In this study, we sought to identify molecular pathways involved in the systemic and arthritic components of SJIA by investigating the gene expression pathways associated with increases in ESR and active joint count. We chose ESR as a marker of systemic inflammation, but we note that SJIA flares associated with elevated ESR may also include arthritis. Further, SJIA flares with macrophage activation syndrome (MAS) may actually lower ESR from fibrinogen consumption as part of the coagulopathy . The latter issue does not confound our analysis, as the three flare samples with low ESR were from patients with mild flares without MAS. Strictly speaking, our approach delineated gene associations with ESR; however, in our group of SJIA samples, ESR typically correlated closely with other evidence of systemic disease.
Several variables that influence transcriptional profiles should be considered in relation to our results. It is possible that some of the observed differences in gene expression are due to differences in cell type composition of PBMC between SJIA and POLY, or between flare and quiescence . Changes in abundance of cell types may be relevant to disease mechanisms. For monocyte-related genes, we  and others  have shown that the differences in transcript abundance are not explained by differences in monocyte numbers alone, but reflect activation state. The use of medication and disease duration at the time of sampling may influence the pattern of gene expression. A larger, likely multi-center, study will be needed to rigorously control for these important variables.
Our analysis revealed overlap in molecular pathways involved in increased ESR and elevated JC in SJIA. This result was not unexpected, given reported correlations between these two parameters [30, 57]. However, the glucocorticoid receptor (GCR) signaling pathway was more significantly related to ESR than JC. Systemic symptoms of SJIA respond to exogenous steroids, suggesting the elevation of GCR signaling may represent an endogenous effort to dampen systemic inflammation. The comparable doses of exogenous steroids in the F and Q groups make it less likely that steroid therapy is inducing this pathway. Notably, polymorphism in the GCR gene is associated with the level of inflammatory activity in JIA . Involvement of GCR signaling in systemic inflammation in SJIA and stronger association of this pathway with inflammation in SJIA versus POLY (at least as reflected in blood cells) is consistent with reduced responses in SJIA patients to non-glucocorticoid drugs that are efficacious in subsets of POLY patients (for example, methotrexate and anti-TNFα [59, 60]).
We also found that the PI3K/Akt signaling pathway is more significantly related to SJIA JC than ESR. This pathway, which is activated by a variety of stimuli, including IL-1β, TNFα and IL-6, is potentially involved in IL-17 production . IL-17 could be an important factor in SJIA arthritis , particularly in the later phase. We did not assess expression of IL-17 in this study, but our preliminary data suggest that CD4+ T cells from SJIA patients secrete higher levels of IL-17 than control cells when cultured in TH17-polarizing conditions [Wong M, Mellins E, unpublished results]. Recently, enrichment of Th17 (and Th1) cells in blood of SJIA patients has been described .
Our findings are consistent with the hypothesis that dysregulation of the innate immune system makes a more prominent contribution to SJIA immunopathology than alterations of the adaptive immune system [17, 63], whereas adaptive responses are thought to drive oligoarticular and polyarticular JIA [64, 65]. However, our results implicate deficiencies in genes associated with T cell-related responses in SJIA pathology, similar to observations in other studies . For example, reduced cytolytic cell activity  and diminished function of T regulatory cells may play roles in SJIA etiology . Down-regulated genes associated with cytolytic function also participate in dendritic cell/NK cell and monocyte/NK cell interaction. Some cytolysis genes are part of the IL-15 signaling pathway, and IL-15 is involved in the development of NK cells .
In the systemic plus arthritic stage of SJIA, we found that expression of IL-10 in PBMC was positively associated with arthritis. In in vitro studies of SJIA monocytes, we and others [26, 29] observe that IL-10 is expressed after TLR stimulation, and IL-10 signaling is intact [Macaubas et al., unpublished]. Given the immunosuppressive effect of IL-10, association of this gene with arthritis in SJIA may represent an attempt by the immune system to reduce inflammation. The level of IL-10 may be inadequate to deal with the inflammatory challenge, as the frequency of a promoter allele associated with low IL-10 expression is increased in SJIA patients [69, 70]. We found that LPS-induced production of IL-10 protein in SJIA monocytes is comparable to controls .
A striking finding of this study is that deficiency in IL-4-related pathways correlates with JC in the arthritic phase of SJIA. IL-4 has been implicated in protection against arthritis. Polymorphism in the IL4Rα gene that confers reduced responsiveness to IL-4 is associated with worse outcome in RA . Low levels of circulating IL-4 are observed in patients with active POLY . IL-4 has been shown to suppress growth factor-induced proliferation of cultured rheumatoid synovial cells by interfering with the cell cycle and by decreasing cell survival . In the murine model of collagen-induced arthritis, IL-4 is protective against cartilage and bone destruction , and neutralization of IL-4 in the same model results in reversal of arthritis suppression . IL-4 is also protective in the model of proteoglycan-induced arthritis . Interestingly, in proteoglycan-induced arthritis, mice deficient in IL-4Rα showed higher IL-1β, IL-6 and MIP1a, whereas levels of IFNγ and autoantibodies were less affected. These results imply that IL-4 suppresses innate immune activity more than the adaptive system in this arthritis model . This might model the arthritis of late stage SJIA.
IL-4 inhibits expression of pro-inflammatory cytokines, such as IL-1β, TNFα [78, 79] and IL-17 . As mentioned, IL-17 is an attractive candidate for a driver of inflammation in the later arthritic phase of SJIA. Th17 cells may become IL-1 independent in SJIA, as seen in an animal model . The IL-1β independence of IL-17 action would be consistent with the decreased efficiency of anti-IL1 therapy in the later arthritic phase of SJIA . The ability of IL-4 to suppress reactivation of committed Th17 cells  may be another mechanism by with IL-4 deficiency could contribute to arthritis in SJIA. Finally, in a small, open label study, oral histone deacetylase inhibitors in patients with mean SJIA duration of five years showed significant therapeutic benefit, specifically for arthritis . This finding is consistent with the idea that distinct biology may be involved in later phase arthritis in SJIA.
We found no gene association or correlation linked with POLY joint count, and a limited number of somewhat different genes were associated with elevated ESR in POLY-JIA subjects. Our gene panel was largely derived from a SJIA-based microarray study and, as such, it has a significant bias towards SJIA-related genes. Further, the systemic nature of SJIA predicts more changes in peripheral blood than for POLY, where pathology is more localized. Our POLY cohort was itself heterogeneous, including RF+ and RF- patients, which were analyzed as one group. Most gene expression studies have analyzed RF- patients only [29, 84, 85]; some have not determined the RF status . Griffin et al. 2009 showed that RF+ and RF- patients can share a similar gene signature .
It will be of interest to determine the cell type within PBMC that is responsible for particular transcripts. Based on correlated expression patterns with more lineage specific genes, it is most likely that IL-4 transcripts derive from CD4 T cells; the IL-4 message expression is correlated with expression of CD40LG and IL2RG (not shown). In contrast, the IL-10 expression correlates with expression of IL-1, IL-1-related genes and IL-6 (not shown), suggesting IL-10 transcripts are expressed in monocytes. Further studies are also needed to determine the specificity of the SJIA gene signature in relation to other acute inflammatory diseases, such as bacterial and viral infections and other rheumatologic pediatric diseases . Nonetheless, our current results add to the growing evidence that different molecular mechanisms distinguish SJIA from other JIA subtypes [10, 11, 26, 29, 32].