This study aimed to identify metabolic changes associated with modeled intestinal inflammation, particularly related to the epithelium. As previous work had demonstrated specific shifts in metabolism during inflammation, we reasoned that such changes could be reflected on a more global basis. An amalgamated approach using NMR-based metabolomics and transcriptional arrays identified shifts in methylation-dependent pathways as a major metabolic fingerprint. These studies significantly extend previous work related to inflammation-associated metabolism and identify changes in methylation as a target signature within the epithelium during mucosal inflammation. Evidence is provided that shifts in methylation associated with inflammation serve an endogenously protective role in murine colitis.
Ongoing mucosal inflammatory responses are characterized by significant shifts in tissue metabolism (10
). These changes include a shift toward a glycolytic phenotype and careful analysis has shown the development of significant hypoxia, termed “inflammatory hypoxia”, particularly prominent within the epithelium (53
). Coinciding with inflammation-associated hypoxia is the stabilization of HIF (32
), and given the central role of HIF in most metabolic processes (54
), we reasoned that overall metabolism would be changed. In support of this hypothesis, global analysis of metabolism using MRS identified significant changes in a number of cellular metabolites. Many of these alterations are in agreement with results from our lab indicating the presence of “inflammatory hypoxia”. These include increased intracellular adenosine, increased glucose uptake and intracellular glucose, increased intracellular lactate and lactate export, and increased turnover of ATP ( and ; Supplemental Figure 1
and Supplemental Tables 1 and 2
). Additionally, significant changes were observed in a number of lipid metabolites including monounsaturated fatty acids (MUFA), triacylglycerol (TAG), polyunsaturated fatty acids (PUFA), phosphatidyl choline, and phosphatidyl ethanolamine (Supplemental Tables 1 and 2
), which may be indicative of dynamic alterations in cellular membranes in response to inflammation. Importantly, this analysis revealed a distinct and specific methylation fingerprint. Cellular methylation reactions include modification of DNA, RNA, proteins and lipids (56
). These reactions all require a methyl donor for the modification of the target. The methyl donor for the majority of these reactions is S-adenosylmethionine (SAM) (11
). SAM is distributed within all cell and tissue compartments and functions as a methyl-donor for a number of different methyltransferases. The donation of methyl groups results in the generation of S-adenosylhomocysteine (SAH). Methyltransferase enzymes have a higher affinity for SAH than SAM, and thus, SAH functions as a potent feedback inhibitor (14
). SAH is rapidly converted to homocysteine and adenosine by SAH hydrolase. Based on this methylation fingerprint, we performed parallel microarray analysis of modeled inflammation using a Th1 cytokine (IFN-γ) as a stimulus and discovered that SAM synthetase (MAT2A) and SAH hydrolase (AHCY) were among the top twenty highest IFN-γ-induced transcripts in T84 intestinal epithelia. The mammalian MAT2A gene promoter is relatively well-characterized. Previous studies have shown that the human MAT2A promoter is basally controlled by three tandem Sp1 sites (58
). Under modeled inflammatory conditions (TNF-α activation), MAT2A is strongly induced through activation of AP-1 and NFκB (59
). These studies support our findings of MAT2A induction by IFN-γ, a known activator of both NFκB and AP-1 (60
). At present, essentially nothing is known about the regulation of the mammalian AHCY gene.
As a proof of principle, we extended these findings to a mucosal inflammation model. Given our understanding of inflammation-associated metabolic changes within the epithelium, we selected DSS colitis as an appropriate animal model to study colitis. DSS functions primarily as an epithelial irritant to drive permeability-induced colonic inflammation (61
). Therefore, inhibition of SAH hydrolase represents a powerful means of inhibiting cellular methylation reactions (15
). It has been known for several years that inhibition of methylation had immunosuppressive influences (16
). This led to the development of more specific, reversible, and less toxic SAH inhibitors for use in animal models of inflammation. Utilizing these compounds, it was demonstrated that SAH hydrolase inhibition particularly down-regulates T cell activation and adaptive immune responses. One of these SAH hydrolase inhibitors, DZ2002, has been found to have potent immunosuppressive effects and ameliorates disease in a number of animal models including delayed-type hypersensitivity (20
), arthritis (20
), and EAE (19
). While we do not know the exact mechanism by which DZ2002, we have shown that inhibition of methylation by DZ2002 inhibits NF-κB in vitro
. NF-κB is a master regulator of the pro-inflammatory response, upregulating a number of genes including cytokines, chemokines, and adhesion molecules, as well as activated under conditions of hypoxia (62
). A number of studies have shown that inhibition of NF-κB may be protective in animal models of IBD ((63
)). Paradoxically, however, NF-κB activation has also been found to be protective in the context of the mucosal epithelium in murine disease models (40
). Our findings with DZ20002 support this hypothesis and demonstrate that parenteral administration of DZ2002 significantly inhibits tissue methylation and that such inhibition is associated with exacerbated DSS colitic responses. Additionally, augmentation of methylation through the administration of folate, ameliorates disease in this colitic model. Whether methylation inhibition would impact other models of intestinal inflammation in the same manner is not currently known.
Taken together, these studies provide a new and compelling role for methylation as an endogenously protective mechanism for mucosal inflammation. Additionally, the findings herein provide a tractable and potentially new therapeutic opportunities for methylation-dependent targets in mucosal diseases such as IBD.