JIA is a prevalent form of arthritis among children that leads to restricted joint mobility caused by both RF-negative pJIA and oJIA. In pursuit of enabling early diagnosis, we conducted a bioinformatic analysis of two datasets, namely, GSE20307 and GSE13501, utilizing various tools. Among these, six pivotal genes were identified, out of which CXCL8, JUN, KRAS, and SOCS3 displayed high diagnostic efficacy in RF-negative pJIA, whereas JUN, CXCL8, PTGS2, NFKBIA, and SOCS3 exhibited similar performance in oJIA, as determined by ROC curve analysis. To validate our findings, we analyzed the expression levels of the hub genes in RF-negative pJIA, oJIA, and healthy controls using the GSE13501 dataset. The outcomes revealed significantly upregulated expression of CXCL8, JUN, KRAS, and SOCS3 in RF-negative pJIA patients and JUN, CXCL8, PTGS2, NFKBIA, and SOCS3 in oJIA patients compared with the control group, which is consistent with our bioinformatics analysis.
As per the PRINTO New Classification Criteria for Juvenile Idiopathic Arthritis, JIA can be classified into six distinct categories: A. Systemic JIA, B. RF-positive JIA, C. Enthesitis/spondylitis-related JIA, D. Early-onset ANA-positive JIA, E. Other JIA, and F. Unclassified JIA5. The revised classification criteria reshuffle the majority of patients who were previously categorized as oligoarthritis and included in the RF-polyarthritis and PsA categories and place them under Category D. Early-onset ANA-positive JIA, which aligns with our study findings. Our observations indicate that hub genes, including JUN, SOCS3, and CXCL8, were shared between RF-negative pJIA and oJIA. The identification of the JUN protein can be traced back to cells carrying avian sarcoma virus 11,12, and it belongs to the proto-oncogene family13,14. The c-Jun amino-terminal kinases (JNKs), which are proline-directed kinases and are activated by UV radiation and oncoproteins, have been identified15,16. Similar to ERKs, JNKs preferentially phosphorylate serine and threonine located within Pro-Xaa-Ser/Thr-Pro sequences, and they participate in the MAPK pathway, which may be related to RF-negative pJIA and oJIA. SOCS3, a member of the SOCS protein family, acts as a classical negative regulator of JAK/STAT pathway signaling and consists of eight similar proteins17,18. The feedback inhibition of the JAK/STAT3 pathway by the SOCS3 protein involves its binding to its corresponding receptor, the primary receptor shared interleukin-6 (IL-6) receptor subunit gp130, to inhibit STAT3 phosphorylation19–21. SOCS3 acts as a negative regulator of cytokine or hormone signaling, but it is capable of positively regulating inflammatory responses in some cases, where it inhibits STAT3. Aberrant expression levels of SOCS3/STAT3 have been found in various bone marrow and lymphocytes, as well as in different nonhematopoietic cells, indicating their involvement in various infectious and inflammatory diseases.
CXCL8 is a well-studied proinflammatory chemokine that has been extensively researched. Neutrophil-activating factor (NAF) was discovered in the late 1980s by Peveri et al., and it was found to activate neutrophil exocytosis and oxidative burst through cell surface receptors in LPS-stimulated blood monocytes22. After its isolation and sequencing, NAF was named interleukin-8 (IL8) and CXCL8 due to the identification of other chemokines23–25. CXCL8 is released by various cells, such as monocytes23,25,26, T lymphocytes27,28, macrophages, synovial cells29, and keratinocytes30, after appropriate stimulation. CXCR1 and CXCR2 are the two well-known receptors for CXCL8 that are expressed not only on leukocytes but also on other cell types, such as endothelial cells, smooth muscle cells, and fibroblasts. Evidence suggests that the activation of these receptors may contribute to several actions, including angiogenesis31. Numerous studies have indicated that CXCL8 and other IL-8 family chemokines induce endothelial cell chemotaxis in vitro and angiogenesis in vivo32. Therefore, CXCL8 might play a critical role in the early synovitis of arthritis that is characterized by vascular proliferation in the synovial membrane where CXCL8 can exert its effect.
This study aimed to further investigate the correlation between three effective biomarkers (JUN, SOCS3, CXCL8) and immune infiltrating cells, which play a crucial role in rheumatoid arthritis (RA), specifically in RF-negative pJIA and oJIA. The results showed a positive correlation between JUN and CXCL8 expression levels and the abundance of B-cell naive and T follicular helper cells. The term T follicular helper cells was coined in a series of studies on human tonsillar germinal center (GC) CD4 + T cells and CXCR5 + CD4 + T cells in the blood33. Previous research has demonstrated that T follicular helper cells are critical in RA progression and are linked to ectopic lymphoid structures (ELSs) in the joints, which consist of B cells, CD4 + T cells, and GCs34. CXCL13-expressing CD4 + T cells are associated with ELSs in joints35, and GC-Tfh cells are the primary source of CXCL13 in human lymphoid tissue36. Therefore, Tfh cells are believed to contribute to the development of ELSs and support the autoantibody response of B cells. However, the biology of the interaction between Tfh cells and ELSs requires further exploration.
The present study has several limitations, as it relied solely on gene transcriptome analysis and lacked multiomics experiments. Furthermore, the data analysis was conducted using only the bioinformatics commit method, and subsequent in vivo and in vitro validation experiments are required to verify the findings. To overcome these limitations, future studies will incorporate a multiomics approach. Additionally, the next study aims to investigate the differences in arthritis between oJIA species with and without uveitis.
In summary, this study successfully identified six critical genes (JUN, SOCS3, CXCL8, KRAS, NFKBIA, and PTGS2) as biomarkers for the early diagnosis of RF-negative pJIA and oJIA. Furthermore, the study provided insights into the landscape of immune cells associated with RF-negative pJIA and oJIA and suggested that Tfh cells may contribute to the early onset of both RF-negative pJIA and oJIA.