Additional file 1 of Fractalkine isoforms differentially regulate microglia-mediated inflammation and enhance visual function in the diabetic retina
Creators: name: Rodriguez, Derek
name: Church, Kaira A.
name: Pietramale, Alicia N.
name: Cardona, Sandra M.
name: Vanegas, Difernando
name: Rorex, Colin
name: Leary, Micah C.
name: Muzzio, Isabel A.
name: Nash, Kevin R.
name: Cardona, Astrid E.
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Cell Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Virology

description: Additional file 1: Fig. S1. rAAV transduction occurs in neurons, without altering peripheral blood immune cell distributions. (A) Schematic presentation of vertical cross section of retinal layers with positions of vascular plexuses. (B) Gating strategy to identify CD45HiCD11b+SSCHi neutrophils, CD45HiCD11b+Ly6C+ inflammatory macrophages, CD45HiCD11b+Ly6C– tissue resident macrophages, CD45HiCD11b–CD11c+ conventional dendritic cells, and CD45HiCD11b+CD11c+ myeloid-derived dendritic cells in blood leukocytes. (C–G) Graphical representation of flow cytometric quantification of CD45HiCD11b+SSCHi neutrophils (C), CD45HiCD11b+Ly6C– tissue-resident macrophages (D), CD45HiCD11b+Ly6C+ inflammatory macrophages (E), CD45HiCD11b–CD11c+ conventional dendritic cells (F), CD45HiCD11b+CD11c+ myeloid-derived dendritic cells (G). Data are shown as mean ± SD, n = 4–6 mice per group, where each data point represents an individual mouse across five experiments. *p &lt; 0.05, **p &lt; 0.01, and ****p &lt; 0.0001 using Student’s t test, Welch’s correction. Fig. S2. Intra-vitreal administration of rAAV does not further alter splenic immune cell distributions. (A) Gating strategy of splenocytes to identify CD45HiCD11b+Ly6G+ granulocytes, CD45HiCD11b+CD11c–Ly6C+ inflammatory macrophages with respective MHC-II antigen presentation, CD45HiCD11b+CD11c–Ly6C– tissue-resident macrophages, CD45HiCD11b–CD11c+ conventional dendritic cells and CD45HiCD11b+CD11c+ myeloid-derived dendritic cells (with respective MHC-II antigen presentation), CD45HiCD11b–CD3+CD4+ T cells, CD45HiCD11b–CD3+CD4+CD44+ activated T cells, CD45HiCD11b–CD3+CD4+CD25+ T regulatory cells, CD45HiCD11b–CD3+CD8+ T cells, CD45HiCD11b–CD3+CD8+CD44+ activated T cells, and CD45HiCD11b–CD3+CD8+CD25+ T regulatory cells. (B–O) Graphical representation of flow cytometric quantification of CD45HiCD11b+Ly6G+ granulocytes (B), CD45HiCD11b+CD11c–Ly6C+ inflammatory macrophages (C), CD45HiCD11b+CD11c–Ly6C+MHCII+ inflammatory macrophages (D), CD45HiCD11b+CD11c–Ly6C– tissue-resident macrophages (E), CD45HiCD11b–CD11c+ conventional dendritic cells (F), CD45HiCD11b–CD11c+MHCII+ activated conventional dendritic cells (G), CD45HiCD11b+CD11c+ myeloid-derived dendritic cells (H), CD45HiCD11b+CD11c+MHCII+ activated myeloid-derived dendritic cells (I), CD45HiCD11b–CD3+CD4+ T cells (J), CD45HiCD11b–CD3+CD4+CD44+ activated T cells (K), CD45HiCD11b–CD3+CD4+CD25+ T regulatory cells (L), CD45HiCD11b–CD3+CD8+ T cells (M), CD45HiCD11b–CD3+CD8+CD44+ activated T cells (N), CD45HiCD11b–CD3+CD8+CD25+ T regulatory cells (O). Data shown as mean ± SD, n = 5 mice per group, where each data point represents an individual mouse across two experimental replicates. *p &lt; 0.05, **p &lt; 0.01, and ****p &lt; 0.0001 using Student’s t test, Welch’s correction. Fig. S3. Non-diabetic WT retinas display higher neuronal densities compared to FKNKO tissues. (A) Confocal images of retinal tissues stained for NeuN (red) and Iba1 (green) (left panel), with representation of cellular tracing for transformation index analysis (Inset). Confocal images of retinal tissues stained for CD31 (red) and fibrinogen (white) (right panel) (Scale bar; 100 µm). (B–E) Quantification of retinal immunofluorescence analysis of NeuN+ cells/mm3 (B) and Iba1+ cells/mm3 (C), immunofluorescence analysis of CD31+ percent immunoreactive area (% IRA) (D) and fibrinogen+ percent immunoreactive area (% IRA) (E). Data shown as mean ± SD, n = 4–11 mice per group, where each dot represents an individual mouse across six experiments. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001 and ****p &lt; 0.0001 using Student’s t test, Welch’s correction. Fig. S4. Intra-vitreal injection of rAAV–sFKN prevented axonal, SYP + loss, and astrogliosis in the diabetic optic nerve. (A) Confocal images of ND and diabetic optic nerves stained for visualization of axons (TUJ1, teal), synaptophysin (SYP, green), and astrocytes (GFAP, magenta) (Scale bar; 75 µm). (B–D) Quantification of the positive immunoreactive area (% IRA) for TUJ1+ (B), SYP+ (C), and GFAP+ (D). Data shown as mean ± SD, n = 4–6 mice per group, each data point represents an individual mouse across four experiments. *p &lt; 0.05, **p &lt; 0.01, and ****p &lt; 0.0001 using Student’s t test, Welch’s correction. Fig. S5. Treatment with rAAV–sFKN shifts the microglial transcriptomic profile to a homeostatic state. (A) Gating strategy to identify CD11b+CD45Lo microglia in brain and spinal cord tissues, CD11b+CD45Hi infiltrating cells, CD11b+CD45LoP2RY12+ homeostatic microglia, and CD11b+CD45LoLy6C+ inflammatory microglia. (B–E) Graphical representation of flow cytometric quantification of CD11b+CD45Lo microglia in brain and spinal cord tissues (B), CD11b+CD45Hi infiltrating cells (C), CD11b+CD45LoP2RY12+ homeostatic microglia, (D), and CD11b+CD45LoLy6C+ inflammatory microglia (E). Data shown as mean ± SD, n = 4–6 mice per group, where each data point represents an individual mouse across four experiments. *p &lt; 0.05 and **p &lt; 0.01 using Student’s t test, Welch’s correction. Fig. S6. Transcriptome analyses of significantly expressed gene in response to rAAV–sFKN in the diabetic retina. Analysis of statistically significant DEGs associated with complement, cell death, and regulation of cell death (A, E), inflammation, (B, F), neuronal and vascular damage (C, G), and microglia activation and DR pathogenesis (D, H) of PBS-treated 4-wk D and 10-wk D mice compared their PBS-treated ND controls. Significant DEGs associated with complement, cell death, and regulation of cell death (I, L), inflammation (J), and neuronal and vascular damage (K) of 4-wk D and 10-wk D mice compared to diabetic rAAV–sFKN-treated groups. n = 4–5 mice per treatment group across two experiments. The level of significance in the DEGs associated with rAAV–sFKN treatment can be found in Table S4. Fig. S7. Bulk mRNA-seq transcriptome gene expression in rAAV–sFKN and rAAV–mFKN treated groups. Graphical representation and volcano plots showing all DEGs of each comparison group without set thresholds (i.e., log2 fold change of 1 and -1 with FDR p value of &lt; 0.05), thresholds are distinguishably marked (dotted line panes and red colored points). Total DEGs of PBS-treated 4-wk D mice versus PBS-treated ND mice (A, B), PBS-treated 10-wk D mice versus PBS-treated ND mice (C, D), rAAV–mFKN-treated 4-wk D mice versus PBS-treated 4-wk D mice (E, F), rAAV–mFKN-treated 10-wk D mice versus PBS-treated 10-wk D mice (G, H), rAAV–sFKN-treated 4-wk D mice versus PBS-treated 4-wk D mice (I, J), and rAAV–sFKN-treated 10-wk D mice versus PBS-treated 10-wk D mice (K, L). n = 4–5 mice per treatment group across two experiments. The complete list of genes with set threshold can be found in Table S4. Fig. S8. Transcriptome gene expression in rAAV–mFKN treated groups. (A–D) Heat map gene expression of all up and down regulated DEGs associated with the complement pathway, cell death and cell death regulation (A), inflammatory cytokines and chemokines (B), vascular and neuronal damage (C) and microglia activation and DR pathogenesis (D). All heat maps compared PBS-treated 4-wk D and 10-wk D mice to PBS-treated ND controls, and PBS-treated 4-wk D and 10-wk D mice compared to rAAV–mFKN-treated mice. (E–P) Analysis of statistically significant DEGs associated with complement, cell death, and regulation of cell death (E, I), inflammation, (F, J), neuronal and vascular damage (G, K), and microglia activation and DR pathogenesis (H, L) of PBS-treated 4-wk D and 10-wk D mice compared their PBS-treated ND controls. Significant DEGs associated with complement, cell death, and regulation of cell death (M), inflammation (N), and neuronal and vascular damage (O), and microglia activation and DR pathogenesis (P) of 4-wk D mice compared to diabetic rAAV–mFKN-treated groups. n = 5 mice per treatment group across two experiments. The level of significance in the DEGs associated with rAAV–sFKN treatment can be found in Table S4. Table S1. Antibodies for immunohistochemistry analysis. Table S2. Combination of antibodies used for analysis.  Table S3. Antibodies for flow cytometry analysis.  Table S4. Level of significance in the differentially expression genes (DEGs) associated with rAAV–mFKN or rAAV–sFKN treatment.