Tim-3 inhibits macrophage control of Listeria monocytogenes by inhibiting Nrf2
Beifen Shen 2 & Gencheng Han
T cell immunoglobulin mucin-3 (Tim-3) is an immune checkpoint inhibitor and its dysregulation has been related to T cell tolerance and many immune disorders, such as tumors and infection tolerance. However, the physiopathology roles of Tim-3 in innate immunity remain elusive. Here, we demonstrate that Tim-3 inhibits macrophage phagocytosis of L. monocytogenes by inhibiting the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway and increases bacterial burden. Tim-3 signaling promotes Nrf2 degradation by increasing its ubiquitination and, as a result, decreasing its nuclear translocation. CD36 and heme oxygenase-1 (HO-1), two downstream molecules in the Tim-3-Nrf2 signaling axis, are involved in the Tim-3- mediated immune evasion of L. monocytogenes both in vitro and in vivo. We here identified new mechanisms by which Tim-3 induces infection tolerance. By modulating the Tim-3 pathway, we demonstrate the feasibility of manipulating macrophage function as a potent tool for treating infectious diseases, such as Listeria infection.Male C57BL/6 mice (6- to 8-weeks-old) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Tim-3 transgenic mice were generated in the Transgenic Core Facility of Cyagen Biosciences Inc., Guangzhou, China by overexpressing Tim-3 under the control of the CMV promoter; incorporation was confirmed by PCR and Tim-3 expression on macrophages and other cells was confirmed using flow cytometry20. All mice were bred and maintained in our facilities under specific pathogen-free conditions and were treated in strict compliance with the guidelines for the care and use of laboratory animals set out by the Beijing Institute of Basic Medical Sciences and the protocol was approved by the Committee on the Ethics of Animal Experiments of the Beijing Institute of Basic Medical Sciences (Permit number: AMMS2015-0165). All efforts were made to minimize suffering.We previously found that Tim-3 inhibits macrophages activation and induces its tolerance. However, it was not known whether Tim-3 regulates the phagocytic activity of macrophages. To test this, we labeled L. monocytogenes with CFSE and added them to cultures of peritoneal macrophages isolated from wild type (WT) or Tim-3 transgenic (Tim-3-TG) mice or to cultured RAW264.7 cells with soluble Tim-3 protein (sTim-3) as soluble Tim-3 is widely used to block the Galectin-9/Tim-3 interaction29,30, then, after 2 h incubation, L. monocytogenes- CFSE- containing cells were measured by flow cytometry as described previously21. The results in Fig. 1A and B show that transgenic overexpression of Tim-3 inhibited macrophage phagocytosis of Listeria, while Fig. 1C and
D show that blockade of Tim-3 signaling by soluble Tim-3 enhanced phagocytosis. These data show that Tim-3 signaling inhibits macrophage phagocytosis of L. monocytogenes in vitro. To test this in a direct way, we labeled L. monocytogenes with CFSE, and after co-culture with macrophages, examined macrophage phagocytosis of L. monocytogenes using confocal laser scanning microscope as described previously24. The data in Fig. 1E showed that blockade of Tim-3 signaling significantly increased macrophage phagocytosis of L. monocytogenes which is consistent with the result of flow cytometry analysis.This study was supported by National Basic Research Program 973 grants (2013CB530506 and 2015CB553704), the National Nature and Science Fund (grants 81272320, 81471529, 81471540, and 81472647), the key program of the Beijing Natural Science Foundation (grant 7141007), and the National Key Research and Development Program of China (2016YFC1202905; AWS16J020).The authors declare no competing financial interests.Author Contributions Zhiding Wang, Dejun Sun and Guojiang Chen wrote the manuscript text. Zhiding Wang, Ge Li and Shuaijie Dou prepared Figures 1, 3 and 4. He Xiao, Renxi Wang and Chunmei Hou prepared Figures 2 and 5. Yan Li and Jiannan Feng prepared Figures 6–7. Beifen Shen and Gencheng Han supervised the project and edited the manuscript. All authors reviewed the manuscript.