Global gene expression analyses of PBMC from a large cohort of polyarticular JIA patients that had not been treated with DMARDs or biologics revealed striking subgroups within an otherwise uniform collection of subjects with recent onset polyarthritis. These gene expression profiles demonstrate biologically significant heterogeneity within the JIA population, with distinct subgroups of patients expressing distinct gene expression signatures. These signatures appear to be manifestations of biological processes that are occurring in some but not all patients, and in the long run they may prove to be valuable tools for classifying and managing individuals with polyarticular JIA.
Three distinct gene expression signatures were identified with variable expression among polyarticular JIA subjects. Signature I was strongly expressed in many RF+ JIA subjects, but also in a number of RF- JIA subjects ( & ). In fact, Signature I may prove useful for identifying a subset of RF- JIA patients that have a disease phenotype similar to RF+ patients, as described by Ravelli and Martini (3
). Signature I contains many genes specifically expressed in monocytes, and thus this signature may be a manifestation of increased abundance and/or activation of peripheral blood monocytes. Furthermore, a number of genes in Signature I have been identified as being regulated in monocytes in other rheumatic diseases. For example, FCGR1A (CD64), the third ranked gene in Signature I, was shown by Wijngaarden, et al. to be dramatically down-regulated in monocytes in rheumatoid arthritis after methotrexate treatment (17
). Likewise, MS4A4A, the top ranked gene in Signature I, and FCGR1A were the top two ranked cell surface marker genes in isolated monocytes that Abe, et al. found to be significantly down-regulated in Kawasaki disease after treatment with intravenous immunoglobulin (18
). Additionally, MS4A4A, CLU, DYSF and IL8RB were found by Ogilvie, et al. to be up-regulated in PBMC of patients with active systemic JIA (7
). A very interesting comparison with 25 genes up-regulated in rheumatoid arthritis finds 16 of these also up-regulated in Group A prototypes, including CD14, AQP9 and several S100 proteins, while in contrast 14 of these up-regulated RA genes were down-regulated in Group C prototypes, emphasizing the contrast between Groups A and C (Supplementary Tables 1 and 3
). Thus, Signature I may be indicative of monocyte activity in a number of autoimmune inflammatory diseases, and it may be useful for assessing disease activity and monitoring response to treatments in these diseases.
Rigorous protocols were followed for isolating and freezing PBMC as quickly as possible to minimize effects of processing on gene expression. Nevertheless, some of the observed gene expression differences that contribute to Signature II were associated with prolonged processing times. Still, this signature does not appear to be solely a function of processing, but also depends on immunological differences between subjects, noting that this signature was observed almost exclusively in JIA subjects and not controls, and many of those JIA subjects also expressed Signatures I and/or III. Signature II contains many immediate-early genes, including several FOS-related genes, JUN and EGR1 (20
), consistent with very recent cellular responses. The association of Signatures I and III with antigen presenting cells (monocytes and plasmacytoid dendritic cells, respectively), suggests that Signature II may develop after phlebotomy in samples that have pathologically primed antigen presenting cells that are able to readily stimulate other cells in the “test tube” environment, leading to rapid induction of immediate-early genes. Furthermore, Signature II's pathological relevance is supported by the presence of many genes that have been previously associated with autoimmune arthritis. For example, NR4A2 (also called NURR1), the first-ranked gene in Signature II, is markedly up-regulated in rheumatoid arthritis synovium (21
). Likewise, OSM (Oncostatin M), the third-ranked gene in Signature II, is detectable in JIA synovial fluid and it can induce joint inflammation in a murine adenoviral gene transfer model (22
). Thus, Signature II may provide valuable information regarding pathological processes in samples with prolonged processing that may not otherwise be observed if processing were prompt.
Signature III was associated with absence of rheumatoid factor (), and appears to be distinct from Signature I. In fact, few subjects expressed both Signatures I and III together (), suggesting that these signatures are manifestations of independent biological processes. Like Group A, the average age of Group C was greater than that of the entire polyarticular JIA population, supporting the concept that Signature III identifies a distinct subset of JIA patients. Additionally, Group C had a trend toward lower ESR, lower CHAQ scores and lower physician global assessment of disease activity (), all of which are consistent Signature III identifying a less inflammatory subset of polyarticular JIA, possibly similar to the “dry synovitis” subset described by Ravelli and Martini (3
). Signature III was associated with low numbers of CD8+
T cells (), increased abundance of BDCA-4+
plasmacytoid dendritic cells, and it contains many genes that are inducible by TGFβ and potentially involved in mediating or regulating TGFβ action. These include the first ranked gene, DAPK1, which is a pro-apoptotic protein that can be induced by TGFβ via SMAD activation (23
). Other Signature III genes that are TGFβ-inducible include SMAD3, BCL2, MAPK1 and FOXO3A (24
). These observations suggest that TGFβ may be responsible for reducing levels of CD8+
T cells via a pro-apoptotic influence, and increasing plasmacytoid dendritic cells via an activating effect. Notably, increased levels of TGFβ have been detected in synovial fluid in juvenile arthritis (28
), where it has been conjectured to serve an immunosuppressive role in countering synovial inflammation (29
). Interestingly, while one might conjecture that PBMC exposure to TGFβ occurs in inflamed synovium, another intriguing possibility is via regulatory T cells that express TGFβ (30
Many polyarticular JIA subjects did not express Signatures I, II or III (24 out of 61, or 39%). These subjects were statistically younger and had a higher rate of ANA positivity than the rest of the polyarticular JIA subjects. It is uncertain as to whether these patients represent a unified group, but one can speculate that they do not have the same disease phenotype as subjects that express either Signature I and/or III, and given their age and ANA status it is likely that many of these subjects comprise a polyarticular JIA subset that closely resembles early-onset ANA positive oligoarticular JIA, described by Ravelli, et al. (3
). It is also noted that a few normal controls clustered into Groups A, B or C. While control subjects were deemed free of inflammatory disease by screening questionnaire, there was no assurance that every control subject was completely healthy and this may account for co-clustering of some controls with JIA subjects.
In summary, PBMC gene expression signatures have been demonstrated within a large population of children with recent onset polyarticular JIA that correlate with differences in disease characteristics. These signatures support the sub-classification of polyarticular JIA offered by Ravelli and Martini (3
), with Signature I identifying both RF+ and RF- patients with disease similar to adult rheumatoid arthritis, Signature III identifying a less inflammatory subset, and patients with ANA+ early-onset disease expressing neither signature. Thus, these signatures offer a molecular classification of polyarticular JIA, and may prove to be valuable tools for assessing disease activity, predicting response to medications and forecasting long-term outcomes.