title: Targeting high mobility group box protein 1 ameliorates testicular inflammation in experimental autoimmune orchitis.
abstract: OBJECTIVE
  Does high mobility group box protein 1 (HMGB1) regulate inflammatory reactions in a rat model of experimental autoimmune orchitis (EAO)?
  
  
  CONCLUSIONS
  HMGB1 appears to be involved in regulating inflammatory reactions in testes, as HMGB1 is translocated from testicular cells during the course of EAO and blocking its action by ethyl pyruvate (EP) reduces disease progression and spermatogenic damage.
  
  
  BACKGROUND
  Despite its immune privileged status, the human testis is prone to inflammatory lesions associated with male factor infertility. Accumulating evidence shows that HMGB1 plays an important role in onset and progression of autoimmune diseases.
  
  
  METHODS
  This is a cross sectional and longitudinal study involving Wistar male rats immunized with testicular homogenates to induce EAO 50 (EAO50; n = 10) and 80 (EAO80; n = 10) days after first immunization. Control adjuvant animals received saline instead of testicular homogenate (n = 16). Untreated animals (n = 10) were also studied. An interventional study was performed to block the action of HMGB1 starting 20 days after first immunization in EAO animals and respective controls (n = 17). Rats were treated i.p. with EP and the effect of EP treatment on testicular pathogenesis was evaluated 30 days later. Moreover, human testicular biopsies from infertile men with focal lymphocytic infiltrates (n = 7) and sections with intact spermatogenesis (n = 6) were probed with antibodies against HMGB1.
  
  
  METHODS
  Testicular RNA and protein extracts from EAO animals, EAO animals treated with EP and relevant controls were used for analysis of cytokine expression by real-time RT-PCR and enzyme-linked immunosorbent assay. HMGB1 was co-localized on rat testicular cross sections with antibodies against testicular macrophages (TM), peritubular cells (PTC) and Sertoli cells (SC). Interaction of HMGB1 and its receptors (RAGE, TLR4) as well signaling pathways after HMGB1 stimulation were studied in isolated TM, PTC and SC by proximity ligation assay and western blot, respectively. Furthermore, HMGB1 immunofluorescence on human testicular biopsies was performed.
  
  
  RESULTS
  HMGB1 was translocated from the nuclei in EAO testes and testes of infertile men with impaired spermatogenesis and lymphocytic infiltrates. Elevated HMGB1 levels were observed during late phase of EAO. In testicular somatic cells HMGB1 receptors Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) were differentially expressed: HMGB1-TLR4 binding was predominant in TM, while HMGB1-RAGE interaction was prevalent in SC and PTC. In support, HMGB1 triggered extracellular signal regulated kinase (ERK)1/2 and cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) activation in SC and PTC, while TM responded to HMGB1 stimulation with p38 mitogen-activated protein kinase (MAPK) and p65 nuclear factor Kappa B (NF-ĸB) phosphorylation followed by increased tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) mRNA levels. In vivo treatment of EAO animals with EP 20 days after induction of disease revealed beneficial effects, as documented by reduced disease progression and spermatogenic damage, lower macrophage numbers, as well as decreased concentrations of HMGB1 and IL-6 in the testis compared with EAO controls.
  
  
  CONCLUSIONS
  The ability of HMGB1 to bind to a wide range of receptors makes it difficult to prevent its action by blockade of a specific receptor; therefore we applied EP, a drug preventing HMGB1 release from cells. Due to its mode of action EP decreases also the secretion of some other pro-inflammatory cytokines. Using isolated primary cells imposes limitations for cell transfection studies. As a compromise between purity and yield primary cells need to be isolated from animals of different age, which has to be considered when comparing their responses.
  
  
  CONCLUSIONS
  HMGB1 could be a promising target in attenuating testicular damage caused by inflammatory reactions.
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.
authors.forename: Ferial
authors.initials: F
authors.lastname: Aslani
authors.affiliation: Department of Urology, Pediatric Urology and Andrology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.
authors.forename: Hans-Christian
authors.initials: HC
authors.lastname: Schuppe
authors.affiliation: Instituto de Investigaciones Biomédicas UBA-CONICET, Facultad de Medicina, Universidad de Buenos Aires, C1121 ABG, Buenos Aires, Argentina.
authors.forename: Vanesa A
authors.initials: VA
authors.lastname: Guazzone
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.
authors.forename: Sudhanshu
authors.initials: S
authors.lastname: Bhushan
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Wahle
authors.affiliation: Institute of Biochemistry, Faculty of Medicine, Justus-Liebig University of Giessen, 35392 Giessen, Germany.
authors.forename: Günter
authors.initials: G
authors.lastname: Lochnit
authors.affiliation: Instituto de Investigaciones Biomédicas UBA-CONICET, Facultad de Medicina, Universidad de Buenos Aires, C1121 ABG, Buenos Aires, Argentina.
authors.forename: Livia
authors.initials: L
authors.lastname: Lustig
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Meinhardt
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany monika.fijak@anatomie.med.uni-giessen.de.
authors.forename: Monika
authors.initials: M
authors.lastname: Fijak
mesh_terms: D000328:Adult; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology / Q000627:therapeutic use*; D001327:Autoimmune Diseases / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D001706:Biopsy; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D024243:HMGB1 Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007248:Infertility, Male / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D008262:Macrophage Activation / Q000187:drug effects; D008297:Male; D058990:Molecular Targeted Therapy; D009920:Orchitis / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D021381:Protein Transport / Q000187:drug effects; D011773:Pyruvates / Q000494:pharmacology / Q000627:therapeutic use; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013737:Testis / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism / Q000473:pathology
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D024243:HMGB1 Protein; C585491:HMGB1 protein, human; C104200:Hbp1 protein, rat; D011773:Pyruvates; D011994:Recombinant Proteins; C046522:ethyl pyruvate
keywords: ethyl pyruvate; experimental autoimmune orchitis; high mobility group box protein-1; receptor for advanced glycation end products; testicular inflammation