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. 2017 Nov 11;82(5):813–827. doi: 10.1002/ana.25085

Figure 4.

Figure 4

Abcd1–/– microglia lack an inflammatory profile, but displayed increased phagocytosis of phosphatidylserine (PS) exposing apoptotic neurons. (A) Proinflammatory genes including IL1b, TNF, NOS2, and Cox2 did not show distinct alterations between wild‐type and Abcd1–/– microglia at basal level, whereas lipopolysaccharide (LPS) treatment for 6 hours dramatically increased expression of these genes in both wild‐type and Abcd1–/– microglia with a greater response from Abcd1–/– cells. (B) The microglial markers, IBA1 and CD11b, showed some increased expression at basal level. (C) Phagocytosis‐related genes including Trem2, MFGE8, Gas6, and Bai1, but not C3 and C1qa, were upregulated in Abcd1–/– mouse microglia at both unstimulated and LPS‐challenged conditions, with MFGE8 showing the most significant increase. LPS challenge decreased the phagocytosis‐related genes except for C3. (D) ELISA of microglia culture medium confirmed higher MFGE8 expression in Abcd1–/– microglia at both basal and LPS‐treated conditions. (E) Representative images show Abcd1–/– microglial (green) phagocytosis of PS‐exposed SH‐Sy5y neurons (CMTPX labeled red, indicated by white arrow). (F) Quantification of red granules within IBA1‐stained microglia (green) shows a trend to increased phagocytosis of PS exposing SH‐SY5Y neuron in Abcd1–/– microglia at both basal and LPS‐stimulated conditions. (G,H) Supplementation of recombinant MFGE8 dramatically increased Abcd1–/– microglial phagocytosis of PS exposing neurons. (I) MFGE8 blocking antibody treatment reduced Abcd1–/– microglia phagocytosis of PS exposing neurons. Bar = 50 µm. Data were expressed as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001. ELISA = enzyme‐linked immunosorbent assay; IBA1 = ionizing calcium‐binding adaptor molecule 1; IgG = immunoglobulin G; SEM = standard error of the mean; WT = wild type.