Myelin-specific Th17 cells induce severe relapsing optic neuritis with irreversible loss of retinal ganglion cells in C57BL/6 mice

Abstract

Purpose: Optic neuritis affects most patients with multiple sclerosis (MS), and current treatments are unreliable. The purpose of this study was to characterize the contribution of Th1 and Th17 cells to the development of optic neuritis.

Methods: Mice were passively transferred myelin-specific Th1 or Th17 cells to induce experimental autoimmune encephalomyelitis (EAE), a model of neuroautoimmunity. Visual acuity was assessed daily with optokinetic tracking, and 1, 2, and 3 weeks post-induction, optic nerves and retinas were harvested for immunohistochemical analyses.

Results: Passive transfer experimental autoimmune encephalomyelitis elicits acute episodes of asymmetric visual deficits and is exacerbated in Th17-EAE relative to Th1-EAE. The Th17-EAE optic nerves contained more inflammatory infiltrates and an increased neutrophil to macrophage ratio. Significant geographic degeneration of the retinal ganglion cells accompanied Th17-EAE but not Th1.

Conclusions: Th17-induced transfer EAE recapitulates pathologies observed in MS-associated optic neuritis, namely, monocular episodes of vision loss, optic nerve inflammation, and geographic retinal ganglion cell (RGC) degeneration.

Figure 1

Experimental design. Cultured, myelin-specific Th1 (interleukin (IL)-12-treated) or Th17 (IL-23-treated) cells were injected into 15 naïve recipient mice each, and optokinetic reflexes (OKTs) and disease severity were assessed daily. At approximately 1, 2, and 3 weeks post-induction, five mice from each group were harvested. The optic nerves were histologically assessed for inflammation, and retinal ganglion cells (RGCs) were counted in retinal flatmounts.

Figure 2

EAE visual deficits are asymmetric and episodic. A: Representative time course of optokinetic reflex (OKT) measurements (purple circles and green squares) and disease severity scores (top, orange diamonds) for a transfer experimental autoimmune encephalomyelitis (EAE) mouse demonstrating the asymmetric and episodic nature of visual deficits, the onset of which coincide with motor deficits. B, C: Means of daily acuity readings (cycles per degree) as measured by OKT for the more affected eyes (purple circles) and less affected eyes (green squares) of the Th1 (B) and Th17 (C) transfer EAE mice. Error bars represent standard error of the mean (SEM). Number of eyes: n = 15 for 1–7 days post-immunization (DPI), n = 10 for 8–12 DPI, and n = 5 for 13–21 DPI. D, E, F: Area under the curve (AUC; presented as % of maximum for each plot) for daily visual acuity values (cycles per degree) as measured by OKT for each Th1 (left, red circles) and Th17 (right, blue squares) mouse for 0–7 DPI (n = 15; D), 0–12 DPI (n = 10; E), and 0–19 DPI (n = 5; F). Eyes from the same mouse are connected by a line. MA = more affected eye and LA = less affected eye for each mouse as determined by the average OKT score for each eye. *p<0.05, **p<0.01, ***p<0.001 with one-tailed Wilcoxon matched-pairs test.

Figure 3

The Th17 transfer EAE model exhibits more severe motor and vision deficits relative to Th1. A: Daily mean experimental autoimmune encephalomyelitis (EAE) severity scores for Th1 (red circles) and Th17 (blue squares) transfer EAE mice. B, C: Daily mean acuity of more (B) or less (C) affected eyes of Th1 (red circles) and Th17 (blue squares) mice as measured by optokinetic reflex (OKT) where the more affected eye of each mouse is classified as that with the lower average OKT value over the course of the study. Error bars represent standard error of the mean (SEM). Number of eyes: n = 15 for −1–7 days post-immunization (DPI), n = 10 for 8–12 DPI, and n = 5 for 13–21 DPI. *p<0.05; **p<0.005 with the linear mixed effect model.

Figure 4

Optic nerve inflammation negatively correlates with visual acuity and is more severe in Th17-EAE relative to Th1. A: Representative hematoxylin and eosin (H&E)-stained paraffin-embedded 5-µm optic nerve sections indicating criterion by which all nerves were graded for inflammation severity on a scale of 0–3. B: Linear regression of average optokinetic reflex (OKT) values on optic nerve inflammation severity scores throughout the study for the corresponding eye for Th1 (red circles, p = 0.0139) and Th17 (blue squares, p = 0.0055) at 8, 13, and 21 days post-immunization (DPI) indicating significant negative correlations by Spearman correlation. n = 30 eyes (from 15 mice) per group. C: Optic nerve inflammation severity scores for Th1 (red circles) and Th17 (blue squares) optic nerves indicating significantly more inflammation in Th17 nerves harvested at only 21 DPI (n = 10 eyes from five mice, p = 0.0281) with an unpaired t test.

Figure 5

Th17-induced optic neuritis consists of a higher neutrophil to macrophage ratio relative to Th1. Quantitation of cell type-specific markers in paraffin-embedded optic nerves from mice harvested at 13 and 21 days post-immunization (DPI). Each nerve is represented as the average value from two separate longitudinal sections. A: Myelination as measured by the percentage of nerves positive for the anti-CNPase signal, p = 0.0770. B: T-cells as measured by the number of CD3-positive cells counted in three representative 20X fields throughout each section, p = 0.2788. C: B-cells as measured by the number of B220-positive cells counted in three representative 20X fields throughout each section, p = 0.3527. D: Macrophage and microglia infiltration as measured by the percentage of nerves positive for the anti-Iba1 signal, p = 0.8583. E: Neutrophil infiltration scored on a scale of 0–3 for the anti-Ly6G signal where 0 = no signal, 1 = 1–15 cells, 2 = 16–30 cells, and 3 = more than 30 cells, p = 0.0540. F: The ratio of the Ly6G:Iba1 values for each optic nerve, p = 0.0050. All comparisons made with a linear mixed effect model. Testing for B220, CD3, and CNPase effects was performed on a log scale. Error bars represent standard deviation (SD). n = 20 eyes from ten mice per group.

Figure 6

Number of RGCs correlates with visual acuity, and RGC death occurs in Th17- but not Th1-induced EAE. A: Representative photomicrograph of a retinal flatmount immunostained with anti-Brn3a. White boxes (labeled 1–12) indicate representative fields taken from peripheral (1, 4, 7, 10), medial (2, 5, 8, 11), and central (3, 6, 9, 12) regions of each quadrant. Automated retinal ganglion cell (RGC) counts in the enhanced fields (shown in surrounding pictures) were performed in Image J. B: Linear regression of the average optokinetic reflex (OKT) on the total number of RGCs counted in the 12 representative retinal fields throughout the study for each Th1 (red circles, p = 0.0396) and Th17 (blue squares, p = 0.0023) experimental autoimmune encephalomyelitis (EAE) eye at 13 and 21 days post-immunization (DPI) indicating significant positive correlations. n = 20 eyes from ten mice per group. C: Average total RGCs counted in each retinal region of all eyes at 13 and 21 dpi for Th1 (red middle bars), Th17 (blue right bars), and healthy, age- and strain-matched control mice (purple left bars). n = 20 eyes from ten mice per group. D: Average total regional RGCs counted in the more affected eyes of each EAE mouse at 21 dpi for Th1 (red middle bars), Th17 (blue right bars), and healthy, age-, and strain-matched control mice (purple left bars). n = 5. Error bars represent standard error of the mean (SEM). *p<0.05 by the linear mixed effect model.