Neutrophil and mononuclear leukocyte pathways and upstream regulators revealed by serum proteomics of adult and juvenile dermatomyositis

Abstract

Objectives: Serum protein abundance was assessed in adult and juvenile dermatomyositis (DM and JDM) patients to determine differentially regulated proteins, altered pathways, and candidate disease activity biomarkers.

Methods: Serum protein expression from 17 active adult DM and JDM patients each was compared to matched, healthy control subjects by a multiplex immunoassay. Pathway analysis and protein clustering of the differentially regulated proteins were examined to assess underlying mechanisms. Candidate disease activity biomarkers were identified by correlating protein expression with disease activity measures.

Results: Seventy-eight of 172 proteins were differentially expressed in the sera of DM and JDM patients compared to healthy controls. Forty-eight proteins were differentially expressed in DM, 32 proteins in JDM, and 14 proteins in both DM and JDM. Twelve additional differentially expressed proteins were identified after combining the DM and JDM cohorts. C-X-C motif chemokine ligand 10 (CXCL10) was the most strongly upregulated protein in both DM and JDM sera. Other highly upregulated proteins in DM included S100 calcium binding protein A12 (S100A12), CXCL9, and nicotinamide phosphoribosyltransferase (NAMPT), while highly upregulated proteins in JDM included matrix metallopeptidase 3 (MMP3), growth differentiation factor 15 (GDF15), and von Willebrand factor (vWF). Pathway analysis indicated that phosphoinositide 3-kinase (PI3K), p38 mitogen-activated protein kinase (MAPK), and toll-like receptor 7 (TLR7) signaling were activated in DM and JDM. Additional pathways specific to DM or JDM were identified. A protein cluster associated with neutrophils and mononuclear leukocytes and a cluster of interferon-associated proteins were observed in both DM and JDM. Twenty-two proteins in DM and 24 proteins in JDM sera correlated with global, muscle, and/or skin disease activity. Seven proteins correlated with disease activity measures in both DM and JDM sera. IL-1 receptor like 1 (IL1RL1) emerged as a candidate global disease activity biomarker in DM and JDM.

Conclusion: Coordinate analysis of protein expression in DM and JDM patient sera by a multiplex immunoassay validated previous gene expression studies and identified novel dysregulated proteins, altered signaling pathways, and candidate disease activity biomarkers. These findings may further inform the assessment of DM and JDM patients and aid in the identification of potential therapeutic targets.

Keywords: Biomarkers; Dermatomyositis; Disease activity; Juvenile dermatomyositis; Monocytes; Multiplex proteomics; Neutrophils.

Fig. 1

Differentially expressed proteins in adult dermatomyositis (DM) and juvenile dermatomyositis (JDM) patients compared to healthy controls. Red indicates upregulation, blue indicates down regulation, and white indicates no change. The row groupings indicate the proteins that had significantly different expression between DM or JDM patients and healthy controls in the adult analysis (adult), the juvenile analysis (juvenile), both the adult and juvenile analyses (shared), as well as the new proteins that arose when the adult and juvenile datasets were combined (combined). Abbreviations: CON, adult control; DM, adult dermatomyositis; JCON, juvenile control; JDM, juvenile dermatomyositis; FC, fold change

Fig. 2

Protein-protein correlations in adult dermatomyositis (DM) patients identified a cluster associated with neutrophil, monocyte, and myeloid dendritic cell expression. (A) DM protein clustering of the 78 differentially expressed proteins. Correlations that met the thresholds of p < 0.05 and |Spearman’s rank correlation coefficient| > 0.4 were clustered. (B) The largest protein cluster, boxed in panel A, was further examined. (C) Relative expression levels of the clustered proteins in immune cell lineages. Abbreviations: DM, adult dermatomyositis; NA, not applicable

Fig. 3

Differentially expressed proteins in adult dermatomyositis (DM) patients are involved in p38 MAPK signaling and immune cell movement. The top of the diagram depicts predicted upstream regulators, below which measured proteins are displayed in ovals. Below the measured proteins, predicted functional outputs of the measured proteins are displayed in three ovals. At the bottom of the diagram, measured proteins associated with the identified functions, but not the upstream regulators, are displayed. Orange boxes and lines indicate predicted activation; light blue boxes and lines indicate predicted inhibition; olive lines indicate the relationship is inconsistent with the state of the downstream molecule; gray lines indicate an association without predicted directionality. Red ovals indicate a positive fold change; dark blue ovals indicate a negative fold change. Abbreviations: DM, adult dermatomyositis

Fig. 4

Correlations between differentially regulated proteins in adult dermatomyositis (DM) and juvenile dermatomyositis (JDM) patients and myositis disease activity measures. (A) Disease activity measures were categorized as measures of global, muscle, or skin disease activity. Correlations that met the thresholds of p < 0.05 and |Spearman’s rank correlation coefficient| > 0.4 are displayed. Red indicates a positive correlation; blue indicates a negative correlation. The left heatmap depicts DM correlations; the right heatmap depicts JDM correlations. (B) Comparisons of the proteins that correlated at least moderately with global, muscle, and skin disease activity in DM versus JDM patients. Shared and distinct correlated proteins from each category are tabulated below their respective Venn diagram. Abbreviations: DM, adult dermatomyositis; JDM, juvenile dermatomyositis; DAS, Disease Activity Score; MDAAT, Myositis Disease Activity Assessment Tool; MMT, Manual Muscle Testing; CMAS, Childhood Myositis Assessment Scale; NA, not applicable