The complement system

The complement system is an integral part of innate immunity with essential roles in protection against foreign intruders via tissue inflammation, cell opsonization, and cytolysis. It is also involved in monitoring and maintaining host tissues by clearing cellular debris, maintaining cellular integrity, tissue homeostasis, and modifying the adaptive immune responses.158

Ever since histopathological studies demonstrated the presence of complement components in drusen,159,160 _{the involvement of the complement system in AMD has been studied}

extensively and genetic evidence showing strong links between components of the alternative pathway of the complement system and AMD followed.12,161 _{Although the complement system}

acts locally, its components can also be detected systemically in serum or plasma. A number of studies have investigated the expression levels of complement regulators, complement components and activation products in AMD patients versus controls. An overview of the alternative pathway of the complement system is provided in Figure 2.

The central molecule of the complement system is complement component 3 (C3). Enzymatic cleavage of C3 results in the generation of its active fragments C3a (a potent proinflammatory molecule) and C3b that, via several digestion steps, leads to C3d.162 _{A number of studies}

measured systemic C3 levels but did not find an association with AMD,163-166 _{whereas higher}

systemic levels of its active fragments, C3a and C3d, were detected in AMD patients.163,164,166,167

These findings suggest that the processing of C3, i.e. its activation, may be associated with AMD and a number of studies have investigated this. Complement activation was measured as the ratio of C3 and its degradation product C3d (C3d/C3),166,168,169 _{or as a cleaved form of}

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Figure 2. Overview of the alternative pathway of the complement system

Spheres are colored to indicate levels in AMD patients compared with controls based on literature: upregulated (green), upregulated/no difference (dark green), upregulated/downregulated (gray), and no difference (yellow). (1) Complement component 3 (C3) splits into C3a and C3b by spontaneous hydrolyzation or by the C3-convertase (C4bC2) resulting from activation of the classical or lection pathway. (2) Factor B (FB) can bind C3b to form C3bB. (3) The bound factor B is then cleaved by factor D (FD) which results in the formation of the C3-convertase: C3bBb (4). This C3-convertase can cleave C3 which leads to more C3b and in turn increased formation of the C3-convertase (known as the C3 amplification loop). The C3-convertase can also bind another C3b molecule to form C3bBb3b, which is a C5-convertase (5). This C5-convertase can convert C5 into C5a and C5b. (6) C5b then sequentially binds C6, C7, C8, and multiple C9 molecules to form the terminal complement complex (SC5b-9), also known as membrane attack complex. * The C3-convertase is inhibited by several complement regulators, among which decay accelerating factor (DAF) and factor H (FH). ** Factor I (FI) can breakdown C3b via several digestion steps to C3c and finally C3d, this protease activity, however, requires a cofactor, such as FH.

activation in AMD, four found higher complement activation levels in AMD patients.166,168-170

An association of C3a-desArg in urine with AMD was not established.37 _{A recent study suggests}

that complement activation levels may decrease at more advanced stages of the disease, but this finding needs to be confirmed in prospective AMD cohorts.171

Besides C3, complement component 5 (C5) is also essential in the activation cascade because it serves as the entry point for the formation of the terminal complement complex (SC5b- 9).162 _{The activation product of C5, C5a, is a potent anaphylatoxin. Increased levels of C5a}

were detected in most, but not all,167 _{studies examining the role of C5a in AMD.}163,164,166

These same studies also tested whether SC5b-9 is associated with AMD. Higher SC5b-9 levels were detected in AMD in one study,164 _{but the other two studies found no evidence for an}

association.163,166

The activity of the complement system is tightly controlled by regulatory factors that ensure appropriate, but not excessive, generation of terminal complexes. Among others, they include complement factor H (FH, encoded by the CFH gene), factor I (FI, encoded by CFI), factor B (FB, encoded by CFB), factor D (FD, encoded by CFD), and decay accelerating factor (DAF/CD55, encoded by CD55).162

Genetic association studies showed strong evidence of an association between the CFH gene and AMD.12 _{Systemic levels of FH have been investigated with mixed results, however. Four}

studies report lower FH levels in AMD,153,163,172,173 _{one study detected higher levels of FH in}

AMD,174 _{and another four studies did not find an association with AMD.}164-166,175

Similar to FH, FI also inhibits the activity of the complement system through inactivation of C3b. Genetic evidence for factor I involvement in AMD has been shown previously, but no conclusive evidence links FI levels to AMD in general. One study reports increased FI levels in AMD,165 _{another reports decreased levels but only in patients carrying a rare genetic variant}

in CFI,176 _{and two did not find any association.}163,166

The findings for FB and FD levels in AMD are also inconsistent. Three studies reported higher FB levels in AMD patients,164,166,167 _{while two others did not detect an association with}

AMD.163,165 _{Similar results were described for FD, where three studies reported higher FD}

levels,164,167,177 _{one study reported lower levels in AMD,}165 _{and another found no association}

with AMD.163 _{Finally, two studies that examined the role of CD55 did not find evidence for an}

association with AMD.178,179

In summary, not only genetic studies but also studies measuring complement components provide evidence that link complement activation to AMD (Table 2). Some factors, however, should be taken into account when considering the use of systemic complement activation levels as a biomarker for AMD in individual patients. Often antibody based tests do not discriminate between the total amount of a specific complement factor and its processed activated part, as cleavage of the pro-form to the active mature form cannot be distinguished by

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the reagent. Moreover, complement activation levels are subject to high variability, and other causes of increased complement activity should be excluded since increased complement activation may reflect immune system activity that is not necessarily connected to disease progression. Linking exacerbated complement activation in an individual patient to his or her genetic blueprint is potentially more useful. For example, haplotypes and combinations of genotypes in several complement genes have been associated with increased complement activation levels.167,180 _{In addition, several investigations have now demonstrated that FI}

Table 2. Overview of studies measuring complement components in AMD patients compared to controls Component Upregulation No difference Downregulation

C3 Scholl et al. 2008164

Reynolds et al. 2009163

Silva et al. 2012165

Smailhodzic et al. 2012166

C3a Scholl et al. 2008164

Reynolds et al. 2009163

C3d Scholl et al. 2008164

Hecker et al. 2010167

Smailhodzic et al. 2012166

C3a des Arg Sivaprasad et al. 2007170 _{Guymer et al. 2011}37

C3d/C3 Smailhodzic et al. 2012166

Ristau et al. 2014168

Ristau et al. 2014169

C5a Scholl et al. 2008164

Reynolds et al. 2009163

Smailhodzic et al. 2012166

Hecker et al. 2010167

SC5b-9 Scholl et al. 2008164 _{Reynolds et al. 2009}163

Smailhodzic et al. 2012166

FH Hakobyan et al. 2008174 _{Scholl et al. 2008}164

Silva et al. 2012165 Smailhodzic et al. 2012166 Guymer et al. 2015175 Reynolds et al. 2009163 Ansari et al. 2013172 Sharma et al. 2013173 Sharma et al 2013153

FI Silva et al. 2012165 _{Reynolds et al. 2009}163

Smailhodzic et al. 2012166

Van de Ven et al. 2013*176

FB Scholl et al. 2008164 Hecker et al. 2010167 Smailhodzic et al. 2012166 Reynolds et al. 2009163 Silva et al. 2012165 FD Scholl et al. 2008164 Hecker et al. 2010167 Stanton et al. 2011177

Reynolds et al. 2009163 _{Silva et al. 2012}165

DAF/CD55 Haas et al. 2011178

Singh et al. 2012179

levels are lower in AMD patients carrying rare genetic variants in the CFI gene.176,181,182 _For

FH levels, there were similar associations with genotype. Some but not all rare variants in the CFH gene were associated with reduced FH levels.183-185 _{Thus, patients carrying rare}

variants in complement genes tend to have higher complement activation levels than AMD patients in general.186 _{These insights may benefit ongoing clinical trials on the effectiveness}

of complement inhibitors and could prioritize patients who carry rare variants in these genes.