Evidence for Anti-Retinal Auto-Antibodies (AAbs) in the Complement Factor H Chimeric Transgenic (Cfh-Tg) Mouse Model of Age-Related Macular Degeneration (AMD)

Abstract

Purpose: To determine if Cfh-Tg mice harboring the H⁴⁰² Cfh variant (Ufret-Vincenty et al. IOVS 2010; 51: 5878-87) develop circulating AAbs recognizing ocular tissue antigens.

Methods: Methods: We compared sera by Western blots (WBs) from 4 groups: 1) Cfh-Tg expressing the AMD-predisposing H⁴⁰² Cfh variant (H-Cfh-Tg); 2) H-Cfh-Tg co-expressing the wild-type (WT) transgene of human C-reactive protein (CRP), which interacts with Cfh in inflammation (H-Cfh-Crp-Tg); 3) mice co-expressing the WT Cfh variant (Y⁴⁰²) and the WT Crp gene (Y-Cfh-Crp-Tg); and 4) control adult C57BL/6 mice. Adult C57BL/6 mouse retina/RPE/BM/choroid tissue lysate (10 µg) was loaded on gels, incubated with serum (5µL) and developed. WB band intensity was quantified with the Odyssey system and compared by Kruskall-Wallis test. Immunohistochemistry was done against anti-mouse IgG Ab by fluorescence microscopy following incubation of adult C57BL/6 retina sections with mouse sera. To identify the autoantigens, mouse sera were immunoprecipitated, followed by 2-dimension electrophoresis (2DE) and Mass-Spectrometry (MS) was performed on spots seen on 2DE following previously reported methods (Lenchik et al. ARVO 2013, Abs. 4103).

Results: H-Cfh-Tg mice showed consistently the highest reactivity compared to controls, Y-Cfh-Crp-Tg and H-Cfh-Crp-Tg. Significantly more intense WB bands were seen in H-Cfh-Tg mice at 91kDa (p=0.001), 17kDa and 13kDa (p=0.004), 31kDa (p=0.012), 54kDa (p=0.039), and 22kDa (p=0.045). The outer nuclear (ONL), inner nuclear (INL), and ganglion cell (GCL) layer of adult C57BL/6 mouse retina sections showed strong staining, with an apparent perinuclear pattern, by both H-Cfh and H-Crp-Cfh sera. Differentially reactive spots between H-Cfh-Tg and control mouse sera were observed also on 2DE and preliminary IDs for some of the spots have been obtained via MS, suggesting that antigens involved in apoptotic mechanisms are targeted.

Conclusions: Similar to what we have seen in human AMD sera (Iannaccone et al. Adv. Exp. Med. Biol. 2012; 723:11-6), AAbs recognizing retinal targets develop also in this allele-specific AMD mouse model. This suggests the existence of an autoimmune response to AMD-like retinal degenerative events triggered by the H-Cfh variant, which could be contributing to the pathology observed in the H-Cfh-Tg AMD model as well as in human AMD.

Keywords: 412 age-related macular degeneration • 557 inflammation • 555 immunomodulation/immunoregulation