Serum proteome analysis of systemic JIA and related pulmonary alveolar proteinosis identifies distinct inflammatory programs and biomarkers

Objectives Recent observations in systemic Juvenile Idiopathic Arthritis (sJIA) suggest an increasing incidence of high-mortality interstitial lung disease, characterized by a variant of pulmonary alveolar proteinosis (PAP). Co-occurrence of macrophage activation syndrome (MAS) and PAP in sJIA suggested a shared pathology, but sJIA-PAP patients also commonly experience features of drug reaction such as atypical rashes and eosinophilia. We sought to investigate immunopathology and identify biomarkers in sJIA, MAS, and sJIA-PAP. Methods We used SOMAscan to measure >1300 analytes in sera from healthy controls and patients with sJIA, MAS, sJIA-PAP and other related diseases. We verified selected findings by ELISA and lung immunostaining. Because the proteome of a sample may reflect multiple states (sJIA, MAS, sJIA-PAP), we used regression modeling to identify subsets of altered proteins associated with each state. We tested key findings in a validation cohort. Results Proteome alterations in active sJIA and MAS overlapped substantially, including known sJIA biomarkers like SAA and S100A9, and novel elevations of heat shock proteins and glycolytic enzymes. IL-18 was elevated in all sJIA groups, particularly MAS and sJIA-PAP. We also identified an MAS-independent sJIA-PAP signature notable for elevated ICAM5, MMP7, and allergic/eosinophilic chemokines, which were all previously associated with lung damage. Immunohistochemistry localized ICAM5 and MMP7 in sJIA-PAP lung. ICAM5’s ability to distinguish sJIA-PAP from sJIA/MAS was independently validated. Conclusions Serum proteins support an sJIA-to-MAS continuum, help distinguish sJIA, sJIA/MAS, and sJIA-PAP, and suggest etiologic hypotheses. Select biomarkers, such as ICAM5, could aid in early detection and management of sJIA-PAP. ### Competing Interest Statement VS reports personal fees from Novartis, grants from Novartis, outside the submitted work. SC reports grants from Systemic JIA Foundation, grants from NICHD, grants from RK Mellon Institute, during the conduct of the study; grants from AB2Bio, Ltd., other from Simcha Therapeutics, grants from IMMvention Therapeutix, outside the submitted work. RG-M reports grants from Systemic JIA Foundation during the conduct of the study; grants from Lilly, grants from SOBI outside the submitted work. EM reports grants from Lucille Packard Foundation for Children Health, grants from CARRA-Arthritis Foundation, during the conduct of the study; grants from Novartis, grants from GSK, outside the submitted work. PK reports personal fees and other from Inflammatix, Inc., personal fees from Cepheid, outside the submitted work. GS reports personal fees from Novartis, personal fees from SOBI, outside the submitted work. GD reports personal fees from Novartis Pharmaceuticals, outside the submitted work. AG reports grants and personal fees from Sobi, grants and personal fees from Novartis, grants and personal fees from AB2Bio, during the conduct of the study. ### Funding Statement The SOMAscan assay was supported by a grant to the Intramural Research Program of the NIAID from the Systemic JIA Foundation. AAdJ and RGM were supported by the NIAID intramural research program. GC is an Eli Lilly Fellow of the Life Science Research Foundation. SJ is supported by Dean Postdoctoral Fellowship, School of Medicine, Stanford. CM and EDM were supported by NIAMS R01 AR066551, AR061297 and Arthritis Foundation Great West Region Arthritis Center of Excellence; VS and EDM were supported by the Lucille Packard Foundation for Children Health and a Childhood Arthritis and Rheumatology Research Alliance/Arthritis Foundation grant. CS and SWC were supported by the RK Mellon Institute for Pediatric Research, NIAID K22 AI123366, and NICHD R01 HD098428. PK is funded in part by the Bill and Melinda Gates Foundation (OPP1113682); NIAID 1U19AI109662, U19AI057229, and 5R01AI125197 grants; Department of Defense contracts W81XWH-18-1-0253 and W81XWH1910235; and the Ralph & Marian Falk Medical Research Trust. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics approval for collection of clinical data and patient serum through institutional review boards at the following institutions: Stanford University, University of Cincinnati, University of Pittsburgh and National Institute of Allergy and Infectious Diseases at the National Institutes of Health. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The SOMAscan data will be uploaded to GEO (Gene Expression Omnibus).