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Pathogenesis of myositis: Lessons learned from animal studies
i n d i a n j o u r n a l o f r h e u m a t o l o g y 8 ( 2 0 1 3 ) 1 7 0 e1 7 8
Available online at www.indianjrheumatol.com and www.sciencedirect.com
Review Article
Pathogenesis of myositis: Lessons learned from animal studies Travis B. Kinder a,c, Sree Rayavarapu a, Kathryn White a,c, Kanneboyina Nagaraju a,b,c,* a
Research Center for Genetic Medicine, Children’s National Medical Center, 111 Michigan Avenue NW, Washington DC, USA; b Department of Integrative Systems Biology; c Institute for Biomedical Sciences, The George Washington University, 2300 Eye Street NW, Ross Hall 605, Washington DC, USA
abstract Keywords:
Recent studies have continued to clarify the pathogenic mechanisms responsible for
Myositis
muscle damage and weakness in inflammatory myopathies. Traditionally, adaptive im-
Inflammation
mune mechanisms such as cell mediated (cytotoxic) and humoral (autoantibodies and
Animal models
complement) components have been implicated in the pathogenesis of polymyositis/in-
Autoimmunity
clusion body myositis and dermatomyositis, respectively. However, recent studies have
Innate immune system
shown a significant overlap of immune components in these disorders. Likewise, studies have provided evidence not only for adaptive immune pathogenic mechanisms but also for innate immune, such as the TLR-NF-kB signaling, and non-immune mechanisms, such as endoplasmic reticulum stress response, autophagy, metabolic deficits in ATP generating pathways and hypoxia. These recent studies indicate that the muscle fiber damage and weakness in myositis may not be solely mediated by an adaptive immune attack (e.g., autoreactive CTLs or autoantibodies) but also mediated through innate immune and metabolic mechanisms. In this review, we have briefly outlined the current developments in immune (adaptive, innate) and non-immune components of disease pathogenesis in inflammatory myopathies. Copyright ª 2013, Indian Rheumatology Association. All rights reserved.
1.
Introduction
Idiopathic inflammatory muscle diseases, such as polymyositis (PM), dermatomyositis (DM), and inclusion body myositis (IBM), are generally considered as autoimmune diseases because many patients have autoantibodies and autoreactive T cells and often respond to immunosuppressive therapies. Traditionally, PM and IBM have been considered T cell
mediated and DM has been considered as humoral (antibody and complement) mediated diseases. These definitions are beginning to change. For example, B cells, plasma cells, and autoantibodies have recently been implicated in IBM.1e3 Similarly, recent studies have provided evidence that changes in innate immune and metabolic mechanisms are responsible for muscle inflammation, damage and weakness. Delineating pathogenic mechanism in humans is complicated
* Corresponding author. Professor of Integrative Systems Biology and Pediatrics, The George Washington University, Children’s National Medical Center, 111 Michigan Avenue NW, Washington DC 20010, USA. Tel.: þ1 202 476 6220; fax: þ1 202 476 6014. E-mail addresses: [email protected] (T.B. Kinder), [email protected] (S. Rayavarapu), kwhite@child rensnational.org (K. White), [email protected] (K. Nagaraju). 0973-3698/$ e see front matter Copyright ª 2013, Indian Rheumatology Association. All rights reserved. http://dx.doi.org/10.1016/j.injr.2013.09.006
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by the heterogeneity, complexity, and chronicity of the disease process. Therefore, several animal models were generated to delineate mechanisms of muscle fiber damage and dysfunction in myositis. Recent studies using human muscle biopsy samples and animal models provide strong evidence not only for adaptive immune pathogenic mechanisms but also for innate immune, such as the TLR-NF-kB signaling, and non-immune mechanisms, such as endoplasmic reticulum stress response, autophagy, metabolic deficits in ATP generating pathways and hypoxia.4e6 We will briefly describe these new developments in the following sections.
2.
Immune mechanisms of muscle damage
2.1.
Innate immune mechanisms
Although myositis has long been considered an autoimmune disease, increasing evidence shows that it also has features of autoinflammatory diseases, which are primarily mediated by the innate immune system.7,8 Muscle biopsies of myositis patients show the hallmarks of innate immune system activation such as the presence of macrophage and dendritic cell infiltrates, the over-expression of Toll-like receptors (TLRs), NF-kB activation, and the abundance of innate immune cytokines, including type 1 interferons (IFNs).9e13 Recent studies using mouse models of myositis further demonstrated that the innate immune system plays a crucial role in mediating skeletal muscle damage in these diseases. For example, presence of muscle disease in the absence of mature T and B cells in the MHC class I transgenic murine models of myositis suggested that non-immune mechanisms, particularly endoplasmic reticulum stress, contribute to muscle disease in the absence of lymphocytes in this model.14 Likewise, Soejima et al demonstrated that intramuscular immunization with histidyl-transfer RNA synthetase (Jo-1) drives muscle inflammation in the absence of T and B cells.5 Thus, studies in mouse models have uncovered the role for innate immune mechanisms in myositis muscle.
2.2.
Innate immune cytokines
Cytokines play a critical role in initiating and perpetuating an inflammatory response to tissue injury. Interestingly, lymphocyte derived cytokines (e.g., IL-2, -4, - 6, -10) were detected sporadically in myositis muscle biopsies, and their presence did not correlate with disease activity or treatment status of the patients, but the data indicated a role for beta chemokines (e.g., MIP-1a, MIP-1b, RANTES) in myositis.15 Several recent studies have highlighted the presence of other innate immune cytokines such as IL-1b, TNF-a, TGF-b, or type 1 IFNs in myositis muscle16e19 (Fig. 1). The sources of these cytokines and chemokines in the muscle microenvironment are not extensively investigated. Our group has previously shown that human skeletal muscle cells are capable of producing a variety of pro-inflammatory cytokines and chemokines.20 More recently, others and we have explored the mechanisms by which the pro-inflammatory cytokine synthesis and release occurs in skeletal muscle cells. We have shown that engaging TLR4 and P2X7 receptor
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with their receptive ligands, LPS and ATP, induce IL-1b secretion in primary mouse muscle cells.21 Other investigators have also reported that muscle precursor cells in the presence of IFNg and TLR3 agonist produce type 1 IFN22 suggesting that not only inflammatory cells but also skeletal muscle is an active contributor of pro-inflammatory cytokines in the muscle microenvironment. Potential endogenous TLR ligands in inflammatory myopathies are currently unknown, but emerging evidence indicates autoantigens like histidyltRNA synthetase might stimulate TLRs and initiate proinflammatory cytokine production.23 It is known that BDCA2þ or CD123þ plasmacytoid dendritic cells (pDCs) produce IFNa suggesting that both skeletal muscle and pDCs contribute to the type 1 IFN secretion.24,25 It appears that the type 1 interferon signature in myositis patients is associated with disease activity, especially for DM patients.26,27 Apart from IFNs, other studies have also demonstrated presence of TNF-a in macrophages and muscle fibers of myositis patient biopsies.28e30 The consequences of excess cytokine presence in the muscle are not explored in details. Experiments performed on mouse skeletal muscle demonstrated that TNF-a compromises contractile function of skeletal muscle and decreases force by blunting the response of muscle myofilaments to calcium activation.31 A recent study by Keller et al further demonstrated that IFNg and TNF-a induced autophagy and increased surface expression of MHC class II in human myoblasts32 suggesting that innate immune cytokines affect skeletal muscle weakness and cell death. One of the most interesting developments in myositis research is the finding that known autoantigens, such as aminoacyl-tRNA synthetases, are proteolytically cleaved and attain cytokine and chemokine properties. For example, human tyrosyl-tRNA synthetase can be cleaved by leukocyte elastase and split into two distinct cytokines; one half activates leukocyte and monocyte chemotaxis and cytokine production (e.g., TNF-a) and the other half binds to IL-8R and stimulates IL-8 signaling.33 Subsequent experiments by Howard et al showed a major autoantigen, histidyl-tRNA synthetase activates the CC chemokine receptor CCR-5 and activates lymphocytes, monocytes, and immature dendritic cells. Thus, release of histidyl-tRNA synthetase from muscle links the cell damage and injury to the inflammatory response in myositis.34 These studies clearly illustrate that autoantigens participate in initiating innate and adaptive immunity as well as inflammatory responses in myositis.
2.3.
Toll-like receptors (TLRs) and NF-kB activation
Proximal events that drive the innate cytokine production in skeletal muscle are not well explored. It is clear that damaged skeletal muscle is likely to release damage-associated molecular patterns (DAMPS) that engage TLRs and trigger inflammation in skeletal muscle.21 There are many reports demonstrating expression of TLR2, TLR3, TLR4, TLR7, and TLR9 in biopsies from patients with IBM, DM, or PM.12,35e38 These receptors are localized mainly on infiltrating inflammatory cells, but some TLRs (TLR3 and TLR7) are also expressed on muscle fibers.35,37 Experiments with cultured muscle cells have shown that stimulation of TLR3 with the agonists Poly (I:C) or necrotic myoblasts elicited the
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Fig. 1 e Mechanisms of muscle damage and myofiber death in myositis. Infiltrating immune cells within the skeletal muscle constitute macrophages, dendritic cells (myeloid and plasmacytoid), T cells (Th1, Th2 and Th17, Tregs, CD28null, cytotoxic T cells (CTL)), B cells, and plasma cells. Tregs influence both effectors as well as T helper cells. Cytotoxic T cells may engage MHC class I and may directly contribute to the muscle injury but the extent of CTL mediated injury to muscle fiber death is currently unknown. Infiltrating immune cells as well as skeletal muscle cells actively secrete a variety of pro-inflammatory cytokines and chemokines. These cytokines and chemokines engage respective receptors on skeletal muscle and induce NF-kB activation, ER stress response, autophagy, inflammasome activation and might also presumably cause deficits in energy generating metabolic pathways. These non-immune mechanisms either alone or in combination contribute to muscle weakness and eventually to muscle fiber death. Skin lesions and loss of capillaries in dermatomyositis are attributable to immune complex mediated damage to capillaries. Loss of capillaries contributes to tissue hypoxia and muscle weakness in dermatomyositis.
production of IFNb22 or the pro-inflammatory cytokines IL-835 or IL-6.37 More importantly, endogenous TLR ligands such as high mobility group box-1 (HMGB-1) and histidyl-tRNA synthetase are identified as DAMPS that stimulate inflammatory response and pathology in skeletal muscle.23,38,39 Stimulation of TLRs or the IL-1b or TNF-a receptors causes the activation of the pro-inflammatory transcription factor NF-kB, which is known to affect inflammation and myogenesis in myositis.40 In skeletal muscle, NF-kB activation impairs myoblast differentiation and causes the secretion of the pro-inflammatory cytokine TNF-a.41 Several groups have observed activated NF-kB within the inflammatory infiltrates, regenerating fibers, and some necrotic fibers in myositis muscle13,42 (Fig. 1). In addition, several downstream NF-kB targets are up-regulated in myositis muscle, including iNOS and TNF-a.28,43 Activation of NF-kB produces proinflammatory cytokines that cause muscle damage and weakness in existing muscle fibers and actively inhibit the
generation of new muscle fibers by blocking the transcription factor MyoD, which is essential for muscle differentiation.44 These studies indicate that innate immune components like cytokines and endogenous TLR agonists (DAMPs) directly act on skeletal muscle and activate downstream proinflammatory pathways that contribute to muscle weakness and damage in myositis.
2.4.
Macrophages
These are the prototypical phagocytic cells, which display remarkable plasticity dependent on the local environment. These cells detect and eliminate invading pathogens, sense tissue damage, clear debris, and dampen the immune response post-inflammation.45 Although few studies have been conducted on macrophages in myositis muscle, they are abundant among the inflammatory infiltrates (Fig. 1). In PM, endomysial macrophages have been observed with markers
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associated with early-activation (MRP14 and 27E10). These markers are displayed on macrophages that have recently migrated into inflamed tissue in the acute phase of injury. Whereas in DM, perimysial macrophages with markers associated with late-activation (25F9) predominate.11 Macrophages displaying the late activation marker 25F9 were previously considered resident, resting cells. However, IHC of patient biopsies has shown that subsets of late-activated macrophages express iNOS, and another subset express TGF-b contributing cell-mediated damage and fibrosis in muscle tissue.46 Even though the relative contribution of macrophages to muscle damage are unknown, the plasticity of macrophage phenotype may lend itself to therapeutic intervention once we have full understanding of their contribution to chronic inflammation and injury to muscle.
2.5.
Adaptive immune mechanisms
Antigen processing and presentation by the professional antigen presenting cells (APCs) is a critical component of adaptive immune response initiation. Dendritic cells are the sentinels of the immune system that coordinate innate and adaptive immunity by acting as professional APCs and by secreting cytokines. Several lineages exist, including myeloid and pDCs, and their phenotypes are dynamic in response to the local microenvironment.47 Both myeloid and pDCs have been observed in the biopsies of patients with PM, DM, or IBM48 (Fig. 1). Although a few immature DCs have been observed in myositis muscle,49 the majority are positive for markers of mature DCs.50,51 Further studies are required to determine whether the DCs mature within the muscle or in the lymph node, but the presence of DCs near the capillaries in DM muscle suggest endothelial cell contact induced local DC maturation.50,51 Recently, pDCs have been implicated in myositis pathology, as they are abundant in the muscle of DM patients.24 Maddur et al found that myeloid DCs cultured with type 1 interferon produce type 1 interferon-induced pro-inflammatory cytokines such as IL-8, IL-6, TNF-a, and IL-1b.52 To fully understand the pathogenesis of myositis we must use animal models to further investigate the function of these observed DCs, especially their potential role in priming autoreactive T and B cells and their contribution to the inflammatory state via innate immune cytokine production. Myositis researchers have contemplated for quite some time about the antigen processing and presentation capabilities of skeletal muscle based on the presence of MHC class I and MHC II antigens (signal 1) on muscle. However, activation of a naı¨ve T cell requires co-stimulatory molecules (signal 2) on skeletal muscle. Despite initial reports of the presence of B7.1 and B7.2 co-stimulatory molecules on human skeletal muscle cells, these findings have not been independently verified and validated.53 MHC class I expression on skeletal muscle fibers is characteristic feature of myositis.54,55 Therefore, it likely that muscle cells expressing MHC class I (signal one) could stimulate memory (signal 2 is not required) but not naı¨ve T cells. Alternatively, it is possible that naı¨ve T cell stimulation can still occur if signal 1 comes from skeletal muscle and signal 2 from infiltrating APCs such as DCs. However, there is no experimental evidence in support of this possibility. The presence of memory cells in myositis biopsies
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supports the hypothesis that muscle is likely to activate memory but not naı¨ve T cells.56
2.6.
B and T lymphocytes
Early studies have demonstrated that infiltrating mononuclear cells in myositis include T cells and B cells. There is ample evidence for the presence of invasive, perforinexpressing cytotoxic CD8þ effector T (a/b) lymphocytes in PM and IBM.57,58 The invasion of non-necrotic muscle fibers by these cytotoxic T lymphocytes is considered a characteristic histological feature of polymyositis. Apart from a/b T cells, there are other reports that demonstrate presence of clonal T cells with g/d T cell receptors.59,60 Recent studies show that these g/d T cells recognize aminoacyl-tRNA synthetases and suggest a link between g/d T cells and antibody responses in autoimmune myositis.61 Case studies indicate that patients with the presence of gamma/delta T cells in muscle tissues appear to be responsive to steroids.59,62 Recent studies have shown some unique subsets of T cells in the muscle microenvironment of myositis patients. One of the subsets is CD28null T Cells, including both CD4þ CD28null and CD8þ CD28null T cells. These apoptosis resistant, proinflammatory cells appear to be present in the muscle at the time of diagnosis as a predominant infiltrating cell type in PM and DM biopsies. Functional studies performed with these effector cells indicate that they are potentially cytotoxic to muscle cells in vitro and in vivo.63,64 T regulatory cells (FOXP3þ cells) are a subset of T cells that actively suppress immune activation by secreting cytokines such as IL-10 and TGF-b. These cells are found in close proximity to effector cells and the numbers correlated with the degree of inflammation within muscle65 (Fig. 1). Some studies have suggested that reduction in regulatory cells and their secreted cytokines contribute to skin lesions in DM patients.66 The proportion of B cells in the perivascular regions of DM is more than in PM.67 So, many suggest a role for humorallymediated immune damage in DM.68 However, subsequent studies showed presence of B cells, plasma cells, and immunoglobulin transcripts in PM and IBM, indicating a role for a humoral component in these disorders also.1,2,69 Further investigations on immunoglobulin transcript sequence analysis demonstrated spatially distributed, clonally-related B cells and plasma cells, suggesting local maturation of B cells into plasma cells in myositis muscle.70,71 The pathological roles of autoantibodies in myositis are unclear; however, there is growing evidence that some of the myositis-specific autoantibodies are associated with distinct clinical phenotypes. For example, anti-synthetase syndrome has a spectrum of phenotypes that includes interstitial lung disease, arthritis, Raynaud’s and mechanic’s hands; anti-Mi2 antibodies are associated with dermatomyositis; anti-P140 antibodies are associated with calcinosis and contractures in juvenile DM. Some autoantibodies, such as anti-SRP, are associated with severe refractory myositis, and anti-MDA5 antibodies are associated with progressive lung disease in some populations but not in others.72 Cause and effect relationship studies between autoantibodies and disease phenotype and activity are few. It is therefore difficult to conclude on the mechanistic basis for the above relationships, but
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existence of such a relationship is immensely useful for the diagnosis and prognosis of this group disorders. Recent studies demonstrated that not only autoantibodies but also B cell activating factor (BAFF), which is known to play a role in type 1 interferon induction and autoantibody production, is strongly associated with disease activity measures in myositis.73
findings from this study suggest that chronic activation of ER stress might induce myofiber degeneration via proteasome dependent mechanisms. Initial studies in mouse models indicate that targeting ubiquitin proteasome mediated muscle degeneration confers benefit on disease phenotype in MHC class I transgenic mouse model of myositis.81
3.2.
3. Non-immune mechanisms of muscle damage Some myositis patients are refractory to potent immunosuppressants and continue to show clinical disease symptoms even after elimination of immune cells from the affected muscle.74,75 These observations suggest a potential role for non-immune mechanisms in myositis pathology. Recent studies using animal models provided several lines of evidence in this regard, including activation of endoplasmic reticulum (ER) stress response, implication of metabolic enzymes, autophagy and hypoxia in myositis muscle (Fig. 1).
3.1.
Endoplasmic reticulum stress
Disturbances in ER homeostasis such as enhanced protein synthesis or accumulation of mis-folded proteins results in the activation of ER stress response pathways. In general, two adaptive mechanisms, the unfolded protein response (UPR) and the ER overload response (EOR) are activated to overcome the stress. Induction of UPR attenuates the translation machinery to reduce the protein synthesis load on the ER. On the other hand, EOR activates NF-kB pathway and the downstream pro-inflammatory processes. In certain extreme conditions, the affected cell initiates the cell death mechanisms by activation of cholesterol oxidase-peroxidase C/EBP homologous protein (CHOP), Bcl-2 family proteins and ERassociated caspases. With respect to myositis, several studies have reported enhanced expression of classical ER stress markers (such as GRP78, GRP94, and calreticulin) in the affected skeletal muscle both in mouse models and human biopsy samples.40,76e78 One recent report also indicated the enhanced expression of heat shock protein (Hsp) families 70 and 90 in myositis muscle.79 One possibility for the induction of ER stress in myositis muscle is the over-expression of MHC class I in the affected muscle fiber. Studies using a transgenic mouse model of myositis reported that muscle specific overexpression of MHC class I and its potential accumulation in the ER induces the stress response.40,80 Although these findings hint at a non-immune role for MHC class I in myositis pathology, it is not clear whether the induction of ER stress is attributable to its forced expression or its immune responses per se. A recent study performed using mature lymphocyte deficient Rag2/ mice showed that MHC class I can induce UPR independent of its immune functions in the myositis muscle suggesting that mere over-expression of MHC class I in muscle might contribute to ER stress and muscle damage.14 More recently, our group established initial evidence suggesting the activation of ER associated degradation mechanisms (ubiquitin proteasome pathway) in myositis muscle using a novel in vivo SILAC proteomics approach.81 Our
Deficit in energy pathways
Metabolic pathways such as mitochondria-associated oxidative metabolism play a critical role in skeletal muscle homeostasis. Emerging evidence indicates a probable disruption in mitochondria and associated metabolic pathways in the chronic stages of inflammatory muscle diseases ultimately leading to muscle weakness.82,83 A recent study suggested an impaired muscle oxidative competence and abnormal blood lactate levels in PM/DM patients.84 Other studies indicate abnormal succinic dehydrogenase and cytochrome c oxidase activities in damaged and atrophic fibers in myositis muscle85,86 suggesting a deficit in energy generating pathways. More recently, our group demonstrated a significant reduction in the levels of adenosine monophosphate deaminase 1 (AMPD1), a rate-limiting enzyme in the purine nucleotide cycle.6 Notably, the reduction of the AMPD1 enzyme levels and the reduction of muscle strength occur prior to the appearance of infiltrating lymphocytes, which indicates that metabolic deficiencies seen in myositis are independent of the action of immune cells. It is possible that reduced AMPD1 might limit the levels of downstream substrates that are needed for producing energy in the affected muscle. At this time, the precise role of dysregulated metabolic pathways in myositis pathology is not completely clear. However, it is possible that pro-inflammatory cytokines and disturbed immune mechanisms might regulate these energy metabolic pathways in myositis muscle.
3.3.
Autophagy
The autophagic process includes degradation of cell’s own proteins or organelles with the aid of lysosomes. This type of cell death is particularly important for skeletal muscle because classical apoptosis is not present in myositis.87 Muscle biopsies from PM and sIBM patients often showed evidence of activation of the autophagic process. Our group demonstrated that TNF-related apoptosis-inducing ligand (TRAIL) and markers of autophagy are up-regulated in myositis muscle fibers.88 On the similar lines, a recent study reported over-expression of a panel of autophagic proteins (clathrin, ATG5, LC3a, LC3b and Beclin 1) in sIBM patient biopsies.89 Moreover, these authors suggested an association between the increased expression of autophagic markers and the reduced clearance of mis-folded proteins. These findings suggest a potential relation between ER stress and autophagy in myositis. Another study reported that human b defensin 3 was overexpressed in sIBM affected muscle tissue and localized near markers of autophagy.90 Even though the role of autophagy in myofiber damage is emerging from these studies, it is important to know how and when these autophagic mechanisms are triggered in the affected muscle in
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order to investigate their validity as therapeutic targets in myositis.
3.4.
Hypoxia
Reduced levels of oxygen and oxidative stress are also implicated in causing muscle fiber damage in inflammatory myositis. Initial evidence indicates a loss of capillaries and a compensatory up-regulation of proteins such as hypoxia inducible factor-1, avb3 integrin, and Flt-1 in the affected muscle tissue.91 Another study reported higher expression of vascular endothelial growth factor, a reduction in the proportion of oxidative, slow twitch fibers suggesting a shift in fiber type composition in the muscle tissue of myositis patients.6,92,93 This shift might be one possible reason for low muscle endurance observed in chronic PM or DM patients. The hypoxia theory causing muscle weakness in myositis is further supported by the beneficial effects of intensive resistance exercise training in myositis muscle.94,95 One recent study reported that 12 weeks of endurance training resulted in clinical improvement in PM and DM patients and this improvement correlated with enhanced aerobic capacity and increased mitochondrial enzyme activities in the myositis muscle.96 These observations highlight the importance of resistance exercise regimens as a treatment modality in addition to immunosuppressive drugs in inflammatory myopathies.
4.
Experimental therapies for myositis
The current gold-standard treatment for patients with IIMs is high-dose glucocorticoids (GCs) often combined with immunosuppressive agents, including azathioprine and methotrexate. However, many patients do not respond to this therapy, and they are accompanied by many side effects. Biologics such as anti-TNF therapies revolutionized treatments for autoimmune diseases like rheumatoid arthritis. Anti-TNF therapies are largely ineffective in myositis. For example, a hand full of patients have been reported to show clinical benefit from the anti-TNF-a monoclonal antibody infliximab, but others have found radiological, clinical, and immunological worsening of myositis.97e99 A randomized, double-blind, placebo-controlled trial of the TNF-a antagonist etanercept showed good tolerability and a steroid-sparing effect in patients with DM.100 Anecdotal evidence exists for the efficacy of the third, most recently FDA approved TNFa antagonist adalimumab for the treatment of DM-associated ILD.101 A mechanistic study of IL-1 blockade by anakinra showed that some patients may respond to the treatment, which seemed to shift the immunological phenotype away from a Th17 response.102 A randomized, double-blind, placebo-phase trial of a B celldepleting antibody rituximab showed that 83% of refractory PM and DM patients attained the definition of improvement.103 Taken together, these studies indicate that therapeutic response to existing biologics is highly variable and highlight the need for developing more targeted therapies. In this regard, animal models play a significant role in identifying therapeutic targets and testing experimental therapies. For example, A C-protein-induced mouse model of myositis
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demonstrated benefit with antagonists of IL-1, TNF-a, and IL6, thereby providing rationale for future clinical studies with such cytokine antagonists.104,105 Treatment of a transgenic mouse model of myositis with the proteasome inhibitor Bortezomib showed a potential therapeutic effect.81 Because most patients with IBM fail to respond to treatment, trials have been undertaken to test therapies specifically for this subtype of IIM. A proof-of principle study in sIBM patients showed that Alemtuzumab, which depletes peripheral blood lymphocytes, slowed down disease progression, improved strength in some, and reduced inflammation and stressor molecules.106 Recently, the FDA granted a break through status for a monoclonal antibody that binds to type II activin receptors and block binding of natural ligands activin and myostatin and improves muscle growth. Therapeutic studies focusing on muscle instead of immune cells are highly encouraging and will pave the way for treating various inflammatory muscle diseases.
Conflicts of interest All authors have none to declare.
Acknowledgments Dr. Nagaraju is supported by NIH (RO1-AR050478; 5U54HD053177; K26OD011171), Muscular Dystrophy Association, and US Department of Defense (W81XWH-05-1-0616). Travis Kinder is supported by NIH (F31-AR065362-01) for his doctoral studies.
references
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8. Baechler EC, Bauer JW, Slattery CA, et al. An interferon signature in the peripheral blood of dermatomyositis patients is associated with disease activity. Mol Med. 2007;13:59e68. 9. Niewold TB, Kariuki SN, Morgan GA, Shrestha S, Pachman LM. Elevated serum interferon-alpha activity in juvenile dermatomyositis: associations with disease activity at diagnosis and after thirty-six months of therapy. Arthritis Rheum. 2009;60:1815e1824. 10. Baechler EC, Bilgic H, Reed AM. Type I interferon pathway in adult and juvenile dermatomyositis. Arthritis Res Ther. 2011;13:249. 11. Rostasy KM, Piepkorn M, Goebel HH, Menck S, Hanefeld F, Schulz-Schaeffer WJ. Monocyte/macrophage differentiation in dermatomyositis and polymyositis. Muscle Nerve. 2004;30:225e230. 12. Brunn A, Zornbach K, Hans VH, Haupt WF, Deckert M. Tolllike receptors promote inflammation in idiopathic inflammatory myopathies. J Neuropathol Exp Neurol. 2012;71:855e867. 13. Creus KK, De Paepe B, Werbrouck BF, Vervaet V, Weis J, De Bleecker JL. Distribution of the NF-kappaB complex in the inflammatory exudates characterizing the idiopathic inflammatory myopathies. Ann N Y Acad Sci. 2009;1173:370e377. 14. Freret M, Drouot L, Obry A, et al. Overexpression of MHC class I in muscle of lymphocyte-deficient mice causes a severe myopathy with induction of the unfolded protein response. Am J Pathol. 2013;183:893e904. 15. Adams EM, Kirkley J, Eidelman G, Dohlman J, Plotz PH. The predominance of beta (CC) chemokine transcripts in idiopathic inflammatory muscle diseases. Proc Assoc Am Physicians. 1997;109:275e285. 16. Authier FJ, Mhiri C, Chazaud B, et al. Interleukin-1 expression in inflammatory myopathies: evidence of marked immunoreactivity in sarcoid granulomas and muscle fibres showing ischaemic and regenerative changes. Neuropathol Appl Neurobiol. 1997;23:132e140. 17. Lundberg I, Ulfgren AK, Nyberg P, Andersson U, Klareskog L. Cytokine production in muscle tissue of patients with idiopathic inflammatory myopathies. Arthritis Rheum. 1997;40:865e874. 18. Bilgic H, Ytterberg SR, Amin S, et al. Interleukin-6 and type I interferon-regulated genes and chemokines mark disease activity in dermatomyositis. Arthritis Rheum. 2009;60:3436e3446. 19. Liao AP, Salajegheh M, Nazareno R, Kagan JC, Jubin RG, Greenberg SA. Interferon beta is associated with type 1 interferon-inducible gene expression in dermatomyositis. Ann Rheum Dis. 2011;70:831e836. 20. Nagaraju K, Raben N, Merritt G, Loeffler L, Kirk K, Plotz P. A variety of cytokines and immunologically relevant surface molecules are expressed by normal human skeletal muscle cells under proinflammatory stimuli. Clin Exp Immunol. 1998;113:407e414. 21. Rawat R, Cohen TV, Ampong B, et al. Inflammasome upregulation and activation in dysferlin-deficient skeletal muscle. Am J Pathol. 2010;176:2891e2900. 22. Tournadre A, Lenief V, Eljaafari A, Miossec P. Immature muscle precursors are a source of interferon-beta in myositis: role of Toll-like receptor 3 activation and contribution to HLA class I up-regulation. Arthritis Rheum. 2012;64:533e541. 23. Fernandez I, Harlow L, Zang Y, et al. Functional redundancy of MyD88-dependent signaling pathways in a murine model of histidyl-transfer RNA synthetase-induced myositis. J Immunol. 2013;191:1865e1872. 24. Greenberg SA, Pinkus JL, Pinkus GS, et al. Interferon-alpha/ beta-mediated innate immune mechanisms in dermatomyositis. Ann Neurol. 2005;57:664e678.
25. Eloranta ML, Barbasso Helmers S, Ulfgren AK, Ronnblom L, Alm GV, Lundberg IE. A possible mechanism for endogenous activation of the type I interferon system in myositis patients with anti-Jo-1 or anti-Ro 52/anti-Ro 60 autoantibodies. Arthritis Rheum. 2007;56:3112e3124. 26. Nasr R, Reed AM, Peterson EJ. Update: biomarkers for idiopathic inflammatory myopathies. Curr Opin Rheumatol. 2012;24:609e615. 27. Zhu W, Streicher K, Shen N, et al. Genomic signatures characterize leukocyte infiltration in myositis muscles. BMC Med Genomics. 2012;5:53. 28. Kuru S, Inukai A, Liang Y, Doyu M, Takano A, Sobue G. Tumor necrosis factor-alpha expression in muscles of polymyositis and dermatomyositis. Acta Neuropathol. 2000;99:585e588. 29. Kuru S, Inukai A, Kato T, Liang Y, Kimura S, Sobue G. Expression of tumor necrosis factor-alpha in regenerating muscle fibers in inflammatory and non-inflammatory myopathies. Acta Neuropathol. 2003;105:217e224. 30. De Bleecker JL, Meire VI, Declercq W, Van Aken EH. Immunolocalization of tumor necrosis factor-alpha and its receptors in inflammatory myopathies. Neuromuscular Disord NMD. 1999;9:239e246. 31. Reid MB, Lannergren J, Westerblad H. Respiratory and limb muscle weakness induced by tumor necrosis factor-alpha: involvement of muscle myofilaments. Am J Respir Crit Care Med. 2002;166:479e484. 32. Keller CW, Fokken C, Turville SG, et al. TNF-alpha induces macroautophagy and regulates MHC class II expression in human skeletal muscle cells. J Biol Chem. 2011;286:3970e3980. 33. Wakasugi K, Schimmel P. Two distinct cytokines released from a human aminoacyl-tRNA synthetase. Science. 1999;284:147e151. 34. Howard OM, Dong HF, Yang D, et al. Histidyl-tRNA synthetase and asparaginyl-tRNA synthetase, autoantigens in myositis, activate chemokine receptors on T lymphocytes and immature dendritic cells. J Exp Med. 2002;196:781e791. 35. Schreiner B, Voss J, Wischhusen J, et al. Expression of tolllike receptors by human muscle cells in vitro and in vivo: TLR3 is highly expressed in inflammatory and HIV myopathies, mediates IL-8 release and up-regulation of NKG2D-ligands. Official Publication of the Federation of American Societies for Experimental Biology. FASEB J. 2006;20:118e120. 36. Kim GT, Cho ML, Park YE, et al. Expression of TLR2, TLR4, and TLR9 in dermatomyositis and polymyositis. Clin Rheumatol. 2010;29:273e279. 37. Tournadre A, Lenief V, Miossec P. Expression of Toll-like receptor 3 and Toll-like receptor 7 in muscle is characteristic of inflammatory myopathy and is differentially regulated by Th1 and Th17 cytokines. Arthritis Rheum. 2010;62:2144e2151. 38. Zong M, Bruton JD, Grundtman C, et al. TLR4 as receptor for HMGB1 induced muscle dysfunction in myositis. Ann Rheum Dis. 2013;72:1390e1399. 39. Park JS, Gamboni-Robertson F, He Q, et al. High mobility group box 1 protein interacts with multiple Toll-like receptors. Am J Physiol Cell Physiol. 2006;290:C917eC924. 40. Nagaraju K, Casciola-Rosen L, Lundberg I, et al. Activation of the endoplasmic reticulum stress response in autoimmune myositis: potential role in muscle fiber damage and dysfunction. Arthritis Rheum. 2005;52:1824e1835. 41. Peterson JM, Bakkar N, Guttridge DC. NF-kappaB signaling in skeletal muscle health and disease. Curr Top Dev Biol. 2011;96:85e119. 42. Monici MC, Aguennouz M, Mazzeo A, Messina C, Vita G. Activation of nuclear factor-kappaB in inflammatory
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tissue of patients with polymyositis and patients with dermatomyositis. Arthritis Rheum. 2008;58:3224e3238. Dastmalchi M, Alexanderson H, Loell I, et al. Effect of physical training on the proportion of slow-twitch type I muscle fibers, a novel nonimmune-mediated mechanism for muscle impairment in polymyositis or dermatomyositis. Arthritis Rheum. 2007;57:1303e1310. Alexanderson H, Dastmalchi M, Esbjornsson-Liljedahl M, Opava CH, Lundberg IE. Benefits of intensive resistance training in patients with chronic polymyositis or dermatomyositis. Arthritis Rheum. 2007;57:768e777. Nader GA, Dastmalchi M, Alexanderson H, et al. A longitudinal, integrated, clinical, histological and mRNA profiling study of resistance exercise in myositis. Mol Med. 2010;16:455e464. Alemo Munters L, Dastmalchi M, Katz A, et al. Improved exercise performance and increased aerobic capacity after endurance training of patients with stable polymyositis and dermatomyositis. Arthritis Res Ther. 2013;15:R83. Riley P, McCann LJ, Maillard SM, Woo P, Murray KJ, Pilkington CA. Effectiveness of infliximab in the treatment of refractory juvenile dermatomyositis with calcinosis. Rheumatology (Oxford). 2008;47:877e880. Dastmalchi M, Grundtman C, Alexanderson H, et al. A high incidence of disease flares in an open pilot study of infliximab in patients with refractory inflammatory myopathies. Ann Rheum Dis. 2008;67:1670e1677. Hengstman GJ, De Bleecker JL, Feist E, et al. Open-label trial of anti-TNF-alpha in dermato- and polymyositis treated concomitantly with methotrexate. Eur Neurol. 2008;59:159e163. A randomized, pilot trial of etanercept in dermatomyositis. Ann Neurol. 2011;70:427e436. Park JK, Yoo HG, Ahn DS, Jeon HS, Yoo WH. Successful treatment for conventional treatment-resistant dermatomyositis-associated interstitial lung disease with adalimumab. Rheumatol Int. 2012;32:3587e3590. Zong M, Dorph C, Dastmalchi M, et al. Anakinra treatment in patients with refractory inflammatory myopathies and possible predictive response biomarkers: a mechanistic study with 12 months follow-up. Ann Rheum Dis. 2013. Oddis CV, Reed AM, Aggarwal R, et al. Rituximab in the treatment of refractory adult and juvenile dermatomyositis and adult polymyositis: a randomized, placebo-phase trial. Arthritis Rheum. 2013;65:314e324. Sugihara T, Okiyama N, Watanabe N, Miyasaka N, Kohsaka H. Interleukin-1 and tumor necrosis factor alpha blockade treatment of experimental polymyositis in mice. Arthritis Rheum. 2012;64:2655e2662. Okiyama N, Sugihara T, Iwakura Y, Yokozeki H, Miyasaka N, Kohsaka H. Therapeutic effects of interleukin-6 blockade in a murine model of polymyositis that does not require interleukin-17A. Arthritis Rheum. 2009;60:2505e2512. Dalakas MC, Rakocevic G, Schmidt J, et al. Effect of alemtuzumab (CAMPATH 1-H) in patients with inclusionbody myositis. Brain A J Neurol. 2009;132:1536e1544.
Available online at www.indianjrheumatol.com and www.sciencedirect.com
Review Article
Pathogenesis of myositis: Lessons learned from animal studies Travis B. Kinder a,c, Sree Rayavarapu a, Kathryn White a,c, Kanneboyina Nagaraju a,b,c,* a
Research Center for Genetic Medicine, Children’s National Medical Center, 111 Michigan Avenue NW, Washington DC, USA; b Department of Integrative Systems Biology; c Institute for Biomedical Sciences, The George Washington University, 2300 Eye Street NW, Ross Hall 605, Washington DC, USA
abstract Keywords:
Recent studies have continued to clarify the pathogenic mechanisms responsible for
Myositis
muscle damage and weakness in inflammatory myopathies. Traditionally, adaptive im-
Inflammation
mune mechanisms such as cell mediated (cytotoxic) and humoral (autoantibodies and
Animal models
complement) components have been implicated in the pathogenesis of polymyositis/in-
Autoimmunity
clusion body myositis and dermatomyositis, respectively. However, recent studies have
Innate immune system
shown a significant overlap of immune components in these disorders. Likewise, studies have provided evidence not only for adaptive immune pathogenic mechanisms but also for innate immune, such as the TLR-NF-kB signaling, and non-immune mechanisms, such as endoplasmic reticulum stress response, autophagy, metabolic deficits in ATP generating pathways and hypoxia. These recent studies indicate that the muscle fiber damage and weakness in myositis may not be solely mediated by an adaptive immune attack (e.g., autoreactive CTLs or autoantibodies) but also mediated through innate immune and metabolic mechanisms. In this review, we have briefly outlined the current developments in immune (adaptive, innate) and non-immune components of disease pathogenesis in inflammatory myopathies. Copyright ª 2013, Indian Rheumatology Association. All rights reserved.
1.
Introduction
Idiopathic inflammatory muscle diseases, such as polymyositis (PM), dermatomyositis (DM), and inclusion body myositis (IBM), are generally considered as autoimmune diseases because many patients have autoantibodies and autoreactive T cells and often respond to immunosuppressive therapies. Traditionally, PM and IBM have been considered T cell
mediated and DM has been considered as humoral (antibody and complement) mediated diseases. These definitions are beginning to change. For example, B cells, plasma cells, and autoantibodies have recently been implicated in IBM.1e3 Similarly, recent studies have provided evidence that changes in innate immune and metabolic mechanisms are responsible for muscle inflammation, damage and weakness. Delineating pathogenic mechanism in humans is complicated
* Corresponding author. Professor of Integrative Systems Biology and Pediatrics, The George Washington University, Children’s National Medical Center, 111 Michigan Avenue NW, Washington DC 20010, USA. Tel.: þ1 202 476 6220; fax: þ1 202 476 6014. E-mail addresses: [email protected] (T.B. Kinder), [email protected] (S. Rayavarapu), kwhite@child rensnational.org (K. White), [email protected] (K. Nagaraju). 0973-3698/$ e see front matter Copyright ª 2013, Indian Rheumatology Association. All rights reserved. http://dx.doi.org/10.1016/j.injr.2013.09.006
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by the heterogeneity, complexity, and chronicity of the disease process. Therefore, several animal models were generated to delineate mechanisms of muscle fiber damage and dysfunction in myositis. Recent studies using human muscle biopsy samples and animal models provide strong evidence not only for adaptive immune pathogenic mechanisms but also for innate immune, such as the TLR-NF-kB signaling, and non-immune mechanisms, such as endoplasmic reticulum stress response, autophagy, metabolic deficits in ATP generating pathways and hypoxia.4e6 We will briefly describe these new developments in the following sections.
2.
Immune mechanisms of muscle damage
2.1.
Innate immune mechanisms
Although myositis has long been considered an autoimmune disease, increasing evidence shows that it also has features of autoinflammatory diseases, which are primarily mediated by the innate immune system.7,8 Muscle biopsies of myositis patients show the hallmarks of innate immune system activation such as the presence of macrophage and dendritic cell infiltrates, the over-expression of Toll-like receptors (TLRs), NF-kB activation, and the abundance of innate immune cytokines, including type 1 interferons (IFNs).9e13 Recent studies using mouse models of myositis further demonstrated that the innate immune system plays a crucial role in mediating skeletal muscle damage in these diseases. For example, presence of muscle disease in the absence of mature T and B cells in the MHC class I transgenic murine models of myositis suggested that non-immune mechanisms, particularly endoplasmic reticulum stress, contribute to muscle disease in the absence of lymphocytes in this model.14 Likewise, Soejima et al demonstrated that intramuscular immunization with histidyl-transfer RNA synthetase (Jo-1) drives muscle inflammation in the absence of T and B cells.5 Thus, studies in mouse models have uncovered the role for innate immune mechanisms in myositis muscle.
2.2.
Innate immune cytokines
Cytokines play a critical role in initiating and perpetuating an inflammatory response to tissue injury. Interestingly, lymphocyte derived cytokines (e.g., IL-2, -4, - 6, -10) were detected sporadically in myositis muscle biopsies, and their presence did not correlate with disease activity or treatment status of the patients, but the data indicated a role for beta chemokines (e.g., MIP-1a, MIP-1b, RANTES) in myositis.15 Several recent studies have highlighted the presence of other innate immune cytokines such as IL-1b, TNF-a, TGF-b, or type 1 IFNs in myositis muscle16e19 (Fig. 1). The sources of these cytokines and chemokines in the muscle microenvironment are not extensively investigated. Our group has previously shown that human skeletal muscle cells are capable of producing a variety of pro-inflammatory cytokines and chemokines.20 More recently, others and we have explored the mechanisms by which the pro-inflammatory cytokine synthesis and release occurs in skeletal muscle cells. We have shown that engaging TLR4 and P2X7 receptor
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with their receptive ligands, LPS and ATP, induce IL-1b secretion in primary mouse muscle cells.21 Other investigators have also reported that muscle precursor cells in the presence of IFNg and TLR3 agonist produce type 1 IFN22 suggesting that not only inflammatory cells but also skeletal muscle is an active contributor of pro-inflammatory cytokines in the muscle microenvironment. Potential endogenous TLR ligands in inflammatory myopathies are currently unknown, but emerging evidence indicates autoantigens like histidyltRNA synthetase might stimulate TLRs and initiate proinflammatory cytokine production.23 It is known that BDCA2þ or CD123þ plasmacytoid dendritic cells (pDCs) produce IFNa suggesting that both skeletal muscle and pDCs contribute to the type 1 IFN secretion.24,25 It appears that the type 1 interferon signature in myositis patients is associated with disease activity, especially for DM patients.26,27 Apart from IFNs, other studies have also demonstrated presence of TNF-a in macrophages and muscle fibers of myositis patient biopsies.28e30 The consequences of excess cytokine presence in the muscle are not explored in details. Experiments performed on mouse skeletal muscle demonstrated that TNF-a compromises contractile function of skeletal muscle and decreases force by blunting the response of muscle myofilaments to calcium activation.31 A recent study by Keller et al further demonstrated that IFNg and TNF-a induced autophagy and increased surface expression of MHC class II in human myoblasts32 suggesting that innate immune cytokines affect skeletal muscle weakness and cell death. One of the most interesting developments in myositis research is the finding that known autoantigens, such as aminoacyl-tRNA synthetases, are proteolytically cleaved and attain cytokine and chemokine properties. For example, human tyrosyl-tRNA synthetase can be cleaved by leukocyte elastase and split into two distinct cytokines; one half activates leukocyte and monocyte chemotaxis and cytokine production (e.g., TNF-a) and the other half binds to IL-8R and stimulates IL-8 signaling.33 Subsequent experiments by Howard et al showed a major autoantigen, histidyl-tRNA synthetase activates the CC chemokine receptor CCR-5 and activates lymphocytes, monocytes, and immature dendritic cells. Thus, release of histidyl-tRNA synthetase from muscle links the cell damage and injury to the inflammatory response in myositis.34 These studies clearly illustrate that autoantigens participate in initiating innate and adaptive immunity as well as inflammatory responses in myositis.
2.3.
Toll-like receptors (TLRs) and NF-kB activation
Proximal events that drive the innate cytokine production in skeletal muscle are not well explored. It is clear that damaged skeletal muscle is likely to release damage-associated molecular patterns (DAMPS) that engage TLRs and trigger inflammation in skeletal muscle.21 There are many reports demonstrating expression of TLR2, TLR3, TLR4, TLR7, and TLR9 in biopsies from patients with IBM, DM, or PM.12,35e38 These receptors are localized mainly on infiltrating inflammatory cells, but some TLRs (TLR3 and TLR7) are also expressed on muscle fibers.35,37 Experiments with cultured muscle cells have shown that stimulation of TLR3 with the agonists Poly (I:C) or necrotic myoblasts elicited the
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Fig. 1 e Mechanisms of muscle damage and myofiber death in myositis. Infiltrating immune cells within the skeletal muscle constitute macrophages, dendritic cells (myeloid and plasmacytoid), T cells (Th1, Th2 and Th17, Tregs, CD28null, cytotoxic T cells (CTL)), B cells, and plasma cells. Tregs influence both effectors as well as T helper cells. Cytotoxic T cells may engage MHC class I and may directly contribute to the muscle injury but the extent of CTL mediated injury to muscle fiber death is currently unknown. Infiltrating immune cells as well as skeletal muscle cells actively secrete a variety of pro-inflammatory cytokines and chemokines. These cytokines and chemokines engage respective receptors on skeletal muscle and induce NF-kB activation, ER stress response, autophagy, inflammasome activation and might also presumably cause deficits in energy generating metabolic pathways. These non-immune mechanisms either alone or in combination contribute to muscle weakness and eventually to muscle fiber death. Skin lesions and loss of capillaries in dermatomyositis are attributable to immune complex mediated damage to capillaries. Loss of capillaries contributes to tissue hypoxia and muscle weakness in dermatomyositis.
production of IFNb22 or the pro-inflammatory cytokines IL-835 or IL-6.37 More importantly, endogenous TLR ligands such as high mobility group box-1 (HMGB-1) and histidyl-tRNA synthetase are identified as DAMPS that stimulate inflammatory response and pathology in skeletal muscle.23,38,39 Stimulation of TLRs or the IL-1b or TNF-a receptors causes the activation of the pro-inflammatory transcription factor NF-kB, which is known to affect inflammation and myogenesis in myositis.40 In skeletal muscle, NF-kB activation impairs myoblast differentiation and causes the secretion of the pro-inflammatory cytokine TNF-a.41 Several groups have observed activated NF-kB within the inflammatory infiltrates, regenerating fibers, and some necrotic fibers in myositis muscle13,42 (Fig. 1). In addition, several downstream NF-kB targets are up-regulated in myositis muscle, including iNOS and TNF-a.28,43 Activation of NF-kB produces proinflammatory cytokines that cause muscle damage and weakness in existing muscle fibers and actively inhibit the
generation of new muscle fibers by blocking the transcription factor MyoD, which is essential for muscle differentiation.44 These studies indicate that innate immune components like cytokines and endogenous TLR agonists (DAMPs) directly act on skeletal muscle and activate downstream proinflammatory pathways that contribute to muscle weakness and damage in myositis.
2.4.
Macrophages
These are the prototypical phagocytic cells, which display remarkable plasticity dependent on the local environment. These cells detect and eliminate invading pathogens, sense tissue damage, clear debris, and dampen the immune response post-inflammation.45 Although few studies have been conducted on macrophages in myositis muscle, they are abundant among the inflammatory infiltrates (Fig. 1). In PM, endomysial macrophages have been observed with markers
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associated with early-activation (MRP14 and 27E10). These markers are displayed on macrophages that have recently migrated into inflamed tissue in the acute phase of injury. Whereas in DM, perimysial macrophages with markers associated with late-activation (25F9) predominate.11 Macrophages displaying the late activation marker 25F9 were previously considered resident, resting cells. However, IHC of patient biopsies has shown that subsets of late-activated macrophages express iNOS, and another subset express TGF-b contributing cell-mediated damage and fibrosis in muscle tissue.46 Even though the relative contribution of macrophages to muscle damage are unknown, the plasticity of macrophage phenotype may lend itself to therapeutic intervention once we have full understanding of their contribution to chronic inflammation and injury to muscle.
2.5.
Adaptive immune mechanisms
Antigen processing and presentation by the professional antigen presenting cells (APCs) is a critical component of adaptive immune response initiation. Dendritic cells are the sentinels of the immune system that coordinate innate and adaptive immunity by acting as professional APCs and by secreting cytokines. Several lineages exist, including myeloid and pDCs, and their phenotypes are dynamic in response to the local microenvironment.47 Both myeloid and pDCs have been observed in the biopsies of patients with PM, DM, or IBM48 (Fig. 1). Although a few immature DCs have been observed in myositis muscle,49 the majority are positive for markers of mature DCs.50,51 Further studies are required to determine whether the DCs mature within the muscle or in the lymph node, but the presence of DCs near the capillaries in DM muscle suggest endothelial cell contact induced local DC maturation.50,51 Recently, pDCs have been implicated in myositis pathology, as they are abundant in the muscle of DM patients.24 Maddur et al found that myeloid DCs cultured with type 1 interferon produce type 1 interferon-induced pro-inflammatory cytokines such as IL-8, IL-6, TNF-a, and IL-1b.52 To fully understand the pathogenesis of myositis we must use animal models to further investigate the function of these observed DCs, especially their potential role in priming autoreactive T and B cells and their contribution to the inflammatory state via innate immune cytokine production. Myositis researchers have contemplated for quite some time about the antigen processing and presentation capabilities of skeletal muscle based on the presence of MHC class I and MHC II antigens (signal 1) on muscle. However, activation of a naı¨ve T cell requires co-stimulatory molecules (signal 2) on skeletal muscle. Despite initial reports of the presence of B7.1 and B7.2 co-stimulatory molecules on human skeletal muscle cells, these findings have not been independently verified and validated.53 MHC class I expression on skeletal muscle fibers is characteristic feature of myositis.54,55 Therefore, it likely that muscle cells expressing MHC class I (signal one) could stimulate memory (signal 2 is not required) but not naı¨ve T cells. Alternatively, it is possible that naı¨ve T cell stimulation can still occur if signal 1 comes from skeletal muscle and signal 2 from infiltrating APCs such as DCs. However, there is no experimental evidence in support of this possibility. The presence of memory cells in myositis biopsies
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supports the hypothesis that muscle is likely to activate memory but not naı¨ve T cells.56
2.6.
B and T lymphocytes
Early studies have demonstrated that infiltrating mononuclear cells in myositis include T cells and B cells. There is ample evidence for the presence of invasive, perforinexpressing cytotoxic CD8þ effector T (a/b) lymphocytes in PM and IBM.57,58 The invasion of non-necrotic muscle fibers by these cytotoxic T lymphocytes is considered a characteristic histological feature of polymyositis. Apart from a/b T cells, there are other reports that demonstrate presence of clonal T cells with g/d T cell receptors.59,60 Recent studies show that these g/d T cells recognize aminoacyl-tRNA synthetases and suggest a link between g/d T cells and antibody responses in autoimmune myositis.61 Case studies indicate that patients with the presence of gamma/delta T cells in muscle tissues appear to be responsive to steroids.59,62 Recent studies have shown some unique subsets of T cells in the muscle microenvironment of myositis patients. One of the subsets is CD28null T Cells, including both CD4þ CD28null and CD8þ CD28null T cells. These apoptosis resistant, proinflammatory cells appear to be present in the muscle at the time of diagnosis as a predominant infiltrating cell type in PM and DM biopsies. Functional studies performed with these effector cells indicate that they are potentially cytotoxic to muscle cells in vitro and in vivo.63,64 T regulatory cells (FOXP3þ cells) are a subset of T cells that actively suppress immune activation by secreting cytokines such as IL-10 and TGF-b. These cells are found in close proximity to effector cells and the numbers correlated with the degree of inflammation within muscle65 (Fig. 1). Some studies have suggested that reduction in regulatory cells and their secreted cytokines contribute to skin lesions in DM patients.66 The proportion of B cells in the perivascular regions of DM is more than in PM.67 So, many suggest a role for humorallymediated immune damage in DM.68 However, subsequent studies showed presence of B cells, plasma cells, and immunoglobulin transcripts in PM and IBM, indicating a role for a humoral component in these disorders also.1,2,69 Further investigations on immunoglobulin transcript sequence analysis demonstrated spatially distributed, clonally-related B cells and plasma cells, suggesting local maturation of B cells into plasma cells in myositis muscle.70,71 The pathological roles of autoantibodies in myositis are unclear; however, there is growing evidence that some of the myositis-specific autoantibodies are associated with distinct clinical phenotypes. For example, anti-synthetase syndrome has a spectrum of phenotypes that includes interstitial lung disease, arthritis, Raynaud’s and mechanic’s hands; anti-Mi2 antibodies are associated with dermatomyositis; anti-P140 antibodies are associated with calcinosis and contractures in juvenile DM. Some autoantibodies, such as anti-SRP, are associated with severe refractory myositis, and anti-MDA5 antibodies are associated with progressive lung disease in some populations but not in others.72 Cause and effect relationship studies between autoantibodies and disease phenotype and activity are few. It is therefore difficult to conclude on the mechanistic basis for the above relationships, but
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existence of such a relationship is immensely useful for the diagnosis and prognosis of this group disorders. Recent studies demonstrated that not only autoantibodies but also B cell activating factor (BAFF), which is known to play a role in type 1 interferon induction and autoantibody production, is strongly associated with disease activity measures in myositis.73
findings from this study suggest that chronic activation of ER stress might induce myofiber degeneration via proteasome dependent mechanisms. Initial studies in mouse models indicate that targeting ubiquitin proteasome mediated muscle degeneration confers benefit on disease phenotype in MHC class I transgenic mouse model of myositis.81
3.2.
3. Non-immune mechanisms of muscle damage Some myositis patients are refractory to potent immunosuppressants and continue to show clinical disease symptoms even after elimination of immune cells from the affected muscle.74,75 These observations suggest a potential role for non-immune mechanisms in myositis pathology. Recent studies using animal models provided several lines of evidence in this regard, including activation of endoplasmic reticulum (ER) stress response, implication of metabolic enzymes, autophagy and hypoxia in myositis muscle (Fig. 1).
3.1.
Endoplasmic reticulum stress
Disturbances in ER homeostasis such as enhanced protein synthesis or accumulation of mis-folded proteins results in the activation of ER stress response pathways. In general, two adaptive mechanisms, the unfolded protein response (UPR) and the ER overload response (EOR) are activated to overcome the stress. Induction of UPR attenuates the translation machinery to reduce the protein synthesis load on the ER. On the other hand, EOR activates NF-kB pathway and the downstream pro-inflammatory processes. In certain extreme conditions, the affected cell initiates the cell death mechanisms by activation of cholesterol oxidase-peroxidase C/EBP homologous protein (CHOP), Bcl-2 family proteins and ERassociated caspases. With respect to myositis, several studies have reported enhanced expression of classical ER stress markers (such as GRP78, GRP94, and calreticulin) in the affected skeletal muscle both in mouse models and human biopsy samples.40,76e78 One recent report also indicated the enhanced expression of heat shock protein (Hsp) families 70 and 90 in myositis muscle.79 One possibility for the induction of ER stress in myositis muscle is the over-expression of MHC class I in the affected muscle fiber. Studies using a transgenic mouse model of myositis reported that muscle specific overexpression of MHC class I and its potential accumulation in the ER induces the stress response.40,80 Although these findings hint at a non-immune role for MHC class I in myositis pathology, it is not clear whether the induction of ER stress is attributable to its forced expression or its immune responses per se. A recent study performed using mature lymphocyte deficient Rag2/ mice showed that MHC class I can induce UPR independent of its immune functions in the myositis muscle suggesting that mere over-expression of MHC class I in muscle might contribute to ER stress and muscle damage.14 More recently, our group established initial evidence suggesting the activation of ER associated degradation mechanisms (ubiquitin proteasome pathway) in myositis muscle using a novel in vivo SILAC proteomics approach.81 Our
Deficit in energy pathways
Metabolic pathways such as mitochondria-associated oxidative metabolism play a critical role in skeletal muscle homeostasis. Emerging evidence indicates a probable disruption in mitochondria and associated metabolic pathways in the chronic stages of inflammatory muscle diseases ultimately leading to muscle weakness.82,83 A recent study suggested an impaired muscle oxidative competence and abnormal blood lactate levels in PM/DM patients.84 Other studies indicate abnormal succinic dehydrogenase and cytochrome c oxidase activities in damaged and atrophic fibers in myositis muscle85,86 suggesting a deficit in energy generating pathways. More recently, our group demonstrated a significant reduction in the levels of adenosine monophosphate deaminase 1 (AMPD1), a rate-limiting enzyme in the purine nucleotide cycle.6 Notably, the reduction of the AMPD1 enzyme levels and the reduction of muscle strength occur prior to the appearance of infiltrating lymphocytes, which indicates that metabolic deficiencies seen in myositis are independent of the action of immune cells. It is possible that reduced AMPD1 might limit the levels of downstream substrates that are needed for producing energy in the affected muscle. At this time, the precise role of dysregulated metabolic pathways in myositis pathology is not completely clear. However, it is possible that pro-inflammatory cytokines and disturbed immune mechanisms might regulate these energy metabolic pathways in myositis muscle.
3.3.
Autophagy
The autophagic process includes degradation of cell’s own proteins or organelles with the aid of lysosomes. This type of cell death is particularly important for skeletal muscle because classical apoptosis is not present in myositis.87 Muscle biopsies from PM and sIBM patients often showed evidence of activation of the autophagic process. Our group demonstrated that TNF-related apoptosis-inducing ligand (TRAIL) and markers of autophagy are up-regulated in myositis muscle fibers.88 On the similar lines, a recent study reported over-expression of a panel of autophagic proteins (clathrin, ATG5, LC3a, LC3b and Beclin 1) in sIBM patient biopsies.89 Moreover, these authors suggested an association between the increased expression of autophagic markers and the reduced clearance of mis-folded proteins. These findings suggest a potential relation between ER stress and autophagy in myositis. Another study reported that human b defensin 3 was overexpressed in sIBM affected muscle tissue and localized near markers of autophagy.90 Even though the role of autophagy in myofiber damage is emerging from these studies, it is important to know how and when these autophagic mechanisms are triggered in the affected muscle in
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order to investigate their validity as therapeutic targets in myositis.
3.4.
Hypoxia
Reduced levels of oxygen and oxidative stress are also implicated in causing muscle fiber damage in inflammatory myositis. Initial evidence indicates a loss of capillaries and a compensatory up-regulation of proteins such as hypoxia inducible factor-1, avb3 integrin, and Flt-1 in the affected muscle tissue.91 Another study reported higher expression of vascular endothelial growth factor, a reduction in the proportion of oxidative, slow twitch fibers suggesting a shift in fiber type composition in the muscle tissue of myositis patients.6,92,93 This shift might be one possible reason for low muscle endurance observed in chronic PM or DM patients. The hypoxia theory causing muscle weakness in myositis is further supported by the beneficial effects of intensive resistance exercise training in myositis muscle.94,95 One recent study reported that 12 weeks of endurance training resulted in clinical improvement in PM and DM patients and this improvement correlated with enhanced aerobic capacity and increased mitochondrial enzyme activities in the myositis muscle.96 These observations highlight the importance of resistance exercise regimens as a treatment modality in addition to immunosuppressive drugs in inflammatory myopathies.
4.
Experimental therapies for myositis
The current gold-standard treatment for patients with IIMs is high-dose glucocorticoids (GCs) often combined with immunosuppressive agents, including azathioprine and methotrexate. However, many patients do not respond to this therapy, and they are accompanied by many side effects. Biologics such as anti-TNF therapies revolutionized treatments for autoimmune diseases like rheumatoid arthritis. Anti-TNF therapies are largely ineffective in myositis. For example, a hand full of patients have been reported to show clinical benefit from the anti-TNF-a monoclonal antibody infliximab, but others have found radiological, clinical, and immunological worsening of myositis.97e99 A randomized, double-blind, placebo-controlled trial of the TNF-a antagonist etanercept showed good tolerability and a steroid-sparing effect in patients with DM.100 Anecdotal evidence exists for the efficacy of the third, most recently FDA approved TNFa antagonist adalimumab for the treatment of DM-associated ILD.101 A mechanistic study of IL-1 blockade by anakinra showed that some patients may respond to the treatment, which seemed to shift the immunological phenotype away from a Th17 response.102 A randomized, double-blind, placebo-phase trial of a B celldepleting antibody rituximab showed that 83% of refractory PM and DM patients attained the definition of improvement.103 Taken together, these studies indicate that therapeutic response to existing biologics is highly variable and highlight the need for developing more targeted therapies. In this regard, animal models play a significant role in identifying therapeutic targets and testing experimental therapies. For example, A C-protein-induced mouse model of myositis
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demonstrated benefit with antagonists of IL-1, TNF-a, and IL6, thereby providing rationale for future clinical studies with such cytokine antagonists.104,105 Treatment of a transgenic mouse model of myositis with the proteasome inhibitor Bortezomib showed a potential therapeutic effect.81 Because most patients with IBM fail to respond to treatment, trials have been undertaken to test therapies specifically for this subtype of IIM. A proof-of principle study in sIBM patients showed that Alemtuzumab, which depletes peripheral blood lymphocytes, slowed down disease progression, improved strength in some, and reduced inflammation and stressor molecules.106 Recently, the FDA granted a break through status for a monoclonal antibody that binds to type II activin receptors and block binding of natural ligands activin and myostatin and improves muscle growth. Therapeutic studies focusing on muscle instead of immune cells are highly encouraging and will pave the way for treating various inflammatory muscle diseases.
Conflicts of interest All authors have none to declare.
Acknowledgments Dr. Nagaraju is supported by NIH (RO1-AR050478; 5U54HD053177; K26OD011171), Muscular Dystrophy Association, and US Department of Defense (W81XWH-05-1-0616). Travis Kinder is supported by NIH (F31-AR065362-01) for his doctoral studies.
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