Skip to main content
SpringerLink
Log in

Toll-Like Receptor 9 Aggravates Pulmonary Fibrosis by Promoting NLRP3-Mediated Pyroptosis of Alveolar Epithelial Cells

  • RESEARCH
  • Published:
Inflammation Aims and scope Submit manuscript

Abstract

The apoptosis-prone property of alveolar epithelial cells plays a crucial role in pulmonary fibrosis(PF), but the role of pyroptosis in it is still unclear. Toll-like receptor 9(TLR9) has been reported to play a vital role in the pathogenesis of many diseases. However, the effect of TLR9 on alveolar epithelial cells in PF has not been fully elucidated. Gene expression microarray related to Idiopathic pulmonary fibrosis(IPF) was obtained from the Gene Expression Omnibus(GEO) database. In the mouse model of bleomycin-induced PF, adeno-associated virus(AAV6) was used to interfere with TLR9 to construct TLR9 knockdown mice to study the role of TLR9 in PF, and the specific mechanism was studied by intratracheal instillation of NLR family pyrin domain containing 3(NLRP3) activator. In vitro experiments were performed using A549 cells. Bleomycin-induced pyroptosis in the lung tissue of PF mice increased, and TLR9 protein levels also increased, especially in alveolar epithelial cells. The levels of fibrosis and pyroptosis in lung tissue of TLR9 knockdown mice were improved. We found that TLR9 can bind to the NLRP3, thereby increasing the activation of the NLRP3/caspase-1 inflammasome pathway. When we use the NLRP3 activator, the levels of fibrosis and pyroptosis in lung tissue of TLR9 knockout mice can be counteracted. Pyroptosis of alveolar epithelial cells plays a vital role in PF, and TLR9 can promote NLRP3-mediated pyroptosis of alveolar epithelial cells to aggravate the progression of PF and may become a feasible target for the prevention and treatment of PF.

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
Fig. 6
Fig. 7

Similar content being viewed by others

AVAILABILITY OF DATA AND MATERIALS

All datasets used in the research can be found in Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/, containing dataset of GSE70866).

Abbreviations

PF:

Pulmonary fibrosis

TLR9:

Toll-like receptor 9

IPF:

Idiopathic pulmonary fibrosis

GEO:

Gene Expression Omnibus

AAV:

Adeno-associated virus

NLRP3:

NLR family pyrin domain containing 3

ECM:

Extracellular matrix

TLR:

Toll-like receptor

PAMPs:

Pathogen-associated molecular patterns

α-SMA:

Alpha smooth muscle actin

MMP-14:

Matrix metalloproteinase-14

ER:

Endoplasmic reticulum

PRGs:

Pyroptosis-related genes

DEGs:

Differentially expressed genes

FDR:

False discovery rate

FC:

Fold-changes

LASSO:

Least Absolute Shrinkage and Selection Operator

ROC:

Receiver operating characteristic

PCA:

Principal component analysis

WGCNA:

Weighted correlation network analysis

BLM:

Bleomycin

NC:

Negative control

Nig:

Nigericin

H&E:

Hematoxylin and eosin

IHC:

Immunohistochemical

WB:

Western blot

BCA:

Bicinchoninic acid

MOI:

Multiplicities of infection

IF:

Immunofluorescence

ELISA:

Enzyme-linked immunosorbent assay

OD:

Optical density

AUC:

Area under curve

NOD:

Nucleotide-binding oligomerization domain

References

  1. Yu, Q.Y., and X.X. Tang. 2022. Irreversibility of Pulmonary Fibrosis. Aging and disease 13 (1): 73–86. https://doi.org/10.14336/AD.2021.0730.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Richeldi, L., H.R. Collard, and M.G. Jones. 2017. Idiopathic pulmonary fibrosis. Lancet (London, England) 389 (10082): 1941–1952. https://doi.org/10.1016/S0140-6736(17)30866-8.

    Article  PubMed  Google Scholar 

  3. Wright, W.A., L.E. Crowley, D. Parekh, A. Crawshaw, D.P. Dosanjh, P. Nightingale, and D.R. Thickett. 2021. Real-world retrospective observational study exploring the effectiveness and safety of antifibrotics in idiopathic pulmonary fibrosis. BMJ open respiratory research 8 (1): e000782. https://doi.org/10.1136/bmjresp-2020-000782.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Bargagli, E., C. Piccioli, E. Rosi, E. Torricelli, L. Turi, E. Piccioli, M. Pistolesi, K. Ferrari, and L. Voltolini. 2019. Pirfenidone and Nintedanib in idiopathic pulmonary fibrosis: Real-life experience in an Italian referral centre. Pulmonology 25 (3): 149–153. https://doi.org/10.1016/j.pulmoe.2018.06.003.

    Article  CAS  PubMed  Google Scholar 

  5. Horowitz, J.C., and V.J. Thannickal. 2006. Idiopathic pulmonary fibrosis : New concepts in pathogenesis and implications for drug therapy. Treatments in respiratory medicine 5 (5): 325–342. https://doi.org/10.2165/00151829-200605050-00004.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Sauler, M., I.S. Bazan, and P.J. Lee. 2019. Cell Death in the Lung: The Apoptosis-Necroptosis Axis. Annual review of physiology 81: 375–402. https://doi.org/10.1146/annurev-physiol-020518-114320.

    Article  CAS  PubMed  Google Scholar 

  7. Elmore, S. 2007. Apoptosis: A review of programmed cell death. Toxicologic pathology 35 (4): 495–516. https://doi.org/10.1080/01926230701320337.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Song, Z., Q. Gong, and J. Guo. 2021. Pyroptosis: Mechanisms and Links with Fibrosis. Cells 10 (12): 3509. https://doi.org/10.3390/cells10123509.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Liu, C., Q. Yao, T. Hu, Z. Cai, Q. Xie, J. Zhao, Y. Yuan, J. Ni, and Q.Q. Wu. 2022. Cathepsin B deteriorates diabetic cardiomyopathy induced by streptozotocin via promoting NLRP3-mediated pyroptosis. Molecular therapy. Nucleic acids 30: 198–207. https://doi.org/10.1016/j.omtn.2022.09.019.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gaul, S., A. Leszczynska, F. Alegre, B. Kaufmann, C.D. Johnson, L.A. Adams, A. Wree, G. Damm, D. Seehofer, C.J. Calvente, D. Povero, T. Kisseleva, A. Eguchi, M.D. McGeough, H.M. Hoffman, P. Pelegrin, U. Laufs, and A.E. Feldstein. 2021. Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis. Journal of hepatology 74 (1): 156–167. https://doi.org/10.1016/j.jhep.2020.07.041.

    Article  CAS  PubMed  Google Scholar 

  11. Lyu, A.K., S.Y. Zhu, J.L. Chen, Y.X. Zhao, D. Pu, C. Luo, Q. Lyu, Z. Fan, Y. Sun, J. Wu, K.X. Zhao, and Q. Xiao. 2019. Inhibition of TLR9 attenuates skeletal muscle fibrosis in aged sarcopenic mice via the p53/SIRT1 pathway. Experimental Gerontology 122 (undefined): 25–33. https://doi.org/10.1016/j.exger.2019.04.008.

    Article  CAS  PubMed  Google Scholar 

  12. Luo, X., Z. Zhai, Z. Lin, S. Wu, W. Xu, Y. Li, J. Zhuang, J. Li, F. Yang, and Y. He. 2023. Cyclophosphamide induced intestinal injury is alleviated by blocking the TLR9/caspase3/GSDME mediated intestinal epithelium pyroptosis. International immunopharmacology 119: 110244. https://doi.org/10.1016/j.intimp.2023.110244.

    Article  CAS  PubMed  Google Scholar 

  13. Lu, P., H. Zheng, H. Meng, C. Liu, L. Duan, J. Zhang, Z. Zhang, J. Gao, Y. Zhang, and T. Sun. 2023. Mitochondrial DNA induces nucleus pulposus cell pyroptosis via the TLR9-NF-κB-NLRP3 axis. Journal of translational medicine 21 (1): 389. https://doi.org/10.1186/s12967-023-04266-5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Yoshida, K., K. Abe, M. Ishikawa, K. Saku, M. Shinoda-Sakamoto, T. Ishikawa, T. Watanabe, M. Oka, K. Sunagawa, and H. Tsutsui. 2019. Inhibition of TLR9-NF-κB-mediated sterile inflammation improves pressure overload-induced right ventricular dysfunction in rats. Cardiovascular research 115 (3): 658–668. https://doi.org/10.1093/cvr/cvy209.

    Article  CAS  PubMed  Google Scholar 

  15. Shintani, Y., A. Kapoor, M. Kaneko, R.T. Smolenski, F. D’Acquisto, S.R. Coppen, N. Harada-Shoji, H.J. Lee, C. Thiemermann, S. Takashima, K. Yashiro, and K. Suzuki. 2013. TLR9 mediates cellular protection by modulating energy metabolism in cardiomyocytes and neurons. Proceedings of the National Academy of Sciences of the United States of America 110 (13): 5109–5114. https://doi.org/10.1073/pnas.1219243110.

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  16. Kirillov, V., J.T. Siler, M. Ramadass, L. Ge, J. Davis, G. Grant, S.D. Nathan, G. Jarai, and G. Trujillo. 2015. Sustained activation of toll-like receptor 9 induces an invasive phenotype in lung fibroblasts: Possible implications in idiopathic pulmonary fibrosis. The American journal of pathology 185 (4): 943–957. https://doi.org/10.1016/j.ajpath.2014.12.011.

    Article  CAS  PubMed  Google Scholar 

  17. Chockalingam, A., J.C. Brooks, J.L. Cameron, L.K. Blum, and C.A. Leifer. 2009. TLR9 traffics through the Golgi complex to localize to endolysosomes and respond to CpG DNA. Immunology and cell biology 87 (3): 209–217. https://doi.org/10.1038/icb.2008.101.

    Article  CAS  PubMed  Google Scholar 

  18. Borok, Z., M. Horie, P. Flodby, H. Wang, Y. Liu, S. Ganesh, A.L. Firth, P. Minoo, C. Li, M.F. Beers, A.S. Lee, and B. Zhou. 2020. Grp78 Loss in Epithelial Progenitors Reveals an Age-linked Role for Endoplasmic Reticulum Stress in Pulmonary Fibrosis. American journal of respiratory and critical care medicine 201 (2): 198–211. https://doi.org/10.1164/rccm.201902-0451OC.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Wang, C., T. Yang, J. Xiao, C. Xu, Y. Alippe, K. Sun, T.D. Kanneganti, J.B. Monahan, Y. Abu-Amer, J. Lieberman, and G. Mbalaviele. 2021. NLRP3 inflammasome activation triggers gasdermin D-independent inflammation. Science immunology 6 (64): eabj3859. https://doi.org/10.1126/sciimmunol.abj3859.

  20. Shen, J., Z. Dai, Y. Li, H. Zhu, and L. Zhao. 2022. TLR9 regulates NLRP3 inflammasome activation via the NF-kB signaling pathway in diabetic nephropathy. Diabetology & metabolic syndrome 14 (1): 26. https://doi.org/10.1186/s13098-021-00780-y.

    Article  CAS  Google Scholar 

  21. Kim, S.K., K.Y. Park, and J.Y. Choe. 2020. Toll-Like Receptor 9 Is Involved in NLRP3 Inflammasome Activation and IL-1β Production Through Monosodium Urate-Induced Mitochondrial DNA. Inflammation 43 (6): 2301–2311. https://doi.org/10.1007/s10753-020-01299-6.

    Article  CAS  PubMed  Google Scholar 

  22. Ritchie, M.E., B. Phipson, D. Wu, Y. Hu, C.W. Law, W. Shi, and G.K. Smyth. 2015. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic acids research 43 (7): e47. https://doi.org/10.1093/nar/gkv007.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wilkerson, M.D., and D.N. Hayes. 2010. ConsensusClusterPlus: A class discovery tool with confidence assessments and item tracking. Bioinformatics (Oxford, England) 26 (12): 1572–1573. https://doi.org/10.1093/bioinformatics/btq170.

    Article  CAS  PubMed  Google Scholar 

  24. Langfelder, P., and S. Horvath. 2008. WGCNA: An R package for weighted correlation network analysis. BMC Bioinformatics 9: 559. https://doi.org/10.1186/1471-2105-9-559.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lu, Y., K. Tang, S. Wang, Z. Tian, Y. Fan, B. Li, M. Wang, J. Zhao, and J. Xie. 2023. Dach1 deficiency drives alveolar epithelium apoptosis in pulmonary fibrosis via modulating C-Jun/Bim activity. Translational research : The journal of laboratory and clinical medicine 257: 54–65. https://doi.org/10.1016/j.trsl.2023.01.006.

    Article  CAS  PubMed  Google Scholar 

  26. Li, J., P. Xu, Y. Hong, Y. Xie, M. Peng, R. Sun, H. Guo, X. Zhang, W. Zhu, J. Wang, and X. Liu. 2023. Lipocalin-2-mediated astrocyte pyroptosis promotes neuroinflammatory injury via NLRP3 inflammasome activation in cerebral ischemia/reperfusion injury. Journal of neuroinflammation 20 (1): 148. https://doi.org/10.1186/s12974-023-02819-5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hübner, R.H., W. Gitter, N.E. El Mokhtari, M. Mathiak, M. Both, H. Bolte, S. Freitag-Wolf, and B. Bewig. 2008. Standardized quantification of pulmonary fibrosis in histological samples. BioTechniques 44 (4): 507–517. https://doi.org/10.2144/000112729.

    Article  CAS  PubMed  Google Scholar 

  28. Jing, G., J. Zuo, Q. Fang, M. Yuan, Y. Xia, Q. Jin, Y. Liu, Y. Wang, Z. Zhang, W. Liu, X. Wu, and X. Song. 2022. Erbin protects against sepsis-associated encephalopathy by attenuating microglia pyroptosis via IRE1α/Xbp1s-Ca2+ axis. Journal of neuroinflammation 19 (1): 237. https://doi.org/10.1186/s12974-022-02598-5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Van De Water, L., S. Varney, and J.J. Tomasek. 2013. Mechanoregulation of the Myofibroblast in Wound Contraction, Scarring, and Fibrosis: Opportunities for New Therapeutic Intervention. Advances in wound care 2 (4): 122–141. https://doi.org/10.1089/wound.2012.0393.

    Article  PubMed  Google Scholar 

  30. Liu, G.Y., G.R.S. Budinger, and J.E. Dematte. 2022. Advances in the management of idiopathic pulmonary fibrosis and progressive pulmonary fibrosis. BMJ (Clinical research ed.) 377: e066354. https://doi.org/10.1136/bmj-2021-066354.

    Article  PubMed  Google Scholar 

  31. Kadono, K., S. Kageyama, K. Nakamura, H. Hirao, T. Ito, H. Kojima, K.J. Dery, X. Li, and J.W. Kupiec-Weglinski. 2022. Myeloid Ikaros-SIRT1 signaling axis regulates hepatic inflammation and pyroptosis in ischemia-stressed mouse and human liver. Journal of hepatology 76 (4): 896–909. https://doi.org/10.1016/j.jhep.2021.11.026.

    Article  CAS  PubMed  Google Scholar 

  32. Koh, E.H., J.E. Yoon, M.S. Ko, J. Leem, J.Y. Yun, C.H. Hong, Y.K. Cho, S.E. Lee, J.E. Jang, J.Y. Baek, H.J. Yoo, S.J. Kim, C.O. Sung, J.S. Lim, W.I. Jeong, S.H. Back, I.J. Baek, S. Torres, E. Solsona-Vilarrasa, L. Conde de la Rosa, and K.U. Lee. 2021. Sphingomyelin synthase 1 mediates hepatocyte pyroptosis to trigger non-alcoholic steatohepatitis. Gut 70 (10): 1954–1964. https://doi.org/10.1136/gutjnl-2020-322509.

    Article  CAS  PubMed  Google Scholar 

  33. Schroder, K., and J. Tschopp. 2010. The inflammasomes. Cell 140 (6): 821–832. https://doi.org/10.1016/j.cell.2010.01.040.

    Article  CAS  PubMed  Google Scholar 

  34. Song, M., J. Wang, Y. Sun, J. Pang, X. Li, Y. Liu, Y. Zhou, P. Yang, T. Fan, Y. Liu, Z. Li, X. Qi, B. Li, X. Zhang, J. Wang, and C. Wang. 2022. Inhibition of gasdermin D-dependent pyroptosis attenuates the progression of silica-induced pulmonary inflammation and fibrosis. Acta pharmaceutica Sinica. B 12 (3): 1213–1224. https://doi.org/10.1016/j.apsb.2021.10.006.

    Article  CAS  PubMed  Google Scholar 

  35. Liang, Q., W. Cai, Y. Zhao, H. Xu, H. Tang, D. Chen, F. Qian, and L. Sun. 2020. Lycorine ameliorates bleomycin-induced pulmonary fibrosis via inhibiting NLRP3 inflammasome activation and pyroptosis. Pharmacological research 158: 104884. https://doi.org/10.1016/j.phrs.2020.104884.

    Article  CAS  PubMed  Google Scholar 

  36. Ma, L., Z. Han, H. Yin, J. Tian, J. Zhang, N. Li, C. Ding, and L. Zhang. 2022. Characterization of Cathepsin B in Mediating Silica Nanoparticle-Induced Macrophage Pyroptosis via an NLRP3-Dependent Manner. Journal of inflammation research 15: 4537–4545. https://doi.org/10.2147/JIR.S371536.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Sun, J., X. Ge, Y. Wang, L. Niu, L. Tang, and S. Pan. 2022. USF2 knockdown downregulates THBS1 to inhibit the TGF-β signaling pathway and reduce pyroptosis in sepsis-induced acute kidney injury. Pharmacological research 176: 105962. https://doi.org/10.1016/j.phrs.2021.105962.

    Article  CAS  PubMed  Google Scholar 

Download references

ACKNOWLEDGEMENTS

The results shown in the study are in part based upon data generated by the Gene Expression Omnibus: https://www.ncbi.nlm.nih.gov/geo/.

Funding

This research received no external funding.

Author information

Authors and Affiliations

  1. Department of Urology, Children’s Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child development and Disorders, China International Science and Technology Cooperation base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing, People’s Republic of China

    Chunnian Ren, Tao Mi, Zhaoxia Zhang & Dawei He

  2. Department of Cardiothoracic Surgery, Children’s Hospital of Chongqing Medical University , National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science and Technology Cooperation base of Child development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing, People’s Republic of China

    Chunnian Ren & Quan Wang

  3. Department of Respiratory Medicine, Second Affiliated Hospital of Chongqing Medical University, Chongqing, China

    Shulei Fan

  4. Second Affiliated Hospital of Chongqing Medical University, Chongqing, China

    Dawei He

Authors
  1. Chunnian Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Quan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shulei Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tao Mi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhaoxia Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dawei He
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.R., Q.W., S.F., D.H.: data curation, statistical analysis, and manuscript preparation. T.M., Z.Z., D.H.: funding acquisition, study design, supervision, and manuscript revision. All authors reviewed the manuscript.

Corresponding author

Correspondence to Dawei He.

Ethics declarations

Ethics Approval and Consent to Participate

Since the information in the GEO database is public and anonymous, patient-informed consent and ethical approval are not required. All animal experiments were approved by the ethics committee of the Chongqing Medical University.

Consent for Publication

None.

Competing Interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

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

Supplementary Information

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

Ren, C., Wang, Q., Fan, S. et al. Toll-Like Receptor 9 Aggravates Pulmonary Fibrosis by Promoting NLRP3-Mediated Pyroptosis of Alveolar Epithelial Cells. Inflammation (2024). https://doi.org/10.1007/s10753-024-02006-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s10753-024-02006-5

KEY WORDS

Search

Navigation