P086 HMGB1 modulates immune responses in a cell-specific manner in the rat autoimmune orchitis

Abstract

Introduction High mobility group box protein 1 (HMGB1) is a non-histone nucleosomal protein. Under inflammatory conditions HMGB1 is secreted from activated immune cells or released from necrotic cells and acts as an endogenous danger signal. Despite its immune privileged status, the testis is prone to inflammatory infertility. In this study we analyzed the role of HMGB1 in mediating autoimmune responses in experimental autoimmune orchitis (EAO) – a rodent model of chronic testicular inflammation. Methods EAO was induced in WKY rats by immunization with testicular homogenate in complete Freund’s adjuvant. HMGB1 localization was analyzed in EAO and adjuvant control rat testes by immunofluorescence staining. Levels of HMGB1, IL-6 and TNF- α were measured in testes and serum using ELISA. Isolated testicular macrophages (TM), Sertoli cells (SC) and peritubular cells (PTC) were stimulated with recombinant HMGB1. Cytokine mRNA levels were measured by qRT-PCR. Testosterone production was evaluated using RIA assay in HMGB1 treated Leydig cells (LC). Expression of RAGE as well as activation of NF-?B (p65), MAPK (p38, ERK1/2) and Akt pathway were investigated in TM, SC and pPTC by Western blot. Possible binding of HMGB1 to TLR4 was investigated in isolated testicular cells using Duolink PLA assay. Results In control testes, HMGB1 was expressed mainly in the nucleus of testicular somatic cells. In contrast, EAO testes showed translocation of HMGB1 from the nucleus into the cytoplasm. Concentrations of IL-6 and TNF- α were increased 50 days after first immunization in EAO testes, whilst HMGB1 levels increased 80 days post-immunization. RAGE was highly expressed in SC as well as PTC and to a lower level in TM. In contrast, TLR4 levels were higher in TM. Our preliminary results show that HMGB1 binding to TLR4 is significantly higher in TM than in PTC. In vitro stimulation of isolated TM using recombinant HMGB1 activates phosphorylation of p38 and p65. However, HMGB1 treated PTC and SC activated ERK1/2. HMGB1 induced a significant increase in mRNA expression of TNF- α and IL-6 in TM. In HMGB1 treated SC the levels of IL-6 and TNF- α mRNA did not show a statistically significant difference. HMGB1 induced a significant increase in testosterone production by LC. Conclusion In control testis, HMGB1 was expressed mainly in the nucleus of testicular somatic cells. In contrast, EAO testes showed translocation of HMGB1 from the nucleus into the cytoplasm. Concentrations of IL-6 and TNF- α were increased 50 days after first immunization in EAO testis, whilst HMGB1 levels increased 80 days post-immunization. RAGE was highly expressed in SC as well as PTC and to a lower level in TM. In contrast, TLR4 levels were higher in TM. Our preliminary results show that HMGB1 binding to TLR4 is significantly higher in TM than in PTC. In vitro stimulation of isolated TM using recombinant HMGB1 activates phosphorylation of p38 and p65. In contrast, HMGB1 treated PTC and SC activated ERK1/2. HMGB1 induced a significant increase in mRNA expression of TNF- α and IL-6 in TM. In HMGB1 treated SC the levels of IL-6 and TNF- α mRNA did not show a statistically significant difference. HMGB1 induced a significant increase in testosterone production by LC.