (J,K) Western blot detection of CD206, CD16, and IL-1β levels in mouse hippocampus, n = 6. (L) qRT-PCR detection of the expression of inflammatory factors Il1b, Il6, Tnfa, and Tgfb in hippocampal tissue, n = 5. (M) qRT-PCR detection of Cx3cl1 and Cxcr1 expressions in hippocampal tissues, n = 6. *p < 0.05, **p < 0.01, ***p < 0.001, and n.s. indicates no statistical difference.

Fig 5: CX3CL1/CX3CR1 signal mediates hypoxia-induced phagocytosis of synapses by microglia. (A) Primary neurons co-cultured with microglia were treated with 1% O2 for 24 h. Iba1 and MAP2 with PSD95 were labeled by Immunofluorescence. (B) CX3CR1-deficient (KO) microglia were treated with 1% O2 for 24 h and incubated with dissociated synapses for 30 min. Immunofluorescence labeling of Iba1 and Synaptophysin. (C) Primary microglia were treated with 1% O2 for 20 h followed by the co-treatment of CX3CL1 for 4 h and incubated with dissociated synapses for 30 min. Immunofluorescence labeling of Iba1 and Synaptophysin. (D) WT and KO microglia were treated with 1% O2 for 24 h. WT microglia were treated with 1% O2 for 20 h followed by CX3CL1 co-treatment for 4 h. Then, the cells were incubated with TRITC-Dextran 40 kDa for 30 min, and confocal images were recorded. (E) Statistics of the total fluorescence intensity of PSD95 in Iba1+ microglia in panel (A), n = 18. (F) Statistics of the total fluorescence intensity of Synaptophysin in Iba1+ microglia in panel (B), n = 80. (G) Statistics of the total fluorescence intensity of Synaptophysin in Iba1+ microglia in panel (C), n = 55–78. (H) Statistics of the total fluorescence intensity of TRITC-Dextran in single cells in panel (D), n = 85–100. Scale bar = 20 μm, *p < 0.05, **p < 0.01, ***p < 0.001, and n.s. indicates no statistical difference.