Investigating a role for TLR signalling and complement deposition in retinal degeneration

Abstract

Age-related macular degeneration (AMD) and Retinitis Pigmentosa (RP) are the most common cause of progressive vision loss in the developed world. AMD is a multifactorial disease with both genetic and environmental risk factors and has a worldwide prevalence of 196 million. RP is a group of inherited retinal degenerations caused by up to 60 mutations which effect the function of photoreceptors. RP affects 1:4000 people worldwide.

Aberrant immune activation, complement deposition and oxidative stress have all been linked to AMD pathogenesis, however, the mechanisms initiating AMD progression are still unknown. Pathological hallmarks of AMD include marked deposition of complement factor 3 (C3) sub-retinal pigment epithelium (RPE) and increased deposition of the oxidative protein modification 2-(-carboxyethyl) pyrrole (CEP). It is now widely accepted that inappropriate activation of the alternative complement cascade is involved in AMD progression, however, the cause of C3 deposition and the outcome remains elusive. Recently, CEP has been identified as an endogenous ligand for TLR2. TLR2 activation has been previously linked to complement deposition in a mouse model of ischemia/reperfusion injury, where blocking TLR2 significantly reduced C3 deposition. Given the evidence of crosstalk between TLRs and complement we hypothesised that TLR2 ligation is responsible for C3 deposition in AMD.

We observed positive staining of TLR2 and its adaptors Mal and MyD88 in normal and AMD donor eyes and have demonstrated TLR2 ligation induces alternative complement factor expression in monocytes, macrophages and the RPE. Interestingly, C3d is released basolateraly from polarised RPE cells corresponding with C3d staining observed sub-RPE in AMD donor eyes. Stimulation with CEP-HSA induced robust secretion of C3 and CFB from the RPE. CFB is an initiating member of the alternative complement pathway and C3 is a central molecule which once activated can further amplify complement activation. In the presence of normal human serum CEP-HSA promoted the alternative complement pathway to completion resulting in terminal sub-lytic Membrane attack complex (MAC) formation and monocyte chemoattractant protein-1 (MCP-1) secretion. Furthermore, we demonstrated that the anaphylatoxins C3a and C5a produced during proteolytic activation of complement can synergize with TLR2 ligands to induce robust pro-inflammatory cytokine secretion from mononuclear cells.

We found that TLR2 induced complement activation was dependent on the presence of its adaptor proteins Mal and MyD88. Furthermore, MAC formation was inhibited using anti-TLR2 blocking antibody and a Mal peptide inhibitor. Demonstrating for the first time that CEP can induce proteolytic complement activation and MAC formation via TLR2 activation. To assess whether blocking TLR2 would be effective in preventing RD we used two mouse models of oxidative retinal degeneration. Both models mimic some aspects of AMD including RPE atrophy, photoreceptor loss and C3 deposition. We have demonstrated that intravitreal injection of anti-TLR2 blocking antibody protects against RD in both models.

A second TLR ligand dsRNA has previously been reported in AMD donor eyes and is known to induce RD by initiating photoreceptor apoptosis and necrosis. We have demonstrated that dsRNA can also induce complement factor expression and secretion from RPE and photoreceptor cells. Moreover, dsRNA in the presence of NHS induced proteolytic complement activation resulting in MAC formation. dsRNA is released from dying cells and is therefore conceivably present in multiple forms of RD including RP. We hypothesised that complement deposition may occur in inherited forms of RD. We studied complement expression in Rho-/- mice, a model of RP. We observed upregulated expression of C3 in the retina and RPE/choroid of Rho-/- mice and increased C3d deposition on the photoreceptor nuclei and surrounding the RPE. In addition, we observed C1q deposition on photoreceptor synapses which co localised with CD68 a marker of activated microglia. C1q, C3 and C4 deposition on synapses has been reported to activate a process of synaptic pruning to target unwanted connections for engulfment by microglia. This process has recently been suggested to play a role in a mouse model of glaucoma. Interestingly, we also observed increased C4 expression in the RPE/choroid and retina of Rho-/- mice. Given our observation of increased C3 expression and specific localisation of C1q to photoreceptor synapses we hypothesise that aberrant reactivation of synaptic pruning plays an important role in RD.

In conclusion, TLR activation by endogenous ligands in the retina can promote complement pathway activation and contribute to the pathogenesis of AMD and RP, two forms of retinal degeneration.