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Cintia S De Paiva, Humberto Hernandez, Rodrigo G. de Souza, Zhiyuan Yu, Fang Bian; Age-associated changes in T effector and regulatory T cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):271.
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Aging is one of the most recognized risk factors for dry eye disease. The purpose of this study was to investigate the T helper (Th) subtypes and regulatory T cells in C57BL/6J mice, which spontaneously develop dry eye with aging.
Young (eight-week-old) and aged (24-months old) C57BL/6J female mice were used. The frequency of CD4+IL-17+ (Th17), CD4+IFN-γ+ (Th1) and CD4+Foxp3+ cells were investigated in lacrimal glands (LG) and cervical lymph nodes (CLN) using flow cytometry. Amnis ImageStreamer cytometer confirmed cytokine production. Young and aged CD4+CD25+ and CD4+CD25- cells were isolated from CLNs and spleens as surrogates for regulatory T cells (Tregs) and T responders (Tresps), respectively. These cells were used in mixed lymphocyte reactions and gene expression analysis. Proliferation and production of IFN-γ in culture supernatants was used as endpoints in mixed lymphocyte reactions.
Aged mice had increased frequency of Th1 cells in LG (P<0.05) and CLNs (P<0.01), while Th17 cells increased only in CLNs (P<0.05). There was an increased frequency of CD4+Foxp3+ cells in LGs (P<0.05), CLN (P<0.01), in aged compared to the young group. Elevated CD4+IFN-γ+Foxp3+ cells (LG [P<0.05], CLN [P<0.001]), and CD4+IL-17+Foxp3+ (CLN, P<0.05) were observed in aged mice compared to the young group. Production of IFN-γ by Foxp3+ cells was confirmed by imaging cytometry in LGs and CLNs. Mixed lymphocyte reactions showed that aged Tregs had decreased suppressive ability in young Tresps compared to young Tregs; however, young Tregs failed to suppress proliferation from aged Tresps. Similar results were observed in IFN-γ protein concentration in supernatants. A significant increase in IFN-γ (9 fold), IL-4 (3 fold) and IL-17 (6.83 fold) mRNA was seen in aged Tresps compared to young Tresps. Aged Tregs expressed higher levels of IFN-γ (4.86 fold), IL-17 (14.37 fold) than young Tregs, but still expressed significantly higher levels of IL-10 (12 fold), CTLA-4 (9 fold) and ICOS (2.9 fold) mRNA transcripts than young Tregs (all P<0.001). Median fluorescence intensity of Foxp3 was maintained in LG and CLNs demonstrating that these cells are not “ex-Foxp3+ cells.”
Aging was accompanied by increased frequency of Th1 cells in periocular tissues.Our results show that aging is associated with dysregulation of both T effectors and T regulatory cells. These findings shed light on the pathogenesis of age-related dry eye.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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