This study aimed to elucidate the role of N6-methyladenosine (m6A) methylation in necrotizing enterocolitis (NEC) pathogenesis, focusing on its regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) expression, and to evaluate PFKFB3 as a therapeutic target for NEC.
Results:
We observed a significant reduction in N6-methyladenosine (m6A) methylation within the 3′-untranslated region (3′-UTR) of PFKFB3 mRNA in human NEC tissues. This epigenetic change stabilized PFKFB3 mRNA, increased protein levels, and accelerated glycolytic flux. In both in vivo (lipopolysaccharide–hypoxia–cold stress) and in vitro (THP-1-differentiated macrophage) NEC models, PFKFB3-driven glycolysis was found to promote M1 macrophage polarization through reactive oxygen species (ROS) accumulation, thereby intensifying intestinal inflammation. Importantly, pharmacological inhibition of PFKFB3 using 3-(3-pyridinyl)−1-(4-pyridinyl)−2-propen-1-one significantly reduced ROS production, limited macrophage infiltration, and mitigated mucosal injury.
Innovation and Conclusion:
This study identifies a critical metabolic–epigenetic axis in NEC pathogenesis, wherein reduced m6A methylation of PFKFB3 mRNA drives intestinal inflammation. Our results demonstrate that pharmacological inhibition of PFKFB3 effectively reduces inflammation and tissue injury in NEC models, positioning PFKFB3 as a novel therapeutic target. This work provides the first evidence of an m6A-mediated mechanism in NEC and highlights the potential of targeting PFKFB3 for clinical intervention. Antioxid. Redox Signal. 43, 765–781.
AkuzumB, LeeJ-Y. Context-dependent regulation of type17 immunity by microbiota at the intestinal barrier. Immune Netw, 2022; 22(6):e46.
2.
AndrewsRE, CoeKL. Clinical presentation and multifactorial pathogenesis of necrotizing enterocolitis in the preterm infant. Adv Neonatal Care, 2021; 21(5):349–355.
3.
ArtyomovMN, Van den BosscheJ. Immunometabolism in the single-cell era. Cell Metab, 2020; 32(5):710–725; doi: 10.1016/j.cmet.2020.09.013
4.
CantelmoAR, ConradiL-C, BrajicA, et al.Inhibition of the glycolytic activator PFKFB3 in endothelium induces tumor vessel normalization, impairs metastasis, and improves chemotherapy. Cancer Cell, 2016; 30(6):968–985.
5.
CaoY, ZhangX, WangL, et al.PFKFB3-mediated endothelial glycolysis promotes pulmonary hypertension. Proc Natl Acad Sci U S A, 2019; 116(27):13394–13403.
6.
ChibaT, MarusawaH, UshijimaT. Inflammation-associated cancer development in digestive organs: Mechanisms and roles for genetic and epigenetic modulation. Gastroenterology, 2012; 143(3):550–563.
7.
DuanS, LouX, ChenS, et al.Macrophage LMO7 deficiency facilitates inflammatory injury via metabolic-epigenetic reprogramming. Acta Pharm Sin B, 2023; 13(12):4785–4800; doi: 10.1016/j.apsb.2023.09.012
8.
EgoziA, OlaloyeO, WernerL, et al.Single-cell atlas of the human neonatal small intestine affected by necrotizing enterocolitis. PLoS Biol, 2023; 21(5):e3002124; doi: 10.1371/journal.pbio.3002124
9.
EtchegarayJ-P, MostoslavskyR. Interplay between metabolism and epigenetics: A nuclear adaptation to environmental changes. Mol Cell, 2016; 62(5):695–711.
10.
FilardyAA, FerreiraJR, RezendeRM, et al.The intestinal microenvironment shapes macrophage and dendritic cell identity and function. Immunol Lett, 2023; 253:41–53.
11.
ForresterSJ, KikuchiDS, HernandesMS, et al.Reactive oxygen species in metabolic and inflammatory signaling. Circ Res, 2018; 122(6):877–902.
GopalakrishnaKP, MacadangdangBR, RogersMB, et al.Maternal IgA protects against the development of necrotizing enterocolitis in preterm infants. Nat Med, 2019; 25(7):1110–1115.
14.
HackamDJ, SodhiCP. Bench to bedside—new insights into the pathogenesis of necrotizing enterocolitis. Nat Rev Gastroenterol Hepatol, 2022; 19(7):468–479.
15.
HalpernMD, ClarkJA, SaundersTA, et al.Reduction of experimental necrotizing enterocolitis with anti-TNF-α. Am J Physiol Gastrointest Liver Physiol, 2006; 290(4):G757–G764.
16.
HeL, LiH, WuA, et al.Functions of N6-methyladenosine and its role in cancer. Mol Cancer, 2019; 18(1):176–115.
17.
HerbM, SchrammM. Functions of ROS in macrophages and antimicrobial immunity. Antioxidants (Basel), 2021; 10(2):313.
18.
HunterCJ, De PlaenIG. Inflammatory signaling in NEC: Role of NF-κB, cytokines and other inflammatory mediators. Pathophysiology, 2014; 21(1):55–65.
19.
KimD-Y, LeemY-H, ParkJ-S, et al.RIPK1 regulates microglial activation in lipopolysaccharide-induced Neuroinflammation and MPTP-induced Parkinson’s disease mouse models. Cells, 2023; 12(3):417.
20.
KonjarŠ, FerreiraC, CarvalhoFS, et al.Intestinal tissue-resident T cell activation depends on metabolite availability. Proc Natl Acad Sci U S A, 2022; 119(34):e2202144119.
21.
KooS-J, GargNJ. Metabolic programming of macrophage functions and pathogens control. Redox Biol, 2019; 24:101198.
22.
LiB, WuRY, HorneRG, et al.Human milk oligosaccharides protect against necrotizing enterocolitis by activating intestinal cell differentiation. Mol Nutr Food Res, 2020; 64(21):e2000519.
23.
LiT, HuPS, ZuoZ, et al.METTL3 facilitates tumor progression via an m(6)A-IGF2BP2-dependent mechanism in colorectal carcinoma. Mol Cancer, 2019; 18(1):112; doi: 10.1186/s12943-019-1038-7
24.
LiuD, XiaoM, ZhouJ, et al.PFKFB3 promotes sepsis-induced acute lung injury by enhancing NET formation by CXCR4hi neutrophils. Int Immunopharmacol, 2023; 123:110737.
25.
MaY, ZhangX, XuanB, et al.Disruption of CerS6-mediated sphingolipid metabolism by FTO deficiency aggravates ulcerative colitis. Gut, 2024; 73(2):268–281; doi: 10.1136/gutjnl-2023-330009
26.
Pålsson-McDermottEM, O’NeillLAJ. Targeting Immunometabolism as an anti-inflammatory strategy. Cell Res, 2020; 30(4):300–314; doi: 10.1038/s41422-020-0291-z
27.
PatelRM, FergusonJ, McElroySJ, et al.Defining necrotizing enterocolitis: Current difficulties and future opportunities. Pediatr Res, 2020; 88(Suppl 1):10–15.
28.
PengZ-P, JiangZ-Z, GuoH-F, et al.Glycolytic activation of monocytes regulates the accumulation and function of neutrophils in human hepatocellular carcinoma. J Hepatol, 2020; 73(4):906–917.
29.
RathS, HawsawiYM, AlzahraniF, et al.Epigenetic regulation of inflammation: The metabolomics connection. Semin Cell Dev Biol, 2024; 154(Pt C):355–363.
30.
SarniakA, LipińskaJ, TytmanK, et al.Endogenous mechanisms of reactive oxygen species (ROS) generation. Postepy Hig Med Dosw (Online), 2016; 70(0):1150–1165.
31.
SchusterS, EwaldJAN, KaletaC. Modeling the energy metabolism in immune cells. Curr Opin Biotechnol, 2021; 68:282–291; doi: 10.1016/j.copbio.2021.03.003
32.
ShockT, BadangL, FergusonB, et al.The interplay between diet, gut microbes, and host epigenetics in health and disease. J Nutr Biochem, 2021; 95:108631.
33.
SinclairTJ, YeC, ChenY, et al.Progressive metabolic dysfunction and nutritional variability precedes necrotizing enterocolitis. Nutrients, 2020; 12(5):1275; doi: 10.3390/nu12051275
34.
TarracchiniC, MilaniC, LonghiG, et al.Unraveling the microbiome of necrotizing enterocolitis: Insights in novel microbial and Metabolomic biomarkers. Microbiol Spectr, 2021; 9(2):e0117621; doi: 10.1128/Spectrum.01176-21
35.
TongJ, FlavellRA, LiH-B. RNA m 6 A modification and its function in diseases. Front Med, 2018; 12(4):481–489.
36.
Van den BosscheJ, O’NeillLA, MenonD. Macrophage Immunometabolism: Where are we (going)? Trends Immunol, 2017; 38(6):395–406; doi: 10.1016/j.it.2017.03.001
37.
WangX, LuZ, GomezA, et al.N 6-methyladenosine-dependent regulation of messenger RNA stability. Nature, 2014; 505(7481):117–120; doi: 10.1038/nature12730
WuY-L, LinZ-J, LiC-C, et al.Epigenetic regulation in metabolic diseases: Mechanisms and advances in clinical study. Signal Transduct Target Ther, 2023; 8(1):98.
40.
XiaoM, LiuD, XuY, et al.Role of PFKFB3-driven glycolysis in sepsis. Ann Med, 2023; 55(1):1278–1289.
41.
YangZ, FujiiH, MohanSVet al.Phosphofructokinase deficiency impairs ATP generation, autophagy, and redox balance in rheumatoid arthritis T cells. J Exp Med, 2013; 210(10):2119–2134; doi: 10.1084/jem.20130252
