Systemic lupus erythematosus (SLE) is a multi-organ rheumatologic disease characterized by immune dysregulation and a heterogeneous disease course, with heightened disease severity in pediatric patients. Conventional clinical and laboratory parameters are not sufficiently sensitive or specific for detecting ongoing disease activity or response to specific immunomodulators, underscoring a need to better delineate the underlying immune changes that drive unpredictable flare-ups of disease activity. The lack of knowledge regarding the precise immune cellular and molecular events leading to SLE disease poses a significant hurdle in the effort to develop accurate prognostic disease biomarkers and selective therapeutic agents. Our long- term goal is to identify immune cellular and molecular mediators that could provide targets for therapeutic intervention. The objective of this proposal is to elucidate the mechanisms by which serum-circulating factors including exosomes modulate immune cellular and cytokine derangements in pediatric SLE pathogenesis. The rationale for the proposed research is that understanding these mechanisms could identify novel disease biomarkers that permit accurate prognosis and provide selective therapeutic targets. To achieve this goal, Dr. Hsieh will use a high-dimensional mass cytometry platform, which offers single-cell analysis of over 40 parameters, utilizing rare earth metal isotopes instead of fluorophores as tags bound to antibodies. In a pilot study, Dr. Hsieh established a quantitative and reproducible mass cytometry assay that allows precise monitoring of phenotypic (surface markers) and functional (cytokines/chemokines) immune derangements detectable in peripheral blood of pediatric SLE patients. The data from this study form the basis of Dr. Hsieh's central hypothesis that pediatric SLE patients share a unique multi-parametric monocyte cytokine signature, which drives disease activity and is propagated by serum factors (including exosomes) through activation of the JAK/STAT signaling pathway. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Determine the association of the cytokine signature (and other immune changes) with SLE disease activity, by integrating mass cytometry immune profile data and clinical parameters from a prospective longitudinal pediatric SLE patient cohort; 2) Elucidate immunomodulatory properties of SLE serum-isolated exosomes, by evaluating the ability of diseased serum exosomes to induce the cytokine signature, and identifying exosome proteomic components essential for such activity; and 3) Determine the capacity of ruxolitinib (JAK1/2 inhibitor) to abrogate expression of the cytokine signature, by evaluating ex vivo immunosuppressive effects (including off-target effects) of ruxolitinib on immune derangements observed in pediatric SLE pathogenesis. Dr. Hsieh is an Assistant Professor at the University of Colorado Denver/Children's Hospital Colorado in the Departments of Immunology and Microbiology, and Pediatrics. As a clinical fellow, she trained in Dr. Garry Nolan's laboratory (Stanford), acknowledged as world-class in the application of mass cytometry to single-cell analysis, where she acquired expertise in this technology. Dr. Hsieh has assembled a mentoring team composed of a multidisciplinary group of scientists with research expertise in basic science immunology, particularly immune signaling alterations in autoimmunity; clinical study design and implementation; exosome biology; and biostatistics. Her primary mentor Dr. John Cambier and co-mentor Dr. V. Michael Holers are well- established scientists with collaborations focused on autoimmune disorders and the immune system. Their extensive experience with trainees will be complemented by the expertise of her scientific advisory committee, co-mentor Dr. Michael Graner, internationally known expert in the field of exosome biology, advisors/collaborators Dr. Jennifer Soep, pediatric rheumatologist with a clinical research focus on SLE, and Dr. Debashis Ghosh, Chairman of Biostatistics with expertise on high-throughput data analysis. Collectively, this team will provide an outstanding training environment that will fill critical gaps in her toolbox and knowledge base to enhance her ability to study immune derangements in pediatric SLE patients. Dr. Hsieh has created a career development plan that will prepare her for successful transition to independence, incorporating didactic and mentored training in: 1) basic science immunology; 2) clinical study design and implementation; 3) exosome biology; and 4) biostatistics and computational biology. The proposed approach is innovative, in the applicant's opinion, because it departs from the status quo by elucidating specific mechanisms underlying inflammatory perturbations in SLE (e.g., serum exosome-mediated immune modulation) and by characterizing these mechanisms as they occur in the context of multiple immune cell phenotypes, intracellular cytokine networks, and remitting/relapsing disease activity, with single-cell level resolution. The proposed research is significant because it will likely identify candidate disease biomarkers for prognostic and therapeutic purposes in SLE, and will likely delineate novel therapeutic targets derived from serum exosomes and their proteomic components. The knowledge gained will have broad translational importance through application of this systems immunology approach to other systemic rheumatologic disorders.

Public Health Relevance

Systemic lupus erythematosus (SLE) is a severe multi-organ rheumatologic disease characterized by immune dysregulation and a heterogeneous disease course, with unpredictable flare-ups of disease activity. Approximately 20 percent of SLE cases are diagnosed in childhood, and children exhibit more active and severe disease than adults, underscoring the need to better understand pediatric disease pathogenesis. The proposed research is relevant to public health because it aims to elucidate cellular and molecular mechanisms that govern immune derangements in pediatric SLE pathogenesis, which could identify novel disease biomarkers that permit accurate prognosis, and provide selective therapeutic targets. Thus, the proposed research is relevant to the NIH mission to acquire fundamental knowledge that will help reduce morbidity and mortality in children with autoimmune disease.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Mentored Patient-Oriented Research Career Development Award (K23)
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Arthritis and Musculoskeletal and Skin Diseases Special Grants Review Committee (AMS)
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Witter, James
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University of Colorado Denver
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