PROGRESS REPORT for Mizutani Foundation Research Grant

Reference Number: 170133
Principal Investigator: Hai-Bin Ruan, PhD
Organization: University of Minnesota
Period: April 1, 2017 through March 31, 2018
Grant Title: Gut microbiota-sensitive O-GlcNAc signaling protects intestinal
inflammation
Progress Report:(a) Abstract:
Inflammatory bowel disease (IBD) is a result ofinteractions between genes and environment. It is
well-recognized that commensal microbiota-derived
signals help maintain barrier function and restrain
inflammation in the intestinal epithelium. In thisproject, we aimed to defining protein O-linked
N-acetylglucosamine (O-GlcNAc) as a novel
mechanism by which intestinal epithelium senses
and integrates microbial signals in health and disease.
We demonstrated that protein O-GlcNAcylationlevels in intestinal epithelial cells (IECs) was
dependent on gut microbiota-derived short-chain fatty acids (SCFAs). In IECs ofhuman IBD patients, levels of protein O-GlcNAcylation and the expression of
O-GlcNAc transferase (OGT), the enzyme adding the O-GlcNAc moiety, werereduced and negatively correlated with disease severity. Deletion of OGT
specifically in IECs in mice resulted in disrupted epithelial barrier, microbial
dysbiosis, Paneth cell dysfunction, and intestinal inflammation. Using fecal
microbiota transplantation in mice, we found that microbial dysbiosis although wasinsufficient to induce spontaneous inflammation but exacerbated chemical-inducedcolitis. Paneth cell-specific deletion of OGT leaded to Paneth cell dysfunction,
which might predispose mice to chemical-induced colitis. On the other hand, theaugmentation of O-GlcNAc signaling by inhibiting O-GlcNAcase, the enzymeremoving O-GlcNAcylation, alleviated chemical-induced colitis. Our data reveals
that protein O-GlcNAcylation in IECs controls key regulatory mechanisms tomaintain mucosal homeostasis.
(b) Objectives:
We hypothesized that, in response to microbiota-derived SCFAs, epithelial
O-GlcNAc maintains intestinal homeostasis by strengthening the barrier function.

We proposed the following specific aims: Aim 1, determine the role of O-GlcNAc
in microbiota-host interactions; Aim 2, assess the effects of elevated intestinal
O-GlcNAc levels on inflammation.
(c) Methods used:
In Aim 1, we treated cultured Caco-2 cells and wildtype mice with SCFAs todetermine if O-GlcNAcylation in IECs is a metabolic sensor for microbial SCFAs.
In Aim 2, we pharmacologically elevated protein O-GlcNAcylation by TMG andgenetically overexpressed OGT specifically in IECs in mice to test if mice wereprotected from chemical-induced colitis.
(d) Results obtained, including negative data:
In Caco-2 cells, we found that SCFAs could not directly increase OGT expression
or global O-GlcNAcylation, suggesting an indirect effect. It is known that SCFAs
are important for immune development and function in the intestinal mucosa, andwe tested several immune cytokines and found that IL-4 could activate OGT andincrease protein O-GlcNAcylation, indicating that microbiota control epithelial
O-GlcNAcylation by indirectly activate the type 2 immune responses.
To determine whether pharmacologically elevated protein O-GlcNAcylation
strengthens barrier function and protects mice from chemical-induced acute colitis,
we orally administered water or TMG, an OGA inhibitor, to C57Bl/6 mice,followed by 2.5% DSS in drinking water. TMG-treated mice lost less body weightand had lower colitis score. In vivo barrier functional assay showed that TMG
treatment reduced intestinal permeability before and after DSS induction.
TMG-treated mice also had longer colon length, accelerated mucosal recovery, andlower levels of inflammatory genes. These data demonstrated that augmenting
global protein O-GlcNAcylation by TMG pretreatment ameliorates
chemical-induced acute colitis. We also obtained IEC-specific OGT overexpression
mice; however, we could not observe any significant increase in protein
O-GlcNAcylation, thus prevented us using the model to test our hypothesis.
In addition, we proposed microbial dysbiosis and Paneth dysfunction observed in
IEC-OGT KO mice precipitated intestinal inflammation cooperatively. Using fecal
microbiota transplantation in mice, we demonstrated that microbial dysbiosis
although was insufficient to induce spontaneous inflammation but exacerbatedchemical-induced colitis. Paneth cell-specific deletion of OGT leaded to Paneth
cell dysfunction, which might predispose mice to chemical-induced colitis.
(e) Discussion:

Here we found that protein posttranslational O-GlcNAc modification in IECs wassensitive to microbiota-derived SCFAs and was compromised in human IBD.
IEC-specific knockout of OGT leaded to intestinal damage and inflammation inmice. O-GlcNAcylation was a regulator of multiple homeostatic modules in theepithelium. Microbial dysbiosis and Paneth cell dysfunction together potentiatedchemical-induced inflammation. Our study will shed light on the future design ofnovel preventions and therapeutics for IBD.
(f) List of publications: Zhao M*, Xiong XW*, Ren K, Xu B, Cheng M, Sahu C, Wu K, Nie Y,Huang Z, Blumberg RS, Han X, Ruan HB. (2018). Deficiency in intestinal
epithelial O-GlcNAcylation predisposes to gut inflammation. EMBO
Molecular Medicine. In press. (*, Equal contribution).


Huang Y, Lia SC, Hu J, Ruan HB, Guo HM, Zhang HH, Wang X, Pei YF,
Pan Y, Fang C. (2018). Gut Microbiota Profiling in Han Chinese with Type
1Diabetes. Diabetes
ResearchandClinical
Practice.
doi: 10.1016/j.diabres.2018.04.032.

(g) Name (signed) & Date:

Hai-Bin Ruan, Ph.D.
June 16, 2018