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Targeting high mobility group box protein 1 ameliorates testicular inflammation in experimental autoimmune orchitis.
Hum Reprod. 2015 Feb; 30(2):417-31.HR

Abstract

STUDY QUESTION

Does high mobility group box protein 1 (HMGB1) regulate inflammatory reactions in a rat model of experimental autoimmune orchitis (EAO)?

SUMMARY ANSWER

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.

WHAT IS KNOWN ALREADY

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.

STUDY DESIGN, SIZE, DURATION

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.

PARTICIPANTS/MATERIALS, SETTING, 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.

MAIN RESULTS AND THE ROLE OF CHANCE

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.

LIMITATIONS, REASONS FOR CAUTION

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.

WIDER IMPLICATIONS OF THE FINDINGS

HMGB1 could be a promising target in attenuating testicular damage caused by inflammatory reactions.

Authors+Show Affiliations

Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.Department of Urology, Pediatric Urology and Andrology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.Instituto de Investigaciones Biomédicas UBA-CONICET, Facultad de Medicina, Universidad de Buenos Aires, C1121 ABG, Buenos Aires, Argentina.Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.Institute of Biochemistry, Faculty of Medicine, Justus-Liebig University of Giessen, 35392 Giessen, Germany.Instituto de Investigaciones Biomédicas UBA-CONICET, Facultad de Medicina, Universidad de Buenos Aires, C1121 ABG, Buenos Aires, Argentina.Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany monika.fijak@anatomie.med.uni-giessen.de.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

25452436

Citation

Aslani, Ferial, et al. "Targeting High Mobility Group Box Protein 1 Ameliorates Testicular Inflammation in Experimental Autoimmune Orchitis." Human Reproduction (Oxford, England), vol. 30, no. 2, 2015, pp. 417-31.
Aslani F, Schuppe HC, Guazzone VA, et al. Targeting high mobility group box protein 1 ameliorates testicular inflammation in experimental autoimmune orchitis. Hum Reprod. 2015;30(2):417-31.
Aslani, F., Schuppe, H. C., Guazzone, V. A., Bhushan, S., Wahle, E., Lochnit, G., Lustig, L., Meinhardt, A., & Fijak, M. (2015). Targeting high mobility group box protein 1 ameliorates testicular inflammation in experimental autoimmune orchitis. Human Reproduction (Oxford, England), 30(2), 417-31. https://doi.org/10.1093/humrep/deu320
Aslani F, et al. Targeting High Mobility Group Box Protein 1 Ameliorates Testicular Inflammation in Experimental Autoimmune Orchitis. Hum Reprod. 2015;30(2):417-31. PubMed PMID: 25452436.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Targeting high mobility group box protein 1 ameliorates testicular inflammation in experimental autoimmune orchitis. AU - Aslani,Ferial, AU - Schuppe,Hans-Christian, AU - Guazzone,Vanesa A, AU - Bhushan,Sudhanshu, AU - Wahle,Eva, AU - Lochnit,Günter, AU - Lustig,Livia, AU - Meinhardt,Andreas, AU - Fijak,Monika, Y1 - 2014/12/01/ PY - 2014/12/3/entrez PY - 2014/12/3/pubmed PY - 2015/9/9/medline KW - ethyl pyruvate KW - experimental autoimmune orchitis KW - high mobility group box protein-1 KW - receptor for advanced glycation end products KW - testicular inflammation SP - 417 EP - 31 JF - Human reproduction (Oxford, England) JO - Hum Reprod VL - 30 IS - 2 N2 - STUDY QUESTION: Does high mobility group box protein 1 (HMGB1) regulate inflammatory reactions in a rat model of experimental autoimmune orchitis (EAO)? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: HMGB1 could be a promising target in attenuating testicular damage caused by inflammatory reactions. SN - 1460-2350 UR - https://www.unboundmedicine.com/medline/citation/25452436/Targeting_high_mobility_group_box_protein_1_ameliorates_testicular_inflammation_in_experimental_autoimmune_orchitis_ L2 - https://academic.oup.com/humrep/article-lookup/doi/10.1093/humrep/deu320 DB - PRIME DP - Unbound Medicine ER -