Abstract
Purpose :
Interleukin 27 (IL-27) is an immunosuppressive cytokine that is constitutively expressed by retinal cells and inhibits experimental autoimmune uveitis (EAU), a mouse model of human uveitis (PNAS. 2021, 118(47):e2109548118). Innate B-1a cells have recently been shown to secrete i27-exosomes (Front Imm. 14: 1071162). In this study we investigated whether retina microglia could suppress uveitis by secreting i27-exosomes.
Methods :
We induced EAU in C57B/6J mice by immunization with IRBP651-670 peptide in CFA. Eyes from EAU mice were perfused, retinas were isolated and enzymatically digested. CD11b+ cells were cultured in L929-supplemented media for 10 days and additional 5 days in exosome-free media containing M-CSF. Microglia cells were identified and confirmed as CD11b+CX3CR1+ cells by FACS analysis. Exosomes were isolated from supernatants of microglia cells cultured in exosome-free media. Apoptotic bodies and cell debris were removed by sequential centrifugation at 300g, 3000g and 12000g. Exosomes were prepared by ultracentrifugation for 120 mins at 110,000g (2x). The pellet was re-suspended in PBS and size distribution of the exosomes was determined by NanoSight tracking analysis. i27-exosomes were validated by Western blot analysis and RNA-seq analysis. EAU mice were treated daily with exosomes (20x109) from day-6 to day-10 and disease was assessed by fundoscopy and optical coherence tomography.
Results :
We show that the CD11b+CX3CR1+ microglia cells secrete i27-exososomes and Western blot confirmed that they express CD81 tetraspanin protein (marker of lymphoid exosomes) and heterodimeric IL-27 (comprised of IL27p28 and Ebi3). Treatment with i27-exosomes ameliorated uveitis by inhibiting expansion of Th17 cells.
Conclusions :
Previous reports showed that IL-27 is constitutively expressed in retina and suppresses uveitis by upregulating IL-27 (Nat Med. 13:711-718; Immunology. 132:492-502). However, producers of IL-27 in the retina were unknown. This study reveals for the first time that microglia constitutively produce IL-27 and secretes i27-exosomes. Suppression of uveitis without alloreactivity or toxicity makes i27-exosomes an attractive therapeutic option for autoimmune uveitis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.