Lack of Goblet Cells in SPDEF KO Mice Increases Retention of IL-12 Producing Dendritic Cells in Conjunctiva

Abstract

Purpose : Goblet cell (GC) mucins have been found to condition tolerogenic properties in intestinal dendritic cells (DCs). Aqueous tear deficiency is associated with GC loss and greater IFN-γ expression in the conjunctiva (Cj). We hypothesize that loss of Cj GCs is associated with greater dendritic cell T helper (Th)1 polarization in the SPDEF knock-out (KO) strain that lacks GCs.

Methods : Six to nine week-old female C57BL/6 wild type (WT) and SPDEF KO mice were used. CD11c⁺ and CD11b⁺ DCs were visualized in whole mount Cj by laser scanning confocal microscope using the Z-stack option. IL-12 expression in DC was quantified by flow cytometry and IL-12⁺ cells were also detected by immunostaining in Cj cryosections. Mixed lymphocyte reactions to evaluate T helper (Th) cell polarization and proliferation were performed with OVA _323-339 peptide pulsed cervical lymph node (CLN) cell suspensions and OT II CD4⁺ T cells. Th cytokine expression (IFN-γ and IL-17) was measured by flow cytometry and immunobead assay. The effects of WT conjunctival conditioned medium (CJCM) on lipopolysaccharide (LPS) -stimulated IL-12 expression in the Cj was evaluated after topical application in SPDEF KO mice.

Results : SPDEF KO had greater frequency of CD11b⁺CD11c^- (p=0.01) and CD11b^-CD11c⁺ conjunctival DCs and CD11b^-CD11c⁺ DCs were more superficially located in WT mice. Immunohistochemistry demonstrated that SPDEF KO mice had a greater number of IL-12⁺ cells in Cj stroma than WT group. A greater percentage of CD11b⁺CD11c^- and CD11b⁺CD11c⁺ cells (p ≤ 0.03) with higher IL-12 median fluorescent intensity (MFI) (both p ≤ 0.03) in the Cj compared to WT control was demonstrated by flow cytometry. Th cells primed by SPDEF KO CLN DCs showed a skewing towards Th1 and generated a lower frequency of CD4⁺Foxp3⁺ cells. Significantly increased IFN-g was measured in the supernatant of these cells (p=0.03). Following topical LPS stimulation, SPDEF KO mice had higher IL-12 expression in CD11b⁺CD11c⁺ Cj DCs than the control group (p=0.02) and reconstitution with topically applied WT CJCM reduced stimulated IL-12 production in CD11b⁺CD11c⁺ cells compared to vehicle control (p=0.04).

Conclusions : Our results support our hypothesis that CJ GCs have a tolerogenic effects on DCs by suppressing IL-12 production and Th1 polarization.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.