Background/Purpose: We aimed to explore the association between HLA-DRB1*15 alleles, eosinophilia, and lung disease (LD) in patients with systemic juvenile idiopathic arthritis (sJIA) through proteomic profiling of circulating immune proteins.

Methods: Peripheral blood (PB) was obtained from children with sJIA (PRINTO criteria) and healthy controls evaluated at our center. Disease status (active/remission) was defined by the treating clinician and confirmed through chart review based on the presence/absence of fever, rash, arthritis, serositis, hepatosplenomegaly, and/or elevated inflammatory markers. Eosinophilia was defined as an absolute eosinophil count (AEC) > 500/μl. A pulmonologist adjudicated the diagnosis of sJIA-LD. HLA typing was done on a clinical basis or through research testing of samples in our institutional biorepository. A proximity extension assay (PEA, Olink Proteomics) was used to profile 45 immune proteins in plasma. Mann-Whitney U tests corrected for an FDR of 0.1 for our discovery analysis were used to compare groups. Volcano plots were generated with GraphPad Prism while PCA plots were created with Clustvis Web Tool.

Results: Table 1 summarizes study participants. Most sJIA patients treated with IL-1 inhibitors at the time of sampling. As expected, broad differences in protein expression were found between active sJIA patients and controls (Fig1A). Previously reported sJIA biomarkers, CCL7, CXCL10, CXCL11, HGF, IL-6, MMP1, and OSM, were confirmed to be significantly elevated in patients with active disease compared to those in remission (Fig1B). We also identified novel proteins differentially expressed in this comparison, including CCL3, CSF3, and TNFSF12/TWEAK (Fig1B). Next, we evaluated sJIA patients with or without eosinophilia at the time of sampling, stratified by disease activity. By univariate testing, IL-33 was elevated in sJIA patients with vs. without eosinophilia, regardless of disease status (Fig2A). In patients with active sJIA, IL-13 and LTA were increased while CXCL12 and OSM were decreased in those with vs. without eosinophilia (univariate testing) (Fig 2A). Levels of circulating immune proteins were not significantly different in patients with and without HLA-DRB1*15:01 alleles when stratified by disease activity and PCA confirmed this finding (Fig2B). Finally, PCA showed that samples obtained from sJIA patients during an episode of eosinophilia closely associated with samples from patients with sJIA-LD who did not have eosinophilia at the time of testing (Fig 2C).

Conclusion: We identified a network of immune proteins in sJIA linked with eosinophilia and type 2-related cytokines. This includes OSM, an IL-6 family member that induces eotaxin-1 release, in sJIA patients with active disease. The IL-1 cytokine family member IL-33, which induces innate lymphoid 2 cells (ILC2s) to produce IL-4, -5, and -13, was associated with eosinophilia in sJIA patients. HLA status did not significantly impact the profile of circulating immune proteins in this cohort. Our studies suggest that the sJIA cytokine milieu may promote type-2 immunity and these responses are likely important in patients who develop eosinophilia and lung disease.

Figure 1. Differential expression of circulating immune proteins in study participants. Volcano plot of immune proteins from plasma in patients with A) active sJIA (n=25) vs. healthy controls (n=51), and B) active sJIA vs. sJIA patients in remission (n=21). Dots in red are significantly higher in patients with active sJIA than the comparison group, with dots in blue representing significantly lower levels. Statistics: Mann-Whitney U tests corrected for an FDR of 0.1.

sJIA, systemic juvenile idiopathic arthritis; CCL, C-C motif chemokine; CSF, colony stimulating factor; CXCL, C-X-C motif chemokine; EGF, pro-epidermal growth factor; FLT3LG, fms-related tyrosine kinase 3 ligand; HGF, hepatocyte growth factor; IFNG, interferon gamma; IL, interleukin; LTA, lymphotoxin-alpha; MMP, matrix metallopeptidase; OLR, oxidized low-density lipoprotein receptor; OSM, oncostatin-M; TGFA, transforming growth factor alpha; TNF, tumor necrosis factor; TNFSF, tumor necrosis factor ligand superfamily; TSLP, thymic stromal lymphopoietin; VEGFA, vascular endothelial growth factor A.

Figure 2. Immune profile of patients with sJIA and eosinophilia.

A) Expression level of the given immune protein found in the plasma of patients with sJIA in remission with (n=5) and without eosinophilia (n=12) or during active disease with (n=6) and without eosinophilia (n=17). Eosinophilia was defined as AEC >500/μL by clinical CBC on the day the research sample was collected. Only the immune proteins that were significantly different based on Mann-Whitney U tests are reported. P value, ns, >0.05; *, ≤ 0.05; **, ≤ 0.01. No significance was found after correction for multiple comparison. B-C) PCA plots using z-scores for all 45 proteins in the PEA were included. 95% confidence level was used for ellipses. B) Groupings of patients during active sJIA with (n=8) and without HLA-DRB1*15:01 alleles (n=11), and patients in remission with (n=7) and without HLA-DRB1*15:01 alleles (n=9) are shown in their respective colors. C) Groupings of patients with active sJIA (n=19), healthy controls (n=51), patients with eosinophilia (n=8), and patients with sJIA-LD (n=4) are shown in their respective colors.

sJIA, systemic juvenile idiopathic arthritis; AEC, absolute eosinophil count; PEA, proximity extension assay; CXCL, C-X-C motif chemokine; IL, interleukin; LTA, lymphotoxin-alpha; OSM, oncostatin-M

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-circulating-immune-profile-of-systemic-jia-patients-with-hla-drb115-alleles-eosinophilia-and-lung-disease/