Abstract
Purpose :
PEDF is ubiquitously expressed 50kD protein with myriad anti-inflammatory functions. In this study, we determined the effect of recombinant PEDF(rPEDF) on dysfunctional Tregs in dry eye disease(DED).
Methods :
MACS sorted CD4+CD25+ T-cells from draining lymph nodes(dLNs) of C57BL/6 mice with DED (induced in controlled environment chamber for 7 days) were cultured with/without rPEDF for 72 hrs. We evaluated Treg(CD4+CD25+FoxP3+ T-cells)frequency and suppressive phenotype by examining expression levels(MFI) of FoxP3, CTLA-4 & GITR using flow cytometry (FC) and mRNA expression levels of IL10 & TGFβ (Treg-associated cytokines) using RT-PCR. We assessed the neutralizing effect of rPEDF on pro-inflammatory cytokine-IL-17A, IFNγ(expressed by CD4+Th17 cells) induced Treg dysfunction, by evaluating Treg frequency and expression of above-mentioned markers with FC & RT-PCR after 72 hrs of co-culture. We treated DED mice(14 days induction) with i.p. injection of 1 µg/ml rPEDF or control(Murine Serum Albumin, MSA) q.d. from day 7. We assessed disease severity by corneal fluorescein staining(CFS), performed FC on dLNs of treated mice to examine changes in Treg frequency and MFI of associated markers.
Results :
The data demonstrated a significantly higher Treg frequency(p=0.001) and expression of FoxP3(p=0.0263),CTLA-4,(p=0.0101),GITR(p=0.0245) and higher mRNA expression of IL10 & TGFβ(p=0.0011,p=0.0009) by Tregs on addition of rPEDF to co-culture. rPEDF reversed the effect of IL-17A, IFNγ induced dysfunction in Tregs by significantly increasing the Treg frequency(p=0.0013),expression of FoxP3(p=0.0001),CTLA-4(p=0.0013),GITR(p=0.019) and significantly higher mRNA expression of IL10 & TGFβ(p=0.002,p=0.005). We observed significant reduction in CFS scores in rPEDF treated mice compared to controls(p=0.05). FC data showed significantly higher Treg frequency(p=0.0002) and expression of FoxP3(p=0.025) in dLNs of rPEDF treated mice compared to controls.
Conclusions :
Our results suggest that rPEDF maintains Treg function in-vitro and nullifies the dysfunctional effect of IL-17A, IFNγ. Our in-vivo data showed a higher Treg frequency and function on rPEDF treatment that may be contributing to disease amelioration. These findings indicate a potential therapeutic role of PEDF associated with promoting regulatory T cell-mediated suppression of Th17 immune response in dry eye disease.
This is a 2020 ARVO Annual Meeting abstract.