Epigenetics has revealed a potential mechanism that may explain how environmental triggers and genetic susceptibility interact in IBD [35]. Evidence from many studies suggests that epigenetic modification, in particular DNA methylation, which exists at cytosines in the cytosine–guanine (CpG) dinucleotide context, plays a key role in paediatric IBD phenotypes, as it is considered a key regulatory mechanism of gene expression in response to environmental cues, without modifying the primary nucleotide sequence [36,37]. However, the little evidence that exists examining histone methylation suggests that this modification takes place in the intestine of paediatric IBD. In CD children, genes exhibiting decreased histone H3-lysine 4 trimethylation (H3K4me3) signatures are found to be associated with the severity of inflammation in IECs [38]. Ileal IECs play a significant role in integrating commensal microbiota-derived cues to regulate immune homeostasis and gene expression. Genes characterized by increased H3K4me3 levels (e.g., DUOX2, NOS2) in IECs from CD children in response to commensal microbiota are enriched in several pathways, including the regulation of reactive oxygen species (ROS), G alpha signalling, digestive system development, and nitric oxide (NO) biosynthesis, suggesting that commensal microbiota may modify histone alterations that reflect intestinal inflammation in CD [38]. Large-scale, genome-wide studies have revealed significant mucosal DNA methylation changes in IBD-associated genes in children. For example, a systematic meta-analysis of 84 genetic studies identified specific genetic variants (rs11209026, rs7517847, rs12521868, rs26313667, rs1800629, rs2241880, rs2066847, rs2066844, and rs2066844) in differential DNA methylated genes (NOD2, IL23R, ATG16L1, IBD5, and TNF-α) known to cause CD and UC [36]. A study that assessed DNA methylation profiles of the colonic mucosa found that 182 CD and 3365 UC susceptible genes (including STAT3, SLPI, ITGB2, SAA1, IFITM1, and S100A9) were associated with differentially methylated regions (DMRs) [39]. Another study detected a number of UC-associated changes in DNA methylation at nine CpG sites located in the TRIM39-RPP21 gene [40]. In respect to the correlation between the colonic mucosal DNA methylation of paediatric IBD and microbiome changes, a study showed that SLC9A3, a gene with decreasing methylation UC-specific DMR, was associated with a reduced abundance of Bacteroides [41].

Loss-of-function mutations in G-protein coupled receptors (GPCRs) may result in reduced ligand-binding affinity to many chemokines/cytokines, local mediators, and neurotransmitters during childhood development [42], which may lead to an increased risk of IBD. Naïve CD4⁺ T cells differ according to cytokine-producing T-helper (Th) subsets, including Th1 (interferon-gamma, IFN-γ, TNF-α), Th2 (IL-4, IL-5, IL-6, IL-13), and Th17 (IL-17) cells, which are implicated in the dysregulation of colonic mucosa in IBD patients [43,44]. Paediatric CD patients were shown to have a mixed Th1/Th2/Th17 cytokine profile, with increased serum levels of IL-1β, IFN-γ, IL-6, TNF-α, C-X-C motif chemokine ligand 10 (CXCL10), and IL-17A observed in both the ileum and colon [45,46]. Increased serum IL-4 and IL-6 levels were detected in the intestinal mucosa of paediatric UC patients, in which the GATA binding protein 3 (GATA3) and signal transducer and activator of transcription-4 (STAT4) signalling molecules were involved [47]. Compared with paediatric CD patients, significantly more mRNAs related to IL5, IL-13, IL-23, and IL17A cytokines were observed in the rectal mucosa of UC patients [48]. Serum levels of IL-6 were found to be higher in the ileum of paediatric CD patients than those of healthy children, whereas the serum levels of IL-22 and IL-17A were higher in UC patients than in CD patients [49]. Although Foxp3⁺T_reg cells are found in higher density in the inflamed mucosa of paediatric IBD patients, they maintain immune homeostasis [49,50]. A higher density of Foxp3⁺ cells in the ileum of untreated paediatric CD patients compared with adult patients may be attributed to the disparate pattern of CD phenotypic expression [51]. In a previous in vitro experiment, serum levels of IL-17 and TNF-α were increased in the peripheral blood of paediatric patients with CD and UC. Patients displayed a decreased expression of Foxp3⁺, CD4⁺, and CD25⁺, and an increased percentage of Th17 cells. Myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCS) expressing CD200, a type I transmembrane glycoprotein, were found to be elevated, and significantly associated with Th17, but negatively associated with regulatory T cells (T_regs). On the contrary, the expression of the CD200 receptor, CD200R1, on mDCs was found to be reduced and negatively associated with Th17 [52]. The mRNA expression of pro-inflammatory cytokines IL-6 and IL-23 expressed at high levels in the colonic mucosa of paediatric patients with CD and UC is found to be associated with a higher frequency of CD4⁺ IL-17a⁺ and a lower frequency of CD4⁺Foxp3⁺T_regs [53]. An experimental study demonstrated that treatment with infliximab, a chimeric monoclonal antibody, does not inhibit the production of TNF-α, but also hampers expansion of FOXP3⁺ T_regs in the colonic mucosa of paediatric CD patients [54]. Another recent experimental study reported that paediatric patients with active CD and UC, though having a high expression of CTLA-4 in FOXP3⁺ T_regs in peripheral blood during pharmacological (aminosalicylates or azathioprine) therapy, showed a significant decrease of CD4⁺Foxp3⁺T_regs levels after therapy compared to the same patients and healthy children at disease onset [55].

In a human IBD model, which is characterised by increased histone deacetylases (HDACs), nuclear factor-κB (NF-κB), nuclear factor kappa-B kinase β (IKKβ), TNF-α, NOD2, and toll-like receptor (TLR) upregulation have been reported to occur in the inflamed IECs, resulting in high pro-inflammatory cytokine expression levels [56]. HDAC1 has been shown to be implicated in several diseases, in which it takes off the acetyl group from lysine residues of histone/non-histone proteins via acetyltransferases (HATs), which results in DNA inaccessibility, gene transcription repression, and chromatin compression [57]. The acetylation of histone H3 lysine 27, which is identified in regions with several risk loci for IBD [58], has been found to be downregulated in the inflamed mucosa of UC patients, resulting in high IL-6 mRNA levels [59]. HDAC1 induces an inflammatory response in the colon epithelium of UC patients by activating NF-κB, and reducing histone H3 acetylation and tight-junction protein, zonula occludens 1 (ZO-1), expression [60]. Loss-of-function NOD2 gene mutations enhance NF-κB activation, which, in turn, bind to the promoters of pro-inflammatory cytokines in paediatric CD patients [61]. This suggests that cytokines with pro-inflammatory effects could play a key role in the pathogenesis of paediatric IBD.