Quantitative Infiltration of Pro-Inflammatory M1 Macrophages Into the Outer Retina Marks the Early Onset of Immune-Mediated Age-Related Macular Degeneration

Abstract

 

Purpose:

 

Recent evidence suggests a role for the immune system in the pathogenesis of age-related macular degeneration (AMD). Macrophages are important effectors of innate immune responses, but their role in AMD remains poorly defined. We aimed to study macrophage recruitment and activation in the retina in a mouse model of immune-mediated AMD at the earliest stages of disease onset, prior to overt retinal damage.

 

Methods:

 

Wild type C57BL/6, WT BALB/c and Ccr2-/- mice were immunized with carboxyethylpyrrole-adducted mouse serum albumin (CEP-MSA). CEP antibody titers were measured by ELISA at different time points post-immunization. At harvest, eyes were prepared for histology and immunohistochemistry while spleens were removed to perform ex vivo stimulation with CEP-MSA. For control comparisons we used mice immunized with sham-adducted MSA or age-matched naïve mice. At least 3 mice were analyzed per group at each time point.

 

Results:

 

CEP autoantibody levels correlate with retinal damage and ex vivo T cell activation. We find a statistically significant and time-dependent increase in the number of macrophage-like infiltrate cells into the rod outer segment (ROS) and the retinal pigment epithelium (RPE) in immunized mice relative to age-matched controls at a young age (3-6 months), most noticeably in the BALB/c background, before extensive damage to the retina. Importantly, old BALB/c immunized mice (after 1.5 yr or older) show signs of geographic atrophy, a hallmark of dry AMD, and extensive loss of photoreceptor cells. Immunohistochemical analysis showed M1 (IL-12-producing) activated macrophages present within the ROS in close proximity to the RPE, but only in immunized mice. Finally, Ccr2-/- mice show a lack of infiltration after immunization.

 

Conclusions:

 

Our data provide strong mechanistic evidence linking immune responses against oxidative damage (CEP) with early presence of macrophages at sites of future AMD and may help to clarify the role of these cells in AMD pathology. Because M1 macrophages are associated with tissue damage, it is likely that these cells are responsible for the retinal degeneration observed in our model. We propose the use of quantitative macrophage recruitment as an early marker of AMD to study the molecular events associated with disease onset as well as potential therapeutic agents.

 

Keywords: age-related macular degeneration • inflammation • autoimmune disease