Immunomodulatory Activity of Tear Protein Lacritin

Abstract

Purpose : Lacritin is a protein found in tear that has been shown to exert cytoprotective effects on human corneal epithelial cells and increase epithelial cell proliferation under stress. Despite the established role of Lacritin in corneal epithelial cell homeostasis, whether Lacritin is capable of acting directly on immune cells has not been studied. The purpose of this study was to investigate the effect of Lacritin on immune cell function, including conventional and regulatory T cell subsets and macrophages.

Methods : CD4⁺ T cells were isolated and pooled from the spleen and cervical draining lymph nodes of C57BL/6 mice using magnetic sorting. Isolated cells were stimulated with anti-CD3 and anti-CD28 antibodies and cultured in the presence or absence of 4μM recombinant Lacritin or control recombinant albumin protein for 72 hours. T cell proliferation was assessed using BrdU incorporation assay. The frequencies of CD4⁺Foxp3⁺ regulatory T cells (Tregs) among cultured cells were determined using flow cytometry. F4/80⁺ macrophages were isolated from mice by peritoneal lavage with ice-cold PBS. Isolated cells were stimulated with IFNγ and cultured in the presence or absence of Lacritin or control protein for 48 hours. Frequencies of MHC-II^hi macrophages were evaluated using flow cytometry.

Results : Stimulation with anti-CD3 and anti-CD28 led to an approximately 9-fold increase in CD4⁺ T cell proliferation (mean±SEM of optical density (OD) values: 8443±258 vs. 951±182; p=0.0001). There was a 13% reduction in CD4⁺ T cell proliferation in cultures treated with Lacritin compared to stimulated T cells cultured with control protein (OD: 7378±575 vs. 8443±258; p=0.16). In addition, Lacritin-treated cultures showed an 11% increase in frequencies of CD4⁺Foxp3⁺ Tregs compared to control-treated cultures (6.12% vs. 5.50%). Furthermore, Lacritin led to a significant (48%) decrease in the frequencies of MHC-II^hi macrophages compared to the control group (49.4±2.1% vs. 95.8±1.6%, p=0.002).

Conclusions : Lacritin significantly suppresses macrophage activation and exerts a moderate regulatory effect on CD4⁺ T cell proliferation and Treg expansion. These findings indicate a possible immunoregulatory role for Lacritin in inflammatory diseases of the ocular surface.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.