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Samantha Dando, Cecilia Naranjo-Golborne, Holly Chinnery, Marc Ruitenberg, Paul G McMenamin; The rd8 mutation in the Crumbs1 gene upregulates activation markers on retinal and brain microglia and CD11c-eYFP+ cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3299. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The rd8 mutation in the Crumbs1 (Crb1) gene is present in C57Bl/6N-derived mouse lines, including CD11c-eYFP transgenic mice. Crb1 is expressed exclusively in the retina and brain of mice, and the Crb1rd8 mutation causes distinctive retinal degenerative lesions that may affect the immunophenotype of resident leukocytes. We hypothesized that the Crb1rd8 mutation would upregulate antigen presentation and/or activation markers on brain and retinal microglia and CD11c-eYFP+ cells.
C57Bl/6N CD11c-eYFP mice (Crb1rd8/rd8) were outcrossed with C57Bl/6J mice to generate CD11c-eYFP Crb1wt/wt mice. At 8 weeks of age, eyes and brains were collected from naïve perfusion-fixed mice (n = 4 - 6) and tissues were processed for confocal microscopy. Single cell suspensions of retina, brain parenchyma (cortex), choroid plexus, pia mater and spleen (n = 7) were analysed by multi-colour flow cytometry; the percentage of positive cells was compared between mouse strains using Chi-square tests.
The density of Iba-1+ microglia and CD11c-eYFP+ cells in the retina and sub-retinal space did not differ between CD11c-eYFP Crb1rd8/rd8 mice and CD11c-eYFP Crb1wt/wt mice (p > 0.05); however, these cells displayed a more amoeboid morphology in the retina of CD11c-eYFP Crb1rd8/rd8 mice. A significantly higher percentage of retinal CD11c-eYFP+ cells from CD11c-eYFP Crb1rd8/rd8 mice expressed CD11c (19% vs. 6.2%) and MHC class II (I-A/I-E, 34.6% vs. 4.8%) compared to CD11c-eYFP Crb1wt/wt mice (p < 0.05). Expression of these markers was also significantly upregulated in cortex CD11c-eYFP+ cells from the same mice (CD11c: 23.1% vs. 11.3%; I-A/I-E: 32.0% vs. 7.4%; p < 0.05). Retinal and cortex microglia also demonstrated an upregulation of CD11c and I-A/I-E in CD11c-eYFP Crb1rd8/rd8 mice compared to CD11c-eYFP Crb1wt/wt mice. Within the choroid plexus and pia mater, mice carrying the Crb1rd8 mutation showed an increased frequency of CD11c-eYFP+ cells expressing I-A/I-E, CD103, CD8α and DEC205 (p < 0.05). In contrast, the Crb1rd8 mutation did not affect the phenotype of splenic CD11c-eYFP+ cells (p > 0.05).
The Crd1rd8 mutation upregulates activation markers on CNS microglia and CD11c-eYFP+ cells within the retina, brain parenchyma, choroid plexus and pia mater, but not spleen. Thus, mice carrying the Crb1rd8 mutation should be used with caution in neuroimmunological studies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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