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Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1α
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a Fake Li a, Jie Luo b, Huan Xu a, Yang Wang a, Wenbin Jiang b, Kai Chang a, Shaoli Deng b, Ming Chen a, c, d, * a
Department of Clinical Laboratory, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China Department of Clinical Laboratory, Special Medical Center, Third Military Medical University (Army Medical University), Chongqing, 400012, China College of Pharmacy and Laboratory Medicine, Third Military Medical University (Army Medical University), Chongqing, 400038, China d State Key Laboratory of Trauma, Burn and Combined Injury, Third Military Medical University (Army Medical University), Chongqing, 400038, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 October 2019 Accepted 6 November 2019 Available online xxx
Early secreted antigenic target 6-kDa protein (ESAT6) is an essential virulence factor of Mycobacterium tuberculosis (MTb). However, ESAT6 helped fighting MTb infection according to vaccine studies. It's unclear whether ESAT6 confers protection via enhancing the innate immunity of macrophages, which are the first-line defense against MTb. We profiled the global transcriptional changes and characterized the innate immunity of THP-1 macrophages treated with ESAT6. We found ESAT6 promoted the phagocytosis ability, enhanced reactive oxygen species (ROS) generation and accelerated glucose metabolism in macrophages. Meanwhile, ESAT6 induced a distinctive phenotype of macrophages with a concurrence of pro-inflammatory and anti-inflammatory cytokines and chemokines. ESAT6 increased the expression of HIF1a mRNA and protein. Interfering HIF1a with siRNA defected the capacity of phagocytosis and ROS generation as well as glucose metabolism. Thus, ESAT6 enhanced the protective innate immunity of macrophages partially via HIF1a. This study provided clues for developing therapies against tuberculosis by targeting ESAT6. © 2019 Elsevier Inc. All rights reserved.
Keywords: Early secreted antigenic target 6-kDa Macrophage activation Innate immune Chemokine Glucose metabolism RNA sequencing
1. Introduction Mycobacterium tuberculosis (MTb) has been responsible for more human deaths than any other pathogens according to Global TB Report from World Health Organization [1]. Early Secreted Antigenic Target 6-kDa protein (ESAT6) is an essential virulence factor of MTb, encoded in region of difference 1 (RD1) which is absent in the genome of vaccine strain M. bovis Bacille CalmetteGu erin (BCG) [2]. ESAT6 is secreted with culture filtrate protein 10kDa (CFP10) in a 1:1 heterodimer manner, but it may function independent of CFP10 [3]. As a highly secreted protein during infection, ESAT6 showed great antigenic capacity and induced high level differentiation of
* Corresponding author. Department of Clinical Laboratory, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China. E-mail address: [email protected] (M. Chen).
ESAT6-specific T cells in mice and human [4,5]. Interestingly, studies found the attenuated live vaccines expressing ESAT6 conferred better protection than BCG [4,6,7]. Mice and guinea pigs vaccinated with recombinant BCG exporting ESAT6, showed less severe pathology and reduced dissemination of the pathogen, as compared with control mice and guinea pigs immunized with BCG alone [7]. The candidate attenuated live vaccine MTBVAC expressing ESAT6, has also presented better protection than BCG [6]. Meanwhile, MTBVAC mutant in exporting ESAT6 showed lower efficacy than the MTBVAC parental strain and similar efficacy to BCG [4]. Training the innate immune cells with pathogen specific antigens (or virulence factors) has opened an effective approach to protest the host against pathogens [8]. Macrophages are hosts’ first-line defense against MTb by utilizing innate immune approaches, including phagocytosis, respiratory bursts (ROS production) and production of inflammatory cytokines and chemokines [9,10]. Accumulated evidence indicated that ESAT6 modulated the apoptosis [11,12], autophagy [13], antigen presentation [14,15] and
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Please cite this article as: F. Li et al., Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.045
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inflammatory activation [12,16,17] of macrophages. However, whether ESAT6 confers protection via enhancing the innate immunity of macrophages and the underlying mechanisms remain largely unclear. To better understand how ESAT6 regulates the innate immunity of macrophage, we systematically profiled the global transcriptional changes and characterized the innate immunity of ESAT6 treated THP-1 macrophages. These characteristics included levels of multiplex cytokines and chemokines, capacity of phagocytosis, ROS production, glucose consumption and lactate generation in macrophages. We also explored the potential regulator involved in the above processes under the regulation of ESAT6. Therefore, this study may provide an integrative view on how ESAT6 potentially enhances the protective innate immunity of macrophages against tuberculosis. 2. Materials and methods
Shanghai, China). The 21-plex panel included IL1b, IL6, IL10, TNFa, GM-CSF, MIF, CCL1, CCL2, CCL3, CCL17, CCL20, CCL22, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL13 and CXCL16. Proper validation of the array system (optics, reporter, classification and fluidics validation) has been performed to ensure the optimal assay performance before sample testing. Levels of IL1b, IL6, IL10, TNFa and CXCL8 were also quantified by IMMULITE® 1000 immunoassay analyzer (Siemens, Germany). Experiments were done in triplicates. 2.5. RNA interference for HIF1A The siRNA targeting HIF1A and negative control siRNA were purchased from Sangon Biotech (Shanghai, China). The siRNA oligonucleotides were transfected into THP-1 macrophages using the Lipofectamine 3000 (Invitrogen) following the manufacturer's protocol. The siHIF1A sequences were: sense (50 -30 ) CUGAUGAC CAGCAACUUGATT, antisense (50 -30 ) UCAAGUUGCUGGUCAUCAGTT.
2.1. Cell culture and treatments 2.6. Phagocytosis capability analysis The human THP-1 monocytes were obtained from Cell Bank of Chinese Academy of Science and with identity confirmed by Short Tandem Repeat (STR) authentication. THP-1 monocytes were cultured in freshly made completed medium at 2-5 105/mL in a humidified, 37 C, 5% CO2 incubator. The completed medium was comprised of RPMI 1640 basic medium (Biological Industries, Israel) supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS) (Biological Industries, Israel) and Penicillin (100 U/ mL)-Streptomycin (100 mg/mL) solution. THP-1 monocytes were primed with phorbol myristate acetate (PMA) (20 ng/mL, SigmaAldrich, USA) for 24 h to differentiate into macrophages. The THP1 macrophages were stimulated with ESAT6 (0.8 or 4.0 mg/mL, Abcam, USA) for 4 h or 24 h. 2.2. RNA isolation, quality evaluation and RNA sequencing THP-1 macrophages were harvested with Trizol (Ambion, Life technologies, USA). The total RNA was isolated with RNeasy mini kit (Qiagen, Germany) and quantified using a Nanodrop 8000 Spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed by Agilent 2200 Bioanalyzer. All samples were in good quality with RNA Integrity Number (RIN) > 8.5. Libraries were done following the procedure of KAPA Stranded mRNA-Seq Kit (Kapa biosystems, USA) and sequenced using the Illumina Hiseq 3000 Sequencer (50 cycles, single read lane). The raw data have been deposited to NCBI's Gene Expression Omnibus (GEO) with accession number GSE107387. 2.3. Quantitative real time PCR RNA samples were reversely transcribed with Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany) following the manufacturer's instructions. cDNA was quantified by qPCR with FastStart Essential DNA Green master (Roche, Germany) using a Bio-Rad C1000 CFX96 thermocycler (Bio-Rad, USA). The relative expression levels were calculated by 2-DDCT method with the ACTB gene as the endogenous control [18]. Primer sequences were listed as Table S1. 2.4. Quantification of cytokines and chemokines in the supernatant of THP-1 macrophages Cytokine and chemokine levels in the culture supernatant of THP-1 macrophages were profiled with a human cytokine and chemokine 21-plex panel under the Bio-Plex® system (Bio-Rad,
Latex beads (0.8 mm in diameter. Aladdin, China) were added to the THP-1 macrophages culture medium for 1 h in a humidified, 37 C 5% CO2 incubator. The free beads were washed away with PBS (pH 7.2) for twice. Then the beads in macrophages were counted under Dm1000 fluorescence microscope (Leica Microsystems, Germany). Pictures were acquired at original magnification 400. Average beads per macrophage were calculated based on 9 fields in microscope for each treatment. 2.7. Detection of ROS generation Levels of ROS generation in THP-1 macrophages were analyzed with ROS Detection Kit (KeyGEN BioTECH, Nanjing, China) following the recommended procedures. Briefly, THP-1 macrophages in 6-well plate (5 105/mL) were loaded with DCFH-DA (20 70 -dichlorodihydrofluorescein diacetate) probes (1:1000 in RPMI 1640 basic medium) for 20 min in a humidified, 37 C, 5% CO2 incubator. After incubation, macrophages were washed, trypsinized, collected and immediately measured with Navios flow cytometry (Beckman Coulter, US). 2.8. Western blotting Whole cell extracts samples were prepared with RIPA buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor (Biotool, China). Protein extracts were separated in sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel (Bio-Rad, China). Then proteins were transferred to PVDF membrane (0.22 mm, Bio-Rad, US) and subjected to immunoblotting with primary antibodies against HIF1a (1:1000) (Rabbit mAb, #D2U3T, Cell Signaling Technology, US) or GAPDH (1:5000) (Rabbit mAb, #D16H11, Cell Signaling Technology, US). 2.9. Bioinformatics analysis RNA sequencing data were analyzed following the standardized workflow described in GEO (GSE107387). Gene Ontology (GO) analysis of differentially expressed genes were performed with R package “clusterProfiler” [19]. Canonical pathway analysis was carried out with Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, US, http://www.ingenuity.com). Regulators for inflammatory effector genes were analyzed with FunRich [20]. The potential interaction networks (medium confidence interaction score 0.4) among effectors were analyzed with STRING (http://www.string-
Please cite this article as: F. Li et al., Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.045
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db.org) [21]. Downstream targets of HIF1a were obtained from GEO (GSE60729 on siHIF1A treated macrophages) and analyzed with online tool GEO2R [22]. Peaks for HIF1a targets in ChIP-seq data (GSE43109) were obtained from Cistrome Data Browser [23] and visualized with UCSC genome browser [24].
beads were engulfed in THP-1 macrophages after ESAT6 treatment for 4 h (P ¼ 0.0020) and 24 h (P < 0.0001) (Fig. 1C & Fig. S1D). To evaluate how ESAT6 influence the cytokine and chemokine production in THP-1 macrophage, we next studied the cytokine and chemokine profile with a 21-multiplex assay.
2.10. Statistical analysis
3.2. ESAT6 induced a distinctive cytokine and chemokine profile in THP-1 macrophages
Statistical analysis was conducted and visualized with the GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA). The simple comparison was done with unpaired Student's t-test and multiple comparisons with the Ordinary one-way ANOVA and Tukey's multiple comparisons test. Statistics were presented as mean ± standard deviation. P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. 3. Results 3.1. ESAT6 enhanced the innate immunity of THP-1 macrophages RNA-seq data showed ESAT6 enhanced the innate immunity of THP-1 macrophages. Biological processes and pathways for phagocytosis, cytokine and chemokine signaling, as well as inflammatory response were enriched for differentially expressed genes of THP-1 macrophages treated with ESAT6 for 4 h (Fig. 1A & Fig. S1AB). We also observed elevated ROS level in THP-1 macrophages treated with ESAT6 for 4 h compared to the untreated (P ¼ 0.0030) (Fig. 1B & Fig. S1C). And increasing number of latex
The 21-multiplex assay data showed these elevated cytokines and chemokines included pro-inflammatory mediators (IL1b, TNFa, CCL2, CCL3, CXCL8, CXCL9 and CXCL10) and anti-inflammatory mediators (IL10, CCL1, CCL17 and CXCL13) (Fig. 2A & Fig. S2). A dose and time dependent manner was observed in the production of pro-inflammatory mediators (CXCL8, TNFa and IL6) and antiinflammatory mediator (IL10) in macrophages treated with ESAT6 (Fig. 2BCDE). Macrophages can be basically induced into the proinflammatory M1 polarization and anti-inflammatory M2 polarization [25]. However, here our data showed the THP-1 macrophages activated with ESAT6 couldn't be simply gridded into either M1 or M2 polarization. It's a concurrence of pro-inflammatory and anti-inflammatory phenotypes. The above mentioned chemokines can be functionally chemotactic to monocytes (CCL1, CCL2, CCL3, CCL20 and CXCL10), neutrophils (CXCL1, CXCL5 and CXCL8) and lymphocytes (Th1: CXCL10; Th2: CCL1, CCL2, CCL3; B cells: CXCL13) [26]. The recruitment of these leukocytes to the infectious loci, plays crucial roles in initiating the adaptive immunity to control MTb infection [26,27].
Fig. 1. ESAT6 enhanced the innate immunity of THP-1 macrophages. (A) Gene Ontology analysis revealed cytokine, chemokine and phagocytosis associated signaling was enriched for differentially expressed genes in macrophages treated with ESAT6 for 4 h. (B) ROS levels was measured in THP-1 macrophages after ESAT6 treatment for 4 h with DCFHDA probe in flow cytometry. (C) Phagocytosis ability of ESAT6 treated THP-1 macrophages for 4 h and 24 h with latex beads (Number of beads/Number of macrophages ¼ 10). **P < 0.01; ****P < 0.0001.
Please cite this article as: F. Li et al., Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.045
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Fig. 2. ESAT6 promoted the production of cytokines and chemokines of THP-1 macrophages in a time and dose dependent manner. (A) Multiplex assay for 21 cytokines and chemokines detection in THP-1 macrophages treated with ESAT6 for 4 h detected with Bio-Plex® system. Chemokine CXCL8 (B), anti-inflammatory cytokine IL10 (C), and proinflammatory cytokines TNFa (D) and IL6 (E) levels were observed in THP-1 macrophages treated with ESAT6 (0.8 mg/ml, 4.0 mg/ml) for 4 h and 24 h by IMMULITE® 1000 immunoassay analyzer. Experiments were done in triplicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
3.3. ESAT6 accelerated the glucose metabolism in THP-1 macrophages partially via enhancing HIF1a expression Inflammatory mediators can be produced or induced along with the glycolysis process or the tricarboxylic acid (TCA) cycle of glucose metabolism [28]. Transcriptome analysis revealed several glycolysis related genes (HK1, PFKFB2/4, PFKM, GAPDH, ENO2, PKM) were highly expressed in ESAT6 treated THP-1 macrophages (Fig. S3AB). The glycolysis process consumes more glucose and generates more lactate compared with oxidative phosphorylation when producing one ATP molecule [28]. We found ESAT6 induced higher levels of glucose consumption and lactate generation in THP-1 macrophages than the untreated (Fig. 3AB). HIF1a has been showed to mediate the glycolysis process in IFNg primed and MTb infected macrophages [29,30]. And several glycolysis related genes were HIF1a targets (HK1, PFKFB2/4, GAPDH, ENO2, PKM) (Figs. S3CeJ). Here we observed ESAT6 promoted HIF1A mRNA and protein levels in THP-1 macrophages (Fig. 3CD). When HIF1A were interfered with siRNA, we found levels of glucose consumption were not changed (Fig. 3E), while the lactate generation levels were significantly reduced (Fig. 3F). These data indicated ESAT6 might accelerate the glucose metabolism in THP-1 macrophages partially via enhancing HIF1a expression. 3.4. HIF1a might participate in the process that ESAT6 enhanced the innate immunity of THP-1 macrophages We next investigated whether HIF1a mediate the process that ESAT6 enhanced the innate immunity of THP-1 macrophages. We interfered the HIF1a expression with siRNA and observed the changes of ROS levels, phagocytosis capacity of macrophages and production of some cytokine and chemokine. The ROS levels in THP-1 macrophages were decreased after siHIF1A treatment
(Fig. 4A). Meanwhile, the phagocytosis ability of THP-1 macrophages was defected after HIF1A was interfered (Fig. 4B). Previously IL1b was reported to be target gene of HIF1a [31]. However, the production of IL1b was not significantly changed in HIF1A interfered macrophages, whereas the levels of IL1b was hiked after ESAT6 treatments (Fig. 4C). Similarly, the production levels of TNF-a and CXCL8 were also not significantly changed in HIF1A interfered macrophages (Fig. 4DE). These above data indicated other regulators but HIF1a might be responsible to produce inflammatory factors (IL1b, TNFa and CXCL8). Integrative analysis of our transcriptome data indicated the existing of upstream regulators (NFIC, IRF1, LHX4, DMBX1, HOXA13, HOXB4 and POU6F1) in the production of inflammatory mediators in THP-1 macrophages treated with ESAT6 (Fig. S4). Roles of the mentioned regulators might require further investigation. Therefore, ESAT6 enhanced the innate immunity of THP-1 macrophages partially via HIF1a. 4. Discussion Innate immunity represents the body's first-line defense against invading pathogens [32]. During the interaction between host innate immune cells and pathogens, innate immune cells acquire the ability to recognize pathogen-associated molecular pattern, subsequently initiate the adaptive immune response [32]. Training the innate immune cells with pathogen specific antigens (or virulence factors) has opened an effective approach to protest the host against pathogens [8,33]. Here we reported ESAT6 from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a. The host innate immune cells quickly produced a great mount of inflammatory mediators upon the pathogen invasion via adjusting their inner metabolic program [34]. Inflammatory mediators can be produced along with the glycolysis process or the tricarboxylic acid
Please cite this article as: F. Li et al., Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.045
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Fig. 3. ESAT6 accelerated the glucose metabolism of THP-1 macrophages partially by enhancing the mRNA and protein levels of HIF1a. Glucose consumption (A) and lactate generation (B) in THP-1 macrophages were enhanced after ESAT6 treatment (0.8 mg/ml for 4 h). (C) HIF1A mRNA expression levels in siHIF1A (48 h) and ESAT6 (0.8 mg/ml for 4 h) treated THP-1 macrophages. (D) HIF1a protein levels in ESAT6 (0.8 mg/ml, 4.0 mg/ml for 4 h and 24 h) treated THP-1 macrophages. Glucose consumption (E) and lactate generation (F) in THP-1 macrophages when HIF1A mRNA was interfered with siRNA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 4. ESAT6 activated THP-1 macrophages via HIF1a partially. (A) Levels of ROS in THP-1 macrophages treated with siHIF1A for 48 h and ESAT6 (0.8 mg/ml for 4 h). (B) Phagocytosis ability THP-1 macrophages treated with siHIF1A for 48 h and ESAT6 (0.8 mg/ml for 4 h) with latex beads (Number of beads/Number of macrophages ¼ 50). IL1b (C), CXCL8 (D) and TNFa (E) in THP-1 macrophages treated with siHIF1A for 48 h and ESAT6 (0.8 mg/ml for 4 h) by IMMULITE® 1000 immunoassay analyzer. (F) Mechanism revealed in this study that ESAT6 enhanced the protective innate immunity of macrophages. *P < 0.05; ***P < 0.001; ****P < 0.0001.
(TCA) cycle of glucose metabolism [28]. Recently, PPAR-g was reported to mediate the vitamin B1 induced protective innate immunity in macrophages to limit MTb [35]. In that study, PPAR-g
integrated the metabolic (mitochondrial respiration and lipid metabolism) and inflammatory signals [35]. Here we observed ESAT6 accelerated the glucose metabolism and induced high levels
Please cite this article as: F. Li et al., Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.045
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of inflammatory mediators. We also observed that expression of HIF1a was regulated by ESAT6, and HIF1a mediated the promotion of protective innate immunity in ESAT6 activated THP-1 macrophage. HIF1a has been reported to mediate the glycolysis process in IFNg primed and MTb infected macrophages [29,30]. Therefore, HIF1a connected the glucose metabolism with the inflammatory activation of macrophages under the context of ESAT6 treatment. Future studies might focus on the mechanisms that how ESAT6 enhances the gene expression and protein stability of HIF1a. Recently, ESAT6 was reported to drive macrophages differentiation toward the pro-inflammatory phenotype and subsequently switch them to the anti-inflammatory phenotype following welldesigned in vitro differentiation procedures [27]. Here we indicated that the macrophages activated with ESAT6 showed a concurrence of pro-inflammatory and anti-inflammatory phenotypes. In our previous patient-based study, tuberculosis patients showed higher levels of cytokines (IL1a, TNFa and IL10) and chemokines (CCL2/MCP-1, CCL3/MIP-1a, CCL11/Eotaxin, CCL22/MDC and CXCL10/IP-10) in their serum comparing to the healthy subjects [36]. All these data indicated the pro-inflammatory and antiinflammatory factors coexisted in the serum of hosts upon MTb infection. And modulating the regulators that balance the proinflammatory and anti-inflammatory factors might determine the outcome of infection. One recent study reported BCG enhanced the antimycobacterial innate immunity in macrophages against tuberculosis by inducing a distinct epigenetic and transcriptomic signature in macrophages [37]. They revealed the involvement of transcription factors IRF and STAT in IFN regulatory networks with increased expression of IFNg, TNFa, and IL1b [37]. The above BCG study has offered quotable experience to explore the detailed mechanisms how ESAT6 enhanced protective innate immunity within macrophages. In sum, this study reported the possible mechanisms that how ESAT6 conferred protection against tuberculosis via enhancing the innate immunity of macrophages (Fig. 4F). ESAT6 induced THP-1 macrophages into a concurrence of pro- and anti-inflammatory phenotypes. ESAT6 also enhanced the phagocytosis capacity, ROS generation and accelerated the glucose metabolism in macrophages to promote the innate immunity of macrophages. These processes might be partially mediated by HIF1a. This study provided clues for developing therapies against tuberculosis by targeting ESAT6. Funding This work was supported by National Natural Science Foundation of China [grant number 81371760], the Major Project of the “Twelfth Five-year Plan” for Medical Science Development of PLA of China [grant number AWS14C003-2], National Infection Disease Prevention and Cure Special Project of China [grant number 2013ZX10003006-003-002], Basic science and advanced technology project of Chongqing [grant number CSTC2017jcyjAX0286] and China Scholarship Council [grant number 201403170243]. Acknowledgments The authors thank Dr. Xueqing Xu for suggestions on improving this manuscript. The first author thanks the corresponding author for seven-year supervision of his PhD work. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.045.
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Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a Fake Li a, Jie Luo b, Huan Xu a, Yang Wang a, Wenbin Jiang b, Kai Chang a, Shaoli Deng b, Ming Chen a, c, d, * a
Department of Clinical Laboratory, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China Department of Clinical Laboratory, Special Medical Center, Third Military Medical University (Army Medical University), Chongqing, 400012, China College of Pharmacy and Laboratory Medicine, Third Military Medical University (Army Medical University), Chongqing, 400038, China d State Key Laboratory of Trauma, Burn and Combined Injury, Third Military Medical University (Army Medical University), Chongqing, 400038, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 October 2019 Accepted 6 November 2019 Available online xxx
Early secreted antigenic target 6-kDa protein (ESAT6) is an essential virulence factor of Mycobacterium tuberculosis (MTb). However, ESAT6 helped fighting MTb infection according to vaccine studies. It's unclear whether ESAT6 confers protection via enhancing the innate immunity of macrophages, which are the first-line defense against MTb. We profiled the global transcriptional changes and characterized the innate immunity of THP-1 macrophages treated with ESAT6. We found ESAT6 promoted the phagocytosis ability, enhanced reactive oxygen species (ROS) generation and accelerated glucose metabolism in macrophages. Meanwhile, ESAT6 induced a distinctive phenotype of macrophages with a concurrence of pro-inflammatory and anti-inflammatory cytokines and chemokines. ESAT6 increased the expression of HIF1a mRNA and protein. Interfering HIF1a with siRNA defected the capacity of phagocytosis and ROS generation as well as glucose metabolism. Thus, ESAT6 enhanced the protective innate immunity of macrophages partially via HIF1a. This study provided clues for developing therapies against tuberculosis by targeting ESAT6. © 2019 Elsevier Inc. All rights reserved.
Keywords: Early secreted antigenic target 6-kDa Macrophage activation Innate immune Chemokine Glucose metabolism RNA sequencing
1. Introduction Mycobacterium tuberculosis (MTb) has been responsible for more human deaths than any other pathogens according to Global TB Report from World Health Organization [1]. Early Secreted Antigenic Target 6-kDa protein (ESAT6) is an essential virulence factor of MTb, encoded in region of difference 1 (RD1) which is absent in the genome of vaccine strain M. bovis Bacille CalmetteGu erin (BCG) [2]. ESAT6 is secreted with culture filtrate protein 10kDa (CFP10) in a 1:1 heterodimer manner, but it may function independent of CFP10 [3]. As a highly secreted protein during infection, ESAT6 showed great antigenic capacity and induced high level differentiation of
* Corresponding author. Department of Clinical Laboratory, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China. E-mail address: [email protected] (M. Chen).
ESAT6-specific T cells in mice and human [4,5]. Interestingly, studies found the attenuated live vaccines expressing ESAT6 conferred better protection than BCG [4,6,7]. Mice and guinea pigs vaccinated with recombinant BCG exporting ESAT6, showed less severe pathology and reduced dissemination of the pathogen, as compared with control mice and guinea pigs immunized with BCG alone [7]. The candidate attenuated live vaccine MTBVAC expressing ESAT6, has also presented better protection than BCG [6]. Meanwhile, MTBVAC mutant in exporting ESAT6 showed lower efficacy than the MTBVAC parental strain and similar efficacy to BCG [4]. Training the innate immune cells with pathogen specific antigens (or virulence factors) has opened an effective approach to protest the host against pathogens [8]. Macrophages are hosts’ first-line defense against MTb by utilizing innate immune approaches, including phagocytosis, respiratory bursts (ROS production) and production of inflammatory cytokines and chemokines [9,10]. Accumulated evidence indicated that ESAT6 modulated the apoptosis [11,12], autophagy [13], antigen presentation [14,15] and
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Please cite this article as: F. Li et al., Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.045
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inflammatory activation [12,16,17] of macrophages. However, whether ESAT6 confers protection via enhancing the innate immunity of macrophages and the underlying mechanisms remain largely unclear. To better understand how ESAT6 regulates the innate immunity of macrophage, we systematically profiled the global transcriptional changes and characterized the innate immunity of ESAT6 treated THP-1 macrophages. These characteristics included levels of multiplex cytokines and chemokines, capacity of phagocytosis, ROS production, glucose consumption and lactate generation in macrophages. We also explored the potential regulator involved in the above processes under the regulation of ESAT6. Therefore, this study may provide an integrative view on how ESAT6 potentially enhances the protective innate immunity of macrophages against tuberculosis. 2. Materials and methods
Shanghai, China). The 21-plex panel included IL1b, IL6, IL10, TNFa, GM-CSF, MIF, CCL1, CCL2, CCL3, CCL17, CCL20, CCL22, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL13 and CXCL16. Proper validation of the array system (optics, reporter, classification and fluidics validation) has been performed to ensure the optimal assay performance before sample testing. Levels of IL1b, IL6, IL10, TNFa and CXCL8 were also quantified by IMMULITE® 1000 immunoassay analyzer (Siemens, Germany). Experiments were done in triplicates. 2.5. RNA interference for HIF1A The siRNA targeting HIF1A and negative control siRNA were purchased from Sangon Biotech (Shanghai, China). The siRNA oligonucleotides were transfected into THP-1 macrophages using the Lipofectamine 3000 (Invitrogen) following the manufacturer's protocol. The siHIF1A sequences were: sense (50 -30 ) CUGAUGAC CAGCAACUUGATT, antisense (50 -30 ) UCAAGUUGCUGGUCAUCAGTT.
2.1. Cell culture and treatments 2.6. Phagocytosis capability analysis The human THP-1 monocytes were obtained from Cell Bank of Chinese Academy of Science and with identity confirmed by Short Tandem Repeat (STR) authentication. THP-1 monocytes were cultured in freshly made completed medium at 2-5 105/mL in a humidified, 37 C, 5% CO2 incubator. The completed medium was comprised of RPMI 1640 basic medium (Biological Industries, Israel) supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS) (Biological Industries, Israel) and Penicillin (100 U/ mL)-Streptomycin (100 mg/mL) solution. THP-1 monocytes were primed with phorbol myristate acetate (PMA) (20 ng/mL, SigmaAldrich, USA) for 24 h to differentiate into macrophages. The THP1 macrophages were stimulated with ESAT6 (0.8 or 4.0 mg/mL, Abcam, USA) for 4 h or 24 h. 2.2. RNA isolation, quality evaluation and RNA sequencing THP-1 macrophages were harvested with Trizol (Ambion, Life technologies, USA). The total RNA was isolated with RNeasy mini kit (Qiagen, Germany) and quantified using a Nanodrop 8000 Spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed by Agilent 2200 Bioanalyzer. All samples were in good quality with RNA Integrity Number (RIN) > 8.5. Libraries were done following the procedure of KAPA Stranded mRNA-Seq Kit (Kapa biosystems, USA) and sequenced using the Illumina Hiseq 3000 Sequencer (50 cycles, single read lane). The raw data have been deposited to NCBI's Gene Expression Omnibus (GEO) with accession number GSE107387. 2.3. Quantitative real time PCR RNA samples were reversely transcribed with Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany) following the manufacturer's instructions. cDNA was quantified by qPCR with FastStart Essential DNA Green master (Roche, Germany) using a Bio-Rad C1000 CFX96 thermocycler (Bio-Rad, USA). The relative expression levels were calculated by 2-DDCT method with the ACTB gene as the endogenous control [18]. Primer sequences were listed as Table S1. 2.4. Quantification of cytokines and chemokines in the supernatant of THP-1 macrophages Cytokine and chemokine levels in the culture supernatant of THP-1 macrophages were profiled with a human cytokine and chemokine 21-plex panel under the Bio-Plex® system (Bio-Rad,
Latex beads (0.8 mm in diameter. Aladdin, China) were added to the THP-1 macrophages culture medium for 1 h in a humidified, 37 C 5% CO2 incubator. The free beads were washed away with PBS (pH 7.2) for twice. Then the beads in macrophages were counted under Dm1000 fluorescence microscope (Leica Microsystems, Germany). Pictures were acquired at original magnification 400. Average beads per macrophage were calculated based on 9 fields in microscope for each treatment. 2.7. Detection of ROS generation Levels of ROS generation in THP-1 macrophages were analyzed with ROS Detection Kit (KeyGEN BioTECH, Nanjing, China) following the recommended procedures. Briefly, THP-1 macrophages in 6-well plate (5 105/mL) were loaded with DCFH-DA (20 70 -dichlorodihydrofluorescein diacetate) probes (1:1000 in RPMI 1640 basic medium) for 20 min in a humidified, 37 C, 5% CO2 incubator. After incubation, macrophages were washed, trypsinized, collected and immediately measured with Navios flow cytometry (Beckman Coulter, US). 2.8. Western blotting Whole cell extracts samples were prepared with RIPA buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor (Biotool, China). Protein extracts were separated in sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel (Bio-Rad, China). Then proteins were transferred to PVDF membrane (0.22 mm, Bio-Rad, US) and subjected to immunoblotting with primary antibodies against HIF1a (1:1000) (Rabbit mAb, #D2U3T, Cell Signaling Technology, US) or GAPDH (1:5000) (Rabbit mAb, #D16H11, Cell Signaling Technology, US). 2.9. Bioinformatics analysis RNA sequencing data were analyzed following the standardized workflow described in GEO (GSE107387). Gene Ontology (GO) analysis of differentially expressed genes were performed with R package “clusterProfiler” [19]. Canonical pathway analysis was carried out with Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, US, http://www.ingenuity.com). Regulators for inflammatory effector genes were analyzed with FunRich [20]. The potential interaction networks (medium confidence interaction score 0.4) among effectors were analyzed with STRING (http://www.string-
Please cite this article as: F. Li et al., Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.045
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db.org) [21]. Downstream targets of HIF1a were obtained from GEO (GSE60729 on siHIF1A treated macrophages) and analyzed with online tool GEO2R [22]. Peaks for HIF1a targets in ChIP-seq data (GSE43109) were obtained from Cistrome Data Browser [23] and visualized with UCSC genome browser [24].
beads were engulfed in THP-1 macrophages after ESAT6 treatment for 4 h (P ¼ 0.0020) and 24 h (P < 0.0001) (Fig. 1C & Fig. S1D). To evaluate how ESAT6 influence the cytokine and chemokine production in THP-1 macrophage, we next studied the cytokine and chemokine profile with a 21-multiplex assay.
2.10. Statistical analysis
3.2. ESAT6 induced a distinctive cytokine and chemokine profile in THP-1 macrophages
Statistical analysis was conducted and visualized with the GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA). The simple comparison was done with unpaired Student's t-test and multiple comparisons with the Ordinary one-way ANOVA and Tukey's multiple comparisons test. Statistics were presented as mean ± standard deviation. P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. 3. Results 3.1. ESAT6 enhanced the innate immunity of THP-1 macrophages RNA-seq data showed ESAT6 enhanced the innate immunity of THP-1 macrophages. Biological processes and pathways for phagocytosis, cytokine and chemokine signaling, as well as inflammatory response were enriched for differentially expressed genes of THP-1 macrophages treated with ESAT6 for 4 h (Fig. 1A & Fig. S1AB). We also observed elevated ROS level in THP-1 macrophages treated with ESAT6 for 4 h compared to the untreated (P ¼ 0.0030) (Fig. 1B & Fig. S1C). And increasing number of latex
The 21-multiplex assay data showed these elevated cytokines and chemokines included pro-inflammatory mediators (IL1b, TNFa, CCL2, CCL3, CXCL8, CXCL9 and CXCL10) and anti-inflammatory mediators (IL10, CCL1, CCL17 and CXCL13) (Fig. 2A & Fig. S2). A dose and time dependent manner was observed in the production of pro-inflammatory mediators (CXCL8, TNFa and IL6) and antiinflammatory mediator (IL10) in macrophages treated with ESAT6 (Fig. 2BCDE). Macrophages can be basically induced into the proinflammatory M1 polarization and anti-inflammatory M2 polarization [25]. However, here our data showed the THP-1 macrophages activated with ESAT6 couldn't be simply gridded into either M1 or M2 polarization. It's a concurrence of pro-inflammatory and anti-inflammatory phenotypes. The above mentioned chemokines can be functionally chemotactic to monocytes (CCL1, CCL2, CCL3, CCL20 and CXCL10), neutrophils (CXCL1, CXCL5 and CXCL8) and lymphocytes (Th1: CXCL10; Th2: CCL1, CCL2, CCL3; B cells: CXCL13) [26]. The recruitment of these leukocytes to the infectious loci, plays crucial roles in initiating the adaptive immunity to control MTb infection [26,27].
Fig. 1. ESAT6 enhanced the innate immunity of THP-1 macrophages. (A) Gene Ontology analysis revealed cytokine, chemokine and phagocytosis associated signaling was enriched for differentially expressed genes in macrophages treated with ESAT6 for 4 h. (B) ROS levels was measured in THP-1 macrophages after ESAT6 treatment for 4 h with DCFHDA probe in flow cytometry. (C) Phagocytosis ability of ESAT6 treated THP-1 macrophages for 4 h and 24 h with latex beads (Number of beads/Number of macrophages ¼ 10). **P < 0.01; ****P < 0.0001.
Please cite this article as: F. Li et al., Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.045
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Fig. 2. ESAT6 promoted the production of cytokines and chemokines of THP-1 macrophages in a time and dose dependent manner. (A) Multiplex assay for 21 cytokines and chemokines detection in THP-1 macrophages treated with ESAT6 for 4 h detected with Bio-Plex® system. Chemokine CXCL8 (B), anti-inflammatory cytokine IL10 (C), and proinflammatory cytokines TNFa (D) and IL6 (E) levels were observed in THP-1 macrophages treated with ESAT6 (0.8 mg/ml, 4.0 mg/ml) for 4 h and 24 h by IMMULITE® 1000 immunoassay analyzer. Experiments were done in triplicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
3.3. ESAT6 accelerated the glucose metabolism in THP-1 macrophages partially via enhancing HIF1a expression Inflammatory mediators can be produced or induced along with the glycolysis process or the tricarboxylic acid (TCA) cycle of glucose metabolism [28]. Transcriptome analysis revealed several glycolysis related genes (HK1, PFKFB2/4, PFKM, GAPDH, ENO2, PKM) were highly expressed in ESAT6 treated THP-1 macrophages (Fig. S3AB). The glycolysis process consumes more glucose and generates more lactate compared with oxidative phosphorylation when producing one ATP molecule [28]. We found ESAT6 induced higher levels of glucose consumption and lactate generation in THP-1 macrophages than the untreated (Fig. 3AB). HIF1a has been showed to mediate the glycolysis process in IFNg primed and MTb infected macrophages [29,30]. And several glycolysis related genes were HIF1a targets (HK1, PFKFB2/4, GAPDH, ENO2, PKM) (Figs. S3CeJ). Here we observed ESAT6 promoted HIF1A mRNA and protein levels in THP-1 macrophages (Fig. 3CD). When HIF1A were interfered with siRNA, we found levels of glucose consumption were not changed (Fig. 3E), while the lactate generation levels were significantly reduced (Fig. 3F). These data indicated ESAT6 might accelerate the glucose metabolism in THP-1 macrophages partially via enhancing HIF1a expression. 3.4. HIF1a might participate in the process that ESAT6 enhanced the innate immunity of THP-1 macrophages We next investigated whether HIF1a mediate the process that ESAT6 enhanced the innate immunity of THP-1 macrophages. We interfered the HIF1a expression with siRNA and observed the changes of ROS levels, phagocytosis capacity of macrophages and production of some cytokine and chemokine. The ROS levels in THP-1 macrophages were decreased after siHIF1A treatment
(Fig. 4A). Meanwhile, the phagocytosis ability of THP-1 macrophages was defected after HIF1A was interfered (Fig. 4B). Previously IL1b was reported to be target gene of HIF1a [31]. However, the production of IL1b was not significantly changed in HIF1A interfered macrophages, whereas the levels of IL1b was hiked after ESAT6 treatments (Fig. 4C). Similarly, the production levels of TNF-a and CXCL8 were also not significantly changed in HIF1A interfered macrophages (Fig. 4DE). These above data indicated other regulators but HIF1a might be responsible to produce inflammatory factors (IL1b, TNFa and CXCL8). Integrative analysis of our transcriptome data indicated the existing of upstream regulators (NFIC, IRF1, LHX4, DMBX1, HOXA13, HOXB4 and POU6F1) in the production of inflammatory mediators in THP-1 macrophages treated with ESAT6 (Fig. S4). Roles of the mentioned regulators might require further investigation. Therefore, ESAT6 enhanced the innate immunity of THP-1 macrophages partially via HIF1a. 4. Discussion Innate immunity represents the body's first-line defense against invading pathogens [32]. During the interaction between host innate immune cells and pathogens, innate immune cells acquire the ability to recognize pathogen-associated molecular pattern, subsequently initiate the adaptive immune response [32]. Training the innate immune cells with pathogen specific antigens (or virulence factors) has opened an effective approach to protest the host against pathogens [8,33]. Here we reported ESAT6 from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a. The host innate immune cells quickly produced a great mount of inflammatory mediators upon the pathogen invasion via adjusting their inner metabolic program [34]. Inflammatory mediators can be produced along with the glycolysis process or the tricarboxylic acid
Please cite this article as: F. Li et al., Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.045
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Fig. 3. ESAT6 accelerated the glucose metabolism of THP-1 macrophages partially by enhancing the mRNA and protein levels of HIF1a. Glucose consumption (A) and lactate generation (B) in THP-1 macrophages were enhanced after ESAT6 treatment (0.8 mg/ml for 4 h). (C) HIF1A mRNA expression levels in siHIF1A (48 h) and ESAT6 (0.8 mg/ml for 4 h) treated THP-1 macrophages. (D) HIF1a protein levels in ESAT6 (0.8 mg/ml, 4.0 mg/ml for 4 h and 24 h) treated THP-1 macrophages. Glucose consumption (E) and lactate generation (F) in THP-1 macrophages when HIF1A mRNA was interfered with siRNA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 4. ESAT6 activated THP-1 macrophages via HIF1a partially. (A) Levels of ROS in THP-1 macrophages treated with siHIF1A for 48 h and ESAT6 (0.8 mg/ml for 4 h). (B) Phagocytosis ability THP-1 macrophages treated with siHIF1A for 48 h and ESAT6 (0.8 mg/ml for 4 h) with latex beads (Number of beads/Number of macrophages ¼ 50). IL1b (C), CXCL8 (D) and TNFa (E) in THP-1 macrophages treated with siHIF1A for 48 h and ESAT6 (0.8 mg/ml for 4 h) by IMMULITE® 1000 immunoassay analyzer. (F) Mechanism revealed in this study that ESAT6 enhanced the protective innate immunity of macrophages. *P < 0.05; ***P < 0.001; ****P < 0.0001.
(TCA) cycle of glucose metabolism [28]. Recently, PPAR-g was reported to mediate the vitamin B1 induced protective innate immunity in macrophages to limit MTb [35]. In that study, PPAR-g
integrated the metabolic (mitochondrial respiration and lipid metabolism) and inflammatory signals [35]. Here we observed ESAT6 accelerated the glucose metabolism and induced high levels
Please cite this article as: F. Li et al., Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.045
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of inflammatory mediators. We also observed that expression of HIF1a was regulated by ESAT6, and HIF1a mediated the promotion of protective innate immunity in ESAT6 activated THP-1 macrophage. HIF1a has been reported to mediate the glycolysis process in IFNg primed and MTb infected macrophages [29,30]. Therefore, HIF1a connected the glucose metabolism with the inflammatory activation of macrophages under the context of ESAT6 treatment. Future studies might focus on the mechanisms that how ESAT6 enhances the gene expression and protein stability of HIF1a. Recently, ESAT6 was reported to drive macrophages differentiation toward the pro-inflammatory phenotype and subsequently switch them to the anti-inflammatory phenotype following welldesigned in vitro differentiation procedures [27]. Here we indicated that the macrophages activated with ESAT6 showed a concurrence of pro-inflammatory and anti-inflammatory phenotypes. In our previous patient-based study, tuberculosis patients showed higher levels of cytokines (IL1a, TNFa and IL10) and chemokines (CCL2/MCP-1, CCL3/MIP-1a, CCL11/Eotaxin, CCL22/MDC and CXCL10/IP-10) in their serum comparing to the healthy subjects [36]. All these data indicated the pro-inflammatory and antiinflammatory factors coexisted in the serum of hosts upon MTb infection. And modulating the regulators that balance the proinflammatory and anti-inflammatory factors might determine the outcome of infection. One recent study reported BCG enhanced the antimycobacterial innate immunity in macrophages against tuberculosis by inducing a distinct epigenetic and transcriptomic signature in macrophages [37]. They revealed the involvement of transcription factors IRF and STAT in IFN regulatory networks with increased expression of IFNg, TNFa, and IL1b [37]. The above BCG study has offered quotable experience to explore the detailed mechanisms how ESAT6 enhanced protective innate immunity within macrophages. In sum, this study reported the possible mechanisms that how ESAT6 conferred protection against tuberculosis via enhancing the innate immunity of macrophages (Fig. 4F). ESAT6 induced THP-1 macrophages into a concurrence of pro- and anti-inflammatory phenotypes. ESAT6 also enhanced the phagocytosis capacity, ROS generation and accelerated the glucose metabolism in macrophages to promote the innate immunity of macrophages. These processes might be partially mediated by HIF1a. This study provided clues for developing therapies against tuberculosis by targeting ESAT6. Funding This work was supported by National Natural Science Foundation of China [grant number 81371760], the Major Project of the “Twelfth Five-year Plan” for Medical Science Development of PLA of China [grant number AWS14C003-2], National Infection Disease Prevention and Cure Special Project of China [grant number 2013ZX10003006-003-002], Basic science and advanced technology project of Chongqing [grant number CSTC2017jcyjAX0286] and China Scholarship Council [grant number 201403170243]. Acknowledgments The authors thank Dr. Xueqing Xu for suggestions on improving this manuscript. The first author thanks the corresponding author for seven-year supervision of his PhD work. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.045.
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Please cite this article as: F. Li et al., Early secreted antigenic target 6-kDa from Mycobacterium tuberculosis enhanced the protective innate immunity of macrophages partially via HIF1a, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.045