Mucosal interleukin-8 expression as a predictor of subsequent relapse in ulcerative colitis patients with Mayo endoscopic subscore 0.

BACKGROUND AND AIM: Complete endoscopic mucosal healing is defined as a Mayo endoscopic subscore of 0. Some patients diagnosed with a Mayo endoscopic subscore 0 may present with subsequent clinical relapse. Here, we aimed to demonstrate mucosal cytokine profile as a predictor of clinical relapse in ulcerative colitis patients with a Mayo endoscopic subscore of 0 as a marker of mucosal healing. METHODS: We conducted prospective observational pilot study to examine the relationship between mucosal cytokine expression and subsequent relapse of UC patients diagnosed with a Mayo endoscopic subscore of 0. We enrolled 55 patients, and expression of cytokines tumor necrosis factor-α, interferon γ, interleukin-1ß, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-12, interleukin-13, interleukin-15, interleukin-17A, interleukin-17F, interleukin-18, interleukin-21, interleukin-22, interleukin-23, interleukin-27, and interleukin-33 was measured by quantitative real-time PCR using rectal mucosa biopsy materials. Cytokine expression levels were compared between patients who relapsed between March 1, 2016, and March 30, 2020, of the study period and those who remained in remission. RESULTS: Ten cytokines, including interleukin-2, interleukin-4, interleukin-8, interleukin-10, interleukin-12, interleukin-15, interleukin-17A, interleukin-21, interleukin-23, and interleukin-33, were significantly elevated in patients with subsequent relapse compared with those who remained in remission. Interleukin-8 expression was the most useful predictor. CONCLUSIONS: In the rectal mucosa of ulcerative colitis patients with Mayo endoscopic subscore 0, levels of several cytokines were elevated in cases of subsequent relapse. Among these, interleukin-8 expression was the most useful for predicting relapse.

Increased mucosal IL-12 expression is associated with relapse of ulcerative colitis.

BACKGROUND: The role of IL-12/23 in the pathogenesis of ulcerative colitis (UC) is unclear. We analyzed mucosal IL-12/23 expression and its relationship with endoscopic severity, histological activity, and UC relapse. METHODS: Rectal biopsies were collected from 70 UC patients with clinical remission. IL-12, IL-23, IFN-γ, IL-17A, and IL-17F mRNA expression was measured by real-time PCR. Endoscopic severity and histological activity were evaluated using the Mayo endoscopic subscore (MES) and the Geboes score, respectively. RESULTS: The longest follow-up period was 51 months. Thirty-four patients relapsed during the study period. Samples from these subsequently relapsed patients formed the "relapse" group, while those from patients that did not relapse formed the "remission" group. IL-12 (P = 0.0003) and IL-23 (P = 0.014) mRNA expression was significantly higher in the relapse than the remission group. Expression of IL-23 (P = 0.015) but not IL-12 (P = 0.374) was correlated with MES. However, in patients with an MES of 0 and 1, IL-12 expression was statistically higher in the relapse than the remission group (P = 0.0015, P = 0.0342). IL-12 and IL-23 expression did not vary significantly between histologically active and inactive mucosa; both were higher in histologically inactive patients in the remission group (IL-12: P = 0.0002, IL-23: P = 0.046). CONCLUSIONS: Rectal IL-12 and IL-23 expression was elevated in the relapse group, but IL-12 was more strongly associated with UC relapse, irrespective of endoscopic severity and histological activity. Mucosal IL-12 was elevated in patients with deep mucosal healing. Our results suggest an important role of IL-12 in UC pathogenesis and the molecular mechanism of UC relapse.

The efficacy of linked color imaging for the endoscopic diagnosis of mucosal healing in quiescent ulcerative colitis.

BACKGROUND AND AIM: The Mayo Endoscopic Subscore (MES) and the Ulcerative Colitis Endoscopic Index of Severity (UCEIS) are used to assess endoscopic mucosal healing in patients suffering from ulcerative colitis. Although mucosal healing is defined by MES 0, relapse of ulcerative colitis is often observed. Over a 48-month period, this study investigated the efficacy of linked color imaging (LCI) in predicting the long-term prognosis of ulcerative colitis patients diagnosed with MES 0. METHODS: Overall, 26 patients in ulcerative colitis remission, diagnosed with MES 0, were enrolled. Using a LASEREO endoscopic system (Fujifilm Co., Tokyo, Japan), endoscopic colonic images were assessed with linked color imaging and the colitis endoscopic index of severity. Endoscopic LCI images were separated into three subgroups (A, no redness; B, redness with visible vessels; and C, redness without visible vessels). The Geboes score was used to evaluate histology; active mucosa was defined as GS > 2B.1. RESULTS: Linked color imaging classification subdivided colonic mucosa, which had been diagnosed with MES 0, into two classes. The LCI-A group did not relapse, and the non-relapse rate was significantly higher (P = 0.018) than that in the LCI-B group. No difference in relapse rates was observed between patients with a colitis endoscopic index of severity of 0 and 1 (P = 0.655). There was no statistical difference between the composition of LCI-A group and the relapse rate between active and inactive mucosa diagnosed by Geboes score. CONCLUSIONS: This methodology can be used to evaluate mucosal healing and predict long-term outcomes in ulcerative colitis patients.

Promotion of wound healing by acetate in murine colonic epithelial cell via c-Jun N-terminal kinase activation.

BACKGROUND AND AIM: Mucosal healing is an important clinical goal in patients with inflammatory bowel disease. Recently, short-chain fatty acids (SCFAs) have been reported to have multifaceted effects to host. However, the effects of SCFAs on wound healing in intestinal epithelial cells are unclear. In the present study, we investigated the effects of acetate, one of the major SCFAs, on the wound healing of murine colonic epithelial cells. METHODS: Young adult mouse colonic epithelial cells were used to determine the effect of acetate using wound healing assay. Mitogen-activated protein kinase and Rho kinase inhibitor were used to elucidate intracellular signal of wound healing treated with acetate. Meanwhile, Rho activation assays were utilized to measure Rho activation levels. To assess in vivo effects, C57B6 mice with dextran sodium sulfate for 7 days were treated with enema administration of acetate for 7 days. Body weight, disease activity index, colon length, and mucosal break ratio in histology were examined. RESULTS: Acetate enhanced wound healing and fluorescence intensity of actin stress fiber compared with control. These effects were canceled with pretreatment of c-Jun N-terminal kinase (JNK) inhibitor or Rho kinase inhibitor. Furthermore, JNK inhibitor reduced the activation of Rho induced by acetate. In the dextran sodium sulfate-induced colitis model, the mice with enema treatment of acetate significantly exhibited recovery. CONCLUSIONS: In this study, we demonstrated that acetate promoted murine colonic epithelial cell wound healing via activation of JNK and Rho signaling pathways. These findings suggested that acetate could have applications as a therapeutic agent for patients with intestinal mucosal damage, such as inflammatory bowel disease.

Ginsenoside Rb1 promotes intestinal epithelial wound healing through extracellular signal-regulated kinase and Rho signaling.

BACKGROUND AND AIM: Daikenchuto, a traditional Japanese herbal medicine, has been reported to exhibit anti-inflammatory effects against intestinal inflammation. However, whether daikenchuto has a therapeutic effect against intestinal mucosal injuries remains unclear. Thus, the aim of this study was to determine the effect of daikenchuto on intestinal mucosal healing. METHODS: Colitis was induced in male Wistar rats by using trinitrobenzenesulfonic acid. Daikenchuto (900 mg/kg/day) was administered for 7 days after the induction of colitis. Thereafter, intestinal mucosal injuries were evaluated by determining the colonic epithelial regeneration ratio ([area of epithelial regeneration/area of ulcer] × 100). Restoration of rat intestinal epithelial cells treated with daikenchuto and its constituent herbs (Zanthoxylum fruit, processed ginger, and ginseng) and ginsenoside Rb1, which is a ginseng ingredient, was evaluated using a wound-healing assay. RESULTS: The colon epithelial regeneration ratio in the daikenchuto-treated rats was significantly higher than that in the control rats. Daikenchuto, ginseng, and ginsenoside Rb1 enhanced wound healing, and the ginsenoside Rb1-induced enhancement was inhibited by extracellular signal-regulated kinase and Rho inhibitors. CONCLUSIONS: Daikenchuto and its constituent, ginsenoside Rb1, promoted wound healing. Because mucosal healing is one of the most important therapeutic targets in patients with inflammatory bowel disease, ginsenoside Rb1 may be a novel therapeutic agent against intestinal mucosal damage such as that occurring in intestinal bowel disease.

Transforming growth factor ß1-induced collagen production in myofibroblasts is mediated by reactive oxygen species derived from NADPH oxidase 4.

Intestinal fibrosis with stricture formation is a severe complication of Crohn's disease (CD). Though new therapeutic targets to enable the prevention or treatment of intestinal fibrosis are needed, markers of this condition and the basic mechanisms responsible have not been established. NADPH oxidase (NOX) 4 has already been reported to play a key role in models of fibrogenesis, including that of the lung. However, its importance in intestinal fibrogenesis remains unclear. In this study, we examined the role of NOX4 in collagen production by intestinal myofibroblasts stimulated with transforming growth factor (TGF)-ß1. Using LmcMF cells, an intestinal subepithelial myofibroblast (ISEMF) line, we first examined the induction of collagen production by TGF-ß1. Subsequently, we investigated the role of NOX4 in TGF-ß1-induced collagen I production in these cells using SB525334 (an SMAD2/3 inhibitor), diphenyleneiodonium (an NOX inhibitor), and Nox4 small interfering RNA (siRNA). Production of collagen was assessed with Sirius red staining, and Nox4 expression was measured by quantitative real-time PCR. Reactive oxygen species (ROS) production was determined using DCFDA and fluorescent microscopy. We observed that TGF-ß1 induced collagen production via NOX4 activation and ROS generation in LmcMF cells. Nox4 siRNA and inhibitors of TGF-ß1 receptor and NOX significantly reduced TGF-ß1-induced ROS and collagen production. Thus, in the present study, we revealed that collagen production in ISEMFs is induced via an NOX4-dependent pathway. This work supports a function for NOX4 in intestinal fibrogenesis and identifies it as a potential therapeutic target in recalcitrant fibrotic disorders of CD patients.

Secreted protein acidic and rich in cysteine functions in colitis via IL17A regulation in mucosal CD4+ T cells.

BACKGROUND AND AIM: Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycol that regulates cell proliferation, tissue repair, and tumorigenesis. Despite evidence linking SPARC to inflammation, the mechanisms are unclear. Accordingly, the role of SPARC in intestinal inflammation was investigated. METHODS: Colitis was induced in wild-type (WT) and SPARC knockout (KO) mice using trinitrobenzene sulfonic acid (TNBS). Colons were assessed for damage; leukocyte infiltration; Tnf, Ifng, Il17a, and Il10 mRNA expression; and histology. Cytokine profiling of colonic lamina propria mononuclear cells (LPMCs) was performed by flow cytometry. Naïve CD4+ T cells were isolated from WT and SPARC KO mouse spleens, and the effect of SPARC on Th17 cell differentiation was examined. Recombination activating gene 1 knockout (RAG1 KO) mice reconstituted with T cells from either WT or SPARC KO mice were investigated. RESULTS: Trinitrobenzene sulfonic acid exposure significantly reduced bodyweight and increased mucosal inflammation, leukocyte infiltration, and Il17a mRNA expression in WT relative to SPARC KO mice. The percentage of IL17A-producing CD4+ T cells among LPMCs from KO mice was lower than that in WT mice when both groups were exposed to TNBS. Th17 cell differentiation was suppressed in cells from SPARC KO mice. In the T cell transfer colitis model, RAG1 KO mice receiving T cells from WT mice were more severely affected than those reconstituted with cells from SPARC KO mice. CONCLUSIONS: Secreted protein acidic and rich in cysteine accelerates colonic mucosal inflammation via modulation of IL17A-producing CD4+ T cells. SPARC is a potential therapeutic target for conditions involving intestinal inflammation.

Low serum albumin at admission is a predictor of early colectomy in patients with moderate to severe ulcerative colitis.

BACKGROUND AND AIM: Several studies have identified postinduction therapy predictors of long-term outcomes of ulcerative colitis (UC) in patients who experienced the first attack of the disease or relapsed after therapy. We aimed to identify the preinduction therapy predictors at admission that predicted early colectomy in patients with moderate to severe UC. METHODS: Ninety-five patients with moderate to severe UC who underwent induction therapy at the Kyoto Prefectural University of Medicine hospital between August 2008 and March 2020 were retrospectively included and categorized into two groups: the colectomy group (n = 27) and the noncolectomy group (n = 68). The clinical parameters (age, gender, disease extent, and disease activity on admission), induction therapies administered [including 5-aminosalicylic acid, steroids, immunomodulators, calcineurin inhibitor, and anti-Tumor Necrosis Factor (TNF)-α antibodies], and laboratory data (hemoglobin, albumin, C-reactive protein, and cytomegalovirus reactivation on admission) were evaluated and compared between the two groups. Multivariate logistic regression analyses were performed to identify significant predictors of early colectomy, and P < 0.05 was considered significant. RESULTS: All clinical parameters were not significant predictors of colectomy. Among laboratory parameters, the serum albumin level on admission was a significant independent predictor of colectomy (odds ratio: 6.097, 95% confidence interval: 1.8310-20.3047). Receiver operating characteristic curves were plotted for the serum albumin levels of the 95 patients at admission. The cut-off value of serum albumin was 2.45 g/dL. CONCLUSIONS: When the serum albumin level of UC patients at admission is below 2.45 g/dL, we should consider presenting the option of surgical treatment to patients.

Carbon monoxide ameliorates murine T-cell-dependent colitis through the inhibition of Th17 differentiation.

Recent studies have identified carbon monoxide (CO) as a potential therapeutic molecule for the treatment of inflammatory diseases including intestinal inflammation. In the present study, we explored the efficacy and the mechanisms of action of CO-releasing molecule (CORM)-A1 in T-cell transfer induced colitis model in mice. In addition, the impact of CORM-A1 on the T helper (Th) cell differentiation was evaluated using naïve CD4+ T cells isolated from the spleens in Balb/c mice. The results showed that CORM-A1 conferred protection against the development of intestinal inflammation and attenuated Th17 cell differentiation. Hence, the observed immunomodulatory effects of CORM-A1 could be useful for developing novel therapeutic approaches for managing intestinal inflammation through the regulation of Th17 differentiation.

Assessment of Endoscopic Mucosal Healing of Ulcerative Colitis Using Linked Colour Imaging, a Novel Endoscopic Enhancement System.

BACKGROUND AND AIMS: Mucosal healing and control of intestinal mucosal inflammation are important treatment goals for maintaining clinical remission in ulcerative colitis [UC] patients. Here, we investigated the efficacy of linked colour imaging [LCI], a novel endoscopic enhancement system, for diagnosing mucosal inflammation in UC patients. METHODS: All examinations were carried out with a LASEREO endoscopic system [FUJIFILM Co., Tokyo, Japan]. Fifty-two patients with UC were enrolled, and 193 areas assessed by LCI were examined. LCI patterns were classified as; A, no redness; B, redness with visible vessels; and C, redness without visible vessels. Regions of interest [ROIs] were set at biopsy sites, and the red colour in the ROI was calculated from the Commission internationale de l'éclairage [CIE] color space and digitized [LCI-index]. Biopsy specimens were taken at each ROI and evaluated with Matts histopathological grade. Thirty months was defined as the time interval between endoscopic diagnosis and relapse of UC. RESULTS: Interobserver agreement for LCI classification was excellent between an expert and non-experts. Among areas with a Mayo endoscopic subscore of 0, 41.8% and 4.6% were classified as LCI-B and LCI-C, respectively. Among areas with a Mayo endoscopic subscore of 1, 60.5% and 34.6% were classified as LCI-C and LCI-B, respectively. The LCI index strongly correlated with the histopathological Matts score. Non-relapse rates significantly correlated with LCI classification [p = 0.0055], but not with Mayo endoscopic subscore [p = 0.0632]. CONCLUSION: Endoscopic LCI classification and LCI index can subdivide samples with the same Mayo endoscopic subscore. LCI may be a novel approach for evaluating colonic mucosal inflammation and for predicting outcome in UC patients.

Rebamipide upregulates mucin secretion of intestinal goblet cells via Akt phosphorylation.

Mucin is produced and secreted by epithelial goblet cells and is a key component of the innate immune system, acting as a barrier in the intestinal tract. However, no studies have been conducted investigating the increase in mucin secretion to enhance the intestinal barrier function. The present study investigated whether rebamipide (Reb) acts as a secretagogue of intestinal mucin and the underlying mechanisms involved, thereby focusing on the effect on goblet cells. The LS174T cell line was used as goblet cell­like cells. Using Reb­treated LS174T cells, the level of mucin content was assessed by periodic acid­Schiff (PAS) staining, and mucin 2, oligomeric mucus/gel­forming (MUC2) mRNA expression was assessed using quantitative polymerase chain reaction (PCR). Furthermore, MUC2 secretion in the supernatant was quantified by the dot blot method. The present study additionally investigated the involvement of the epidermal growth factor receptor/Akt serine/threonine kinase 1 (Akt) pathway in mucin secretion by western blotting. The results suggested that Reb strongly enhanced the positivity of PAS staining in LS174T cells, thereby suggesting increased intracellular mucin production. The PCR results indicated that Reb significantly increased MUC2 mRNA in whole cell lysate of LS174T cells. In order to assess the subsequent secretion of mucin by LS174T, MUC2 protein expression in the supernatant was assessed using the dot blot method and it was demonstrated that Reb significantly increased the secretion of MUC2 in a concentration­dependent manner. The p­Akt was significantly increased by Reb treatment, and an Akt inhibitor specifically suppressed MUC2 secretion. Overall, Reb increased mucin secretion directly via p­Akt. Reb­increased mucin may act as a strong non­specific barrier against pathogenic stimulants in various intestinal diseases.

Partially hydrolyzed guar gum enhances colonic epithelial wound healing via activation of RhoA and ERK1/2.

BACKGROUND AND AIMS: Healing of the intestinal mucosal epithelium was found to be a critical factor in the treatment of inflammatory bowel disease (IBD). In this study, we provide further evidence that partially hydrolyzed dietary fiber (PHGG) enhances colonic epithelial cell wound healing, and partially characterize the mechanism that governs this process. MATERIALS AND METHODS: Young adult mouse colonic (YAMC) epithelial cells were scraped with a 10 µl micro-pipette tip to denude a round of the monolayer and were incubated with PHGG. The area of cell migration was measured using Image J software. Meanwhile, Rho activation assays were utilized to monitor Rho activation levels. To assess in vivo effects, C57B6 mice were treated with DSS for 7 days and then provided food supplemented with PHGG for 8 days. RESULTS: YAMC cells treated with PHGG exhibited significantly enhanced wound healing compared to the control cells; however, this enhancement was inhibited by both Y-27632 (RhoA inhibitor) and U0126 (ERK1/2 inhibitor). Likewise, there was a PHGG-dependent increase in F-actin accumulation and Rho kinase activity that was blocked by U0126. Meanwhile, PHGG-dependent ERK1/2 activity was not inhibited by Y-27632. In the DSS-induced mouse colitis model, animals that received food supplemented with PHGG exhibited significant recovery of the colonic mucosa. CONCLUSIONS: In this study, we demonstrate that PHGG promotes colonic epithelial cell wound healing via activation of RhoA, which occurs downstream of ERK1/2 activation. These findings indicate that PHGG could be utilized as a therapeutic agent for patients with intestinal mucosal damage such as those with IBD.