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. 2020 Feb 11:11:154.
doi: 10.3389/fimmu.2020.00154. eCollection 2020.

A Systematic Investigation on Complement Pathway Activation in Diabetic Retinopathy

Shahna Shahulhameed  1 , Sushma Vishwakarma  1 , Jay Chhablani  2   3 , Mudit Tyagi  2 , Rajeev R Pappuru  3 , Saumya Jakati  4 , Subhabrata Chakrabarti  1 , Inderjeet Kaur  1
Affiliations

A Systematic Investigation on Complement Pathway Activation in Diabetic Retinopathy

Shahna Shahulhameed et al. Front Immunol. .

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.

Keywords: angiogenesis; complement pathway; diabetic retinopathy; inflammation; microglia; retina; vitreous humor.

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Figures

Figure 1
Figure 1
(A) Representative Western blot of C3 in PDR and no-DM vitreous. (B) Quantification of total C3 in PDR and in no-DM control vitreous by densitometry (PDR, n = 38 and Control, n = 38). (C) Quantification of C3bα' (110 kDa) in PDR (n = 16) vitreous compared to control vitreous (n = 16). **p = 0.004, *p = 0.03, respectively; data represented as mean ± SEM, C, control vitreous; D, PDR vitreous; L, protein ladder.
Figure 2
Figure 2
Representative Western blots of (A) C1q, (B) C4b, and (C) Factor Bb in PDR and No-DM controls, (D). Quantification of C1q in PDR (n = 17) and no-DM control (n = 17) vitreous, p > 0.05, C4b in PDR (n = 8) and no-DM control (n = 8) vitreous, p > 0.05 and Bb in PDR (n = 22) and no-DM control (n = 22) vitreous, *p = 0.03. Data represented as mean ± SEM, D, PDR; C, controls; L, protein ladder; n.s., not significant.
Figure 3
Figure 3
Representative Western blot of (A) CFH (150 kDa) in PDR and No-DM controls and (B) CFH (150 kDa) in PDR, NPDR, and no-DM control serum. Quantitative estimates of (C) CFH band in PDR (n = 31) vs. no-DM control (n = 31) vitreous, ***p < 0.0004 and (D) CFH band in PDR (n = 12), NPDR (n = 12), and no-DM control (n = 12) serum, p > 0.05 (not significant). Data represented as mean ± SEM, D, PDR vitreous; C, control vitreous; L, protein ladder; DS, PDR serum; NS, NPDR; CS, Control serum; n.s., not significant.
Figure 4
Figure 4
Representative photomicrograph showing thickened and dilated blood vessels in diabetic retina (B) in comparison to control retina (A) (Periodic acid-Schiff, Magnification: 40X). (C) Quantification of number of capillaries in diabetic vs. control retinal tissues. (D) Quantification of capillary thickness in diabetic retina vs. control retina. *p = 0.01, ***p = 0.0006.
Figure 5
Figure 5
Representative images of the 10X magnified H and E sections and post-immunofluorescence showing the localization of C3 and GFAP in retinal tissues collected from (A) control and (B) diabetic retina, C3, and CD11b in (C) control and (D) diabetic retina, and CFH and CD11b in (E) control and (F) diabetic retina, magnification 20X, scale bar 200 μm. Co-localized expression of CFH and CD11b is highlighted in 40X magnification in panel (F).
Figure 6
Figure 6
Representative image of (A,B) immunofluorescence of CXCR4 in retinal tissues collected from control and diabetic retina, respectively, magnification 20X. (C) Representative Western blotting of CD11b in PDR vitreous (n = 5) compared to controls (n = 4); (D) Representative gelatin zymography of vitreous samples from PDR (n = 10) vs. controls (n = 10) (C, control; D, PDR). (E) Scatter plot with individual data points showing differential levels of soluble VEGF (***p = 0.0002) and VEGFR2 (*p = 0.03) (PDR, n = 8, Control, n = 8) and VEGF (*p = 0.04) (PDR, n = 6, Control, n = 6) by multiplex ELISA.
Figure 7
Figure 7
Scatter plot with individual data points showing the quantitative estimation of inflammation in the vitreous samples based on Multiplex ELISA from PDR (n = 8) vs. controls (n = 8) (C, control; D, PDR) for (A) sPECAM and IL-8 and (B) IL-10 in PDR vs. control vitreous. Differential expression based on quantitative PCR for (C) complement and angiogenic genes from blood in PDR (n = 20) and NPDR (n = 20) vs. no-DM controls (n = 20), (D) early (diabetic retina) vs. late changes, and (E) ERM tissues in the proteins involved in the activation of the alternative complement pathway. Data represented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns, p > 0.05.
Figure 8
Figure 8
A Schematic summary of the role of complement activation by microglial cells in diabetic retinopathy.
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