The role of autophagy in asparaginase-induced immune suppression of macrophages
Erwinia asparaginase, a bacteria-derived enzyme drug, has been used in the treatment of various cancers, especially acute lymphoblastic leukemia (ALL). One of the most significant side effects associated with asparaginase administration is immune suppression, which limits its application in clinic. Macrophages are phagocytic immune cells and have a central role in inflammation and host defense. We reported here that asparaginase disturbed the function of macrophages including phagocytosis, proliferation, ROS and nitric oxide secretion, interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) secretion, and major histocompatibility complex II (MHC-II) molecule expression, thus induced immune suppression in interferon-γ and lipopolysaccharide-stimulated macrophages. We also observed that asparaginase inhibited autophagy in macrophages via activating Akt/mTOR and suppressing Erk1/2 signaling pathway as evidenced by less formation of autophagosomes, downregulation of autophagy-related protein LC3-II, and decreased number of autophagy-like vacuoles. Further study discovered that treatment with autophagy inhibitor 3-MA in place of asparaginase on activated macrophages could also downregulate phagocytosis, cytokine secretion, and MHC-II expression. Moreover, incubation with autophagy inducer trehalose restored the capacity of phagocytosis, IL-6 and TNF-α secretion, and MHC-II expression in macrophages. These results prove the important role of autophagy in the function of macrophages, and activation of autophagy can overcome asparaginase-induced immune suppression in macrophages.First, the effect of asparaginase on macrophages phagocytic function was determined in activated Ana-1 and RAW264.7 cells. Phagocytosis is a basic cellular function of macrophages having an important role in innate immunity.31 As shown in Figure 1a and Supplementary Figure 1, Ana-1 and RAW264.7 cells stimulated by IFN-γ and LPS for 24 h showed significantly increased phagocytic activity measured as zymosan-Alexa Fluor 488 particles internalization (green fluorescence). However, the macrophage phagocytic function was significantly impaired when treated with asparaginase, indicating the phagocytosis was inhibited by asparaginase in activated Ana-1 and RAW264.7 cells.Second, we labeled macrophages with carboxyfluorescein succinimidyl ester (CFSE) and investigated the effect of asparaginase on proliferation of Ana-1 and RAW264.7 cells.32 As shown in Figure 1b, at 0 h in control, Ana-1 and RAW264.7 cells showed a potent fluorescence intensity (right side), and at 24 h in both control and IFN-γ/LPS-treated cells the fluorescence intensity attenuated owing to cell division (left side). After treating with asparaginase, the curve shifted from left to the right as compared with control and IFN-γ/LPS-treated cells, indicating that asparaginase inhibited proliferation of macrophages.Finally, we investigated the effect of asparaginase on cell cycle distribution in Ana-1 and RAW264.7 cells. Cell cycle is a repeating series of events that take place in a cell leading to its division and DNA replication to produce two daughter cells. As shown in Figure 1c, upon asparaginase treatment, Ana-1 and RAW264.7 cells at G1 phase increased with reduced cells at S phase when compared with IFN-γ and LPS-stimulated macrophages, indicating asparaginase could induce G1 arrest to decelerate the cell cycle, and prevent the cells from entering the S phase and proliferating.These results demonstrate that asparaginase inhibits phagocytosis and proliferation in M1 macrophages, and support our hypothesis that asparaginase might influence the function of M1 macrophages.Asparaginase (derived from Erwinia) was purchased from Baiyunshan Mingxing Pharmaceutical Co., Ltd. (Guangzhou, Guangdong Province, China). The autophagy inhibitor 3-MA was obtained from EMD Chemicals, Inc. (San Diego, CA, USA). The autophagy inducer Tre was purchased from Sigma-Aldrich (St. Louis, MO, USA). IFN-γ was obtained from Cyagen Biosciences Inc. (Guangzhou, Guangdong Province, China). LPS was obtained from Sigma-Aldrich. The antibodies including anti-β-actin, anti-LC3B, anti-p62, anti-mTOR (Ser2448), anti-phospho-mTOR (Ser2448), anti-Akt (Ser473), anti-phospho-Akt (Ser473), anti-p70S6 Kinase (pS371), anti-p70S6 Kinase Phospho (pS371), anti-4EBP1-pT45, anti-phospho-4EBP1-pT45, anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), and anti-p44/42 MAPK (Erk1/2) were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies horseradish peroxidases (HRP)-conjugated goat anti-mouse and anti-rabbit immunoglobulin G were purchased from MR Biotech (Shanghai, China).This study was supported by National Key Basic Research Program of China (2015CB931800 and 2013CB932502), the National Natural Science Foundation of China (81573332 and 81572979), Shanghai Science and Technology Funds (14431900200), and Special Research Foundation of State Key Laboratory of Medical Genomics and Collaborative Innovation Center of Systems Biomedicine.Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis)Edited by GM FimiaThe authors declare no conflict of interest.