Immune‐Mediated Necrotizing Myopathy (IMNM): A Story of Antibodies

1. Anti-SRP and Anti-HMGCR Autoantibody as a Hallmark of Disease

Anti-SRP aAbs were first discovered in 1986 by Reeves et al. in patients diagnosed with PM [12]. The Signal Recognition Particle (SRP) is a complex composed of six proteins, SRP9, SRP14, SRP19, SRP54, SRP68, SRP72, and 7SL RNA respectively. This complex, along with its receptor (SR), facilitates the translocation of neo synthetized proteins from the cytosol to the endoplasmic reticulum (ER). The process occurs in three steps. First, while the protein is translated by the ribosome, SRP54 recognizes the hydrophobic signal sequence of the protein. With the involvement of 7SL RNA, it stops in a transitory manner its elongation. Then the SRP complex binds to its receptor on the ER membrane. Finally, GTP hydrolysis, which occurs following interaction with the SR, releases the SRP from the ribosome-signal peptide complex, allowing it to bind to another signal peptide. Protein translation then resumes at the translocation channel, called sec61, guiding the protein into the lumen of the ER [13]. Anti-SRP54 aAbs are found in 15% of diagnosed patients [8]. We demonstrated in 21 sera from patients with anti-SRP positive IMNM that 81% of the patients had isotype 1 IgG antibodies, and 71% of these IgG1 antibodies were present alone while the others were associated with one or two other isotypes. IgG4 was found in 29% of the patients, while IgG3 was only found in one sample, and IgG2 was not detected anywhere [14]. Four different antibodies associated with IMNM were identified and target SRP19, SRP54, SRP72 proteins and 7SL RNA [15,16,17,18]. Anti-SRP54 is the most dominant antibody found in patients with anti-SRP IMNM. It has been shown that anti-SRP54 antibodies can bind to the amino-terminal SRP54 N-domain and the central SRP54 G-domain. However, they cannot bind to the carboxy-terminal M-domain which is responsible for binding ER signal sequences [16]. To our knowledge, the epitope mapping of other anti-SRP aAbs has not been performed.

Anti-HMGCR aAbs were discovered in 2010 by Christopher-Stine et al. [19]. HMGCR was initially considered as a target of aAbs in patients with IMNM due to evidence that several individuals developed IMNM following statin exposure [20,21]. Statins are used as medications to lower cholesterol levels by targeting HMGCR, a protein involved in the synthesis of cholesterol. HMGCR is a 97kDa glycoprotein localized in the ER. This enzyme contains a hydrophobic N-terminal domain that crosses the ER membrane and a soluble domain into the cytosol. The C-terminal domain is the effector domain of the enzyme and catalyzes the transformation of HMG-CoA to mevalonate, the precursor of cholesterol [22]. The anti-HMGCR antibody has been identified in more than 60% of cohorts in some studies [10]. It has been demonstrated that anti-HMGCR aAbs recognize a portion of the protein spanning a part of the C-terminal domain and intracellular portion (amino acids 340 to 888) [23].

Seronegative IMNM, described as IMNM without anti-SRP or anti-HMGCR aAbs, represents between 10 and 12% of patients [10]. Allenbach et al. described this subclass “by default” with poorly studied characteristics [10]. The diagnosis of seronegative patients remains a difficult task. Possibly, sera from these patients with seronegative IMNM contain an undiscovered autoantibody.

2. Autoantibody Assays at the Heart of Diagnosis

To diagnose IIM, aAbs are systematically tested. Several methods are available to detect aAbs: immunoprecipitation (IP), line blot, dot blot, enzyme-linked immunosorbent assay (ELISA), Addressable Laser Bead ImmunoAssay (ALBIA), or chemiluminescence assay (CLIA) and they could be completed by Indirect Immunofluorescence (IFI) on human epithelial type 2 (Hep2) cells.

The reference method for detecting IIM autoantibodies is IP. This method has allowed the discovery of many specific aAbs in autoimmune diseases as anti-SRP and anti-HMGCR aAbs in IMNM [12,19]. Moreover, IP is sensitive and specific. However, the use of IP in routine is not relevant because it is rarely available in medical biology laboratories [24]. Several detection methods are now available in routine practice. aAbs can be detected by mono- or multispecific (detection of multiple antibodies at the same time) immunoassays [25]. These methods can be qualitative and/or quantitative. Commercial multispecific immunoassays correspond to immunoblot (line or dot blot) and are qualitative. Their main advantage is being able to detect all specific myositis antibodies in a single test run. In contrast, monospecific immunoassays are quantitative. They also have excellent analytical and diagnostic performance. However, their disadvantage is that they only allow the detection of one antibody at a time. ELISA and ALBIA tests using SRP54 as antigen are used to detect anti-SRP aAbs, and ELISA, CLIA and ALBIA for detecting anti-HMGCR aAbs. Only ELISA and CLIA are commercially available. Moreover, anti-SRP aAbs are well detected using IFI on Hep2 cells, a method that reveals the localization of autoantigens which are associated with specific staining patterns, making them easily detectable. SRP appears with a finely granular cytoplasmic staining making IFI on Hep2 cells an interesting element to support anti-SRP immunoassay results [26,27]. However, efforts towards harmonization and standardization among the different proposed methods are still necessary.

The assessment of the analytical and clinical quality of anti-SRP and anti-HMGCR tests was discussed by the ENMC and the European Autoimmunity Standardization Initiative (EASI) working groups [25,28]. It was noted that the comparison between anti-SRP-54 ELISA and IP revealed strong agreement between the methods with ELISA exhibiting high specificity (100%) and sensitivity (88%) while the comparison between line blot and ALBIA revealed 87% agreement. In contrast, immunoblot presented some false negative. Anti-HMGCR ELISA test showed a sensitivity of 94.4% and a specificity of 99.3% compared to IP. Furthermore, the evaluation using ELISA, CLIA and ALBIA on positive and negative sera for anti-HMGCR antibodies demonstrated an excellent concordance agreement (close to 100%) among these different methods. Immunoblots offer a broader screening capability, yet they remain limited to qualitative analysis. Monospecific assays, on the other hand, enable precise determination of antibody titers that can be a critical parameter as studies have indicated a correlation between aAb levels and disease severity [14].

IIM aAbs are more than diagnosis markers, they are also prognosis markers, of cancer for instance. For anti-HMGCR IMNM, results were conflicting, suggesting a mild association with cancer. No association was found for anti-SRP. In contrast seronegative IMNM was found at increased risk of malignancy [11]. More than just diagnosis and prognosis markers of the disease, anti-SRP and anti-HMGCR aAbs play a pathogenic role in IMNM.

3. Autoantibodies at the Heart of Pathophysiology

In systemic lupus erythematosus (SLE), anti-dsDNA antibody levels were shown to be correlated with disease activity and their decrease led to the remission of patients, suggesting their pathogenicity [29]. In a similar manner, the pathogenic role of anti-SRP and anti-HMGCR antibodies was suggested when anti-SRP and anti-HMGCR antibody levels in sera were correlated with CK levels in sera, and muscle weakness indicating the extent of muscle breakdown and hence the severity of the disease [14,30]. Additionally, several studies demonstrated the efficacy of plasmapheresis or immunosuppressive treatments as plasmapheresis, prednisone or rituximab to reduce the number of antibodies, resulting in a recovery of muscle strength in patients [14,31,32]. All these data provide compelling evidence suggesting a pathogenic role of these antibodies.

The correlation between aAb titer and CK levels, along with the effects of depleting treatments suggests the involvement of autoantibodies. Experimental in vitro evidence further supports this hypothesis. Rojana-udomsart et al. demonstrated that the culture of human myoblasts in the presence of serum from patients with anti-SRP IMNM, with a high titer, resulted in a decrease in cell survival [33]. Subsequently, another study showed that plasma from patients with anti-SRP positive or anti-HMGCR positive IMNM led to a decrease in myotube surface area in culture. Similar results were obtained after incubating the myotubes with purified total IgG, as well as in the presence of purified aAbs against SRP or HMGCR. Furthermore, the expression of TRIM63/MURF1 and MAFbx/ATROGIN-1 mRNA, which are markers of atrophy, were increased. This increase appears to be linked to an upregulation of pro-inflammatory molecules such as IL6, TNF, and ROS, factors known to induce muscle atrophy [34]. Moreover, plasma, purified total IgG, or purified anti-SRP or anti-HMGCR aAbs from patients with IMNM decreased the fusion of myoblasts into myotubes reflecting a negative effect of these aAbs on in vitro muscle regeneration. The secretion of IL-4 and IL-13, known to play a major role in myotube formation from myoblasts, is also decreased. Myotube formation was then restored after the addition of these cytokines to the medium [34]. Thus, anti-SRP or anti-HMGCR aAbs have a direct pathogenic role in vitro by (i) reducing cell survival, (ii) inducing atrophy of already mature fibers, and (iii) preventing optimal regeneration from myoblasts.

The interaction between these aAbs and their target was investigated further by Romish et al. These authors studied the impact of anti-SRP54 on the function of the SRP54 protein and of the SRP complex in vitro. They found a significant and targeted inhibitory impact of anti-SRP54 aAbs on the in vitro translocation of the secretory protein preprolactin. These aAbs disrupt the binding of signal sequence of protein to SRP54, likely through steric hindrance. Moreover, the aAbs impeded the SRP receptor-mediated release of ER signal sequences from the SRP54 subunit [16]. To our knowledge, only the impact of anti-SRP54 was studied and not the other anti-SRP or anti-7SL RNA aAbs.

Unlike anti-SRP54 aAbs, which inhibit the function of the protein as presented above, the inhibitory activity of anti-HMGCR aAbs on protein function has not been yet demonstrated to our knowledge. In 2003, a study demonstrated that disruption of the HMGCR gene in mice was lethal, with the mice not surviving beyond the E8.5 stage underscoring the importance of this protein in embryonic development [35]. Additionally, it has been shown that a specific homozygous invalidation of the HMGCR gene in skeletal muscle induced postnatal myopathy, characterized by high levels of CK and muscle necrosis [36]. Thus, it appears that the absence of HMGCR in muscle is the root cause of the myopathy. Therefore, it is plausible to hypothesize that aAbs could inhibit the function of the protein and induce myopathy.

As in vitro studies demonstrated the impact of aAbs on muscle cells and to clarify the effects of aAbs, we evaluated their pathogenic role by i) an active immunization of the animal with recombinant autoantigens, and ii) a passive transfer of purified antibodies from patients with IMNM. First, immunization of mice with recombinant SRP and HMGCR proteins led to the detection of anti-HMGCR and anti-SRP antibodies in the serum on day 7 and day 28, respectively, and a decrease in grip strength and in situ muscle strength [37]. In the same way, the passive transfer of plasma or purified IgG from patients with IMNM, but not IgG-depleted plasma to mice, resulted in a decrease in grip strength and in situ muscle strength in mice. This muscle impairment was associated with an increased number of necrotic myofibers and macrophage infiltration [37]. This active and passive immunization demonstrated that anti-SRP and anti-HMGCR antibodies were pathogenic in vivo. This murine model could be useful in understanding further the pathophysiological mechanisms. However, it has several limitations. Indeed, the murine model requires the administration of purified patient antibodies for several consecutive days to develop the disease [37]. These purified antibodies come from patient plasmapheresis, which is a limitation as it is a finite resource. The development of standardized antibodies produced in cells could be an interesting prospect to obtain a robust and sustainable murine model. Furthermore, another limitation of this model is that the mice receive an immunosuppressant injection to prevent xenogeneic responses towards the injected human-origin IgG. However, this injection eliminates immune cells that could potentially play a role in the pathological reaction. It would be interesting to consider using animals that are tolerant to human IgG.

Knowing that aAbs have a pathogenic effect, we sought to understand their mechanism of action. The hypothesis that the complement system could play a role in the physiopathology of IMNM has been highlighted. Initially, it was observed in muscle biopsies from patients’ complement deposits at the sarcolemma of non-necrotic muscle fibers [38]. Moreover, aAbs associated with IMNM are predominantly of the IgG1 isotype [14], an isotype known to activate the complement system. These observations suggest a mechanism mediated by complement-dependent antibodies. To determine the involvement of complement in myolysis, human myoblasts were cultured in the presence of plasma from patients with SRP-positive IMNM, and a decrease in cell survival was observed [33]. Interestingly, this survival was even more affected when human complement was added to the culture [33]. Furthermore, a co-localization of SRP and C3c at the cell membrane on myoblasts was observed after pre-incubation with serum from patients with anti-SRP positive IMNM. Additionally, a positive staining for membrane attack complex C5b-9 was observed [33]. Finally, in vivo, it has been demonstrated that the administration of total IgG from patients with anti-SRP positive or anti-HMGCR positive IMNM to complement-deficient mice does not induce muscle deficit. Moreover, supplementation with human complement in mice worsens the muscle deficit in the presence of total IgG from patients. Associated with this muscle deficit, necrotic fibers and infiltration of macrophages were observed in muscle biopsies from mice and complement deposits were detected within the sarcolemma of diseased mice [37]. All these findings suggest the involvement of the complement system in antibody-mediated pathogenicity in patients and bring new keys to develop more specific therapeutic strategies for patients.

SRP and HMGCR are intracellular and ubiquitous targets. To date, it is still unknown how these antibodies manage to specifically target these proteins, and why this leads to muscle-related manifestations exclusively. Allenbach et al. hypothesized a potential ectopic expression of these proteins on the cell membrane, which would make them accessible to antibodies following labeling on muscle cells. However, this labeling does not precisely define the protein's location, as it has not been observed under a confocal microscope and has not been performed with co-labeling with a strictly membrane-bound protein [38]. Other hypotheses can be considered, including the possibility that antibodies may enter cells to reach their target. It has indeed been described that the TMab4 antibody, which is a human IgG1, can access the cytosol of cells. This penetration is possible after interaction with the heparan sulfate proteoglycan (HSPG) located on the surface, acting as a receptor. This complex is then internalized into endosomes. The acidification of the endosome induces conformational changes in the antibody due to a specific sequence located on the CDR3 light chain. These conformational changes lead to an interaction with the endosomal membranes, resulting in the formation of a pore allowing the antibody to escape [39]. Although the phenomenon of intracellular penetration is currently poorly documented, it should not be ruled out in the case of aAbs in IMNM. Finally, it is also possible to suspect that anti-SRP or anti-HMGCR antibodies may recognize another protein, based on sequence homology, located on the membrane.