Figure 1: mRNA expression in conjunctival cellsof VKC.
Fig 1A: Enzymes of sphingolipid metabolism
UGCG: UDP-Glucose Ceramide-Glucosyltransferase; CERK: Ceramide Kinase; SPHK1: Sphingosine Kinase-; ACER: Alkaline Ceramidase; SGMS2: Sphingomyelin Synthase; ASMA: Acid Sphingomyelinase; ASAH1: Acid Ceramidase; SGPL: Sphingosine-1-phosphate lyase; Control: n=60; VKC: n=87
Fig 1B: S1P1R and S1P3R receptor
S1P1R: Control& VKC n=13
S1P3R: Control n=13; VKC n=9
Fig 1C: α-SMA, Vimentin, BCl2 and Bax
Control, n=22; VKC, n=19
Figure 1D: Serum C1P, S1P and IgE in VKC by ELISA
Control: n=14; VKC: 29; NR-VKC: n= 6; R-VKC: n=23
(S1P, IgE: Control: n=12)
*p<0.05; **p<0.01;***p<0.01: control versus VKC
Altered levels of sphingolipids in the ocular surface was evaluated by estimation in the pooled sets of Schirmer collected tears(6) by LC-MS/MS analysis that showed significant alterations in the VKC cases when classified as Refractory (R-VKC, n= 22 sets) and Non-Refractory (NR-VKC, n=6 sets). Accordingly, 4 of the specific sphingolipids namely,S1P (d17:0); S1P (d20:1); S1P (d17:1) and Cer (d18:/17:0) were significantly lowered in R-VKC. While Cer (d18:1/17:0) was significantly increased (by 30%) and C1P (d18:1/8:0) (by 65%) in NR-VKC than control, this response was not observed in R-VKC (Fig 2A, B). A lower level of ceramide in the conjunctival epithelium probably alters barrier permeability and reduced apoptosis may further contribute to the refractory behavior in a select group of VKC patients. Looking at the cumulative levels of tear sphingolipids a relatively lower level of total sphingolipids, S1P, C1P and ceramide (d18:/17:0) were seen in R-VKC compared to NR-VKC, possibly indicating the defense in NR-VKC which R-VKC fails to elicit. Of the refractory cases classified based on intervention, those who were on topical immunomodulators > 8 weeks and still active, showed maximal lowering of tear ceramide and raised S1P/ceramide ratio thus correlating with the more severe/refractory grade of VKC (Fig 2C).