Abstract
Purpose :
Goblet cells (GCs) are in close proximity to dendritic cells (DCs) in the conjunctival epithelium and stroma. We hypothesized that GCs produce retinoic acid (RA) that modulates cytokine signaling in DCs.
Methods :
Primary murine conjunctival and corneal epithelia grown from explants were cultured for 7 days or added diethylaminobenzaldehyde (DEAB) on day 5, then switched to IMDM for 4 days when conditioned media from conjunctiva (CjCM) and cornea (KCM) were collected. Bone marrow-derived dendritic cells (BMDCs) were cultured for 6 days in IMDM, then conditioned for 2 days with CjCM or exogenous RA. Quantitative real-time PCR (RT-PCR) and ALDEFLUOR activity assay were performed to compare ALDH1 expression and ALDH (aldehyde dehydrogenase) activity. RA concentration in supernatants from cultures with or without retinol palmitate（RP）supplementation was detected with F9-RARE-lacZ reporter cells. Expression of the RA inducible gene SOCS3, IFN-a1 and Th1 associated genes was measured by RT-PCR in control BMDCs and those treated with CjCM or exogenous RA with or without adding 1mM mm11253 (RARγ antagonist) or 1µg/µl lipopolysaccharide (LPS).
Results :
Conjunctival GCs had greater production of RA than cornea (Fig.1a), supported by higher levels of ALDH1 and had higher ALDEFLUOR activity in GCs than cornea. RA in CjCM increased with RP supplementation.
CjCM increased SOCS3 and down regulated IFN-γ and IFN-a1 in DCs, similar to exogenous RA (Fig.1b). DCs in CjCM showed a tolerogenic phenotype with reduced expression of co-stimulatory molecules CD86 and increased anti-inflammatory (IL-10) cytokines. Furthermore, CjCM suppressed LPS stimulated MHC class II and pro-inflammatory cytokine (IL-1ß, IL-12, IL-23) expression. However, DEAB (the ALDH inhibitor) added to GC culture reduced the effect. DEAB also inhibited an increase in ALDH activity with CjCM in DCs. Addition of the RAR-γ antagonist decreased expression of SOCS3 (0.71±0.11 P<0.05; 0.47±0.01 P<0.01) and increase IL-1ß (2.17; 2.51±0.19) in response to CjCM or RA, respectively.
Conclusions :
Our study demonstrates that GCs can convert tear retinol to RA, which increases SOCS3 expression and inhibits Th1 inducing cytokines in DCs. GCs appear to play an important role in regulating DC maturation and maintaining ocular surface immune tolerance.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.