Effects of Inflammatory Cytokines on Human Retinal Endothelial Cells

Author: Lisia Barros Ferreira

Barros Ferreira, Lisia, 2023 Effects of Inflammatory Cytokines on Human Retinal Endothelial Cells, Flinders University, College of Medicine and Public Health

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Uveitis is among the leading causes of blindness worldwide. The treatment of non-infectious uveitis is not ideal, especially for those forms of the disease that involve the posterior segment of the eye. In this setting, retinal endothelial dysfunction results in vascular leakage and macular oedema, consequently leading to visual loss.

Cytokines are major mediators of uveitis, and some are also known to alter endothelial cell function, potentially contributing to loss of the inner blood-retinal barrier which is mainly composed of tightly interconnected retinal endothelial cells. In research documented in this thesis, the core mechanisms involved in cytokine-induced human retinal endothelial cell dysfunction, the presence of a key cytokine receptor in these cells, and the expression of long non-coding RNAs (lncRNAs) potentially involved in the cell activation were examined.

The effects of selected cytokines on the electrical impedance of human retinal endothelial cell monolayers were tested with a real-time biosensor, demonstrating that tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 induced the breakdown of the retinal endothelial barrier, while IL-8, IL-17 and C-C motif chemokine ligand 2 had no significant effect on barrier function. Moreover, IL-6 did not enhance the impairment of the endothelial barrier when applied in combination with TNF-α or IL-1β.

The decline of the cellular electrical impedance provoked by IL-6 suggested that the IL-6 receptor (IL-6R) was expressed by human retinal endothelial cells, a surprising finding considering the available literature. Reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric studies were employed to examine IL-6R expression in human retinal endothelial cells, and in silico methodology combined with small interfering RNA experiments were employed to assess IL-6R regulation by transcription factors. Expression of IL-6R was confirmed in human retinal endothelial cells, and TNF-α, IL-1β and lipopolysaccharide were shown to downregulate IL-6R in these cells. Also, the transcription factor, ETS proto-oncogene 1, was revealed to play a role in IL-6R regulation.

To address the underlying mechanisms of the observed cytokine-induced barrier changes in the retinal endothelial cell monolayers, RT-PCR and immunolabeling studies, as well as a cell survival assay and flow cytometric analyses of apoptosis and necrosis, were performed. Both TNF-α and IL-1β disturbed the intercellular junctional components and induced cell death, in the case of TNF-α by necrosis, while IL-6 mildly affected the integrity of the junctional complexes, without being cytotoxic.

Long non-coding RNAs influence endothelial cell function, but the literature on retinal endothelial lncRNAs is limited, and lncRNAs have not been investigated in the context of uveitis. A number of lncRNAs putatively expressed in human retinal endothelial cells were identified. Due to the documented pronounced effects of TNF-α and IL-1β on the retinal endothelial cell monolayer, the impact of these cytokines on retinal endothelial lncRNA expression was assessed by RT-PCR, and differential expression of a cluster of lncRNA, especially MIR155HG, was identified in activated human retinal endothelial cells.

The work presented in this thesis progresses knowledge of uveitis mechanisms in unveiling the central cytokines that mediate retinal endothelial dysfunction during inflammation as TNF-α, IL-1β and IL-6, demonstrating the expression and regulation of IL-6R in human retinal endothelial cells, and linking specific lncRNAs to retinal endothelial cell activation. The uncovering of essential targets in retinal endothelial cell dysfunction may translate clinically to improved management of vascular leakage in uveitis, thereby reducing the visual loss ensuing from this condition.

Keywords: retinal endothelial cells, uveitis, cytokines, intraocular inflammation

Subject: Medicine thesis

Thesis type: Doctor of Philosophy
Completed: 2023
School: College of Medicine and Public Health
Supervisor: Justine R. Smith