IL-10- and IL-35-expressing cells are highly enriched among peripheral blood B cells during experimental autoimmune uveitis

Abstract

Purpose : IL-10 and IL-35 are regulatory cytokines that suppress host inflammatory immune responses. IL-35- and IL-10-producing regulatory B cells (Bregs) were recently found to play essential roles in negatively regulating development of experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (Nature Med. 20:633, 2014, Nature 507:366, 2014). However, it is not clear whether they migrate to peripheral tissues to directly modulate inflammatory response and how they are important in IL-10 and IL-35 production in comparison with T cells and monocytes. In this study, we have investigated the source of Breg cells and whether they traffic to peripheral retina during EAU.

Methods : EAU was induced in C57BL/6J mice by active immunization with IRBP (interphotoreceptor retinoid-binding protein) in CFA (complete Freund’s adjuvant). EAU progression was monitored by fundoscopy and histology. Single cell suspensions isolated from retina, peripheral blood, dLN (draining-lymph-nodes), and spleen were used for FACS to detect the expression of CD3, CD4, CD19, B220, CD11b, CXCR4, CD1d, CD5, CD38, CD138, IL-12p35, EBI3, IL-10, IFN-g, IL-17, and Foxp3. CD19+ B cells isolated from unimmunized and EAU-immunized mice or human healthy PBMC (peripheral blood mononuclear cells) were stimulated with rIL-35 and detection of IL-35- and IL-10-expressing Bregs was by FACS, RT-PCR, ELISA, and/or JAK-STAT gene-array.

Results : We show that the percentages of IL-10-expressing cells increased by 2-times from 1% to 3% of total PBMC during EAU. IL-10-expressing B cells in PBMC were 5-10 times higher compared to T cells or monocytes. In addition, IL-10- and/or IL-35-expressing cells were highly enriched in B cells in dLN and spleen. Interestingly, retinal-infiltrated B-cells were predominantly CD19⁺CD138^HiCD38^HiCD1d^Hi CD44^HiCXCR4^Lo. We also demonstrated that human IL-35 induced IL-35 and/or IL-10-expressing Breg cells.

Conclusions : Peripheral B-cells are important producers of IL-10 and IL-35 in vivo and Breg cells suppress uveitis through production of these anti-inflammatory cytokines.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.