Immune regulation in the aging retina

Abstract

The retina is an immune privileged tissue, which is protected from external and internal insults by its blood-retina barriers and immune suppressive microenvironment. Apart from the avoidance and tolerance strategies, the retina is also protected by its own defense system, i.e., microglia and the complement system. The immune privilege and defense mechanisms work together to maintain retinal homeostasis. During aging, the retina is at an increased risk of developing various degenerative diseases such as age-related macular degeneration, diabetic retinopathy, and glaucomatous retinopathy. Previously, we have shown that aging induces a para-inflammatory response in the retina. In this review, we explore the impact of aging on retinal immune regulation and the connection between homeostatic control of retinal immune privilege and para-inflammation under aging conditions and present a view that may explain why aging puts the retina at risk of developing degenerative diseases.

Keywords: Aging; Blood-retina barrier; Complement; Inflammation; Microglia; Retinal degeneration.

Figure 1. Immune cells in the aged retina.

Retinal flatmounts from 24 months old C57BL/6J mice were stained for Iba-1 (green), Isolectin B4 (blue), and CD3 (red in A) or B200 (red in B) and imaged by confocal microscopy. Arrows indicating intravascular leukocytes.

Figure 2. Tight-junctions and subretinal immune cells in retinal pigment epithelial cells of the aged eye.

(A) RPE-choroidal flatmounts from 24 months old C57BL/6J mice were stained for claudin-1 (Green), ZO-1 (red), and Phalloidin (blue) and imaged by confocal microscopy. Arrows indicate area of ZO-1 loss with no defect in claudin-1 and F-actin (by phalloidin labelling) expression. (B, C) RPE-choroidal flatmounts from 24 months old C57BL/6J mice were stained for Iba-1 (Green) and CD3 (red in B) or isolectin B4 (red in C) and imaged by confocal microscopy. Arrowheads: cells dual-positive for Iba-1 and isolectin B4.

Figure 3. The expression of CD200-CD200R and CD47-SIRP in mouse retina.

(A, B) Confocal images showing CD200 (A) and CD200R (B) expression in the retina from 3 months old C57BL/6J mice. (C, D) mRNA expression levels of CD200 (C) and CD200R (D) in the retina from 3 months (young) and 24 months (old) C57BL/6J mice. (E, F) Confocal images showing CD47 (E) and SIRP (F) expression in the retina from 3 months old C57BL/6J mice. (G, H) mRNA expression levels of CD47 (G) and SIRP (H) in the retina from 3 months (young) and 24 months (old) C57BL/6J mice. Mean ± SEM, n = 5.

Figure 4. Age-related changes in endocannabinoids 2-AG and AEA in mouse retina.

Retinal tissues from 3, 18 and 27 months old C57BL/6J and CCL2^-/- mice were snap-frozen and processed for measuring of 2-AG and AEA using liquid chromatography/mass spectrometry. The values of 2-AG and AEA were normalized by tissue weights and total protein content. (A) 2-AG levels in the retina of WT mice at different ages. (B) AEA levels in the retina of WT mice at different ages. (C, D) Comparison of retinal 2-AG (C) and AEA (D) levels between WT and CCL2^-/- mice at different ages. N ≥ 6 mice. *, P<0.05.

Figure 5. The effect of RPE on macrophage gene expression.

(A, B) Bone marrow-derived macrophages (BMDMs) and primary RPE cells were cultured from 3 months old C57BL/6J mice. RPE cells were then treated with oxidized photoreceptor outer segments (oxPOS) for 24 h. BMDMs were co-cultured with RPE cells for 7 h. Cells were detached in ice-cold PBS with 2mM EDTA. Macrophages were then isolated by CD11b MACS kit (Miltenyi Biotec, UK) and processed for real-time RT-PCR. (A) Genes related to the inflammatory M1 phenotype. (B) Genes related to the M2 phenotype. Data shown are gene fold changes compared with macrophages without RPE treatment. RPE(-) – BMDMs alone; RPE(+) – BMDMs co-cultured with normal RPE cells; _oxPOSRPE(+) – BMDMs co-cultured with oxPOS-treated RPE cells. Mean ± SEM, n = 3. *, P<0.05; **, P<0.01 compared with macrophages without RPE treatment. †, P<0.05 compared with normal RPE treated macrophages.

Figure 6. The effect of RPE cells on macrophage phagocytosis.

CD4⁺ T cells were isolated from 3 months old C57BL/6J mice and were labeled with MitoTracker Orange. Apoptosis was induced by serum deprivation. Primary RPE cells were treated with oxPOS for 24 h. Bone marrow-derived macrophages, either untreated, or pre-treated with normal RPE, or oxPOS-treated RPE, were labeled with Calcein AM. Macrophages were then supplemented with apoptotic T cells (macrophage: T cell ratio = 1:10) for 24h. Remaining T cells were counted at the end of the study. (A) Apoptotic T cells without macrophages. (B) Apoptotic T cells incubated with normal macrophages for 24 h. (C) Apoptotic T cells incubated with normal RPE pre-treated macrophages. (D) Apoptotic T cells incubated with macrophages that were pre-treated with oxPOS-treated RPE cells. apoCD4 – apoptotic CD4 T cells; Mac – macrophages; Mac(RPE) – macrophages pre-treated with normal RPE cells; Mac(oxRPE) – macrophages pre-treated with oxPOS-treated RPE cells. Mean ± SEM, n = 3. *, P<0.05. Scale bar = 50 μm.

Figure 7. Age-related alterations in retinal microglial activation and function.

(A) Under normal physiological conditions, microglia patrol retinal microenvironment through crawling dendrites. Microglia may be activated after engaging with Damage Associated Molecular Patterns (DAMPs) by cell surface receptors such as TLRs, NLRs and IL-1Rs. This activation process is tightly controlled by various signals from surrounding neurons (e.g., CX3CL1-CX3CR1, CD200-CD200R, CD47-SIRP-1, etc.). (B) During aging, oxidative stress damages retinal neurons leading to the weakening of negative regulatory signals and the accumulation of DAMPs. This results in low-levels of sustained microglial activation. In the meantime, sustained chronic oxidative stress also damages microglial function.

Figure 8. Age-related changes in retinal complement expression.

(A) Under normal physiological conditions, retinal cells, including microglia, RPE and neurons express various complements and complement regulators. The expression levels of complement inhibitors such as CFH, C1INH and CD59 is relatively higher than that of complement components such as C3 and C5. Low levels of complement expression and activation is critical for retinal homeostasis. (B) During aging, oxidative stress and low-levels of inflammation reduce the expression of complement inhibitors but increase the expression of complement components (e.g., CFB and C3). BM – Bruch’s Membrane; RPE – retinal pigment epithelium; CFB – complement factor B; CFH – complement factor H, C1INH – C1 inhibitor; C4BP – C4 binding protein; Mϕ - macrophage