Biochanin a Enhances the Defense Against Salmonella enterica Infection Through AMPK/ULK1/mTOR-Mediated Autophagy and Extracellular Traps and Reversing SPI-1-Dependent Macrophage (MΦ) M2 Polarization

ORIGINAL RESEARCH published: 11 September 2018 doi: 10.3389/fcimb.2018.00318 Biochanin a Enhances the Defense Against Salmonella enterica Infection Through AMPK/ULK1/mTOR-Mediated Autophagy and Extracellular Traps and Reversing SPI-1-Dependent Macrophage (M8) M2 Polarization Edited by: Igor Brodsky, University of Pennsylvania, United States Reviewed by: Vishvanath Tiwari, Central University of Rajasthan, India Travis Bourret, Creighton University, United States *Correspondence: Yang Wang Lu Yu † These authors have contributed equally to this work Specialty section: This article was submitted to Bacteria and Host, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 31 January 2018 Accepted: 21 August 2018 Published: 11 September 2018 Citation: Zhao X, Tang X, Guo N, An Y, Chen X, Shi C, Wang C, Li Y, Li S, Xu H, Liu M, Wang Y and Yu L (2018) Biochanin a Enhances the Defense Against Salmonella enterica Infection Through AMPK/ULK1/mTOR-Mediated Autophagy and Extracellular Traps and Reversing SPI-1-Dependent Macrophage (M8) M2 Polarization. Front. Cell. Infect. Microbiol. 8:318. doi: 10.3389/fcimb.2018.00318 Xingchen Zhao 1,2† , Xudong Tang 3 , Na Guo 1† , Yanan An 1† , Xiangrong Chen 1 , Ce Shi 1 , Chao Wang 1 , Yan Li 1 , Shulin Li 1 , Hongyue Xu 1 , Mingyuan Liu 1,4 , Yang Wang 1* and Lu Yu 1* 1 Key Laboratory for Zoonosis Research, Department of Infectious Diseases, First Hospital of Jilin University, Ministry of Education, College of Veterinary Medicine, College of Food Science and Engineering, Institute of Zoonosis, Jilin University, Changchun, China, 2 Department of Food Quality and Safety, College of Food Science and Engineering, Tonghua Normal University, Tonghua, China, 3 Key Lab for New Drug Research of TCM, Research Institute of Tsinghua University in Shenzhen, Shenzhen, China, 4 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China A novel treatment regimen for bacterial infections is the pharmacological enhancement of the host’s immune defenses. We demonstrated that biochanin A (BCA), an isoflavone constituent in some plants, could enhance both intra- and extracellular bactericidal activity of host cells. First, BCA could induce a complete autophagic response in nonphagocytic cells (HeLa) or macrophages (M8) via the AMPK/ULK1/mTOR pathway and Beclin-1-dependent manner, and BCA enhanced the killing of invading Salmonella by autophagy through reinforcing ubiquitinated adapter protein (LRSAM1, NDP52 and p62)-mediated recognition of intracellular bacteria and through the formation of autophagolysosomes. Second, we demonstrated that BCA could enhance the release of M8 extracellular traps (METs) to remove extracellular Salmonella also via the AMPK/ULK1/mTOR pathway, not through reactive oxygen species (ROS) pathway. Furtherly, in a Salmonella-infected mouse model, BCA treatment increased intra- and extracellular bactericidal activity through the strengthening autophagy and MET production, respectively, in peritoneal M8, liver and spleen tissue. Additionally, our findings showed that BCA downregulated SPI-1 (Salmonella pathogenicity island 1) expression during Salmonella infection in vitro and in vivo to reverse the M8 M2 polarization, which was different from the M8 M1 phenotype caused by most of bacteria infection. Together, these findings suggest that BCA has an immunomodulatory effect on Salmonella-infected host cells and enhances their bactericidal activity in vitro and in vivo through autophagy, extracellular traps and regulation of M8 polarization. Keywords: reactive oxygen species, autophagy, extracellular traps, polarization, Salmonella Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 September 2018 | Volume 8 | Article 318 Zhao et al. BCA Defense Against Salmonella INTRODUCTION expression of Salmonella pathogenicity island 1 (SPI-1) (Kyrova et al., 2012), the M2 polarization caused by Salmonella infection was different from other bacteria infection that caused M8 M1 phenotype polarization. Until recently, few agents were found to enhance bacterial clearance by promoting anti-bacterial autophagy (Conway et al., 2013) or ETs (Chow et al., 2010). In this study, we found that Biochanin A (BCA), a major isoflavone constituent found in red clover, cabbage, alfalfa and some other herbal dietary supplements, could enhance bactericidal intra- and extracellular activity. BCA also has putative benefits in dietary cancer prophylaxis (Medjakovic and Jungbauer, 2008). BCA has potential antimicrobial activity against several types of bacteria, but these cases include only high minimum inhibitory concentration (MIC) values (Liu et al., 2011). BCA is a common product extracted from natural plant and is considered to be innocuous (Sklenickova et al., 2010). We found that BCA induced the autophagic response in epithelial cells and M8s or induced ET formation of M8s in vitro and in an in vivo mouse model. We also investigated the influence of BCA on SPI-1-dependent M8 polarization during bacteria infection. The wide spread of drug-resistant bacteria and the slow development of new antimicrobial agents has created a desperate scarcity of new therapeutic approaches of bacterial infections (Viveiros et al., 2012). The enhancement of the host’s immune defenses through pharmacological treatment is a novel area of study (Ankomah and Levin, 2014). There is an urgent need to discover agents that can enhance both extra- and intracellular bactericidal action in the host (Viveiros et al., 2012). The innate immune system is the first line of defense against invading microorganisms through immune response elements such as reactive oxygen species (ROS), autophagy and extracellular traps (ETs) (Medzhitov, 2010). Autophagy is the natural, regulated mechanism of the cell, in which cytoplasmic components are delivered to lysosomes and autophagosomes for degradation (Yuan et al., 2012). The classical autophagy mechanism depends on two ubiquitin-like conjugation systems: Atg4-Atg7-Atg8 or Atg7-Atg12-Atg5. These two systems are important for the formation of an early complex containing class III phosphoinositide 3-kinase, which then forms the autophagosome (Yuan et al., 2012). Recent reports have demonstrated that AMP-activated protein kinase (AMPK) and mTOR coordinate autophagy initiation in mammalian cells (Shang and Wang, 2011). ETs were recognized recently (Brinkmann et al., 2004) and can be produced by many innate effector cells including macrophages (M8), eosinophils, mast cells and neutrophils. ETs are fiber-like extracellular structures that can defend against infections by trapping extracellular bacteria or fungi (Brinkmann et al., 2004). NADPH oxidase (NOX2)-dependent or NOX2-independent oxidative bursts have been reported to mediate ET formation (Remijsen et al., 2011). According to the tissue microenvironment, peripheral monocytes could polar (...truncated)