Rev. Neurosci. 2014; 25(4): 575–583
Ruksana Huda, Erdem Tüzün and Premkumar Christadoss*
Targeting complement system to treat myasthenia gravis Abstract: While the complement system is desired for protective immunity, antibody- and complement-mediated neuromuscular junction (NMJ) destruction, a hallmark of myasthenia gravis (MG) or experimental autoimmune MG (EAMG), is a significant concern. Evidence suggests that the binding of complement factors to the pathogenic antiacetylcholine receptor (AChR) autoantibody induces the formation of membrane attack complexes (MAC), which ultimately lead to NMJ destruction and muscle weakness. Studies corroborating the evidence show that the complement (C3–C6)-deficient or complement inhibitor (anti-C1q, soluble CR1, anti-C6, and C5 inhibiting peptide)-treated animals are highly resistant to EAMG induction, whereas the deficiency of the naturally occurring complement inhibitors, such as the decay-accelerating factor (DAF), increases EAMG susceptibility. Notably, the complementinhibited animals do not exhibit significant immunosuppression but only a marginal reduction in the production of certain cytokines and immunoglobulin isotypes. A preliminary clinical trial using antibody-based C5 inhibitor eculizumab has been shown to be of potential use for MG treatment. The inhibition of the classic complement pathway (CCP) alone appears to be enough to suppress EAMG, suggesting that the complement inhibitors targeting specifically the classic pathway could effectively treat MG without causing immunosuppressive and other side effects. For instance, a recent non-antibody-based therapeutic approach selectively targeting the CCP component C2 by small interfering RNA (siRNA) has proven useful in EAMG treatment. The treatment strategies developed for MG might also be beneficial for other complement-mediated autoimmune diseases. Keywords: autoimmunity; complement; complement regulators; experimental autoimmune myasthenia gravis; myasthenia gravis; siRNA.
*Corresponding author: Premkumar Christadoss, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA, e-mail: [email protected] Ruksana Huda: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA Erdem Tüzün: Institute of Experimental Medicine, Department of Neuroscience, Istanbul University, Istanbul, Turkey
DOI 10.1515/revneuro-2014-0021 Received March 6, 2014; accepted March 26, 2014; previously published online April 12, 2014
Introduction Myasthenia gravis (MG) is a T-cell-dependent and antibody-mediated autoimmune disease of the neuromuscular junction (NMJ), manifesting with muscle weakness as a result of acetylcholine receptor (AChR) deficiency and/ or malfunctioning. MG is a classic antibody-mediated disease and one of the best characterized autoimmune disorders. About 85–90% of MG patients display AChR antibodies (Vincent and Drachman, 2002; Conti-Fine et al., 2006), which induce NMJ dysfunction by initiating a complement-mediated muscle membrane destruction, by crosslinking and down-regulating AChRs or blocking the acetylcholine binding sites of the AChR. Clinical, pathological, and animal model studies suggest that the complement-mediated mechanism of action plays the major pathogenic role in MG (Drachman et al., 1980; Engel et al., 1981). Both clinical and animal model studies have shown that Th1 and Th17 cells are important mediators of AChR immunity. Th17 cells shift the balance from regulatory T cells to Th1 cells, and Th1 cytokines promote the presentation of AChR peptides to T helper cells, the activation of B cells by T helper cells, and the proliferation of AChR antibody-producing B cells. Some Th2-type cytokines such as interleukin (IL)-5 and IL-10 also contribute to AChR immunity probably by enhancing the proliferation of AChR reactive B cells (Conti-Fine et al., 2008; Tüzün et al., 2011). Although T-cell- and antibody-mediated mechanisms play a crucial role in AChR-related MG, the complement system perhaps plays an even more profound role in MG development. This is exemplified by the striking resistance of complement-deficient mice or rats to experimental autoimmune MG (EAMG) induced by AChR immunization or the passive transfer of AChR antibodies (Christadoss, 1988; Tüzün et al., 2003; Chamberlain-Banoub et al., 2006). Experimental animals, which are inherently cytokine deficient or treated with cytokine inhibitors, generally show 40–80% reduction in EAMG incidence and/or
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576 R. Huda et al.: Myasthenia and complement (Engel et al., 1977; Sahashi et al., 1980). The experiments performed with a series of complement KO animal strains, as well as in vitro studies, have shown that, aside from the formation of MAC, various complement factors influence the immunoresponse by enhancing the proinflammatory cytokine production, clearing of the circulating immunocomplexes, modulation of IgG production, regulation of T cells, and isotype switching (Zachrau et al., 2004; Roumenina et al., 2011; Liszewski et al., 2013).
severity compared to controls (Tüzün et al., 2011), whereas mice or rats of the same strain often show 75–100% reduction in EAMG incidence when their complement systems are inhibited (Table 1). These findings put emphasis on the complement system as a potential target for future treatment trials. The complement system is composed of a group of proteins with a large variety of biological activities. The complement cascade is classically divided into three pathways: the classic complement pathway (CCP), the alternative complement pathway, and the mannose binding lectin (MBL) pathway (Figure 1). While the alternative and MBL pathways are usually activated by microorganisms and thus are generally involved in the defense against pathogens, the CCP pathway is mainly activated by the antigen-antibody complexes and thus is involved in the pathogenesis of a variety of immunocomplex and antibody-associated diseases (Ricklin and Lambris, 2013). The muscle pathology findings of MG patients and EAMG experiments conducted with complement knockout (KO) mice have suggested that the accumulation of anti-AChR IgG and the initial components of the complement cascade at the NMJ result in the assembly of membrane attack complex (MAC; a complex of C5b and C6–C9 factors) (Figure 1), thereby damaging the muscle membrane and reducing the number of functional AChRs
Involvement of the complement system in MG Clinical and pathological evidence from MG patients and EAMG animals The significance of the complement system in the pathogenesis of MG has first been recognized by studies showing alterations in the levels of various complement factors (C3, sC5b-9, etc.) in the sera of MG patients, suggesting the utilization of the complement system during the pathogenic processes (Nastuk et al., 1960). Moreover, almost all MG patients have the complement-fixing antiAChR IgG1 and/or IgG3 isotypes in their sera (Rodgaard et al., 1987).
Table 1 A comparison of EAMG clinical incidences and immunological effects of mice or rats that were treated with complement inhibitors. Treatment
CVF
Animal
Anti-C6 antibody Anti-C5 antibody C5 inhibiting protein Soluble CR1 Crry-Ig Anti-C1q antibody C2 siRNA
EAMG method
Lewis rats Wistar rats Lewis rats Lewis rats Lewis rats Lewis rats B6 mice
B6 mice
Passive transfer Immunization Passive transfer Passive transfer
Type of Percent reduction Immunopathological treatmenta in clinical effects incidenceb
Passive transfer Immunization Passive transfer Passive transfer Immunization
Immunization
Prevention Prevention Prevention Prevention Treatment Prevention Treatment Prevention Prevention Prevention Treatment
Treatment
100% 75% 100% 100% 100% 100% 100% 83% 100% 75% 60%
None reported None reported None reported Reduced serum IgG1 levels
None reported Reduction in complement deposition Increased serum immunocomplex levels Renal IgG and complement deposits (high dose) and reduced IL-6 production 80% Reduction in complement deposition, functional muscle AChR preservation, and reduction in serum anti-AChR IgG2b
a In the prevention experiments, the complement inhibitors are administered before or during the induction of EAMG, whereas, in the treatment studies, the complement inhibitors are administered after the establishment of clinical muscle weakness. b Relative EAMG incidences of animals with noticeable muscle weakness, when EAMG incidences of the animals treated with the control reagent are taken as 100% (prevention experiments) or EAMG incidences of the animals that showed clinical amelioration (treatment studies).
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Figure 1 Schematic illustration of complement activation pathways. The complement factors in circles have been found to participate in EAMG pathogenesis and some of these factors have been targeted in EAMG treatment trials. The immunological factors that are suppressed following the inhibition of the reciprocal complement factors are shown in squares. Green arrows indicate activation and red arrows indicate inhibition. FB, factor B; FD, factor D; MASP, MBLassociated serine protease.
To date, one of the most convincing proofs for the involvement of complement system in the development of myasthenic muscle weakness is the demonstration of IgG, C3, and MAC deposits on the degenerating postsynaptic muscle membranes of MG patients. IgG and complement deposits not only colocalize with AChR at the NMJ, but also the numbers of these deposits show an inverse correlation with NMJ AChR levels (Engel et al., 1977; Sahashi et al., 1980). MG patients with higher serum anti-AChR antibody levels have been shown to display lower serum C3 and C4 levels (Romi et al., 2005), indicating the participation of CCP as well as the common complement pathway in the NMJ destruction. In the latter study, the serum levels of C3 or C4 were not associated with the presence of anti-titin and anti-ryanodine receptor (RyR) antibodies, suggesting that the complement factors were only utilized by AChR antibodies. In two other studies, the circulating C3 and C3c levels were found to be correlated with the clinical severity of MG (Kamolvarin et al., 1991; Liu et al., 2009). In one of these studies, the serum C3 levels were inversely correlated with the serum AChR antibody levels, further confirming the complement consumption hypothesis (Liu et al., 2009). Complement and IgG deposits are also found at the NMJ of EAMG animals, whether the disease is induced by passive transfer or active immunization with AChR. The numbers of NMJs with complement and IgG deposits almost always correlate with clinical disease severity.
The EAMG-resistant animal strains display much lower amounts of NMJ deposits than the EAMG susceptible ones (Tüzün et al., 2006a, 2007; Yang et al., 2007). Also, both serum complement levels and NMJ deposits increase in parallel after AChR immunization in a time-dependent manner (Tüzün et al., 2006b). In addition to the C3 and MAC deposits, the CCP factor C1q deposits have also been observed at the NMJs of AChR-immunized C57BL/6 (B6) mice (Tüzün et al., 2006b). Overall, these findings suggest that AChR antibodies activate the CCP upon binding AChR at the NMJ and initiate the complement cascade, ultimately leading to the formation of MAC. Due to the difficulty in obtaining biopsy samples from extraocular muscles, there is no substantial evidence for the involvement of the complement system in extraocular muscle weakness, the hallmark symptom of MG. Nevertheless, in a recent EAMG model induced by the immunization of HLA-DQ8 transgenic mice with recombinant human AChR α or γ subunits and characterized with severe ptosis and mild generalized muscle weakness, complement and IgG deposits have been identified in abundance at the extraocular muscle NMJs. Moreover, the severity of extraocular symptoms has been found to be correlated with the amount of NMJ complement deposits (Yang et al., 2007; Wu et al., 2012). The extraocular muscles of mice express reduced amounts of complement regulators compared to limb muscles, and complement regulator expression is even further reduced after EAMG induction (Kaminski et al., 2004), presumably rendering extraocular muscles more susceptible to autoimmune attack than limb muscles. In line with this hypothesis, a single nucleotide polymorphism in the decay-accelerating factor (DAF) regulatory region has been found in MG patients with extraocular muscle involvement. This polymorphism has been shown to prevent the lipopolysaccharide-induced up-regulation of DAF (Heckmann et al., 2010), suggesting that the genetic defects leading to the reduced expression of complement regulators might render individuals susceptible to ocular MG. The role of the complement system in muscle-specific receptor tyrosine kinase (MuSK) antibody-related MG (MuSK-MG) is less well understood. Serum MuSK antibodies are predominantly of the IgG4 isotype, which in contrast with the IgG1 and IgG3 isotypes (that are prevalently found in AChR-related MG) that do not activate the complement cascade (Plomp et al., 2012). While the passive transfer of anti-MuSK IgG4 elicits severe muscle weakness, the passive transfer of IgG1–3 antibodies from MuSK-MG patients does not induce any clinical signs (Klooster et al., 2012). Moreover, C3 and C5-deficient mice, which are resistant to AChR-induced EAMG, are highly
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578 R. Huda et al.: Myasthenia and complement susceptible to EAMG induced by MuSK immunization (Mori et al., 2012). Nevertheless, about 25% of MuSK-MG patients appear to bear complement deposits at their limb NMJs (Shiraishi et al., 2005). This ratio might plausibly be even higher in bulbar muscles that are more prominently affected in MuSK-related MG. Also, most but not all MuSK-MG patients exhibit anti-MuSK IgG1, although its levels are often much lower than those of anti-MuSK IgG4 (Leite et al., 2008). Likewise, the immunization of B6 mice with MuSK generates complement activating anti-MuSK IgG2 (albeit at levels lower than the noncomplement activating anti-MuSK IgG1) and NMJ complement deposits (Viegas et al., 2012). These findings suggest that although the complement system is not the main culprit in MuSK-MG, it might still to some extent be involved in MuSK-MG patients, especially in those with severe muscle weakness or myasthenic crisis. The significance of the complement system in the recently identified lipoproteinrelated protein 4 (LRP4) antibody-associated MG has not been established yet. However, LRP4 antibodies have been shown to be predominantly of the IgG1 isotype and to mediate complement-dependent cell lysis, suggesting that the complement system might also be involved in this MG subtype (Higuchi et al., 2011; Shen et al., 2013).
EAMG studies with complement KO and deficient rodent strains An essential line of evidence supporting the involvement of the complement system in MG pathogenesis comes from EAMG studies using mouse or rat strains with inborn deficiency of complement factors or complement regulators. The AChR-immunized, C5-deficient (B10.D2/ oSn), C3 KO and C4 KO mice show significantly reduced EAMG incidence and disease severity compared to the wild-type (WT) littermates. Only 5% of the C5-deficient mice developed EAMG, whereas 78% of the C5-sufficient mice showed signs of EAMG. Also, the C5-sufficient mice showed 37% muscle AChR loss as opposed to 14% AChR loss of the C5-deficient mice (Christadoss, 1988). Likewise, while the C3 KO and C4 KO mice demonstrated 0% and 5.8% EAMG incidence, respectively, the WT littermates had 86% and 88% EAMG incidence (Tüzün et al., 2003). The C6-deficient rats also showed a remarkable resistance (100% reduction in clinical incidence) to EAMG induced by passive transfer of AChR antibodies and the administration of C6 rendered the C6-deficient rats susceptible to EAMG (Chamberlain-Banoub et al., 2006). Notably, the serum anti-AChR IgG levels of the C5-deficient and C4 KO mice were comparable to those
of the control WT animals, whereas the C3 KO mice showed reduced anti-AChR IgG levels presumably due to the involvement of C3 in cytokine production and T-cell responses (Liszewski et al., 2013). Moreover, the C4 KO mice showed NMJ IgG deposits just like the WT mice (Christadoss, 1988; Tüzün et al., 2003). These results indicate that the deficiency of CCP or MAC factors does not interrupt the development of anti-AChR autoimmunity. These results also show that AChR antibodies alone are not highly pathogenic in the absence of a fully functioning complement system. While C5b is a component of MAC, C5a is an anaphylactic and chemotactic agent, which mediates various immunological functions such as the production of cytokines IL-1β, IL-6, and tumor necrosis factor (TNF)-α that are actively involved in EAMG pathogenesis (Liang et al., 2011). The AChR-immunized C5a receptor KO mice show high susceptibility to EAMG (Qi et al., 2008), suggesting that C5a is not involved in AChR immunity and C5 deficiency prevents EAMG induction through impaired MAC production due to the absence of the C5b component. The C3 convertase C4bC2a is activated by both classic and MBL pathways, suggesting that EAMG resistance of the C4 KO mice might be due to the deficient functioning of both classic and lectin pathways. However, the MBL KO mice are not resistant to EAMG induced by AChR immunization and the serum MBL levels of MG patients are comparable to those of the healthy individuals, showing that the lectin pathway does not participate in MG pathogenesis (Li et al., 2009). The role of the alternative pathway in EAMG pathogenesis is still to be characterized.
EAMG and the complement regulators The increased EAMG incidence observed in complement regulator-deficient mice is another supporting evidence for the participation of complement factors in NMJ destruction. Mouse skeletal muscle tissues express several complement regulators such as Daf1 (the mouse equivalent of human DAF), CD59, and Crry (Kaminski et al., 2004; Soltys et al., 2008). Among these regulators, the deficiency of Daf1 has been shown to increase the susceptibility to EAMG induction. Daf1 accelerates the breakdown of C3 and C5 convertases, which are the crucial enzymes for MAC formation (Ruiz-Argüelles and Llorente, 2007). The Daf1 KO mice show increased EAMG incidence and clinical severity, elevated NMJ C3 deposit counts, and reduced muscle AChR concentration (Lin et al., 2002). The mouse CD59 gene has two promoters controlling the expression of mCD59a and mCD59b transcripts (Qin
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et al., 2006). Notably, in both passive transfer and immunization studies, the CD59a KO mice have shown equal susceptibility to EAMG induction as the WT mice (Tüzün et al., 2006c). Likewise, EAMG incidence, severity, and NMJ complement deposition of the Crry KO mice were identical to those of the WT mice (Soltys and Wu, 2012). The CD59 KO mice showed reduced serum anti-AChR IgG levels and decreased IL-2 production in response to AChR stimulation. The Crry KO mice showed increased serum anti-AChR IgG levels and AChR-stimulated IL-4 and interferon (IFN)-γ production. These findings indicate the involvement of these two regulators in the modulation of adaptive immunoresponses and AChR immunity (Tüzün et al., 2006c; Soltys and Wu, 2012). Although the CD59a/b double-KO mice showed moderately increased EAMG susceptibility, their EAMG incidence and severity were lower than those of the Daf1 KO mice (Soltys et al., 2012). These results suggest that the NMJ levels of CD59 and Crry are probably too low to confer a robust protection against EAMG.
Treatment trials performed with complement inhibitors The current treatment methods of MG and other autoimmune diseases generally include steroids and cytotoxic drugs that lead to significant side effects and global immunosuppression (Kumar and Kaminski, 2011). A large body of research on cytokine-based treatment methods has failed to produce a useful medication for MG. The absence of a relatively safe immunosuppressive treatment method for MG has prompted treatment trials with complement inhibitors. The major reasons for choosing the complement system as a potential target for MG treatment were the significantly high reduction ( ≥ 90%) in EAMG clinical incidence and the absence of substantial immunological deficiencies in complement KO studies (Tüzün et al., 2011). The therapeutic strategies based on the inhibition of the complement system include the use of complement-inhibiting recombinant proteins, complementinhibiting chemicals, monoclonal antibodies against complement components, soluble isoforms of complement receptor, and small interfering RNA (siRNA). These treatment trials have widely utilized EAMG models induced by immunization or passive transfer. These studies can be classified as those that inhibit the entire complement system and those that specifically inhibit the CCP.
Inhibition of the whole complement system Muscle weakness of mice and rats with EAMG induced by active AChR immunization or the passive transfer of AChR antibodies has been shown to improve following the inhibition of the entire complement system by several complement inhibitors including cobra venom factor (CVF), soluble complement receptor 1 (sCR1), C5 binding protein, anti-C5 antibody, and anti-C6 antibody (Lennon et al., 1978; Biesecker and Gomez, 1989; Piddlesden et al., 1996; Zhou et al., 2007; Soltys et al., 2009). The anti-C6 antibody administration before the passive transfer of AChR antibodies has achieved 100% prevention of EAMG induction in rats and has inhibited the accumulation of MAC components C6 and C9 at the NMJ. Anti-C6 treatment has not reduced serum C3 and C5 levels, suggesting that the antibody did not have any effect on the earlier components of the complement system; therefore, EAMG inhibition was solely associated with the inhibition of MAC formation (Biesecker and Gomez, 1989). EAMG symptoms induced by passive transfer and/or immunization were significantly ameliorated (75–100%) by sCR1 or CVF treatment. In concert with clinical results, these reagents have also maintained the preservation of muscular AChR concentrations (Lennon et al., 1978; Piddlesden et al., 1996). Both sCR1 and CVF inhibit the classic and alternative pathways, which ultimately stimulate the MAC formation. Therefore, unlike the anti-C6 antibody study, the EAMG-preventing effects of these reagents could be related with the inhibition of proinflammatory effects of early complement factors in addition to the prevention of MAC formation. The anti-C5 monoclonal antibody administered before the passive transfer of AChR antibodies or 24 h after EAMG induction effectively restored muscle strength in all rats, while all control rats had to be terminated due to severe weakness. Clinical amelioration was accompanied with reduction of NMJ C9 deposits. Similarly, rEV576, a novel 17-kDa C5 inhibiting protein, was found to be effective in the prevention and treatment of EAMG in both passive transfer and immunization experiments. Notably, in these studies, clinical amelioration was paralleled with the reduction of NMJ MAC deposits and preservation muscle AChR concentrations despite the presence of IgG deposits at the NMJ (Zhou et al., 2007; Soltys et al., 2009), confirming once again that AChR antibodies are not capable of inducing NMJ damage in the absence of an active complement system. Nevertheless, C5 inhibitor-treated rats showed a marginal reduction in serum IgG1 levels (Soltys et al., 2009), suggesting that C5 is involved in adaptive immunoresponses as well.
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580 R. Huda et al.: Myasthenia and complement In other trials utilizing complement regulators, Crry coupled with rat IgG2a Fc (rCrry-Ig) and DAF coupled with a single-chain antibody directed against the α-subunit of the AChR (scFv-35-DAF) were effectively used in the treatment of EAMG induced by passive transfer of AChR antibodies. The rats treated with rCrry-Ig were rendered entirely resistant to EAMG induction and this effect was accompanied with a significant reduction in NMJ complement deposition (Hepburn et al., 2008). Likewise, the rats treated with scFv-35-DAF showed significantly lower clinical muscle weakness scores and a reduction of complement deposition compared to the nontreated control rats (Kusner et al., 2013).
Inhibition of the CCP A potential drawback of the reagents that block the common complement pathway (e.g., C5 inhibitors and anti-C6 antibody) or both alternative and classic pathways (e.g., sCR1 and CVF) is the possible susceptibility to opportunistic infections, since the complement system is required for host defense against invading microorganisms. To overcome this problem, we performed EAMG treatment trials using specific inhibitors of CCP, which has been shown to be primarily involved in AChR antibodyrelated MG (Tüzün et al., 2003). With this approach, we anticipated that the alternative and lectin pathways would be left intact in order to be utilized by the host defense mechanisms against the invading microorganisms. To show that the acquired deficiency of CCP may render mice resistant to EAMG, we administered anti-C1q antibodies before and after induction of EAMG by AChR immunization. In these experiments, anti-C1q treatment achieved a significant reduction in EAMG clinical incidence compared to AChR immunized mice treated with the control nonspecific antibody (Table 1). The anti-C1q administered mice also showed reduced NMJ C3, IgG, and MAC deposits and lymph node cell IL-6 production in response to AChR and immunodominant peptide α146– 162 challenge (Tüzün et al., 2006a, 2007). This experiment showed for the first time that an autoimmune disease could be prevented by CCP inhibition. However, anti-C1q antibody-treated mice developed renal C3 and IgG deposits and showed increased serum immunocomplex levels (Tüzün et al., 2007). To overcome the problem of immunocomplex formation and to improve the effectiveness of CCP inhibition, we decided to utilize a non-antibody-mediated treatment method targeting a CCP component that has fewer immunological functions. A non-vector-based, chemically
modified siRNA-based gene silencing was anticipated not to induce any harmful side effects. C2 was thought to be a good candidate target since it is expressed at a very low level and there are no major immunological functions for C2 other than MAC formation. Also, congenital C2 deficiency is less frequently associated with immunocomplex disorders than C1q and C4 deficiency (Klint et al., 2000). The B6 mice with established severe EAMG were treated with either C2 siRNA or a control nontargeting (NT) siRNA. The efficacy of in vivo C2 siRNA treatment was confirmed with the evaluation of liver and peripheral blood cell C2 mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR). While 40% of the NT siRNA-treated mice died and 20% showed improvement of muscle weakness, only 10% of the C2 siRNA-treated mice died and 80% showed significant improvement in muscle weakness as assessed by clinical evaluation. The clinical amelioration in the C2 siRNA-treated group was associated with a transient reduction in serum anti-AChR IgG and IgG2b (but not IgG1 and IgM) levels, reduced NMJ C3 and MAC deposits, and higher muscle AChR levels compared to the NT siRNAtreated group. The serum C3 levels were comparable in the C2 and NT siRNA-treated groups, confirming that C2 inhibition does not affect the remaining components of the complement cascade. This study was the first example of utilization of siRNA-mediated complement inhibition for the treatment of an autoimmune disease with the preservation of the alternative pathway to fight against infection (Huda et al., 2013).
Concluding remarks As reviewed herein, many complement inhibitors lead to a significant reduction in EAMG incidence and clinical severity without causing noticeable toxicity or making a remarkable change in other immunological functions. These findings prompt the utilization of complement inhibitors as a novel therapeutic approach in MG treatment. Although there is a small number of complementinhibiting therapeutics in the market such as C1 inhibitor and sCR1, they have mostly been approved or are in the stage of approval for non-autoimmune disorders (Rioux, 2001; de Zwaan et al., 2003). Currently, the only complement-inhibiting therapeutic that can be used for autoimmune disorders and is available in the market is the C5 inhibiting monoclonal antibody eculizumab, which has been approved for another complement-mediated autoimmune disease, paroxysmal nocturnal hemoglobinuria
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(Risitano, 2013). In a recent randomized, double-blind, placebo-controlled trial performed on patients with severe, refractory generalized MG, eculizumab treatment has achieved a significant amelioration (at least a threepoint reduction in the quantitative MG score) in 86% of the participants (Howard et al., 2013). Although the common complement pathway inhibitors are expected to be effective in complement-mediated autoimmune disorders, they are also predicted to cause opportunistic infections since they prevent the major host defense mechanism against the invading pathogens to function. Not surprisingly, eculizumab and pexelizumab (another C5 inhibiting antibody used for coronary heart disease) are both associated with a high risk of meningococcal infection; therefore, patients are recommended to receive a vaccine before starting therapy. Upper respiratory system infections, nasopharyngitis, urinary tract infections, wound infections, and fungal infections are encountered in some patients. Eculizumab has also been associated with severe skin eruptions, rash, and kidney failure (Brodsky et al., 2008; Davis, 2008; Knoll et al., 2008). Some of these adverse effects might also be associated with the immunocomplex disease induced by the monoclonal antibody used for the inhibition of the complement cascade. Another problem that might emerge due to the usage of monoclonal antibodies is immunogenicity. These findings suggest that a non-antibody-based complement-inhibiting strategy targeting the CCP might effectively treat autoimmune disorders without causing infections or immunocomplex disorders and inducing immunogenicity that would decrease the efficacy. An effective suppression of EAMG in mice by the inhibition of a CCP factor C2 using a siRNA-based treatment method and without causing any major side effects suggests that this treatment approach might prove useful in patients with MG and other complement-mediated autoimmune disorders. Apart from MAC formation, the complement system is involved in several immunological functions that are of vital importance in the development of anti-AChR immunoresponse. Experimental animal studies suggest that the complement system is a part of a complex network of cytokines and IgGs activating one’s and other’s production, thus forming an autocrine feedback loop that promotes chronic autoimmunity. The findings of EAMG studies suggest that C1q might mediate the IL-6 production, CD59 might interfere with the IL-2, C3, C4, IgG1, and IgG2b production, and C3 might be enhancing the synthesis of IgG, IgG2b, IL-6, IL-10, and IFN-γ (Tüzün et al., 2003, 2006a,c, 2007). Moreover, C2 and C4 appear to be involved in the IgG2b production, whereas C5 might be involved in
the IgG1 production (Tüzün et al., 2003; Soltys et al., 2009; Huda et al., 2013) (Figure 1). These results suggest that complement-based treatment methods are not entirely devoid of immune system suppression. Nevertheless, the effects of complement inhibitors on the immunological functions appear to be rather marginal and they do not prevent the formation of a robust immunoresponse to immunogens. This is exemplified by the formation of AChR antibodies and the accumulation of IgG deposits at the NMJs of complement KO or complement-inhibited EAMG mice. Therefore, a complement inhibition could probably be safely used without causing significant immunosuppression.
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Lin, F., Kaminski, H.J., Conti-Fine, B.M., Wang, W., Richmonds, C., and Medof, M.E. (2002). Markedly enhanced susceptibility to experimental autoimmune myasthenia gravis in the absence of decay-accelerating factor protection. J. Clin. Invest. 110, 1269–1274. Liszewski, M.K., Kolev, M., Le Friec, G., Leung, M., Bertram, P.G., Fara, A.F., Subias, M., Pickering, M.C., Drouet, C., Meri, S., et al. (2013). Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation. Immunity 39, 1143–1157. Liu, A., Lin, H., Liu, Y., Cao, X., Wang, X., and Li, Z. (2009). Correlation of C3 level with severity of generalized myasthenia gravis. Muscle Nerve 40, 801–808. Mori, S., Kubo, S., Akiyoshi, T., Yamada, S., Miyazaki, T., Hotta, H., Desaki, J., Kishi, M., Konishi, T., Nishino, Y., et al. (2012). Antibodies against muscle-specific kinase impair both presynaptic and postsynaptic functions in a murine model of myasthenia gravis. Am. J. Pathol. 180, 798–810. Nastuk, W.L., Plescia, O.J., and Osserman, K.E. (1960). Changes in serum complement activity in patients with myasthenia gravis. Proc. Soc. Exp. Biol. Med. 105, 177–184. Piddlesden, S.J., Jiang, S., Levin, J.L., Vincent, A., and Morgan, B.P. (1996). Soluble complement receptor 1 (sCR1) protects against experimental autoimmune myasthenia gravis. J. Neuroimmunol. 71, 173–177. Plomp, J.J., Huijbers, M.G., van der Maarel, S.M., and Verschuuren, J.J. (2012). Pathogenic IgG4 subclass autoantibodies in MuSK myasthenia gravis. Ann. NY Acad. Sci. 1275, 114–122. Qi, H., Tüzün, E., Allman, W., Saini, S.S., Penabad, Z.R., Pierangeli, S., and Christadoss, P. (2008). C5a is not involved in experimental autoimmune myasthenia gravis pathogenesis. J. Neuroimmunol. 196, 101–106. Qin, X., Ferris, S., Hu, W., Guo, F., Ziegeler, G., and Halperin, J.A. (2006). Analysis of the promoters and 5′-UTR of mouse Cd59 genes, and of their functional activity in erythrocytes. Genes Immun. 7, 287–297. Ricklin, D. and Lambris, J.D. (2013). Complement in immune and inflammatory disorders: pathophysiological mechanisms. J. Immunol. 190, 3831–3838. Rioux, P. (2001). TP-10 (AVANT Immunotherapeutics). Curr. Opin. Investig. Drugs 2, 364–371. Risitano, A.M. (2013). Paroxysmal nocturnal hemoglobinuria and the complement system: recent insights and novel anticomplement strategies. Adv. Exp. Med. Biol. 735, 155–172. Rodgaard, A., Nielsen, F.C., Djurup, R., Somnier, F., and Gammeltoft, S. (1987). Acetylcholine receptor antibody in myasthenia gravis: predominance of IgG subclasses 1 and 3. Clin. Exp. Immunol. 67, 82–88. Romi, F., Kristoffersen, E.K., Aarli, J.A., and Gilhus, N.E. (2005). The role of complement in myasthenia gravis: serological evidence of complement consumption in vivo. J. Neuroimmunol. 158, 191–194. Roumenina, L.T., Sène, D., Radanova, M., Blouin, J., HalbwachsMecarelli, L., Dragon-Durey, M.A., Fridman, W.H., and Fremeaux-Bacchi, V. (2011). Functional complement C1q abnormality leads to impaired immune complexes and apoptotic cell clearance. J. Immunol. 187, 4369–4373. Ruiz-Argüelles, A. and Llorente, L. (2007). The role of complement regulatory proteins (CD55 and CD59) in the pathogenesis of autoimmune hemocytopenias. Autoimmun. Rev. 6, 155–161.
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R. Huda et al.: Myasthenia and complement 583 Sahashi, K., Engel, A.G., Lambert, E.H., and Howard, F.M., Jr. (1980). Ultrastructural localization of the terminal and lytic ninth complement component (C9) at the motor end-plate in myasthenia gravis. J. Neuropathol. Exp. Neurol. 39, 160–172. Shen, C., Lu, Y., Zhang, B., Figueiredo, D., Bean, J., Jung, J., Wu, H., Barik, A., Yin, D.M., Xiong, W.C., et al. (2013). Antibodies against low-density lipoprotein receptor-related protein 4 induce myasthenia gravis. J. Clin. Invest. 123, 5190–5202. Shiraishi, H., Motomura, M., Yoshimura, T., Fukudome, T., Fukuda, T., Nakao, Y., Tsujihata, M., Vincent, A., and Eguchi, K. (2005). Acetylcholine receptors loss and postsynaptic damage in MuSK antibody-positive myasthenia gravis. Ann. Neurol. 57, 289–293. Soltys, J. and Wu, X. (2012). Complement regulatory protein Crry deficiency contributes to the antigen specific recall response in experimental autoimmune myasthenia gravis. J. Inflamm. (Lond.) 9, 20. Soltys, J., Gong, B., Kaminski, H.J., Zhou, Y., and Kusner, L.L. (2008). Extraocular muscle susceptibility to myasthenia gravis: unique immunological environment? Ann. N. Y. Acad. Sci. 1132, 220–224. Soltys, J., Kusner, L.L., Young, A., Richmonds, C., Hatala, D., Gong, B., Shanmugavel, V., and Kaminski, H.J. (2009). Novel complement inhibitor limits severity of experimentally myasthenia gravis. Ann. Neurol. 65, 67–75. Soltys, J., Halperin, J.A., and Xuebin, Q. (2012). DAF/CD55 and Protectin/CD59 modulate adaptive immunity and disease outcome in experimental autoimmune myasthenia gravis. J. Neuroimmunol. 244, 63–69. Tüzün, E., Scott, B.G., Goluszko, E., Higgs, S., and Christadoss, P. (2003). Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis. J. Immunol. 171, 3847–3854. Tüzün, E., Saini, S.S., Yang, H., Alagappan, D., Higgs, S., and Christadoss, P. (2006a). Genetic evidence for the involvement of Fcgamma receptor III in experimental autoimmune myasthenia gravis pathogenesis. J. Neuroimmunol. 174, 157–167.
Tüzün, E., Saini, S.S., Ghosh, S., Rowin, J., Meriggioli, M.N., and Christadoss, P. (2006b). Predictive value of serum anti-C1q antibody levels in experimental autoimmune myasthenia gravis. Neuromuscul. Disord. 16, 137–143. Tüzün, E., Saini, S.S., Morgan, B.P., and Christadoss, P. (2006c). Complement regulator CD59 deficiency fails to augment susceptibility to actively induced experimental autoimmune myasthenia gravis. J. Neuroimmunol. 181, 29–33. Tüzün, E., Li, J., Saini, S.S., Yang, H., and Christadoss, P. (2007). Pros and cons of treating murine myasthenia gravis with antiC1q antibody. J. Neuroimmunol. 182, 167–176. Tüzün, E., Huda, R., and Christadoss, P. (2011). Complement and cytokine based therapeutic strategies in myasthenia gravis. J. Autoimmun. 37, 136–143. Viegas, S., Jacobson, L., Waters, P., Cossins, J., Jacob, S., Leite, M.I., Webster, R., and Vincent, A. (2012). Passive and active immunization models of MuSK-Ab positive myasthenia: electrophysiological evidence for pre and postsynaptic defects. Exp. Neurol. 234, 506–512. Vincent, A. and Drachman, D.B. (2002). Myasthenia gravis. Adv. Neurol. 88, 159–188. Wu, X., Tuzun, E., Li, J., Xiao, T., Saini, S.S., Qi, H., Allman, W., and Christadoss, P. (2012). Ocular and generalized myasthenia gravis induced by human acetylcholine receptor γ subunit immunization. Muscle Nerve 45, 209–216. Yang, H., Wu, B., Tüzün, E., Saini, S.S., Li, J., Allman, W., Higgs, S., Xiao, T.L., and Christadoss, P. (2007). A new mouse model of autoimmune ocular myasthenia gravis. Invest. Ophthalmol. Vis. Sci. 48, 5101–5111. Zachrau, B., Finke, D., Kropf, K., Gosink, H.J., Kirchner, H., and Goerg, S. (2004). Antigen localization within the splenic marginal zone restores humoral immune response and IgG class switch in complement C4-deficient mice. Int. Immunol. 16, 1685–1690. Zhou, Y., Gong, B., Lin, F., Rother, R.P., Medof, M.E., and Kaminski, H.J. (2007). Anti-C5 antibody treatment ameliorates weakness in experimentally acquired myasthenia gravis. J. Immunol. 179, 8562–8567.
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Ruksana Huda, Erdem Tüzün and Premkumar Christadoss*
Targeting complement system to treat myasthenia gravis Abstract: While the complement system is desired for protective immunity, antibody- and complement-mediated neuromuscular junction (NMJ) destruction, a hallmark of myasthenia gravis (MG) or experimental autoimmune MG (EAMG), is a significant concern. Evidence suggests that the binding of complement factors to the pathogenic antiacetylcholine receptor (AChR) autoantibody induces the formation of membrane attack complexes (MAC), which ultimately lead to NMJ destruction and muscle weakness. Studies corroborating the evidence show that the complement (C3–C6)-deficient or complement inhibitor (anti-C1q, soluble CR1, anti-C6, and C5 inhibiting peptide)-treated animals are highly resistant to EAMG induction, whereas the deficiency of the naturally occurring complement inhibitors, such as the decay-accelerating factor (DAF), increases EAMG susceptibility. Notably, the complementinhibited animals do not exhibit significant immunosuppression but only a marginal reduction in the production of certain cytokines and immunoglobulin isotypes. A preliminary clinical trial using antibody-based C5 inhibitor eculizumab has been shown to be of potential use for MG treatment. The inhibition of the classic complement pathway (CCP) alone appears to be enough to suppress EAMG, suggesting that the complement inhibitors targeting specifically the classic pathway could effectively treat MG without causing immunosuppressive and other side effects. For instance, a recent non-antibody-based therapeutic approach selectively targeting the CCP component C2 by small interfering RNA (siRNA) has proven useful in EAMG treatment. The treatment strategies developed for MG might also be beneficial for other complement-mediated autoimmune diseases. Keywords: autoimmunity; complement; complement regulators; experimental autoimmune myasthenia gravis; myasthenia gravis; siRNA.
*Corresponding author: Premkumar Christadoss, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA, e-mail: [email protected] Ruksana Huda: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA Erdem Tüzün: Institute of Experimental Medicine, Department of Neuroscience, Istanbul University, Istanbul, Turkey
DOI 10.1515/revneuro-2014-0021 Received March 6, 2014; accepted March 26, 2014; previously published online April 12, 2014
Introduction Myasthenia gravis (MG) is a T-cell-dependent and antibody-mediated autoimmune disease of the neuromuscular junction (NMJ), manifesting with muscle weakness as a result of acetylcholine receptor (AChR) deficiency and/ or malfunctioning. MG is a classic antibody-mediated disease and one of the best characterized autoimmune disorders. About 85–90% of MG patients display AChR antibodies (Vincent and Drachman, 2002; Conti-Fine et al., 2006), which induce NMJ dysfunction by initiating a complement-mediated muscle membrane destruction, by crosslinking and down-regulating AChRs or blocking the acetylcholine binding sites of the AChR. Clinical, pathological, and animal model studies suggest that the complement-mediated mechanism of action plays the major pathogenic role in MG (Drachman et al., 1980; Engel et al., 1981). Both clinical and animal model studies have shown that Th1 and Th17 cells are important mediators of AChR immunity. Th17 cells shift the balance from regulatory T cells to Th1 cells, and Th1 cytokines promote the presentation of AChR peptides to T helper cells, the activation of B cells by T helper cells, and the proliferation of AChR antibody-producing B cells. Some Th2-type cytokines such as interleukin (IL)-5 and IL-10 also contribute to AChR immunity probably by enhancing the proliferation of AChR reactive B cells (Conti-Fine et al., 2008; Tüzün et al., 2011). Although T-cell- and antibody-mediated mechanisms play a crucial role in AChR-related MG, the complement system perhaps plays an even more profound role in MG development. This is exemplified by the striking resistance of complement-deficient mice or rats to experimental autoimmune MG (EAMG) induced by AChR immunization or the passive transfer of AChR antibodies (Christadoss, 1988; Tüzün et al., 2003; Chamberlain-Banoub et al., 2006). Experimental animals, which are inherently cytokine deficient or treated with cytokine inhibitors, generally show 40–80% reduction in EAMG incidence and/or
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576 R. Huda et al.: Myasthenia and complement (Engel et al., 1977; Sahashi et al., 1980). The experiments performed with a series of complement KO animal strains, as well as in vitro studies, have shown that, aside from the formation of MAC, various complement factors influence the immunoresponse by enhancing the proinflammatory cytokine production, clearing of the circulating immunocomplexes, modulation of IgG production, regulation of T cells, and isotype switching (Zachrau et al., 2004; Roumenina et al., 2011; Liszewski et al., 2013).
severity compared to controls (Tüzün et al., 2011), whereas mice or rats of the same strain often show 75–100% reduction in EAMG incidence when their complement systems are inhibited (Table 1). These findings put emphasis on the complement system as a potential target for future treatment trials. The complement system is composed of a group of proteins with a large variety of biological activities. The complement cascade is classically divided into three pathways: the classic complement pathway (CCP), the alternative complement pathway, and the mannose binding lectin (MBL) pathway (Figure 1). While the alternative and MBL pathways are usually activated by microorganisms and thus are generally involved in the defense against pathogens, the CCP pathway is mainly activated by the antigen-antibody complexes and thus is involved in the pathogenesis of a variety of immunocomplex and antibody-associated diseases (Ricklin and Lambris, 2013). The muscle pathology findings of MG patients and EAMG experiments conducted with complement knockout (KO) mice have suggested that the accumulation of anti-AChR IgG and the initial components of the complement cascade at the NMJ result in the assembly of membrane attack complex (MAC; a complex of C5b and C6–C9 factors) (Figure 1), thereby damaging the muscle membrane and reducing the number of functional AChRs
Involvement of the complement system in MG Clinical and pathological evidence from MG patients and EAMG animals The significance of the complement system in the pathogenesis of MG has first been recognized by studies showing alterations in the levels of various complement factors (C3, sC5b-9, etc.) in the sera of MG patients, suggesting the utilization of the complement system during the pathogenic processes (Nastuk et al., 1960). Moreover, almost all MG patients have the complement-fixing antiAChR IgG1 and/or IgG3 isotypes in their sera (Rodgaard et al., 1987).
Table 1 A comparison of EAMG clinical incidences and immunological effects of mice or rats that were treated with complement inhibitors. Treatment
CVF
Animal
Anti-C6 antibody Anti-C5 antibody C5 inhibiting protein Soluble CR1 Crry-Ig Anti-C1q antibody C2 siRNA
EAMG method
Lewis rats Wistar rats Lewis rats Lewis rats Lewis rats Lewis rats B6 mice
B6 mice
Passive transfer Immunization Passive transfer Passive transfer
Type of Percent reduction Immunopathological treatmenta in clinical effects incidenceb
Passive transfer Immunization Passive transfer Passive transfer Immunization
Immunization
Prevention Prevention Prevention Prevention Treatment Prevention Treatment Prevention Prevention Prevention Treatment
Treatment
100% 75% 100% 100% 100% 100% 100% 83% 100% 75% 60%
None reported None reported None reported Reduced serum IgG1 levels
None reported Reduction in complement deposition Increased serum immunocomplex levels Renal IgG and complement deposits (high dose) and reduced IL-6 production 80% Reduction in complement deposition, functional muscle AChR preservation, and reduction in serum anti-AChR IgG2b
a In the prevention experiments, the complement inhibitors are administered before or during the induction of EAMG, whereas, in the treatment studies, the complement inhibitors are administered after the establishment of clinical muscle weakness. b Relative EAMG incidences of animals with noticeable muscle weakness, when EAMG incidences of the animals treated with the control reagent are taken as 100% (prevention experiments) or EAMG incidences of the animals that showed clinical amelioration (treatment studies).
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R. Huda et al.: Myasthenia and complement 577
Figure 1 Schematic illustration of complement activation pathways. The complement factors in circles have been found to participate in EAMG pathogenesis and some of these factors have been targeted in EAMG treatment trials. The immunological factors that are suppressed following the inhibition of the reciprocal complement factors are shown in squares. Green arrows indicate activation and red arrows indicate inhibition. FB, factor B; FD, factor D; MASP, MBLassociated serine protease.
To date, one of the most convincing proofs for the involvement of complement system in the development of myasthenic muscle weakness is the demonstration of IgG, C3, and MAC deposits on the degenerating postsynaptic muscle membranes of MG patients. IgG and complement deposits not only colocalize with AChR at the NMJ, but also the numbers of these deposits show an inverse correlation with NMJ AChR levels (Engel et al., 1977; Sahashi et al., 1980). MG patients with higher serum anti-AChR antibody levels have been shown to display lower serum C3 and C4 levels (Romi et al., 2005), indicating the participation of CCP as well as the common complement pathway in the NMJ destruction. In the latter study, the serum levels of C3 or C4 were not associated with the presence of anti-titin and anti-ryanodine receptor (RyR) antibodies, suggesting that the complement factors were only utilized by AChR antibodies. In two other studies, the circulating C3 and C3c levels were found to be correlated with the clinical severity of MG (Kamolvarin et al., 1991; Liu et al., 2009). In one of these studies, the serum C3 levels were inversely correlated with the serum AChR antibody levels, further confirming the complement consumption hypothesis (Liu et al., 2009). Complement and IgG deposits are also found at the NMJ of EAMG animals, whether the disease is induced by passive transfer or active immunization with AChR. The numbers of NMJs with complement and IgG deposits almost always correlate with clinical disease severity.
The EAMG-resistant animal strains display much lower amounts of NMJ deposits than the EAMG susceptible ones (Tüzün et al., 2006a, 2007; Yang et al., 2007). Also, both serum complement levels and NMJ deposits increase in parallel after AChR immunization in a time-dependent manner (Tüzün et al., 2006b). In addition to the C3 and MAC deposits, the CCP factor C1q deposits have also been observed at the NMJs of AChR-immunized C57BL/6 (B6) mice (Tüzün et al., 2006b). Overall, these findings suggest that AChR antibodies activate the CCP upon binding AChR at the NMJ and initiate the complement cascade, ultimately leading to the formation of MAC. Due to the difficulty in obtaining biopsy samples from extraocular muscles, there is no substantial evidence for the involvement of the complement system in extraocular muscle weakness, the hallmark symptom of MG. Nevertheless, in a recent EAMG model induced by the immunization of HLA-DQ8 transgenic mice with recombinant human AChR α or γ subunits and characterized with severe ptosis and mild generalized muscle weakness, complement and IgG deposits have been identified in abundance at the extraocular muscle NMJs. Moreover, the severity of extraocular symptoms has been found to be correlated with the amount of NMJ complement deposits (Yang et al., 2007; Wu et al., 2012). The extraocular muscles of mice express reduced amounts of complement regulators compared to limb muscles, and complement regulator expression is even further reduced after EAMG induction (Kaminski et al., 2004), presumably rendering extraocular muscles more susceptible to autoimmune attack than limb muscles. In line with this hypothesis, a single nucleotide polymorphism in the decay-accelerating factor (DAF) regulatory region has been found in MG patients with extraocular muscle involvement. This polymorphism has been shown to prevent the lipopolysaccharide-induced up-regulation of DAF (Heckmann et al., 2010), suggesting that the genetic defects leading to the reduced expression of complement regulators might render individuals susceptible to ocular MG. The role of the complement system in muscle-specific receptor tyrosine kinase (MuSK) antibody-related MG (MuSK-MG) is less well understood. Serum MuSK antibodies are predominantly of the IgG4 isotype, which in contrast with the IgG1 and IgG3 isotypes (that are prevalently found in AChR-related MG) that do not activate the complement cascade (Plomp et al., 2012). While the passive transfer of anti-MuSK IgG4 elicits severe muscle weakness, the passive transfer of IgG1–3 antibodies from MuSK-MG patients does not induce any clinical signs (Klooster et al., 2012). Moreover, C3 and C5-deficient mice, which are resistant to AChR-induced EAMG, are highly
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578 R. Huda et al.: Myasthenia and complement susceptible to EAMG induced by MuSK immunization (Mori et al., 2012). Nevertheless, about 25% of MuSK-MG patients appear to bear complement deposits at their limb NMJs (Shiraishi et al., 2005). This ratio might plausibly be even higher in bulbar muscles that are more prominently affected in MuSK-related MG. Also, most but not all MuSK-MG patients exhibit anti-MuSK IgG1, although its levels are often much lower than those of anti-MuSK IgG4 (Leite et al., 2008). Likewise, the immunization of B6 mice with MuSK generates complement activating anti-MuSK IgG2 (albeit at levels lower than the noncomplement activating anti-MuSK IgG1) and NMJ complement deposits (Viegas et al., 2012). These findings suggest that although the complement system is not the main culprit in MuSK-MG, it might still to some extent be involved in MuSK-MG patients, especially in those with severe muscle weakness or myasthenic crisis. The significance of the complement system in the recently identified lipoproteinrelated protein 4 (LRP4) antibody-associated MG has not been established yet. However, LRP4 antibodies have been shown to be predominantly of the IgG1 isotype and to mediate complement-dependent cell lysis, suggesting that the complement system might also be involved in this MG subtype (Higuchi et al., 2011; Shen et al., 2013).
EAMG studies with complement KO and deficient rodent strains An essential line of evidence supporting the involvement of the complement system in MG pathogenesis comes from EAMG studies using mouse or rat strains with inborn deficiency of complement factors or complement regulators. The AChR-immunized, C5-deficient (B10.D2/ oSn), C3 KO and C4 KO mice show significantly reduced EAMG incidence and disease severity compared to the wild-type (WT) littermates. Only 5% of the C5-deficient mice developed EAMG, whereas 78% of the C5-sufficient mice showed signs of EAMG. Also, the C5-sufficient mice showed 37% muscle AChR loss as opposed to 14% AChR loss of the C5-deficient mice (Christadoss, 1988). Likewise, while the C3 KO and C4 KO mice demonstrated 0% and 5.8% EAMG incidence, respectively, the WT littermates had 86% and 88% EAMG incidence (Tüzün et al., 2003). The C6-deficient rats also showed a remarkable resistance (100% reduction in clinical incidence) to EAMG induced by passive transfer of AChR antibodies and the administration of C6 rendered the C6-deficient rats susceptible to EAMG (Chamberlain-Banoub et al., 2006). Notably, the serum anti-AChR IgG levels of the C5-deficient and C4 KO mice were comparable to those
of the control WT animals, whereas the C3 KO mice showed reduced anti-AChR IgG levels presumably due to the involvement of C3 in cytokine production and T-cell responses (Liszewski et al., 2013). Moreover, the C4 KO mice showed NMJ IgG deposits just like the WT mice (Christadoss, 1988; Tüzün et al., 2003). These results indicate that the deficiency of CCP or MAC factors does not interrupt the development of anti-AChR autoimmunity. These results also show that AChR antibodies alone are not highly pathogenic in the absence of a fully functioning complement system. While C5b is a component of MAC, C5a is an anaphylactic and chemotactic agent, which mediates various immunological functions such as the production of cytokines IL-1β, IL-6, and tumor necrosis factor (TNF)-α that are actively involved in EAMG pathogenesis (Liang et al., 2011). The AChR-immunized C5a receptor KO mice show high susceptibility to EAMG (Qi et al., 2008), suggesting that C5a is not involved in AChR immunity and C5 deficiency prevents EAMG induction through impaired MAC production due to the absence of the C5b component. The C3 convertase C4bC2a is activated by both classic and MBL pathways, suggesting that EAMG resistance of the C4 KO mice might be due to the deficient functioning of both classic and lectin pathways. However, the MBL KO mice are not resistant to EAMG induced by AChR immunization and the serum MBL levels of MG patients are comparable to those of the healthy individuals, showing that the lectin pathway does not participate in MG pathogenesis (Li et al., 2009). The role of the alternative pathway in EAMG pathogenesis is still to be characterized.
EAMG and the complement regulators The increased EAMG incidence observed in complement regulator-deficient mice is another supporting evidence for the participation of complement factors in NMJ destruction. Mouse skeletal muscle tissues express several complement regulators such as Daf1 (the mouse equivalent of human DAF), CD59, and Crry (Kaminski et al., 2004; Soltys et al., 2008). Among these regulators, the deficiency of Daf1 has been shown to increase the susceptibility to EAMG induction. Daf1 accelerates the breakdown of C3 and C5 convertases, which are the crucial enzymes for MAC formation (Ruiz-Argüelles and Llorente, 2007). The Daf1 KO mice show increased EAMG incidence and clinical severity, elevated NMJ C3 deposit counts, and reduced muscle AChR concentration (Lin et al., 2002). The mouse CD59 gene has two promoters controlling the expression of mCD59a and mCD59b transcripts (Qin
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R. Huda et al.: Myasthenia and complement 579
et al., 2006). Notably, in both passive transfer and immunization studies, the CD59a KO mice have shown equal susceptibility to EAMG induction as the WT mice (Tüzün et al., 2006c). Likewise, EAMG incidence, severity, and NMJ complement deposition of the Crry KO mice were identical to those of the WT mice (Soltys and Wu, 2012). The CD59 KO mice showed reduced serum anti-AChR IgG levels and decreased IL-2 production in response to AChR stimulation. The Crry KO mice showed increased serum anti-AChR IgG levels and AChR-stimulated IL-4 and interferon (IFN)-γ production. These findings indicate the involvement of these two regulators in the modulation of adaptive immunoresponses and AChR immunity (Tüzün et al., 2006c; Soltys and Wu, 2012). Although the CD59a/b double-KO mice showed moderately increased EAMG susceptibility, their EAMG incidence and severity were lower than those of the Daf1 KO mice (Soltys et al., 2012). These results suggest that the NMJ levels of CD59 and Crry are probably too low to confer a robust protection against EAMG.
Treatment trials performed with complement inhibitors The current treatment methods of MG and other autoimmune diseases generally include steroids and cytotoxic drugs that lead to significant side effects and global immunosuppression (Kumar and Kaminski, 2011). A large body of research on cytokine-based treatment methods has failed to produce a useful medication for MG. The absence of a relatively safe immunosuppressive treatment method for MG has prompted treatment trials with complement inhibitors. The major reasons for choosing the complement system as a potential target for MG treatment were the significantly high reduction ( ≥ 90%) in EAMG clinical incidence and the absence of substantial immunological deficiencies in complement KO studies (Tüzün et al., 2011). The therapeutic strategies based on the inhibition of the complement system include the use of complement-inhibiting recombinant proteins, complementinhibiting chemicals, monoclonal antibodies against complement components, soluble isoforms of complement receptor, and small interfering RNA (siRNA). These treatment trials have widely utilized EAMG models induced by immunization or passive transfer. These studies can be classified as those that inhibit the entire complement system and those that specifically inhibit the CCP.
Inhibition of the whole complement system Muscle weakness of mice and rats with EAMG induced by active AChR immunization or the passive transfer of AChR antibodies has been shown to improve following the inhibition of the entire complement system by several complement inhibitors including cobra venom factor (CVF), soluble complement receptor 1 (sCR1), C5 binding protein, anti-C5 antibody, and anti-C6 antibody (Lennon et al., 1978; Biesecker and Gomez, 1989; Piddlesden et al., 1996; Zhou et al., 2007; Soltys et al., 2009). The anti-C6 antibody administration before the passive transfer of AChR antibodies has achieved 100% prevention of EAMG induction in rats and has inhibited the accumulation of MAC components C6 and C9 at the NMJ. Anti-C6 treatment has not reduced serum C3 and C5 levels, suggesting that the antibody did not have any effect on the earlier components of the complement system; therefore, EAMG inhibition was solely associated with the inhibition of MAC formation (Biesecker and Gomez, 1989). EAMG symptoms induced by passive transfer and/or immunization were significantly ameliorated (75–100%) by sCR1 or CVF treatment. In concert with clinical results, these reagents have also maintained the preservation of muscular AChR concentrations (Lennon et al., 1978; Piddlesden et al., 1996). Both sCR1 and CVF inhibit the classic and alternative pathways, which ultimately stimulate the MAC formation. Therefore, unlike the anti-C6 antibody study, the EAMG-preventing effects of these reagents could be related with the inhibition of proinflammatory effects of early complement factors in addition to the prevention of MAC formation. The anti-C5 monoclonal antibody administered before the passive transfer of AChR antibodies or 24 h after EAMG induction effectively restored muscle strength in all rats, while all control rats had to be terminated due to severe weakness. Clinical amelioration was accompanied with reduction of NMJ C9 deposits. Similarly, rEV576, a novel 17-kDa C5 inhibiting protein, was found to be effective in the prevention and treatment of EAMG in both passive transfer and immunization experiments. Notably, in these studies, clinical amelioration was paralleled with the reduction of NMJ MAC deposits and preservation muscle AChR concentrations despite the presence of IgG deposits at the NMJ (Zhou et al., 2007; Soltys et al., 2009), confirming once again that AChR antibodies are not capable of inducing NMJ damage in the absence of an active complement system. Nevertheless, C5 inhibitor-treated rats showed a marginal reduction in serum IgG1 levels (Soltys et al., 2009), suggesting that C5 is involved in adaptive immunoresponses as well.
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580 R. Huda et al.: Myasthenia and complement In other trials utilizing complement regulators, Crry coupled with rat IgG2a Fc (rCrry-Ig) and DAF coupled with a single-chain antibody directed against the α-subunit of the AChR (scFv-35-DAF) were effectively used in the treatment of EAMG induced by passive transfer of AChR antibodies. The rats treated with rCrry-Ig were rendered entirely resistant to EAMG induction and this effect was accompanied with a significant reduction in NMJ complement deposition (Hepburn et al., 2008). Likewise, the rats treated with scFv-35-DAF showed significantly lower clinical muscle weakness scores and a reduction of complement deposition compared to the nontreated control rats (Kusner et al., 2013).
Inhibition of the CCP A potential drawback of the reagents that block the common complement pathway (e.g., C5 inhibitors and anti-C6 antibody) or both alternative and classic pathways (e.g., sCR1 and CVF) is the possible susceptibility to opportunistic infections, since the complement system is required for host defense against invading microorganisms. To overcome this problem, we performed EAMG treatment trials using specific inhibitors of CCP, which has been shown to be primarily involved in AChR antibodyrelated MG (Tüzün et al., 2003). With this approach, we anticipated that the alternative and lectin pathways would be left intact in order to be utilized by the host defense mechanisms against the invading microorganisms. To show that the acquired deficiency of CCP may render mice resistant to EAMG, we administered anti-C1q antibodies before and after induction of EAMG by AChR immunization. In these experiments, anti-C1q treatment achieved a significant reduction in EAMG clinical incidence compared to AChR immunized mice treated with the control nonspecific antibody (Table 1). The anti-C1q administered mice also showed reduced NMJ C3, IgG, and MAC deposits and lymph node cell IL-6 production in response to AChR and immunodominant peptide α146– 162 challenge (Tüzün et al., 2006a, 2007). This experiment showed for the first time that an autoimmune disease could be prevented by CCP inhibition. However, anti-C1q antibody-treated mice developed renal C3 and IgG deposits and showed increased serum immunocomplex levels (Tüzün et al., 2007). To overcome the problem of immunocomplex formation and to improve the effectiveness of CCP inhibition, we decided to utilize a non-antibody-mediated treatment method targeting a CCP component that has fewer immunological functions. A non-vector-based, chemically
modified siRNA-based gene silencing was anticipated not to induce any harmful side effects. C2 was thought to be a good candidate target since it is expressed at a very low level and there are no major immunological functions for C2 other than MAC formation. Also, congenital C2 deficiency is less frequently associated with immunocomplex disorders than C1q and C4 deficiency (Klint et al., 2000). The B6 mice with established severe EAMG were treated with either C2 siRNA or a control nontargeting (NT) siRNA. The efficacy of in vivo C2 siRNA treatment was confirmed with the evaluation of liver and peripheral blood cell C2 mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR). While 40% of the NT siRNA-treated mice died and 20% showed improvement of muscle weakness, only 10% of the C2 siRNA-treated mice died and 80% showed significant improvement in muscle weakness as assessed by clinical evaluation. The clinical amelioration in the C2 siRNA-treated group was associated with a transient reduction in serum anti-AChR IgG and IgG2b (but not IgG1 and IgM) levels, reduced NMJ C3 and MAC deposits, and higher muscle AChR levels compared to the NT siRNAtreated group. The serum C3 levels were comparable in the C2 and NT siRNA-treated groups, confirming that C2 inhibition does not affect the remaining components of the complement cascade. This study was the first example of utilization of siRNA-mediated complement inhibition for the treatment of an autoimmune disease with the preservation of the alternative pathway to fight against infection (Huda et al., 2013).
Concluding remarks As reviewed herein, many complement inhibitors lead to a significant reduction in EAMG incidence and clinical severity without causing noticeable toxicity or making a remarkable change in other immunological functions. These findings prompt the utilization of complement inhibitors as a novel therapeutic approach in MG treatment. Although there is a small number of complementinhibiting therapeutics in the market such as C1 inhibitor and sCR1, they have mostly been approved or are in the stage of approval for non-autoimmune disorders (Rioux, 2001; de Zwaan et al., 2003). Currently, the only complement-inhibiting therapeutic that can be used for autoimmune disorders and is available in the market is the C5 inhibiting monoclonal antibody eculizumab, which has been approved for another complement-mediated autoimmune disease, paroxysmal nocturnal hemoglobinuria
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(Risitano, 2013). In a recent randomized, double-blind, placebo-controlled trial performed on patients with severe, refractory generalized MG, eculizumab treatment has achieved a significant amelioration (at least a threepoint reduction in the quantitative MG score) in 86% of the participants (Howard et al., 2013). Although the common complement pathway inhibitors are expected to be effective in complement-mediated autoimmune disorders, they are also predicted to cause opportunistic infections since they prevent the major host defense mechanism against the invading pathogens to function. Not surprisingly, eculizumab and pexelizumab (another C5 inhibiting antibody used for coronary heart disease) are both associated with a high risk of meningococcal infection; therefore, patients are recommended to receive a vaccine before starting therapy. Upper respiratory system infections, nasopharyngitis, urinary tract infections, wound infections, and fungal infections are encountered in some patients. Eculizumab has also been associated with severe skin eruptions, rash, and kidney failure (Brodsky et al., 2008; Davis, 2008; Knoll et al., 2008). Some of these adverse effects might also be associated with the immunocomplex disease induced by the monoclonal antibody used for the inhibition of the complement cascade. Another problem that might emerge due to the usage of monoclonal antibodies is immunogenicity. These findings suggest that a non-antibody-based complement-inhibiting strategy targeting the CCP might effectively treat autoimmune disorders without causing infections or immunocomplex disorders and inducing immunogenicity that would decrease the efficacy. An effective suppression of EAMG in mice by the inhibition of a CCP factor C2 using a siRNA-based treatment method and without causing any major side effects suggests that this treatment approach might prove useful in patients with MG and other complement-mediated autoimmune disorders. Apart from MAC formation, the complement system is involved in several immunological functions that are of vital importance in the development of anti-AChR immunoresponse. Experimental animal studies suggest that the complement system is a part of a complex network of cytokines and IgGs activating one’s and other’s production, thus forming an autocrine feedback loop that promotes chronic autoimmunity. The findings of EAMG studies suggest that C1q might mediate the IL-6 production, CD59 might interfere with the IL-2, C3, C4, IgG1, and IgG2b production, and C3 might be enhancing the synthesis of IgG, IgG2b, IL-6, IL-10, and IFN-γ (Tüzün et al., 2003, 2006a,c, 2007). Moreover, C2 and C4 appear to be involved in the IgG2b production, whereas C5 might be involved in
the IgG1 production (Tüzün et al., 2003; Soltys et al., 2009; Huda et al., 2013) (Figure 1). These results suggest that complement-based treatment methods are not entirely devoid of immune system suppression. Nevertheless, the effects of complement inhibitors on the immunological functions appear to be rather marginal and they do not prevent the formation of a robust immunoresponse to immunogens. This is exemplified by the formation of AChR antibodies and the accumulation of IgG deposits at the NMJs of complement KO or complement-inhibited EAMG mice. Therefore, a complement inhibition could probably be safely used without causing significant immunosuppression.
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