Despite its immune privileged status, the testis is prone to inflammatory infertility. However, sterile inflammation of the testis (autoimmune orchitis) is neglected as a cause of male factor infertility due to lack of non-invasive diagnostic tools. Accumulating data shows that high mobility group box protein 1 (HMGB1), a non-histone chromosomal protein, plays an important role in onset and chronification of autoimmune diseases once released from the nuclei. In this study, we analyzed how HMGB1 can regulate inflammatory reactions in vivo using a rat model of experimental autoimmune orchitis (EAO) and in vitro in primary testicular cells. HMGB1 was translocated from the nuclei in EAO testis and in the testis of infertile men with leukocytic infiltrates. Interestingly, HMGB1 levels in EAO testis were elevated at the late chronic phase of disease as compared to early proinflammatory cytokines such as IL-6 and TNF-a. We found that testicular somatic cells show a cell-specific expression profile of HMGB1 receptors TLR4 and Receptor for Advanced Glycation End products (RAGE). The highly sensitive and specific proximity ligation assay was used to analyze HMGB1 receptor binding in testicular cells. HMGB1-TLR4 binding was dominant in testicular macrophages. However, Sertoli and peritubular cells showed higher levels of HMGB1-RAGE interaction. In support, HMGB1 triggered RAGE-dependent ERK1/2 MAPK and CREB activation in Sertoli and peritubular cells, whilst in testicular macrophages HMGB1 induced TLR4-signaling as evidenced by p38 MAPK and p65 NF-ĸB phosphorylation which stimulated an increase in mRNA levels of TNF-a and IL-6. Recent studies showed that RAGE induced ERK activation leads to enhanced autophagy levels. In line with these data, in our study extracellular HMGB1 triggered formation of autophagosomes in Sertoli cells. Increased autophagy levels in isolated Sertoli cells may explain how these cells survive the inflammatory environment in EAO testis. Considering HMGB1 s late phase of action, inhibition of HMGB1 may be a putative target for therapeutic intervention in treatment of chronic testicular inflammation.
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