Intestinal enteroids/organoids: A novel platform for drug discovery in inflammatory bowel diseases

Table 1 Enteroids/organoids retain the tissue specific transcriptional and epigenetic profiles

	Enteroidsderived from	Distinct genes	Transcriptional / epigenetic	Culture period / passage number of enteroids	Human / mouse	Ref.
Region-specific	Duodenum	Gata4, Cybrd1, Slc40a1	Increased mRNA	12 weeks	Mouse	[25]
	Jejunum	Gata4, Lct
	Ileum	Slc10a2, Ostb
Region- specific	Small bowel	CHGA, ATOH1	Increased mRNA	Not mentioned	Human fetus	[172]
	Large bowel	LGR5, MYC
Region-specific	Terminal ileum	SLC5A1, CLDN15	Increased mRNA	3 months / 11 passages	Human children and adults	[26]
		SATB2AS1	Increased methylation
	Sigmoid colon	CFTR, SATB2	Increased mRNA
		IL6R, CLDN15	Increased methylation
Disease-specific	Normal rectum	GATA2	Increased methylation	3-5 passages	Human children	[26]
	Gastric heterotopia in rectum	SATB2AS1
Disease-specific	Colon of non-IBD patients	PLA2G2A, ZG16, CLCA1, AQP8, MUC12	Increased mRNA	1 passage	Human	[14]
	Colon of UC patients	LYZ, ANXA10CLDN18
Disease-specific	Sigmoid colon of non-IBD patients	LYZ (low)	mRNA expression	Not mentioned	Human	[14]
	Sigmoid colon of CD patients	LYZ (High)
Disease-specific	Healthy control	GREB1, TMEM173	Increased methylation	Not mentioned	Human children: Terminal ileum, sigmoid colon	[28]
	CD patients	PDE1B

Gata4: GATA binding protein 4, transcription factor specific to duodenum and jejunum; Cybrd1: Cytochrome b reductase 1 specific for iron uptake; Slc40a1: Solute carrier family 40 member 1, Fe2⁺ transporter ferroportin for iron uptake; Lct: Lactase-phlorizin hydrolase for dissacharide digestion; Slc10a2: Solute carrier family 10 member 2, apical sodium-dependent bile acid transporter; Ostb: Organic solute transporter beta for bile acid uptake; CHGA: Chromogranin A, a marker of enteroendocrine cells; ATOH1: Atonal BHLH Transcription Factor 1, key regulator of the Notch pathway; LGR5: Leucine rich repeat containing G protein-coupled receptor 5; MYC: MYC Proto-Oncogene, BHLH Transcription Factor; SLC5A1: Solute carrier family 5 member 1; CLDN15: Claudin 15; SATB2AS1: Special AT-rich sequence-binding protein 2 antisense 1; CFTR: Cystic fibrosis transmembrane conductance regulator; SATB2: Special AT-rich sequence-binding protein 2; IL6R: Interleukin-6 receptor; GATA2: GATA binding protein 2; PLA2G2A: Phospholipase A2 Group IIA, antibacterial enzyme; ZG16: Zymogen Granule Protein 16, mucus secreting cell marker; CLCA1: Chloride Channel Accessory 1, mucus secreting cell marker; AQP8: Aquaporin-8, water transporter; MUC12: Transmembrane mucin 12; LYZ: Lysozyme C; ANXA10: Annexin-10; CLDN18: Claudin 18; GREB1: Growth regulation by estrogen in breast cancer 1; TMEM173: Transmembrane protein 173; PDE1B: Phosphodiesterase 1B.

Table 2 Current therapeutic approaches in inflammatory bowel diseases focused on immune cells

Effects on immune cells	Drug type	Drug name/approach	Mechanism of action	Ref.
Inhibits pro-inflammatory cytokine production	Classic anti-inflammatory drugs	5-ASA Corticosteroid	NF-kB inactivation	[77,78]
	JAK inhibitors	Tofacitinib (JAK1/2/3)	Inhibits differentiation of Th1, Th2, and Th17 cells by inhibition of JAK/STAT pathway	[90,91]
		Filgotinib (JAK1)		[173]
	ROR-γt inhibitor	GSK805	Inhibits differentiation of Th17 cells but not ILC3s by inhibition of transcription factor ROR-γt	[92]
	GATA3 specific DNAzyme (DNA antisense molecule)	Rectal delivery	Inhibits differentiation of Th2 cells by inhibition of transcription factor GATA3 expression	[93]
	Anti-p40 subunit IL-12/IL-23	Ustekinumab	Inhibits differentiation of Th1 and Th17 cells by neutralization of IL-12/IL-23	[5,86]
	PDE4 inhibitors	Apremilast, roflumilast	Increases intracellular cAMP which inhibits NF-kB (PDE4 degrades cAMP)	[174-176]
Induces apoptosis	Classic immunosuppressive agents	Azathioprine 6-mercaptopurine	Metabolized purine analogue induces apoptosis by small GTPase RAC1 inactivation	[79]
		Cyclosporine A	Activates caspase 8 signaling	[80,81]
		Tacrolimus (FK506)	Inhibits calcineurin/NFAT pathway	[177]
		Methotrexate	Cleavage of PARP but not caspase 3 activation	[82]
	Anti-IL-6/IL-6R	PF-04236921 (anti-IL-6)	Inhibits IL-6/STAT3 signaling which mediates resistance against apoptosis	[87-89]
		Tocilizumab (anti-IL-6R)
Blocks pro-inflammatory cytokines released from immune cells	Anti-TNF-α	Infliximab, adalimumab, golimumab, certolizumab pegol	Neutralizing antibody against TNF-α	[83]
Blocks trafficking or homing of immune cells toward inflamed gut	Gut specific anti-integrin	Vedolizumab (anti-α4β7) Etrolizumab (anti-β7)	Neutralizing antibody against integrin	[6-8,94,95]
	Chemokine receptor (CCR9) inhibitor	Vercirnon (CCX282-B)	inhibits CCL25 (CCR9 ligand) directed chemotaxis	[96,97]
	Anti-cell adhesion molecules	PF-00547659	Neutralizing antibody against MAdCAM-1 (gut specific ligand for integrin α4β7) in vascular endothelial cells	[98,99]
		Alicaforsen	ICAM-1 antisense oligonucleotide which inhibits ICAM-1 production in vascular endothelial cells	[100]
	S1P1/S1P5 receptor agonist/functional antagonist	Ozanimod	Induces internalization and degradation of S1P receptor, and subsequently inhibit S1P/NF-kB/STAT3 pathway	[101-103]
Activates endogenous anti-inflammatory mechanisms	Anti-SMAD7 oligonucleotide	Mongersen	Activates anti-inflammatory TGFβ/SMAD3 signaling by degradation of SMAD7 mRNA	[104]
	Mesenchymal stem cells	Transplantation	Directs T cell differentiation toward anti-inflammatory phenotype (Th2 cells, Treg) and inhibits T cell proliferation, cytokine production, and migration	[77,105,106,178]
	Treg	Transplantation	Secretion of anti-inflammatory cytokines (TGFβ and IL-10)	[107]

5-ASA: 5-Aminosalicylic acid; NF-kB: Nuclear factor kappa B; JAK: Janus kinase; STAT: Signal transducer and activator of transcription; ROR: RAR-related orphan receptor; Th: T helper; ILC3: Group3 innate lymphoid cells; GATA3: GATA binding protein 3; PDE4: Phosphodiesterase 4; cAMP: cyclic adenosine 3’5’-monophosphate; RAC1: Rac family small GTPase 1; NFAT: Nuclear factor of activated T cells; PARP: Poly (ADP-ribose) polymerase; CCR9: C-C motif chemokine receptor 9; MAdCAM-1: Mucosal vascular addressin cell adhesion molecule 1; ICAM-1: Intercellular adhesion molecule 1; S1P: Sphingosine-1-phosphate; SMAD: SMAD family member; Treg: Regulatory T cells.

Table 3 Current therapeutic approaches in inflammatory bowel diseases focused on intestinal epithelial cells

Effects on IECs	Drug type	Drug name/approach	Mechanism of action	Ref.
Promotes physical barrier functions (by increasing proliferation and survival of IECs)	Anti-IL-22BP	Infliximab or adalimumab	Anti-TNF-α therapy may block the expression of IL-22BP, the endogenous inhibitor of IL-22 (IL-22 is known to promote IEC proliferation via STAT3 activation in ISCs)	[108-111]
	ER stress reducers	TUDCA, PBA	Reduce apoptosis by increasing protein folding capacity of ER	[112]
	Human recombinant EGF	Enema	Promotes proliferation and reduces apoptosis of ISCs	[113,114]
Promotes physical barrier functions (by promoting expression and assembly of TJ proteins)	Inhibitor of para-cellular permeability	AT-1001	Inhibits TJ disassembly by interfering with cytoskeletal rearrangement	[115]
	Vitamin D	Calcitriol, vitamin D3 analogue	Induces expression of TJ proteins and E-cadherin	[117,179]
	Sex hormone	Estradiol, diarylpropionitrile (estrogen receptor-β agonist)	Induces expression of TJ proteins (occludin, junctional adhesion molecule-A)	[118]
	AMPK activator	Metformin	Induces expression and assembly of TJ proteins via AMPK dependent pathway	[119]
Promotes biochemical barrier functions (by promoting mucus secretion or production)	PGE2 receptor (subtype EP4) agonist	KAG-308, AGN205203	Promotes mucus secretion and proliferation of goblet cells (Promotes proliferation and reduces apoptosis of IECs)	[120,123,145,180,181]
	Short chain fatty acid	Butyrate	Stimulates mucin production by HDAC inhibitor activity	[121,124]
	Recombinant anti-microbial peptide	Cathelicidin	Stimulates mucin production by MAPK dependent pathway	[122,182]
	Phosphatidylcholine (PC)	LT-02	Mucus component supplement for UC patients who have reduced PC in rectal mucus	[125,126,183]
Promotes biochemical barrier functions (by promoting anti-microbial peptide secretion or production)	TLR7 ligand	Imiquimod	Promotes anti-microbial peptide (defensin) production in IECs	[127]
	Probiotics	Escherichia coli Nissle 1917	Promotes anti-microbial peptide (defensin) production in IECs by NF-kB activation	[128,184]
Corrects innate immune barrier dysfunctions (by inhibiting the retention of T cells in inflamed site)	Gut specific anti-integrin	Etrolizumab (anti-β7)	Blocks the interaction between αEβ7 integrin on T cells with E-cadherin on IECs (Neutralizing antibody against integrin)	[129]

IECs: Intestinal epithelial cells; IL-22BP: Interleukin-22 binding protein; STAT3: Signal transducer and activator of transcription 3; ISCs: Intestinal stem cells; ER: Endoplasmic reticulum; TUDCA: Tauroursodeoxycholate; PBA: 4-phenyl butyrate; EGF: Epidermal growth factor; TJ: Tight junction; AMPK: Adenosine monophosphate-activated protein kinase; PGE2: Prostaglandin E2; EP4: Prostaglandin E2 receptor 4; HDAC: Histone deacetylase; MAPK: Mitogen activated protein kinase; UC: Ulcerative colitis; TLR7: Toll-like receptor 7; NF-kB: Nuclear factor kappa B; IECs: Intestinal epithelial cells.