The rd8 mutation in the Crumbs1 gene upregulates activation markers on retinal and brain microglia and CD11c-eYFP⁺ cells

Abstract

Purpose : 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 Crb1^rd8 mutation causes distinctive retinal degenerative lesions that may affect the immunophenotype of resident leukocytes. We hypothesized that the Crb1^rd8 mutation would upregulate antigen presentation and/or activation markers on brain and retinal microglia and CD11c-eYFP⁺ cells.

Methods : C57Bl/6N CD11c-eYFP mice (Crb1^rd8/rd8) were outcrossed with C57Bl/6J mice to generate CD11c-eYFP Crb1^wt/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.

Results : The density of Iba-1⁺ microglia and CD11c-eYFP⁺ cells in the retina and sub-retinal space did not differ between CD11c-eYFP Crb1^rd8/rd8 mice and CD11c-eYFP Crb1^wt/wt mice (p > 0.05); however, these cells displayed a more amoeboid morphology in the retina of CD11c-eYFP Crb1^rd8/rd8 mice. A significantly higher percentage of retinal CD11c-eYFP⁺ cells from CD11c-eYFP Crb1^rd8/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 Crb1^wt/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 Crb1^rd8/rd8 mice compared to CD11c-eYFP Crb1^wt/wt mice. Within the choroid plexus and pia mater, mice carrying the Crb1^rd8 mutation showed an increased frequency of CD11c-eYFP⁺ cells expressing I-A/I-E, CD103, CD8α and DEC205 (p < 0.05). In contrast, the Crb1^rd8 mutation did not affect the phenotype of splenic CD11c-eYFP+ cells (p > 0.05).

Conclusions : The Crd1^rd8 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 Crb1^rd8 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.