Skip to main content

Advertisement

SpringerLink
Log in

Single-cell RNA sequencing of intestinal immune cells in neonatal necrotizing enterocolitis

  • Original Article
  • Published:
Pediatric Surgery International Aims and scope Submit manuscript

Abstract

Purpose

Necrotizing enterocolitis (NEC) causes fatal intestinal necrosis in neonates, but its etiology is unknown. We analyzed the intestinal immune response to NEC.

Methods

Using single-cell RNA sequencing (scRNA-seq), we analyzed the gene expression profiles of intestinal immune cells from four neonates with intestinal perforation (two with NEC and two without NEC). Target mononuclear cells were extracted from the lamina propria of the resected intestines.

Results

In all four cases, major immune cells, such as T cells (15.1–47.7%), B cells (3.1–19.0%), monocytes (16.5–31.2%), macrophages (1.6–17.4%), dendritic cells (2.4–12.2%), and natural killer cells (7.5–12.8%), were present in similar proportions to those in the neonatal cord blood. Gene set enrichment analysis showed that the MTOR, TNF-α, and MYC signaling pathways were enriched in T cells of the NEC patients, suggesting upregulated immune responses related to inflammation and cell proliferation. In addition, all four cases exhibited a bias toward cell-mediated inflammation, based on the predominance of T helper 1 cells.

Conclusion

Intestinal immunity in NEC subjects exhibited stronger inflammatory responses compared to non-NEC subjects. Further scRNA-seq and cellular analysis may improve our understanding of the pathogenesis of NEC.

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

Similar content being viewed by others

Data availability

Original dataset of this article is available after requesting the corresponding author.

References

  1. Neu J, Walker WA (2011) Necrotizing enterocolitis. N Engl J Med 364:255–264

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Okuyama H, Kubota A, Oue T, Kuroda S, Ikegami R, Kamiyama M (2002) A comparison of the clinical presentation and outcome of focal intestinal perforation and necrotizing enterocolitis in very-low-birth-weight neonates. Pediatr Surg Int 18:704–706

    PubMed  Google Scholar 

  3. Shah J, Singhal N, da Silva O, Rouvinez-Bouali N, Seshia M, Lee SK, Shah PS (2015) Intestinal perforation in very preterm neonates: risk factors and outcomes. J Perinatol Off J Calif Perinat Assoc 35:595–600

    CAS  Google Scholar 

  4. Adams-Chapman I (2018) Necrotizing enterocolitis and neurodevelopmental outcome. Clin Perinatol 45:453–466

    PubMed  Google Scholar 

  5. Hickey M, Georgieff M, Ramel S (2018) Neurodevelopmental outcomes following necrotizing enterocolitis. Semin Fetal Neonatal Med 23:426–432

    PubMed  Google Scholar 

  6. Amin SC, Pappas C, Iyengar H, Maheshwari A (2013) Short bowel syndrome in the NICU. Clin Perinatol 40:53–68

    PubMed  Google Scholar 

  7. Nowicki PT (2005) Ischemia and necrotizing enterocolitis: where, when, and how. Semin Pediatr Surg 14:152–158

    PubMed  Google Scholar 

  8. Chen Y, Chang KT, Lian DW, Lu H, Roy S, Laksmi NK, Low Y, Krishnaswamy G, Pierro A, Ong CC (2016) The role of ischemia in necrotizing enterocolitis. J Pediatr Surg 51:1255–1261

    PubMed  Google Scholar 

  9. Patel RM, Underwood MA (2018) Probiotics and necrotizing enterocolitis. Semin Pediatr Surg 27:39–46

    PubMed  Google Scholar 

  10. Neu J, Pammi M (2018) Necrotizing enterocolitis: the intestinal microbiome, metabolome and inflammatory mediators. Semin Fetal Neonatal Med 23:400–405

    PubMed  Google Scholar 

  11. Schreurs R, Baumdick ME, Sagebiel AF, Kaufmann M, Mokry M, Klarenbeek PL, Schaltenberg N, Steinert FL, van Rijn JM, Drewniak A, The SML, Bakx R, Derikx JPM, de Vries N, Corpeleijn WE, Pals ST, Gagliani N, Friese MA, Middendorp S, Nieuwenhuis EES, Reinshagen K, Geijtenbeek TBH, van Goudoever JB, Bunders MJ (2019) Human fetal TNF-α-cytokine-producing CD4(+) effector memory T cells promote intestinal development and mediate inflammation early in life. Immunity 50:462-476.e468

    CAS  PubMed  Google Scholar 

  12. Denning TL, Bhatia AM, Kane AF, Patel RM, Denning PW (2017) Pathogenesis of NEC: role of the innate and adaptive immune response. Semin Perinatol 41:15–28

    PubMed  Google Scholar 

  13. Shi N, Li N, Duan X, Niu H (2017) Interaction between the gut microbiome and mucosal immune system. Mil Med Res 4:14

    PubMed  PubMed Central  Google Scholar 

  14. Zhang X, Zhivaki D, Lo-Man R (2017) Unique aspects of the perinatal immune system. Nat Rev Immunol 17:495–507

    CAS  PubMed  Google Scholar 

  15. Patman G (2016) Paediatrics: T cells in necrotizing enterocolitis. Nat Rev Gastroenterol Hepatol 13:63

    PubMed  Google Scholar 

  16. Sawa S, Cherrier M, Lochner M, Satoh-Takayama N, Fehling HJ, Langa F, Di Santo JP, Eberl G (2010) Lineage relationship analysis of RORgammat+ innate lymphoid cells. Science (New York, NY) 330:665–669

    CAS  Google Scholar 

  17. Yu JC, Khodadadi H, Malik A, Davidson B, Salles ÉDSL, Bhatia J, Hale VL, Baban B (2018) Innate immunity of neonates and infants. Front Immunol 9:1759

    PubMed  Google Scholar 

  18. MohanKumar K, Namachivayam K, Ho TT, Torres BA, Ohls RK, Maheshwari A (2017) Cytokines and growth factors in the developing intestine and during necrotizing enterocolitis. Semin Perinatol 41:52–60

    PubMed  Google Scholar 

  19. Mara MA, Good M, Weitkamp JH (2018) Innate and adaptive immunity in necrotizing enterocolitis. Semin Fetal Neonatal Med 23:394–399

    PubMed  Google Scholar 

  20. Senger S, Ingano L, Freire R, Anselmo A, Zhu W, Sadreyev R, Walker WA, Fasano A (2018) Human fetal-derived enterospheres provide insights on intestinal development and a novel model to study necrotizing enterocolitis (NEC). Cell Mol Gastroenterol Hepatol 5:549–568

    PubMed  PubMed Central  Google Scholar 

  21. Hodzic Z, Bolock AM, Good M (2017) The role of mucosal immunity in the pathogenesis of necrotizing enterocolitis. Front Pediatr 5:40

    PubMed  PubMed Central  Google Scholar 

  22. Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM 3rd, Hao Y, Stoeckius M, Smibert P, Satija R (2019) Comprehensive Integration of single-cell data. Cell 177:1888-1902.e1821

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Aran D, Looney AP, Liu L, Wu E, Fong V, Hsu A, Chak S, Naikawadi RP, Wolters PJ, Abate AR, Butte AJ, Bhattacharya M (2019) Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nat Immunol 20:163–172

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Mabbott NA, Baillie JK, Brown H, Freeman TC, Hume DA (2013) An expression atlas of human primary cells: inference of gene function from coexpression networks. BMC Genomics 14:632

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102:15545–15550

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Prabhu SB, Rathore DK, Nair D, Chaudhary A, Raza S, Kanodia P, Sopory S, George A, Rath S, Bal V, Tripathi R, Ramji S, Batra A, Aggarwal KC, Chellani HK, Arya S, Agarwal N, Mehta U, Natchu UC, Wadhwa N, Bhatnagar S (2016) Comparison of human neonatal and adult blood leukocyte subset composition phenotypes. PLoS One 11:e0162242

    PubMed  PubMed Central  Google Scholar 

  27. Szabo PA, Levitin HM, Miron M, Snyder ME, Senda T, Yuan J, Cheng YL, Bush EC, Dogra P, Thapa P, Farber DL, Sims PA (2019) Single-cell transcriptomics of human T cells reveals tissue and activation signatures in health and disease. Nat Commun 10:4706

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Mousset CM, Hobo W, Woestenenk R, Preijers F, Dolstra H, van der Waart AB (2019) Comprehensive phenotyping of T cells using flow cytometry. Cytom Part A J Int Soc Anal Cytol 95:647–654

    Google Scholar 

  29. Caccamo N, Joosten SA, Ottenhoff THM, Dieli F (2018) Atypical human effector/memory CD4(+) T cells with a naive-like phenotype. Front Immunol 9:2832

    PubMed  PubMed Central  Google Scholar 

  30. Zhu X, Zhu J (2020) CD4 T helper cell subsets and related human immunological disorders. Int J Mol Sci 21(21):8011

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Egan CE, Sodhi CP, Good M, Lin J, Jia H, Yamaguchi Y, Lu P, Ma C, Branca MF, Weyandt S, Fulton WB, Niño DF, Prindle T Jr, Ozolek JA, Hackam DJ (2016) Toll-like receptor 4-mediated lymphocyte influx induces neonatal necrotizing enterocolitis. J Clin Investig 126:495–508

    PubMed  Google Scholar 

  32. Ma F, Li S, Gao X, Zhou J, Zhu X, Wang D, Cai Y, Li F, Yang Q, Gu X, Ge W, Liu H, Xiao X, Hao H (2019) Interleukin-6-mediated CCR9(+) interleukin-17-producing regulatory T cells polarization increases the severity of necrotizing enterocolitis. EBioMedicine 44:71–85

    PubMed  PubMed Central  Google Scholar 

  33. Melchers F (2015) Checkpoints that control B cell development. J Clin Investig 125:2203–2210

    PubMed  PubMed Central  Google Scholar 

  34. Oleinika K, Mauri C, Salama AD (2019) Effector and regulatory B cells in immune-mediated kidney disease. Nat Rev Nephrol 15:11–26

    CAS  PubMed  Google Scholar 

  35. Kapellos TS, Bonaguro L, Gemünd I, Reusch N, Saglam A, Hinkley ER, Schultze JL (2019) Human monocyte subsets and phenotypes in major chronic inflammatory diseases. Front Immunol 10:2035

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Cormican S, Griffin MD (2020) Human monocyte subset distinctions and function: insights from gene expression analysis. Front Immunol 11:1070

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Chinetti-Gbaguidi G, Colin S, Staels B (2015) Macrophage subsets in atherosclerosis. Nat Rev Cardiol 12:10–17

    CAS  PubMed  Google Scholar 

  38. Glezeva N, Horgan S, Baugh JA (2015) Monocyte and macrophage subsets along the continuum to heart failure: Misguided heroes or targetable villains? J Mol Cell Cardiol 89:136–145

    CAS  PubMed  Google Scholar 

  39. Ahmed I, Ismail N (2020) M1 and M2 macrophages polarization via mTORC1 influences innate immunity and outcome of ehrlichia infection. J Cell Immunol 2:108–115

    PubMed  PubMed Central  Google Scholar 

  40. Gardner A, Ruffell B (2016) Dendritic cells and cancer immunity. Trends Immunol 37:855–865

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Nutt SL, Chopin M (2020) Transcriptional networks driving dendritic cell differentiation and function. Immunity 52:942–956

    CAS  PubMed  Google Scholar 

  42. Yang C, Siebert JR, Burns R, Gerbec ZJ, Bonacci B, Rymaszewski A, Rau M, Riese MJ, Rao S, Carlson KS, Routes JM, Verbsky JW, Thakar MS, Malarkannan S (2019) Heterogeneity of human bone marrow and blood natural killer cells defined by single-cell transcriptome. Nat Commun 10:3931

    PubMed  PubMed Central  Google Scholar 

  43. Björkström NK, Strunz B, Ljunggren HG (2022) Natural killer cells in antiviral immunity. Nat Rev Immunol 22(2):112–123

    PubMed  Google Scholar 

  44. Amorim A, De Feo D, Friebel E, Ingelfinger F, Anderfuhren CD, Krishnarajah S, Andreadou M, Welsh CA, Liu Z, Ginhoux F, Greter M, Becher B (2022) IFNγ and GM-CSF control complementary differentiation programs in the monocyte-to-phagocyte transition during neuroinflammation. Nat Immunol 23:217–228

    CAS  PubMed  Google Scholar 

  45. Santarlasci V, Cosmi L, Maggi L, Liotta F, Annunziato F (2013) IL-1 and T helper immune responses. Front Immunol 4:182

    PubMed  PubMed Central  Google Scholar 

  46. Mahnke YD, Brodie TM, Sallusto F, Roederer M, Lugli E (2013) The who’s who of T-cell differentiation: human memory T-cell subsets. Eur J Immunol 43:2797–2809

    CAS  PubMed  Google Scholar 

  47. Golubovskaya V, Wu L (2016) Different subsets of T cells, memory, effector functions, and CAR-T immunotherapy. Cancers 8(3):36

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to acknowledge all participating patients, their families, and the doctors who made this study possible. This work was supported by numerous grants and programs: the JSPS Grant-in-Aid for Scientific Research (B) Grant Number JP17H04235 for YT; the Grant-in-Aid for Young Scientists (B) Grant Number JP17K17000 for KO; the Grant-in-Aid for Challenging Research (Exploratory) Grant Number JP18K19503 for SS; the Joint Research Program of the Institute for Genetic Medicine in Hokkaido University for KO; the Cooperative Research Project Program of the Medical Institute of Bioregulation in Kyushu University for YT; and the Joint Research of the Exploratory Research Center on Life and Living Systems (ExCELLS) (program No. 19-308, 20-316) in the National Institutes of Natural Sciences for SS.

Author information

Authors and Affiliations

  1. Department of Pediatric Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan

    Kazuo Oshima, Akinari Hinoki, Hiroo Uchida, Yujiro Tanaka, Chiyoe Shirota, Takahisa Tainaka, Wataru Sumida, Kazuki Yokota, Satoshi Makita, Aitaro Takimoto & Yoko Kano

  2. Department of Pediatric Surgery, Saitama Medical University, Saitama, Japan

    Kazuo Oshima & Yujiro Tanaka

  3. Department of Virology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

    Yusuke Okuno

  4. Cognitive Genomics Research Group, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Okazaki, Japan

    Yasuhiro Go

  5. Department of System Neuroscience, National Institute for Physiological Science, Okazaki, Japan

    Yasuhiro Go

  6. Department of Physiological Science, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan

    Yasuhiro Go

  7. Division of Mucosal Immunology, Research Center for Systems Immunology, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Shinichiro Sawa

Authors
  1. Kazuo Oshima
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Akinari Hinoki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hiroo Uchida
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yujiro Tanaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yusuke Okuno
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yasuhiro Go
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chiyoe Shirota
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Takahisa Tainaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Wataru Sumida
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Kazuki Yokota
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Satoshi Makita
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Aitaro Takimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yoko Kano
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Shinichiro Sawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

KO, YT, YO, AH, HU and SS: wrote the main manuscript text and prepared figures. All authors reviewed the manuscript.

Corresponding author

Correspondence to Shinichiro Sawa.

Ethics declarations

Conflict of interest

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.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1150 KB)

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

Oshima, K., Hinoki, A., Uchida, H. et al. Single-cell RNA sequencing of intestinal immune cells in neonatal necrotizing enterocolitis. Pediatr Surg Int 39, 179 (2023). https://doi.org/10.1007/s00383-023-05461-7

Download citation

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00383-023-05461-7

Keywords

Search

Navigation