TLR4-Dependent DUOX2 Activation Triggered Oxidative Stress and Promoted HMGB1 Release in Dry Eye

Abstract

Dry eye disease (DED) is one of the most common ocular surface diseases worldwide. DED has been characterized by excessive accumulation of reactive oxygen species (ROS), following significant corneal epithelial cell death and ocular surface inflammation. However, the key regulatory factor remains unclear. In this study, we tended to explore whether DUOX2 contributed to DED development and the underlying mechanism. Human corneal epithelial (HCE) cells were treated with hyperosmolarity, C57BL/6 mice were injected of subcutaneous scopolamine to imitate DED. Expression of mRNA was investigated by RNA sequencing (RNA-seq) and quantitative real-time PCR (qPCR). Protein changes and distribution of DUOX2, high mobility group box 1 (HMGB1), Toll-like receptor 4 (TLR4), and 4-hydroxynonenal (4-HNE) were evaluated by western blot assays and immunofluorescence. Cell death was assessed by Cell Counting Kit-8 (CCK8), lactate dehydrogenase (LDH) release, and propidium iodide (PI) staining. Cellular ROS levels and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. RNA-seq and western blot assay indicated a significant increase of DUOX2 dependent of TLR4 activation in DED both in vitro and in vivo. Immunofluorescence revealed significant translocation of HMGB1 within corneal epithelial cells under hyperosmolar stress. Interestingly, after ablated DUOX2 expression by siRNA, we found a remarkable decrease of ROS level and recovered MMP in HCE cells. Moreover, knockdown of DUOX2 greatly inhibited HMGB1 release, protected cell viability and abolished inflammatory activation. Taken together, our data here suggest that upregulation of DUOX2 plays a crucial role in ROS production, thereafter, induce HMGB1 release and cell death, which triggers ocular surface inflammation in DED.

Diabetic Keratopathy: Redox Signaling Pathways and Therapeutic Prospects

“…Notably, HMGB1, an alternative ligand of the receptor for RAGE, and dual oxidase 2 (DUOX2), a subtype of NOX, play crucial roles in DED. Hyperosmolarity-induced DUOX2 activation, mediated by toll-like receptor 4 (TLR4)-dependent signaling and exacerbated by ROS excess, leads to HMGB1 release, cell death, and inflammation in a positive loop in human corneal epithelial cells exposed to DED conditions [136]. In a diabetic murine model, hyperglycemia induces a more severe mitochondrial bioenergetic deficiency in lacrimal gland cells compared to corneal cells.…”

Potential New Target for Dry Eye Disease—Oxidative Stress

“…ROS production has been detected in various models of in vitro studies [23,24]. RNA-seq analysis of corneal epithelial cells exposed to hyperosmolarity has revealed significant modulation in the activation of ROS metabolism processes [25]. In humans, numerous exogenous and endogenous factors are sources of ROS production, which was particularly emphasized in a recent cross-sectional study on population aging and lifestyle factors, where it was found that DED is associated with aging, reduced sleep duration, prolonged exposure to digital device screens, and increased psychological stress [26].…”

Inflammation of Dry Eye Syndrome: A Cellular Study of the Epithelial and Macrophagic Involvement of NFAT5 and RAGE

“…Following such recognition, PRRs lead to a microbe-free inflammatory response or a “sterile” inflammation. Thus, it has been determined that TF, in the case of DED, contains many of these alarmins, such as S100A4, S100A6, S100A8, S100A9, S100A11, and HMGB1 and many cytokines, which are accompanied by a recruitment of numerous immune cells in the ocular surface [ 5 , 6 , 7 , 8 ]. Moreover, regarding PRRs, they seem to have an increase of TLR expression and recruitment, such as TLR4 and TLR2, in surface epitheliums [ 9 , 10 , 11 , 12 ], with a particular emphasis on TLR4 implication in inflammation and on immune cell recruitment [ 11 ].…”

“…This activation will induce cytokine expression and release and the attraction of immune cells to an inflammation site [ 18 , 19 , 20 ]. In dry eye syndrome, many potential RAGE ligands are released [ 5 , 6 , 7 ], but their involvement has not yet been studied at the ocular epithelium level. Thus, our study intends to provide new information about the molecular actors implicated in inflammation by focusing on the similarity/difference of response of surface epithelial cells (cornea and conjunctiva) in triggering the DED inflammation.…”