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.

  • Basic Science Article
  • Published:

CCL3 aggravates intestinal damage in NEC by promoting macrophage chemotaxis and M1 macrophage polarization

Pediatric Research volume 94, pages 119–128 (2023)Cite this article

Abstract

Background

NEC is a life-threatening gastrointestinal disease in neonates, the pathogenesis of which remains poorly understood.

Methods

CCL3 levels in intestinal tissue of mice were measured and analyzed. HE staining was used to assess pathological changes in intestinal tissue. FCM was used to detect the proportion and phenotype of macrophages. RNA-seq and RT-PCR were used to evaluate the effect of CCL3 on macrophages.

Results

CCL3 was highly expressed in the intestinal tissues of mice with NEC and induced macrophage infiltration. Transcriptome data showed that CCL3 strongly induced a transition in the phenotype of macrophages into a proinflammatory one. Mechanistically, in vivo experiments confirmed that CCL3 induced M1 macrophage polarization in NEC intestinal tissue, thereby aggravating inflammatory injury of intestinal tissue, which was alleviated by anti-CCL3 treatment. In addition, in vitro experiments showed that CCL3 significantly enhances the expression of M1-related genes in both PMφ and BMDM while inhibiting the expression of M2-related genes, which was also alleviated by anti-CCl3 treatment.

Conclusions

Our data elucidated the involvement of CCL3 in the pathogenesis of NEC, in which upregulated CCL3 expression exacerbated inflammatory intestinal damage by regulating macrophage chemotaxis and M1 phenotype polarization, suggesting that blocking CCL3 may be a potential strategy for effective intervention in NEC.

Impact

  • Our study represents an important conceptual advancement that CCL3 may be one of the key culprits of intestinal tissue damage in patients with NEC.

  • CCL3 aggravates inflammatory intestinal injury and intestinal mucosal barrier imbalance by regulating the chemotaxis, polarization, and function of macrophages.

  • Blocking CCL3 significantly reduced NEC-mediated intestinal injury, suggesting a new potential therapeutic strategy.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: The expression and role of CCL3 in NEC.
Fig. 2: The chemotaxis and inflammatory regulation of CCL3 in NEC.
Fig. 3: Gene expression profiles of BMDMs treated with rCCL3 and anti-CCL3.
Fig. 4: CCL3 mediates M1 macrophage polarization in NEC.
Fig. 5: The role of CCL3 in inducing M1 Mφ polarization.

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in this published article.

References

  1. Neu, J. & Walker, W. A. Necrotizing enterocolitis. N. Engl. J. Med. 364, 255–264 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Frost, B. L. et al. New medical and surgical insights into neonatal necrotizing enterocolitis: a review. JAMA Pediatr. 171, 83–88 (2017).

    PubMed  Google Scholar 

  3. Ou, J. et al. Nutrition in necrotizing enterocolitis and following intestinal resection. Nutrients 12, 520 (2020).

  4. Niño, D. F., Sodhi, C. P. & Hackam, D. J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms. Nat. Rev. Gastroenterol. Hepatol. 13, 590–600 (2016).

    PubMed  PubMed Central  Google Scholar 

  5. Hunter, C. J. et al. Understanding the susceptibility of the premature infant to necrotizing enterocolitis (NEC). Pediatr. Res. 63, 117–123 (2008).

    PubMed  Google Scholar 

  6. Reisinger, K. W. et al. Breast-feeding improves gut maturation compared with formula feeding in preterm babies. J. Pediatr. Gastroenterol. Nutr. 59, 720–724 (2014).

    CAS  PubMed  Google Scholar 

  7. Heida, F. H. et al. Risk factors associated with postnecrotizing enterocolitis strictures in infants. J. Pediatr. Surg. 51, 1126–1130 (2016).

    CAS  PubMed  Google Scholar 

  8. Amin, S. C. et al. Short bowel syndrome in the NICU. Clin. Perinatol. 40, 53–68 (2013).

    PubMed  Google Scholar 

  9. Elfvin, A. et al. Low birthweight, gestational age, need for surgical intervention and gram-negative bacteraemia predict intestinal failure following necrotising enterocolitis. Acta Paediatr. 104, 771–776 (2015).

    PubMed  Google Scholar 

  10. Collado, M. C. et al. Factors influencing gastrointestinal tract and microbiota immune interaction in preterm infants. Pediatr. Res. 77, 726–731 (2015).

    PubMed  Google Scholar 

  11. Good, M., Sodhi, C. P. & Hackam, D. J. Evidence-based feeding strategies before and after the development of necrotizing enterocolitis. Expert Rev. Clin. Immunol. 10, 875–884 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhou, Y. et al. Inflammation and apoptosis: dual mediator role for toll-like receptor 4 in the development of necrotizing enterocolitis. Inflamm. Bowel Dis. 23, 44–56 (2017).

    PubMed  Google Scholar 

  13. MohanKumar, K. et al. Gut mucosal injury in neonates is marked by macrophage infiltration in contrast to pleomorphic infiltrates in adult: evidence from an animal model. Am. J. Physiol. Gastrointest. Liver Physiol. 303, G93–G102 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Wei, J. et al. Irf5 deficiency in myeloid cells prevents necrotizing enterocolitis by inhibiting M1 macrophage polarization. Mucosal Immunol. 12, 888–896 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Liu, X. et al. Geraniin inhibits LPS-induced THP-1 macrophages switching to M1 phenotype via SOCS1/NF-κB pathway. Inflammation 39, 1421–1433 (2016).

    CAS  PubMed  Google Scholar 

  16. Wei, J. & Besner, G. E. M1 to M2 macrophage polarization in heparin-binding epidermal growth factor-like growth factor therapy for necrotizing enterocolitis. J. Surg. Res. 197, 126–138 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Deniffel, D. et al. Otitis media and nasopharyngeal colonization in mice. Infect. Immun. 85, e00148–17 (2017).

  18. Russell, C. D. et al. The human immune response to respiratory syncytial virus infection. Clin. Microbiol. Rev. 30, 481–502 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Gibaldi, D. et al. CCL3/macrophage inflammatory protein-1α is dually involved in parasite persistence and induction of a TNF- and IFNγ-enriched inflammatory milieu in Trypanosoma cruzi-induced chronic cardiomyopathy. Front. Immunol. 11, 306 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Schaller, T. H. et al. Chemokines as adjuvants for immunotherapy: implications for immune activation with CCL3. Expert Rev. Clin. Immunol. 13, 1049–1060 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Ishida, Y. et al. Prevention of CaCl-induced aortic inflammation and subsequent aneurysm formation by the CCL3-CCR5 axis. Nat. Commun. 11, 5994 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Koike, Y. et al. Remote ischemic conditioning counteracts the intestinal damage of necrotizing enterocolitis by improving intestinal microcirculation. Nat. Commun. 11, 4950 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Kovler, M. L., Sodhi, C. P. & Hackam, D. J. Precision-based modeling approaches for necrotizing enterocolitis. Dis. Model Mech. 13, dmm044388 (2020).

  24. Allen, F. et al. CCL3 augments tumor rejection and enhances CD8(+) T cell infiltration through NK and CD103(+) dendritic cell recruitment via IFNγ. Oncoimmunology 7, e1393598 (2018).

    PubMed  Google Scholar 

  25. Soares, D. M. et al. CCL3/MIP-1 alpha is not involved in the LPS-induced fever and its pyrogenic activity depends on CRF. Brain Res. 1269, 54–60 (2009).

    CAS  PubMed  Google Scholar 

  26. Llorián-Salvador, M. et al. Involvement of CC chemokine receptor 1 and CCL3 in acute and chronic inflammatory pain in mice. Basic Clin. Pharm. Toxicol. 119, 32–40 (2016).

    Google Scholar 

  27. Yuan, X. et al. Novel therapeutic targeting of CCL3-CCR4 axis mediated apoptotic intesitnal injury in necrotizing enterocolitis. Front. Immunol. 13, 859398 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Pineda-Torra, I. et al. Isolation, culture, and polarization of murine bone marrow-derived and peritoneal macrophages. Methods Mol. Biol. 1339, 101–109 (2015).

    CAS  PubMed  Google Scholar 

  29. Weischenfeldt, J. & Porse, B. Bone marrow-derived macrophages (BMM): isolation and applications. CSH Protoc. 2008, pdb.prot5080 (2008).

  30. Van Dyken, S. J. & Locksley, R. M. Interleukin-4- and interleukin-13-mediated alternatively activated macrophages: roles in homeostasis and disease. Annu. Rev. Immunol. 31, 317–343 (2013).

    PubMed  PubMed Central  Google Scholar 

  31. Shapouri-Moghaddam, A. et al. Macrophage plasticity, polarization, and function in health and disease. J. Cell Physiol. 233, 6425–6440 (2018).

    CAS  PubMed  Google Scholar 

  32. Mosser, D. M., Hamidzadeh, K. & Goncalves, R. Macrophages and the maintenance of homeostasis. Cell Mol. Immunol. 18, 579–587 (2021).

    CAS  PubMed  Google Scholar 

  33. Rosas, M. et al. The transcription factor Gata6 links tissue macrophage phenotype and proliferative renewal. Science 344, 645–648 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Yu, X. et al. Identifying Prokineticin2 as a novel immunomodulatory factor in diagnosis and treatment of sepsis. Crit. Care Med. 50, 674–684 (2021).

  35. Schreurs, R. et al. Human fetal TNF-α-cytokine-producing CD4(+) effector memory T cells promote intestinal development and mediate inflammation early in life. Immunity 50, 462–476.e8 (2019).

    CAS  PubMed  Google Scholar 

  36. Egan, C. E. et al. Toll-like receptor 4-mediated lymphocyte influx induces neonatal necrotizing enterocolitis. J. Clin. Invest. 126, 495–508 (2016).

    PubMed  Google Scholar 

  37. Ma, F. et al. Melatonin ameliorates necrotizing enterocolitis by preventing Th17/Treg imbalance through activation of the AMPK/SIRT1 pathway. Theranostics 10, 7730–7746 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Parkhomchuk, D. et al. Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res. 37, e123 (2009).

    PubMed  PubMed Central  Google Scholar 

  39. Arlauckas, S. P. et al. Arg1 expression defines immunosuppressive subsets of tumor-associated macrophages. Theranostics 8, 5842–5854 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Corbin, A. L. et al. IRF5 guides monocytes toward an inflammatory CD11c macrophage phenotype and promotes intestinal inflammation. Sci. Immunol. 5, eaax6085 (2020).

  41. Zhu, L. et al. TSC1 controls macrophage polarization to prevent inflammatory disease. Nat. Commun. 5, 4696 (2014).

    CAS  PubMed  Google Scholar 

  42. Horbar, J. D. et al. Mortality and neonatal morbidity among infants 501 to 1500 grams from 2000 to 2009. Pediatrics 129, 1019–1026 (2012).

    PubMed  Google Scholar 

  43. Emami, C. N. et al. Role of neutrophils and macrophages in the pathogenesis of necrotizing enterocolitis caused by Cronobacter sakazakii. J. Surg. Res. 172, 18–28 (2012).

    CAS  PubMed  Google Scholar 

  44. Gourlay, D. M. The good and the bad of the innate immune response in necrotizing enterocolitis. J. Surg. Res. 175, 51–53 (2012).

    PubMed  Google Scholar 

  45. Li, C. et al. MicroRNA regulated macrophage activation in obesity. J. Transl. Int. Med. 7, 46–52 (2019).

    PubMed  PubMed Central  Google Scholar 

  46. Li, C. et al. AtheroSpectrum reveals novel macrophage foam cell gene signatures associated with atherosclerotic cardiovascular disease risk. Circulation 145, 206–218 (2022).

    CAS  PubMed  Google Scholar 

  47. Pelisch, N. et al. CCL3 contributes to secondary damage after spinal cord injury. J. Neuroinflammation 17, 362 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Sica, A. & Mantovani, A. Macrophage plasticity and polarization: in vivo veritas. J. Clin. Investig. 122, 787–795 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Na, Y. R. et al. Macrophages in intestinal inflammation and resolution: a potential therapeutic target in IBD. Nat. Rev. Gastroenterol. Hepatol. 16, 531–543 (2019).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr Jinfeng Hou (Department of Gastrointestinal and Neonatal Surgery, Children’s Hospital of Chongqing Medical University) and Dr Xiaohong Die (Department of Gastrointestinal and Neonatal Surgery, Children’s Hospital of Chongqing Medical University) for clinical samples collection.

Funding

The study was supported, in part, by the National Natural Science Foundation of China (Grant Number: 81801956 to Z.S.), Chongqing Municipal Health Commission (Grant Number: 2020MSXM050 to D.C.), Chongqing Science and Technology Commission (Grant Number: cstc2019jcyj-msxmX0364 to H.T.), Distinguished Young Scholars of the Children’s Hospital of Chongqing Medical University (to Z.S.).

Author information

Author notes

  1. These authors contributed equally: Xi Yuan, Wei Liu.

Authors and Affiliations

  1. Department of Clinical Laboratory, 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, Chongqing Key Laboratory of Child Infection and Immunity, Chongqing, China

    Xi Yuan, Zhixin Song & Dapeng Chen

  2. Department of Gastrointestinal and Neonatal Surgery, Children’s Hospital of Chongqing Medical University, Chongqing, China

    Wei Liu

  3. Molecular Medicine and Cancer Research Center, College of Basic Medical Sciences, Chongqing Medical University, Chongqing, China

    Yue Li

  4. Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine, Chongqing Medical University, Chongqing, 400016, China

    Kai Chen & Yibing Yin

  5. Emergency Department, Children’s Hospital of Chongqing Medical University, Chongqing, 400016, China

    Hongdong Li

  6. Department of Critical Care Medicine, Department of Surgical Intensive Care Unit, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China

    Hong Tang

Authors
  1. Xi Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yue Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kai Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongdong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hong Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yibing Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zhixin Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Dapeng Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z.S. and D.C. conceived and designed the experiments. X.Y., W.L., Y.L., H.T. and H.L. performed the experiments. W.L. and Y.Y. collected and analyzed the data. Z.S. and X.Y. wrote of the manuscript. All authors reviewed and approved the final submitted and published versions.

Corresponding authors

Correspondence to Zhixin Song or Dapeng Chen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics approval

This study was approved by the Ethics Committee of Children’s Hospital of Chongqing Medical University and the Animal Ethics Committee of Chongqing Medical University.

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

Yuan, X., Liu, W., Li, Y. et al. CCL3 aggravates intestinal damage in NEC by promoting macrophage chemotaxis and M1 macrophage polarization. Pediatr Res 94, 119–128 (2023). https://doi.org/10.1038/s41390-022-02409-w

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41390-022-02409-w

Search

Advanced search

Quick links