The relationship between epithelial cell fate, microbiota composition and inflammation in the intestine

Keratins and intestinal inflammation

The epithelial cytoskeleton of intermediate filaments is composed of keratins; filaments are formed through the non‑covalent, heteropolymerisation of type I (keratins K9 to K20) and type II (keratins K1 to K8) keratins. The main keratin proteins in the intestinal epithelium are K19 and K8. The importance of the intermediate filament network was illustrated by the gene targeting experiments of Baribault and Oshima, who showed that inactivation of the Krt8 gene was embryonic lethal for some mouse strains (C57BL/6) [1], whereas other strains (FVB/n) demonstrated viability and went on to develop colitis [2].

The type II keratin genes are located within the IBD2 locus at chromosome 12q, one of the multiple risk loci for inflammatory bowel disease (IBD) identified in humans. In patients with IBD, point mutations in KRT8 have been identified that result in a single amino acid substitution; for example, K8(G62C). In an elegant series of in vitro studies, Owens and co‑workers showed that mutant Keratins, such as K8(G62C), demonstrate a reduced rate of heterodimerisation with the type I Keratin, K18 [3]. Also, Keratin heterodimers involving K8(G62C) took longer to assemble into polymer filaments, initially forming more aggregate structures. In part, this reduced rate of filament assembly is attributable to the property of mutant K8(G62C) molecules to homodimerise, as shown in cells transfected with K8(G62C) and exposed to oxidative stress. The in vivo consequences of K8 mutation could be a general disruption of the ability of the Keratin filament network to support epithelial cell responses to cellular stress/injury.

The occurrence of spontaneous colitis in K8-/- FVB/n mice was further investigated by Habtezion and colleagues [4]. The observed colonic pathology in these mice was associated with the infiltration of CD4+ T cells into the mucosa and a Th2 cytokine profile, with increased expression of IL‑4, IL‑5 and IL‑13. The colitis occurs despite apparently normal tight junction permeability and paracellular transport in the K8-/- mice, although they do demonstrate abnormal ion transport [5].

K8 and dysbiosis

Heterozygous, K8+/- mice do not demonstrate a colitis phenotype; however, it has been recently shown by Lui and co-workers that these mice are more susceptible to colitis and chronic colitis‑associated carcinogenesis, induced by treatment with Dextran Sulfate (DSS) or azoxymethane (AOM) and DSS, respectively [6]. K8+/- mice demonstrated more acute weight loss, greater colon shortening and more mucosal damage in response to DSS treatment than wild-type (wt) control mice. These effects were associated with higher levels of pro‑inflammatory cytokine gene transcripts and greater colonic permeability, although in the absence of stress (DSS or AOM/DSS chemical insult), cytokine levels and permeability were almost identical in K8+/+ wt and K8+/- heterozygous mice. Changes in faecal microbiota were also examined in the model of chronic colitis‑associated carcinogensis. Identical species abundance was observed in healthy wt and heterozygous mice; however, following AOM/DSS treatment, K8+/- mice demonstrated increased abundance of Firmicutes and Proteobacteria and decreased Bacteroidetes and Verrucomicrobia, relative to wt mice. Through administration of either antibiotics (broad spectrum cocktail) or the TLR‑4 antagonist, TAK‑242, the researchers were able to provide evidence to support the hypotheses that the more severe colitis symptoms seen in K8+/- mice were driven by TLR‑4 signalling, and that the increased tumour burden observed in the carcinogenesis model was microflora‑dependent. In both the acute colitis and chronic colitis/carcinogenesis settings, data was obtained which showed increase levels of NF‑κB transcriptional activity. In conclusion, under conditions of stress, K8 deficiency was associated with dysbiosis in the colon, with an associated increase in TLR‑4 signalling, driving the expression of NF‑κB transcriptional targets, including pro‑inflammatory cytokines.

K8, Notch1 signalling and epithelial cell fate

Lähdeniemi and co-workers have shown another important role for keratins in regulating epithelial cell fate in the large bowel, via Notch1 signalling [7]. The authors demonstrated that K8/K18 co‑immunoprecipitated with Notch1 via its intracellular domain (NICD), using both lysates derived from colon tissue and from a genetically‑modified cell line. Co‑localisation of K8 and Notch1 was visualised by immunofluorescence microscopy in colon and breast cancer cell lines. K8/K18 were also shown to stabilise cellular levels of NICD and enhance the expression of Notch target genes. Complementary to these findings, K8-/- mice demonstrated reduced levels of full‑length Notch (FLN) and NICD, and reduced transcript levels of Notch target genes. Identical data were generated using CRISPER/Cas9 to specifically delete KRT8 in Caco‑2 cells, which were then be re‑transfected with K8 and K18 to reverse the phenotype.

In vivo, Notch and Wnt signals are integrated to provide balanced cellular output from intestinal stem cell populations [8] [9] [10], and maintain mucosal epithelial architecture and function. Notch signalling is known to regulate the activity of the bHLH transcription factors, Hes1 and Math1. In the presence of Notch signalling, Hes1 predominates, promoting the commitment of proliferative transit amplifying cells in the intestinal crypts to being absorptive enterocytes; in contrast, in the absence of Notch, Math1 drives commitment towards a secretory cell lineage i.e., goblet cell or enteroendocrine cell, or (in the small bowel) Paneth cell (dependent on other, integrated transcriptional events). Canonical Wnt signalling stabilises cellular β‑catenin, which can subsequently form active transcriptional complexes with TCF/LEF proteins (such as TCF4), driving the expression of Wnt targets governing cell cycle regulation, differentiation and survival. Blocking Wnt signalling inhibits secretory lineage commitment.

In K8-/- mice, an increase in goblet cell and enteroendocrine cell markers (alcian blue staining and synaptophysin expression, respectively) were observed in colonic crypts, together with decreased absorptive enterocyte markers (carbonic anhydrase and villin). Accompanying these changes in cell fate, there was an increase in the number of proliferative transit amplifying cells. These finding presumably reflect the increased Wnt signalling influence on the crypt epithelial cells, in the environment of decreased Notch signalling associated with the K8 deficiency.

Notch signalling and dysbiosis

The relationship between aberrant Notch signalling and dysbiosis (both features of mice with K8 insufficiency) was defined in a recent publication from Guo and colleagues [11], in studies on mice deficient in the Notch1 target gene, Hes1. Like K8+/- mice, the Hes1‑knockout (Hes1‑KO) mice demonstrated dysbiosis in the terminal ileum and colon, in samples of both mucosal‑associated bacteria and bacteria isolated from flushed stool. A decrease in Bacteroidetes was observed (and in some animals, Firmicutes), which was specific to the terminal ileum; in the colon, an increase in Verrucomicrobia was observed. At the bacterial species level, the changes included increases in E. coli (a Proteobacteria: both terminal ileum and colon) and A. mucinophilia (a Verrucomicrobia: colon only). Mice with targeted deletion of Hes1 in intestinal epithelial cells were employed to demonstrate that the observed dysbiosis was specifically dependent on altered epithelial cell function. Hes1‑KO mice showed reduced expression three known Hes1 target genes coding for antimicrobial peptides (Saa1, Reg3b, Reg3g) in the terminal ileum (but not colon); however, their expression was still induced by oral administration of LPS. The authors went on to demonstrate the enhanced colonisation of Hes1‑KO mice intestine by two pathogenic bacterial species: C. rodentium and E. coli O157:H7. Also, in common with K8‑deficient mice, an increase in the severity of DSS‑induced colitis was shown; in addition, faecal‑microbiota transplant from Hes1‑KO mice to wt mice enhanced the severity of DSS‑induced colitis in the wt mice. The other main similarity to K8‑deficient mice observed was an increase in the size of the transit amplifying cell population and goblet cell hyperplasia, with associated mucin overproduction. Guo and colleagues hypothesised that, in part, that dysbiosis was a consequence of the altered mucosal environment resulting from the increased mucin.

Increased Notch signalling in intestinal epithelial cells is also associated with the development of spontaneous colitis in mice with dendritic cell‑specific deletion of the TGF‑β receptor II subunit (CD11c‑cre Tgfbr2fl/fl mice), as shown by Ihara and co‑workers [12]. These mice showed an opposite phenotype to that described for Hes1‑KO and K8-/- mice, with goblet cell depletion and an upregulation of the Th1 cytokines, TNF‑α and IFN‑γ [12]. In common with K8+/- heterozygote mice, CD11c‑cre Tgfbr2fl/+ mice did not have a colitis phenotype, but deletion of the single Tgfbr2 allele did confer increased sensitivity to DSS‑induced colitis, relative to wt mice. Analysis of faecal bacteria from CD11c‑cre Tgfbr2fl/fl mice and DSS-treated CD11c‑cre Tgfbr2fl/+ mice showed, in each case, that colitis was associated with increased numbers of Enterobacteriaceae family bacteria (in particular, E. coli). Increased amounts of E. coli were also demonstrated at the luminal surface of the colonic epithelium; in wt mice, E. coli were unable to penetrate the mucus layer above the epithelium. The authors demonstrated that the Notch ligand genes, Jag1 and Jag2, and the Notch target gene, Hes1, were upregulated in colon tissue lysates from CD11c‑cre Tgfbr2fl/fl mice. The presence of CD11c+ DCs showing Jagged1 and Jagged2 immunoreactivity, in close juxtaposition with colonic epithelial cells, was confirmed by immunofluorescent labelling; increased numbers of Hes1‑positive epithelial cells were also confirmed within the proliferative zone of the colonic crypts.

The secondary proteolytic processing of Notch by γ‑secretase (following ligand binding), to yield NICD, can be inhibited by dibenezepine (DBZ). Treatment of CD11c‑cre Tgfbr2fl/fl mice with DBZ partly reversed their colitic phenotype, with decreased crypt hyperplasia and increased goblet cell number and mucin secretion. DBZ also ameliorated DSS‑induced colitis in CD11c‑cre Tgfbr2fl/+ mice, attenuating body weight loss and inhibiting mucosal ulceration. The authors hypothesised that TGF‑β1 production and activation (principally by epithelial and dendritic cells [13]) within the intestinal mucosa is promoted by the gut microbiota. Subsequently, TGF‑βR signalling in DCs suppresses their expression of the Notch1 ligands, Jagged1 and Jagged2, thus modifying Notch1 signalling in adjacent intestinal epithelial cells. This permits epithelial cells to adopt a secretory lineage fate, maintaining production of mucin and anti‑microbial peptides. Absence of adequate TGF‑βR signalling decreases the production of these mucosal defences, altering the luminal environment local to the apical surface of the epithelium, resulting in dysbiosis and inflammation.

Summary

Keeping a balance in the network of signals between the mucosal epithelium, the mucosal immune system and the intestinal microbiota is essential in order to maintain epithelial homeostasis. The highlighted research illustrates the opposing but complementary roles played by the Notch and Wnt signalling pathways in the epithelium, and how genetic defects in other cell types, and in non‑signal transduction proteins within epithelial cells themselves, impact on Notch/Wnt‑directed cell fate.

References

[1] Baribault H, Price J, Miyai K and Oshima RG. Mid-gestational lethality in mice lacking keratin 8. Genes Dev. 7, 1191-202 (1993). doi:10.1101/gad.7.7a.1191

[2] Baribault H, Penner J, Iozzo RV and Wilson-Heiner M. Colorectal hyperplasia and inflammation in keratin 8-deficient FVB/N mice. Genes Dev. 8, 2964-73 (1994). doi:10.1101/gad.8.24.2964

[3] Owens DW, Wilson NJ, Hill AJM, Rugg EL, Porter RM, Hutcheson AM, Quinlan RA, van Heel D, Parkes M, Jewell DP, Campbell SS, Ghosh S, Satsangi J and Lane EB. Human keratin 8 mutations that disturb filament assembly observed in inflammatory bowel disease patients. J Cell Sci. 117, 1989-1999 (2004). doi:10.1242/jcs.01043

[4] Habtezion A, Toivola DM Butcher EC and Omary MB. Keratin-8-deficient mice develop chronic spontaneous Th2 colitis amenable to antibiotic treatment. J Cell Sci. 118, 1971-1980 (2005). doi:10.1242/jcs.02316

[5] Toivola DM, Krishnan S, Binder HJ, Singh SK and Omary MB. Keratins modulate colonocytes electrolyte transport via protein mistargeting. J Cell Biol. 164, 911-921 (2004). doi:10.1083/jcb.200308103.

[6] Liu C, Liu E-D, Meng Y-X, Dong X-M, Bi Y-L, Wu H-W, Jin Y-C, Zhao K, Li J-J, Yu M, Zhan Y-Q, Chen Hui, Ge C-H, Yang Z-M and Li C-Y. Keratin 8 reduces colonic permeability and maintains gut microbiota homeostasis, protecting against colitis and colitis-associated tumorigenesis. Oncotarget. 8, 96774-96790 (2017). doi:10.18632/oncotarget.18241

[7] Lähdeniemi IAK, Misiorek JO, Antila CJM, Landor SK-J, Stenvell C-GA, Fortelius LE, Bergström LK, Sahlgren C and Toivola DM. Keratins regulate colonic epithelial cell differentiation through the Notch1 signalling pathway. Cell Death Differ. 34, 984-996 (2017). doi:10.1038/cdd.2017.28

[8] Yang Q, Bermingham NA, Finegold MJ and Zoghbi HY. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine. Science. 294, 2155-2158 (2001). doi:10.1126/science.1065718

[9] Tian H, Biehs B, Chiu C, Siebel C, Wu Y, Costa M, de Sauvage FJ and Klein OD. Opposing activities of Notch and Wnt signaling regulate intestinal stem cells and gut homeostasis. Cell Rep. 11, 33–42 (2015). doi:10.1016/j.celrep.2015.03.007

[10] Buske P, Galle J, Barker N, Aust G, Clevers H and Loeffler M. A comprehensive model of the spatio-temporal stem cell and tissue organisation in the intestinal crypt. PLoS Comput Biol. 7, e1001045 (2011). doi: 10.1371/journal.pcbi.1001045

[11] Guo X-K, Ou J, Liang S, Zhou X and Hu X. Epithelial Hes1 maintains gut homeostasis by preventing microbial dysbiosis. Mucosal Immunol. Advance online publication 3 January 2018. doi:10.1038/mi.2017.111

[12] Ihara S, Hirata Y, Serizawa T, Suzuki N, Sakitani K, Kinoshita H, Hayakawa Y, Nakagawa H, Ijichi H, Tateishi K and Koike K. TGF-β Signaling in Dendritic Cells Governs Colonic Homeostasis by Controlling Epithelial Differentiation and the Luminal Microbiota. J Immunol. 196, 4603-13 (2016). doi:10.4049/jimmunol.1502548

[13] Bauché D and Marie J. Transforming growth factor β: a master regulator of the gut microbiota and immune cell interactions. Clin Transl Immunology. 6, e136 (2017). doi: 10.1038/cti.2017.9

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