Transcriptional profiling of PBMCs unravels B cell mediated immunopathogenic imprints of HCV vasculitis

RESEARCH ARTICLE Transcriptional profiling of PBMCs unravels B cell mediated immunopathogenic imprints of HCV vasculitis Emily Comstock1, Cheol-Woo Kim2, Alison Murphy3, Benjamin Emmanuel1, Xi Zhang3, Michael Sneller3, Bhawna Poonia1, Shyamasundaran Kottilil1,3* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Division of Clinical Care and Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, United States of America, 2 Department of Internal Medicine, Inha University, Incheon, South Korea, 3 Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, United States of America * Abstract OPEN ACCESS Citation: Comstock E, Kim C-W, Murphy A, Emmanuel B, Zhang X, Sneller M, et al. (2017) Transcriptional profiling of PBMCs unravels B cell mediated immunopathogenic imprints of HCV vasculitis. PLoS ONE 12(12): e0188314. https:// doi.org/10.1371/journal.pone.0188314 Editor: Ranjit Ray, Saint Louis University, UNITED STATES Received: June 26, 2017 Accepted: November 3, 2017 Published: December 11, 2017 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported in whole by the Intramural Research Program of the NIH, [National Institute of Allergy and Infectious Diseases]. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. B cell depletion therapy using rituximab has been shown to be effective in achieving remission in patients with HCV-mixed cryoglobulinemic (MC) vasculitis. Previously, we have demonstrated abnormalities in peripheral immune cells involving neutrophils, chemotaxis, and innate immune activation among patients with HCV-MC vasculitis when compared to HCV patients without vasculitis. In this study, we evaluated the effect of B cell depletion therapy on transcriptional profiles of peripheral blood mononuclear cells before and after riruximab therapy, in order to unravel the pathogenic mechanism involved in HCV-MC vasculitis induced by abnormal B cell proliferation. DNA microarray analysis was performed using RNA from PBMCs from seven patients with HCV-MC vasculitis and seven normal volunteers. DNA was hybridized to Affymetrix U133A chips. After normalization, differentially expressed gene list with treatment was generated using partitional clustering. RT-PCR, flow cytometry, and enzyme immunoassay (EIA) was used to validate DNA microarray findings. Differentially expressed genes included B cells and non-B cell genes. Validation of genes using purified cell subsets demonstrated distinct effect of B cell depletion therapy on non-B cells, such as monocytes, T cells, and NK cells. Notably, B lymphocyte stimulator (BLyS) levels were persistently elevated in patients who subsequently relapsed. In conclusion, pathogenesis of HCV-MC vasculitis is mediated by abnormal proliferation of B cells, driven by BLyS, leading to significant effects on non-B cells in mediating symptomatology. Future therapeutics using a combination approach of B cell depletion and proliferation may be desired to achieve long-term remission. Introduction While estimates vary, chronic hepatitis C (CHC) infection is present in approximately 71 to 170 million people globally [1–2]. Hepatitis C virus (HCV) is a single-stranded RNA Flavivirus that preferentially infects human hepatocytes [3]. Over time, CHC can lead to progressive liver fibrosis and cirrhosis of the liver. CHC is also the leading cause of hepatocellular carcinoma PLOS ONE | https://doi.org/10.1371/journal.pone.0188314 December 11, 2017 1 / 14 HCV vasculitis and B cell effects and liver transplantation [4–5]. A unique feature of CHC is the association with several extrahepatic manifestations, among which most commonly include: mixed cyroglobulinemic (MC) vasculitis, lymphoproliferative disorders, and insulin resistance [6–7]. Of these, Type II MC vasculitis is the most strongly associated with, and directly attributed to, CHC as more than 80% of patients with persistent MC vasculitis are seropositive for HCV [8–10]. Additionally, MC vasculitis is known to be a negative prognostic factor of virological response to HCV treatment and is generally associated with a high morbitity and mortality rate [11–12]. The pathogenesis of HCV-associated MC vasculitis is characterized by a preferential expansion of B cells, which are presumably triggered by HCV antigens or epitopes [8, 13–14]. These clonally expansive B cells produce soluble IgM with rheumatoid factor activity that has been shown to develop into immune complexes [15]. These complexes subsequently deposit in small vessels, ultimately resulting in vasculitis [8, 13]. The disease manifests with tissue and organ damage, particularly of the kidneys (glomeruli) and the skin. As a result, common clinical manifestations include membranoproliferative glomerulonephritis and cutaneous vasculitis [6, 16–17]. Various studies have demonstrated that patients diagnosed with MC vasculitis can be effectively treated with B cell depletion therapy [17–23]. B lymphocyte stimulator (BLyS, also known as the B cell–activating factor belonging to the TNF family, or BAFF) plays a major role in B cell homeostasis [24]. The BLyS protein is expressed as a trimer on monocytes, activated neutrophils, T cells, and dendritic cells [25–27], but can also be released into the circulation. Leading to the scertion of inflammatory cytokines, such as IL-2, TNF-α, and IFN-γ [26, 28– 29]. BLyS can bind to 3 receptors: BLyS receptor 3 (BR3; also known as BAFF-R), transmembrane activator–1 and calcium modulator and cyclophilin ligand–interactor (TACI), and B cell maturation antigen (BCMA). BLyS is the sole ligand for BR3, whereas TACI and BCMA each can bind either BLyS or another TNF family ligand known as a proliferation-inducing ligand (APRIL) [30]. These ligand-receptor interactions vary in affinity: BLyS binds more strongly to BR3 than to TACI or BCMA, whereas APRIL displays the reverse affinity hierarchy. Elevated serum BLyS levels are frequently observed in patients with autoimmune Systemic lupus erytematosus (SLE). The use of a fully human monoclonal antibody that binds soluble BLyS (i.e., belimumab) in serologically active SLE patients has resulted in reductions in disease activity and B cell populations, resulting in symptomatic relief for most patients [31]. However, these effects are not indefinitely sustained, as many patients experien (...truncated)