4-HNE induces proinflammatory cytokines of human retinal pigment epithelial cells by promoting extracellular efflux of HSP70

https://doi.org/10.1016/j.exer.2019.107792Get rights and content

Highlights

  • Oxidative 4-HNE induces production of proinflmmatory cytokines of RPE cells.

  • Intracellular HSP70 inhibited 4-HNE-induced production of proinflammatory cytokines.

  • 4-HNE exerts proinflammatory effects by enhancing extracellular release of HSP70.

  • Arimoclomol suppresses the function of 4-HNE via increasing intracellar HSP70.

  • MBC coordinates with arimoclomol to inhibit proinflammatory effects of 4-HNE.

Abstract

Oxidative stress and subsequent chronic inflammation result in dysfunction of the retinal pigment epithelium (RPE) and represent therapeutic targets in the context of age-related macular degeneration (AMD). However, molecular mechanisms that linked oxidative stress and inflammation still unclear. As an important byproduct of oxidative stress, 4-hydroxynonenal (4-HNE) induces apoptosis and lysosome dysregulation of RPE cells. In the present study, we evaluated cytokines production of RPE cells induced by 4-HNE by using cytokine array and confirmed that 4-HNE induced IL-6, IL-1β and TNF-α production in a concentration dependent manner. Specifically, 4-HNE also induced IL-10 and TGF-β production in low concentration. Molecular analysis revealed that intracellular HSP70 inhibited 4-HNE-induced production of pro-inflammatory cytokines, and 4-HNE exerted proinflammatory effects in RPE cells by enhancing extracellular release of HSP70, as efflux inhibitor Methyl-β-cyclodextrin (MBC) treatment significantly blocked the release of HSP70 and decreased IL-6 production of RPE cells induced by 4-HNE. Meanwhile, HSP70 inducer arimoclomol increased intracellular HSP70 production, but showed no influence on its extracellular level, also performed anti-inflammatory effects in 4-HNE-stimulated RPE cells. Whereas the anti-inflammatory effects of paeoniflorin, an HSP70 inducer simultaneously promoted its extracellular efflux, was lower than arimoclomol. In addition, we further confirmed that MBC exhibited synergetic effect with both paeoniflorin and arimoclomol to inhibit the production of proinflammatory cytokines induced by 4-HNE. Taken together, these results indicate that HSP70 plays a vital role in regulating inflammation of RPE cells induced by oxidative stress and might be a potential novel target for clinical treatment of AMD.

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).

References (43)

  • T. Paimela et al.

    Celastrol regulates innate immunity response via NF-κB and Hsp70 in human retinal pigment epithelial cells

    Pharmacol. Res.

    (2011)
  • V. Tarallo et al.

    DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88

    Cell

    (2012)
  • W.W. Yu et al.

    Heat shock protein 70 suppresses neuroinflammation induced by α-synuclein in astrocytes

    Mol. Cell. Neurosci.

    (2018)
  • R. Agrawal et al.

    Cytokine profiling in patients with exudative age-related macular degeneration and polypoidal choroidal vasculopathy

    Invest. Ophthalmol. Vis. Sci.

    (2019)
  • J. Ambati et al.

    Immunology of age-related macular degeneration

    Nat. Rev. Immunol.

    (2013)
  • M.M. Barreca et al.

    Extracellular Hsp70 enhances mesoangioblast migration via an autocrine signaling pathway

    J. Cell. Physiol.

    (2016)
  • I. Bellezza

    Oxidative stress in age-related macular degeneration: Nrf2 as therapeutic target

    Front. Pharmacol.

    (2018)
  • L. Bonet-Ponce et al.

    Rotenone induces the formation of 4-hydroxynonenal aggresomes. Role of ROS-mediated tubulin hyperacetylation and autophagic flux disruption

    Mol. Neurobiol.

    (2016)
  • J. Chen et al.

    Phosphatidylinositol 3 kinase pathway and 4-hydroxy-2-nonenal-induced oxidative injury in the RPE

    Invest. Ophthalmol. Vis. Sci.

    (2009)
  • C.M. Ethen et al.

    Age-related macular degeneration and retinal protein modification by 4-hydroxy-2-nonenal

    Invest. Ophthalmol. Vis. Sci.

    (2007)
  • J. Gautam et al.

    4-Hydroxynonenal-induced GPR109A (HCA2 receptor) activation elicits bipolar responses, Galphai -mediated anti-inflammatory effects and Gbetagamma -mediated cell death

    Br. J. Pharmacol.

    (2018)
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