The Role of Inflammation in Age-Related Macular Degeneration

Abstract

Age-related macular degeneration (AMD) is a blinding eye disease which incidence gradually increases with age. Inflammation participates in AMD pathogenesis, including choroidal neovascularization and geographic atrophy. It is also a kind of self-protective regulation from injury for the eyes. In this review, we described inflammation in AMD pathogenesis, summarized the roles played by inflammation-related cytokines, including pro-inflammatory and anti-inflammatory cytokines, as well as leukocytes (macrophages, dendritic cells, neutrophils, T lymphocytes and B lymphocytes) in the innate or adaptive immunity in AMD. Possible clinical applications such as potential diagnostic biomarkers and anti-inflammatory therapies were also discussed. This review overviews the inflammation as a target of novel effective therapies in treating AMD.

Introduction

Age-related macular degeneration (AMD), as the name suggests, is an eye disease closely related to aging with an average onset at around 60 years of age, which causes severe vision loss and blindness, especially in developed countries ^1-3. The number of population with AMD is expected to be 196 million by 2020, and will increase to 288 million by 2040 ⁴.

There are mainly two types of AMD: dry (also named non-neovascular, non-exudative or atrophic) AMD and wet (also named neovascular or exudative) AMD (nAMD) ⁵. As the most common type, dry AMD is characterized by the increase of extracellular deposits called drusen, along with advanced-stage geographic atrophy (GA) which is characterized by decreasing of retinal pigment epithelium (RPE) cells, photoreceptors and choroidal capillaries ^{5, 6}. Currently, there is no effective treatment for GA, and the complement cascade is expected to be a potential therapeutic option ⁷. On the other hand, in patients with wet AMD, which is characterized by choroidal neovascularization (CNV), leading to severe and fast vision impairment, accompanied by hard exudate, leaking fluid or retinal hemorrhage, RPE detachments or develop fibrosis around neovascular tufts ^{5, 8}. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents such as ranibizumab ⁹ and aflibercept ¹⁰, have been widely and effectively used worldwide in the clinical treatment of nAMD via targeting CNV. It has been suggested that anti-VEGF therapy significantly improved vision and quality of life for patients with nAMD ^{11, 12}. Nevertheless, about one-third of patients do not get effects from anti-VEGF therapy owing to macular fibrosis or atrophy ¹³. In addition, there is a heavy demand for repeated intravitreal injections to maintain efficacy ^{14, 15}, which leads to a heavy economic burden. Because of the limitation of anti-VEGF therapies, the development of novel alternative therapies is urgently needed. Some novel molecules were reported to be potentially therapeutic targets, including secretogranin III ¹⁶, tenascin-C ¹⁷, vitamin D ¹⁸, prorenin receptor ¹⁹, galactin-1 ²⁰, etc. However, further validations of clinical application have not been reported.

Aging participates in the accumulation of oxidative damage, and it is believed that the primary trigger of age-related degenerative diseases is oxidative damage ²¹. Numerous studies paid attention to the crosstalk between oxidative stress and inflammation. It has been indicated that oxidative stress induces inflammation during the AMD pathological process ²². Pathological oxidative damage contributes to damaged proteins, lipids and DNA, as well as dysfunction of mitochondria, and generates “oxidation-specific epitopes” (such as AGEs and MDA), induced pro-inflammatory responses, and promoted macrophage infiltration and polarization ^{22, 23}. Inflammation caused by tissue damage is considered to be essential in the protective immune response ²¹. It is well known that chronic inflammation involves many age-related diseases such as cancer ²⁴ and Alzheimer's Disease ²⁵. In this review, we summarize and discuss the role and mechanism(s) of inflammation, as well as inflammatory cytokines and leukocytes in the pathogenesis of AMD.

Inflammation in AMD pathogenesis

AMD is the consequence of a multifactorial interaction of metabolism, functions, genetics and the environment, and these multiple factors foster a stage conducive for the chronic structural changes in the macular area (choriocapillaries, Bruch's membrane (BM), RPE, photoreceptor) ^{2, 26}. Early signs of AMD contain the appearance of drusen and changes in retinal pigmentation, while advanced stages show CNV or atrophy of photoreceptor cells and RPE ²⁷. Local inflammation leads to drusogenesis, RPE/photoreceptor degeneration, BM disruption and the development of CNV ²⁶. Thus, inflammation is believed to play indispensable roles in the pathogenesis of both dry and wet AMD.

The occurrence of CNV is the main feature of nAMD, which is associated with inflammatory cytokines, complement system activation, and promotion/inhibition of macrophages/microglia ²⁸. Anti-VEGF therapy is mainly used for treating wet AMD, rather than dry AMD. Cytokines such as IL-6 and IP-10 were significantly altered after intravitreal injection of anti-VEGF agents in wet AMD ²⁹. In dry AMD, with the accumulation of lipofuscin and destruction of phagocytic activity of lysosomal enzymes, photoreceptors and RPE cells are damaged. Inflammatory cells release cytokines to attract more inflammatory cells ³⁰. Therefore, it is speculated that inflammation plays different roles in the pathogenesis of wet and dry AMD, respectively.

In 2001, Hageman et al. proved that the inflammatory immune response is associated with drusen, ascribed to multiple components found in drusen, including classic acute phase reactants, complement cascade components, etc ³¹. Besides, it has been demonstrated that RPE and dendritic cells (DCs) play vital roles in drusogenesis. Choroidal DCs are “activated and recruited” by locally injured and/or sublethal damaged RPE cells, related to RPE blebs, fragments, and debris. It can maintain and enhance the local inflammation by multiple mechanisms, such as forming an immune complex, activating complement and choroidal T-cells or phagocytic cells, collectively contributing to the development of AMD ^{31, 32}.

RPE plays a series of indispensable roles in the eye, such as blood-retinal barrier formation, ocular immune privilege establishment, and secretion of soluble immunomodulatory factors that mediate immunogenic inflammation ^{33, 34}. The breakdown of ocular immune tolerance involves blood-retinal barrier, anti-inflammatory and anti-immune proteins, resulting in the specific attack by effector T cells on autoantigens ^{34, 35}. When the blood-ocular barrier is broken, another defense system, called the local ocular immune system, inhibits pathogenic T cells ³⁶. RPE cells play a regulatory role by secreting soluble inhibitory molecules (transforming growth factor (TGF)-β and thrombospondin (TSP)-1) and transforming T cells into regulatory T cells (Tregs) ^{36, 37}. In addition to RPE cells, microglia, DCs and perivascular macrophages also participate in immunomodulatory ³⁸. On the other hand, under the stimulation of inflammatory mediators, such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-1β, RPE cells produce cytokines and chemokines, including IL-4, -5, -6, -8, -10, -13, -17, IFN-β, IFN-γ, TGF-β, MCP-1 and VEGF. The interaction of pro-inflammatory and anti-inflammatory cytokines ultimately leads to chronic inflammatory responses ^{39, 40}. Inflammatory cytokines can also enhance the secretion of VEGF, which can initiate and cause the pathological CNV and retinal neovascularization of AMD, and macrophages and lymphocytes were found in the active CNV stage ⁴¹. Altered expression levels of inflammatory factors were revealed in AMD ^42-46. Besides, RPE cells express a series of necessary cytokine receptors such as IL-1R, -4R, -6R, -8RA, -10RB, IFN-AR1, indicated the sensitivity to systemic and retinal inflammatory signals ⁴⁰. Moreover, RPE can transport nutrients to photoreceptors and dispose waste products, such as the outer segment is detached and then swallowed by RPE before a new outer segment is formed during the renewal of the photoreceptor membranes ³³. However, accompanied with the degeneration of RPE cells, photoreceptor cells are gradually and irreversibly destroyed, which leads to vision loss ⁴⁷. BM is another vital change, which is characterized by increased thickness, basal layer deposits accumulation and/or drusen formation, and there is irregular pigmentation caused by RPE cell hypertrophy, hyperplasia or atrophy. The pathological changes of BM with age further contribute to RPE cell dysfunction and choriocapillaris disorders ³⁸.

Taken together, current evidence indicated that inflammation plays an integral role in the entire pathogenesis of AMD (Figure 2), especially in CNV or GA. We summarize the mechanisms of inflammation-related cytokines and leukocytes, and look forward to getting more inspiration for clinical treatment in AMD.

Figure 2.

Inflammation plays role in the pathogenesis of AMD. Innate immune cells (macrophages, DCs, Neutrophils) can stimulate adaptive immune cells (B cells and T cells), and participate in CNV pathogenesis. Cytokines, include IL-1β, IL-6, IL-8, IL-10, IL-17, TGF-β, IFN-γ, TNF-α, etc, have angiogenic property. Cytokines, such as IL-4, IL-12, IFN-β, inhibit angiogenesis. In the late stage of AMD, photoreceptor cells are gradually damaged.

Cytokines

In aging eyes, due to the regulation of pro- and anti-inflammatory cytokines by RPE, low-grade chronic inflammation may be induced by these and continue for a long time, and then promote AMD pathogenesis ⁴⁵. A variety of cytokines have been found to study the relationship between inflammation and the progression of AMD. Regrettably, it is not found that stable trends in different organizations about related cytokines, so we have summarized the present literature about the changes in cytokines in clinical characteristics in Table 1, and their mechanisms in Table 2.

Table 1.

Expression of different cytokines in samples of patients with AMD.

Cytokine	AMD subtype	Source	Expression
IL-1	Wet AMD	Serum	(IL-1β)↑ 150, (IL-1α+IL-1β)↑151
	Wet AMD	Plasma	(IL-1β) ↓54, ↑152, No change 44
	Wet AMD	Vitreous	(IL-1β)↑51
	Wet AMD	Aqueous humor	(IL-1α)↑153, 154, (IL-1β)No change 153
	Dry AMD	Plasma	(IL-1β)↓54
	NA	Aqueous humor	No change 155
IL-2	Wet AMD	Aqueous humor	No change 153, 154, 156
	Dry AMD	Plasma	↓ 54
IL-3	Wet AMD	Aqueous humor	↑ 153
IL-4	Wet AMD	Serum	↑ 151
	Wet AMD	Aqueous humor	No change 153, 154, 156
	NA	Aqueous humor	No change 155
IL-5	Wet AMD	Serum	↑ 151
	Wet AMD	Plasma	↓ 54
	Wet AMD	Aqueous humor	No change 153, 156
	Dry AMD	Plasma	↓ 54
IL-6	Wet AMD Wet AMD	Plasma	↑ 44, 54, 152
		Aqueous humor	↑ 46, 153, ↓ 29, No change 154, 156
	Wet AMD	Serum	No change 150
	Wet AMD	Blood	↑ 157
	Dry AMD	Plasma	↑ 54
	Dry AMD	Serum	↑ 158
	NA	Aqueous humor	No change 155, 159
IL-8	Wet AMD	Aqueous humor	↑ 46, 153, 160, No change 29, 154, 156
	Wet AMD	Plasma	No change 44, 152
	Dry AMD	Serum	↑ 158
	NA	Aqueous humor	No change 155, ↑159
IL-10	Wet AMD	Serum	↑ 151
	Wet AMD	Plasma	↓ 54, ↑ 44, 152
	Wet AMD	Aqueous humor	No change 153, 154, 156
	Dry AMD	Plasma	↓ 54
	NA	Aqueous humor	No change 155
IL-12	Wet AMD	Plasma	↓ 54
	Wet AMD	Aqueous humor	(IL-12p40)↑ 153, (IL-12p70)↓161, No change 29, 154, (IL-12p70)No change 46, 153, 156
	Dry AMD	Plasma	↓ 54
	NA	Aqueous humor	(IL-12p70)No change 155
IL-13	Wet AMD	Serum	↑ 151
	Wet AMD	Aqueous humor	↓ 156, No change 29, 46, 154
IL-17	Wet AMD	Serum	↑74, 151
	Wet AMD	Aqueous humor	↓156, No change 154
	NA	Aqueous humor	No change 155
	NA	Macular lesion	↑77
IL-23	Wet AMD	Aqueous humor	No change 156
GM-CSF	Wet AMD	Plasma	↑ 54
	Wet AMD	Aqueous humor	↓ 162, No change 156
	Dry AMD	Plasma	↑ 54
IFN	Wet AMD Wet AMD	Plasma	(IFN-γ)↑ 54
		Serum	(IFN-β)↑ 163
	Wet AMD	Aqueous humor	(IFN-α+IFN-β+IFN-γ)No change 153
	Dry AMD	Serum	(IFN-β)No change163
	Both	Serum	(IFN-α+IFN-γ)No change 163
	NA	Plasma	(IFN-γ)No change 164
	NA	Aqueous humor	(IFN-γ)No change 155
TGF	Wet AMD	Aqueous humor	(TGF-β1)↑ 165, (TGF-β2)↓ 166, (TGF-α+TGF-β)No change 153
	Wet AMD	Vitreous	(TGF-β1)↑ 167
TNF-α	Wet AMD	Blood	No change 157
	Wet AMD	Plasma	No change 168
	Wet AMD	Aqueous humor	↓ 161, No change 46, 160
	Dry AMD	Plasma	↓ 54
	NA	Aqueous humor	No change 155

Table 2.

The mechanisms of different cytokines in AMD.

Cytokine	Research type	Mechanism(s)
IL-1	In vitro	IL-1α induces inflammasome which increases the sensitivity of RPE cell to cell death mediated by photooxidative damage and the mechanism of cell death becomes pyroptosis 55
	In vivo	IL-1Ra therapy signally suppresses CNV 56, 57
IL-2	In vitro	IL-2 contributes to cell migration, ECM synthesis and TGF-β2 expression via JAK/STAT3 and NF-κB signaling pathways 59
IL-4	In vivo + in vitro	lL-4 suppresses angiogenesis via Arg-1+ macrophage sFlt-1 80
IL-6	In vitro	Proteasome inhibitor MG132 upregulates IL-6 secretion by activating of P38 MAPKs 61
	In vivo + in vitro	IL-6, expressed by activated macrophages, promotes subretinal fibrosis 60 IL-6R-mediated activation of STAT3 contributes to CNV 62
IL-8	In vitro	Intracellular calcium mobilization promotes IL-8 secretion through NF-κB pathway 63 CRP can induce IL-8 expression by multiple pathways 64 25-OH causes IL-8 production through AP-1 and NF-κB pathways 65
IL-10	In vivo + in vitro	IL-10/STAT3 signaling contributes to pathological angiogenesis in senescent macrophages 81 IL-10(-/-) mice can reduce CNV with increased macrophage infiltrates 82 HSP70 induces IL-10 production through TLR2 and TLR4, and reduces subretinal fibrosis 84
	In vivo	CNV is inhibited by low-dose LPS pretreatment through IL-10 secretion by macrophages 83
IL-13	In vitro	IL-13 suppresses ARPE-19 cell proliferation and promotes fibrogenesis 85
IL-17	In vitro	IL-17 involves in choroidal angiogenesis via PI3K-Rac1 and RhoA-mediated actin cytoskeleton remodeling 76
	In vivo + in vitro	IL-17A causes the death of RPE cells by activating Casepase-9 and Casepase-3 77 and activates IL-1β production 78. IL-17 contributes to CNV, and IL-17 mainly produced by γδT cells not Th17 cells in the ocular lesions 75 IL-17 involves inflammation in CNV lesions, through producing γδT cells to strengthen the immune response and probably in a C5a-dependent manner 50.
IFN	In vitro	IFN-γ induces VEGF secretion by PI-3K/mTOR/translational pathway 73
	In vivo	IFN-β therapy weaken microgliosis and macrophage responses in the early AMD and decreased CNV size in the late AMD 71
TGF-β	In vivo	The inhibition of TGF-β/Smad signaling suppresses CNV via down-regulation of VEGF and TNF-α 88 Lack of TGF-β signaling promotes CNV 89, 90
TNF	In vivo +in vitro	BMP4 is down-regulated by TNF by activating JNK pathways in CNV 68 TNF-α promotes CNV by upregulating VEGF secretion via ROS-dependent activation of β-catenin signaling 66
	In vivo	Anti-TNF-α therapy reduces CNV 67

Pro-inflammatory cytokines include IL-1β, IL-2, IL-6, IL-8, IL-12, IL-17, TNF-α, IFN-γ, colony-stimulating factor (CSF) -1 ^48-50. Inflammasome connects the sensing of pathogen and danger signals with pro-IL-1β activation, and NLR family pyrin domain-containing3 (NLRP3) inflammasome is closely associated with IL-1β maturation. IL-1β can initiate innate immunity related to inflammation, infection and autoimmunity, such as macrophage recruitment, IL-6 activation and chemokine expression modulation ^51-53. If the retina was damaged for a long time, the overreactive neurotoxic microglia would release numerous kinds of pro-inflammatory and cytotoxic factors, including IL-1β, further create a pro-inflammatory environment which is beneficial to the recruitment of retinal microglia and exogenous infiltrating monocytes, and eventually result in progressive photoreceptor degeneration ^{53, 54}. Evidence has been accumulated that IL-1α induced inflammasome increases the susceptibility of RPE cell to cytotoxicity mediated by photooxidative damage ⁵⁵. On the other hand, IL-1β is also known as a pro-angiogenic factor through stimulating VEGF secretion. IL-1 receptor antagonist (IL-1Ra) treatment that inhibits IL-1β significantly suppressed CNV ^56-58. IL-2 participates in RPE cell migration, extracellular matrix (ECM) synthesis, TGF-β2 expression, indicating that IL-2 makes a constructive effect on the fibrosis of macular degeneration ⁵⁹. IL-6 is a key mediator for promoting subretinal fibrosis, which is considered as an injured repair in damaged organs ⁶⁰, and serum IL-6 level correlates with GA ⁶¹. Besides, it has been reported that IL-6 receptor-mediated activation of STAT3 promotes CNV, and the level of IL-6 is associated with the size and activity of CNV in the aqueous humor of AMD patients ^60-62. In the pathological mechanism of AMD, intracellular calcium mobilization, C-reactive protein (CRP) and 25-OH are able to induce IL-8 production and secretion. IL-8 participates in acute and chronic inflammation and has a potent proangiogenic ability. IL-8 causes tissue destruction by further attracting neutrophils and neutrophil-mediated inflammation ^63-65. TNF-α promotes CNV formation via upregulating VEGF production through reactive oxygen species (ROS)-dependent β-catenin activation, while the treatment of anti-TNF-α can reduce the size and leakage of CNV in mice ^{66, 67}. A recent study indicated that the TNF level is negatively related to the level of bone morphogenetic protein-4 in CNV lesions, which was increased in the dry AMD and decreased in the wet AMD. Moreover, the reduced level of bone morphogenetic protein-4 by TNF may promote the angiogenic environment of the active CNV lesion ⁶⁸. Besides, it is found that CSF-1 receptor inhibitor PLX5622 treatment can greatly reduce retinal microglia and the CNV lesion size in mice ⁶⁹. As another pro-inflammatory cytokine, IL-12 can activate T cells and NK cells, thereby inducing Th1-related inflammation related to wet AMD ⁷⁰. Interestingly, IL-12 may act as an important anti-angiogenic factor to suppress CNV ⁷⁰. Likewise, IFN-β therapy can limit microglia/macrophage activation, vessel leakage and the development of CNV in the laser-damage model of nAMD ⁷¹. IFN-β and IFN-γ have an antagonistic effect ⁷². IFN-γ promotes the inflammatory response through the activation of pro-inflammatory cytokines and chemokines, then recruits immune cells like macrophages and T cells ⁷². IFN-γ is also beneficial to the polarization of M1 macrophages, and synergistically increases the production of IL-6. Moreover, IFN-γ also restrains immune cells associated with autoimmune response and up-regulates anti-inflammatory factors ⁷². In the pathological process of AMD, blocking IFN-γ may weaken the protective effect of Th2 response, thereby strengthen the destruction of Th1 cells ⁷². Besides, it has been revealed that IFN-γ induces VEGF secretion in RPE cells, and the progression involves the activation of the Phosphoinositide 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR)/ translational pathway ⁷³.

As a Th17 cytokine, IL-17 has a beneficial effect on inflammation of CNV lesions, through producing γδT cells to strengthen the immune response and probably in a C5a-dependent manner ⁵⁰. It is indicated that the C5a enhanced secretion of Th17 cytokines from CD4+ T cells and is possibly involved in nAMD ⁷⁴. Meanwhile, IL-17 contributes to CNV pathogenesis and the effective IL-17 is mainly produced by γδT cells, rather than Th17 cells in the ocular lesions ⁷⁵. IL-17 is also involved in cell migration and tube formation, thereby exerting angiogenesis effect on choroidal endothelial cells (CECs) via PI3K-Rac1 and RhoA-mediated actin cytoskeleton remodeling ⁷⁶. IL-17A causes the death of RPE cells ⁷⁷, and activates IL-1β via NLRP3 in RPE ⁷⁸. Another study demonstrated that IL-23 is able to induce IL-17 production from γδT cells ⁷⁵.

On the other hand, anti-inflammatory cytokines include IL-4, IL-10, IL-13 and TGF-β ⁷⁹. IL-4, as a Th2 cytokine, directly drives macrophage soluble fms-like tyrosine kinase 1 (sFlt-1) secretion in Arg-1+ macrophages, and inhibits angiogenesis ⁸⁰. IL-10 and its downstream STAT3 signaling activation are major regulators of the aging macrophages mainly toward M2 phenotype, and promote ocular angiogenesis ⁸¹. In IL-10¯/¯ mice, CNV is significantly reduced ⁸². Matsumura et al. suggested that the pretreatment of low-dose lipopolysaccharide (LPS) inhibited CNV formation through IL-10 secreted by macrophages ⁸³. Exogenous HSP70 induces IL-10 production via both TLR2 and TLR4 in RPE cells, thereby attenuates the formation of subretinal fibrosis ⁸⁴. IL-13, mainly produced by Th2 cells and monocytes/macrophages, suppressed ARPE-19 cell proliferation in vitro and promoted epithelial-mesenchymal transition (EMT) ⁸⁵. The higher level of IL-13 is presented in aqueous humor of AMD ⁸⁵.

TGF-β is an important promoter of immune homeostasis and tolerance, which inhibits the expansion and function of many components of the immune response ⁸⁶. TGF-β superfamily members involves angiogenesis, inflammatory reactions, vascular fibrosis, immune responses and crosstalk with other signaling pathways in AMD pathogenesis ⁸⁷. TGF-β plays a vital role in the formation and development of CNV by Smad2/3-VEGF/TNF-α signaling pathway in wet AMD ⁸⁸. Interestingly, other studies indicated that deficient of TGF-β signaling leads to retinal degeneration and exacerbates CNV ^{89, 90}. Furthermore, TGF-β promotes the EMT of RPE cells, induces subretinal fibrosis and production of IL-6 ⁶⁰.

Subretinal fibrosis is a clinical manifestation of later period of nAMD ⁹¹, which is a wound healing response after CNV, together with the damage of photoreceptors, RPE and choroidal blood vessels, causing irreparable visual impairment ¹⁹. Cellular and ECM constituents, and the growth factor mediated EMT act as important roles in the RPE and the complex signaling networks of fibrosis in AMD ²⁸. There are two important processes, including EMT and endothelial-mesenchymal transition (EndMT), and TGF-β is the main regulator and the Snail superfamily are key transcription factors ⁹¹. Snail superfamily can bind to the DNA promoter region and stimulate the mesenchymal changes, cell migration and proliferation of different epithelial cells, thereby inhibiting the effects of epithelial molecules ⁹². TGF-β can upregulate the expression of Snail ⁹², and suppression of TGF-β reduced the size of subretinal fibrosis in vivo ⁹³. Moreover, knockdown of both TGF-β2 and Snail suppressed the EMT process of RPE cells more obviously compared to either single gene silencing ⁹⁴.

In summary, a series of cytokines play constructive roles in the pathogenesis of AMD, including the formation of CNV and subretinal fibrosis. The development of novel targeted therapies could potentially be considered for further investigations.

Leukocytes

Leukocytes are immune cells that are closely correlated with AMD pathogenesis. Both innate and adaptive immune cells play key roles in AMD.

Microglia/Macrophages

A hallmark of AMD development is the recruitment of the innate immune cells in the subretinal area with age ⁹⁵. The resident immune cells are the microglia in the retina, similar to tissue macrophages, which maintain normal retinal function, including the monitor and phagocytosis of damaged cell components ⁹⁶. Senescent microglia respond slowly to the injury and microglial dysfunction is a key factor in early AMD ⁹⁵. When retinal microglia migrate to the subretinal space, they may cause obvious changes in RPE cells, including further accumulation of microglia, increasing inflammation in the outer layer of the retina, and contributing to the formation of new blood vessels in wet AMD ⁹⁷. Besides, activated microglia maybe neurotoxic, and cause the degeneration of photoreceptors, along with phagocytizing dead photoreceptor cells ⁹⁸. The infiltration of microglia and macrophages to the injured retina, contribute to the development of retinal neovascularization ⁹⁶.

It is well-known that macrophages are a key modulator of tissue repair, regeneration and fibrosis. There are two major functional subtypes of macrophages, namely classically activated macrophages (M1) and alternatively activated macrophages (M2). M1, or pro-inflammatory macrophages, are anti-tumoral and cause tissue injury. M2, or anti-inflammatory macrophages, facilitate tissue repair and angiogenesis, as well as tumorigenesis and tumor metastasis. However, these two phenotypes can transform into each other as the microenvironment changes ^99-101. IL-1β, IL-12, IL-23, IFN-γ, LPS, and TNF-α induce the M1 macrophages that express CCL3, CCL5, CD80, CCR7, and iNOS. M2 macrophages, induced by IL-4, IL-10, IL-13, TGF-β, can express CCL22, CD206, CD163. ROCK signal can determine the polarization of macrophages to M1 and M2 phenotypes, and aging increases ROCK2 signal transduction, leading to the overexpression of pro-angiogenic M2 macrophages ¹⁰². Yang et al. demonstrate that M1 macrophages participate in the initial stage of CNV, while M2 phenotype plays an important role in the middle and late stages of CNV development and remodeling, thus, M2 is considered to be more important in the progress of CNV ¹⁰¹. However, Zhou et al. indicated that M1 macrophages have a more direct effect in suppressing CNV development, and M1 macrophages were primarily present in the RPE-choroid, while M2 were mainly located in the retina ¹⁰³. Both macrophage recruitment to BM and polarization of resident choroidal macrophages were related to extracellular deposits, including soft drusen and thick, continuous basal laminar deposits ¹⁰⁴. In nAMD patients, a large number of macrophages are involved with considerable florid CNV formations in the submacular choroid ¹⁰⁵. Activated macrophages are significantly increased in the submacular choroid related to RPE atrophy in GA eyes ¹⁰⁵. It is demonstrated that M2 macrophage polarization and CNV formation are induced by chitinase-3-like-1 (CHI3L1) that can also increase VEGFA expression ¹⁰⁶. Besides, there are higher levels of phosphorylated signal transducer and activator of transcription3 (pSTAT3) and higher VEGF secretion in monocytes, promoting the development of CNV ¹⁰⁷. Apte et al. proved that IL-10 suppressed the recruitment of macrophage to neovascular lesions and enhanced CNV formation ⁸². Macrophages and microglia may be closely related to RPE degeneration. It was found that even if the number of macrophages in the subretinal space is lower, it may contribute to the apoptosis of RPE cells, thereby promoting the development of AMD ¹⁰⁸. It was also discovered that local component 1q (C1q) produced by microglia/macrophages plays a role in inflammasome activation and inflammation, and neutralizing effects of C1q may slow retinal atrophy ¹⁰⁹.

Dendritic Cells

DCs, which are effective antigen-presenting cells (APCs), have the special ability to activate B and T lymphocytes. In other words, while DCs are innate immune cells, they are also closely related to the adaptive immune response ¹¹⁰. Without obvious damage, retinal DCs promote homeostasis, but they respond quickly once an injury occurs (the number increases dramatically and supports T cell activation) ¹¹¹. Nakai et al. revealed that DCs have the pro-angiogenic effect in CNV model, and intravenously injected immature DCs, rather than mature DCs, increased CNV size in vivo ¹¹². In the case of RPE cell injury, DCs are presented in drusen-related changes in the retina. Furthermore, it has been discovered that autophagy-related dying RPE cells would gradually be engulfed by macrophages, DCs and living RPE cells in vitro ¹¹³. DCs in choroid may lose the tolerogenic functions and develop into effective APCs, when pro-inflammatory cytokines like GM-CSF, TNF-α or IL-1 were presented without immunomodulatory cytokines such as MCP-1. Moreover, other immune cells can interact with DCs. For instance, macrophages can synergistically promote antigen presentation by DCs, NK cells and DCs can mutually promote activation and maturity, and produce cytokines ^{114, 115}. Accumulating evidence shows that the occurrence of AMD may be the consequence of the dysregulation of choroidal DCs ¹¹⁴.

Neutrophils

Neutrophils are the frontline effective cells in the innate immune system, with complex biological functions including regulating acute injury and repair, autoimmunity, and chronic inflammation ¹¹⁶. Once neutrophils are recruited, second-wave inflammation occurs, and leads to the recruitment of monocytes/macrophages ¹¹⁷. It can also stimulate T cell activation by expressing MHC class II¹¹⁸. A stronger correlation has been shown between nAMD and neutrophil-to-lymphocyte ratio (NLR) elevation compared with healthy controls ¹¹⁹, and NLR is related to disease severity as well as CNV and lesion size ^{120, 121}. Neutrophils are associated with retinal angiogenesis in laser-induced CNV. Neutrophils produce matrix metalloproteinase 9 (MMP-9), which degrades and reshapes the extracellular matrix (a key process of angiogenesis) and destroys the integrity of the RPE barrier ¹¹⁸. Besides, neutrophils cause angiogenesis by producing pro-angiogenic factors such as VEGF and IL-8, VEGF recruit neutrophils which secret more MMP-9 in turn ¹¹⁸. Furthermore, increased infiltration of lipocalin-2 (LCN-2)-positive neutrophils were found in the choroid and retina of patients with early AMD ¹²². AKT2/nuclear factor-kB (NF-kB)/LCN-2 signaling axis can mediate inflammation activation in AMD ¹²². Inhibiting AKT2 decreases LCN-2-mediated neutrophil infiltration into the retina and reverses early AMD-like phenotype changes ¹²³.

T lymphocytes

T cells are an important part of the adaptive immune system. The evidence of adaptive immunity involved in AMD derives from anti-retinal autoantibodies in AMD patients ¹²⁴. Th cells activate B cells to produce antibodies, macrophages to damage ingested microbes, and cytotoxic T-cells to destroy infected target cells ¹²⁵. Carboxyethylpyrrole (CEP) - specific T cells secret pro-inflammatory cytokines, leading to M1 polarization, and link innate immunity and adaptive immunity at the beginning of AMD ¹²⁴. Th1 cells are associated with IL-2, IL-12, IFN-γ, TNF, while IL-4, IL-10 and IL-13 are involved in Th2 response, and IL-17 is a Th17 cytokine ¹²⁶. The relevance of Th cells, cytokines and macrophages has been summarized in Figure 1.

Figure 1.

Links between Th cells and macrophages by cytokines in inflammation of AMD. Inflammation includes pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8, IL-12, IL-17, TNF-α, IFN-γ, etc.) and anti-inflammatory cytokines (IL-4, IL-10, IL-13, TGF-β, etc.).

Th1 cytokines increased in the vitreous and aqueous humor, while TGF-β can block Th1 cell activation and promote ocular immune tolerance ⁴⁰. Th1 and Th17 cells can produce pro-inflammatory cytokines, promote the polarization of M1 macrophages, and participate in the process of neovascularization and neurodegeneration ^{125, 127}. In nAMD patients, there are a lower frequency of Th1 cells and CXCR3+CD4+T cells. CXCR3 inhibits angiogenesis, so a lower level of CXCR3 may contribute to angiogenesis and cause CNV formation and growth ^{125, 128}. Th2 and Th17 cells may be involved in the development of subretinal fibrosis ¹¹⁸. Circulating Th1 cells and Th2 cells participate in the pathogenesis of nAMD ¹²⁹. Patients present a higher level of follicular helper T (Tfh) cells which modulate B cells secreting Ig¹³⁰. It has been recognized that circulating CD56(+) CD28(-) T cells are increased, and CD56(+) is a marker of T cell aging. nAMD is related to T cell immunosenescence ^{131, 132}. Shi et al. demonstrated that A2E inhibited the regulatory effects of RPE cells in Th1 cell differentiation by producing IL-1β and suppressing PGE2 ¹³³.

B lymphocytes

The number of B lymphocytes changes with age, which may be because of increased autoimmunity in the elderly population. But there are no significant differences in AMD compared with healthy individuals ¹³⁴. A study suggested that B cells from advanced AMD patients secret higher levels of antibodies to fight bacterial antigens, especially including IgM, IgG and IgA, and more sensitive to the more diluted concentration of bacterial antigens ¹³⁵. However, the relationship and mechanisms between B lymphocytes and AMD still remain unclear and further investigations are needed.

Potential applications in AMD

There are multiple auxiliary diagnostic methods for AMD. Local and systemic inflammatory molecules have been proposed as AMD potential biomarkers, such as CRP, active monocytes, NLR ^{120, 136-138}, but no specific and reliable markers have been found so far.

For early AMD patients, it is suggested to control relevant risk factors, such as quit smoking and keep a balanced and healthy diet ¹³⁹. The application of anti-angiogenic drugs by blocking VEGF, is a major breakthrough for nAMD patients ¹⁴⁰. However, some patients have a poor response. And anti-VEGF therapy cannot change the process of the disease at all, but only resist its effects over time and delays its development ^{15, 141}. Therefore, some scholars have turned their attention to anti-inflammatory therapy which is crucial for AMD pathogenesis. Drugs such as Lampalizumb, Eculizumab, Zimura and Iluvien have initially shown potential effectiveness in clinical trials, and need to be further verified ^142-144. As some popular emerging technologies, small interfering RNAs (siRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) could selectively disrupt the VEGF gene ^145-149. Several inflammatory-related cytokines also act on VEGF or VEGF-related mechanisms, so we hypothesized whether can use CRISPR/ Cas9 or siRNAs to target these cytokines and achieved the therapeutic effect on AMD. It is necessary to explore applications of pro- and anti-inflammation in future investigations.

Conclusion

Although the pathogenesis of AMD is undoubtedly an interaction of multiple factors, significant evidence has emerged implicating inflammation contribute to the development of AMD. RPE releases a large number of inflammatory mediators, contributing to an inflammatory cascade. When the long-term struggle between pro-inflammatory and anti-inflammatory responses eventually loses balance, AMD occurs. Both pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, IL-17, TNF-α, CSF-1, IFN-β and IFN-γ, and anti-inflammatory cytokines IL-4, IL-10 and TGF-β, play important roles in CNV formation through different signaling mechanisms. Similarly, pro-inflammatory cytokines IL-2, IL-6 and anti-inflammatory cytokine IL-10 and TGF-β are involved in the process of fibrosis. Besides, the inflammatory response is inseparable from the accumulation of various inflammatory cells in the eye, mainly innate immune cells such as macrophages, DCs, neutrophils and adaptive immune cells such as T lymphocytes and B lymphocytes. Immune cells can secret cytokines, and are affected by cytokines in turn. Mounting evidence supports the notion that inflammation is involved in AMD. Our review provides an overview of inflammation-related factors that may provide a feasible basis for better treatment options for AMD.

Abbreviations

AMD

Age-related macular degeneration

nAMD

neovascular AMD

GA

geographic atrophy

RPE

retinal pigment epithelium

CNV

choroidal neovascularization

VEGF

vascular endothelial growth factor

BM

Bruch's membrane

DCs

dendritic cells

TGF

transforming growth factor

TSP

thrombospondin

Tregs

regulatory T cells

TNF

tumor necrosis factor

IFN

interferon

IL

interleukin

CSF

colony-stimulating factor

NLRP3

NLR family pyrin domain-containing3

IL-1Ra

IL-1 receptor antagonist

ECM

extracellular matrix

CRP

C-reactive protein

ROS

reactive oxygen species

PI-3K

Phosphoinositide 3-kinase

mTOR

mammalian target of rapamycin

CECs

choroidal endothelial cells

sFlt-1

soluble fms-like tyrosine kinase 1

LPS

lipopolysaccharide

EMT

epithelial-mesenchymal transition

EndMT

endothelial-mesenchymal transition

CHI3L1

chitinase-3-like-1

pSTAT3

phosphorylated signal transducer and activator of transcription3

C1q

component 1q

APCs

antigen-presenting cells

NLR

neutrophil-to-lymphocyte ratio

MMP-9

matrix metalloproteinase 9

LCN-2

lipocalin-2

NF-kB

nuclear factor-kB

CEP

Carboxyethylpyrrole

Tfh

follicular helper T

siRNAs

small interfering RNAs

CRISPR

clustered regularly interspaced short palindromic repeats

Cas9

CRISPR-associated protein 9