Adult and juvenile dermatomyositis (DM) are autoimmune disorders characterized by proximal muscle weakness, muscle inflammation, and hyperkeratotic skin rash. Current pathogenetic models suggest that a combination of genetic and environmental factors confers susceptibility to disease (1). Susceptibility to DM is associated with inheritance of the HLA alleles DQA1*0501 (2) and DR3 (3), and polymorphisms in the tumor necrosis factor α (TNFα) gene are associated with disease severity (4). Viral infection has long been suspected to play a causative role in DM (5). Despite advances in the understanding of pathogenetic factors in DM, monitoring of disease activity is heavily dependent on the physician's clinical assessment. Few reliable indicators of prognosis, disease activity, or potential response to treatment have been identified.
Accumulating data from our group and other investigators suggest that cells from the muscle tissue and blood of patients with DM carry distinct immune “fingerprints.” Several studies have demonstrated up-regulation of genes related to antigen processing (6, 7), immunoglobulin-encoding genes (7), and type I interferon-α/β (IFNα/β)–regulated genes in the muscle tissue of patients with DM (6, 8). Type I IFN–inducible proteins (IFNα/β-inducible myxovirus resistance protein [MxA] and IFN-stimulated transcription factor 15 [ISGF-15], and IFN regulatory factor 7 [IRF-7]) were detected in the muscle fibers and capillaries of patients with adult or juvenile DM (8–10). Numerous type I IFN–inducible genes are up-regulated in the peripheral blood, and these form a type I IFN gene “signature” in DM (11, 12). In addition, we have observed elevated serum levels of IFN-inducible chemokines in patients with DM (11). In aggregate, these data documenting type I IFN induction at both the transcriptional and translational levels suggest a major pathogenetic role for type I IFNs in DM.
Histopathologic studies have revealed that both lymphoid and myeloid immune cell infiltrates are present in the muscles of patients with DM. Perivascular and perifascicular infiltrates display B cell and CD4+ T cell predominance in adult DM, and display a combination of B, CD4+, and CD8+ T cells in juvenile DM (13), suggesting that muscle tissue may be a target for both humoral- and cell-mediated autoimmune responses. The functions of both the CD8+ and CD4+ T cell lineages are likely important for causing inflammation in the muscle and other tissues in DM. A number of distinct CD4+ T cell subsets, including Th1, Th2, and Treg, may regulate human autoimmune processes.
The presence of inflammatory cells in affected tissue in DM suggests a potential for regulation of the disease process by soluble cytokine networks. Recent work in a mouse model of inflammatory myositis identified the cytokine interleukin-6 (IL-6) as a critical mediator of tissue-destructive processes (14). IL-6 is a proinflammatory cytokine that has been targeted therapeutically for human rheumatic disease. IL-6 plays central roles in the regulation of both innate and adaptive inflammatory and immune responses, as well as both humoral and cell-mediated autoimmune reactions. IL-6 is a multifunctional cytokine that was originally identified as a B cell differentiation factor. IL-6 is produced by various types of lymphoid and nonlymphoid cells, such as T cells, B cells, monocytes, fibroblasts, keratinocytes, endothelial cells, mesangial cells, and several tumor cells. In addition to B cell differentiation activity, IL-6 induces T cell growth and T cell differentiation (15).
Elevated levels of IL-6 have been documented in a variety of rheumatic diseases, including Castleman's disease, primary Sjögren's syndrome, rheumatoid arthritis, colitis, and Crohn's disease (16–19). Blockade of IL-6 and of IL-6 signaling have been shown to be effective in treating several of these inflammatory diseases (e.g., inflammatory arthritis and colitis) (19).
We tested the hypotheses that multiple proinflammatory pathways are active in DM, and that detection of peripheral blood chemokines and proinflammatory cytokines marking these pathways will permit improved disease activity assessments. Using genomic and proteomic approaches, we observed robust activation of the IFNα pathway in the peripheral blood of patients with DM. Prominent type I IFN signatures, manifesting as both transcript up-regulation and elevated levels of serum proteins, correlated strongly with DM disease activity. In addition, elevated IL-6 levels in the patients' serum correlated strongly with both DM disease activity and IFN-driven chemokine levels. These data suggest that genes and proteins in the type I IFN– and IL-6–related pathways are coregulated in DM, and that monitoring dysregulation within these pathways will help guide diagnostic and treatment decisions.
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Our results demonstrate that the levels of IL-6 and type I IFN–regulated transcripts and proteins are elevated in the peripheral blood of patients with adult or juvenile DM. Type I IFN gene and protein signatures and serum IL-6 levels are correlated significantly with indicators of DM disease activity and with each other. Our results suggest the possibility that coordinated dysregulation of type I IFN signaling and IL-6 production may be a characteristic pathogenic feature of DM.
Dysregulated type I IFN has also been implicated in the pathogenesis of other autoimmune diseases, including human SLE (28, 29), Sjögren's syndrome (30), and scleroderma (31). A key aspect of the current model for IFN production in autoimmunity is the triggering of IFN production after ligation, by viral and bacterial nucleic acids, of Toll-like receptors (TLR-7 and TLR-9) in plasmacytoid dendritic cells (DCs) (32, 33). Plasmacytoid DCs comprise a DC subset that has important functions in the regulation of innate and adaptive immune responses, particularly those in response to viral infections. Plasmacytoid DCs are relatively infrequent in the peripheral blood, but they are found in the lesional skin of patients with SLE (34).
Both plasmacytoid DCs and type I IFN–induced proteins (MxA, ISGF-15, and IRF-7) are found in the affected muscle and skin of patients with DM (10, 35–37), suggesting a potential pathophysiologic function for plasmacytoid DCs. Indeed, CD4+ plasmacytoid DCs comprise a large proportion (30–90%) of what had previously been thought to be CD4+ T cells in DM muscle (8, 37). Viral infection has long been suspected as an inciting factor in DM (5), but the specific pathogen-derived TLR ligands that might signal elaboration of IFN by plasmacytoid DCs remain unknown.
Type I IFNs have pleiotropic functions that may contribute to the pathogenesis of DM. Type I IFNs up-regulate class I major histocompatibility complex (MHC) expression, activate natural killer cell cytotoxicity, promote activated T cell survival, and support DC maturation (36). The regulatory effect of IFN on class I MHC expression may be particularly relevant in human DM, since class I MHC levels are elevated in the muscle cells of patients with DM (36). Furthermore, animals with enforced muscle-specific overexpression of class I molecules exhibit inflammation and muscle necrosis (38). IFN-regulated proteins have been shown to play a role in recruitment of lymphocytes to sites of inflammation in the muscle (39, 40) and skin (41). These proteins (IP-10, I-TAC, MCP-1, and MCP-2) were highly expressed in our DM cohort, and this comprised the type I IFN chemokine score.
Not all patients with active DM according to the clinical disease activity score displayed significant elevation in the type I IFN chemokine score (Figure 1B). In fact, we observed a significantly large population of patients in the VAShigh/IFNlow subset, but saw a near absence of patients in the converse VASlow/IFNhigh population. This trend may suggest that 1) IFN gene and chemokine scores are less sensitive than the global VAS score for detecting clinically important disease, 2) the global VAS score overestimates disease activity in DM, 3) a subset of DM disease is driven by an IFN-independent pathophysiologic mechanism, or 4) treatment may have been recently initiated. The level of IL-6 was also elevated in those with the VAShigh/IFNhigh profile (details available from the corresponding author upon request), suggesting that activation of IL-6 and type I IFN may describe a clinical subset of patients with DM whose disease has a unique pathophysiologic pathway. Distinguishing between these possibilities and establishing the degree to which IFN-driven events may serve as disease activity biomarkers for the DM patient population as a whole will require longitudinal studies that prospectively evaluate correlations between disease flare and type I IFN gene or chemokine score elevation.
IL-6 is implicated in the regulation of both innate and adaptive immune processes in a number of human autoinflammatory and autoimmune diseases (16–19). Elevated serum IL-6 levels are found in Castleman's disease, a rare disorder characterized by fevers, anorexia, anemia, hypergammaglobulinemia, and plasma cell accumulation in enlarged lymph nodes (18). In rheumatoid arthritis, IL-6 levels are elevated in both the serum and synovial fluid (42). IL-6 is a major contributor both to the constitutional symptoms of fatigue and fever, and, via activation of osteoclasts, to the bony erosions that characterize the rheumatoid joint. Antagonism of IL-6 function through genetic or pharmacologic manipulation has been shown to be beneficial in the treatment of experimental arthritis in murine models, as well as in the treatment of human Castleman's disease, colitis, rheumatoid arthritis, and systemic-onset juvenile arthritis (15, 17, 18).
Evidence in the literature concerning a pathogenic role for IL-6 in inflammatory myositis is sparse. In the experimental mouse model of myosin-induced myositis, deficiency of IL-6 leads to marked amelioration of the clinical signs and pathologic findings of muscle injury (14). However, there are no descriptions of IL-6 overexpression at the level of skeletal muscle in humans with DM.
Few reports to date have suggested the utility of IL-6 as a biomarker for disease activity in the idiopathic inflammatory myopathies (IIMs), including DM. Interestingly, Tucci et al reported no significant differences in the serum levels of IL-6 between controls and patients with active IIM (43). The contrast between the latter results and the present findings of elevated IL-6 levels may be due to the more limited, yet more diverse patient population in that study (n = 8 patients, comprising both those with polymyositis and those with DM). Our findings suggest that IL-6 levels are positively correlated with overall disease activity and with specific manifestations of DM, as well as with the levels of type I IFN–regulated chemokines (Figure 3). The latter finding suggests that there is coregulation of the type I IFN– and IL-6–related pathways, and possibly a regulatory cross-talk between the pathways. Notably, IL-6 and MCP-1, a type I IFN–regulated chemokine, have both been shown to be potent inducers of inflammation, acting through mononuclear cell accumulation at a site of tissue injury (19), and both are coregulated by the same transcription factors (44). Whether the level of IL-6 is a sufficiently sensitive clinical marker for current or impending disease flare is a question that must be addressed by longitudinal, prospective studies.
The majority of patients with DM in this study were receiving treatment with antimetabolite, antiproliferation, or corticosteroid agents at the time of enrollment. We observed a nonsignificant trend toward both a lower global VAS score and lower levels of candidate biomarker analytes (the type I IFN chemokine score) in the treatment groups compared with those not taking immunosuppressive medication; for example, IL-6 levels were lower in the treated group compared with the untreated group (median 3.32 pg/ml versus 8.05 pg/ml; P = 0.04). Previously, Walsh et al reported a marked reduction in type 1 IFN signature levels in patients with DM after treatment with prednisone, intravenous immunoglobulin, mycophenolate mofetil, methotrexate, or azathioprine (12). Although the low sample size does not allow definite conclusions, we speculate that the higher disease activity and higher values for laboratory parameters among the untreated subjects were due to a high prevalence of new-onset, treatment-naive disease in that group. However, the cross-sectional design of the present study did not allow an assessment of analyte fluctuation in response to treatment in individual subjects over time. Longitudinal studies are required to elucidate the exact relationship between immunosuppressive agents, disease activity, and the expression of candidate biomarkers in DM.
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- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Peterson had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Bilgic, Ytterberg, McNallan, Koeuth, Bauer, Peterson, Baechler, Reed.
Acquisition of data. Bilgic, Ytterberg, Amin, McNallan, Wilson, Koeuth, Ellingson, Newman, Bauer, Peterson, Reed.
Analysis and interpretation of data. Bilgic, Koeuth, Newman, Bauer, Peterson, Baechler, Reed.