Idiopathic inflammatory myopathies: from immunopathogenesis to new therapeutic targets

INVITED REVIEW

Idiopathic inflammatory myopathies: from

immunopathogenesis to new therapeutic targets

Syed A. HAQ

and Anne TOURNADRE

1_{BSM Medical University, Dhaka, Bangladesh, and}2_{Rheumatology department CHU Clermont-Ferrand and UMR 1019 INRA/}

University of Auvergne, Clermont-Ferrand, France

Abstract

Pathogenesis of idiopathic inflammatory myositis (IIM) involves strong interactions between dendritic cells (DCs), activated Th1 and Th17 cells, B cells, muscle cells, genes and environment. Local maturation of DCs per-mit the activation and polarization of CD4+ T cells into TH1 and TH17 that play a key role in maintaining

chronic muscle inflammation. T-cell mediated myocytotoxicity promotes the liberation of specific muscle autoantigens from regenerating muscle cells with production of myositis-specific autoantibodies. Type I inter-feron signature is a key characteristic of IIM. Type I IFN that can be induced by immune complexes containing myositis-specific autoantibodies is produced by scattered plasmacytoid DCs but also by muscle cells particularly regenerating muscle cells. These immature muscle precursors appear to be critical in the pathogenesis of IIM as they up-regulate muscle autoantigens, type I IFN, HLA class I antigens and TLR3-7, all together involved in maintaining chronic muscle inflammation. In addition to the role of immune and muscle cells, genome-wide association studies have confirmed the importance of several MHC and non-MHC genes in IIM. Environmental factors can contribute to the pathogenesis of IIM. In sIBM, distinct features suggest both degenerative and inflammatory processes. In addition to our better understanding of the pathogenesis, identify molecular path-way leads to consider new targeted therapies including cytokine inhibition, B-cell and T-cell costimulation blockade, type I IFN neutralization or inhibition of the ubiquitin proteasome pathway.

Key words: dermatomyositis, idiopathic inflammatory myositis, inclusion body myositis, pathogenesis, polymyositis, treatment.

INTRODUCTION

Idiopathic inflammatory myositis (IIM) is a heteroge-neous group of autoimmune myopathies. It comprises at least five conditions, namely, polymyositis (PM), der-matomyositis (DM), juvenile derder-matomyositis (JDM), sporadic inclusion body myositis (sIBM) and immune-mediated necrotizing myopathy (IMNM). In addition, new classifications distinguish overlap myositis and cancer-associated myositis.1,2However, these classifica-tions are limited by clinical, histological and serological

overlap. Not much was known about the etiopathogen-esis of this condition. The evidence for an immune-me-diated disease comes from the presence of cellular infiltrates in muscle biopsies, a T cell-mediated myocy-totoxicity, autoantibodies in the peripheral blood and association with class I major histocompatibility (MHC) overexpression. On the basis of these under-standings, so far, IIMs have been treated with glucocor-ticoids and other conventional immunosuppressive agents, including methotrexate and azathioprine. The treatment is unsuccessful in some cases and is often fraught with the consequences of non-specific immuno-suppression. Therapies targeting specific cytokines, receptors and intracellular molecules have shown pro-mise in the treatment of other rheumatic conditions, like rheumatoid arthritis (RA), ankylosing spondylitis

Correspondence: Dr Anne Tournadre, Rheumatology

Department, Gabriel Montpied Hospital, 58 rue Montalembert BP69 63003 Clermont-Ferrand cedex 1, France

(AS) and systemic lupus erythematosus (SLE). It has become imperative that the pathogenesis of IIMs be explored in detail so that more specific and targeted therapies that are likely to be more effective and safe may be developed. In this communication, keeping the sequence of events in consideration, the roles of den-dritic cells, lymphocytes and cytokines will be presented with transcriptional regulation. Critical contribution of regenerating muscle cells in pathophysiology will be discussed as well as the role of genetics and environ-ment. Finally, we will address potential therapeutic tar-gets.

DENDRITIC CELLS, LYMPHOCYTES AND

CYTOKINES

Dendritic cells (DCs) are professional antigen-present-ing cells that support adaptive and innate immune responses. Their activation in the lymphoid and periph-eral tissues leads to their migration, maturation and to the activation of CD4+ and CD8+T cells as well as B cells. It appears from histological studies that CD4+ T cells and B cells play a predominant role in the patho-genesis of DM, whereas this CD8+T cells in the patho-genesis of PM. Immature CD1a+ and mature LAMP3+ (lysosome-associated membrane glycoprotein 3) den-dritic cells are found in the inflammatory infiltrates from endomysial areas in PM and perimysial areas in DM.3DCs are of two types: myeloid (or conventional) DCs (mDCs or cDCs) supporting specific adaptive immune response and plasmacytoid DCs (pDCs) which produce type I IFN (interferon) and have a key role in innate immunity. pDCs are found mainly in DM.4

mDCs predominate in PM and sIBM.5The local matu-ration of DCs is suggested by the presence of CCL20 involved in the migration of immature DCs with the lack of the C–C motif chemokines CCL19 and CCL21 involved in the migration of mature DCs.6Activation of toll-like receptors (TLR) and C-type lectin receptors (CLR) on the surface of immature DCs by pathogen-as-sociated molecular patterns (PAMPs) and muscle auto-antigens could favor their maturation.7,8Once mature, mDCs can activate CD4+T cells. An inverse correlation of peripheral blood T cell count with disease activity and limited pattern expression of T cell receptors (TCR) in muscle tissue hint at homing of T cells during active disease with local proliferation and possibly presence of a pathogenic antigen in the muscle tissue.9,10

Activated CD4+T cells develop into distinct types of T helper (TH) cells that can be identified according to

the types of cytokines T cells produce when they are

stimulated to differentiate. TH1 cells produce large

amount of IFN-c, induce delayed hypersensitivity reac-tions, target macrophages, and are essential for the defense against intracellular pathogens. TH2 cells

mainly produce interleukin 4 (IL4) and contribute to immunoglobulin E (IgE) production and eosinophil recruitment to clear parasites. TH17 cells preferentially

produce IL17, but not IFN-c as TH1 cells, or IL-4 as TH2

cells. The TH17 pathway plays a critical role in inducing

and maintaining chronic inflammation and autoimmu-nity. TH1 cells develop in presence of IL12 and IL18.

Differentiation of human TH17 cells depends on key

cytokines, namely IL-6, IL-23 and transforming growth factor (TGF)-b. All these cytokines are locally produced by macrophages, monocytes, endothelial cells and DCs. IL18 is over-expressed in endomysial areas in PM and in the perivascular areas in DM.11TH1 cells secrete

IFN-c. In addition to enhancing natural killer (NK) cell activity, antigen processing and presentation by antigen presenting cells (APCs), IFN-c increases expression of MHC class I and TLR3 on myoblasts.7,8 The role of muscle cell, human leukocyte antigen (HLA) class I and TLR3 is elaborated in the next section. TH17 cells

elabo-rate IL17. In IIMs, IL17 is detected by immunohisto-chemistry in muscle lymphocytic infiltrates.3 In combination with IL1, it induces production of IL6 and CCL20 by myoblasts.12 The IL-23–TH17 pathway is

important for maintaining chronic tissue inflammation. Locally IL17 amplifies the initial response by acting in synergy with pro-inflammatory cytokines produced by macrophages and DCs to promote the production of the b chemokine CCL20, adhesion molecules and IL6 that facilitate in turn migration of mononuclear cells to the endomysial and perimysial spaces, maturation of DCs and differentiation of T cells.13

Mature mDCs also activate CD8+cells by presenting antigen in the groove of HLA class I antigens. It leads to activation, proliferation and differentiation of cytotoxic T cells. In PM, endomysial CD8+T cells surround and invade muscle fibers that express MHC class I anti-gens.14IL15 and IL15 receptors upregulated in muscle tissue of IIMs may be responsible for local T cell activa-tion and expansion.15It is assumed that an interaction between myocytotoxic CD8+ T cells and regenerating muscle cells expressing MHC class I antigen leads to lib-eration of cytotoxic effector molecules, including gran-zyme B. Granzyme B generates myositis-specific autoantigens after cleavage of autoantigenic peptides such as histidyl-tRNA (transfer RNA) synthetases and exposition of new dominant epitopes.16 In addition to antibody response initiation, autoantigenic tRNA

synthetases are chemotactic for lymphocytes and imma-ture DCs17 leading to a positive feedback loop for maintaining the inflammation.

In PM and DM, a large proportion of T cells are of CD28nullphenotype.18 They are thought to arise from repeated antigenic stimulation, and are terminally dif-ferentiated and apoptosis resistant. Both CD4+CD28null and CD8+CD28null T cells contain granzyme and perforin, having NK/cytotoxic properties and are easily stimulated to produce cytokines.19 In autologous muscle T cell co-culture both types of CD28null cells were found to be highly toxic to mytubes.20

mDCs play a key role in humoral activity with activa-tion of B cells and producactiva-tion of autoantibodies. B cells and plasma cells in muscle tissue, large numbers of B cells and auto-antibodies in peripheral blood, high serum B cell activating factor (BAFF) also known as B lymphocyte stimulator (BLyS), evidences of in situ dif-ferentiation of B cells, including somatic mutation, iso-type switching, clonal expansion and intraclonal variation, hint at the role of these cells in the pathogen-esis.21The majority of patients with IIMs have at least one myositis specific autoantibody (MSA), provided sensitive techniques are used.22

Scattered pDCs are found in the infiltrates in IIMs.23 There are evidences that immune complexes containing anti–Jo-1 or anti–Ro 52/anti–Ro 60 autoantibodies and RNA may activate IFNa production by pDCs.23 _IFNa

activates specific transcription activators including STATA. It stimulates transcription of genes responsible for synthesis of a series of pro-inflammatory cytokines, particularly those driving TH1 and TH17 cell lines. It

also enhances expression of HLA class I and class II molecules. The expression of type I IFN inducible genes and their protein products are upregulated in blood and in muscle tissue in IIMs (type 1 IFN signature).24 Beside the endogenous IFNa production by pDCs, immature muscle precursors may act as a local source of IFNb.7

TRANSCRIPTIONAL REGULATION

Micro-RNAs (miRNAs) are key components for post-transcriptional regulation. They function via base-pair-ing with complementary sequences within mRNA molecules. As a result, these mRNA strands are silenced because they can no longer be translated into proteins by ribosomes, and such complexes are often actively disassembled by the cell (“target degradation”).25It is plausible that certain miRNAs may modulate

inflamma-tion through silencing mRNAs coding for pro-inflam-matory cytokines. In IIM, a decreased expression of such miRNAs (miR-1, miR-133a, miR-133b) has been reported and miRNAs and could influence the NFjB pathway.26,27

The signal transducer and activator of transcription 4 (STAT4) is activated by IL-1, IL-23, IFN-a and IFN-b. It induces transcription of genes that drive the TH1 and

TH17 responses. Polymorphisms within the STAT4 gene

have been associated with IIMs in a Japanese popula-tion.28

REGENERATING MUSCLE CELLS

Chronic muscle inflammation leading to muscle wast-ing is a typical feature of IIMs and contrast with the presence in muscle tissue of immature muscle precur-sors involved in regeneration. Unlike differentiated muscle fibers, the regenerating muscle cells in IIMs over-express class I HLA antigens, TLR3, TLR7 and myositis-specific antigens such ashistidyl-tRNA synthetase, Mi-2 or 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCR) associated with statin-induced IMNM.8,29–32

In addition to making the cells vulnerable to cyto-toxic T cell injury, overexpression of HLA class I anti-gens leads to protein accumulation and misfolding in the endoplasmic reticulum (ER). Such misfolding acti-vates the ER stress pathway leading to activation of the NFjB pathway with enhanced synthesis of pro-inflam-matory cytokines and endogenous class I MHC up-regu-lation, and ER death pathway through caspase 12, leading to muscle degeneration.33,34 In presence of necrotic cell materials, muscle autoantigens released from regenerating muscles may activate IFN.35Further studies suggest that PAMPs or alarmins, notably high mobility group box 1 (HMB1), released from degener-ated and necrotic muscle cells, activate myoblasts through the TLR3 pathway.8,35 Activation of TLR3 on myoblasts leads to release of IL6 and CCL20.7 This is further enhanced in the presence of IL17. IL6 and CCL20 contribute to recruitment and differentiation of DCs and T cells, particularly TH17 cells. The

chemotac-tic effect of muscle autoantigens on DCs and lympho-cytes have been mentioned earlier. Moreover, TLR3 activation leads to expression of IFNb by the muscle cells.7 Contrary to the expectation from above-men-tioned activation of pDCs, IFNb, and not IFNa induci-ble transcripts, are upregulated in IIMS.30 _{It may}

indicate that the major source of type I IFN in IIMs may be immature muscle cells rather than plasmacytoid DCs. IFNb in turn activates nuclear mechanisms leading

to inflammation and also further up-regulates class I HLA expression on myocytes.7

GENETICS AND ENVIRONMENT

A genome-wide association study in DM has confirmed the importance of the MHC complex but also suggested non-MHC genetic features overlapped with other autoimmune diseases.36 Further studies support the link between HLA genotype and IIM serological or clini-cal phenotype. The 8.1 ancestral haplotype (HLA-A1-B8-Cw7-DRB1*0301-DQA1*0501-C4A*Q0), which is carried by most Caucasians with HLA-B8 is associated with various immune-mediated disorders, including IIM.37Alleles of the 8.1 ancestral haplotype are impor-tant risk markers for the development of IIM with dis-tinct allelic profiles associated with different myositis antibodies.38,39 _{In a study in the Han population in}

China, specific HLA class II alleles were found to be associated with DM and its pulmonary complications and dysphagia.40 An association between HLA-DRB1 alleles and anti-melanoma differentiation-associated gene 5 antibodies has been reported in a Japanese pop-ulation.41HLA-DR11 allele has been reported to confer risk for anti-HMGCR positive statin-induced myopa-thy.42In sporadic IBM, strongest association has been reported with HLA-DRB1*03:01/*01:01 genotype.43

There are evidences that environmental triggers play an important role in the pathogenesis of IIMs. A large proportion of T cells in the blood and muscles of IIMs bears CD28nullphenotype.18Circulating CD28nullwere significantly more common in cytomegalovirus (CMV) seropositive IIM patients, suggesting that viral infec-tions may be important triggers in IIMs.18 In a recent report, the causation of PM has been linked with recur-rent infection in bronchiectasis.44 The frequency of smoking was higher in anti-Jo-1 positive IIM patients compared to controls. In these smokers, the frequency of HLA-DRB1*03 was increased.45

It not only indicates an association between smoking and pathogenesis of IIMs, but also provides a testimony to interaction between environment and gene. The association between statins and IIMs has already been referred to.32,42Low serum vitamin D level is found to be asso-ciated with IIMs.46

SPECIAL CASE OF SPORADIC INCLUSION

BODY MYOSITIS

It appears from a recently reported study that the prevalence of sIBM has increased in recent decades.47

sIBM has certain distinctive features for which its pathogenesis merits a separate discussion. There are evidences of both degenerative and inflammatory pro-cesses in its pathogenesis. Evidences of adaptive immunity include presence of mDCs, CD4+ T cells, cytotoxic CD8+ T cells and plasma cells in muscle tis-sue,48,49 clonally restricted maturation of intramuscu-lar T and B cells,21 and a sIBM-specific autoantibody directed against cytosolic 50-nucleotidase 1A (cN1A).50 Evidences of degeneration include appear-ance of vacuoles and deposits of abnormal proteins (b-amyloid, tau and a-synuclein) in the cells and in filamentous inclusions. It has been postulated that in genetically predisposed individuals, effects of ageing (maybe acting through oxidative stress-related muta-tion in mitochondrial DNA)51 and environmental factors, including viral infection52 may reduce the sir-tuin 1 (SIRT1) activity.53_{It leads to increase in}

mem-brane-bound amyloid b precursor protein (AbPP).53 AbPP has three-fold effect: (i) it inhibits proteasome and thus leads to accumulation of misfolded pro-teins; (ii) it leads to increase in myostatin leading to muscle fiber atrophy; and (iii) abnormal processing of AbPP generates a cleavage product Ab.54 _Ab

mole-cules can aggregate to form flexible soluble oligomers which can induce other Ab molecules to also take the misfolded oligomeric form, leading to a chain reaction. These oligomer aggregates may precipitate as congophilic materials. Ab may cause damage to the cells by generating reactive oxygen species, lipid per-oxidation and so on. Misfolded Ab can induce tau to misfold. Misfolded proteins accumulated as a result of overproduction of AbPP and Ab as well as inhibi-tion of proteasome result in ER stress.54 As men-tioned earlier, ER stress may lead to activation of the NFjB pathway and enhanced synthesis of pro-inflam-matory cytokines, and to apoptosis through the cas-pase pathway.33 ER stress-related activation of NFjB may be one of the linkages between degenerative and inflammatory mechanisms found in sIBM. Myonu-clear breakdown is evidenced by lining of the rimmed vacuoles by nuclear membrane proteins like laminin A/C, 12 emerin and histone 120.55 Accumu-lation of nuclear nucleic acid binding protein trans-activation response DNA protein 43 in the extra nuclear sarcoplasm may represent myonuclear break-down or increased shuttling of nuclear proteins to sarcoplasm.56 _{Looked from the perspective of}

innu-merable sketchy and mutually contradictory research observations, the above scheme of pathogenesis of sIBM sounds an oversimplified one. There is much

controversy around most of the steps described in this scheme and the pathogenesis of this disease still remains a mystery.57

EMERGING THERAPEUTIC TARGETS

Cytokine inhibition through TNFa inhibitors broadly used with efficacy in RA and spondyloarthritis is still controversial in IIM.58,59 It should not be recom-mended in regard to the possibility of muscle disease exacerbation. IL1 blockade by anakinra, an IL1 recep-tor antagonist, has been used in 15 patients for 12 months with clinical response in six patients. IL1RA could act on TH1/TH17 balance, reducing TH17

pathway activation.60 No improvement with anakinra was noted in four patients with sIBM.61The therapeu-tic effect of intravenous immunoglobulins may be mediated by the TH17 lymphocyte pathway.3 In

addi-tion, development of monoclonal antibodies targeting IL12/IL23 (ustekinumab) or IL17 pathway (secuk-inumab and brodalumab) tested in RA, spondy-loarthritis and psoriasis, may be beneficial in IIM as well. Tocilizumab is a monoclonal antibody that acts as an IL6 receptor antagonist developed to treat RA. In addition to inhibiting IL-6 activity, it blocks its autocrine growth activity on B cells. Its efficacy for refractory PM was reported in two patients.62 Treat-ment with tocilizumab requires further investigations in IIM and a randomized controlled study is ongoing (Clinical Trials NCT02043548).

Rituximab (RTX), an CD20 (B cell) anti-body, is found to be effective in IIMs.63–65The national French registry, the Auto-immunity and Rituximab (AIR) registry, which contains data from patients with autoimmune diseases treated with RTX since 2005, ana-lyzed the efficacy and tolerance of RTX in 30 patients with refractory IIMs. RTX was well tolerated, effective in 16 patients, time of improvement after the first infusion was 3.2 months (2–5 months).64

Unfortunately, RTX has not shown efficacy in the only randomized pla-cebo-phase trial performed including 200 patients.63 Seventy-six PM, 76 DM and 48 JDM refractory to ster-oids and immunosuppressive therapy, were random-ized to receive either rituximab at the beginning of the study or rituximab 8 weeks later. Although there were no significant differences between the arms arms at the endpoint (week 8), 83% of patients improved at the end of the follow-up (week 44). While the trial itself was negative, the overall response rate in a group of patients with refractory myositis suggest that RTX was effective but the study design made identification of

such an effect difficult. The long time to improvement after RTX and the assessment of the response after only 8 weeks of placebo, made it difficult to distinguish the response in the two treatment arms. Presence of antisynthetase and anti-Mi-2 autoantibodies, juvenile DM subset, and lower disease damage were predictors of good response, indicating a subset of patients in whom B cells might play a more pivotal role.66

Targeting T cells might be an effective option for the treatment of refractory myositis. Alemtuzumab, a mon-oclonal antibody (MAb) targeting CD52, a protein pre-sent on CD28nullT cells, is reported to be effective in IBM67 and polymyositis.68 T cell costimulation block-ade with abatacept has been discussed in one case report.69

Sifalimumab, an anti-IFN-a monoclonal antibody, suppressed the type I IFN gene signature and appeared to be promising in a phase 1b clinical trial in IIMs.70

Fifty-one patients were included in a randomized, dou-ble-blind placebo-controlled study. Neutralization of the type I IFN gene signature correlated with improve-ment in muscle testing.

Inhibition of the ubiquitin proteasome pathway with bortezomib improved muscle function in a mouse model of myositis.34

From the pathogenesis that has been discussed above, targeting BAFF (BlyS), IL15, IL18 or miRNA offers potential therapeutic alternatives. The recently approved belimumab, a monoclonal antibody, effectively targets the soluble form of BlyS and has shown efficacy in sys-temic lupus erythematosus. One trial with belimumab started in January 2015 in IIMs (ClinicalTrials.gov: NCT02347891). Other trials with experimental treat-ments are ongoing, such as inhibition of complement activity by humanized anti-C5 antibody in DM, (Clini-calTrials.gov: NCT00005571), siponimod as elective sphingosine 1-phosphate (S1P)-1 and -5 receptor that regulates lymphocyte migration in PM and DM (Clini-calTrials.gov: NCT01148810), bimagrumab that modu-lates myostatin and activin signaling (ClinicalTrials.gov: NCT01925209) or arimoclomol that could enhance heat shock protein expression with neuroprotective effects in IBM (ClinicalTrials.gov: NCT00769860).

CONCLUSIONS

An interaction between dendritic cell-lymphocyte path-way and regenerating myocytes may be responsible for the pathogenesis of the IIMs. An appropriate genetic predisposition and a chance encounter with some environmental trigger also appear to provide the

background. Currently identified molecular targets are ushering an advent of more specific therapeutic options. While no biologic agent is currently consis-tently effective in IIMs, it appears that we are heading toward discovering mechanisms responsible for hom-ing of autoimmunity specifically to the muscles. How-ever, a highly specific, effective and individualized therapy remains beyond our dreams.

ACKNOWLEDGMENTS

The authors wish to acknowledge Syed Jamil Abdal and Tanveer Hasan for their help in preparation of the manuscript.

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