Abstract
Purpose :
The blood retinal barrier (BRB) breakdown in diabetic retinopathy (DR) is induced in part by increased paracellular permeability. Interleukin-6 (IL-6) plays an important role in the regulation of paracellular permeability in human retinal endothelial cells (HRECs). This pleiotropic cytokine acts via two distinct mechanisms, including “classical” signaling through a membrane-bound IL-6 receptor and “trans-signaling” through a soluble IL-6 receptor. The purpose of this study was to compare the effects of these two IL-6 pathways on paracellular permeability in HRECs.
Methods :
To activate IL-6 classical signaling, HRECs were treated with IL-6 (50 ng/mL), and to activate IL-6 trans-signaling, cells were treated with IL-6 (50 ng/mL) and sIL-6R (150 ng/mL) overnight. Trans-endothelial electrical resistance (TEER) was measured using electrical cell impedance sensing (ECIS). Gene expression changes were determined using RNA-Seq.
Results :
IL-6 trans-signaling activation caused a significant drop in TEER, while there was no effect of classical signaling. Among the 3 parameters of endothelial barrier function, paracellular resistance was significantly decreased, cell membrane capacitance was significantly increased, and no change was observed in basal adhesion. We found distinct effects on gene expression with each treatment, with more genes significantly altered following trans-signaling activation. Significant changes in the expression of 7 tight junctions were observed after trans-signaling activation: CLDN1 (-3.27-fold), CLDN3 (-4.44-fold), CLDN11 (-1.85-fold), CLDN12 (1.25-fold), CLDN14 (-1.36-fold), TJP1 (1.17-fold) and TJP2 (-1.31-fold). Among the adherens junctions, genes which showed significant changes were CDH6 (-1.66-fold), CDH11 (1.74-fold), CDH12 (1.97-fold), CDH24 (-1.67-fold), CTNNAL1 (-1.21-fold), and CTNNBIP1 (-1.29-fold). The 3 differentially expressed gap junctions included GJA1 (1.49-fold), GJA4 (-2.41-fold) and GJC1 (-1.19-fold). Only CLDN15 (1.48-fold) was significantly upregulated by classical signaling.
Conclusions :
IL-6 trans-signaling caused a significant increase in paracellular permeability in HRECs, while classical signaling had no significant effect. Specific gene expression changes in intercellular junctions were identified that will aid our future understanding of the molecular mechanisms of IL-6 trans-signaling mediated BRB breakdown in HRECs.
This is a 2021 ARVO Annual Meeting abstract.