Our initial 2D-DIGE results indicated differential plasma protein profiles between active and inactive SJIA. To our knowledge, this is the first study to describe a unique proteomic profile of SJIA F using 2D-DIGE. Because >50% of plasma protein content is accounted for by albumin and other abundant proteins, such as IgG and transferrin, we performed an initial depletion step, removing six of the most abundant proteins. This step allowed us to detect less abundant proteins, such as serum amyloid p [31
]. The DIGE technique has a dynamic range of ~5 orders of magnitude in protein concentration [32
], whereas plasma protein concentrations vary over ~10 orders of magnitude, with the highest concentrations reaching mgs/ml [33
]. Even with the depletion step, protein detection by our 2D-DIGE system is limited to proteins whose plasma concentrations are >10 ug/ml, clearly influencing the composition of the SJIA flare signature we detected. In addition, potentially informative low molecular weight proteins may bind to albumin and thus be removed at the depletion step [34
]. Nonetheless, as levels of some abundant plasma proteins are reduced (e.g., APO A-1, TTR) or increased (e.g. CRP) significantly during inflammatory states, and other proteins that are not found in normal control plasma rise to a level detectable by 2D-DIGE, such as S100A9, a flare signature is observable. Moreover, specific protein species are altered abundance during active SJIA and are sufficient to produce signatures that robustly differ from the protein pattern at SJIA quiescence and from other inflammatory conditions we tested (see more below).
From the 2-D DIGE, we evaluated 89 spots, representing 26 proteins, as candidate components of the SJIA F flare signature. Among these, some proteins have individually been associated with SJIA flare, such as SAA, CRP and the inflammation-associated S100A8/S100A9 complex [13
]. In addition, our observation of reduced levels of APO A-1 at SJIA flare confirms previous investigations of JIA subjects [37
]. The fact that these (expected) proteins were identified by our analyses increases confidence in the use of DIGE as a platform to detect plasma proteins differentially expressed in association with SJIA disease activity.
Particular isoforms/derivatives of 15 proteins gave rise to a robust SJIA flare signature that differentiated flare from quiescence and from acute febrile illnesses. These 15 proteins can be assigned to different functional groups, including proteins involved in the classical acute phase response, the innate immune system (S100 proteins), the complement cascade, the coagulation system and lipid/cholesterol metabolism. There is the substantial evidence supporting crosstalk between these pathways in inflammatory states. For example, CRP, a quintessential positive acute phase protein, binds molecular patterns typically found on the surface of pathogens and also activates the classical complement pathway [38
]. A2M, a thrombin (and other protease) inhibitor, is also a component of the innate immune system, acting as a scavenger of novel proteases introduced by pathogens [40
]. APO A-1, the major protein component of high density lipoprotein (HDL), also has anti-inflammatory and anti-thrombotic properties [39
]. Acute phase HDL, where SAA is exchanged for APO A-1, are lipid transport particles, but also function in innate immune responses, for example, by promoting monocyte chemotaxis [42
]. APO A-IV, the major protein component of intestinal triacylglycerol-rich lipoproteins, is a positive acute phase protein involved in lipid homeostasis, but also reduces Toll-like receptor 4-induced pro-inflammatory cytokines [43
Our data show that the differentially expressed plasma proteins at SJIA F compared to Q have a substantial degree of specificity for SJIA F, compared to poly JIA F or FI; this is the case for both proteins detected by 2D-DIGE and by the ELISA panel. These observations are in line with other evidence indicating that specific patterns of acute phase reactants are associated with certain diseases [45
]. In a relevant example, Yu et al [49
] described a unique 2D protein fingerprint in KD versus non-KD febrile control subjects, with increases in protein spots, representing fibrinogen β and γ chains, α-1-antitrypsin, CD5 antigen-like precursor, and clusterin, and decreases in spots from immunoglobulin light chains. This pattern differs from SJIA flare, although we confirmed a significant difference in fibrinogen β between KD and FI, and noted a KD-specific increase in APO-D compared to FI subjects (Supplementary Table 6
). Similarly, a study of gene expression in peripheral blood mononuclear cells (PBMC) showed that the list of genes differentially expressed in SJIA patients compared to controls had more overlap (35/286) with PBMC gene expression in an autoinflammatory condition (neonatal onset multisystem inflammatory disease, NOMID) than with PBMC gene expression in poly JIA (6/286) or KD (17/286) [50
]. Disease-associated variation in acute phase proteins implies their independent regulation and is thought to reflect differences in the driving cytokines and their endogenous modulators [51
]. This idea finds support within childhood rheumatic diseases in the apparent roles for IL-1β and IL-6 in SJIA, as compared to TNFα/sTNFR in poly JIA or interferon α in SLE [52
Pathway analyses of the 15 proteins in the SJIA flare signature corroborate growing evidence implicating IL-1 as a key mediator of this disease. This is in line with recent findings that IL-1 beta is a central mediator of the arthritic matrix derived FSTL-1, which appears to be a biomarker of SJIA disease activity [56
]. IL-1β and TNFα, pro-inflammatory cytokine products of monocyte/macrophages, are known to stimulate IL-6 production by monocyte/macrophages and endothelial cells. These cytokines, and IL-6 especially, act on hepatocytes to induce production of classical acute phase proteins, such as SAA and CRP, complement components and fibrinogen and suppress production of proteins such as APO A-1 [39
]. Notably, the evidence of IL-1 activity, as reflected in the pattern of proteins in SJIA plasma at flare, is consistent with recent reports of the therapeutic effects of IL-1 inhibition in SJIA patients [52
Differences in profiles of PBMC transcripts or plasma proteins from active SJIA and acute KD are of particular interest because, at disease onset, these two conditions can present a diagnostic dilemma. Interestingly, two new molecular links appear, suggesting processes that differ between SJIA and at least a subset of KD subjects: IL-23, a cytokine associated with Th17 cells, and CD163, a scavenger receptor on alternatively activated macrophages, CD163 is known to bind and clear haptoglobin/hemoglobin complexes and monocyte/macrophages expressing this receptor have been implicated in SJIA, particularly in association with a life-threatening complication of the disease termed “macrophage activation syndrome” [57
We detected a highly discriminatory SJIA flare signature by identifying the particular protein species (spot) most highly associated with disease activity. Different post-translational modifications, particularly altered glycosylation, and/or proteolysis of plasma proteins associated with active disease most likely underlie this observation [51
]. These changes are likely cytokine-driven. For example, it is known that matrix metallo-proteinases (MMPs), especially MMP-1, -3, -9 and -13 are induced by IL-1β [59
]. In a separate study of low concentration plasma proteins, we have found that increased circulating MMP9 is associated with SJIA flare (Ling, XB et al, manuscript in preparation). More work is warranted to investigate the molecular events that generate specific protein modifications and intermediates in inflammatory states. Of note, increased levels of SAA-related derivatives are found in supernatants of IL-1β-activated human monocytes and are thought to reflect a block in SAA degradation [61
]. These in vitro
results are consistent with our observation of increased circulating levels of isoforms of SAA in SJIA flare. A biomarker panel based on the unique protein derivatives we identified as optimal for SJIA will require generation of specific detection reagents.
We validated a subset of our DIGE results using ELISA as an independent method. ROC curve analysis suggests that the 7 ELISA panel may aid in diagnosis of SJIA, as it was better than CRP or S100A8/9 at classifying SJIA versus acute FI. However, an important caveat is that the SJIA F subjects studied with our panel were not all new onset, untreated cases, which would be the best comparator group. The ELISA panel also might be useful to distinguish SJIA flare from inter-current infection in a febrile child with known SJIA. A prospective study with SJIA subjects will be required to address this potential clinical utility. Nonetheless, our panel appears to provide stronger classifying power than any single biomarker alone.
Our data suggest that certain changes in plasma protein profiles occur in advance of clinically detectable disease activity. In unsupervised analysis of our DIGE data, one SJIA F sample clustered with the Q samples. This subject had active disease at the time of sample draw, but entered clinical quiescence over the next 2 months. Based on the DIGE analysis, SAA had already normalized in the flare sample from this subject, suggesting this protein changes earlier than others. APO A-1 spots were also similar to a quiescent pattern; this protein may contribute to resolution of a flare by inhibiting monocyte activation and synthesis of pro-inflammatory cytokines [62
]. The 7-member ELISA panel also classified 4 out of 5 quiescent samples correctly as “pre-flare”. This is in line with the previous observation of serum S100 protein level change in advance of clinical flare [63
], supporting the notion that disease flares can be predicted because local disease activity may be present before flares become clinically apparent. Larger studies are needed to determine whether the ELISA panel can reliably predict flare prior to clinically detectable disease activity. If so, it will be important to test the hypothesis that earlier treatment leads to a better short- or long-term outcome in SJIA.
In addition to the diagnostic challenges associated with fevers of unknown origin and fever in children with SJIA, prognostic challenges are prominent in SJIA. The clinical course is variable, ranging from a monocyclic episode with recovery in about 50% of subjects to a chronic, either polycyclic or persistent, condition, often with severe joint damage [6
]. Only subsets of SJIA patients respond to currently available therapies [66
]. Complications of SJIA include growth failure, macrophage activation syndrome and amyloidosis, the latter two being potentially life-threatening [68
]. Proteomic strategies provide an attractive approach to discover prognostic biomarkers in SJIA. We have found a preliminary suggestion that our ELISA panel may identify those subjects at onset who will have a monocyclic course (JLP, unpublished data). In this regard, it is encouraging that a recent 2D-DIGE analysis of synovial fluid provided evidence for markers that predict the transition from oligoarticular to polyarticular disease in a subset of oligoarticular-onset JIA subjects [32