| Background and Objectives:Although the testis is an immunoprivileged site, infections of the testis can be usually overcome. Therefore, the local cell-initiated testicular innate immune responses play a crucial role in defense against microbial infections. In this paper, we investigate the contribution of Leydig cells to the testicular innate immunity. Mechanism underlying low immune response of testicular macrophages to infections remains to be clarified. We examine effect of Leydig cells on macrophage responses to stimulation.Materials and Methods:Leydig cells were isolated from C57/BL6 mice. Gene expression at mRNA level was analyzed by quantitative real-time RT-PCR (Q-PCR). Proteins were determined by Western blotting and immunohistochemistry. The concentration of cytokines and testosterone in medium was determined by ELISA. The mouse mutants for Axl and Mer RTKs were used to study the function of TAM RTKs in regulating TLR signaling.Results:TLR3 and TLR4 were expressed in Leydig cells at relatively high level compared to other TLR members. Both TLR3 and TLR4 can be activated by their agonists—polyinosinic:polycytidylic acid (poly (I:C)) and lipopolysaccharide (LPS). TLR signaling activates various transcription factors, including nuclear factor-KB (NF-κB) and interferon regulatory factor 3 (IRF3), which are characterized by their translocation from the cytoplasms into the nuclei of cells, and subsequently induce the production of inflammatory factors such as interleukin (IL)-1β,L-6, tumor necrosis factor alpha (TNF-a) and type 1 interferons (IFN-a and IFN-(3). Inflammatory factors and type 1 interferons should participate in defense against invading pathogens. TLR signaling must be tightly controlled because unrestrained activation of TLRs generates a chronic inflammatory milieu that is harmful to host. We detected expression of Axl and Mer, but not Tyro3 receptor tyrosine kinases (RTKs) in primary Leydig cells. Axl and Mer RTKs negatively regulate TLR signaling in a redundant manner in Leydig cells. In response to Poly (I:C) or LPS, Axl and Mer knockout (Mer-/-) Leydig cells expressed significantly high levels of cytokines in comparison with wild-type (WT) controls. Further, Gas6, a common ligand of TAM RTKs, substantially inhibited TLR-mediated phosphorylation of NF-κB and IRF-3 in WT and Axl or Mer single mutant (Axl-/- or Mer-/-) Leydig cells. In contrast, this inhibitory effect of Gas6 was abolished in Axl-/-Mer-/- Leydig cells. These results indicate that Axl and Mer RTKs negatively regulate TLR signaling in a redundant manner in Leydig cells. Further, we provide evidence that activation of TLR3 and TLR4 in primary Leydig cells significantly inhibited testosterone production. Finally, we demonstrate that Leydig cells inhibit pathogen-induced inflammatory cytokine production by macrophages during co-culture.Conclusions:We demonstrate that Leydig cells initiate testicular innate immune responses through activation of TLR3 and TLR4. Moreover, TLR3 and TLR4 signaling pathways in Leydig cells can be negatively regulated by Axl and Mer RTKs. We provide evidence that Leydig cells inhibit macrophage responses to pathogen stimulation. Our data provide insights into mechanisms of the testicular cell-initiated innate immunity and immunoprivilege property of the testis. |
