Connexin43 hemichannel block inhibits NLRP3 inflammasome activation in a human retinal explant model of diabetic retinopathy

https://doi.org/10.1016/j.exer.2020.108384Get rights and content

Highlights

  • The inflammasome pathway is activated in a human retinal explant model of diabetic retinopathy.

  • Connexin43 hemichannel block prevents inflammasome activation in the human retina explant model.

  • Connexin43 hemichannel block inhibits inflammatory cytokine release in the human retinal explant model.

Abstract

Diabetic retinopathy (DR), the most common ocular complication associated with diabetes, is a chronic vascular and inflammatory disease that leads to vision loss. The inflammasome pathway, a key part of the innate immune system, is required to activate chronic inflammation in DR. Unfortunately, current therapies for DR target pathological signs that are downstream of the inflammasome pathway, making them only partly effective in treating the disease. Using in vitro and in vivo DR models, it was discovered that connexin43 hemichannel blockers can inhibit activation of the inflammasome pathway. However, those studies were conducted using in vitro cell culture and in vivo animal disease models that are predictive but do not, of course, like any model, completely replicate the human condition. Here, we have developed an addition to our armamentarium of useful models, an ex vivo human organotypic retinal culture model of DR by exposing human donor retinal explants to a combination of high glucose (HG) and pro-inflammatory cytokines, interleukin-1 beta (IL-1β) and tumour necrosis factor alpha (TNF-α). We hypothesized that in this model, connexin43 hemichannel block would protect against NLRP3 inflammasome complex assembly which would in turn decrease signs of inflammation characteristic of DR. To test our hypothesis, molecular changes in the inflammatory and inflammasome pathway were assessed using immunohistochemistry and a Luminex cytokine release assay. Our results showed that the human retinal explant DR model was associated with increased inflammation and activation of the inflammasome pathway, characteristic of the human condition. Furthermore, we showed that by blocking connexin43 hemichannels with the hemichannel modulator, tonabersat, we were able to prevent NLRP3 inflammasome complex assembly, Müller cell activation, as well as release of pro-inflammatory cytokines and VEGF. This further supports the possible use of connexin43 hemichannel blockers as potential new therapies for DR.

Introduction

In 2017, it was estimated that 451 million people (age 18–99 years) suffered from diabetes globally with this number expected to increase to 693 million by 2045 (Cho et al., 2018). The most common ocular complication associated with diabetes is diabetic retinopathy (DR), a chronic vascular disease that leads to vision loss. It is estimated that every individual suffering from diabetes has a 100% risk of developing some form of DR over the course of the disease, making DR the leading cause of blindness in the industrialised world (Durham and Herman, 2011). For this reason, a good understanding of the pathology along with the development of effective therapeutics is required to target the underlying causes of the retinopathy.

Previous studies have shown that the inflammasome pathway, a key part of the innate immune system, is required to activate chronic inflammation through pro-inflammatory cytokine secretion, vascular leak, and disruption of the retinal pigment epithelium (RPE) barrier, hallmark molecular pathologies that occur in DR (Mugisho et al., 2018a). Specifically, the NOD-like receptor protein-3 (NLRP3) inflammasome has been implicated in DR and is known to be upregulated and activated in DR patients (Chaurasia et al., 2018; Chen et al., 2018; Loukovaara et al., 2017). Unfortunately, current pharmacological therapies for DR generally target pathological signs that are significantly downstream of the NLRP3 inflammasome pathway. This includes anti-vascular endothelial growth factors (anti-VEGF), the current gold standard for DR treatment (Stefanini et al., 2014; Takamura et al., 2018). Anti-VEGF drugs only target the VEGF protein, one of many cytokines activated following inflammasome assembly. This could explain why other pro-inflammatory cytokines remain elevated in the vitreous and serum of DR patients even after anti-VEGF treatment (Comyn et al., 2019). Furthermore, the incomplete reduction of inflammation by these agents might explain why only 50% of patients respond to the treatment (Tranos et al., 2013). For these reasons, inhibitors of the pathological connexin43 hemichannel which prevents NLRP3 inflammasome assembly, are currently being investigated as a treatment for chronic inflammatory eye diseases since they act upstream of and will potentially modify the disease process.

Our group and others have previously characterised two new inflammasome inhibitors, Peptide5 and tonabersat, important in several respects, including that they do not act directly on the inflammasome. These upstream inhibitors act by blocking connexin43 hemichannels whose opening mediates an ATP-dependent activation of the NLRP3 inflammasome. Using in vitro and in vivo models of DR (Mugisho et al., 2018a, 2018b, 2019a), it has been shown that both drugs are able to prevent NLRP3 complex assembly inhibiting chronic inflammation and blood vessel breakdown, to more effectively address the clinical signs of DR. Of these two inhibitors, tonabersat is of interest because it is an orally available drug, removing the need for intravitreal injections potentially having a bilateral effect on the retinopathy. However, these studies were conducted using cell culture and animal disease models that, while important and predictive, may not entirely replicate the human condition and some organotypic human retinal explant models are being evaluated for the study of ocular diseases and identification of novel therapeutic agents (Valdes et al., 2016).

Here, we have developed an ex vivo human organotypic retinal culture model of DR by exposing human donor retinal explants to a combination of high glucose (HG) and pro-inflammatory cytokines, interleukin-1 beta (IL-1β) and tumour necrosis factor alpha (TNF-α). We hypothesized that in this model, the hemichannel modulator tonabersat would protect against NLRP3 inflammasome complex assembly which would in turn decrease signs of inflammation characteristic of DR. Molecular changes in the inflammatory and inflammasome pathway were assessed using immunohistochemistry and a Luminex cytokine release assay.

Section snippets

Preparation of human retinal explants

Human donor eye cups were obtained from the New Zealand National Eye Bank following corneal excision for transplantation. The use of human donor explants for research was approved by the Northern B Health and Disability Ethics Committee (NTX/06/19/CPD). Three pairs of donor eye cups were used in this study with the donor information outlined in Table 1. The average age of the donors was 70.0 ± 11.5 years with an average post-mortem delay of 17.5 ± 5.1 h with no systemic or eye diseases reported.

Tonabersat treatment prevents HG + Cyt induced GFAP immunoreactivity

Results showed that in normal culture conditions (basal group), GFAP expression was observed in the GCL and IPL layers only, even after 72 h in culture (Fig. 2A). In the presence of HG + Cyt, there was both an increase in overall GFAP labelling and with GFAP-positive processes now spanning from the GCL to the ONL, consistent with both astrocytosis and Müller cell activation (Fig. 2A). Quantification confirmed that this increase (12.0 + 0.6%) compared to normal conditions (6.9 + 1.1%) was

Discussion

A combination of HG and pro-inflammatory cytokines, IL-1β and TNF-α, has previously been used to model signs of DR in vitro and in vivo (Mugisho et al., 2018a, Mugisho et al., 2018b, Mugisho et al., 2019a). In a recent study, mouse retinal explants when cultured under these conditions also exhibited biochemical and molecular changes akin to the human DR condition (Shivashankar et al., 2020). In that study, D-mannitol did not have any effect on the biochemical properties of the retina, including

Funding

This work was supported by the Maurice and Phyllis Paykel Trust and the Auckland Medical Research Foundation [1117015]. CRG holds the W&B Chair in Ophthalmology and IDR's directorship is supported by the Buchanan Charitable Foundation.

Acknowledgement

The authors would like to thank the generous donors of eye tissues, and the team from the New Zealand National Eye Bank for their support with this project.

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