Skip to main content
SpringerLink
Log in

Mycobacterium tuberculosis virulence protein ESAT-6 influences M1/M2 polarization and macrophage apoptosis to regulate tuberculosis progression

  • Research Article
  • Published:
Genes & Genomics Aims and scope Submit manuscript

Abstract

Background

Tuberculosis (TB) is an infectious disease caused by infection with Mycobacterium tuberculosis (Mtb), and it remains one of the major threats to human health worldwide. To our knowledge, the polarization of M1/M2 macrophages were critical innate immune cells which play important roles in regulating the immune response during TB progression.

Objective

We aimed to explore the potential mechanisms of M1/M2 macrophage polarization in TB development.

Methods

THP-1 macrophages were treated with early secreted antigenic target of 6 kDa (ESAT-6) protein for an increasing time. The polarization profiles, apoptosis levels of M1 and M2 macrophages were detected by RT-qPCR, immunofluorescence, Western blot and flow cytometry.

Results

ESAT-6 initially promoted the generation of pro-inflammatory M1-polarized macrophages in THP-1 cells within 24 h, which were suppressed by further ESAT-6 treatment at 30–42 h. Interestingly, ESAT-6 continuously promoted M2 polarization of THP-1 cells, thereby maintaining the anti-inflammatory response in a time-dependent manner. In addition, ESAT-6 promoted apoptotic cell death in M1-polarized macrophages, which had little effects on apoptosis of M2-phenotype of macrophages. Then, the potential underlying mechanisms were uncovered, and we verified that ESAT-6 increased the protein levels of TLR4, MyD88 and NF-κB to activate the TLR4/MyD88/NF-κB pathway within 24 h, and this signal pathway was significantly inactivated at 36 h post-treatment. Interestingly, the following experiments confirmed that ESAT-6 TLR4/MyD88/NF-κB pathway-dependently regulated M1/M2 polarization and apoptosis of macrophage in THP-1 cells.

Conclusion

Our study investigated the detailed effects and mechanisms of M1/M2 macrophages in regulating innate responses during TB development, which provided a new perspective on the development of treatment strategies for this disease.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data Availability

All available data supporting the results of this study are included in the article.

References

  • Araujo Z, Fernández de Larrea C, López D, Isern-Kebschull J, de Waard JH, Hagel I, Camargo M, Vanegas M, Patarroyo MA (2021) ESAT-6 and Ag85A Synthetic Peptides as Candidates for an Immunodiagnostic Test in Children with a Clinical Suspicion of Tuberculosis. Dis Markers 2021:6673250

  • Bade P, Simonetti F, Sans S, Laboudie P, Kissane K, Chappat N, Lagrange S, Apparailly F, Roubert C, Duroux-Richard I (2021) Integrative Analysis of Human Macrophage Inflammatory Response Related to Mycobacterium tuberculosis Virulence. Front Immunol 12:668060

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bussi C, Gutierrez MG (2019) Mycobacterium tuberculosis Infection of host cells in space and time. FEMS Microbiol Rev 43:341–361

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • C ÓM, Cox DJ, Phelan JJ, Mitermite M, Murphy DM, Leisching G, Thong L, O’Leary SM, Gogan KM, McQuaid K et al (2021) Lactate alters metabolism in human macrophages and improves their ability to kill Mycobacterium tuberculosis. Front Immunol 12:663695

    Article  Google Scholar 

  • Chai Q, Wang L, Liu CH, Ge B (2020) New insights into the evasion of host innate immunity by Mycobacterium tuberculosis. Cell Mol Immunol 17:901–913

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen SN, Tan Y, Xiao XC, Li Q, Wu Q, Peng YY, Ren J, Dong ML (2021) Deletion of TLR4 attenuates lipopolysaccharide-induced acute liver injury by inhibiting inflammation and apoptosis. Acta Pharmacol Sin 42:1610–1619

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Clemmensen HS, Knudsen NPH, Billeskov R, Rosenkrands I, Jungersen G, Aagaard C, Andersen P, Mortensen R (2020) Rescuing ESAT-6 specific CD4 T cells from terminal differentiation is critical for Long-Term Control of Murine Mtb Infection. Front Immunol 11:585359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Coppola M, Arroyo L, van Meijgaarden KE, Franken KL, Geluk A, Barrera LF, Ottenhoff THM (2017) Differences in IgG responses against Infection phase related Mycobacterium tuberculosis (Mtb) specific antigens in individuals exposed or not to Mtb correlate with control of TB Infection and progression. Tuberculosis (Edinb) 106:25–32

    Article  CAS  PubMed  Google Scholar 

  • de Martino M, Lodi L, Galli L, Chiappini E (2019) Immune Response to Mycobacterium tuberculosis: a narrative review. Front Pediatr 7:350

    Article  PubMed  PubMed Central  Google Scholar 

  • Faridgohar M, Nikoueinejad H (2017) New findings of toll-like receptors involved in Mycobacterium tuberculosis Infection. Pathog Glob Health 111:256–264

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gopalaswamy R, Shanmugam S, Mondal R, Subbian S (2020) Of Tuberculosis and non-tuberculous mycobacterial Infections - a comparative analysis of epidemiology, diagnosis and treatment. J Biomed Sci 27:74

    Article  PubMed  PubMed Central  Google Scholar 

  • Grover A, Izzo AA (2012) BAT3 regulates Mycobacterium tuberculosis protein ESAT-6-mediated apoptosis of macrophages. PLoS ONE 7:e40836

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hsieh WY, Zhou QD, York AG, Williams KJ, Scumpia PO, Kronenberger EB, Hoi XP, Su B, Chi X, Bui VL et al (2020) Toll-like receptors induce Signal-Specific reprogramming of the macrophage lipidome. Cell Metab 32:128–143e125

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang X, Li Y, Fu M, Xin HB (2018) Polarizing macrophages in Vitro. Methods Mol Biol 1784:119–126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jha V, Pal R, Kumar D, Mukhopadhyay S (2020) ESAT-6 protein of Mycobacterium tuberculosis increases holotransferrin-mediated Iron Uptake in macrophages by Downregulating Surface hemochromatosis protein HFE. J Immunol 205:3095–3106

    Article  CAS  PubMed  Google Scholar 

  • Ju M, Liu B, He H, Gu Z, Liu Y, Su Y, Zhu D, Cang J, Luo Z (2018) MicroRNA-27a alleviates LPS-induced acute lung injury in mice via inhibiting inflammation and apoptosis through modulating TLR4/MyD88/NF-κB pathway. Cell Cycle 17:2001–2018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Khanna M, Rady H, Dai G, Ramsay AJ (2021) Intranasal boosting with MVA encoding secreted mycobacterial proteins Ag85A and ESAT-6 generates strong pulmonary immune responses and protection against M. Tuberculosis in mice given BCG as neonates. Vaccine 39:1780–1787

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Korbecki J, Bajdak-Rusinek K (2019) The effect of palmitic acid on inflammatory response in macrophages: an overview of molecular mechanisms. Inflamm Res 68:915–932

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lam A, Prabhu R, Gross CM, Riesenberg LA, Singh V, Aggarwal S (2017) Role of apoptosis and autophagy in Tuberculosis. Am J Physiol Lung Cell Mol Physiol 313:L218–l229

    Article  PubMed  PubMed Central  Google Scholar 

  • Li C, Yang S, Ma H, Ruan M, Fang L, Cheng J (2021a) Influence of icariin on inflammation, apoptosis, invasion, and Tumor immunity in Cervical cancer by reducing the TLR4/MyD88/NF-κB and Wnt/β-catenin pathways. Cancer Cell Int 21:206

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li Y, Zhang L, Ren P, Yang Y, Li S, Qin X, Zhang M, Zhou M, Liu W (2021b) Qing-Xue-Xiao-Zhi formula attenuates Atherosclerosis by inhibiting macrophage lipid accumulation and inflammatory response via TLR4/MyD88/NF-κB pathway regulation. Phytomedicine 93:153812

    Article  CAS  PubMed  Google Scholar 

  • Liu S, Wu M, Wu AE, Geng S, Li S, Li Z, Li M, Pang L, Kang Y W et al (2021) Factors associated with differential T cell responses to antigens ESAT-6 and CFP-10 in pulmonary Tuberculosis patients. Med (Baltim) 100:e24615

    Article  CAS  Google Scholar 

  • Ma B, Athari SS, Mehrabi Nasab E, Zhao L (2021) PI3K/AKT/mTOR and TLR4/MyD88/NF-κB signaling inhibitors attenuate pathological mechanisms of allergic Asthma. Inflammation 44:1895–1907

    Article  CAS  PubMed  Google Scholar 

  • Mandala JP, Ahmad S, Pullagurla A, Thada S, Joshi L, Ansari MSS, Valluri VL, Gaddam SL (2020) Toll-like receptor 2 polymorphisms and their effect on the immune response to ESAT-6, Pam3CSK4 TLR2 agonist in pulmonary Tuberculosis patients and household contacts. Cytokine 126:154897

    Article  CAS  PubMed  Google Scholar 

  • Marongiu L, Donini M, Toffali L, Zenaro E, Dusi S (2013) ESAT-6 and HspX improve the effectiveness of BCG to induce human dendritic cells-dependent Th1 and NK cells activation. PLoS ONE 8:e75684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mily A, Kalsum S, Loreti MG, Rekha RS, Muvva JR, Lourda M, Brighenti S (2020) Polarization of M1 and M2 human monocyte-derived cells and analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection. J Vis Exp

  • Mir SA, Sharma S (2022) Immunotherapeutic potential of n-terminally formylated ESAT-6 protein in murine Tuberculosis. Int J Mycobacteriol 11:108–112

    Article  CAS  PubMed  Google Scholar 

  • Omar RA, Verma N, Arora PK (2021) Development of ESAT-6 based Immunosensor for the detection of Mycobacterium tuberculosis. Front Immunol 12:653853

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pathak SK, Basu S, Basu KK, Banerjee A, Pathak S, Bhattacharyya A, Kaisho T, Kundu M, Basu J (2007) Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nat Immunol 8:610–618

    Article  CAS  PubMed  Google Scholar 

  • Poulakis N, Gritzapis AD, Ploussi M, Leventopoulos M, Papageorgiou CV, Anastasopoulos A, Constantoulakis P, Karabela S, Vogiatzakis E, Tsilivakos V (2016) Intracellular ESAT-6: a new biomarker for Mycobacterium tuberculosis Infection. Cytometry B Clin Cytom 90:312–314

    Article  CAS  PubMed  Google Scholar 

  • Powers M, Sanchez TR, Welty TK, Cole SA, Oelsner EC, Yeh F, Turner J, O’Leary M, Brown RH, O’Donnell M et al (2020) Lung function and respiratory symptoms after Tuberculosis in an American Indian Population. The strong heart study. Ann Am Thorac Soc 17:38–48

    Article  PubMed  PubMed Central  Google Scholar 

  • Refai A, Gritli S, Barbouche MR, Essafi M (2018) Mycobacterium tuberculosis virulent factor ESAT-6 drives macrophage differentiation toward the pro-inflammatory M1 phenotype and subsequently switches it to the anti-inflammatory M2 phenotype. Front Cell Infect Microbiol 8:327

    Article  PubMed  PubMed Central  Google Scholar 

  • Saba K, Sameeullah M, Asghar A, Gottschamel J, Latif S, Lössl AG, Mirza B, Mirza O, Waheed MT (2020) Expression of ESAT-6 antigen from Mycobacterium tuberculosis in broccoli: an edible plant. Biotechnol Appl Biochem 67:148–157

    Article  CAS  PubMed  Google Scholar 

  • Samten B, Wang X, Barnes PF (2011) Immune regulatory activities of early secreted antigenic target of 6-kD protein of Mycobacterium tuberculosis and implications for Tuberculosis vaccine design. Tuberculosis (Edinb) 91(Suppl 1):S114–118

    Article  CAS  PubMed  Google Scholar 

  • Sánchez D, Rojas M, Hernández I, Radzioch D, García LF, Barrera LF (2010) Role of TLR2- and TLR4-mediated signaling in Mycobacterium tuberculosis-induced macrophage death. Cell Immunol 260:128–136

    Article  PubMed  Google Scholar 

  • Satoh T, Akira S (2016) Toll-like receptor signaling and its inducible proteins. Microbiol Spectr 4

  • Sawai M, Miyauchi Y, Ishida T, Takechi S (2022) Dihydropyrazine suppresses TLR4-dependent inflammatory responses by blocking MAPK signaling in human hepatoma HepG2 cells. J Toxicol Sci 47:381–387

    Article  CAS  PubMed  Google Scholar 

  • Shneerson JM (2004) Respiratory Failure in Tuberculosis: a modern perspective. Clin Med (Lond) 4:72–76

    Article  PubMed  Google Scholar 

  • Sreejit G, Ahmed A, Parveen N, Jha V, Valluri VL, Ghosh S, Mukhopadhyay S (2014) The ESAT-6 protein of Mycobacterium tuberculosis interacts with beta-2-microglobulin (β2M) affecting antigen presentation function of macrophage. PLoS Pathog 10:e1004446

    Article  PubMed  PubMed Central  Google Scholar 

  • Su Q, Li L, Sun Y, Yang H, Ye Z, Zhao J (2018) Effects of the TLR4/Myd88/NF-κB signaling pathway on NLRP3 inflammasome in Coronary Microembolization-Induced Myocardial Injury. Cell Physiol Biochem 47:1497–1508

    Article  CAS  PubMed  Google Scholar 

  • Swanson L, Katkar GD, Tam J, Pranadinata RF, Chareddy Y, Coates J, Anandachar MS, Castillo V, Olson J, Nizet V et al (2020) TLR4 signaling and macrophage inflammatory responses are dampened by GIV/Girdin. Proc Natl Acad Sci U S A 117:26895–26906

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Valizadeh A, Imani Fooladi AA, Sedighian H, Mahboobi M, Gholami Parizad E, Behzadi E, Khosravi A (2022) Evaluating the performance of PPE44, HSPX, ESAT-6 and CFP-10 factors in Tuberculosis Subunit vaccines. Curr Microbiol 79:260

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Y, Chen H, Chen Q, Jiao FZ, Zhang WB, Gong ZJ (2018) The Protective Mechanism of CAY10683 on Intestinal Mucosal Barrier in Acute Liver Failure through LPS/TLR4/MyD88 Pathway. Mediators Inflamm 2018:7859601

  • Wang L, Yang JW, Lin LT, Huang J, Wang XR, Su XT, Cao Y, Fisher M, Liu CZ (2020a) Acupuncture attenuates inflammation in Microglia of vascular Dementia rats by inhibiting miR-93-Mediated TLR4/MyD88/NF-κB signaling pathway. Oxid Med Cell Longev 2020:8253904

    PubMed  PubMed Central  Google Scholar 

  • Wang J, Gao Y, Lin F, Han K, Wang X (2020b) Omentin-1 attenuates lipopolysaccharide (LPS)-induced U937 macrophages activation by inhibiting the TLR4/MyD88/NF-κB signaling. Arch Biochem Biophys 679:108187

    Article  CAS  PubMed  Google Scholar 

  • Wang P, Xiao T, Li J, Wang D, Sun J, Cheng C, Ma H, Xue J, Li Y, Zhang A et al (2021) miR-21 in EVs from pulmonary epithelial cells promotes myofibroblast differentiation via glycolysis in arsenic-induced pulmonary fibrosis. Environ Pollut 286:117259

    Article  CAS  PubMed  Google Scholar 

  • Weiss G, Schaible UE (2015) Macrophage defense mechanisms against intracellular bacteria. Immunol Rev 264:182–203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Welin A, Eklund D, Stendahl O, Lerm M (2011) Human macrophages infected with a high burden of ESAT-6-expressing M. Tuberculosis undergo caspase-1- and cathepsin B-independent necrosis. PLoS ONE 6:e20302

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wu L, Du L, Ju Q, Chen Z, Ma Y, Bai T, Ji G, Wu Y, Liu Z, Shao Y et al (2021) Silencing TLR4/MyD88/NF-κB signaling pathway alleviated inflammation of corneal epithelial cells infected by ISE. Inflammation 44:633–644

    Article  CAS  PubMed  Google Scholar 

  • Xie X, Lv H, Liu C, Su X, Yu Z, Song S, Bian H, Tian M, Qin C, Qi J et al (2021) HBeAg mediates inflammatory functions of macrophages by TLR2 contributing to hepatic fibrosis. BMC Med 19:247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yabaji SM, Dhamija E, Mishra AK, Srivastava KK (2020) ESAT-6 regulates autophagous response through SOD-2 and as a result induces intracellular survival of Mycobacterium bovis BCG. Biochim Biophys Acta Proteins Proteom 1868:140470

    Article  CAS  PubMed  Google Scholar 

  • Yang S, Li F, Jia S, Zhang K, Jiang W, Shang Y, Chang K, Deng S, Chen M (2015) Early secreted antigen ESAT-6 of Mycobacterium Tuberculosis promotes apoptosis of macrophages via targeting the microRNA155-SOCS1 interaction. Cell Physiol Biochem 35:1276–1288

    Article  CAS  PubMed  Google Scholar 

  • Zhan L, Liu H, Zheng J, Meng J, Fu D, Pang L, Ji C (2022) Electroacupuncture at Zusanli Alleviates Sepsis by Regulating the TLR4-MyD88-NF-Kappa B Pathway and Diversity of Intestinal Flora. Evid Based Complement Alternat Med 2022:6706622

  • Zhang L, Zhang H, Zhao Y, Mao F, Wu J, Bai B, Xu Z, Jiang Y, Shi C (2012) Effects of Mycobacterium tuberculosis ESAT-6/CFP-10 fusion protein on the autophagy function of mouse macrophages. DNA Cell Biol 31:171–179

    Article  PubMed  Google Scholar 

  • Zhang L, Jiang X, Pfau D, Ling Y, Nathan CF (2021) Type I interferon signaling mediates Mycobacterium tuberculosis-induced macrophage death. J Exp Med 218

  • Zhao R, Luo T, Ma P, Ge L, Chen Z, Wang X, Liao W, Bao L (2021) Improvement of the immunogenicity of ESAT-6 via fusion with the dodecameric protein dodecin of Mycobacterium tuberculosis. Microb Pathog 155:104890

    Article  CAS  PubMed  Google Scholar 

  • Zhu L, Yang T, Li L, Sun L, Hou Y, Hu X, Zhang L, Tian H, Zhao Q, Peng J et al (2014) TSC1 controls macrophage polarization to prevent inflammatory Disease. Nat Commun 5:4696

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This study was supported by the State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia Fund (SKL-HIDCA-2021-JH2) and the Youth Fund of Natural Science Foundation of Xinjiang Uygur Autonomous Region (2018D01C207).

Author information

Authors and Affiliations

  1. State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China

    Feng Sun

  2. Pulmonary and Critical Care Medicine Center, The First Affiliated Hospital of Xinjiang Medical University, No.137, South Liyu Shan Road, Urumqi, 830054, China

    Feng Sun & Cunzi Yan

  3. Xinjiang Medical University, Urumqi, China

    Jiangbo Li

  4. Inspection Center, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, China

    Ling Cao

Authors
  1. Feng Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiangbo Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ling Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cunzi Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Feng Sun: conception, study design and wrote the manuscript. Jiangbo Li: performed the experiments and statistical analysis. Cunzi Yan and Ling Cao: collected, analyzed and interpretated the data. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Cunzi Yan.

Ethics declarations

Consent to participate

All authors approved and were directly involved in the planning, execution, and analysis of the data presented here.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sun, F., Li, J., Cao, L. et al. Mycobacterium tuberculosis virulence protein ESAT-6 influences M1/M2 polarization and macrophage apoptosis to regulate tuberculosis progression. Genes Genom 46, 37–47 (2024). https://doi.org/10.1007/s13258-023-01469-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13258-023-01469-4

Keywords

Search

Navigation