A Systematic Investigation on Complement Pathway Activation in Diabetic Retinopathy

Shahna Shahulhameed,Mudit Tyagi,Jay Chhablani,Saumya Jakati,Subhabrata Chakrabarti,Inderjeet Kaur,Sushma Vishwakarma,Rajeev R Pappuru

doi:10.3389/fimmu.2020.00154

Shahna Shahulhameed, Mudit Tyagi + Show 6 more

Open Access

https://doi.org/10.3389/fimmu.2020.00154

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Abstract

The complement system plays a crucial role in retinal homeostasis. While the proteomic analysis of ocular tissues in diabetic retinopathy (DR) has shown the deposition of complement proteins, their exact role in the pathogenesis of DR is yet unclear. We performed a detailed investigation of the role of the complement system by evaluating the levels of major complement proteins including C3, C1q, C4b, Complement Factor B (CFB), and Complement Factor H (CFH) and their activated fragments from both the classical and alternative pathways in vitreous humor and serum samples from proliferative DR (PDR) patients and controls. Further, the expressions of complements and several other key pro- and anti-angiogenic genes in the serum and vitreous humor were analyzed in the blood samples of PDR and non-PDR (NPDR) patients along with controls without diabetes. We also assessed the pro-inflammatory cytokines and matrix metalloproteinases in the vitreous humor samples. There was a significant increase in C3 and its activated fragment C3bα' (110 kDa) along with a corresponding upregulation of CFH in the vitreous of PDR patients, which confirmed the increased activation of the alternative complement pathway in PDR. Likewise, a significant upregulation of angiogenic genes and downregulation of anti-angiogenic genes was seen in PDR and NPDR cases. Increased MMP9 activity and upregulation of inflammatory markers IL8 and sPECAM with a downregulation of anti-inflammatory marker IL-10 in PDR vitreous indicated the possible involvement of microglia in DR pathogenesis. Further, a significantly high C3 deposition in the capillary wall along with thickening of basement membranes and co-localization of CFH expression with CD11b+ve activated microglial cells in diabetic retina suggested microglia as a source of CFH in diabetic retina. The increased CFH levels could be a feedback mechanism for arresting excessive complement activation in DR eyes. A gradual increase of CFH and CD11b expression in retina with early to late changes in epiretinal membranes of DR patients indicated a major role for the alternative complement pathway in disease progression.

Highlights

Diabetic retinopathy (DR), characterized by pathological ocular angiogenesis in retina, is a major cause of irreversible vision loss worldwide, with a global prevalence of 34.6% [1]
The role of the complement system in angiogenesis is of prime importance, since there are several eye diseases associated with abnormal ocular angiogenesis and neurodegeneration, such as retinopathy of prematurity (ROP), age-related macular degeneration (AMD), and proliferative diabetic retinopathy (PDR) [12,13,14]
The levels of total C3 molecules and of individual C3 activated fragments in PDR patients and controls were separated on PAGE and evaluated by quantifying the mean band intensity using Image StudioTM Lite quantification software (LI-COR)

Summary

Introduction

Diabetic retinopathy (DR), characterized by pathological ocular angiogenesis in retina, is a major cause of irreversible vision loss worldwide, with a global prevalence of 34.6% [1]. Low-level activation of the innate immune mechanisms, the complement system, is required to preserve normal eye homeostasis and maintain retinal integrity while aging [4]. This protective mechanism can have a detrimental impact if the insults persist for a longer duration and leads to irreversible functional loss, as is seen in neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and age-related macular degeneration (ARMD) [5]. The complement system, besides having a major role as an immune defense mechanism, is involved in several tissue-remodeling processes, such as liver regeneration and synaptic pruning during development, and in retinal angiogenesis and neurodegenerative diseases [8,9,10,11]. The role of the complement system in angiogenesis is of prime importance, since there are several eye diseases associated with abnormal ocular angiogenesis and neurodegeneration, such as retinopathy of prematurity (ROP), age-related macular degeneration (AMD), and proliferative diabetic retinopathy (PDR) [12,13,14]

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