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Neutralization of CX3CL1 Attenuates TGF-β-Induced Fibroblast Differentiation Through NF-κB Activation and Mitochondrial Dysfunction in Airway Fibrosis

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Abstract

Background

Severe asthma, characterized by inflammation and airway remodeling, involves fibroblast differentiation into myofibroblasts expressing α-SMA. This process leads to the production of fibronectin and connective tissue growth factor (CTGF), driven by factors such as transforming growth factor (TGF)-β. Furthermore, the persistent presence of myofibroblasts is associated with resistance to apoptosis and mitochondrial dysfunction. The chemokine (C-X3-C motif) ligand 1 (CX3CL1) plays a role in tissue fibrosis. However, it is currently unknown whether neutralization of CX3CL1 decreases TGF-β-induced fibroblast differentiation and mitochondrial dysfunction in normal human lung fibroblasts (NHLFs).

Methods

CX3CL1/C-X3-C motif chemokine receptor 1 (CX3CR1), CX3CL1 was analyzed by immunofluorescence (IF) or immunohistochemical (IHC) staining of ovalbumin-challenged mice. CX3CL1 release was detected by ELISA. TGF-β-induced CTGF, fibronectin, and α-SMA expression were evaluated in NHLFs following neutralization of CX3CL1 (TP213) treatment for the indicated times by Western blotting or IF staining. Mitochondrion function was detected by a JC-1 assay and seahorse assay. Cell apoptosis was observed by a terminal uridine nick-end labeling (TUNEL) assay.

Results

An increase in CX3CL1 expression was observed in lung tissues from mice with ovalbumin-induced asthma by IF staining. CX3CR1 was increased in the subepithelial layer of the airway by IHC staining. Moreover, CX3CR1 small interfering (si)RNA downregulated TGF-β-induced CTGF and fibronectin expression in NHLFs. CX3CL1 induced CTGF and fibronectin expression in NHLFs. TGF-β-induced CX3CL1 secretion from NHLFs. Furthermore, TP213 decreased TGF-β-induced CTGF, fibronectin, and α-SMA expression in NHLFs. Mitochondrion-related differentially expressed genes (DEGs) were examined after CX3CL1 neutralization in TGF-β-treated NHLFs. TP213 alleviated TGF-β-induced mitochondrial dysfunction and apoptosis resistance in NHLFs. CX3CL1 induced p65, IκBα, and IKKα phosphorylation in a time-dependent manner. Furthermore, CX3CL1-induced fibronectin expression and JC-1 monomer were decreased by p65 siRNA. TP213 reduced TGF-β-induced p65 and α-SMA expression in NHLFs.

Conclusions

These findings suggest that neutralizing CX3CL1 attenuates lung fibroblast activation and mitochondrial dysfunction. Understanding the impacts of CX3CL1 neutralization on fibroblast mitochondrial function could contribute to the development of therapeutic strategies for managing airway remodeling in severe asthma.

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Abbreviations

ADAM17:

A disintegrin and metalloproteinase 17

α-SMA:

α-Smooth muscle actin

BSA:

Bovine serum albumin

CTGF:

Connective tissue growth factor

CX3CL1:

(C-X3-C motif) ligand 1

CX3CR1:

C-X3-C motif chemokine receptor 1

DEG:

Differentially expressed gene

DMEM:

Dulbecco’s modified minimal essential/Eagle medium

ECM:

Extracellular matrix

EGFR:

Epidermal growth factor receptor

ELISA:

Enzyme-linked immunosorbent assay

EMT:

Epithelial-mesenchymal transition

ETC:

Electron transport chain

FBS:

Fetal bovine serum

HRP:

Horseradish peroxidase

IF:

Immunofluorescence

IHC:

Immunohistochemical

IgG:

Immunoglobulin G

IL:

Interleukin

mAb:

Monoclonal antibody

NES:

Normalized enrichment score

OVA:

Ovalbumin

NEAAs:

Nonessential amino acids

NF-κB:

Nuclear factor-κB

NHLF:

Normal human lung fibroblast

phospho:

Phosphorylated

OXPHOS:

Oxidative phosphorylation

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

PDGF:

Platelet-derived growth factor

PVDF:

Polyvinylidene difluoride

SDS-PAGE:

Sodium dodecylsulfate polyacrylamide gel electrophoresis

siRNA:

Small interfering RNA

TGF:

Transforming growth factor

TH2:

T helper 2

TUNEL:

Terminal uridine nick-end labeling

References

  1. Vos T, Lim SS, Abbafati C et al (2020) Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet 396:1204–1222

    Article  Google Scholar 

  2. Hough KP, Curtiss ML, Blain TJ et al (2020) Airway remodeling in asthma. Front Med 7:191

    Article  Google Scholar 

  3. Hinz B, Lagares D (2020) Evasion of apoptosis by myofibroblasts: a hallmark of fibrotic diseases. Nat Rev Rheumatol 16:11–31

    Article  CAS  PubMed  Google Scholar 

  4. Bueno M, Calyeca J, Rojas M, Mora AL (2020) Mitochondria dysfunction and metabolic reprogramming as drivers of idiopathic pulmonary fibrosis. Redox Biol 33:101509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lee M, Lee Y, Song J et al (2018) Tissue-specific role of CX3CR1 expressing immune cells and their relationships with human disease. Immune Netw 18:e5

    Article  PubMed  PubMed Central  Google Scholar 

  6. Cormican S, Griffin MD (2021) Fractalkine (CX3CL1) and its receptor CX3CR1: a promising therapeutic target in chronic kidney disease? Front Immunol 12:664202

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zheng C, Xuan W, Chen Z et al (2022) CX3CL1 worsens cardiorenal dysfunction and serves as a therapeutic target of canagliflozin for cardiorenal syndrome. Front Pharmacol 13:848310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dong J, Ma Q (2019) In vivo activation and pro-fibrotic function of NF-κB in fibroblastic cells during pulmonary inflammation and fibrosis induced by carbon nanotubes. Front Pharmacol 10:1140

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wright JG, Christman JW (2003) The role of nuclear factor kappa B in the pathogenesis of pulmonary diseases: implications for therapy. Am J Respir Med 2:211–219

    Article  CAS  PubMed  Google Scholar 

  10. Edwards MR, Bartlett NW, Clarke D et al (2009) Targeting the NF-κB pathway in asthma and chronic obstructive pulmonary disease. Pharmacol Ther 121:1–13

    Article  CAS  PubMed  Google Scholar 

  11. Korbecki J, Simińska D, Kojder K et al (2020) Fractalkine/CX3CL1 in neoplastic processes. Int J Mol Sci 21:3723

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Reddy PH (2011) Mitochondrial dysfunction and oxidative stress in asthma: implications for mitochondria-targeted antioxidant therapeutics. Pharmaceuticals 4:429–456

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Qian L, Mehrabi Nasab E, Athari SM, Athari SS (2022) Mitochondria signaling pathways in allergic asthma. J Investig Med 70:863–882

    Article  PubMed  PubMed Central  Google Scholar 

  14. Huang M, Nasab EM, Athari SS (2021) Immunoregulatory effect of mesenchymal stem cell via mitochondria signaling pathways in allergic asthma. Saudi J Biol Sci 28:6957–6962

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Liu R-M, Desai LP (2015) Reciprocal regulation of TGF-β and reactive oxygen species: a perverse cycle for fibrosis. Redox Biol 6:565–577

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li X, Zhang W, Cao Q et al (2020) Mitochondrial dysfunction in fibrotic diseases. Cell Death Discov 6:80

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Chen J-Y, Cheng W-H, Lee K-Y et al (2021) Abnormal ADAM17 expression causes airway fibrosis in chronic obstructive asthma. Biomed Pharmacother 140:111701

    Article  CAS  PubMed  Google Scholar 

  18. Cheng W-H, Lee K-Y, Yu M-C et al (2021) Pref-1 induced lung fibroblast differentiation by hypoxia through integrin α5β1/ERK/AP-1 cascade. Eur J Pharmacol 909:174385

    Article  CAS  PubMed  Google Scholar 

  19. Feng K, Meng P, Zou X et al (2022) IL-37 protects against airway remodeling by reversing bronchial epithelial–mesenchymal transition via IL-24 signaling pathway in chronic asthma. Respir Res 23:1–17

    Article  Google Scholar 

  20. Mionnet C, Buatois V, Kanda A et al (2010) CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung. Nat Med 16:1305–1312

    Article  CAS  PubMed  Google Scholar 

  21. Julia V (2012) CX3CL1 in allergic diseases: not just a chemotactic molecule. Allergy 67:1106–1110

    Article  CAS  PubMed  Google Scholar 

  22. Rimaniol A-C, Till SJ, Garcia G et al (2003) The CX3C chemokine fractalkine in allergic asthma and rhinitis. J Allergy Clin Immunol 112:1139–1146

    Article  CAS  PubMed  Google Scholar 

  23. Green G, Johnson SM, Costello H et al (2021) CX3CR1 is a receptor for human respiratory syncytial virus in cotton rats. J Virol 95:10–1128

    Article  Google Scholar 

  24. Cunoosamy D, Zhou X-H, Rehnberg M et al (2014) Expression of CX3CL1 and CX3CR1 in human asthmatic lung in relation to cell survival. Eur Respir J 44:3863

    Google Scholar 

  25. Jones BA, Beamer M, Ahmed S (2010) Fractalkine/CX3CL1: a potential new target for inflammatory diseases. Mol Interv 10:263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Moss ML, Minond D (2017) Recent advances in ADAM17 research: a promising target for cancer and inflammation. Mediators Inflamm 2017:9673537

    Article  PubMed  PubMed Central  Google Scholar 

  27. Ishida Y, Kimura A, Nosaka M et al (2017) Essential involvement of the CX3CL1-CX3CR1 axis in bleomycin-induced pulmonary fibrosis via regulation of fibrocyte and M2 macrophage migration. Sci Rep 7:1–12

    Article  Google Scholar 

  28. Guo R, Gu J, Zong S et al (2018) Structure and mechanism of mitochondrial electron transport chain. Biomed J 41:9–20

    Article  PubMed  PubMed Central  Google Scholar 

  29. Sebag SC, Koval OM, Paschke JD et al (2017) Mitochondrial CaMKII inhibition in airway epithelium protects against allergic asthma. JCI Insight 2:e88297

    Article  PubMed  PubMed Central  Google Scholar 

  30. Zhang Y, Do DC, Hu X et al (2021) CaMKII oxidation regulates cockroach allergen-induced mitophagy in asthma. J Allergy Clin Immunol 147:1464–1477

    Article  CAS  PubMed  Google Scholar 

  31. Susin SA, Lorenzo HK, Zamzami N et al (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397:441–446

    Article  CAS  PubMed  Google Scholar 

  32. Beere HM (2005) Death versus survival: functional interaction between the apoptotic and stress-inducible heat shock protein pathways. J Clin Investig 115:2633–2639

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Drakopanagiotakis F, Xifteri A, Polychronopoulos V, Bouros D (2008) Apoptosis in lung injury and fibrosis. Eur Respir J 32:1631–1638

    Article  CAS  PubMed  Google Scholar 

  34. Albensi BC (2019) What is nuclear factor kappa B (NF-κB) doing in and to the mitochondrion? Front Cell Dev Biol 7:154

    Article  PubMed  PubMed Central  Google Scholar 

  35. Galán-Ganga M, García-Yagüe ÁJ, Lastres-Becker I (2019) Role of MSK1 in the induction of NF-κB by the chemokine CX3CL1 in microglial cells. Cell Mol Neurobiol 39:331–340

    Article  PubMed  Google Scholar 

  36. Mizutani S, Nishio J, Kondo K et al (2021) Treatment with an anti-Cx3cl1 antibody suppresses M1 macrophage infiltration in interstitial lung disease in Skg mice. Pharmaceuticals 14:474

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This study was supported by grant (NSTC112-2320-B-038-001 and NSTC112-2320-B-038-022) from the National Science and Technology Council of Taiwan, R.O.C. DP2-TMU-112-T-05 from the Ministry of Education (MOE) in Taiwan, R.O.C. TMU112-AE1-B10 from the Taipei Medical University (TMU), Taiwan, R.O.C.

Funding

NSTC112-2320-B-038-001 and NSTC112-2320-B-038-022 from the National Science and Technology Council of Taiwan, R.O.C. DP2-TMU-112-T-05 from Ministry of Education (MOE) in Taiwan, R.O.C. TMU112-AE1-B10 from the Taipei Medical University (TMU), Taiwan, R.O.C.

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Authors and Affiliations

  1. School of Respiratory Therapy, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei, 11031, Taiwan

    Wun-Hao Cheng, Yu-Chih Wu, Shao-An Wang, Chia-Ling Chen, Feng-Lin Hsu, Mei-May Neoh & Bing-Chang Chen

  2. Respiratory Therapy, Division of Pulmonary Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan

    Wun-Hao Cheng

  3. Division of Thoracic Medicine, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

    Wun-Hao Cheng, Yu-Chih Wu, Shao-An Wang, Chia-Ling Chen, Chien-Huang Lin & Bing-Chang Chen

  4. School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

    Pao-Lung Chang & Lee-Yuan Lin

  5. Department of Pharmacology and Therapy, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia

    Fara Silvia Yuliani

  6. Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei, 11031, Taiwan

    Chien-Huang Lin

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Contributions

Conceptualization: B-CC. Data curation: W-HC and P-LC. Formal analysis: W-HC and FSY. Funding acquisition: B-CC. Investigation: W-HC, S-AW, F-LH, M-MN, and P-LC. Methodology: B-CC, Y-CW, S-AW and C-LC. Project administration: B-CC. Resources: B-CC, Y-CW. Software: W-HC and B-CC. Supervision: B-CC and C-HL. Validation: W-HC, P-LC, L-YL, F-SY, and B-CC. Visualization: W-HC, F-SY, L-YL, C-HL, and B-CC. Writing—original draft: W-HC. Writing—review and editing: B-CC, W-HC, C-LC and C-HL.

Corresponding authors

Correspondence to Chien-Huang Lin or Bing-Chang Chen.

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Conflict of interest

The authors declare no conflict of interest.

Ethics Approval and Consent to Participate

All animal protocols were approved by the Animal Ethics Committee of Taipei Medical University (approval No. LAC-2019-0042).

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Cheng, WH., Chang, PL., Wu, YC. et al. Neutralization of CX3CL1 Attenuates TGF-β-Induced Fibroblast Differentiation Through NF-κB Activation and Mitochondrial Dysfunction in Airway Fibrosis. Lung 202, 343–356 (2024). https://doi.org/10.1007/s00408-024-00701-6

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