Untargeted and temporal analysis of retinal lipidome in bacterial endophthalmitis

Abstract

Bacterial endophthalmitis is a blinding infectious disease typically acquired during ocular surgery. We previously reported significant alterations in retinal metabolism during Staphylococcus (S) aureus endophthalmitis. However, the changes in retinal lipid composition during endophthalmitis are unknown. Here, using a mouse model of S. aureus endophthalmitis and an untargeted lipidomic approach, we comprehensively analyzed temporal alterations in total lipids and oxylipin in retina. Our data showed a time-dependent increase in the levels of lipid classes, sphingolipids, glycerolipids, sterols, and non-esterified fatty acids, whereas levels of phospholipids decreased. Among lipid subclasses, phosphatidylcholine decreased over time. The oxylipin analysis revealed increased prostaglandin-E2, hydroxyeicosatetraenoic acids, docosahexaenoic acid, eicosapentaenoic acid, and α-linolenic acid. In-vitro studies using mouse bone marrow-derived macrophages showed increased lipid droplets and lipid-peroxide formation in response to S. aureus infection. Collectively, these findings suggest that S. aureus-infection alters the retinal lipid profile, which may contribute to the pathogenesis of bacterial endophthalmitis.

Keywords: Endophthalmitis; Inflammation; Lipidomic; Oxylipins; Retina; S. aureus.

Figure 1.. Untargeted retinal lipidomic analysis in bacterial endophthalmitis

(A) Schematic of experimental design for lipidomic analysis in bacterial endophthalmitis. Retinal tissue from uninfected (mock) or S. aureus (SA)-infected eyes (n=4) at three different time points (12, 24 or 48 h) were used for lipidomic analysis. (B) Principal component analysis (PCA) of untargeted lipidomic data shows distinct clustering of various experimental groups at different time intervals with QC (Quality control). (C) Schematic of the lipid classification used in this study indicating five major lipid classes and their subclasses and species. (D) Lipidomic analysis showing temporal percentage changes in the mock (c) versus SA-infected retinal tissue. Statistical analysis was performed using one-way ANOVA, with significance levels of (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001, ns indicating non-significance. Comparisons were made between uninfected control (C) vs. SA-infected retina.

Figure 2.. Alterations in lipid subclasses during S. aureus endophthalmitis.

(A) Heatmap of lipid subclasses showing significant alterations at indicated time points (12, 24, and 48 h) post-infection. (B) Bar diagram showing temporal changes in some representative lipid subclasses in mock (c) versus S. aureus (SA)- infected mouse retinal tissue. Statistical analysis was performed using one-way ANOVA, with significance levels of (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001, ns indicating non-significance. Comparisons were made between the uninfected control (C) vs. SA-infected retina.

Figure 3.. Alterations in lipid species during S. aureus endophthalmitis.

(A) Heatmap of lipid species showing significant alterations at indicated time points (12, 24, and 48h) post-infection. (B) Bar diagram showing temporal changes in some representative lipid species in mock (c) versus S. aureus (SA)-infected mouse retinal tissue. Statistical analysis was performed using one-way ANOVA, with significance levels of (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001, ns indicating non-significance. Comparisons were made between uninfected control (C) vs. SA- infected retina.

Figure 4.. Targeted retinal oxylipin analysis in bacterial endophthalmitis.

(A) Schematic representation of generation of oxylipins based on their sources and metabolic pathways regulated by three key enzymes, COX, LOX, and CYP450. (B) Principal component analysis (PCA) of retinal oxylipins from mock-infected and S. aureus (SA) infected mouse retinas (n=4) at 12, 24, and 48 h post-infection.

Figure 5.. Alterations in retinal oxylipin production during S. aureus endophthalmitis.

(A) Heat map showing temporal changes in the relative abundance of retinal oxylipins during endophthalmitis. (B) Line graph showing fold changes in oxylipins with significant alterations at indicated time post infection. (C) Volcano plot showing the differentially produced oxylipins with red dots indicating increased and blue dots showing reduced levels of oxylipins from the centerline (y-axis) and above a certain significance threshold. Statistical analysis was performed using one-way ANOVA, with significance levels of (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001, ns indicating non-significance. Comparisons were made between uninfected control (C) vs. SA- infected retina.

Figure 6.. S. aureus infection induced lipid droplets and lipid peroxidation in BMDMs.

BMDMs were isolated from the bones of B6 mice and cells were infected with S. aureus (SA) for 6 h. (A) Cells were stained with specific dye to visualize lipid droplets (green). (B) Semi-quantitative analysis was performed by counting lipid droplets ~50 cells/image. (C) The formation of lipid peroxidation (LPO) in ratiometric dye which gives shift from red (reduced lipids) to green (oxidized lipids) indicating lipid peroxidation, Scale bar; 100μm. Images are representative of two independent experiments. (D) Quantitative analysis was of lipid peroxidation was performed by measuring mean fluorescence intensity (MFI). Statistical analysis was performed using an unpaired, t-test, with significance levels of (****) p<0.0001. Comparisons were made between uninfected control (C) vs. SA- infected BMDM.

Figure 7.. Oxylipin formation in S. aureus infected BMDMs.

(A) Heatmap showing the changes in production of various oxylipins in BMDMs (n=3) in response to S. aureus (SA) infection for 6h. (B) Principal Component Analysis (PCA) of oxylipins, showing the distinct clustering of control and SA-infected BMDMs. (C) Volcano plot showing differentially expressed lipids represented by dots located above the significance threshold and far from the centerline (y-axis). The upregulated oxylipins (red) located on the right-hand side of the plot. (D) Violin plot showing the most significantly upregulated oxylipins, plotted based on the heatmap analysis. Statistical analysis was performed using an unpaired, t-test, with significance levels of (*) p<0.05, (**) p<0.01. Comparisons were made between uninfected control (C) vs. SA- infected BMDM.