T-helper-associated cytokines expression by peripheral blood mononuclear cells in patients with polypoidal choroidal vasculopathy and age-related macular degeneration

Age-related macular degeneration (AMD) is a leading cause of irreversible visual disability and blindness among elderly in developed countries [1]. An advanced stage of AMD is characterized by choroidal neovascularization (neovascular AMD, nAMD) or geographic atrophy. Polypoidal choroidal vasculopath (PCV) exhibits commonalities with nAMD in that both are choroidal vasculopathy associated with subretinal hemorrhage, scars and fibrosis [2]. However, PCV is characterized by inner choroidal vascular networks ending in polypoidal structures at the borders of the lesion, while nAMD is characterized by choroidal neovascularization [3]. PCV still remains a subject of debate as to whether it is a subtype of AMD or its own distinct entity. The differences between the pathogeneses of PCV and nAMD are still unknown.

Recent information supports the notion that immune mechanisms play an important and perhaps central role in the pathogenesis of AMD [4]. CD4⁺ T cells are key players in immune responses. Among the T-helper cells, each subset produces different effector cytokines. Th1 cells predominantly secrete Interferon-gamma (IFN-γ), Th2 cells primarily produce IL-4 [5], CD4⁺ Th cells also produce IL-17, denoted as Th17 cells [6]. It is suggests that the underlying mechanism leading to AMD is the decline of the ocular down-regulatory immune environment [7]. The subsequent activation of the immune system would lead to T-cell sensitization. The accumulation of complement component 5a (C5a) and C3a in drusen promote choroidal neovascularization (CNV), which is the hallmark of wet AMD [8]. C5a promotes interleukin (IL)-22 and IL-17 expression by human CD4⁺ T cells [9]. IL-17A was initially reported to be mainly expressed by activated CD4⁺ T cells. It was implicated in the pathogenesis of various autoimmune diseases including uveitis, arthritis, multiple sclerosis and inflammatory bowel disease [10]. IL-17A rs2275913G/A and rs3748067C/T polymorphisms are associated with increased risk of AMD [11]. IFN-γ, a soluble cytokine associated with innate and adaptive immunity, is considered to be a pro-inflammatory factor. Constituents of drusen such as amyloid beta and advanced glycation endproducts (AGE) are capable of activating the IFN-γ pathway [12]. IFN-γ promotes proinflammatory responses by activating proinflammatory cytokines and chemokines, thereby recruiting immune cells such as macrophages, microglia, NK and T cells into inflammatory sites [13, 14]. IFN-γ induces a dose- and time-dependent increase in complement factor B mRNA expression in primary macrophages and macrophage cell lines [15] and increases CXCL11 mRNA expression and CXCL11 protein secretion by ARPE-19 cells [16]. IFN-γ also induces VEGF expression in human retinal pigment epithelial cells. Unexpectedly, IFNγ-induced VEGF secretion in human retinal pigment epithelial cells (RPE) was not through the classic IFN-γ Jak/Stat1 pathway, but through the PI-3 K/mTOR/p70-S6K-translational regulation pathway [17]. In laser-induced CNV model, NK cell depletion leads to decreased areas of CNV, macrophage infiltration and reduced mRNA expression of VEGFs and IFN-γ in the choroid, suggesting that IFN-γ-secreting NK cells promote angiogenesis by enhancing VEGF expression via macrophage activation [18]. IFN-γ may involve in AMD pathogenesis through macrophage polarization. INF-γ can selectively promote polarization into M1 subtype [19]. M1 subtype is predominantly proinflammatory and there is a pathological shift towards M1 subtype with the development of AMD [13].

Although IFN-γ and IL-17 are shown to be the major product of inflammasome cascade activation, few study focus on the immune-inflammatory response of PCV. The Th-cytokine levels of PCV and nAMD in peripheral blood mononuclear cells (PBMCs) have not been previously studied. In the present study, we studied the production of IFN-γ, IL-17A, and IL-4 in cultures of PBMCs from PCV and nAMD patients.

There is more and more evidence for immune response involve in the pathogenesis and progression of AMD. Retinal pigment epithelial (RPE) cell is one of the most important systems that regulate the immune response in the eye. RPE cell represents a primary site of pathology in AMD and their function and phenotype may modify by cytokines and inflammatory mediators. Dysfunction of RPE cells could trigger the inflammatory response, which then could trigger the photoreceptor degeneration and neovascularization in the retinas [22]. This might be one of the mechanisms of PCV and nAMD development. A few data on these immuno-inflammatory cytokines expression in PCV were reported. We therefore examined the levels of Th cytokines of PCV and nAMD patients. Our result showed that IFN-γ levels in the supernatants of cultured PBMCs in PCV and nAMD patients were markedly elevated compared with normal controls, indicating that IFN-γ related inflammatory pathway may involve in the pathogenesis of PCV and nAMD.

Recent studies point towards an emerging relationship between IFN-γ and mechanisms underlying the pathogenesis of AMD. The interaction of pathways activated by IFN-γ is complex and not fully understood. IFN-γ is classically considered as a pro-inflammatory factor, yet in recent years, multiple studies find IFN-γ to mediate an immune-modulatory and protective function. IFN-γ increases complement factor H (CFH) expression in cultured human RPE [23]. CFH is a major inhibitor of the alternative complement activation pathway that plays a critical role in driving the inflammatory responses in outer retina and is associated with AMD pathogenesis [24]. In human endothelial cells IFN-γ inhibits the angiogenic activity of VEGF through activation of STAT1 pathway. This may inhibit excess angiogenesis process in wet AMD [25]. Another study suggests that IFN-γ induces VEGF expression in human retinal pigment epithelial cells [17]. The activation of the PI-3 K/mTOR/translational pathway is important for IFN-γ-mediated VEGF expression in RPE cells. It is known that STAT1-deficient mouse are highly susceptible to autoimmune disorders, thus STAT1 activation by IFN-γ may be important in mitigating AMD progression [13].

IFN-γ may also play a beneficial role by regulating Th17 cells. IL-17A is an important cytokine implicated in the pathogenesis of autoimmune disease [26]. Our data indicate that no significant difference in IL-17A levels in PHA-stimulated cultures among PCV, nAMD and control groups. It is reported that in vitro stimulated PBMCs carrying rs2275913AA genotype produced significantly more IL-17 than those with the GG genotype, and similar data were observed in both controls and AMD patients [11]. In paraffin-embedded tissues, expression of IL17A mRNA and the receptor IL17RC mRNA are higher in the macula of AMD patients than in control [27]. In vitro, IL17A induces RPE cell death characterized by the accumulation of cytoplasmic lipids and autophagosomes with subsequent activation of pro-apoptotic Caspase-3 and Caspase-9 [27]. Aqueous humor levels of IL-17 in eyes with nAMD or PCV are not statistically different from those in control eyes [28]. This result is consistent with our results. The different results are owing to different sample tissue, target population, detection mean and so on. Further studies are needed to clarify the role of Th17 in these patients.

In our analyses of serum cytokine profiles, IL-4 level in PHA-stimulated cultures was higher in PCV and nAMD patients than that in healthy controls. IL-4, a cytokine of Th2 subtype, plays an important role in humoral and cell-mediated immunity [29]. AGEs are a constituent of drusen and accumulate in Bruch’s membrane with age. IL-4 in supernatant samples from AGE stimulation of human RPE cells is 5.5 fold changes over controls [30]. Aqueous humor levels of IL-4 in eyes with nAMD or PCV are not statistically different from those in control eyes [28]. The levels of IL-4 in the aqueous humor samples from PCV patients are significantly higher than in the controls.

However, we were unable to distinguish nAMD and PCV from PHA-stimulated PBMCs based the expression of IFN-γ and IL-4. The reason remains unclear as the number of subjects enrolled in our present study was limited. One possibility is that IFN-γ and IL-4 is upregulated in both PCV and nAMD conditions but with different level and time window which may further trigger different signaling pathways and lead to different abnormal angiogenesis process. Another possibility is that PCV may have similar immune character with nAMD. PCV has many similarities with neovascular AMD, including clinical manifestations, genetic background, and epidemiological features [28]. It remains controversial as to whether or not PCV represents a subtype of nAMD. In clinical, some patients have possibility having CNV we cannot distinguish PCV from neovascular AMD. ‘Probable’ cases were determined if at least one of the following criteria were met: 1. only an abnormal vascular network is seen in ICGA. 2. Recurrent hemorrhagic and/or serous detachments of the RPE were observed [2]. From view of immune character, aqueous humor concentrations of CRP and IP-10 were elevated in eyes with PCV or nAMD. None of the 14 cytokines, including VEGF, IL-1α, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, monocyte chemoattractant protein 1, were significantly different between eyes with nAMD and those with PCV [28]. It is reported that PCV and nAMD have common associations with the complement factor H (CFH) and the HtrA serine peptidase 1 (HTRA1) genes [31, 32], which suggests some genetic similarity between these two entities. ARMS2/HTRA1 locus has a strong and consistent association with both exudative AMD and PCV, suggesting the two disorders share, at least partially, similar molecular mechanisms [33].

In our previous study, we identified a variant in chromosome 9p21, well known to be associated with coronary artery disease and type 2 diabetes, was significantly associated with PCV but not with neovascular AMD in the Chinese Han population [34]. The rs42524 polymorphism is a risk allele for nAMD in a Han Chinese population [35]. However, rs42524 in COL1A2 confers different levels of susceptibility to nAMD and PCV [35] Thus, the different role of IFN-γ and IL-4 in the development of PCV and nAMD needs to be verified in further studies.

The major limitation of this study is the relatively small sample size. Additionally, no mechanistic and functional evaluations were conducted in this study. Extended cohorts with both PCV and nAMD patients offering higher statistical power will be necessary to confirm our results.

In summary, our data show that circulating IFN-γ-producing Th1 cells and IL-4-producing Th2 cells is associated with the pathogenesis of PCV and nAMD. There is no significant difference in IFN-γ, IL-17A and IL-4 levels in the supernatants of cultured PBMCs between PCV and nAMD, indicating PCV may have the similar immune character with nAMD.

AMD, age-related macular degeneration; CFH, complement factor H; CNV, choroidal neovascularization; IL, interleukin; INF-γ, interferon-gamma; PBMCs, peripheral blood mononuclear cells; PCV, polypoidal choroidal vasculopath; RPE, retinal pigment epithelial; VEGF, vascular endothelial growth factor.