Interleukin-6 (IL-6) mediates protection against glucose toxicity in human Müller cells via activation of VEGF-A signaling

https://doi.org/10.1016/j.bbrc.2019.07.044Get rights and content

Highlights

  • Human Müller cells express mIL-6R and gp130 allowing for classical IL-6 signaling.

  • Hyperglycemia upregulates mIL-6R and gp130 expression.

  • IL-6 via classical signaling induces VEGF-A signaling in human Müller cells.

  • IL-6-induced VEGF-A production protects human Müller cells from glucose toxicity.

  • Human Müller cells only weakly respond to IL-6 trans-signaling.

Abstract

Interleukin-6 (IL-6) has become a target of interest for drug development aiming to treat diabetic retinopathy. Since IL-6 signaling can promote beneficial as well as detrimental effects via two different signaling pathways, the objective of the present study was to investigate the effects of classical IL-6 and IL-6 trans-signaling on human Müller cells (HMC), which are important for the development of diabetic retinopathy. HMCs were cultured in normal (5 mmol/L) and high (25 mmol/L) glucose plus or minus IL-6 or IL-6/sIL-6R. IL-6 receptor expression using immunohistochemistry and flow cytometry and cytokine release using magnetic bead assays were determined. HMCs express the membrane bound form of the IL-6 receptor (mIL-6R), gp130, and can release the soluble forms sIL-6R and sgp130 demonstrating that HMCs are capable of responding to classical IL-6 and IL-6 trans-signaling. IL-6 protected HMCs from glucose toxicity via VEGF-A signaling. IL-6/sIL-6R caused only modest protection, which was not mediated by VEGF-A. Our data show for the first time that classical IL-6 signaling exerts its beneficial effects through VEGF-A action contrary to IL-6 trans-signaling, which was VEGF-A independent. These results have clinical implications for drug development targeting IL-6 since strict anti-IL-6 therapies might further decrease neuroretinal functions in the diabetic retina.

Introduction

Neovascularization and retinal edema are two vision threatening complications for patients with diabetic retinopathy (DR). The current treatment strategy of choice for these patients is anti-VEGF therapy; however, this therapy is not reliable and is only effective for a small portion of the patient population [1]. Since the diabetic population is steadily rising, there is a pressing need for additional and more reliable therapies.

Recently, interleukin-6 has risen to the forefront as a target for drug development either as an anti-IL-6 therapy alone or in combination with anti-VEGF-A therapy. IL-6 is a pleiotropic cytokine known to participate in both acute and chronic inflammatory events. Its effects are dependent on microenvironment, cell type, and receptor expression [2]. Depending on which receptors are being expressed IL-6 can have beneficial or detrimental actions. The diverse effects of IL-6 are potentially due to activation of different IL-6 signaling pathways: classical IL-6 signaling and IL-6 trans-signaling. Classical IL-6 signaling, originally thought to be a protective pathway, is mediated by the binding of IL-6 to the membrane-bound IL-6 receptor (mIL-6R) and its association with membrane-bound glycoprotein 130 (gp130). On the other hand, IL-6 trans-signaling, which is has been suggested to be more pro-inflammatory in nature, is mediated by IL-6 binding to the soluble form of the IL-6 receptor (sIL-6R) and forming the IL-6/sIL-6R complex. This complex then interacts with gp130 to promote downstream signaling [[3], [4], [5]].

In the retina, IL-6 has been shown to be protective with regards to the neuroretina. Previous studies have shown that IL-6 is a key player in protecting photoreceptors from retinal detachment-induced cell death, retinal ganglion cells from pressure-induced cell death, and retinal Müller cells from high glucose-induced cell death [[6], [7], [8]]. In contrast, detrimental outcomes linked with IL-6, such as retinal barrier disruption, have been associated with the retinal vasculature [[9], [10], [11], [12]]. However, the full outcomes of the diverse actions of IL-6 in the retina have yet to be determined. Nevertheless, new treatment strategies are being developed towards targeting IL-6 as a potential treatment for diabetic retinopathy.

In the present study, we focused on characterizing expression and regulation of receptors that are associated with IL-6 signaling (mIL-6R, sIL-6R, gp130, and sgp130) and gaining more insight into the effects of IL-6 signaling on human Müller cells under hyperglycemic conditions. Human Müller cells play an important role in the development of diabetic retinopathy [[13], [14], [15], [16]]. They maintain the inner blood retinal barrier and are the only other retinal cell type besides photoreceptors found within the macula making them a strong candidate for promoting neovascularization as well as edema in the diabetic retina. They are also a well-known source for pro-inflammatory cytokines and VEGF-A production [13,17,18]. Therefore, the purpose of this study was to evaluate the ability of human Müller cells to signal through either classical IL-6 or IL-6 trans-signaling and to identify potential mechanisms of action for the protective effects of IL-6 on neuroretinal cells, such as Müller cells.

Section snippets

Antibodies and recombinant proteins

IL-6, IL-6/sIL-6R, VEGF-A, VEGFR inhibitor (Axitinib), human BD Fc Block, mouse anti-mIL-6R-APC antibody, and mouse anti-gp130-PE antibody were purchased from R&D Systems (Minneapolis, MN). Rabbit anti-mIL-6R and rabbit anti-gp130 antibodies were obtained from Abcam (Cambridge, MA). Anti-rabbit Texas Red antibody was purchased from Invitrogen (Carlsbad, CA). Human Milliplex MAP magnetic bead panels were purchased from MilliporeSigma (Burlington, MA).

Tissue culture of human retinal Müller cells

Handling of human tissue conformed to the

Determination of mIL-6R and gp130 levels on human Müller cells

Human Müller cells (HMCs) express the membrane bound form of the IL-6 receptor (mIL-6R) and the co-receptor gp130 under normal glucose conditions. Two methods were used to determine expression of mIL-6R and gp130, immunohistochemistry and flow cytometry. Fig. 1A and C shows that HMCs express mIL-6R and gp130 as demonstrated by immunofluorescence. Interestingly, some of the gp130 seems to be located around or within the nucleus. The expression levels of mIL-6R and gp130 were also verified using

Discussion

The primary objective of the present study was to determine the effects of IL-6 on human retinal Müller cells. This is the first study looking in detail at consequences of the activation of the two distinct IL-6 signaling pathways, classical IL-6 signaling versus IL-6 trans-signaling, under normal and hyperglycemic conditions in human Müller cells, which are hugely important for the development of diabetic retinopathy. The major findings of our study are that human Müller cells strongly respond

Conflicts of interest

BAC, BTT, and SM have no financial disclosures that would be potential conflicts of interests with this manuscript.

Acknowledgement

This project was supported by DFI (Discretionary Funding Initiative; SM) funding from Michigan State University and grant NIFA MICL02375 (SM).

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      Therefore, although systemically produced IL-6 is known to be capable of crossing the blood-brain barrier as an acute phase reactant, its elevation in the retina is most likely from secretion by retinal immune-competent cells. In vitro evidence suggests that IL-6 protects Müller cells from hyperglycemia via a VEGF-dependent mechanism (Coughlin et al., 2019); this may help to explain why steroids are neuroprotective but detrimental to Müller cells (Pereiro et al., 2018). This aligns with the finding that IL-6 reprograms Müller cells for retinal regeneration in zebrafish; however, IL-6 has also been implicated in contributing to choroidal neovascularization (Izumi-Nagai et al., 2007; Zhao et al., 2014).

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