Fig 2: Immunofluorescent analysis of RPE/choroidal flat mounts and western blot analysis of RPE/Choroid and retinal material. Alpha-crystallin B (red) immunofluorescent staining in RPE/choroidal complexes. (A) Strong Alpha-crystallin B signals were detected in CXCR5-/- animals; (B,D) noise levels of red fluorescence were detected in WT controls; z-stack depth analysis in (C) CXCR5-/- flat mounts indicate sub-RPE localization of the signals in CXCR5-/- RPE/choroidal complexes, forming a distinctive layer under the RPE cells. Marked amyloid beta (Aß) staining (green) was detected in RPE/choroidal complexes in the (E) CXCR5-/- sub-RPE localization of Aß was identified by (F) z-stack depth analysis, no amyloid beta expression detected in (G) WT animals and (H) it's z-stacks. CXCR5-/- mice also indicated an increased presence of IBA-1 (green) and CD4 (red) positive cells within the (I) RPE/choroidal complex; (J) z-stack analysis revealed the presence of IBA-1 positive signals on the surface of the RPE layer and sub-RPE localization of CD4 positive cells; (K,L) WT samples demonstrated minimal IBA-1 fluorescence and no CD4 positive cells. Nuclear counterstaining by DAPI (blue) in fluorescent images. Protein expression of (M) ZO-1, COX-2, CD4, Cryab, CXCL13, and VEGF-a; (N) Arg-1, expression CXCR5-/- and age-matched WT control RPE/choroid and retina lysate. ß-Actin used as a loading control. (O) Cleavage of Caspase-1 in retina lysate of CXCR5-/- and age-matched WT control animals. All samples were obtained from 24 m.o. female mice.

Fig 3: Expression level of protein of COX-2 and 5-LOX through western blot and levels of gene contributor through quantitative RT-PCR. Immunoblotting of respective individual group [I-Control (Normal saline, 3 ml/kg, p.o.), II- Toxic control (MNU 47 mg/kg, i.v.), III- Zaltoprofen (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.), IV- Zileuton (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.) and V- Zaltoprofen + Zileuton (5 + 5 mg/kg, p.o. + MNU 47 mg/kg, i.v.)] for COX-2 and 5-LOX. Excised mammary gland tissue sample lysed in trizol for RNA extraction and analyzed for the mRNA expression of COX-2 and 5-LOX by qRT-PCR. ß-actin was used as loading control. Each experiment was performed in triplicate. Values are presented as Mean ± SD. Comparisons were made by the one-way ANOVA followed by Bonferroni multiple test. All groups were compared to the MNU treated group (**p < 0.01, ***p < 0.001).

Fig 4: COX-2, phopho-I?Ba, and reactive oxygen species expression in LPS-stimulated BV-2 cells with and without L-VGCC blockade. Effects of L-VGCC blockade on COX-2 expression (a) and detection of phosphorylated I?Ba (b) in BV-2 cells stimulated with LPS. Quantitative RT-PCR analysis of COX-2 expression in BV-2 cells treated with DMSO control (2 µl/ml), Zinc20267861 (2.0 µM), Zinc18204217 (2.0 µM), and Zinc33254827 (2.5 µM) stimulated with LPS 1 µg/ml (c). Arg-1 expression with the same treatments in BV-2 cells stimulated with IL-4 (20 ng/ml) (d) (n = 3 with 3 technical replicates). A one-way ANOVA test with Tukey multiple comparisons was used to determine statistical significance. p > 0.05; ***p < 0.001. Target genes were normalized to cyclophilin and presented as the fold change of DMSO control. Detection of reactive oxygen species in BV-2 cell culture, minimal oxidation of dihydroethidium in DMSO 2 µl/ml untreated control cells (e); strong oxidized ethidium signals detected when stimulated with LPS 1 µg/ml (e). Treatment with 3.5 µM of amlodipine (g), Zinc20267861 (2.0 µM) (h), Zinc18204217 (2.0 µM) (i), and Zinc33254827 (2.5 µM) (j) demonstrated marked reduction of the oxidized ethidium signals in BV-2 cell culture.

Fig 5: Conceptual summary of the proposed cellular events in aged CXCR5–/– retinas and choroid based on the findings of the current study. (A) CXCR5-/- microglia cells demonstrated upregulation of Arg-1 in the retina. Increased production of CXCL13-activated microglia cells migrating toward the RPE, releasing COX-2, TNFa, and excessive amounts of Arg-1. RPE and photoreceptor cells were affected by cytokine signaling, triggering cell death. The loss of photoreceptors and subsequent neuronal connections led to reduced MAP-2 signaling within the retina. RPE65 positive cellular debris of dying photoreceptors subjected to phagocytosis by microglia cells and eventually deposited in the sub-RPE space, triggering the accumulation of amyloid beta, Cryaa, Cryab, and other AMD-associated proteins. Constant proinflammatory signaling within the RPE layer affected the tight junction proteins, including ZO-1, compromising the BRB and making drusen components accessible to the immune system and production of specific autoantibodies. The binding of autoantibodies to the components of sub-RPE deposits triggered infiltration with CD4-positive inflammatory cells. Constant VEGF signaling within the choroid led to the release of VEGF-a and choroidal neovascularization. (B) Healthy retina and RPE layers of WT animal.