Download PDF
Am J Perinatol
DOI: 10.1055/s-0044-1787739
Original Article

Reduced Expression of REG4 as a Sign of Altered Goblet Cell Function in Necrotizing Enterocolitis

Alice Hoffsten
1   Department of Women's and Children's Health, Uppsala University, Uppsala, Sweden
,
Laszlo Markasz
1   Department of Women's and Children's Health, Uppsala University, Uppsala, Sweden
2   Neonatology Division, University Children's Hospital, Uppsala, Sweden
,
Helene Engstrand Lilja
1   Department of Women's and Children's Health, Uppsala University, Uppsala, Sweden
3   Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden
,
Hamid Mobini-Far
4   Department of Pathology, Uppsala University Hospital, Uppsala, Sweden
5   Department of Surgical Sciences, Uppsala University, Uppsala, Sweden
,
Richard Sindelar
1   Department of Women's and Children's Health, Uppsala University, Uppsala, Sweden
2   Neonatology Division, University Children's Hospital, Uppsala, Sweden
› Author Affiliations Funding This study was funded by HKH Kronprinsessan Lovisas Stiftelse (Grant No. 2018-00459); Stiftelsen Samariten (2022-11-24); Gillbergska stiftelsen (2022-11-15); Födelsefonden (2022-12-15).
› Further Information
Also available at
    Permissions and Reprints

Abstract

Objective Defective Goblet cells have been proposed to be involved in necrotizing enterocolitis (NEC). The aim was to study the expression of the Goblet cell marker REG4 and its potential involvement in NEC in preterm infants with and without NEC.

Study Design Seventy histologically intact intestinal biopsies were studied: 43 were collected during surgery due to NEC (NEC group: 26.5 ± 3.0 weeks' gestational age [wGA]), and 27 from individuals who underwent surgery due to other conditions (Control group; 36.1 ± 4.5 wGA). The tissue samples were immunohistochemically stained for REG4. REG4 expression was quantified with a semiautomated digital image analysis and with clinical data compared between the groups.

Results REG4 expression was lower in the NEC group than in the Control group (p = 0.035). Low REG4 expression correlated to the risk of NEC (p = 0.023). In a multivariable logistic regression analysis including GA and REG4 expression for NEC risk, only GA (p < 0.001) and not REG4 expression (p = 0.206) was associated with NEC risk.

Conclusion This study concludes that Goblet cell dysfunction may be involved in NEC development, as low expression of the Goblet cell marker REG4 was related to an increased NEC risk in preterm infants. Maturity could however not be excluded as a potential confounder for REG4 expression.

Key Points

  • REG4 is a specific Goblet cell marker not yet studied in NEC.

  • REG4 was quantified in intestinal biopsies from infants with and without NEC.

  • REG4 expression was lower in infants with NEC, and expression seems to be maturity dependent.

Keywords

necrotizing enterocolitis - Goblet cell - REG4

Authors' Contributions

A.H. performed the collection of data and the statistical analysis and wrote and edited the manuscript. L.M. set up the study, performed data collection, data analysis, and partook in writing and revising the manuscript. H.E.L. partook in data analysis and in writing and revising the manuscript. R.S. performed data analysis, statistical analysis, as well as writing and revising of the manuscript. H.M.F. participated in data collection and data analysis, as well as writing and revising the manuscript.


Ethical Approval

Written informed consent was required from the parents of the infants and from the adult participants included in this study. The Regional Ethical Review Board in Sweden approved this study (approval no.: 2019-00,437).




Publication History

Received: 18 March 2024

Accepted: 27 May 2024

Article published online:
15 July 2024

© 2024. Thieme. All rights reserved.

Thieme Medical Publishers, Inc.
333 Seventh Avenue, 18th Floor, New York, NY 10001, USA

 

  • References

  • 1 Neu J, Walker WA. Necrotizing enterocolitis. N Engl J Med 2011; 364 (03) 255-264
    CrossrefPubMedGoogle Scholar
  • 2 Hoffsten A, Markasz L, Ericson K, Nelin LD, Sindelar R. The value of autopsy in preterm infants at a Swedish tertiary neonatal intensive care unit 2002-2018. Sci Rep 2021; 11 (01) 14156
    CrossrefPubMedGoogle Scholar
  • 3 Neu J. Necrotizing enterocolitis: the future. Neonatology 2020; 117 (02) 240-244
    CrossrefPubMedGoogle Scholar
  • 4 Berrington JE, Hearn RI, Bythell M, Wright C, Embleton ND. Deaths in preterm infants: changing pathology over 2 decades. J Pediatr 2012; 160 (01) 49-53.e1
    CrossrefPubMedGoogle Scholar
  • 5 Bazacliu C, Neu J. Necrotizing enterocolitis: long term complications. Curr Pediatr Rev 2019; 15 (02) 115-124
    CrossrefPubMedGoogle Scholar
  • 6 Liu D, Xu Y, Feng J, Yu J, Huang J, Li Z. Mucins and tight junctions are severely altered in necrotizing enterocolitis neonates. Am J Perinatol 2021; 38 (11) 1174-1180
    Article in Thieme ConnectPubMedGoogle Scholar
  • 7 Moore SA, Nighot P, Reyes C. et al. Intestinal barrier dysfunction in human necrotizing enterocolitis. J Pediatr Surg 2016; 51 (12) 1907-1913
    CrossrefPubMedGoogle Scholar
  • 8 Benkoe TM, Mechtler TP, Weninger M, Pones M, Rebhandl W, Kasper DC. Serum levels of interleukin-8 and gut-associated biomarkers in diagnosing necrotizing enterocolitis in preterm infants. J Pediatr Surg 2014; 49 (10) 1446-1451
    CrossrefPubMedGoogle Scholar
  • 9 Weitkamp JH, Koyama T, Rock MT. et al. Necrotising enterocolitis is characterised by disrupted immune regulation and diminished mucosal regulatory (FOXP3)/effector (CD4, CD8) T cell ratios. Gut 2013; 62 (01) 73-82
    CrossrefPubMedGoogle Scholar
  • 10 Boron WF, Boulpaep EL. Medical Physiology. 2nd updated ed.. Elsevier; 2012
    Google Scholar
  • 11 Hoffsten A, Lilja HE, Mobini-Far H, Sindelar R, Markasz L. Paneth cell proteins DEFA6 and GUCA2A as tissue markers in necrotizing enterocolitis. Eur J Pediatr 2023; 182 (06) 2775-2784
    CrossrefPubMedGoogle Scholar
  • 12 Birchenough GMH, Johansson ME, Gustafsson JK, Bergström JH, Hansson GC. New developments in goblet cell mucus secretion and function. Mucosal Immunol 2015; 8 (04) 712-719
    CrossrefPubMedGoogle Scholar
  • 13 Pelaseyed T, Bergström JH, Gustafsson JK. et al. The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system. Immunol Rev 2014; 260 (01) 8-20
    CrossrefPubMedGoogle Scholar
  • 14 Denning TL, Bhatia AM, Kane AF, Patel RM, Denning PW. Pathogenesis of NEC: role of the innate and adaptive immune response. Semin Perinatol 2017; 41 (01) 15-28
    CrossrefPubMedGoogle Scholar
  • 15 Rasiah V, Yajamanyam PK, Ewer AK. Necrotizing enterocolitis: current perspectives. Res Rep Neonatol 2014; 40 (01) 32
    PubMedGoogle Scholar
  • 16 Clark JA, Doelle SM, Halpern MD. et al. Intestinal barrier failure during experimental necrotizing enterocolitis: protective effect of EGF treatment. Am J Physiol Gastrointest Liver Physiol 2006; 291 (05) G938-G949
    CrossrefPubMedGoogle Scholar
  • 17 McElroy SJ, Prince LS, Weitkamp JH, Reese J, Slaughter JC, Polk DB. Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: a potential role in neonatal necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 2011; 301 (04) G656-G666
    CrossrefPubMedGoogle Scholar
  • 18 The Human Protein Atlas Project. REG4 protein expression summary—The Human Protein Atlas. Published 2022. Accessed October 27, 2022 at: https://www.proteinatlas.org/ENSG00000134193-REG4
    PubMedGoogle Scholar
  • 19 Hartupee JC, Zhang H, Bonaldo MF, Soares MB, Dieckgraefe BK. Isolation and characterization of a cDNA encoding a novel member of the human regenerating protein family: Reg IV. Biochim Biophys Acta 2001; 1518 (03) 287-293
    CrossrefPubMedGoogle Scholar
  • 20 Dieckgraefe BK, Crimmins DL, Landt V. et al. Expression of the regenerating gene family in inflammatory bowel disease mucosa: Reg Ialpha upregulation, processing, and antiapoptotic activity. J Investig Med 2002; 50 (06) 421-434
    CrossrefPubMedGoogle Scholar
  • 21 Kämäräinen M, Heiskala K, Knuutila S, Heiskala M, Winqvist O, Andersson LC. RELP, a novel human REG-like protein with up-regulated expression in inflammatory and metaplastic gastrointestinal mucosa. Am J Pathol 2003; 163 (01) 11-20
    CrossrefPubMedGoogle Scholar
  • 22 van Beelen Granlund A, Østvik AE, Brenna Ø, Torp SH, Gustafsson BI, Sandvik AK. REG gene expression in inflamed and healthy colon mucosa explored by in situ hybridisation. Cell Tissue Res 2013; 352 (03) 639-646
    CrossrefPubMedGoogle Scholar
  • 23 Bell MJ, Ternberg JL, Feigin RD. et al. Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging. Ann Surg 1978; 187 (01) 1-7
    CrossrefPubMedGoogle Scholar
  • 24 Shu J, Dolman GE, Duan J, Qiu G, Ilyas M. Statistical colour models: an automated digital image analysis method for quantification of histological biomarkers. Biomed Eng Online 2016; 15: 46
    CrossrefPubMedGoogle Scholar
  • 25 Leaphart CL, Cavallo J, Gribar SC. et al. A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair. J Immunol 2007; 179 (07) 4808-4820
    CrossrefPubMedGoogle Scholar
  • 26 Mihi B, Good M. Impact of Toll-like receptor 4 signaling in necrotizing enterocolitis: the state of the science. Clin Perinatol 2019; 46 (01) 145-157
    CrossrefPubMedGoogle Scholar
  • 27 Sodhi CP, Neal MD, Siggers R. et al. Intestinal epithelial toll-like receptor 4 regulates goblet cell development and is required for necrotizing enterocolitis in mice. Gastroenterology 2012; 143 (03) 708-718.e5
    CrossrefPubMedGoogle Scholar
  • 28 Zhang YW, Ding LS, Lai MD. Reg gene family and human diseases. World J Gastroenterol 2003; 9 (12) 2635-2641
    CrossrefPubMedGoogle Scholar
  • 29 Granlund Av, Beisvag V, Torp SH. et al. Activation of REG family proteins in colitis. Scand J Gastroenterol 2011; 46 (11) 1316-1323
    CrossrefPubMedGoogle Scholar
  • 30 Planell N, Lozano JJ, Mora-Buch R. et al. Transcriptional analysis of the intestinal mucosa of patients with ulcerative colitis in remission reveals lasting epithelial cell alterations. Gut 2013; 62 (07) 967-976
    CrossrefPubMedGoogle Scholar
  • 31 Sun C, Wang X, Hui Y, Fukui H, Wang B, Miwa H. The potential role of REG family proteins in inflammatory and inflammation-associated diseases of the gastrointestinal tract. Int J Mol Sci 2021; 22 (13) 7196
    CrossrefPubMedGoogle Scholar
  • 32 Chaaban H, Patel MM, Burge K. et al. Early antibiotic exposure alters intestinal development and increases susceptibility to necrotizing enterocolitis: a mechanistic study. Microorganisms 2022; 10 (03) 519
    CrossrefPubMedGoogle Scholar
  • 33 He W, Wang ML, Jiang HQ. et al. Bacterial colonization leads to the colonic secretion of RELMbeta/FIZZ2, a novel goblet cell-specific protein. Gastroenterology 2003; 125 (05) 1388-1397
    CrossrefPubMedGoogle Scholar
  • 34 Neu J, Pammi M. Necrotizing enterocolitis: the intestinal microbiome, metabolome and inflammatory mediators. Semin Fetal Neonatal Med 2018; 23 (06) 400-405
    CrossrefPubMedGoogle Scholar
  • 35 Olm MR, Bhattacharya N, Crits-Christoph A. et al. Necrotizing enterocolitis is preceded by increased gut bacterial replication, Klebsiella, and fimbriae-encoding bacteria. Sci Adv 2019; 5 (12) eaax5727
    CrossrefPubMedGoogle Scholar
  • 36 Warner BB, Deych E, Zhou Y. et al. Gut bacteria dysbiosis and necrotising enterocolitis in very low birthweight infants: a prospective case-control study. Lancet 2016; 387 (10031): 1928-1936
    CrossrefPubMedGoogle Scholar
  • 37 Qi H, Wei J, Gao Y. et al. Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut. Commun Biol 2020; 3 (01) 483
    CrossrefPubMedGoogle Scholar
  • 38 Wang W, Wang Y, Lu Y. et al. Reg4 protects against Salmonella infection-associated intestinal inflammation via adopting a calcium-dependent lectin-like domain. Int Immunopharmacol 2022; 113 (Pt A): 109310
    CrossrefPubMedGoogle Scholar
  • 39 Fawkner-Corbett D, Antanaviciute A, Parikh K. et al. Spatiotemporal analysis of human intestinal development at single-cell resolution. Cell 2021; 184 (03) 810-826.e23
    CrossrefPubMedGoogle Scholar
  • 40 Tourneur E, Chassin C. Neonatal immune adaptation of the gut and its role during infections. Clin Dev Immunol 2013; 2013: 270301
    CrossrefPubMedGoogle Scholar
  • 41 Barker N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration. Nat Rev Mol Cell Biol 2014; 15 (01) 19-33
    CrossrefPubMedGoogle Scholar
  • 42 Thiagarajah JR, Yildiz H, Carlson T. et al. Altered goblet cell differentiation and surface mucus properties in Hirschsprung disease. PLoS One 2014; 9 (06) e99944
    CrossrefPubMedGoogle Scholar