gms | German Medical Science

Background: Age-related macular degeneration (AMD) – a leading cause of blindness in the western world – is characterized by chronic inflammation, mediated by a loss of immune barrier function of the retinal pigment epithelium (RPE) and the infiltration of monocytes into the retinal space. A risk variant of complement factor H (CFH) and the deletion of the related proteins FHR1 and FHR3 are highly associated with AMD. While the CFH risk variant showed increased alternative complement pathway activation on surfaces, the deletion of FHR1 is protective (Data et al., 2017), but its molecular role in AMD is still unclear. Recent studies showed that by binding to necrotic cell surfaces in atherosclerosis, FHR1 triggers systemic sterile inflammation via monocyte activation (Irmscher et al., 2019) which could be a potential mechanism also in AMD.

Methods: Using immunohistochemistry, we visualized muFHR1 (FHR1 mouse homologue), its corresponding receptor Emr1 (EMR2 mouse homologue), evaluated cellular inflammation (quantifying Iba1+ cells, pro-inflammatory factors) in mice models of AMD (mouse laser induced choroidal neovascularization in WT and muFHR1-/- mice, Cx3CR1GFP/GFP mice, TRE2 model). Furthermore, we labelled AMD human donor eye tissue with antibodies directed against FHR1. To determine the FHR1-EMR2 pathway, we performed in vitro experiments of Ca2+ imaging, qPCR, and immunoblotting.

Results: In all mouse models with AMD relevance, we observed robust Emr1 expression within both RPE and Iba1+ cells. Strikingly, the expression of Emr1 was not restricted to necrotic/apoptotic cells but also found in structurally normal cells. We detected muFHR1 accumulation in proximity to Emr1 – likely forming protein-receptor complexes. Cx3CR1GFP/GFP mice revealed an increase in Emr1-muFHR1 staining with progressing age, underscoring the dynamic nature of Emr1 in pathogenesis of inflammation. Iba1+ cells bound via Emr1-muFHR1 complexes to RPE cells. Accordingly, a significant decrease of Emr1+ cells (both RPE and Iba1+ cells) was observed in muFHR1-/- animals upon laser CNV. FHR1 reactivity was observed in Bruch’s membrane punctae, basal laminar deposits and rare RPE cells of AMD patients, supporting the relevance of FHR1 in human AMD. A distinct intracellular second-messenger signaling in RPE cells observed in in vitro experiments that involved increases in intracellular free Ca2+ with subsequent cytokine secretion could explain the activation via EMR2-FHR1.

Conclusion: Our data suggest a dominant role of FHR1/E in AMD as: i) EMR2/1 receptor activation through FHR1/E significantly contributes to parainflammation; ii) not only, but already stressed cells enter the FHR1/E signaling; iii) FHR1/E permits physical interaction between Iba1+ cells and RPE; iv) FHR1/E contributes to the Iba1+ cells accumulation in the retina, and ignites intracellular second-messenger signaling with the potential change of RPE’s functional phenotype.