Discussion
Fungi are widely distributed across the globe and have been identified as a rich source of new pharmacologically active ingredients. The members of the Aspergillus genus are already an important source of novel pharmacological metabolites such as alkaloids, meroterpenoids, and lignans. Secoemestrin C is an epitetrathiodioxopiperazine extracted from Aspergillus nidulans . The biological activity of secoemestrin C and its analog has only been reported in terms of inhibiting tumor cell proliferation (Onodera et al. , 2004). Here, we first present evidence to show that secoemestrin C can inhibit Con A-induced mouse splenocyte proliferation as well as hepatic MNC activation and proliferation in vitro , and then we demonstrate that this molecule has a protective effect in vivo on Con A-induced autoimmune hepatitis in mice.
Accumulating evidence suggests that CD4+ T cells (Mizuhara et al. , 1994; Wang et al. , 2012), CD8+ T cells (Watanabeet al. , 1996), and NKT cells (Takeda et al. , 2000a; Kumar, 2013) are critical components of Con A-induced liver damage. After Con A challenge, NKT cells and conventional T cells are activated and either directly participate in perforin/granzyme- and/or Fas/Fas ligand-mediated cytotoxicity or indirectly release large amounts of proinflammatory cytokines, thus leading to hepatocyte death. Therefore, agents that can inhibit the activation of T cells and/or NKT cells are most likely capable of alleviating hepatitis induced by Con A. In our in vitroexperiments, we found that secoemestrin C can potently suppress the proliferation of splenocytes and activation of hepatic MNCs induced by Con A, indicating that it may have a significant protective effect on Con A-induced liver damage. As predicted, pretreatment with secoemestrin C markedly protected against liver injury in Con A-challenged mice, as evidenced by decreased serum levels of liver enzymes and proinflammatory cytokines, attenuated bridging coagulative necrosis of hepatocytes, and a lessened apoptotic response in the livers. In addition, the levels of serum transaminase and hepatocyte apoptosis in secoemestrin C-treated mice were similar to those in normal control mice, indicating that secoemestrin C has limited hepatic toxicity. Thus, secoemestrin C appears to be a good prospect for clinical application in immunosuppression.
In addition to aggravating inflammation and liver injury, activation of NKT cells also leads to Fas/FasL-mediated apoptotic elimination of NKT cells during Con A challenge (Takedaet al. , 2000b). Our results have confirmed the rapid disappearance of activated NKT cells in Con A-challenged livers. Although we did not detect any expression of FasL on NKT cells, our observation of a lowered CD69 expression level, together with a restored NKT cell ratio, suggest that the activation-induced cell death (AICD) of NKT cells is largely abolished in the secoemestrin C-treated mice.
Given that the depletion of NKT cells begins as early as 4 h after Con A administration (Takeda et al. , 2000b), it appears that an NKT cell-derived proinflammatory cytokine response, rather than NKT cell-mediated cytotoxicity, plays a particularly important role in the latter phase of inflammation induced by Con A. In the treatment of autoimmune disease, targeting the production of proinflammatory cytokines has been approved as a potential therapy (Danese et al. , 2019; Salvi et al. , 2019). It has been reported that SOCS3 suppresses IFN-γ production from NKT and T cells, thereby protecting against Con A-induced liver injury (Nakaya et al. , 2009). Our results show that secoemestrin C can down-regulate IFN-γ production by NKT and conventional T cells after Con A challenge. In addition, we have found that secoemestrin C is able to suppress cytokine production by Con A-stimulated hepatic MNCs. The regulatory mechanism(s) by which secoemestrin C inhibits IFN-γ production remain to be further explored.
Overall, we have demonstrated for the first time that secoemestrin C can inhibit NKT- and conventional T-cell activation stimulated by Con A and thus can protect against Con A-induced autoimmune hepatitis in mice. Our results provide evidence for the immunosuppressive activity of secoemestrin C and offer novel insights regarding the development of fungi-derived metabolites for the treatment of T cell-dependent autoimmune diseases.
Author contributions: X. Tan and G. Chen conceived and designed the experiments. X. Tan, L. Sun, C. Qi, C. Fu, X. Yang. H. Feng, and Y. Li performed the experiments. Q. Li and Y. Zhang isolated and identified secoemestrin C. X. Tan, L. Sun, Y. Zhang, and G. Chen analyzed the data. X. Tan, L. Sun, C. Qi, Y. Zhang, and G. Chen wrote the manuscript.