Inflammatory Bowel Disease (IBD) is an idiopathic and relapsing inflammatory disorder occurring in the gastrointestinal (GI) tract, and has two major subtypes, namely Crohn’s disease and ulcerative colitis (1, 2). Despite symptom similarity, Crohn’s disease and ulcerative colitis show distinctive characteristics in damaged regions, pro-inflammatory cytokine profiles and histological phenotypes (1, 2). Crohn’s disease shows a non-continuous damaged region in the GI tract (3-5). Conversely, ulcerative colitis involves an extensive area, from the proximal to distal colon, in a continuous fashion (3-5). Various animal models have become useful tools to investigate the mediator of the mucosal immune response and immunological processes underlying acute or chronic intestinal inflammation, improving the understanding of IBD (6, 7). The dextran sulfate sodium (DSS)-induced colitis model is the most widely used in IBD research, even though most IBD experimental models are not appropriate due to disease complexity (8, 9).
DSS is a negatively charged sulfated polysaccharide that can be easily and rapidly administered as a water-soluble agent (10, 11). Although the exact mechanism underlying colitis induction by DSS is unclear, the administration of DSS solution damages the epithelial monolayer cells and mucosal layer in the large intestine. Consequently, gut microbiota enter the colonic lumen and induce a radical immunity imbalance (9). Massive pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α, are also released in mouse colon tissues with DSS-induced acute or chronic colitis (6, 12). Therefore, pro-inflammatory cytokines have become a useful maker for colitis in animal models.
Nuclear factor-κB (NF-κB) plays a key role in the regulation of intestinal inflammation and colitis-associated cancer (13-15). The aberrant regulations of canonical and non-canonical NF-κB signaling have been well studied in IBD patients (13-15). This condition induces the release of vast amounts of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which induces tissue damage and immune cell infiltration (16, 17). Post-translational modification of NF-κB by LSD1, NSD1/FBXL11, and PRMT5 is associated with its diverse functions (18-20).
Lysine-specific demethylase 1 (LSD1, also called KDM1A) catalyzes the demethylation of histone H3 (21). H3K4me2 is generally associated with gene activation, while H3K9me2 is associated with gene suppression. Thus, LSD1 acts as a transcriptional activator or inhibitor, depending on the residues of the target histone H3 (22-24). In addition to its well-known H3 demethylase function, LSD1 also demethylates non-histone proteins, including p53, HIF-1α, and the p65 subunit of NF-κB (25-27). LSD1 cellular function is controlled by phosphorylation, mediated by several kinases including PLK1, CK2/WIP1, and PKCα (18, 28, 29). Particularly, LSD1 phosphorylation on the 112th serine residue in mice (analogous to 111th serine in human LSD1) by PKCα, was identified as a critical regulatory point for LSD1 functioning in circadian rhythm and inflammatory signaling (18, 30). In inflammatory signaling, lipopolysaccharide (LPS) stimulation of immune cells like macrophages activates PKCα, which then phosphorylates LSD1 on serine-112 (18, 28, 29). Although LSD1 phosphorylation does not alter demethylase activity, it modulates interaction of LSD1 with substrate (18). Phosphorylated LSD1 interacts with mono-methylated p65 and removes the methyl group, leading to the stabilization of NF-κB, consequently mediating prolonged inflammatory gene expression (18). Subsequently,
We investigate whether the suppression of the LSD1-NF-κB pathway could reduce colitis and whether an LSD1 inhibitor could alleviate ulcerative colitis symptoms in mice. Consequently, we found that both lack of LSD1 phosphorylation and inhibition of LSD1 activity reduced colitis symptoms, infiltration of immune cells, and pro-inflammatory gene expression in mice.
Our previous study revealed the involvement of LSD1 phosphorylation on 112th serine residue in the prolonged activation of the NF-κB (18). Here, we investigated whether the phosphorylation plays a role in colitis (where uncontrolled inflammation is the main cause of the disease) using a DSS-induced colitis mouse model.
We then obtained the colon tissues from four groups of mice (
We then subjected the colon tissues, which had undergone the injury and recovery phases, to hematoxylin & eosin (H&E) staining and examined the detailed histological features. Most of the colonic epithelium of
We then used GSK-LSD1, a demethylase inhibitor of LSD1, to investigate whether the inhibition of LSD1 activity could alleviate colitis symptoms. GSK-LSD1 (5 mg/kg body weight) was injected intraperitoneally on days 0, 1, and 3 of DSS administration (Fig. 3A). The control group was injected with the same volume of PBS instead of GSK-LSD1. The GSK-LSD1 injection group showed milder symptoms than the PBS group 8 days after acute colitis induction, based on body weight recovery and DAI scores (Fig. 3B and 3C). Colon length shortening was also less severe in the GSK-LSD1 injection group compared to the PBS control (Fig. 3D and 3E). Consistent with the less severe colitis symptoms, Ki-67 expression (representing cell proliferation in colonic epithelium) decreased in the GSK-LSD1 injection group (Fig. 3F). Thus, these results demonstrate the protective effect of LSD1 inhibition in mice, against DSS-induced acute colitis.
Considering that GSK-LSD1 injections produced similar results to
Uncontrolled inflammation is a well-known trigger of human IBD (7, 8). In this study, we observed the relief of colitis symptoms along with the downregulation of the inflammatory response in
Recent studies indicated that LSD1 phosphorylation on 112th serine (111th in human) is crucial for a variety of functions, including epithelial-mesenchymal transition (EMT), cancer metastasis, circadian rhythmicity and inflammation (18, 30, 32, 33). Ectopic expression of the LSD1 S111D mutant, mimicking the phosphorylated form of LSD1, promoted the metastatic capacity of MDA-MB-231 breast cancer cells in nude mice (32). Additionally, PKCq-mediated phosphorylation of LSD1 on the residue was critical for its demethylase and EMT promoting activity in breast cancer (33). PKCα is the first enzyme identified as a kinase, responsible for LSD1 phosphorylation on the residue, in the regulation of the circadian cycle (30). LSD1 phosphorylation by PKCα is also critical for the prolonged activation of NF-κB signaling (18). Intestinal epithelial cells express at least 10 PKC isoforms; not only “classic” PKC isoforms like PKCα, but also “novel” PKC isoenzymes like PKCq (34). Although PKCα phosphorylated LSD1 upon LPS stimulation (18), DSS-induced colitis is a complex process involving a number of proinflammatory agents. We therefore cannot rule out the possibility that LSD1 is phosphorylated by PKCα as well as other kinases, including PKCq, in colitis models.
In the previous study we successfully used GSK-LSD1, an inhibitor of LSD1 demethylase activity, to reduce systemic inflammation induced by LPS or CLP (cecal ligation and puncture) (18). The demethylase activity of LSD1 is required for demethylation of methyl-p65, a subunit of the NF-κB transcription factor, resulting in the stabilization of p65 and prolonged activation of NF-κB. Inhibition of LSD1 by GSK-LSD1 resulted in the accumulation of mono-methylated p65, which was eventually degraded by the ubiquitin-proteasome system resulting in premature termination of the NF-κB-mediated inflammatory responses. In this study, we identified the beneficial effect of GSK-LSD1 on the DSS-induced colitis mouse model. Relief of colitis symptoms was similar to what we saw in
Currently, LSD1 is considered a validated epigenetic target for anticancer drug development, with many LSD1 inhibitors in clinical trials as anticancer agents for AML (acute myeloid leukemia), SCLC (small-cell-lung cancer) and other solid tumors (35, 36). In this study, we showed the potential of LSD1 inhibitors as anti-inflammatory agents that can alleviate colitis symptoms. Since high levels of LSD1 expression have been identified in inflammation-associated cancers, including colorectal cancer (36), the LSD1 inhibitor, GSK-LSD1, has therapeutic potential in colitis and may be good candidates for limiting colitis-associated cancer (CAC) progression, by target ing the microenvironment as well as the cancer itself.
Male mice with corresponding age (7-9 weeks) and body weight (20-25?g) received 2% DSS (MP Biomedicals, M.W. 36-50 kDa) in drinking water for 5 days, followed by normal drinking water for 5 days. In the LSD1 inhibition groups, wild-type mice were intraperitoneally injected with the LSD1 inhibitors, GSK-LSD1 (5 mg/kg body weight, Sigma) or PBS, on days 0, 1, and 3 of 2% DSS administration. Control mice received DSS-free drinking water. The DAI of colitis was monitored daily, based on Supplementary Table 1.
Mouse colon tissues were fixed in a 4% paraformaldehyde solution at 4°C for 2 days and embedded in paraffin. All samples were cut into 4 μm sections for H&E staining. Microscopic images were obtained through light microscopy (Olympus IX71 and Leica M80). Histological scoring was based on three categories, namely, mucosal hyperemia, surface epithelium loss, and crypt damage (Supplementary Table 2). Immune cell infiltration into colon epithelium was analyzed with immunofluorescence staining. The primary antibodies used were anti-Ki67 (Abcam, ab15580), anti-CD4 (BD Biosciences, #553047), anti-Gr-1 (Serotech, #MCA2387GA), and anti-F4/80 (Serotech, #MCA497GA).
Mouse colon tissues were homogenized in a Trizol reagent (Life Technologies) and total RNA was isolated using the GeneJET RNA Purification Kit (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the cDNA synthesis kit (Thermo Fisher Scientific). Primers used for mouse genes are in Supplementary Table 3.
Colon tissues were washed with ice-cold PBS and solution B (2.7 mM KCl, 150 mM NaCl, 1.2 mM KH2PO4, 680 mM Na2HPO4, 1.5 mM EDTA, and 1 mM DTT). Fragments of the distal portion were then homogenized in RIPA buffer (150 mM NaCl, 20 mM Tris pH 7.5, 0.1% SDS, 1% NP-40, and 2 mM EDTA). Proteins were resolved on 8-15% gradient gels and transferred onto a nitrocellulose membrane (GE Healthcare). The primary antibodies used were anti-β-actin (AB Frontier), anti-iNOS (Merck Millipore), and anti-PCNA (Abcam).
Statistical analyses were performed with GraphPad Prism v5.01 software. The statistical significance of the two groups was analyzed by two-way ANOVA (Body weight and DAI) or Mann-Whitney test (Histological score). One-way ANOVA (Newman keule’s test) was used for the statistical analysis among four groups. Data were expressed as the mean ± SEM.
This work was supported by the Science Research Center Program (Cellular Heterogeneity Research Center, NRF-2016 R1A5A1011974) and by Basic Science Research Program (NRF-2018R1A2B6004112) to K.I.K. through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT. We would like to thank Editage (www.editage.co.kr) for English language editing.
The authors have no conflicting interests.