Differential Activation of Inflammatory Pathways in Testicular Macrophages Provides a Rationale for Their Subdued Inflammatory Capacity

Abstract

Spermatogenic cells express cell-specific molecules with the potential to be seen as “foreign” by the immune system. Owing to the time difference between their appearance in puberty and the editing of the lymphocyte repertoire around birth, local adaptations of the immune system coined immune privilege are required to confer protection from autoattack. Testicular macrophages (TM) play an important role in maintaining testicular immune privilege and display reduced proinflammatory capacity compared with other macrophages. However, the molecular mechanism underlying this macrophage phenotype remained elusive. We demonstrate that TM have a lower constitutive expression of TLR pathway–specific genes compared with peritoneal macrophages. Moreover, in TM stimulated with LPS, the NF-κB signaling pathway is blocked due to lack of IκBα ubiquitination and, hence, degradation. Instead, challenge of TM with LPS or polyinosinic-polycytidylic acid induces MAPK, AP-1, and CREB signaling pathways, which leads to production of proinflammatory cytokines such as TNF-α, although at much lower levels than in peritoneal macrophages. Pretreatment of TM with inhibitors for MAPKs p38 and ERK1/2 suppresses activation of AP-1 and CREB signaling pathways and attenuates LPS-induced TNF-α and IL-10 secretion. High levels of IL-10 production and activation of STAT3 by LPS stimulation in TM indicate a regulatory macrophage phenotype. Our results suggest that TM maintain testicular immune privilege by inhibiting NF-κB signaling through impairment of IκBα ubiquitination and a general reduction of TLR cascade gene expression. However, TM do maintain some capacity for innate immune responses through AP-1 and CREB signaling pathways.

Characterization of the Micro-Environment of the Testis that Shapes the Phenotype and Function of Testicular Macrophages

“…Similarly, the majority of resident macrophages of the testis are also categorized *Institute of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, Giessen 35392, Germany; as M2 macrophages. In support, several in vitro studies showed that the treatment of TM with inflammatory stimuli induced the secretion of large amounts of IL-10 and a low amount of TNF-a (4,13). Moreover, TM activate the anti-inflammatory STAT3 signaling pathway (4).…”

“…In support, several in vitro studies showed that the treatment of TM with inflammatory stimuli induced the secretion of large amounts of IL-10 and a low amount of TNF-a (4,13). Moreover, TM activate the anti-inflammatory STAT3 signaling pathway (4).…”

“…Quantitative real time PCR (qRT-PCR) was performed as previously described (4,25). The primer sequences are listed in Table I.…”

“…In this regard, TM exhibit an attenuated inflammatory response by expression of low levels of TLR signaling pathway genes and by inhibiting the activation of the NF-kB signaling pathway by impairing the ubiquitination of phosphorylated IkBa (4). Although in TM the NF-kB signaling pathway is blocked, they paradoxically secrete proinflammatory cytokines through the AP-1 and CREB signaling pathways, albeit at lower levels than peritoneal macrophages (PM), for example (4). Moreover, TM retain bactericidal and phagocytic activities.…”

“…Notably, recent advances now imply that testicular macrophages (TM), which represent the largest pool of immune cells in the testis, also contribute to the maintenance of the testes' immune privilege by skewing innate immune responses (3)(4)(5). In this regard, TM exhibit an attenuated inflammatory response by expression of low levels of TLR signaling pathway genes and by inhibiting the activation of the NF-kB signaling pathway by impairing the ubiquitination of phosphorylated IkBa (4). Although in TM the NF-kB signaling pathway is blocked, they paradoxically secrete proinflammatory cytokines through the AP-1 and CREB signaling pathways, albeit at lower levels than peritoneal macrophages (PM), for example (4).…”