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The importance of Reg4 in the response of the colonic epithelium to inflammation

The regenerating islet-derived gene (Reg) family was initially identified by Okamoto and co-workers in a cDNA library prepared from rat regenerating islet cells [Terazono et al., Diabetolgia 33, 250-252: 1990]. Subsequently, homologous genes were identified in mouse and human.

Expression of Reg family members has been shown to be associated with:

  • cell survival and proliferation [1,2]
  • poor prognosis in a range of human cancers [3,4]
  • chemo- and radio-resistance of tumour cells [5,6].

Some of these activities are associated with the activation of specific signalling pathways, such as the EGFR/AKT/AP‑1 pathway [7]. Reg proteins have also been shown to act as antimicrobial peptides, like Defensins, with human Reg3α being shown to form pores in bacterial membranes [8].

Reg family proteins are members of the C‑type (calcium-dependent) lectin family [9]. C‑type lectins possess N‑terminal carbohydrate-recognition domains which allow them to bind glycans, including glycosylated hormones and proteins derived from microorganisms. There are many different types of C‑type lectin, most of which also possess a transmembrane domain linking the extracellular carbohydrate-recognition domain to intracellular signalling machinery, including type IV C‑lectins (the Selectins) and type V C‑lectins (NK cell receptors). Some C‑type lectins are secreted proteins, lacking the transmembrane domain; these include the Reg proteins (type VII C‑lectins) and Eosinophil Major Basic Protein (type XII C‑lectin).

Reg4, which is the subject of this Spotlight article, was recently identified as a marker for enteroendocrine and Paneth cells in the mouse small intestine [10] and subsequently, as a marker of deep crypt secretory (DCS) cells in the colon [11], which are important for providing the niche environment and maintaining stem cell numbers in the colonic epithelial crypts.

Xiao and co-workers have now presented evidence in a publication earlier this year that Reg4 gene expression can modulate intestinal inflammation and that this is associated with changes in the colonic microbiome [12]. In their initial studies, the authors examined the expression of Reg4 gene and protein expression in a cohort of paediatric patients (mean age, 6 months) with Intestinal Failure (IF), in mice treated with dextran sulphate (DSS) to induce colitis and in monolayers of the human colorectal cell line, Caco2, treated with the inflammatory stimulus, lipopolysaccharide (LPS; 100 μg/ml). In each case, it was demonstrated that inflammation was associated with increased Reg4 gene and protein expression. In DSS-treated mice and IF patients, expansion of Reg4-positive cells was observed in both small and large intestinal crypts, which was co-incident with the expression of MUC2 and activation of the transcription factor, STAT3 (phospo‑STAT3). Data from Caco2 cells transfected with a Reg4 reporter vector, provided evidence that Reg4 gene expression could also be driven by the activation of the transcription factor, ATF2.

In order to further characterise the role of Reg4 in the inflammatory response of the intestinal mucosa, a conditional knockout mouse was generated, with specific deletion of Reg4 expression in intestinal epithelial cells. The Reg4ΔIEC mice did not display any overt harmful phenotype, although they demonstrated reduced body weight gain, relative to Reg4fl/fl control mice. Reg4ΔIEC mice also showed reductions in the number of goblet cells and proliferative cells within the crypts and in particular, significantly reduced expression of phospho‑STAT3 and intestinal alkaline phosphatase (Alpi). Despite these reductions, the authors reported no significant change in small intestinal villus height or number of crypts per unit length; however, an obvious reduction in the length of the mucosal folds in the proximal large bowel was evident in H&E-stained sections.

Following treatment with DSS, Reg4fl/fl control mice demonstrated body weight loss and inflammation of the colonic mucosa, as expected. Accompanying these changes were the increased expression of genes coding for pro-inflammatory cytokines (such as Ifng, Il1b, Il6, Il22 and Tnf) and chemokines (Ccl28, Cx3cl1 and Cxcl2). An epithelial regenerative response was associated with the mucosal damage, with increased expression of stem cell-associated genes (such as Lrg5) and markers of proliferation (Mki67). In contrast, Reg4ΔIEC mice showed no body weight loss and less damage to the colonic mucosa (c.40% reduction) following DSS treatment. This amelioration of DSS-induced colitis was associated with no increase in the relative expression of genes coding for pro-inflammatory cytokines, chemokines, or markers of stem cells or proliferative cells. Colonic lysates from Reg4ΔIEC mice demonstrated barely detectable levels of Reg4 or phospho‑STAT 3 by Western blotting, whereas Reg4fl/fl control mice had shown strong expression of both proteins. Although intestinal alkaline phosphatase was expressed at much lower levels (at both RNA and protein level) in Reg4ΔIEC mice, ALPI gene expression was still inducible by DSS treatment. This retained ability for induction of ALPI could be important for the health phenotype of Reg4ΔIEC mice, as inactivating mutations in ALPI have been associated with inflammatory bowel disease in humans, with the enzyme's role in the detoxification of LPS being hypothesised as a critical factor in modulating disease risk [13].

Xiao and co-workers were keen to investigate the effect of Reg4 status on the colonic microbiome, given the role of other C‑type lectins, such as Dectins 1 and 2, Mincle (macrophage inducible calcium-dependent lectin receptor) and other Reg proteins, in host-microbiome interactions. Bacterial cultures established from homogenised colon tissue obtained from both Reg4ΔIEC and Reg4fl/fl mice demonstrated some obvious differences in the abundance of different bacterial families (based on 16S rRNA analysis), with Reg4ΔIEC mice showing reductions in both Gram-positive and Gram-negative organisms, with the largest relative reductions in Escherichia, Lactobacillus and Prevotella; however, overall bacterial load in the colon was not altered. Immunofluorescent staining of colonic sections from DSS-treated mice appeared to demonstrate that bacteria were less closely associated with the mucosal surface in Reg4ΔIEC mice, as compared to Reg4fl/fl control mice, in which bacteria were found adjacent to the epithelium and showed co-incident Reg4 immunoreactivity.

Smaller changes in the relative size of different bacterial families were observed in the faeces of Reg4ΔIEC and Regfl/fl mice. Reductions in Staphylococcus, Eschericia and Proteus were observed for Reg4ΔIEC mice, together with increases in Bifidobacterium and Clostridium. In contrast, use of an in vitro bacteria:intestinal cell binding assay revealed increased levels of adherent bacteria in the faecal samples from Reg4ΔIEC mice, including Lactobacillus and Staphylococcus, two of the three bacterial families that showed the greatest reduction in mucosal association in these mice. In this assay, the adherence of the different bacterial families could be inhibited by treatment of the Caco2 cells with Reg4 siRNA, except for Bacteroides, which required addition of exogenous Reg4 protein to the cultures to modulate their adherence.

Finally, the authors looked at the growth of intestinal organoids derived from the colonic mucosa of both Reg4ΔIEC and Regfl/fl mice, in order to gain insight into the importance of Reg4 for epithelial regeneration. It was found that fewer organoids could be established from colonic crypts isolated from the Reg4ΔIEC mice; the organoids from the knockout mice also appeared smaller and less branched in photomicrographs of the cultures. Complementary to this, Reg4ΔIEC organoids demonstrated reduced expression of the intestinal stem cell marker, Lgr5, and showed a lower level of cell proliferation (determined by EdU-incorporation and expression of Mki67). Addition of exogenous Reg4 protein to organoid cultures from either Reg4ΔIEC or Regfl/fl mice, resulted in increased numbers of organoids, enhanced EdU incorporation and increased levels of Lgr5 and Mki67. This effect of exogenous Reg4 was blocked by treatment with the STAT3 inhibitor, Stattic.

In summary, the secreted C‑type Lectin, Reg4, has previously been identified as a marker of deep crypt secretory cells in the colon, which are important in defining the stem cell niche in colonic epithelium. Expression of Reg4 has also been shown to promote cell survival and proliferation via activation of specific signalling pathways, and is a prognostic indicator in various cancers. In the new publication from Xiao and colleagues, their generation of a conditional knockout mouse allowed them to demonstrate that the upregulation of, or treatment with Reg4 was associated with the following activities:

  • the maintenance and proliferation of colonic organoid cultures;
  • the epithelial response to mucosal injury in an in vivo model of chemically-induced acute colitis (DSS) and in paediatric patients with Intestinal Failure;
  • the response of intestinal epithelial cells to an inflammatory stimulus (LPS) in vitro.

In each of the first two cases, the action of Reg4 was allied to the activation of STAT3 and upregulation of the stem cell marker, Lgr5; in the third, Reg4 expression was associated with the activation of ATF2. It is not clear at this stage, precisely how Reg4 interacts with cellular signalling machinery. Does it have a direct signalling role, or does is modulate signalling by other paracrine or autocrine factors in the mucosa?

Both in vivo and in vitro studies showed that Reg4 status could modulate the adherence of different bacterial families to colonic epithelial surfaces and change the abundance of the different microbiome components in the faecal compartment. This is clearly an important effect in vivo, that may change important host-microbiome interactions and have implications for mucosal function, as perhaps illustrated by the altered sensitivity of the Reg4ΔIEC mice to DSS.

References

[1] Zhao D, Kim YH, Jeong S, Greenson JK, Chaudhry MS, Hoepting M, Anderson ER, van den Brink MR, Peled JU, Gomes AL, Slingerland AE, Donovan MJ, Harris AC, Levine JE, Ozbek U, Hooper LV, Stappenbeck TS, Ver Heul A, Liu TC, Reddy P and Ferrara JL. J Clin Invest. 128, 4970-4979: 2018 DOI: 10.1172/JCI99261

[2] Rafa L, Dessein AF, Devisme L, Buob D, Truant S, Porchet N, Huet G, Buisine MP and Lesuffleur T. Int J Oncol. 36, 689-699: 2010. DOI: 10.3892/ijo_00000544

[3] Sasaki Y, Minamiya Y, Takahashi N, Nakagawa T, Katayose Y, Ito A, Saito H, Motoyama S and Ogawa J. Ann Surg Oncol. 15, 32244-3251: 2008. DOI: 10.1245/s10434-008-0137-2

[4] Astrosini C, Roeefzaad C, Dai YY, Dieckgraefe BK, Jöns T and Kemmner W. Int J Cancer 123, 409-413: 2008. DOI: 10.1002/ijc.23466

[5] Eguchi H, Ishikawa O, Ohigashi H, Takahashi H, Yano M, Nishiyama K, Tomita Y, Uehara R, Takehara A, Nakamura Y and Nakagawa H. Pancreas 38, 791-798: 2009. DOI: 10.1097/MPA.0b013e3181ac5337

[6] Kobunai T, Watanabe T and Fukusato T. Anticancer Res. 31, 4147-41: 2011. http://ar.iiarjournals.org/content/31/12/4147.long

[7] Bishnupuri KS, Luo Q, Murmu N, Houchen CW, Anant S and Dieckgraefe BK. Gastroenterol. 130, 137-149: 2006. DOI: 10.1053/j.gastro.2005.10.001

[8] Mukherjee S, Zheng H, Derebe MG, Callenberg KM, Partch CL, Rollins D, Propheter DC, Rizo J, Grabe M, Jiang QX and Hooper LV. Nature 505, 103-107: 2014. DOI: 10.1038/nature12729

[9] Brown GD, Willment JA and Whitehead L. Nature Rev Immunol. 18, 374-389: 2018. DOI: 10.1038/s41577-018-0004-8

[10] Grün D, Lyubimova A, Kester L, Wiebrands K, Basak O, Sasaki N, Clevers H and van Oudenaarden A. Nature 525, 251-273: 2015. DOI: 10.1038/nature14966

[11] Sasaki N, Sachs N, Wiebrands K, Ellenbroek SI, Fumagalli A, Lyubimova A, Begthel H, van den Born M, van Es JH, Karthaus WR, Li VS, López-Iglesias C, Peters PJ, van Rheenen J, van Oudenaarden A and Clevers H. Proc. Natl. Acad Sci USA 113, E5399-E5407: 2016. DOI: 10.1073/pnas.1607327113

[12] Xiao Y, Lu Y, Wang Y, Yan W and Cai W. Mucosal Immunol. 12, 919-929: 2019. DOI: 10.1038/s41385-019-0161-5

[13] Parlato M, Charbit-Henrion F, Pan J, Romano C, Duclaux-Loras R, Le Du MH, Warner N, Francalanci P, Bruneau J, Bras M, Zarhrate M, Bègue B, Guegan N, Rakotobe S, Kapel N, De Angelis P, Griffiths AM, Fiedler K, Crowley E, Ruemmele F, Muise AM and Cerf-Bensussan N. EMBO Mol Med. 10, e8483: 2018. DOI: 10.15252/emmm.201708483

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