The epithelium has many functions, not least of which are digestion, secretion, and absorption. The intestinal epithelium forms tight junctions between enterocytes that can exclude small molecules, thus forming a mucosal barrier to entry of most substances. Tight junctions are regulated by cytokines and other stimuli and dynamically respond to the local microenvironment. This exclusion of nutrients and small molecules is usually referred to as barrier function, however epithelial barrier function subsumes a broader role in that the epithelium also forms a barrier to the microbiota in the lumen. Positioned between the microbiota in the lumen and the immune cells in the intestinal lamina propria, the epithelium communicates with and is signaled by both. For example, the microbiota signals enterocytes as well as innate cells in the lamina propria via pattern recognition molecules via receptors such as the TLR, NOD, etc. In fact, such microbiota signaling via TLRs is required for normal epithelial organization and resistance to injury.34
Immune cytokines such as IFNγ, IL-17, and IL-22 have substantial effects on the epithelium. In turn, the epithelium secretes certain mediators, such as TSLP, IL-33, and IL-25, that “instruct” intestinal dendritic cells to become tolerogenic, i.e., to preferentially induce lamina propria Treg cells and thus maintain intestinal homeostasis.35
It should not be too surprising that genes linked to epithelial renewal, repair, stress response, and to antimicrobial peptide secretion have all been linked to IBD susceptibility in humans or mice.
The epithelial layer is renewed every 2–3 d by a process involving proliferation of epithelial cells in the crypts with migration up on the villi in the small intestine or onto the surface epithelium in the colon. The proliferation is, in turn, balanced by enterocyte apoptosis and shedding of the epithelium. Disruption of these processes impairs the epithelial barrier and results in chronic inflammation. NF-κB signaling in intestinal epithelial cells stimulates important anti-apoptotic molecules. Intestinal epithelial cell-specific deletion of NEMO (IKKγ) or of both IKKα and IKKß in mice results in apoptosis of colon epithelial cells, impaired expression of antimicrobial peptides, translocation of intestinal bacteria, and chronic colitis.36
Concomitant deficiency of MYD88 prevented the colitis due to a reduction in TNFα-induced apoptosis.36
A second molecule regulating epithelial apoptosis is A20 (TNFAIP3), an E3 ligase-editing enzyme required for inhibition of TLR and TNFα signaling. A20 gene variants have been linked to Crohn’s disease. Epithelial-specific deletion of A20 did not result in spontaneous colitis, but rendered the epithelial cells highly sensitive to TNFα-induced apoptosis and thus a high mortality upon challenge with DSS.37
Generalized deletion of A20 in mice results in spontaneous diffuse inflammation, including the intestine, resulting in cachexia and early death. This inflammation is dependent on microbiota-mediated homeostatic TLR-MYD88 signaling in myeloid cells.38
A SNP variant of the multi-drug resistance I gene (MDR1) has been linked to human IBD susceptibility.3
The homologous gene in mice, MDR1α, is expressed in intestinal epithelial cells and in some lamina propria myeloid cells. MDR1α, or p-glycoprotein, is an ATP binding cassette transporter that is postulated to pump toxic xenobiotic molecules out of epithelial cells and other cells, but its exact role in intestinal epithelial cells is not known. Mice deficient in MDR1α develop colitis spontaneously and antibiotic treatment prevents this colitis.39
T cells from MDR1 α colitic mice demonstrate increased responsiveness to the microbiota. Based on bone marrow chimera experiments, the disease is caused by MDR1α deficiency in non-hemopoietic, likely epithelial, cells making this an interesting example on which an epithelial cell defect results in an abnormal adaptive T-cell response to the microbiota.39
Colitis onset is accelerated by colonization with Helicobacter bilus
. Interestingly, co-infection with H. bilus
and murine norovirus 4 increased severity of the colitis, demonstrating an environment-microbiota-immune interaction leading to severe IBD.40
Variants in the gene encoding the transcription factor XBP1 have been linked to susceptibility to IBD in humans.3,33
XBP1 is part of the unfolded protein response (UPR), which, in turn, is important for the maintenance of cellular integrity, particularly of secretory epithelial cells. The UPR is activated by accumulation of unfolded or misfolded proteins in the endoplasmic reticulum. Conditional deletion of XBP1 in intestinal epithelial cells resulted in apoptosis of Paneth cells, reduced numbers of small intestinal goblet cells, and small intestinal inflammation.33
XBP1-deficient mice were more susceptible to DSS colitis, an effect that was dependent on the colonic microbiota.33
Human ileal and colonic biopsies from patients with IBD have signs of ER stress, demonstrating the relevance of this mouse model data for human IBD.
Another pathway that is activated by cell stress is autophagy.41
Autophagy is best known as a cellular process whereby a cell can recycle its components via the lysosomes, a process that is particularly important during starvation. However, autophagy is present widely in nature and provides host resistance to intracellular pathogens such as mycobacteria. Two autophagy genes, ATG16L1 and IRGM, are linked to Crohn’s disease.3
An ATG16L1 hypomorphic mouse line that expresses about 1% of the normal level of ATG16L1 demonstrates Paneth cell granule abnormalities that are very similar to those found in ileal resections in patients with Crohn’s disease who carry the relevant ATG16L1 gene variant.42
Although these ATG16L1HM
do not develop IBD themselves, they are more susceptible to DSS-induced injury. When ATG16L1HM
mice were re-derived virus-free, they lost the Paneth cell phenotype and the increased susceptibility to DSS-induced colitis. Infection with norovirus restored both the Paneth cell phenotype and the DSS colitis susceptibility, although the latter was prevented with treatment with broad spectrum antibiotics.43
This is another example of a virus-immune-microbiota interaction having a profound effect on disease expression. The pattern recognition receptor, NOD2, is highly expressed in Paneth cells and regulates their secretion of antimicrobial peptides.44
NOD2 has recently been found to regulate autophagy, including the autophagy pathway involving ATG16L145
linking these two IBD susceptibility loci. Moreover, ATG16L1 deficiency in mice has been found to increase endotoxin-induced inflammasome activation, resulting in high production of IL-1β and IL-18.46
In these studies, mice lacking ATG16L1 on in hematopoietic cells were highly susceptible to DSS colitis, which was mediated by IL-1β and IL-18. Thus, autophagy also appears to regulate also inflammasome-mediated inflammation.
Recent studies have identified the inflammasome as critical to resistance to innate injury with DSS and maintenance of homeostasis. Inflammasomes are multimeric proteins in the cytoplasm that form complexes that act as sensors of damage-associated molecular patterns and regulate the maturation and secretion of IL-1β and IL-18. NLRP3 inflammasomes, which include apoptosis-associated spec-like protein (ASC), activate caspase-1, which, in turn, cleaves pro-IL-1β and pro-IL-18 into their active forms. NLRP3 inflammasomes have been found to play an important role in resistance to DSS colitis. Deletion of NLRP3, ASC, caspase-1, or IL-18 all result in an increased severity and mortality to DSS colitis.47,48
NLRP3 gene variants have been linked to human IBD susceptibility, which may involve a similar mechanism.3
NLRP6-containing inflammasomes in colon enterocytes also provide resistance to DSS colitis, but surprisingly appear to regulate the composition of the colonic microbiota. Mice deficient in NLRP6 in colon enterocytes had reduced IL-18 levels and an altered fecal microbiota characterized by increases in the Provotellaceae
of the Bacteroidetes
phylum and increases in TM7.49
This microbiota conferred increased susceptibility to DSS colitis, which could be transferred to neonatal or adult wild type mice by co-housing. The mechanism for this shift in the microbiota and which members of the altered microbiota account for the increased DSS-colitogenicity are unknown. This highlights an important feature of DSS-induced injury, namely that DSS acts in concert with the microbiota to induce injury to the epithelial layer. The mechanism of such injury and the specific role of the microbiota in this process are unknown.