Abstract
Purpose :
Substance P (SP) is a neuropeptide released by peripheral nerves that mediate pain perception. Recent studies have reported that SP and its receptor NK1R are expressed on immune cells. Here, we aimed to investigate whether murine regulatory T (Treg) cells express the SP receptor (NK1R) and whether they respond to SP in healthy and dry eye mice.
Methods :
Dry eye disease (DED) was induced in wild-type (WT) C57BL/6 mice using the controlled environment chamber for 14 days. The draining lymph nodes (DLN) of WT (n=5) and DED (n=10) mice were harvested to assess NK1R and Foxp3 expression in Tregs (CD4+CD25+) using flow cytometry analysis at days 7 and 14. The DLNs and spleens of WT mice (n=5) were harvested and Treg cells (CD4+CD25+) were sorted using magnetic-activated cell sorting (MACS). Sorted Treg cells were co-cultured with interleukin (IL)-2 and SP, and Foxp3 expression of NK1R+ Tregs was analyzed after 18 hours using flow cytometry.
Results :
Using flow cytometry analysis we found that 15 ± 4 of Tregs were NK1R+ and that NK1R+ Tregs express lower Foxp3 levels than NK1R- Tregs in WT mice. In vitro treatment of sorted naive Treg cells (CD4+CD25+) with SP led to statistically significant reduction in Foxp3 levels in NK1R+ Tregs. Mice with DED showed increased NK1R expression (WT: 4631 ± 401; DED: 6048 ± 603; P < 0.005), but decreased Foxp3 expression in NK1R+ Tregs (WT: 2896 ± 168; DED: 2249 ± 198; P < 0.005) on day 14.
Conclusions :
Our data demonstrate that SP directly suppresses Foxp3 expression in NK1R+ Treg cells. Increased SP receptor expression and decreased Foxp3 expression in DED mice suggest that SP amplifies T cell mediated immunity in dry eye disease.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.