Fig. 1
From: Splenic monocytes drive pathogenic subretinal inflammation in age-related macular degeneration
ANGII mobilizes spleMos and promotes subretinal inflammation spleen dependently. A RT-PCR results of Agtr1a mRNA normalized with Rps26 mRNA of fresh FACS-sorted Ly6Chigh bone marrow monocytes (BMMos), Ly6Chigh spleen monocytes (SpleMos), and retinal microglial cells, as well as freshly homogenized retina, RPE/choroid, and kidney (n = 5, samples from three independent experiments). B Representative image of an RNAscope in situ hybridization of Itgam mRNA (CD11b) (red) and Agtr1a mRNA (green) of a kidney (positive control) and spleen cryosection. Three independent experiments gave similar results. Scale bar = 1 mm. C Representative image of a CD11b (red) and Ly6C (green) double-immuno-labeled spleen cryosection. Scale bar = 1 mm. D, E Quantification by flow cytometry of CD11b+Ly6Chigh cells in the spleen (D) and in the BM (E) 3 h after PBS or ANGII intraperitoneal injections (n = 10; Mann–Whitney test, D PBS vs ANGII: *p = 0.0434). F Representative images of IBA1 (green, MP marker) and CD102 (red) immunostained RPE flatmounts 7 days after laser-injury of sham-operated or splenectomized (− 30 days) mice that were transplanted with subcutaneous pumps (− 3 day) filled with PBS or ANGII (1 µg/kg/min), as indicated in the cartoon. Quantification of IBA1+ MPs surrounding laser impacts and CD102+ CNV area (n = 5–6; one-way ANOVA, PBS Sham vs ANGII Sham: MPs *p = 0.0495; ANGII Sham vs ANGII splenectomy: MPs #p = 0.0023, CD102+ area #p = 0.0124). Scale bar = 20 µm. G Representative images of IBA1 (green) and CD102 (red) immunostained RPE flatmounts 7 days after laser impact of mice treated by intraperitoneal injection of PBS or ATR1 antagonist losartan (10 mg/kg) as indicated. Quantification of IBA1+ MPs surrounding laser impacts and CD102+ CNV area of these mice. (n = 8; Mann–Whitney test, PBS vs Losartan, MPs *p = 0.0172; CD102+ area *p < 0.0001). Scale bar = 20 µm. RPE: retinal pigment epithelium; BMMo: bone marrow monocyte; spleMo: splenic monocyte; IP injection: intraperitoneal injection; BM: bone marrow; ANGII: angiotensin II; ATR1: angiotensin II receptor type 1