Person:

Stevenson, William

Purpose.

To investigate the corneal expression of toll-like receptor (TLR) 4 and determine its contribution to the immunopathogenesis of dry eye disease (DED).

Methods.

Seven to 8-week-old female C57BL/6 mice were housed in a controlled environment chamber and administered scopolamine to induce experimental DED. Mice received intravenous TLR4 inhibitor (Eritoran) to block systemic TLR4-mediated activity. The expression of TLR4 by the corneal epithelium and stroma was evaluated using real-time polymerase chain reaction and flow cytometry. Corneal fluorescein staining (CFS) was performed to evaluate clinical disease severity. The corneal expression of proinflammatory cytokines (IL-1β, IL-6, TNF, and CCL2), corneal infiltration of CD11b+ antigen-presenting cells, and lymph node frequency of mature MHC-IIhi CD11b+ cells were assessed.

Results.

The epithelial cells of normal corneas expressed TLR4 intracellularly; however, DED significantly increased the cell surface expression of TLR4. Similarly, flow cytometric analysis of stromal cells revealed a significant increase in the expression of TLR4 proteins by DED-induced corneas as compared with normal corneas. DED increased the mRNA expression of TLR4 in corneal stromal cells, but not epithelial cells. TLR4 inhibition decreased the severity of CFS and significantly reduced the mRNA expression of IL-1β, IL-6, and TNF. Furthermore, TLR4 inhibition significantly reduced the corneal infiltration of CD11b+ cells and the lymph node frequency of MHC-IIhi CD11b+ cells.

Conclusions.

These results suggest that DED increases the corneal expression of TLR4 and that TLR4 participates in the inflammatory response to ocular surface desiccating stress.

Purpose.

A majority of experimental data on dry eye disease (DED) immunopathogenesis have been derived from a murine model of DED that combines desiccating environmental stress with systemic muscarinic acetylcholine receptor (mAChR) inhibition. However, to our knowledge the effects of pharmacologic mAChR blockade on the pathogenesis of experimental DED have not been evaluated systemically. The purpose of our study was to investigate the differential effects of desiccating environmental stress and mAChR inhibition on the pathogenesis of DED.

Methods.

DED was induced in female C57BL/6 mice by exposure to a desiccating environment in the controlled-environment chamber or to systemic scopolamine, or by performing extraorbital lacrimal gland excision. Clinical disease was assessed using corneal fluorescein staining (CFS) and the cotton thread test (CTT). Corneal CD11b+ and conjunctival CD3+ T-cell infiltration were evaluated by flow cytometry. T-cells from draining cervical lymph nodes (CLN) and distant inguinal lymph nodes (ILN) were analyzed for Th1, Th2, Th17, and Treg responses by flow cytometry and ELISA.

Results.

Desiccating environmental stress and systemic mAChR blockade induced similar clinical signs of DED. However, desiccating environmental stress imparted higher conjunctival CD3+ T-cell infiltration, and greater Th17-cell activity and Treg dysfunction than mAChR blockade, while mAChR blockade decreased tear secretion to a greater extent than desiccating environmental stress. Systemic mAChR blockade attenuated Th17 activity and enhanced Th2 and Treg responses without affecting Th1 activity.

Conclusions.

In vivo inhibition of mAChRs variably affects CD4+ T-cell subsets, and desiccating environmental stress and systemic mAChR blockade induce DED through different primary pathogenic mechanisms.