Figure 4.

Oral antibiotic-treated mice have reduced retinal vascular disruption and increased Tregs in the eye. Lectin staining of the retinal vasculature from (A) PO H₂O immunized mouse showing marked retinal endothelial cell disruption compared with (B) PO Abx mice. Immunofluorescently labeled Foxp3+, CD4+ cells are more abundant in antibiotic-treated mouse retinas (D) compared with (C) water-fed mice, at 3 weeks after immunization. (E) High-magnification views of Foxp3+ and negative CD4+ cells from white boxes shown in (C) to confirm localization of staining. (F) Mann-Whitney U test of Foxp3+ cell proportions (of total CD4+ cells) in water versus antibiotic-treated mouse retinas from retinal whole mounts using Image J quantitation could be performed only at week 4 (n = 5), whereas only two mice at weeks 2 and 3 had enough inflammation to count cells. (G) Flow cytometric analysis of Foxp3+, CD4+ cells from whole eye preparations of water-fed versus antibiotic-fed EAU mice at 2 weeks after immunization. (H) Mann-Whitney U test of Foxp3+ cell proportions (of total CD4+ cells) in water versus single-antibiotic–treated mice from retinal whole mounts using Image J quantitation. (neomycin is not shown because of poor image quality due to severe inflammation); *P < 0.05, n = 2 to 5 per treatment group (± SEM shown); total cell counts are shown rather than percentages in (G). Statistics in (G) could not be performed because data were obtained from pooled eyes from n = 3 mice and run in duplicate.