Figure 2. NLRP3 inflammasome activation in non-RPE cells but not in RPE cells promotes CNV.

(A) Nuclear Cre immunostaining (white) in choroidal flat mounts from 6-weeks-old Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mice confirms uniform Cre expression in the RPE. Inset shows higher magnification image. No Cre staining is seen in Vegfa^hyperCre^negNlrp3^A350V/A350V mice ( = Vegfa^hyperNlrp3^-/- mice). Scale bars, 200 μm. (B-C). CNV lesion numbers and average CNV lesion area (in μm²/lesion) were measured in 6-weeks-old mice. Vegfa^hyperNlrp3^-/- mice (group 2) and Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mice (group 3) show significantly decreased CNV lesion numbers compared to Vegfa^hyper mice (group 1), but no significant difference in CNV lesion sizes. CNV lesion sizes and numbers between Vegfa^hyperNlrp3^-/- mice and Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mice are similar. No marked differences in lesion sizes or lesion numbers were observed among male or female groups of Vegfa^hyperNlrp3^-/- mice and Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mice. Vegfa^hyperCasp1^-/-Casp11^-/- mice (group 4) have fewer CNV lesions than Vegfa^hyperNlrp3^-/- mice or Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mice. Values represent total CNV lesion numbers/eye for each mouse and average lesion area/eye for each mouse. Absolute numbers of mice per group are indicated in parentheses. Graphs show mean ± SEM. P-values are shown and were determined by a Kruskal-Wallis test followed by a Dunn’s test.

Figure 2—figure supplement 1. RPE-specific Cre expression in Best1^Cre/+ mice mediates the removal of floxed alleles in the RPE.

(A) A choroidal flat mount of a Best1^Cre/+ mouse is shown (Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mouse) that was labeled with phalloidin-Alexa488 (green) and with an anti-Cre antibody (white). Nuclear staining was performed with DAPI. Phalloidin staining (green) highlights cell membranes and shows the honeycomb pattern of RPE cells (white arrows). Best1 expression occurs in the eye only in RPE cells, which can be unambiguously identified by their hexagonal cell structure and the doublet cell nuclei (yellow arrows). Nuclear Best1-specific Cre signal (white; red arrows) is only seen in RPE cells. Scale bars, 20 µm. 6-weeks-old mouse. (B) Nuclear Cre⁺ immunolabeling (white, yellow arrow) is seen in RPE cells of Best1^Cre/+ mice, outlined by immunolabeling for active β-catenin (red). Scale bar, 20 µm. 6-weeks-old mouse. (C) Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mice show Cre-mediated removal of the floxed neoR cassette in their RPE cells. RPE cells were isolated from the eyes of an adult mouse of each strain: WT, Vegfa^hyper, Vegfa^hyperNlrp3^-/-, and Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V. PCR result of isolated genomic DNA from RPE cells using primers that flank the floxed neoR cassette in intron 2 of Nlrp3 shows the presence of a PCR product without the neoR cassette (Nlrp3^A350V allele from the subset of RPE cells in which Cre was active) in addition to a PCR product containing the neoR cassette (Nlrp3^-/- allele from the subset of RPE cells in which Cre was not active) in Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mice. In contrast, RPE cells from Vegfa^hyperNlrp3^-/- mice without the Cre show only the PCR product containing the floxed neoR cassette in intron 2 of the Nlrp3 gene (Nlrp3^-/- allele). RPE cells from WT and Vegfa^hyper mice show neither PCR product (only WT allele is seen). This demonstrates that Cre recombinase is not only expressed in the RPE of Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mice but also active and mediates Cre-mediated excision of floxed alleles that lead to the Nlrp3^A350V/A350V mutant alleles. Here, result for a Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mouse is shown where Cre activity occurs only in a subset of RPE cells to demonstrate the size difference between the mutant alleles with and without the neoR cassette, but for CNV lesion quantifications we only used eyes of Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mice that showed uniform Cre-expression in the RPE (confirmed by immunolabeling for Cre). No Cre-mediated excision of the floxed neoR cassette was observed in the retina, lens, or choroid in eyes of the identical mice and only the Nlrp3^-/- allele was detected in these other eye tissues of Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mice and Vegfa^hyperNlrp3^-/- mice, consistent with an RPE-specific Cre activity in eyes of Vegfa^hyperBest1^Cre/+Nlrp3^A350V/A350V mice. 2% agarose gel is shown.