Cx3cr1 Deficiency Alters Cell Replenishment Rates in BALB/c Mice but Does Not Affect Disease Onset and Severity of EAU in C57Bl/6 Mice

Abstract

Purpose: : Cx3cr1, expressed by monocyte-derived cells including macrophages and dendritic cells, mediates leukocyte migration and adhesion in homeostatic and inflammatory conditions. This study examined the role of Cx3cr1 in the rate of replenishment of monocyte-derived cells in the uveal tract and retina during ‘homeostatic’ conditions, and in the course of Experimental Autoimmune Uveoretinitis (EAU). It was hoped this model would also allow us to determine the origin of the monocytic infiltrate in the retina in EAU.

Methods: : Chimeras were created whereby bone marrow (BM) from BALB/c Cx3cr1^+/gfp (heterozygous) or Cx3cr1^gfp/gfp (homozygous) transgenic mice (in which one or both copies of the Cx3cr1 gene have been replaced by the eGFP reporter gene) was used to reconstitute irradiated WT BALB/c mice. Donor eGFP⁺ cell densities were compared in the chimeras at wks 4 and 8 post-transplantation. To induce EAU, WT, Cx3cr1^+/gfp and Cx3cr1^gfp/gfp C57Bl/6 mice were administered a subcutaneous injection of IRBP_1-16 in CFA, with simultaneous i.p injection of pertussis toxin (PTX). Mice were sacrificed 14, 16, 21 and 28 days post injection and eyes were embedded in Glycol Methacrylate and serial sections cut along the pupillary-optic nerve axis. Sections were graded for disease using a customised histopathology grading system. Immunohistochemistry was performed on retinal wholemounts using a range of anti-leukocyte phenotypic markers and analysed by confocal microscopy.

Results: : In BM chimeras, there were significantly less Cx3cr1^gfp/gfp donor cells when compared to Cx3cr1^+/gfp donor cell densities in the iris stroma at wk 4, and in the posterior iris and choroid at wk 8 post-reconstitution. In the retina, replenishment appeared similar in mice that had received either Cx3cr1^+/gfp or Cx3cr1^gfp/gfp bone marrow. During EAU, disease scores in WT, Cx3cr1^+/gfp and Cx3cr1^gfp/gfp mice did not differ significantly, however, a higher incidence of disease and trend for greater disease severity in Cx3cr1^+/gfp and Cx3cr1^gfp/gfp mice was evident at day 28 when compared to WT mice.

Conclusions: : Whilst the absence of Cx3cr1 alters the rate of replenishment of monocyte-derived cells in the iris and choroid in BM chimeras, Cx3cr1 does not significantly influence disease onset or severity of EAU in Cx3cr1^+/gfp or Cx3cr1^gfp/gfp versus WT mice.

Keywords: uveitis-clinical/animal model • cytokines/chemokines • autoimmune disease