Attenuated CSF-1R signalling drives cerebrovascular pathology (2020)
Figure 3

Figure 3. Peripheral Variant CSF-1R Macrophages Have Altered Identity and Function

A, B) FACS of control and ΔA781_N783 PBMCs using lineage markers for CD3 (T-cells), CD14 (monocytes/macrophages), CD19 (B-cells), CD16, CD20 and CD56. Cells negative for all 6 makers were determined to be lineage-negative (Lin-).

C) Immunocytochemistry of macrophages differentiated in vitro from control, ΔA781_N783 or P824R PBMCs. Cells were stained for DAPI (blue) and CD68 (red). White arrows indicate dysmorphic macrophages.

D) Immunocytochemistry of macrophages differentiated in vitro from control, ΔA781_N783 or P824R PBMCs. Macrophages were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker (red) and DAPI (blue).

E) Quantification of phagocytic activity expressed as percentage bead+ cells. P values were calculated using (** p = 0.008, * p = 0.0141, one-way ANOVA with Tukey's multiple comparison test, n = 4 assays, error bars indicate SEM).

F) Quantification of macrophages displaying an aberrant morphology as indicated in (c), displayed as percentage of dysmorphic cells per image (*** p < 0.0005, one-way ANOVA with Dunnet's multiple comparison test, data representative of n = 2 independent differentiations, with two fields of view imaged per well, error bars indicate SEM).

G) Western blot for CSF-1R in untreated (Unt) BV2 microglia, or BV2 microglia transfected with non-targeting or Csf-1r targeting siRNA. Corresponding densitometry (below) representative of technical replicate of n = 3 blots (One-way ANOVA with Dunnett's multiple comparison, *** p = 0.0003, error bars indicate SEM).

H) Immunocytochemistry of untreated and Csf-1r siRNA treated BV2 microglia. BV2 microglia were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker (red) and DAPI (blue). Arrowheads (white) indicate bead+ cells.

I) Quantification of BV2 phagocytic activity expressed as percentage bead+ cells (data representative of n = 2 independent differentiations, with two fields of view imaged per well, error bars indicate SEM).

J) Immunocytochemistry of microglia isolated from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ P0 mouse pup brains. Microglia were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker(red). Arrowheads (white) indicate bead+ cells.

K) Quantification of mouse microglial phagocytic activity expressed as percentage bead+ cells (n = 4 assays, error bars indicate SEM).

L) Immunocytochemistry of macrophages differentiated in vitro from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mouse bone marrow. Macrophages were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker (red) and DAPI (blue). Arrowheads (white) indicate bead+ cells.

M) Quantification of mouse macrophage phagocytic activity expressed as percentage bead+ cells. (* p = 0.024, Student's t-test with Welch's correction, n = 4 assays, error bars indicate SEM).