Abstract
Purpose:
Conjunctival goblet cells (GC) confer ocular surface protection via mucin secretion. Previously we reported their immunomodulatory role based on their ability to release soluble factors like active TGFβ and support tolerogenic phenotype of dendritic cells. In this study we determined the effect of cytotoxic vs. commensal bacteria on the immunomodulatory function of conjunctival GCs.
Methods:
Primary cultures of GCs were generated from WT (C57BL/6) conjunctival explants. Cultured GCs were challenged with heat-inactivated cytotoxic (Pseudomonas aeruginosa, PA14) or commensal (Streptococcus sp.) bacterial strains for 24h. Proliferation and apoptosis of GCs was assessed by BrdU/7-AAD staining and flow cytometry. Culture supernatants were evaluated for levels of MUC5AC by ELISA and active TGFβ using MFB11 reporter assay. Expression of TGFβ2 and thrombospondin-1 (TSP1) in GCs was determined by RT-PCR. Expression of activation markers MHC class II, CD80, CD86 and CD40 was assessed by RT-PCR on bone marrow derived dendritic cells (BMDCs) co-cultured with GCs in a transwell system for 24h.
Results:
Viability of GCs challenged with either the cytotoxic or the commensal strain at MOI=60 was comparable to that in untreated control cells. At this concentration, the commensal strain induced 38% increase in GC proliferation and no change in apoptosis as compared to untreated control cells. However, the challenge with the cytotoxic strain induced a 17% increase in GC apoptosis without changing proliferation as compared to untreated control cells. Supernatants from GCs challenged with the cytotoxic strain contained significantly diminished levels of MUC5AC and active TGFβ compared to untreated controls, while the commensal strain did not induce such change. The decline in active TGFβ correlated with significantly reduced expression of TSP1 in GCs challenged with the cytotoxic but not the commensal strain. Consistently, a significant increase in the expression of MHC class II and co-stimulatory molecules was detected in BMDCs co-cultured with GCs challenged with the cytotoxic but not the commensal strain.
Conclusions:
Cytotoxic strains of bacteria, but not commensals, disrupt ocular surface homeostasis by altering mucin secretion as well as the immunomodulatory function of conjunctival goblet cells. These results underscore the significant contribution of conjunctival goblet cells in preventing ocular surface inflammation.