Homocysteine Induces Inflammation in Retina and Brain

Abstract

Homocysteine (Hcy) is an amino acid that requires vitamins B₁₂ and folic acid for its metabolism. Vitamins B₁₂ and folic acid deficiencies lead to hyperhomocysteinemia (HHcy, elevated Hcy), which is linked to the development of diabetic retinopathy (DR), age-related macular degeneration (AMD), and Alzheimer's disease (AD). The goal of the current study was to explore inflammation as an underlying mechanism of HHcy-induced pathology in age related diseases such as AMD, DR, and AD. Mice with HHcy due to a lack of the enzyme cystathionine-β-synthase (CBS) and wild-type mice were evaluated for microglia activation and inflammatory markers using immuno-fluorescence (IF). Tissue lysates isolated from the brain hippocampal area from mice with HHcy were evaluated for inflammatory cytokines using the multiplex assay. Human retinal endothelial cells, retinal pigment epithelial cells, and monocyte cell lines treated with/without Hcy were evaluated for inflammatory cytokines and NFκB activation using the multiplex assay, western blot analysis, and IF. HHcy induced inflammatory responses in mouse brain, retina, cultured retinal, and microglial cells. NFκB was activated and cytokine array analysis showed marked increase in pro-inflammatory cytokines and downregulation of anti-inflammatory cytokines. Therefore, elimination of excess Hcy or reduction of inflammation is a promising intervention for mitigating damage associated with HHcy in aging diseases such as DR, AMD, and AD.

Keywords: Alzheimer’s disease; age-related macular degeneration; diabetic retinopathy; homocysteine; inflammation.

Figure 1

Hcy activates microglia in retina and brain. Microglia activation in retinal flat-mounts of cbs^−/−, cbs^+/− mice and brains of cbs^−/− and cbs^+/− mice (A) RPE flat-mount (B) inner retinal flat-mount, and (C) brain frozen sections compared to cbs^+/+ mice. Iba1 microglia marker (green) shows quiescent ramified microglia in cbs^+/+ and rod-like microglia devoid of branching processes stains for both Iba1 and isolectin-B4 (white arrows, yellow stain) in the cbs^−/− and cbs^+/– retinas and brains indicating microglia activation. In addition, Hcy activated microglia (green) in the retinal section from normal wild type mice injected intravitreal with Hcy (D). Samples were representative to at least three mice for each immuno-fluorescence (IF) experiment. Scale bars: 20, 20, 50, and 20 μm, respectively. Abbreviations: GCL = ganglion cell layer, INL = inner nuclear layer, ONL = outer nuclear layer.

Figure 2

(A) Multiplex assay for human monocyte cell line U937 treated with and without Hcy 20 μM for 24 h revealed significant increase in pro-inflammatory cytokines (IP-10, TNFα, IL-B, and IL-6). (B–E). HHcy activates NF-κB transcription factor in cultured HRECs and ARPE-19 cells. Western blot analysis for HRECs (B,D) and ARPE-19 cells (C,E) treated with 100 µM Hcy, showing significant increase in the NF-κB nuclear to cytoplasmic ratio compared to the control untreated cells. NF-κB is a transcription factor, which controls the transcription of DNA and cytokine production. (F) IF staining for NF-κB, showed that Hcy activated and translocated NF-κB from the cytoplasmic to the nuclear side in HRECs treated with Hcy (green). (G) Primary RPE cells isolated from cbs^+/− mice retina (red). (H–I) ELISA measurement of NFκB (P65) in HRECs (H) and ARPE-19 (I) treated with and without Hcy (50 μM and 100 μM) for 24 h. Samples were representative to at least three mice for each experiment. Data are expressed as mean ± SD. (* p < 0.05, ** p < 0.01). Scale bar: 50 and 20 μm, respectively.

Figure 3

HHcy activates IL-1β in retina and TNF-α in retina and hippocampus area of the cbs mice brain. (A) IF staining for retinal frozen section from the cbs^−/−, cbs^+/−, and cbs^+/+ mice for IL1β (green); (B) TNFα (green, retina); (C) IL1β (green, brain); (D) TNFα (green, brain), and isolectin-B4 (ISB) for the blood vessels (red, A–D) showing the activation of retinal IL1β and TNFα in the retinal and brain by HHcy. Samples were representative to at least three mice for each experiment. Scale bar: 20, 50, 50, and 50 μm, respectively. Abbreviations: GCL = ganglion cell layer, INL = inner nuclear layer, ONL = outer nuclear layer.

Figure 4

Cytokine array analysis for retinal cells treated with Hcy for 24 h. (A–F) Cytokine array analysis of ARPE cells and HRECs treated with Hcy for 24 h. (A,B) Membranes showing the change in the inflammatory cytokines. The intensities of the spot were quantified densitometrically and compared to the density of the internal standards, yielding an integrated density value (IDV) for each cytokine. (C–E) Analysis showing significant increase of pro-inflammatory cytokines (IL-1β, IL5, IFN-γ, MIP-α, TNF-α, GM-CSF, IL-12, G-CSF, IL6, and Rantes) and downregulation of anti-inflammatory cytokines (IL-4 and IL10) when ARPE-19 cells were treated with 100 µM Hcy for 24 h. (F) HRECs treated with Hcy-50 µM for 24 h showed significant increase in pro-inflammatory and pro-angiogenic cytokines (TNF-α, PDGF BB, IFN-γ, IL8, IL10, IL-1 β, IL-α, and Rantes) and a marked increase in leptin and VEGF. Data are expressed as mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 5

Multiplex assay for cytokines secreted from retinal cells treated with Hcy and cbs^+/− mice brain. Multiplex cytokine assay analysis from conditioned media of HRECS and ARPE-19 cells in response to 50 µM and 100 µM Hcy, respectively. (A,B) Showed and confirmed the increased of the pro-inflammatory cytokines and downregulation of the anti-inflammatory cytokine (IL4), while brain cytokine array from the hippocampal area of the cbs^+/− mice brain (C) showed a significant decrease in IL13, MCP-1, and increase in Exotoxin and IL3. Three replicates per sample and two replicates per cytokine were performed (* p < 0.05, ** p < 0.01, *** p < 0.001). Data are expressed as mean ±SD.