Deficits in Monocyte Function in Age Related Macular Degeneration: A Novel Systemic Change Associated With the Disease

Abstract

Age-related macular degeneration (AMD) is characterized by the accumulation of debris in the posterior eye. In this study we evaluated peripheral blood monocyte phagocytic function at various stages of AMD and in aged matched control participants. Real-time tri-color flow cytometry was used to quantify phagocytic function of peripheral blood monocyte subsets (non-classic, intermediate and classic) isolated from subjects with intermediate or late AMD and compared with age matched healthy controls. Assessment of phagocytic function of monocytes isolated from those with and without reticular pseudodrusen was also made, and the effect of glatiramer acetate on phagocytic function assessed. Phagocytic function was reduced in all subjects with AMD, irrespective of stage of disease. However, there was no correlation between phagocytic function and drusen load, nor any difference between the level of phagocytosis in those with or without reticular pseudodrusen. Treatment with glatiramer acetate increased phagocytosis of classical and non-classical monocytes, normalizing the reduction in phagocytosis observed in those with AMD. These findings suggest that defective systemic phagocytosis is associated with both intermediate and late stages of AMD, highlighting a potential role in the accumulation of debris that occurs early in the disease process. Assessing peripheral monocyte phagocytic function provides further insights into the etiology of this disease and offer a novel therapeutic target.

Keywords: drusen; glatiramer acetate; monocytes; phagocytosis; reticular pseudodrusen.

Figure 1

Quantification of phagocytosis across the different types of monocytes. Whole blood leukocytes were labeled with different surface markers in four-color flow cytometry panels. (A) A typical gate strategy used in YO bead uptake for gating CD14^dimCD16⁺ nonclassical monocytes (red), CD14⁺CD16⁺ intermediate monocytes (blue) and CD14⁺CD16⁻ classic monocytes (green). (B) The corresponding kinetic curve of YO beads uptake in gated populations are shown.

Figure 2

Monocyte phagocytosis in subjects with AMD. (A) Representative fundus images from a healthy control (normal) subject, person with intermediate AMD (iAMD), geographic atrophy and choroidal neovascularization. (B) A typical example of YO beads uptake curve by three monocyte subsets from a healthy control (HC; blue) a patient with intermediate AMD (green) and a subject with late AMD (red) Fresh human peripheral blood monocytes (PBMCs) were labeled with APC-conjugated CD14 and FITC-conjugated CD16 before the addition of 1 μm YO beads. The YO beads fluorescence intensity was analyzed by real time flow cytometry. (C) Graph showing mean ± standard deviation basal phagocytic function of monocytes subsets isolated from healthy control subjects (HC) (n = 35), subjects with iAMD (iAMD) (n = 61) and subjects with late AMD (n = 30). P-values from One-way ANOVA analysis and Tukey's multiple comparisons tests are shown for comparison between specific groups. (D) Correlations of basal phagocytic function for the three monocyte subtypes compared to drusen area. There was no significant correlation between phagocytosis and drusen area for any of the monocytes examined.

Figure 3

Phagocytic function in participants with and without reticular pseudodrusen (A) Fundus auto-fluorescence (FAF; top) and near Infrared reflectance (NIR; bottom) spectroscopy images showing reticular pseudodrusen (RPD) area outlined using Image J software. (B) Graph showing mean+standard deviation of monocyte phagocytosis in healthy controls (HC, n = 35), subjects with intermediate AMD without RPD (RPD⁻; n = 42) and intermediate AMD with RPD (RPD⁺; n = 18). P-values from One-way ANOVA analysis and Tukey's multiple comparisons tests are shown for comparison between specific groups. (C) Correlations of basal phagocytic function of three monocyte subsets with RPD area (n = 60). There was no significant correlation between phagocytosis and drusen area for any of the monocytes examined.

Figure 4

The effect of glatiramer acetate treatment on phagocytosis and its correlation with drusen size. (A) Graph of monocyte phagocytosis before and after 10 min treatment with glatiramer acetate 100 μg/mL for healthy controls, subjects with intermediate AMD (iAMD) and subjects with late AMD. P-values from a Two-way ANOVA analysis with post-hoc Tukey's-test are shown on top of each panel. (B) Correlations of drusen area with phagocytic function following glatiramer acetate treatment in subsets of monocytes. There was a significant correlation between drusen area and glatiramer acetate stimulated phagocytosis for non-classical and intermediate monocytes. ns: no significance.

Figure 5

The effect of glatiramer acetate treatment on monocytes phagocytosis isolated from those with and without RPD and its correlation with RPD area. (A) Graph showing phagocytic function before and after 10-min application of glatiramer acetate in monocytes isolated from healthy controls, subjects with intermediate AMD without RPD (RPD⁻) and subjects with intermediate AMD with RPD (RPD⁺). P-values from a Two-way ANOVA with post-hoc Tukey's analysis analysis are shown on top of each panel. (B) Correlations of RPD area with phagocytic function following glatiramer acetate treatment in subsets of monocytes. There was no significant correlation between RPD area and glatiramer acetate stimulated phagocytosis. ns: no significance.

Figure 6

Surface expression of phagocytosis related molecules in leukocyte from HC, early and late AMD. Whole blood leukocytes were labeled with different surface markers in four-color flow cytometry panels. Samples were from healthy controls (n = 52), early AMD (n = 39) and late AMD (n = 22). Parameters that showed significant differences are shown. P-values from One-way ANOVA analysis and Tukey's multiple comparisons tests are shown for comparison between specific groups. In B, the p-value for the One-Way ANOVA across all three groups is shown.

Figure 7

Changes in proportions of monocyte subtypes in healthy control, early and late AMD subjects Graph of the mean ± SD proportion of (A) non-classical monocytes (CD14⁻CD16⁺), (B) intermediate monocytes (CD14⁺CD16⁺), (C) classic monocytes (CD14⁺CD16⁻) and (D) and ratio of neutrophils to monocytes. Samples were from healthy controls (n = 35), intermediate AMD (n = 42) and late AMD (n = 30). Parameters that showed significant differences are shown. P-values from One-way ANOVA analysis and Tukey's multiple comparisons tests are shown for comparison between specific groups.

Figure 8

Phagocytic function and leukocyte biomarkers with potential diagnosis values for AMD. Top potential variables for diagnosis of AMD are shown in the table. AUC, area under curve of Receiver Operating Characteristic (ROC); Mono, monocytes; exp, surface expression; SD, standard deviation; phago, phagocytic function. Binary logistic regression was performed among selected multiple variables to create predicted probabilities for ROC analysis. ROC curves distinguishing between (A) healthy controls (HC) and all AMD (intermediate, GA and CNV) and (B) HC and intermediate AMD are shown. P-values are for asymptotic significance calculated by SPSS v24.

Figure 9

Leukocyte proportion and surface biomarkers with potential prognostic values for AMD. Top 6 potential variables for prognosis of AMD are shown in the table. AUC, area under curve of Receiver Operating Characteristic (ROC); Mono, monocytes; exp, surface expression; SD, standard deviation. Binary logistic regression was performed among multiple parameters to create predicted probabilities for ROC analysis. ROC curves distinguishing between (A) early stages (intermediate) and late stages of AMD (GA and CNV) and between (B) RPD⁻ and RPD⁺ AMD patients are shown. P-values are for asymptotic significance calculated by SPSS v24.