The concurrent collection of RNA microarray data and detailed PBMC cell phenotyping provided powerful means to determine associations between different gene-expression signatures and PBMC subpopulations. This is based on the fact that differences in gene-expression patterns seen in the peripheral blood are due not only to the up- and downregulation of gene expression but also to the over- or underrepresentation of certain cell populations. The data presented in this study demonstrate that specific gene-expression signatures in sJIA are associated with the expansion of immature PBMC subpopulations and the active disease state. Furthermore, an expansion of precursor cells and upregulation of an erythropoiesis gene-expression signature occurred predominantly in individuals with sJIA and anemia. Strikingly, a discrepancy was noted with strong erythropoiesis-related gene expression and expansion of early precursor cells in peripheral circulation and bone marrow but an absence of reticulocytosis.
The etiology and pathogenesis of sJIA are poorly understood. The lack of specific biomarkers makes difficult the diagnosis and differentiation from other febrile conditions, and, therefore, the development of new diagnostic tests is highly desirable. The availability of whole-genome gene-expression analysis technology has allowed the discovery of sJIA-specific gene-expression signatures, opening a window into disease pathogenesis and diagnosis [14
In a previous study of sJIA, we demonstrated strong PBMC gene-expression signatures that allowed the distinction between patients with new-onset sJIA and healthy controls [14
]. Although several signatures were apparent, the most robust gene-expression signature was an overexpressed 67-probe-set erythropoiesis signature consisting of a large number of strongly erythropoiesis-related mRNAs. "Erythropoiesis" gene-expression signatures have been reported by others (for example, by transcriptional profiling during in vitro
lineage-specific differentiation of bone marrow-derived CD34+
precursor cells) [23
]. In addition, the reticulocyte transcriptome derived from human umbilical cord blood reticulocytes and adult reticulocytes contains many of the genes described in our erythropoiesis signature [24
]. In the context of disease and in the peripheral blood, however, reports of erythropoiesis-specific gene-expression signatures have been scarce. The underexpression of erythropoiesis-specific signatures has been reported predominantly [25
]. Chua et al.
] demonstrated underexpression of an 11-gene erythropoiesis cluster in anemia of chronic renal allograft rejection [25
]; of these 11 underexpressed genes, four are found overexpressed within our 67-gene erythropoiesis signature. Ebert et al.
] demonstrated underexpression of a 47-gene erythropoiesis signature in patients with lenalidomide-responsive myelodysplastic syndrome; of those 47 underexpressed genes, 16 are found overexpressed within our 67-gene erythropoiesis signature. Therefore, the presence of a "negative" gene-expression signature in the disease state indicates that the corresponding genes likely are expressed in the healthy state. To our knowledge, overexpression or upregulation of a similar signature was not reported in the literature before our initial description. Notably, in gene-expression studies of sickle cell anemia, which is characterized by severe hemolysis and subsequent expanded erythropoiesis, little to no overlap is found with our gene-expression signature [27
Our data suggest that the origin of the erythropoiesis-related gene-expression signature may lie within immature and precursor cell subpopulations, most likely CD71+
, based on the overlap of the signature with the gene-expression pattern observed in isolated CD71+
cells. One flaw of our study is that the study was not designed a priori
to investigate erythrocyte precursors directly (by flow cytometry) and that some observations were made a posteriori
. Therefore, more-detailed phenotyping of erythrocyte precursors should be considered in future studies to prove the suggested link. Another strong signature, the "innate immune response cluster," also correlated strongly with the immature cell populations, suggesting that they may have a central role in the pathogenesis of sJIA. The expansion of these cell populations and upregulation of the erythropoiesis-related signature was found most prominently in patients with sJIA and anemia, and it was not seen in patients with other types of JIA and anemia. The signature therefore appears to be a characteristic feature of patients with sJIA. Replication in an independent cohort is the gold standard to validate findings. Our data were confirmed by a non-overlapping cohort previously published by Allantaz et al.
] that included patients with other febrile diseases such as bacterial infections, SLE, and PAPA syndrome. The analysis suggests that the erythropoiesis signature is rather specific for the presence of active sJIA (fever present), whereas it was not present in patients with inactive sJIA (no fever and no arthritis). Of note, a significant number of patients with bacterial infections also overexpressed the erythropoiesis signature, pointing toward a common pathogenic mechanism responsible for the occurrence of the signature.
An important discrepancy with strong erythropoiesis-related gene expression and expansion of precursor cells is found in the context of severe anemia, but conversely, an absence of reticulocytosis, consistent with ineffective erythropoiesis. Ineffective erythropoiesis is the consequence of the premature destruction of erythrocyte precursors either within the bone marrow or shortly after they reach the peripheral blood [28
]. According to Cazzola et al.
], the phenotype of the severe anemia in sJIA is characterized by microcytosis, hypoferremia, hyperferritinemia, normal adjusted serum erythropoietin levels, and normal, unadjusted erythroid blast-forming unit levels, which the authors attribute to high circulating levels of interleukin-6 (IL-6). IL-6 is markedly increased in sJIA [29
], and it has a prominent effect on erythropoiesis [31
] by inducing hepatic expression of hepcidin and subsequent decreased intestinal absorption of iron and by inducing ferritin expression in monocytes/macrophages, causing iron sequestration. The overall effect of these actions is a markedly diminished delivery of iron to the proliferating erythrocyte precursor pool. Impaired iron delivery could potentially explain the divergence between the presence of a strong erythropoiesis expression signature and the absence of reticulocytosis as a "maturation block" may occur. In addition, IL-6 has a strong stimulatory effect on other aspects of hematopoiesis [32
].This suggests that IL-6 may contribute to the expansion of CD34+
cells and the erythropoiesis signature in active sJIA. However, the absence of strong correlation between serum levels of IL-6 and the strength of the signature in our cohort suggests that other factors contribute to the development of this phenomenon. The presence of increased ineffective erythropoiesis has previously been reported in patients with adult rheumatoid arthritis and anemia [28
]. It is possible that the severe anemia observed in sJIA has similar underlying pathogenic mechanisms, but detailed erythrokinetic studies have not been performed in sJIA.
Another striking feature is the similarity between sJIA/MAS, FHLH, and bacterial infection/sepsis in many aspects, with findings of hypercytokinemia, expansion of cytokine-driven macrophages, hemophagocytosis, and an increase in sCD163 levels [36
]. These similarities suggest a pathogenetic link between these conditions [39
] and may suggest that hemophagocytosis plays an important role in sJIA as well. This view is supported by the fact that 30% to 50% of patients with new-onset sJIA have evidence for subclinical hemophagocytosis [7
]. The close clustering of sJIA, sJIA/MAS, and FHLH patients by using the erythropoiesis signature and the evidence for overexpression of the erythropoiesis signature in some patients with bacterial infection further support the hypothesis of a shared pathogenic mechanism that may include hypercytokinemia, hemophagocytosis, ineffective erythropoiesis, and expansion of immature precursor cells.