Role of Inflammatory Cells in Pathophysiology and Management of Diabetic Retinopathy

Abstract

Diabetic retinopathy is a potentially sight-threatening complication of diabetes mellitus. Several inflammatory cells and proteins, including macrophages, and microglia, cytokines, and vascular endothelial growth factors are found to play a significant role in the development and progression of DR. Inflammatory cells play a significant role in the earliest changes seen in DR including the breakdown of the blood retinal barrier leading to leakage of blood into the retina. They also have an important role in the pathogenesis of more advanced stage of proliferative diabetic retinopathy leading to neovascularization, vitreous hemorrhage, and tractional retinal detachment. In this review, we examine the function of numerous inflammatory cells involved in the pathogenesis, progression, and role as a potential therapeutic target in DR. Additionally, the role of inflammation following treatment of DR has also been explored.

I. Introduction

Diabetic retinopathy (DR) is a complication that is common and specifically seen in diabetics and is one of the leading causes of preventable blindness.[1] Clinically, DR is divided into two stages: non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). NPDR is characterized by increased vascular permeability and capillary occlusion, forming microaneurysms and hemorrhages in the retinal vasculature, while PDR is characterized by neovascularization and tractional retinal detachment.[2] One-third of people with DR develop diabetic macular edema (DME) secondary to the breakdown of the blood-retinal barrier (BRB), leading to vision loss.[3] Improvement in our understanding of the pathophysiology of DR can lead to earlier detection and a newer therapeutic approach to this vision-threatening disease. Here in this review, we explore the evidence for the hypothesis that inflammatory cells play a critical role in pathophysiology, the prognosis, and as a potential therapeutic target in DR.

II. Overview of Diabetic Retinopathy

A. Role of the neurovascular unit in Diabetic Retinopathy

Traditionally DR has been considered a microvascular complication of diabetes and is associated with the breakdown of the BRB.[3] The damage to the endothelial cells, basement membrane thickening, and loss of pericytes can cause increased capillary permeability by disrupting the BRB. However, the damage to the microvasculature cannot fully explain the damage to the retina and the loss of retinal function. Several studies have looked at the retina as a neurovascular unit which constitutes the delicate functional interactions between the blood vessels, neurons, and the glial cells in the retina. [4] Neurons exhibit changes in biochemical signaling and in cases of uncontrolled diabetes, undergo apoptosis. Glial cells and astrocytes also undergo changes, as do the microglial cells which have a predominant role as the local macrophage. This gives rise to the idea that DR could be a form of neurovascular degeneration and not merely a microvascular dysfunction. [4] Visual function, including contrast sensitivity and microperimetry, has been shown to be affected in the early stages of DR even when visual acuity is relatively intact. [5] During the early stages, the retina has the ability to adapt its high metabolic activity to a lower steady-state. [6] Continuing metabolic and inflammatory stress to the neurovascular unit over a period of 5–10 years leads to the clinical features of DR, including microaneurysms, venous dilation, exudation, hemorrhages, and diabetic macular edema.

B. Role of inflammation in the pathophysiology of DR

A low level of inflammation (para-inflammation) through activation of the microglia and complement system is proposed to maintain homeostasis in the very early stages of DR when the BRB is still intact. [7] Stimulation of the damage associated molecular patterns over a long period of time can lead to dysregulation of the immune systems leading to the development of DR. Additionally vascular dysfunction has also long been the focus of interest in the pathogenesis of DR. One of the key factors which play a role in the increased vascular permeability and angiogenesis is the overexpression of vascular endothelial growth factor (VEGF). [3] Lupo et al in their study showed that high or fluctuating levels of glucose could induce a breakdown in the blood retinal barrier, and also increased the levels of VEGF and Phospholipase A2. Inhibition of Phospholipase A2 decreased the level of VEGF to normal levels. [8] There is also associated thickening of the vascular basement membrane due to the upregulation of fibronectin, collagen, and laminin, which produces changes in the factors mediating the growth and function of the pericytes and endothelial cells. [9] Pericytes play a significant role in the delineation, proliferation and migration of endothelial cells and the loss of pericytes is believed to be a characteristic feature of DR. The pericytes are highly susceptible to the variations in the metabolic environment and loss of pericytes play a significant role in the development of microaneurysms, and capillary nonperfusion. [10] High levels of glucose can cause appostosis and reduce the viability of retinal pigment epithelial cells and these effects have been shown to be counteracted by curcumin in vitro.[11]

Patients with no clinical signs of DR have shown glial activation or gliosis, and increased levels of glial fibrillary acidic protein (GFAP), aquaporin 1 (AQP1), and aquaporin 4 (AQP4) have been reported in the aqueous humor of these patients.[12] Müller glial cells are an important source of numerous factors, including inflammatory modulators, and their activation may have a role in the onset of the inflammatory process, which is responsible for retinal damage and a valuable contributor for the development of DR.[12] (Figure 1) Various inflammatory cytokines and chemokines (interleukin-(IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-10, interferon-γ, tumor necrosis factor-α (TNF-α)) are found to be elevated in serum and ocular samples of both vitreous and aqueous humor, from diabetic patients with DR. [13, 14] One of the initiating events in the release of these proinflammatory mediators is the activation of purinergic ionotropic receptor P2XN by extracellular adenosine tri phosphate. [15] In the pathogenesis of macular edema, hyperglycemia produces rupture of BRB secondary to vasogenic changes. This is associated with low-grade inflammation, which can produce permanent damage to the retina. [3] The response of macular edema to various forms of steroids also implies the significant role of inflammation in this process.

Figure 1.

Th = T helper cell; Treg = regulatory T cells; Tfh = T follicular helper cells; miRNA = microRNA; IL = interleukin; TNF-α = tumor necrosis factor α; CTLA-4 = cytotoxic T lymphocyte-associated protein 4; RBC = red blood cells; NETs = neutrophil extracellular traps; ICAM-1 = intracellular adhesion molecule 1; IFN-α = interferon alpha; VEGF-A = vascular endothelial growth factor-A; ERK = extracellular signal-regulated kinase.

III. Cell types involved in pathogenesis and progression of DR

A. Leukocytes

One of the earliest identifiable features in DR is leukostasis, with increased adherence of leukocytes to the retinal vasculature. [16, 17] A reduction in retinal perfusion and narrowing of capillaries was initially thought of as contributing factors. However, mounting evidence points towards a chronic low-grade inflammation that induces adhesion of the leukocyte to the endothelium. This leads to damage to the endothelium, eventually impairing the BRB. Under glycemic conditions, the adhesion of leukocytes to retinal capillaries contributes to endothelial damage and the subsequent increase in vascular permeability results in macular edema and capillary occlusion. [16, 17] The intravascular coagulation occurs as a result of several rheological alterations associated with diabetes: these include activation of leucocytes, reduced deformity of these activated leukocytes, and increased expression of adhesion molecules of vascular cell adhesion molecule-1 (VCAM-1) intercellular adhesion molecule-1 (ICAM-1) and P-selectin in vascular endothelial cells. [18] There is an increase in the levels of various adhesion molecules in circulation as the retinopathy progresses due to shedding from the leukocytes and the damaged endothelium. L-selectin (CD62L) an adhesion molecule found on the surface of lymphocytes, granulocytes, and monocytes, plays a significant role in the trapping and subsequent rolling of leukocytes along the endothelium. [19] MacKinnon et al in their study found a significant reduction in expression of L-selectin levels on lymphocytes (CD3+) in patients with DR (p=0.004). They also found that L-selectin mRNA levels (p = 0.007) and serum L-selectin are significantly higher (p = 0.04) in patients with DR. [15] This observation lends credibility to associate leukocyte activation more specifically with the onset of microvascular disease of the retina in DR.

ICAM1 has been shown to be upregulated under conditions of hyperglycemia. It acts as a mediator facilitating the adhesion of leukocytes to the endothelium leading to changes in the vascular endothelium, increased vascular permeability and areas of capillary non-perfusion. [20] The source of the stimulus for the ICAM-1molecule expression has been evaluated and it was demonstrated that it is seen associated with an increase in retinal 12/15-lipoxygenase expression in the inflammatory response, which is seen in early DR. This has been demonstrated in the vitreous in patients with DR [21] and in the human retinal endothelial cells incubated with high glucose. [22] The endothelial 12/15-lipoxygenase and not the monocytic or macrophagic 12/15 lipoxygenase that plays the most important role in the expression of ICAM-1 leading to leukocyte adhesion followed by breakdown of the BRB. [23]

1. T cells

Chronic low-grade inflammation has been identified as an immunopathologic change found in DR. [24] This is associated with multiple events, including upregulation of inflammatory mediators as well as the activation and trafficking of immune cells, especially CD4+ T cells, [25] which are recruited along with other immune cells and cause inflammation and accelerate vascular injuries. [26] Additionally, advanced glycation end products have been found to promote CD4+ T cells differentiation into a pro-inflammatory state, [27] while there was a reversal of insulin resistance in T2DM by the T regulatory cells. [28] The main CD4+ effector T cells include T helper 1 (Th1), Th2, and Th17 cells. A new subset called follicular helper T cells (Tfh) were found to be reduced in normal subjects but significantly expanded in patients with DR. [29] Bcl-6, a crucial transcription factor in the development of Tfh cells, when inhibited, reduces retinal vascular leakage suggesting that Tfh may play a role in retinal inflammation and angiogenesis in DR. [29]

The non-coding RNA, microRNA (miRNA), has been identified as an important regulator of numerous biological tasks. There are over 2000 human miRNA listed in miRBase which regulate gene transcription. An alteration in the level of expression of miRNA can lead to a reduction and dysfunction of the number of pancreatic islets β cells causing resistance to insulin and can lead to chronic inflammatory responses in diabetics. [30] The miRNA is believed to regulate the proliferation and functions of cytokines to play a role in intracellular signal transduction of the T regulatory (Treg) cells. [31] Regulatory T cells can express Foxp3, the key transcriptional factor, which is important in the differentiation and maintenance of the function of Treg cells. This in turn can lead to the secretion of TGF-β and Interleukin-10 (IL-10). [32] The Treg cells have anti-inflammatory properties through contact inhibition and immune suppressive function. [33] Although miR-155 can regulate Treg cell differentiation and Foxp3 expression, it was not found to play a role in its immune suppressive function. [34] Yang et al in their study evaluated the Treg cell levels in patients with DR and were found to be significantly lower than those in healthy controls. The levels were observed to vary dynamically with the severity of the disease. [35] In patients with PDR, the Treg cell count was reduced significantly, and there was a downregulation of the expression of transcriptional factor TGF-β and other pro-inflammatory factors.[36] TGFβ1 has been proposed as a diagnostic and prognostic biomarker for the stages of DR as the serum levels of TGFβ1 was shown to be predictive of progression of the disease from NPDR to PDR.[37]

Cytokines, which are implicated in the pathogenesis and complications of T2DM are vital for the maintenance of the balance of the immune responses. T cell proliferation and activation are believed to be an important step in the pathogenesis of T2DM and IL-1, IL-6 and TNF-α are known to trigger the elevation of inflammatory markers. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which is an inhibitory regulator of T cell activation and proliferation, plays a significant role in regulating immune responses. It is a transmembrane glycoprotein which plays a role in T-cell activation as a co-stimulatory molecule, [38] and there are over 100 single nucleotide polymorphisms (SNP) in its genetic coding which regulate its expression and activity. The SNPs of the CTLA-4 gene has been related to the development of T1DM. [39] However, an association with T2DM and the CTLA-4 SNPs has revealed inconsistent results. [40, 41] Shih et al evaluated the role of the negative regulator of immune response, CTL-4, in regulation of lipid metabolism and the onset and evolution of diabetes. T2DM subjects carrying C/T genotype CTLA-4-318 SNPs, showed significantly low levels of serum triglycerides. Control subjects showed an association between −318 genotypes with fasting glucose and CTLA-4 +49 genotypes with CHO/HDL although this was not statistically significant. This suggests a possible role of CTLA-4 in lipid metabolism and in turn affects T2DM disease progression. [42] This is supported by yet another study which reported a correlation between CTL-4 genotype and progression of diabetic disease, development of microangiopathy and earlier onset of insulin requirement. [43]

The retinal vascular endothelium is a part of the BRB and any damages to the vascular endothelium can affect the remodeling repair of these cells. The vascular endothelial cells can secrete nitric oxide, [44] cytokines and play a role in regulation of inflammation. [45] IL-4, a Th2 differentiation cytokine, affects and regulates the vascular endothelial cells. [46] The proliferation of umbilical vein endothelial cells has been shown to be inhibited by IL-4 through the expression of p53, p21, cyclin D1, and cyclin E.[47] Upon application of the IL-4 antibody, the abilities of proliferation and migration was restored in the human umbilical vein endothelial cells. [48]

a. The Natural killer T cells

The Natural killer T (NKT) cells are cytotoxic and contain small granules containing perforin and proteases like granzyme B which upon release forms pores in the target cell inducing apoptosis and have an important role in immune regulation. They are heterogenous immune cells which resemble the actions of both T cells and NK cells. [49] They have T lymphocyte markers which bind to ligands to activate the NKT cells to secrete cytokines (like IFN-γ, IL-4, and IL-17) contributing to the immune cascade and Th1/2 conversion. [50] Diabetic patients exhibit several functional abnormalities including atypical activation of the NKT cells, disproportionate Th1/Th2 ratio and vascular remodeling via macrophages. [51] A significant increase has been noted in the ratio of CD3+CD56+ NKT cells and an increase in the ratio of CD3−CD56+ NK cells in peripheral blood of T2DM. [48] Additionally, it was also seen that the CD4+ and CD4−CD8− NKT cells values were significantly elevated whereas the number of CD8+ NKT cells were significantly reduced. The production of IL-4 and IFN γ secondary to TCR activation was significantly elevated in diabetics.[48]

2. B Cells

T cells have been traditionally considered to be the major destroyers of pancreatic β-cells in patients with T1DM, but there is increasing evidence for the role of B cells in destruction of pancreatic β-cell in T1DM pathogenesis. Delay in β cell-depletion is shown to delay disease progression in patients with newly diagnosed T1DM. Majority of B cells which are newly synthesized in the bone marrow are highly reactive and self-destructive as they have the capacity to bind self-antigens like DNA or insulin via their B-cell receptor. [52] These self-reactive B-cells must be culled and rehabilitated to avert the development of autoimmunity. However, the role of B cells in T1DM is still under investigation. It has been suggested that B cells present islet autoantigens to diabetogenic T cells, in addition to cytokine production and autoantibodies to islet cell antigens. [53] It is believed that the T cells are activated when the B cells present the antigen and this in turn leads to destruction of pancreatic β cells resulting in T1DM.

In patients with T1DM, autoreactive B cells are thought to be more numerous in the blood when compared to healthy individuals. [54] Autoreactive B cells are checked at two barriers- the immature cells undergo receptor editing/deletion in the bone marrow and anergy in the periphery. [52] An impairment of these checkpoints leads to an increase in the number of autoreactive B cells at the transitional stage and the naïve mature B cells. [54] Smith et al evaluated patients with islet autoantibodies but without DM, and patients with new onset T1DM (< 1 year) and compared them with healthy controls. The levels of insulin-binding anergic B cells in blood of these patients were found to be significantly lower compared to controls. [55] Additionally, it is also seen that depletion of B cells using anti-CD20 or anti-CD22 monoclonal antibodies, can prevent the development of diabetes. [56] Also, Rituximab, a monoclonal antibody, when used to deplete B-cells in T1DM patients, has been shown to preserve β-cell function and postponed insulin requirement following 1 year of treatment. [57] Thus B cell depletion may be of some benefit to persons who produce multiple islet-cell antibodies and are at a high risk of developing T1DM.

The nuclear factor kappa-light-chain-enhancer of activated B cells play a significant role in transcriptional changes in activated microglia. These along with extra-cellular signal regulated kinase signaling pathways can lead to the release of several mediators of inflammation like cytokines and chemokines. These changes in the microglia can lead to apoptosis of the affected retinal neurons and thinning of the retinal nerve fiber layer leading to loss of vision. [58] It has also been found that the expression of IL10 in peripheral B cells is suppressed by IL-17 by enhancing microRNA-19A expression and Histone deacetylase 11 in patients with DR. Thus these may be considered to be novel therapeutic targets for the management of DR.[59]

3. Neutrophils

Hyperglycemia is a promoter of DR and plays a role in activating other downstream pathways with chronic inflammation and oxidative stress. The process of releasing neutrophil extracellular traps (NETs) into the extracellular space to defend against pathogens known as NETosis is a neutrophil-specific cell death process. [60, 61] NETs are composed of a decondensed chromatin structural framework containing proteins such as myeloperoxidase, neutrophil elastase, proteinase, and histones. Several studies have shown that neutrophils can trigger NETosis in the presence or absence of reactive oxygen species (ROS), [62, 63] which in turn may provide a link between hyperglycemia induced NETosis and NADPH oxidase pathway. (Table 1) Neutrophils in peripheral blood infiltrate and adhere to vascular endothelial cells following breakdown of the BRB leading to NET formation in the diabetic rat retina and the vitreous bodies of DR patients. Long-term hyperglycemia provides peripheral neutrophils with more opportunities to cross the BRB, form NETs and deposit in the vitreous body as well as the retina. The NETs are found to be elevated in the serum of diabetic individuals and are induced by classical NETosis inducer: phorbol myristate acetate (PMA) and high glucose. [62, 64] Additionally, it is also seen that the NET production increased with worsening stages of DR. [62] ROS levels are found to be significantly higher in hyperglycemia induced, which suggests the involvement of NADPH oxidase activation. It has been reported that ROS promotes the production of VEGF, and VEGF in turn promotes ROS production derived from NADPH oxidase leading to a vicious cycle. Besides, ROS are critical chemical species in VEGF receptor 2-mediated signaling, which leads to neovascularization in DR. [65, 66] These findings indicate that NADPH oxidase-derived ROS may be another signaling pathway involved in anti-VEGF therapy.

Table 1.

Cells involved in pathogenesis and progression of Diabetic Retinopathy

Cell	Subtype	Factor	DM	NPDR	PDR	Location	Study	Host
Neutrophil – Lymphocyte ratio	-	-	↑	↑	-	Blood	Wang et al 2015	Human
Neutrophil (CD11b+MPO+)	NET	NADPH oxidase	↑	↑	↑↑	Blood	Wang et al 2019	Human
			-	-	↑	Vitreous		Human
			↑	-	-	Blood, Retina, Vitreous		Rat
	NET	IL-6	↑	-	-	Blood	Menegazzo et al 2015	Human
T lymphocytes (Th1, Th2, Th17, Treg, Tfh)	Tfh cells (PD-1+ CXCR5+CD4+)	Bcl-6, IL-21	↑	-	-	Blood	Liu et al 2020	Humans and mice
	Th	IL-17 and IL-22	↑	-	-	Blood	Taguchi et al 2020	Mice
		VEGF, I-CAM	↑			Vitreous
	Th	IL 17A	↑	↓	↓	Blood	Chen et al 2016	Human
	Th	IL-4, IL-17A, IL-22, IL-31, TNFα	-		↑↑	Vitreous	Takeuchi et al 2015
	Th	IL-17, IL-10, IL-22, TNFα	-	↑	↑↑	Vitreous	Takeuchi et al 2017
	Th 17	IL-35	-	-	↓	Blood	Yan et al 2022
		IL-17			↑
	Treg (CD4+CD25+ Foxp3+)	Percentage of Treg	↓	↓↓	↓↓↓	Blood	Yang et al 2015
		miR-155,	↑	↑↑	↑↑↑
		TGF-β	↓	↓↓	↓↓↓
	Treg	CTLA-4 gene polymorphism	↑	-	-	Blood	Douroudis et al 2009
	Treg	CTLA-4 49A/G polymorphism	↑	-	-	Blood	Caputo et al 2005
	Treg	CTLA-4 polymorphism	↑	-	-	Blood	Shih et al 2018
	Treg	CTLA-4 49A/G polymorphism	Not a risk factor	-	-	Blood	Uzer et al 2010
	Treg	CTLA4 Alanine 17	Not a risk factor	-	-	Blood	Rau et al 2001
	NK T cells (CD3+CD56+)	IL-4, IFNγ	↑	-	-	Blood	Lv et al 2018
	CD3+	L-Selectin	↑	-	-	Blood	MacKinnon et al 2004
B lymphocyte (CD19+CD20+)	Autoreactive B cells	-	↑	-	-	Blood	Menard et al 2011	Human
	B cell	IgA, IgM,	↑	-	-	Vitreous	Liu 2020
	B cell	IL-10	↓	-	-		Wang et al 2017
		miR-19a	↑
Monocytes/Macrophage (CD11b+CD68+)	MIF, MCP-1	-	-	↑	↑↑	Aqueous	Tashimo et al 2004	Human
	CD11b+CD68+	CCL2, F4/80	↑	↑	-	Retina	Rangasamay et al 2014	Rats
	MCP-1	TNF-α	↑	-	-	Retina	Dong et al 2014
	Monocyte	Cathepsin D	↑	↑	↑	Retina	Monickraj et al 2016	Humans and mice

DM - Diabetes Mellitus

NPDR - Non-proliferative diabetic retinopathy

PDR - Proliferative diabetic retinopathy

NET - neutrophil extracellular traps

NADPH - nicotinamide adenine dinucleotide phosphate

IL - Interleukin

Tfh - follicular helper T cells

Bcl6 - B-cell lymphoma 6

Treg - regulatory T cells

miR - microRNA

ICAM-1-intercellular adhesion molecule-1

VEGF - Vascular endothelial growth factor

TGF β-Transforming Growth Factor β

CTLA - Cytotoxic T lymphocyte antigen

NK T cells - Natural killer T cells

IFNγ - Interferon γ

MIF - Macrophage migration inhibiting factor

CCL2 - Chemokine ligand 2

F4/80 - Macrophage marker

MCP-1 - Monocyte chemotactic protein-1

TNF-α-Tumor necrosis factor-α

4. Monocytes / macrophages

The activated monocytes are observed adhering to the outer surface of the retinal capillaries as a part of the active process of extravasation and in the extravascular spaces providing evidence of breach of the BRB by diapedesis. [67] These activated monocytes then differentiate into macrophages which in turn secrete cytokines and other growth factors like VEGF, TNFα and interleukin. The breakdown of BRB causing an increase in the permeability of retinal vasculature can be ascribed to an increase in these cytokines and growth factors. VEGF has been considered the mediator for increase in vascular permeability in DR and hence it has been the target of several clinical studies exploring the management of diabetic macular edema. [68, 69] The improvement seen following the use of anti-VEGF agents are transient and recurrence is common even after one year of monthly injections of anti-VEGF agents. [70] VEGF acts by inducing the phosphorylation of occludin which is dependent on Protein kinase C and this in turn increases permeability of the BRB.[71] The role of inhibiting Protein kinase C in an effort to prevent the breakdown of BRB is a potential avenue of study.

Macrophage migration inhibitory factor levels are found to be elevated in the vitreous of patients with PDR. It is a proinflammatory and proangiogenic cytokine which plays a significant role in ischemia induced retinal neovascularization. [72] Monocytes are also found to secrete increased levels of aspartyl proteinase Cathepsin D which causes an increase in a mechanical breakdown of the endothelial barrier. Thus, a potential avenue for the management of diabetic macular edema by targeting Cathepsin D on its own or combined with anti-VEGF agents.[73]

One of the strongest chemotactic factors and their receptors which attract the monocytes into the tissues is the CCL2, also known as the monocyte chemoattractant protein, is found to be elevated in patients with diabetic retinopathy.[74] The role of CCL-2 in altering the BRB either directly through effects on the retinal vasculature or indirectly through the recruitment of leukocytes in diabetic retina has been evaluated. The CCL-2 gene is found to be significantly upregulated in human retinal endothelial cells treated with glucose and the retina of diabetic rats. This was associated with an abundant influx of perivascular monocytes into the retinal tissues.[67] Additionally the levels of F4/80mRNA (monocyte/macrophage marker) in the retina was found to be doubled in diabetic animals compared to non-diabetic animals. It was also seen that intraocular injection of CCL2 in non-diabetic rats led to an increase in F4/80 expression in the retina showing the ability of this chemokine to attract monocytes/macrophages to the retina. Thus CCL-2 plays an indirect role in increasing the vascular permeability in diabetics.[67]

B. Glial cells

1. Microglia

Hyperglycemia-induced microvascular damage induces activation of the advanced glycation end products and superoxide production in excess secondary to cellular oxidative stress. This is followed by production of free radicals in the mitochondria, which can worsen the oxidative stress. [75–77] Oxidative stress via generation of reactive oxygen species (ROS) has been found to play a major role in activation of microglia.[78] ROS has also been found to induce nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and phosphorylation of extracellular signal–regulated kinase (ERK). NFκB which has been found to be increased in activated microglia produces more cytokines and plays a significant role in retinal angiogenesis. [79] ERK activation also plays a prominent role in expression of TNFα and in survival and proliferation of endothelial cells. [80, 81] Microglial activation can be observed distinctly in the various stages of DR. In the early stages the microglia are found in the inner retinal layers, collected around the intra-retinal hemorrhages and microaneurysms. [82] They later migrate to the plexiform layer and in the stage of PDR, the microglia are found to be significantly increased and are seen surrounding the ischemic areas. [82, 83]

2. Müller cells

Diabetes being a pro-inflammatory condition, induces Müller glia to produce IL-17A leading to neuronal apoptosis and vascular leukostasis.[38] IL-17A plays a significant role in retinal inflammation, oxidative stress, subsequent vascular leakage and capillary nonperfusion leading to proliferative diabetic retinopathy (PDR).[24] Additionally, some studies have revealed that the BRB breakdown is enhanced by intravitreal injections of IL-17A, and the diabetes-mediated retinal pathogenesis was decreased by blockade of the IL-17RA portion of the IL-17A receptor.[38]

IV. Clinical implications of inflammation in diabetic retinopathy

Inflammation has long been proposed as a contributing factor for the development of DR.[84] Consequently, aspirin was used to try and treat the disease in the early days, but however, it was seen that there was no effect on DR with aspirin,[85] and later studies have shown that the dosing was not high enough to produce the anti-inflammatory effect.[86] There are several clinical signs which are observed in patients with diabetic retinopathy. NPDR is characterized by microaneurysms, dot hemorrhages, and hard exudates in the retina. Microaneurysms of the retinal vessels are considered the hallmark of diabetic retinopathy and they are biomarkers of injury to the microvasculature. Activation of NFκB can lead to nonperfusion of the capillaries, loss of pericytes and these inflammatory cells play a significant role in the formation of microaneurysms.[87] TGFβ signaling in the pericytes plays a significant role in the thickening of the basement membrane in pre-clinical diabetic retinopathy, and complement activation leads to functional loss and death of these pericytes.[10] The microaneurysms that are formed following these damages are easily detected by fluorescein angiography and also by clinical ophthalmoscopy. Larger microaneurysms are found in areas with significant nonperfusion. Increased size of the microaneurysm, coupled with increased RBC adhesion, promotes the movement of platelets into the lumen of the microaneurysm, increasing the chance of thrombosis within the microaneurysm.[88] The dot hemorrhages seen in DR are secondary to increased vascular permeability following the breakdown of the BRB.[89] Growth factors and cytokines-VEGF, TNFα and interleukin, play a significant role in the breakdown of the BRB.[67] The monocyte chemoattractant protein CCL2, also plays an indirect role in increasing the vascular permeability. This increased vascular permeability causes the plasma proteins and lipids to leak into the retina forming hard exudates which appear as small white-yellow spots.[90] There are small areas of retinal ischemia surrounding the microaneurysms, which may gradually increase in size and develop into intraretinal microvascular abnormalities as the disease progresses. Further hypoxia and ischemia can produce microthrombosis with retinal microinfarcts producing cotton wool spots. Additionally larger flame-shaped hemorrhages may be seen especially if associated with hypertension. As the disease advances, PDR develops when the pericyte density is <50%. [10] IL-17A induced by Muller cells plays a significant role in oxidative stress, vascular leakage, and capillary nonperfusion, leading to PDR, [24]where larger areas of retinal ischemia lead to the development of fragile new vessels. Additionally, ROS via VEGF receptor 2 mediated signaling[65] and activated microglia play a significant role in the development of these new vessels. NFκB which has been found in these activated microglia produce cytokines which play a significant role in angiogenesis.[79] These vessels tend to bleed very easily leading to the development of large retinal and vitreous hemorrhages. [90] IL-6 plays a significant role in the infiltration of monocyte and lymphocytes, which subsequently activates the fibroblasts, which leads to the formation of tractional retinal detachment.[91]

Macular edema which is the most common cause of vision loss in DR, occurs due to an imbalance in the reabsorption of fluid into the retinal vessels from the extracellular space. In the hyperglycemic state seen in diabetes, leukocytes adhere to the endothelial cells facilitated by ICAM-1 and VCAM-1, and increase the vascular permeability by damaging the capillaries leading to a reduction in number of pericytes, endothelial cells and muller cells.[16] This increases the permeability of the retinal vessels leading to macular edema.[3] The Muller cells play a significant role in the transport of fluid from the extracellular space into the retinal vessels. Damage to these Muller cells, as a result of the breakdown of the blood retinal barrier will prevent the reabsorption of the fluid. IL-1b plays a significant role in this breakdown of the blood retinal barrier,[92] leading to increased fluid in the extracellular space which can later form cysts in the macular region.

V. Role of Inflammation following treatment of DR

Pharmacologic approach in DR is primarily aimed to treat macular edema. These include both systemic and ocular agents. Systemic agents that promote intensive glycemic control, control of dyslipidemia and antagonists of the renin-angiotensin system demonstrate beneficial effects for DR. Ocular therapies include anti-VEGF agents and corticosteroids. In hyperglycemic state, retinal ganglion cell survival was found to reduce significantly (54%) when grown alone and further decreased to 33% when co-cultured with Muller cells. Cytokines, including IL-1β, IL-6 and TNF-α which were found to be elevated under hyperglycemic conditions, reverted to basal concentration when dexamethasone was added. [93] Additionally, dexamethasone was found to salvage the retinal ganglion cells and Muller cells. However, currently the steroids are only used to address macular edema with intraocular injections and have no clinical use in preventing the development and progress of diabetic retinopathy.

Sitagliptin, an oral medication, has been found to prevent the diabetes induced rise in dipeptidyl peptidase-IV activity in both the serum and retina. It also promoted migration and capillary morphogenesis in the retinal endothelial cells challenged with TNF-α in animal models. [94] Sitagliptin has been found to inhibit the breakdown of the BRB by preventing the changes in the tight junctions and plays a role in protecting against inflammation and apoptosis.

Anti-VEGF therapy is evolving as the mainstream method, both in preventing the progression of and treating the macular edema. Recently, DRCR.net protocol S showed excellent 2-year clinical outcome for patients with PDR treated with anti-VEGF, questioning the common notion that pan retinal photocoagulation is the gold standard in these patients. The role of VEGF in neovascularization has been studied extensively, and it has also been shown to have effect in inflammation and reduction of inflammatory markers (VEGFR-1, placental growth factor, monocyte chemoattractant protein 1, soluble intercellular adhesion molecule-1, interleukin (IL)-6, and interferon-inducible 10-kDa protein). [95]

VI. Conclusion and future direction

Clinical and animal studies suggest that inflammation plays a pivotal role in the pathogenesis of DR. In this review we have explored the role of various inflammatory cells such as T cells, B cells, microglia, macrophages and their secretory products such as cytokines, and growth factors in the development and progression of DR. Early alteration in the endothelium and the permeability of the BRB by these inflammatory cells play a key role, including upregulation of inflammatory mediators as well as the activation and trafficking of immune cells especially CD4+ T cells. CCL2 is one of the strongest chemotactic factors for monocytes and the level of CCL-2 in the vitreous is elevated in patients with diabetic retinopathy. ROS are critical chemical species in VEGF receptor 2-mediated signaling, which lead to neovascularization in DR. They activate microglia which increase NFκB and activate ERK leading to increased angiogenesis and progression of DR. In patients with PDR, the Treg cell count is reduced and there is a down regulation of the expression of transcriptional factor TGF-β and other pro-inflammatory factors. These patients also exhibit high levels of VEGF-A and Placental growth factor (PIGF) mRNA and this is reduced following treatment with Aflibercept.

Additional studies are warranted to comprehend the specific roles of these inflammatory cells in the immunopathogenesis of DR. Although most studies have inferred inflammation as a causal factor of DR, further studies are needed to characterize whether observed inflammation is a cause or a consequence of DR as each may necessitate different treatment approaches as secondary and/or tertiary prevention in the clinical setting. Moreover, identifying the precise molecular mechanisms and function of each type of inflammatory cell in the onset and development of DR could pave the way for targeted medicine. As such, preclinical studies showing the pathogenesis of DR following the knockdown of each cell population may provide a more definite role of each cell type in the immunopathogenesis of ocular inflammation in DR. Of note, T cells have been implicated in the pathogenesis of T2DM, suggesting the involvement of antigen-specific inflammation. A study characterizing the antigen-specificity of these T cells for auto/altered antigens will provide a deeper understanding of their role in DR pathogenesis. An in-depth understanding will help physicians to tailor the specific management to each patient based on their risks for progression, bringing precision medicine into a reality.

VII. Methods of Literature Search

We used PubMed and Medline and searched for the terms “diabetic retinopathy” in combination with the following terms: “inflammation”, “immune cells”, “vascular proliferation”, “intravitreal injection”. We primarily selected articles within the past ten years. We also searched the reference lists of the articles identified in our search strategy and selected those we judged relevant. Out of the 79 articles, we selected 61 publications between 2010–2021, 15 publications between 2000–2009, and 3 publications between 1994–1999.