title: The HLA-B*35 allele modulates ER stress, inflammation and proliferation in PBMCs from Limited Cutaneous Systemic Sclerosis patients.
abstract: BACKGROUND
  HLA-B*35 is associated with increased risk of developing pulmonary hypertension in SSc patients. We previously reported that HLA-B*35 induces endothelial cell dysfunction via activation of ER stress/UPR and upregulation of the inflammatory response. Because PBMCs from lcSSc-PAH patients are also characterized by activation of ER stress/UPR and inflammation, the goal of this study was to assess whether the presence of HLA-B*35 contributes to those characteristics.
  
  
  METHODS
  PBMCs were purified from healthy controls (n = 49 HC) and lcSSc patients, (n = 44 with PAH, n = 53 without PAH). PBMCs from each group were stratified for the presence of HLA-B*35. Global changes in gene expression in response to HLA-B*35, HLA-B*8 or empty lentivirus were investigated by microarray analysis in HC PBMCs. Total RNA was extracted and qPCR was performed to measure gene expression.
  
  
  RESULTS
  ER stress markers, in particular the chaperones BiP and DNAJB1 were significantly elevated in PBMC samples carrying the HLA-B*35 allele. IL-6 expression was also significantly increased in HLA-B*35 lcSSc PBMCs and positively correlated with ER stress markers. Likewise, HMGB1 was increased in HLA-B*35-positive lcSSc PBMCs. Global gene expression analysis was used to further probe the role of HLA-B*35. Among genes downregulated by HLA-B*35 lentivirus were genes related to complement (C1QB, C1QC), cell cycle (CDNK1A) and apoptosis (Bax, Gadd45). Interestingly, complement genes (C1QC and C1QB) showed elevated expression in lcSSc without PAH, but were expressed at the low levels in lcSSc-PAH. The presence of HLA-B*35 correlated with the decreased expression of the complement genes. Furthermore, HLA-B*35 correlated with decreased expression of cyclin inhibitors (p21, p57) and pro-apoptotic genes (Bax, Gadd45) in lcSSc B35 subjects. FYN, a tyrosine kinase involved in proliferation of immune cells, was among the genes that were positively regulated by HLA-B*35. HLA-B*35 correlated with increased levels of FYN in lcSSc PBMCs.
  
  
  CONCLUSIONS
  Our study demonstrates that HLA-B*35 contributes to the dysregulated expression of selected ER stress, inflammation and proliferation related genes in lcSSc patient PBMCs, as well as healthy individuals, thus supporting a pathogenic role of HLA-B*35 in the development of PAH in SSc patients.
authors.affiliation: Arthritis Center, Boston University School of Medicine, 72 East Concord Street, E-5, Boston, MA, 02118, USA. slenna@bu.edu.
authors.forename: Stefania
authors.initials: S
authors.lastname: Lenna
authors.affiliation: Division of Rheumatology, University of Texas Health Science Center at Houston, Houston, TX, 77030, USA. shervin.assassi@uth.tmc.edu.
authors.forename: Shervin
authors.initials: S
authors.lastname: Assassi
authors.affiliation: Arthritis Center, Boston University School of Medicine, 72 East Concord Street, E-5, Boston, MA, 02118, USA. farina@bu.edu.
authors.forename: G Alessandra
authors.initials: GA
authors.lastname: Farina
authors.affiliation: Arthritis Center, Boston University School of Medicine, 72 East Concord Street, E-5, Boston, MA, 02118, USA. jmantero@bu.edu.
authors.forename: Julio C
authors.initials: JC
authors.lastname: Mantero
authors.affiliation: Referral Center for Systemic Autoimmune Diseases, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico and University of Milan, Milan, 20122, Italy. scorzarunimi@gmail.com.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Scorza
authors.affiliation: Arthritis Center, Boston University School of Medicine, 72 East Concord Street, E-5, Boston, MA, 02118, USA. lafyatis@bu.edu.|University of Pittsburgh Medical Center, Pittsburgh, PA, 15213, USA. lafyatis@bu.edu.
authors.forename: Robert
authors.initials: R
authors.lastname: Lafyatis
authors.affiliation: Pulmonary Center, Boston University School of Medicine, Boston, MA, 02118, USA. hfarber@bu.edu.
authors.forename: Harrison W
authors.initials: HW
authors.lastname: Farber
authors.affiliation: Arthritis Center, Boston University School of Medicine, 72 East Concord Street, E-5, Boston, MA, 02118, USA. trojanme@bu.edu.
authors.forename: Maria
authors.initials: M
authors.lastname: Trojanowska
mesh_terms: D000483:Alleles; D049109:Cell Proliferation / Q000502:physiology*; D002478:Cells, Cultured; D059865:Endoplasmic Reticulum Stress / Q000502:physiology*; D005260:Female; D015797:HLA-B35 Antigen / Q000502:physiology*; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000502:physiology*; D008297:Male; D012595:Scleroderma, Systemic / Q000235:genetics* / Q000378:metabolism
chemical_list: D015797:HLA-B35 Antigen