Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Transcriptional and ubiquitinative suppression of macrophage CST3 disrupts colonic homeostasis through defective efferocytosis

Cell Death & Differentiation (2026)Cite this article

Subjects

Abstract

To elucidate the role and regulatory mechanisms of macrophage-derived cystatin C (CST3) in Crohn’s disease (CD), focusing on colonic inflammation, macrophage–epithelial interactions, and barrier dysfunction. Colonic samples from CD patients, including inflamed and non-inflamed regions, were subjected to scRNA-seq. In vitro macrophage–epithelial co-culture models and untargeted metabolomics were employed, and the findings were validated using macrophage-specific CST3 knockout (KO) and overexpression mice under TNBS-induced and IL-10 KO colitis conditions. Mechanistic investigations included Co-IP, ChIP-qPCR, ubiquitination assays, rescue experiments, and functional analyses of efferocytosis, macrophage polarization, and barrier integrity. CST3 expression was considerably reduced in macrophages from inflamed CD tissues through suppressor of SMAD5-dependent transcriptional repression and MYCBP2-mediated K48-linked ubiquitination and degradation. Loss of CST3 impaired efferocytosis and M2 polarization by inhibiting the ACVR1C/TGF-β/SMAD pathway. CST3 deficiency also disrupted intestinal epithelial proliferation, compromised barrier function, and increased apoptosis via enhanced NAMPT-INSR signaling and accumulation of the inflammatory cytokines. In mice, macrophage-specific CST3 deletion exacerbated colitis, whereas its overexpression alleviated inflammation and restored epithelial integrity. These findings establish macrophage CST3 as a key regulator of immune–metabolic–epithelial crosstalk in CD, and indicate that restoring CST3 function or targeting its regulatory axis may represent a novel therapeutic strategy for CD.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: scRNA-seq reveals enrichment of pro-inflammatory macrophages in inflamed CD tissues.
The alternative text for this image may have been generated using AI.
Fig. 2: CST3 enhances TGF-β signaling via ACVR1C to regulate macrophage function.
The alternative text for this image may have been generated using AI.
Fig. 3: Reciprocal regulation between CST3 and SMAD5 in intestinal macrophages.
The alternative text for this image may have been generated using AI.
Fig. 4: MYCBP2 mediates CST3 ubiquitination and degradation through the ubiquitin-proteasome pathway.
The alternative text for this image may have been generated using AI.
Fig. 5: CST3 mediates macrophage–epithelial crosstalk via the NAMPT–INSR ligand–receptor pathway.
The alternative text for this image may have been generated using AI.
Fig. 6: CST3 mediates macrophage–epithelial crosstalk via the inflammatory cytokines.
The alternative text for this image may have been generated using AI.
Fig. 7: Macrophage-specific CST3 deficiency exacerbates colitis and disrupts intestinal barrier function.
The alternative text for this image may have been generated using AI.
Fig. 8: CST3-overexpressing macrophages mitigate colitis and restore the intestinal barrier in IL-10 KO mice.
The alternative text for this image may have been generated using AI.
Fig. 9: Biological function of CST3 in mouse and human colonic organoids.
The alternative text for this image may have been generated using AI.
Fig. 10: Mechanistic diagram of CST3 regulating colitis through macrophage–epithelial cell crosstalk.
The alternative text for this image may have been generated using AI.

Similar content being viewed by others

Data availability

Single-cell sequences and RNA-sequences were deposited to GEO database (GSE319204, GSE318527). Please contact the corresponding author upon reasonable request.

References

  1. Torres J, Mehandru S, Colombel JF, Peyrin-Biroulet L. Crohn’s disease. Lancet. 2017;389:1741–55.

    Article  PubMed  Google Scholar 

  2. Rogler G, Singh A, Kavanaugh A, Rubin DT. Extraintestinal manifestations of inflammatory bowel disease: current concepts, treatment, and implications for disease management. Gastroenterology. 2021;161:1118–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gisbert JP, Chaparro M. De-escalation of biologic treatment in inflammatory bowel disease: a comprehensive review. J Crohns Colitis. 2024;18:642–58.

    Article  PubMed  Google Scholar 

  4. Dharmasiri S, Garrido-Martin EM, Harris RJ, Bateman AC, Collins JE, Cummings J, et al. Human intestinal macrophages are involved in the pathology of both ulcerative colitis and Crohn disease. Inflamm Bowel Dis. 2021;27:1641–52.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Han X, Ding S, Jiang H, Liu G. Roles of macrophages in the development and treatment of gut inflammation. Front Cell Dev Biol. 2021;9:625423.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Elliott TR, Hudspith BN, Rayment NB, Prescott NJ, Petrovska L, Hermon-Taylor J, et al. Defective macrophage handling of Escherichia coli in Crohn’s disease. J Gastroenterol Hepatol. 2015;30:1265–74.

    Article  CAS  PubMed  Google Scholar 

  7. Pastorelli L, De Salvo C, Mercado JR, Vecchi M, Pizarro TT. Central role of the gut epithelial barrier in the pathogenesis of chronic intestinal inflammation: lessons learned from animal models and human genetics. Front Immunol. 2013;4:280.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Zhao J, Li Y, Ying P, Zhou Y, Xu Z, Wang D, et al. ITLN1 exacerbates Crohn’s colitis by driving ZBP1-dependent PANoptosis in intestinal epithelial cells through antagonizing TRIM8-mediated CAPN2 ubiquitination. Int J Biol Sci. 2025;21:3705–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Best WR, Becktel JM, Singleton JW, Kern F Jr. Development of a Crohn’s disease activity index. National Cooperative Crohn’s Disease Study. Gastroenterology. 1976;70:439–44.

    Article  CAS  PubMed  Google Scholar 

  10. Daperno M, D'haens G, Van Assche G, Baert F, Bulois P, Maunoury V, et al. Development and validation of a new, simplified endoscopic activity score for Crohn’s disease: the SES-CD. Gastrointest Endosc. 2004;60:505–12.

    Article  PubMed  Google Scholar 

  11. Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell. 2015;161:1202–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018;36:411–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Raudvere U, Kolberg L, Kuzmin I, Arak T, Adler P, Peterson H, et al. g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. 2019;47:W191–W8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Prianichnikov N, Koch H, Koch S, Lubeck M, Heilig R, Brehmer S, et al. MaxQuant Software for Ion Mobility Enhanced Shotgun Proteomics. Mol Cell Proteomics. 2020;19:1058–69.

    Article  PubMed  PubMed Central  Google Scholar 

  15. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021. 49: D480-D9.

  16. Wirtz S, Popp V, Kindermann M, Gerlach K, Weigmann B, Fichtner-Feigl S, et al. Chemically induced mouse models of acute and chronic intestinal inflammation. Nat Protoc. 2017;12:1295–309.

    Article  CAS  PubMed  Google Scholar 

  17. Wirtz S, Neufert C, Weigmann B, Neurath MF. Chemically induced mouse models of intestinal inflammation. Nat Protoc. 2007;2:541–6.

    Article  CAS  PubMed  Google Scholar 

  18. Chiriac MT, Hracsko Z, Günther C, Gonzalez-Acera M, Atreya R, Stolzer I, et al. IL-20 controls resolution of experimental colitis by regulating epithelial IFN/STAT2 signalling. Gut. 2024;73:282–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Huang S, Xie Z, Han J, Wang H, Yang G, Li M, et al. Protocadherin 20 maintains intestinal barrier function to protect against Crohn’s disease by targeting ATF6. Genome Biol. 2023;24:159.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Singh UP, Singh S, Taub DD, Lillard JW Jr. Inhibition of IFN-gamma-inducible protein-10 abrogates colitis in IL-10-/- mice. J Immunol. 2003;171:1401–6.

    Article  CAS  PubMed  Google Scholar 

  21. Yu M, Luo Y, Cong Z, Mu Y, Qiu Y, Zhong M. MicroRNA-590-5p Inhibits Intestinal Inflammation by Targeting YAP. J Crohns Colitis. 2018;12:993–1004.

    Article  PubMed  Google Scholar 

  22. Arrieta MC, Madsen K, Doyle J, Meddings J. Reducing small intestinal permeability attenuates colitis in the IL10 gene-deficient mouse. Gut. 2009;58:41–8.

    Article  CAS  PubMed  Google Scholar 

  23. Looijer-van Langen M, Hotte N, Dieleman LA, Albert E, Mulder C, Madsen KL. Estrogen receptor-β signaling modulates epithelial barrier function. Am J Physiol Gastrointest Liver Physiol. 2011;300:G621–6.

    Article  CAS  PubMed  Google Scholar 

  24. Putt KK, Pei R, White HM, Bolling BW. Yogurt inhibits intestinal barrier dysfunction in Caco-2 cells by increasing tight junctions. Food Funct. 2017;8:406–14.

    Article  CAS  PubMed  Google Scholar 

  25. Ahmad R, Rah B, Bastola D, Dhawan P, Singh AB. Obesity-induces organ and tissue specific tight junction restructuring and barrier deregulation by claudin switching. Sci Rep. 2017;7:5125.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Zhao J, Wang H, Zhou J, Qian J, Yang H, Zhou Y, et al. miR-130a-3p, a Preclinical Therapeutic Target for Crohn’s Disease. J Crohns Colitis. 2021;15:647–64.

    Article  PubMed  Google Scholar 

  27. Wang L, Li H, Huang A, Zhao Y, Xiao C, Dong J, et al. Mutual regulation between TRIM21 and TRIM8 via K48-linked ubiquitination. Oncogene. 2023;42:3708–18.

    Article  CAS  PubMed  Google Scholar 

  28. Li Z, Zhang W, Wei XY, Hu JZ, Hu X, Liu H, et al. TRIM15 drives chondrocyte senescence and osteoarthritis progression. Sci Transl Med. 2025;17:eadq1735.

    Article  CAS  PubMed  Google Scholar 

  29. Jiang X, Wang J, Lin L, Du L, Ding Y, Zheng F, et al. Macrophages promote pre-metastatic niche formation of breast cancer through aryl hydrocarbon receptor activity. Signal Transduct Target Ther. 2024;9:352.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Hong H, Han H, Wang L, Cao W, Hu M, Li J, et al. ABCF1-K430-Lactylation promotes HCC malignant progression via transcriptional activation of HIF1 signaling pathway. Cell Death Differ. 2025;32:613–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Cerda MB, Lloyd R, Batalla M, Giannoni F, Casal M, Policastro L. Silencing peroxiredoxin-2 sensitizes human colorectal cancer cells to ionizing radiation and oxaliplatin. Cancer Lett. 2017;388:312–9.

    Article  CAS  PubMed  Google Scholar 

  32. Rana N, Privitera G, Kondolf HC, Bulek K, Lechuga S, De Salvo C, et al. GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis. Cell. 2022;185:283–98.e17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wang Y, Liu XY, Wang Y, Zhao WX, Li FD, Guo PR, et al. NOX2 inhibition stabilizes vulnerable plaques by enhancing macrophage efferocytosis via MertK/PI3K/AKT pathway. Redox Biol. 2023;64:102763.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Fritsch SD, Sukhbaatar N, Gonzales K, Sahu A, Tran L, Vogel A, et al. Metabolic support by macrophages sustains colonic epithelial homeostasis. Cell Metab. 2023;35:1931–43.e8.

    Article  CAS  PubMed  Google Scholar 

  35. Flannagan RS, Jaumouillé V, Grinstein S. The cell biology of phagocytosis. Annu Rev Pathol. 2012;7:61–98.

    Article  CAS  PubMed  Google Scholar 

  36. Fan W, Qu Y, Yuan X, Shi H, Liu G, Loureirin B. Accelerates diabetic wound healing by promoting TGFβ/Smad-dependent macrophage M2 polarization: a concerted analytical approach through single-cell RNA sequencing and experimental verification. Phytother Res. 2024;39:5450–63 .

  37. Geng K, Ma X, Jiang Z, Gu J, Huang W, Wang W, et al. WDR74 facilitates TGF-β/Smad pathway activation to promote M2 macrophage polarization and diabetic foot ulcer wound healing in mice. Cell Biol Toxicol. 2023;39:1577–91.

    Article  CAS  PubMed  Google Scholar 

  38. Ramos GP, Papadakis KA. Mechanisms of disease: inflammatory bowel diseases. Mayo Clin Proc. 2019;94:155–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Liu J, Chen Y. Cell-cell crosstalk between fat cells and immune cells. Am J Physiol Endocrinol Metab. 2024;327:E371–E83.

    Article  CAS  PubMed  Google Scholar 

  40. Serretti A, Olgiati P, De Ronchi D. Genetics of Alzheimer’s disease. A rapidly evolving field. J Alzheimers Dis. 2007;12:73–92.

    Article  CAS  PubMed  Google Scholar 

  41. Gren ST, Janciauskiene S, Sandeep S, Jonigk D, Kvist PH, Gerwien JG, et al. The protease inhibitor cystatin C down-regulates the release of IL-β and TNF-α in lipopolysaccharide activated monocytes. J Leukoc Biol. 2016;100:811–22.

    Article  CAS  PubMed  Google Scholar 

  42. Lin Z, Zhuang J, He L, Zhu S, Kong W, Lu W, et al. Exploring Smad5: a review to pave the way for a deeper understanding of the pathobiology of common respiratory diseases. Mol Med. 2024;30:225.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Allaire JM, Darsigny M, Marcoux SS, Roy SA, Schmouth JF, Umans L, et al. Loss of Smad5 leads to the disassembly of the apical junctional complex and increased susceptibility to experimental colitis. Am J Physiol Gastrointest Liver Physiol. 2011;300:G586–97.

    Article  CAS  PubMed  Google Scholar 

  44. Szondy Z, Garabuczi E, Joós G, Tsay GJ, Sarang Z. Impaired clearance of apoptotic cells in chronic inflammatory diseases: therapeutic implications. Front Immunol. 2014;5:354.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Kawano M, Nagata S. Efferocytosis and autoimmune disease. Int Immunol. 2018;30:551–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wu J, Jiang L, Wang S, Peng L, Zhang R, Liu Z. TGF β1 promotes the polarization of M2-type macrophages and activates PI3K/mTOR signaling pathway by inhibiting ISG20 to sensitize ovarian cancer to cisplatin. Int Immunopharmacol. 2024;134:112235.

    Article  CAS  PubMed  Google Scholar 

  47. Su J, Song Y, Zhu Z, Huang X, Fan J, Qiao J, et al. Cell-cell communication: new insights and clinical implications. Signal Transduct Target Ther. 2024;9:196.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Naik S, Larsen SB, Gomez NC, Alaverdyan K, Sendoel A, Yuan S, et al. Inflammatory memory sensitizes skin epithelial stem cells to tissue damage. Nature. 2017;550:475–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Zhao D, Ravikumar V, Leach TJ, Kraushaar D, Lauder E, Li L, et al. Inflammation-induced epigenetic imprinting regulates intestinal stem cells. Cell Stem Cell. 2024;31:1447–64.e6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was supported by National Natural Science Foundation of China (82300609, 82000492), Jiangsu Provincial Commission of Health and Family Planning (No.M2025006), Changzhou Sci&Tech Program (2022CZBJ066, CJ20245024), Changzhou Medical Center of Nanjing Medical University Program (CMC2024PY17), Yanzhen Talent Program for Emerging Academic Leaders (YZ-HBDTR-SJ-2025), Taizhou School of Clinical Medicine, Nanjing Medical University (TZKY20230308, TZKY20230102).

Author information

Author notes

  1. These authors contributed equally: Honggang Wang, Chenyang Jiao, Hailin Xing, Chao Cheng.

Authors and Affiliations

  1. Department of General Surgery, Affiliated Taizhou People’s Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine, Nanjing Medical University, Taizhou, China

    Honggang Wang, Chao Cheng, Shaoqi Cheng & Wenliang Jiang

  2. Department of Gastroenterology, Affiliated Taizhou People’s Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine, Nanjing Medical University, Taizhou, China

    Chenyang Jiao

  3. Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, China

    Chenyang Jiao

  4. Department of Anesthesiology, The Affiliated Taizhou People’s Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine, Nanjing Medical University, Taizhou, Jiangsu, China

    Hailin Xing

  5. Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China

    Ziwei Xu

  6. Department of General Surgery, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China

    Jianbo Yang

  7. Department of Gastroenterology, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, China

    Jing Sun

  8. Department of Gastrointestinal Surgery, The Affiliated Changzhou No.2 People’s Hospital of Nanjing Medical University, The Third Affiliated Hospital of Nanjing Medical University, Changzhou Medical Center, Nanjing Medical University, Changzhou, China

    Jie Zhao

Authors
  1. Honggang Wang
    View author publications

    Search author on:PubMed Google Scholar

  2. Chenyang Jiao
    View author publications

    Search author on:PubMed Google Scholar

  3. Hailin Xing
    View author publications

    Search author on:PubMed Google Scholar

  4. Chao Cheng
    View author publications

    Search author on:PubMed Google Scholar

  5. Shaoqi Cheng
    View author publications

    Search author on:PubMed Google Scholar

  6. Wenliang Jiang
    View author publications

    Search author on:PubMed Google Scholar

  7. Ziwei Xu
    View author publications

    Search author on:PubMed Google Scholar

  8. Jianbo Yang
    View author publications

    Search author on:PubMed Google Scholar

  9. Jing Sun
    View author publications

    Search author on:PubMed Google Scholar

  10. Jie Zhao
    View author publications

    Search author on:PubMed Google Scholar

Contributions

HGW, CYJ, HLX, and JZ: Study design and data analysis. SQC, CC, WLJ, and ZWX: Patient recruitment, data collection and writing up of the first draft of the paper. HGW, JBY, JS, and JZ: scientific advice, supervision and drafting of the manuscript.

Corresponding authors

Correspondence to Jianbo Yang, Jing Sun or Jie Zhao.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics approval

The clinical study was approved by the ethics committee of The First Affiliated Hospital of Nanjing Medical University (No. 2023-SR-115). All the experimental protocols were approved by the Ethics Committee of the Laboratory Animal Center of Nantong University (NO.S20200323-289) according to the guidelines by the Chinese Council on Animal Care.

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

Wang, H., Jiao, C., Xing, H. et al. Transcriptional and ubiquitinative suppression of macrophage CST3 disrupts colonic homeostasis through defective efferocytosis. Cell Death Differ (2026). https://doi.org/10.1038/s41418-026-01750-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Version of record:

  • DOI: https://doi.org/10.1038/s41418-026-01750-5

Search

Advanced search

Quick links