4-HNE induces proinflammatory cytokines of human retinal pigment epithelial cells by promoting extracellular efflux of HSP70
Introduction
With the ability to maintain the regular activity of both photoreceptors and choriocapillaris, a substantial amount of evidence has confirmed that dysfunction of retinal pigment epithelium (RPE) cells is a major contributor of age-related macular degeneration (AMD) progression (Li et al., 2018). Due to intensive oxygen metabolic activity, continuous exposure to environmental (e.g. UV) or cellular factors (e.g. photoreceptor outer segments and lipofuscin), RPE cells are especially sensitive to oxidative stress (Kauppinen et al., 2016). Chronic oxidative stress eventually leads to RPE cell death and subsequent inflammation, which in turn form a vicious cycle to aggravate oxidative stress (Hollyfield et al., 2008). Thus, oxidative damage and inflammation are strongly associate with the degeneration of RPE cells and identifying molecular mechanisms that link oxidative stress to inflammation will have central roles in developing novel therapy options for clinical AMD management (Hollyfield et al., 2008; Ambati et al., 2013).
Cytokines are mediators of various inflammatory responses, and several cross-sectional studies have showed that patients with AMD have higher levels of proinflammatory cytokines, like IL-6, IL-1β and IL-8 (Spindler et al., 2018; Agrawal et al., 2019; Krogh Nielsen et al., 2019). Specifically, AMD patients with higher systemic concentrations of IL-6 and TNF-α had worse pathological changes in the RPE, and a higher progression rate of geographic atrophy (Cao et al., 2013; Krogh Nielsen et al., 2019). Toll-like receptor 3 (TLR3) activation and its subsequent IL-1β promotes both neovascularization and RPE degeneration (Yang et al., 2008; Kleinman et al., 2012; Zhao et al., 2015). RPE cells have been shown to express multiple TLRs (1–7, 9 and 10), and secreted large amount of proinflammatory cytokines, like IL-1β and IL-6, in response to stimulation of oxidative products, like Alu RNA, advanced glycation end products (AGEs), A2E (N-retinyl-N-retinylidene ethanolamine), amyloid-β, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) (Paimela et al., 2011; Tarallo et al., 2012; Liu et al., 2014).
4-HNE is one of the primary end products during lipid peroxidation, and its accumulation in RPE cells is associated with AMD progression (Hytti et al., 2015). Owning to have an abundance of lipid material and a high metabolic demand, RPE cells are prone to lipid peroxidation (Ambati et al., 2013). Numerous studies have confirmed that 4-HNE can induce apoptosis, lysosomal dysregulation and lipofuscin generation of RPE cells by activating multiple molecules, like NF-κB, p53, Caspase-3 or NOX4, and thus disrupt the photoreceptor maintenance function (Bae et al., 2018). In particular, our previous studies found that the Nrf2-dependent antioxidant response protects against 4-HNE toxicity in RPE cells, and this protective mechanism is dependent on the functions of the PI3K pathway (Chen et al., 2009). With regard to inflammation, 4-HNE has been reported to induce significant amount of IL-6, IL-1β and IL-8 production in RPE cells (Kauppinen et al., 2012; Hytti et al., 2015). However, the underlying molecular mechanisms that link 4-HNE to cytokines production in RPE cells is still unknown.
HSP70, one of 4-HNE targeted proteins (Krogh Nielsen et al., 2019), has been confirmed with the ability to inhibit IL-6 production in RPE cells (Subrizi et al., 2015). In the present study, we evaluated the role of HSP70 in cytokines production of RPE cells induced by 4-HNE by using cytokine array. Our results confirmed that 4-HNE induces IL-6, IL-1β and TNF-α production by promoting the extracellular efflux of HSP70. In addition, we also observed that a well-described HSP70 co-inducer arimoclomol, which has already in phase II/III clinical trials for several degenerative diseases (Fog et al., 2018), together with an efflux inhibitor Methyl-β-cyclodextrin (MBC), synergenically inhibits production of proinflammatory cytokines and enhances anti-inflammatory cytokines production of RPE cells stimulated by 4-HNE.
Section snippets
Reagents
4-HNE was purchased from BioVision (CA, USA). HSP70 co-inducer paeoniflorin and arimoclomol were from Sigma (Shanghai, China) and MCE (Shanghai, China), respectively. MBC was from Sigma. DMSO was used as solvent control. Human Th1/Th2/Th17 Array was from RayBiotech Life (GA, USA). Human IL-1β, IL-6, TNF-α, IL-10 and TGF-β1 ELISA kits were from DAKEWEI Biotechnology Co., Ltd. (Shenzhen, China). Human HSP70 ELISA kit was from Invitrogen (Shanghai, China). Lipofectamine™ RNAiMAX transfection
4-HNE induces production of inflammatory cytokines in ARPE-19 cells
To determine the effects of 4-HNE on cytokine production, ARPE-19 cells were stimulated with 4-HNE for 24 h at different concentrations (0, 5, 10 μM). At the concentrations of 5 and 10 μM, 4-HNE showed no significant cytotoxicity to ARPE-19 cells (data not shown). Then, Human Th1/Th2/Th17 cytokines array was used to measure the cytokines level in supernatant of ARPE-19 cells. A total of 20 cytokines was included in this array. 4-HNE induced IL-1β, IL-6, IL-10, CCL-20, TGF-β1 and TNF-α
Discussion
AMD is the leading cause of vision loss and blindness in individuals over 50 year old with a global incidence of 8.7%, and the projected number of individuals affected in 2020 is 196 million and 288 million in 2040 (Wong et al., 2014). The etiology of AMD is multifactorial and mainly affected by aging, environmental stresses and genetic susceptibility, which trigger oxidation and subsequent inflammation in RPE cells, ultimately leading to vision impairment (Huang et al., 2015). Thus,
Conflicts of interest
The authors have no financial conflicts of interest.
Funding
This project was supported by grants from Natural Science Foundation of Hubei Province, China (No. 2009CDB115, 2012FKB02444 and 2018CFB463), and Natural Science Foundation of Health and Family Planning Commission of Wuhan Municipality, Hubei Province, China (No. WX18Q03).
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These authors contributed equally to this work.