Abstract
Purpose :
Substance P (SP) is a proinflammatory neuropeptide that has been implicated in the pathogenesis of various inflammatory conditions. Previous work by our group has demonstrated increased levels of SP in the course of dry eye disease (DED). However, the precise role of SP in the pathogenesis of DED, in particular, its effect on regulatory T cell (Treg) function, is still unclear. This study aimed to assess the phenotypic and functional changes in Tregs in response to SP, and to evaluate the role of blocking neurokinin 1 receptor (NK-1R) in restoring Treg function in a mouse model of DED.
Methods :
CD4+CD25+Foxp3+ Tregs were isolated from draining lymph nodes (DLN) of naïve female C57BL/6 mice. Isolated Tregs were co-cultured with SP (1uM) with or without Spantide I (10uM) for 48 hours. Frequencies and phenotype of Tregs were evaluated using flow cytometry, and the capacity of Tregs to suppress effector T cell proliferation was investigated using suppression assay. DED was induced in mice by housing them in the controlled environment chamber (CEC) for 14 days. To evaluate the effect of NK-1R blockade, Spantide I or PBS (control) was administered intraperitoneally (36ug/day) from one day before DED induction until day 14. Tregs were isolated from DLN of normal and DED mice and Treg suppressive function were evaluated using the suppression assay.
Results :
Addition of SP led to a 20.8±9.4% reduction in frequencies of Tregs (p<0.01) and a 16.8±2.5% decrease in their expression of Foxp3 (p=0.021) compared to the control group. This suppression was reversed to control levels in the presence of Spantide I. Additionally, SP-treated Tregs showed significantly lower ability to suppress effector T cells in vitro compared to the control group (52.2±4.5% vs. 38.6±3.4%; p=0.033), which was reversed by the addition of Spantide I (56.3±3.7%; p<0.01). While Tregs derived from DLN of mice with DED demonstrated a lower ability to suppress effector T cell proliferation compared to normal mice (41.8±7.4% vs. 56.5±4.4%, p<0.01), treatment of DED mice with Spantide I led to a 12.2% increase in Treg suppressive function (p=0.13).
Conclusions :
Our results show that SP reduces Treg frequencies and leads to Treg dysfunction in vitro, and Spantide I reverses this suppressive effect. Treatment of DED mice with Spantide I tend to restore the immunoregulatory function of Tregs.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.