The objective of this study was to determine the role of individual NFAT
isoforms in TNFα-induced
retinal leukostasis. To this end, human
retinal microvascular endothelial cells (HRMEC) transfected with
siRNA targeting individual NFAT
isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each
isoform to the TNFα-induced upregulation of leukocyte adhesion
proteins. This showed that NFATc1
siRNA increased ICAM1 expression, NFATc2
siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression,
NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4
siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of
isoform-specific knockdown on TNFα-induced
cytokine production was also measured using
protein ELISAs and conditioned cell culture medium, and showed that NFATc4
siRNA reduced CXCL10, CXCL11, and MCP-1
protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced
retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced
retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role.