The Role of Retinal Antigen-Presenting Cells in Spontaneous Retinal Autoimmunity

Abstract

Purpose: We reported earlier that induction of spontaneous autoimmune uveoretinitis (SAU) correlated with the recruitment of circulating antigen-presenting cells (APCs) into the retina. Here, we investigated the role of resident retinal APCs on the course of SAU.

Methods: R161H+/- mice (B10.RIII), which spontaneously and rapidly develop severe autoimmune uveoretinitis, were crossed with CD11cDTR/GFP mice (B6/J). R161H+/- mice on the B6/J background develop slower, less severe SAU than R161H+/--B10.RIII mice, allowing assessment of disease development relative to the depletion or activation of retinal or systemic APCs. The course of SAU was established in a cohort of control R161H+/- × CD11cDTR/GFP F1 mice and then reanalyzed in test cohorts following treatment with diphtheria toxin or stimulation by optic nerve crush (ONC) injury. Analysis was done by retinal imaging, flow cytometry, and histology.

Results: Systemic depletion of APCs halted the progression of SAU in R161H+/- × CD11cDTR/GFP F1 mice and, if commenced early in the disease process, would reduce SAU severity. However, following depletion of APCs specifically from the retina, SAU in R161H+/- × CD11cDTR/GFP F1 mice progressed in a manner similar to that of control mice. In contrast, SAU in R161H+/- × CD11cDTR/GFP F1 mice was exacerbated following the activation of retinal APCs by ONC.

Conclusions: Our observations that local depletion of retinal APCs failed to inhibit SAU progression and that depletion of circulating APCs not only limited SAU progression but also, under defined circumstances, reduced clinical SAU support the idea that circulating APCs are crucial for the induction and progression of SAU.

Figure 1.

Course of pathology in R161H^+/− mice. Mouse retinas at the indicated age were evaluated by clinical (fundoscopy) and histopathological analysis and scored for SAU. (A) Representative images of clinical and histopathological SAU at 40 days of age in R161H^+/− × CD11c^DTR/GFP F₁ mice. Arrows on histology panels indicate specific areas of inflammatory infiltrate or photoreceptor cell damage. (B) Collective SAU pathology scores with incidence of SAU (SAU-positive eyes/total eyes examined) indicated for each time point. For comparison of R161H^+/− × CD11c^DTR/GFP F₁ mice at different days: ns, not significant; *P < 0.0001 for clinical incidence and severity and P < 0.001 for histopathological incidence and severity. For comparison of R161H^+/− × CD11c^DTR/GFP F₁ mice to R161H^+/−–B10.RIII mice, P < 0.01 for severity at any day of age and for incidence at days 30 and 40 of age.

Figure 2.

Depletion of retinal GFP^hi cells from CD11c^DTR/GFP mice given an ONC following a single intraocular (2 ng) or a single systemic injection (100 ng) of DTx. (A) Representative fundoscopy images of CD11c^DTR/GFP mice given a bilateral ONC and then injected with DTx at 10 days post-ONC (day 0). (B) Representative fluorescence-activated cell sorting (FACS) plots of retinal CD45^medCD11b⁺ cells analyzed for GFP expression. (C) Collective FACS analysis for GFP^hi cell levels in retinal CD45^medCD11b⁺ cells. GFP^hi cell levels are expressed as percent GFP^hi cells observed in control (no DTx) CD11c^DTR/GFP mice 20 days post-ONC (day 10 post-DTx injection). (D) Percent of CD11b⁺ cells in blood 3 days post-DTx injection that are GFP^hi in control (no DTx) and intraocular-injected CD11c^DTR/GFP mice. For C and D, GFP^hi cell levels are given as mean ± SD (n ≥ 3 for all groups).

Figure 3.

Inhibition of SAU progression in R161H^+/− × CD11c^DTR/GFP F₁ mice following systemic depletion of APCs. (A) Collective clinic SAU scores (mean ± SD) for control (no DTx, blue) and systemic APC-depleted (test, red) cohorts. Incidence of SAU/total number of eyes for each group is indicated. (B) Percent increase (mean) in clinical SAU incidence (I) and severity (S) from age day 40 to age day 50 in the control and systemically depleted cohorts. (C) Comparison of histopathological SAU in control and systemically depleted cohorts. P values are indicated.

Figure 4.

Depletion of circulating APCs at the time of acute, early SAU reduces the disease. Cohorts of R161H^+/− × CD11c^DTR/GFP F₁ mice with moderate SAU at day 30 were given a single injection of DTx (100 ng) on day 30 and reexamined on days 40 and 50 (test, red) or not given DTx and reexamined on days 40 and 50 (control, blue). (A) Representative fundus and histopathology images from a test mouse showing reduction of clinical SAU and loss of infiltrating GFP^hi cells between days 30 and 50. Histopathology image shows a trace amount of inflammatory infiltrate (arrow) but overall lack of SAU pathology. (B) Cumulative SAU pathology scores. SAU scores are given as mean ± SD. *P < 0.015, **P < 0.001; n.s., not significant.

Figure 5.

Depletion of retinal APCs does not inhibit SAU. R161H^+/− × CD11c^DTR/GFP F₁ test mice (red) were analyzed for clinical SAU at 40 days of age, given a single interocular injection of 2 µL (2 ng), reanalyzed for SAU at 50 days of age, and compared to control R161H^+/− × CD11c^DTR/GFP F₁ mice (blue, non-injected). The incidence of SAU/total number of eyes for each group is indicated under the group label. Scores are given as mean ± SD. P values are indicated for incidence (i) and severity (s) of SAU.

Figure 6.

Enhancement of SAU in R161H^+/− × CD11c^DTR/GFP F₁ mice following unilateral ONC injury. (A) Representative clinical (fundoscopy) and histopathological images of the retinas from a single mouse at 30 days of age (pre-ONC) and 40 days of age (10 days post-ONC). Scoring of representative pathology is indicated. Arrows on histology panels indicate specific areas of pathology. (B) Collective SAU incidence and pathology scores for R161H^+/− × CD11c^DTR/GFP F₁ control (blue) and test (ONC, red) mice. *P < 0.02 for incidence and P < 0.01 for severity.