Complement component C5a Promotes Expression of IL-22 and IL-17 from Human T cells and its Implication in Age-related Macular Degeneration

Age related macular degeneration (AMD) is clinically characterized by degenerative changes in the macula, the region of the retina that permits fine central vision. One of the key pathological features of AMD is the development of large drusen, extracellular deposits located between Bruch's membrane and the retinal pigment epithelium (RPE). These large drusen and the associated RPE changes are the major risk factors for the development of advanced AMD, which can be classified into two subtypes: dry (geographic atrophic) and wet (neovascular) [1]. Inflammation has been suggested to play an important role in AMD pathogenesis [2, 3].

Genetic studies have demonstrated strong associations between AMD and several gene variants in genes coding for complement proteins, including complement factor H (CFH), factor B/C2, and C3 [4‐12]. CFH is a factor that down-regulates complement activation. It is commonly thought that CFH polymorphism leads to dysregulation of alternative complement activation which may contributes to AMD pathogenesis [13]. However, the mechanism by which CFH regulates AMD progress is still not clear. Systemic activation of the complement cascade has been implicated in AMD patients [14‐16]. C5a, among many alternative complement activation molecules, are elevated in peripheral blood of AMD patients [15, 16]. Locally, C5a and C3a accumulate in drusen and are shown to promote choroidal neovascularization (CNV) [17], which is the hallmark of wet AMD.

Recently, a subset of effector helper T cells, IL-17-producing T cell (Th17), is implicated in the pathogenesis of various autoimmune diseases including uveitis, arthritis, multiple sclerosis, psoriasis and inflammatory bowel disease [18‐20]. Proinflammatory cytokines, including IL-1β, IL-6, IL-23, IL-21 and TNFα, as well as transcription factor RORC, are responsible for differentiation and maintenance of Th17 cells within human body [21‐24]. Recent evidence from the mouse suggests that C5a provides both costimulatory and survival signals to CD4⁺ T cells and induces Th17 cytokine expression [25, 26]. However, it is still not clear if C5a can impact human T cells and if Th17 cells are associated with AMD.

Here we showed that C5a protected human CD4⁺ T cells from undergoing apoptosis and C5a promoted IL-22 and IL-17 expression from CD4⁺ T cells of AMD patients and normal subjects as well. Intriguingly, consistent with previous observation of elevated C5a expression in the serum of AMD patients [15, 16], we found significantly increased levels of IL-22 and IL-17 in the sera of AMD patients, suggesting possible roles of IL-22 and IL-17 in the inflammation that contributes to AMD.

We listed the demographic, clinical information for both controls and AMD patients in Table 1 and Table 2. Ocular therapies, co-morbidities and complement related genetic variance were also included to AMD patients for later data analysis. These information will be used to evaluate confounding factors. All the subjects in this study are Caucasians. There are 45 controls and the age range was from 59 to 87. Fifty-three percent (53%) are females and 47% are males. There are 40 AMD patients in this study and the age range was from 57 to 97. Fifty percent (50%) are females and 50% are males.

In this study, we have provided evidence that C5a induced IL-22 and IL-17A expression from human CD4⁺ T cells. Importantly, consistent with previous observations of elevated C5a expression in the serum of AMD patients from different cohorts [15, 16], we observed significantly increased levels of IL-22 and IL-17A in the sera of AMD patients. However, so far, we do not have direct evidence showing that the elevated Th17 cytokine levels in AMD patients' sera are due to higher C5a expression in AMD patients. C5a may be one of the many factors related to this observed effect. Other unknown factors may also contribute to this T cell activation seen in AMD patients. Interestingly, the findings that C5a specifically promoted the Th17 family cytokine production, but not IFNκ nor IL-4, also correlated with the fact that there were similar IFNκ and IL-4 levels in the sera of AMD patients as compared to controls. The dysregulation of the complement system has been linked to multiple neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, as well as AMD [29]. The induction of inflammatory Th17 cytokines, including IL-22 and IL-17, by complement component C5a could potentially elucidate the general mechanism by which inflammation contributes to the pathogenesis of these diseases previously referred to as degenerative. Our results support a role for C5a in protecting CD4⁺ T cells from undergoing apoptosis (Figure 3). These findings suggest that the enhanced effector T cell function by C5a is at least partially mediated by limiting pro-inflammatory cell death.

We found that monoctytes are necessary for C5a induced Th17 cytokine production through two mechanisms: 1) promoting the direct interaction between monocytes and T cells; 2) indirectly stimulating the production of IL-1β and IL-6 from monocytes. C5a can bind to the trans-membrane receptors C5aR/CD88 and C5L2 (GPR77) which are expressed on monocytes. C5L2 is expressed at much lower levels as compared to CD88. C5a binding to CD88 leads to a number of functional changes including activation of inflammation. However, the pathophysiological role of C5L2 is currently controversial with both pro-inflammatory and anti-inflammatory roles reported [30]. Previous reports from rodent models have shown that C5a has a direct effect on T cells by interacting with the C5a receptor expressed on T cells, a finding which is different from what we have observed in humans [26]. Fang et al. recently demonstrated that C5a itself has no effect on Th17 cytokine production in mouse [31]. However, C5a synergizes with TLR4 to produce serum factors that drive Th17 cell differentiation [31]. Liu et al. reported that local interactions among C3a/C5a, C3aR/C5aR, antigen presenting cells (APC) and T cells are important for IFNκ and IL-17 production of T cells in a murine EAE (Experimental autoimmune encephalomyelitis) model [32]. In another murine sepsis model, Xu et al. shows that C5a affects the crosstalk between DC and gamma/delta T cells and results in a large production of IL-17[33]. In a human study, Hueber and colleagues showed that C5a induces IL-17 from human mast cells [34]. Our work is the first human study showing that monocytes play an essential role in C5a promoted expression of Th17 cytokines from CD4⁺ T cells.

Several research teams have reported that a common SNP of CFH, Tyr⁴⁰²His, has a particularly strong association with AMD [4, 6, 8]. We sub-grouped IL-22/IL-17 expression based on the subjects' CFH SNP genotypes and found that AMD patients with higher IL-22/IL-17 cytokine expression were likely to have the risk CFH allele (TC/CC) (Figure 4). However, serum IL-22/IL-17 cytokine levels showed no difference between the two CFH genotype groups (TT versus TC/CC) in controls. These results suggest that this CFH SNP does not explain the elevated Th17 cytokine expression. However, this genetic variant may be one of the many factors influencing Th17 cytokine expression.

Dysregulation of alternative complement activation has been reported to be involved in AMD pathogenesis. The drusen of AMD donor eyes contain almost all molecules of the alternative complement pathway, including CFH, C3, C5, C3a, C5a, and the membrane attack complex (MAC) [35‐37]. These results suggest the role of the complement system in the eye. The products of complement activation can also be detected in the blood of AMD patients. Scholl et al.[16] found higher levels of alternative complement activation molecules in the blood from an AMD cohort, including Ba, C3d, MAC, C3a, and C5a. A subsequent study in a larger independent cohort of patients and controls confirmed these results, showing the activation of the alternative pathway of complement in blood is associated with genetic polymorphisms in complement factor B and increases with age [14]. Reynolds and colleagues also found an increased plasma concentration of C5a and Bb in advanced AMD [15]. In addition, a recent report has shown that immunization with carboxyethylpyrrole generated by oxidative damage to DHA (Docosahexaenoic acid) present in the drusen and plasma from AMD-affected individuals is sufficient to produce AMD like lesions in mice and antibody titers of carboxyethylpyrrole correlates with disease pathology, suggesting the involvement of the acquired immune pathway in disease pathology [38]. In this study, we found C5a induced Th17 cytokine expression from human T cells in vitro, which correlates with the increased levels of Th17 cytokines in AMD blood. IL-22 has been shown to induce apoptosis of fetal retinal pigment epithelium (RPE) cells and reduce RPE cell electrical resistance in culture [39]. However, whether systemic observations reflects pathological events in the eye and how systemic activation may ultimately be manifest in the eye remain to be defined.

To date there is no effective treatments other than attempts to slow the progression of geographic atrophy form of AMD, while neovascular AMD is treated with anti-VEGF medications injected directly into the eye [40, 41]. Previous attempts at controlling the wet form of AMD with corticosteroid therapy have shown that the beneficial effect is transient with a significant side-effect risk profile [42]. Health improving behavior (no-smoking), diet, and exercise may be preventive measures for AMD [43]. Several compounds targeting complement pathway are currently in clinical trials [13]. We recently reported that immunotherapy directed against T-cell activation resulted in patients with recurrent CNV requiring fewer injections of anti-VEGF [44]. The dysregulation of the acquired immune pathway we describe here may provide us with new therapeutic strategies.

In conclusion, we have shown that C5a promoted expression of Th17 cytokines from human CD4⁺ T cells. Consistent with several cohort observation of elevated C5a expression in the serum of AMD patients [14‐16], our results support the notion that C5a may be one of the factors contributing to the elevated serum IL-22 and IL-17 levels in AMD patients. Targeting adaptive immune system, more specifically the Th17 family of cytokines, may have beneficial effect on the course of AMD.