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Jennifer A E Williams, John Greenwood, Judy Latcham, Peter S Adamson, Stephen E Moss; Comparative analysis of retinal phenotype in Cfh-/-, Cfb-/- and Cfh-/-Cfb-/- double knock-out mice. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3450. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Single nucleotide polymorphisms in genes of the alternative complement pathway are linked to susceptibility to developing age-related macular degeneration. The Y402H mutation in complement factor H (CFH) predisposes to the disease whereas the R32Q mutation in complement factor B (CFB) is protective. The purpose of this study was to use Cfh-/- and Cfb-/- mice, together with the Cfh-/-Cfb-/- double knock-out mouse, to investigate the functional interplay between CFH and CFB in the context of retinal phenotype.
Retinas were isolated from wild type (WT), Cfh-/-, Cfb-/-, Cfh-/-Cfb-/- and Cfh-/-Cfb+/- mice. Retinal morphology was assessed using toluidine blue stained semi-thin sections. Fixed frozen sections were stained with antibodies to C3 and the C3 breakdown products C3b, iC3b and C3c. Neuroretinal whole mounts were stained with collagen IV to visualise the retinal vessels and F4/80 for microglia/macrophages.
WT mice showed C3 staining in the retinal vasculature and along the basal surface of the retinal pigment epithelium (RPE) where C3 breakdown products were also detected. Cfb-/- mice also exhibited C3 staining in the retinal vasculature but less C3 staining along the basal surface of the RPE compared to WT and no accumulation of C3 breakdown products. Loss of CFH caused secondary depletion of C3 from the serum, and in the retina staining for C3 and its breakdown products was absent along the basal surface of the RPE. Deletion of both Cfb and Cfh in Cfh-/-Cfb-/- double knock-out mice restored C3 to levels similar to those observed in WT mice, however this reversal of phenotype was not observed in Cfh-/-Cfb+/- mice. Cfh-/-Cfb-/- mice differed from WT mice in having very low C3 breakdown products at the basal surface of the RPE. Overall the retinal morphology, retinal vasculature and number of infiltrating macrophages did not appear different across the 5 different genotypes.
C3 accumulates at the basal surface of the RPE in WT, Cfb-/- and Cfh-/-Cfb-/- mice, but is absent in Cfh-/- and Cfh-/-Cfb+/- mice, consistent with its consumption in the serum of mice lacking CFH when CFB is present. The partial or complete absence of CFB in Cfh-/-Cfb+/- and Cfb-/- mice was associated with a lack of C3 breakdown products at the basal RPE suggesting that the C3b, iC3b and C3c observed in WT mice are correlates of an active and functionally normal alternative pathway.
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