Person:

Amouzegar, Afsaneh

Objective: To evaluate dry eye disease (DED)-induced alterations in subsets of corneal antigen presenting cells (APCs) in a mouse model of experimental DED. Methods and Analysis Seven to 8-week-old female C57BL/6 mice were housed in a controlled environment chamber and were treated with subcutaneous scopolamine to induce DED. Normal mice were used as controls. The frequencies of different subsets of dendritic cells (DCs) and macrophages in the cornea were evaluated using immunohistochemistry and flow cytometry at days 2, 7 and 14 after DED induction. Real-time PCR was used to assess the functional phenotype of macrophages in the cornea of DED mice. Results: Our results demonstrated significant corneal infiltration of CD11b+ and CD11c+ cells on days 7 and 14. Further analysis of different DC subsets revealed non-significant changes in the frequencies of total CD11b+CD11c+ cells at different time points. However, frequencies of CD11c+CD11b- DCs, CD11c+ Langerin (CD207)+ DCs and macrophages were significantly increased on both days 7 and 14 after DED induction. Real-time PCR data demonstrated increased expression of M1 macrophage markers, iNOS and TNF-α, and reduced expression of M2 macrophage markers, Arg1 and IL-10, by corneal F4/80+ macrophages at day 7. Conclusion: Although the frequencies of total CD11b+CD11c+ cells do not significantly change in the course of DED, CD11c+CD11b- DCs and Langerin+ DCs do show a significant increase. Interestingly, macrophages exhibit a predominant inflammatory M1 phenotype and suppressed anti-inflammatory M2 phenotype early after induction of DED, which are restored to near baseline levels in later stages of the disease.

Purpose Neutrophil-secreted effector molecules are one of the primary causes of tissue damage during corneal inflammation. In the present study, we have investigated the effect of stromal cells in regulating neutrophil expression of tissue-damaging enzymes, myeloperoxidase (MPO), and N-elastase (ELANE). Methods: Bone marrow–purified nonhematopoietic mesenchymal stromal cells and formyl-methionyl-leucyl-phenylalanine–activated neutrophils were cocultured in the presence or absence of Transwell inserts for 1 hour. Neutrophil effector molecules, MPO and ELANE, were quantified using ELISA. In mice, corneal injury was created by mechanical removal of the corneal epithelium and anterior stroma approximating one third of total corneal thickness, and mesenchymal stromal cells were then intravenously injected 1 hour post injury. Corneas were harvested to evaluate MPO expression and infiltration of CD11b+Ly6G+ neutrophils. Results: Activated neutrophils cocultured with mesenchymal stromal cells showed a significant 2-fold decrease in secretion of MPO and ELANE compared to neutrophils activated alone (P < 0.05). This suppressive effect was cell–cell contact dependent, as stromal cells cocultured with neutrophils in the presence of Transwell failed to suppress the secretion of neutrophil effector molecules. Following corneal injury, stromal cell–treated mice showed a significant 40% decrease in MPO expression by neutrophils and lower neutrophil frequencies compared to untreated injured controls (P < 0.05). Reduced MPO expression by neutrophils was also accompanied by normalization of corneal tissue structure following stromal cell treatment. Conclusions: Mesenchymal stromal cells inhibit neutrophil effector functions via direct cell–cell contact interaction during inflammation. The current findings could have implications for the treatment of inflammatory ocular disorders caused by excessive neutrophil activation.