Fig 2: Effect of macrophage (M?) depletion on corneal nerve regeneration. A: Anti–ß-III tubulin NL557 and CD64 fluorescein isothiocyanate antibody costaining of cornea. B: RT-PCR evaluation of flow cytometry–sorted CCR2- and CCR2+ corneal macrophages for the expression of neurotrophin genes. C and E: The change of neurotrophin gene expression in corneal tissues at 8 days after epithelial abrasion. CCR2- corneal macrophages were depleted by subconjunctival injection of anti–colony stimulating factor 1 receptor (CSF1R) antibody, and CCR2+ corneal macrophages were depleted by i.p. injection of CCR2 antagonist BMS CCR2 22. D and F: Change in the density of subbasal nerves at 8 days after epithelial abrasion in mice depleted of CCR2- or CCR2+ macrophages. G and H: The change in corneal sensitivity after corneal epithelial abrasion in mice depleted of CCR2- or CCR2+ macrophages. Data are expressed as means ± SD. n = 3 independent experiments [10 mice per experiment (B) and 6 mice per experiment (C and E)]; n = 6 mice in each group (D and F–H). *P < 0.05, **P < 0.01. Scale bar = 100 µm (A).

Fig 3: Isotype controls for justifying the gating strategies. A: Isotype controls for antibodies [CD45–fluorescein isothiocyanate (FITC), CD64-BV421, and CCR2–allophycocyanin (APC)] used in Figure 4A. B: Isotype controls for antibodies [Ly6G-FITC and Ly6C–phosphatidylethanolamine (PE)–Cy7] used in Supplemental Figure S4A. SSC, side scatter.

Fig 4: CX3CL1-Fc reduces atherosclerotic lesions in a prevention model of atherosclerosis. (A) Circulating CX3CL1 levels after one injection of CX3CL1-Fc (30 mg/kg) in Ldlr KO mice. After injection, CX3CL1 levels were rapidly increased with stabilization at ~2 nmol/L after 24 h (n = 4). (B) A schematic of prevention study design. CX3CL1-Fc/VEH treatment was started when mice were provided the high-fat/high-cholesterol Western Diet (WD) for 4 months. (C–F) Serum CX3CL1 and lipid content at sacrifice. (C) CX3CL1-Fc-treated mice have ~14 times more serum CX3CL1 in their blood (n = 7–10). Body weight (D), serum cholesterol (E), FPLC cholesterol profile of Ldlr KO mice after 4 months of WD treated with CX3CL1-Fc/VEH (n = 13). (G) En face analysis of total aorta. Percent Sudan IV positive staining over total aortic area in Ldlr KO mice treated with CX3CL1-Fc/VEH, representative images are shown (n = 4–7). Cross-sectional aortic root analyses of lesion size (H) and necrotic area within lesions (I), representative images are shown to the right (n = 4–7). AUC, area under the curve. A.U., arbitrary unit. (J–M) Flow cytometry analysis of aortic cell digests showed no difference the in percentage of macrophages of CD45+ Live cells (J). However, there was a decrease in CD86+ M1-like polarized macrophages (K) and no change in CD206+ M2-like polarized macrophages (L) as a percent of total macrophages (CD64+ CD11b+) in mice treated with CX3CL1-Fc (n = 9). Also, a decrease in CD3+ T cells (M) as a percent of CD45+ Live cells was observed in aortic cell digests of CX3CL1-Fc treated mice (n = 9). Data are presented as mean ± SEM. #p < 0.05 (one-tailed t-test). For all panels, values are mean ± SD. *p < 0.05 and ***p < 0.001.

Fig 5: DNGR-1 lineage tracing marks non-cDCs lining the brain ventricular system and the spinal cord central canal(A) Brain cryosection from a Clec9aCreRosaLSLtdTomato animal stained with antibodies against IBA-1 (green), MHC II (white); zooms: (Ai) choroid plexus (Aii) ventricle wall. Arrowheads indicate DNGR-1-traced cells (red) in the ventricle wall lacking IBA-1 and MHC II staining.(B) Brain coronal cuts showing DNGR-1-traced cells (red, arrowheads) in the ventricle walls of all CSF-filled compartments. No DNGR-1-traced cells (red) were observed in olfactory bulbs or dentate gyrus. OB, olfactory bulb; LV, lateral ventricle; TV, third ventricle; DG, dentate gyrus; Aq, aqueduct; FV, fourth ventricle.(C) Multiphoton image of vibratome spinal cord sections showing DNGR-1-traced cells (red, arrowheads) around the central canal in different rostral-caudal levels. Second harmonic signal, blue.(D) Spinal cord cryosections labeled with antibodies against MHC II, CD64, or CD45 (all green).(E and F) Optically clarified (E) and light-sheet imaged (F) whole-CNS with DNGR-1-traced cells (red) lining the entire ventricular compartment. Bottom, zoomed view of indicated areas (Fi) brain (Fii) spinal cord.Scale bars, 5 mm (E), 1 mm (F), 100 µm (A–D and zoom F), and 20 µm (zooms A and C). Sections from at least 3 animals were analyzed per experiment.