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Glutathione S‐transferase omega 2 (GSTO2) lacks any appreciable GST activity, but it exhibits thioltransferase activity. The significance of GSTO2 in lung function has been reported; however, the precise expression and molecular function of GSTO2 in the lungs remain unclear. In the present study, we found that GSTO2 is expressed in airway basal cells, non–ciliated, columnar Clara cells, and type II alveolar cells, which have self‐renewal capacity in the lungs. Contrastingly, no GSTO2 expression was observed in 94 lung squamous cell carcinoma (LSCC) samples. When human LSCC cell lines were treated with 5‐aza‐2′‐deoxycytidine, a DNA‐methyltransferase inhibitor, GSTO2 transcription was induced, suggesting that aberrant GSTO2 hypermethylation in LSCC is the cause of its downregulation. Forced GSTO2 expression in LSCC cell lines inhibited cell growth and colony formation in vitro. In a subcutaneous xenograft model, GSTO2‐transfected cells formed smaller tumors in nude mice than mock‐transfected cells. Upon intravenous injection into nude mice, the incidence of liver metastasis was lower in mice injected with GSTO2‐transfected cells than in those injected with mock‐transfected cells. In addition, GSTO2 induction suppressed the expression of β‐catenin and the oxygen consumption rate, but it did not affect the extracellular acidification rate. Furthermore, GSTO2‐transfected cells displayed lower mitochondrial membrane potential than mock‐transfected cells. When GSTO2‐transfected cells were treated with a p38 inhibitor, β‐catenin expression and mitochondrial membrane potential were recovered. Our study indicated that the loss of GSTO2 via DNA hypermethylation contributes to the growth and progression of LSCC, probably by modulating cancer metabolism via the p38/β‐catenin signaling pathway., Glutathione S‐transferase omega 2 (GSTO2) is expressed in the stem cells of the bronchial and alveolar epithelium. Loss of GSTO2 might contribute to lung squamous cell carcinoma growth by modulating cancer metabolism via the p38/β‐catenin signaling pathway.

Liver cancer is the fourth leading cause of cancer-related death. Hepatocellular carcinoma (HCC) is a primary liver cancer that results from chronic hepatitis caused by multiple predisposing factors such as viral infection, alcohol consumption, and non-alcoholic fatty liver disease. Accumulating studies have indicated that dysfunction of the gut epithelial barrier and hepatic translocation of gut microbes may be implicated in the pathogenesis of HCC. However, the translocated bacteria in HCC patients remains unclear. Here, we characterised tumour-associated microbiota in patients with liver cancer and focused on HCC. We observed that the number of amplicon sequence variants in tumour-associated microbiota was significantly higher compared with that in non-tumour regions of the liver. The tumour-associated microbiota consisted of Bacteroidetes, Firmicutes, and Proteobacteria as the dominant phyla. We identified an unclassified genus that belonged to the Bacteroides, Romboutsia, uncultured bacterium of Lachnospiraceae as a signature taxon for primary liver cancer. Additionally, we identified Ruminococcus gnavus as a signature taxon for HCC patients infected with hepatitis B and/or hepatitis C viruses. This study suggests that tumour microbiota may contribute to the pathology of HCC.

Glutathione S-transferase omega 2 (GSTO2) lacks any appreciable GST activity but presents thioltransferase activity and therefore plays an important role in regulating the glutathione redox balance. In addition, GSTO2 polymorphisms are strongly associated with lung function and the risk of chronic obstructive pulmonary disease. We recently reported that GSTO2 is exclusively expressed in airway basal cells, Clara cells, and type II alveolar cells, which have self-renewal capacity in the lungs. In this study, we examined the expression of GSTO2 in non-small cell lung cancer (NSCLC) and analyzed the relationship between GSTO2 expression and clinicopathological features. We enrolled 233 patients who were diagnosed with NSCLC and underwent surgery, and immunohistochemically evaluated the expression of GSTO2 using formalin-fixed, paraffin-embedded sections of surgical specimens. While all 94 squamous cell carcinoma samples showed no GSTO2 expression, 58 of 139 lung adenocarcinoma (LAC) samples exhibited GSTO2 expression. The expression of GSTO2 in LAC samples was significantly associated with never or light (Brinkman Index < 400) smoking history (P=0.0027) and histological subtypes (lepidic 85/110, 77%; acinar 30/68, 44%; papillary 18/64, 22%; micropapillary 1/6, 16.7%; solid 3/31, 9.6%; P Citation Format: Ryusuke Sumiya, Hiroto Hatano, Teruki Hagiwara, Satoshi Nagasaka, Hiromu Suzuki, Kazuhiko Yamada, Norihiro Kokudo, Yuki I. Kawamura. Loss of glutathione S-Transferase omega 2 is associated with PD-L1 expression and poor prognosis in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2602.

A previous study conducted a transcriptome analysis of paired normal and esophageal squamous cell carcinoma (ESCC) tissue samples. The results showed that the expression of serine protease 27 (PRSS27) was perturbed in tumor samples. Hence, this retrospective study aimed to validate the prognostic significance of PRSS27 in patients with preoperative treatment for ESCC. We enrolled 86 patients who received preoperative treatment before esophagectomy for ESCC. The expression of PRSS27 in resected ESCC and biopsy tissue samples obtained before preoperative treatment was evaluated via immunostaining, and its relationship with clinicopathological features and prognosis was analyzed. In normal esophageal mucosa tissue samples, PRSS27 was expressed in the cytoplasm of spinous cells in the suprabasal layer and basal cells in the basal layer. Of 64 resected ESCC tissue samples, 35 (54.7%) expressed PRSS27 and 29 (45.3%) did not. Moreover, ectopic nuclear expression of PRSS27 was observed. Based on multivariate analysis, PRSS27 expression in resected tumor samples was a predictor of poor prognosis. In cases in which PRSS27 expression was observed in biopsy samples, patients with PRSS27-negative resected tumors had a better postoperative prognosis than those with PRSS27-positive resected tumors. PRSS27 expression in resected ESCC tissue samples is a poor prognostic factor in ESCC patients with preoperative treatment. Furthermore, conversion of PRSS27 expression from positive in biopsy samples to negative in resected tumor samples is a predictor of good prognosis in these patients. Hence, PRSS27 status is an effective tool for decision making regarding adjuvant treatment in ESCC patients.

Glutathione S-transferase omega 2 (GSTO2), which belongs to the superfamily of GST omega class, lacks any appreciable GST activity. Although GSTO2 exhibits thioltransferase and glutathione dehydrogenase activities, its precise expression and physiological functions are still unclear. In the present study, we found that GSTO2 is exclusively expressed in the basal cell layer in Ki67-negative non-proliferative cells in the human esophageal mucosa. GSTO2 overexpression in esophageal squamous cell carcinoma (ESCC) cell lines inhibited cell growth and colony formation, and GSTO2-transfected cells formed smaller tumors in nude mice compared with mock-transfected cells. Interestingly, GSTO2 induction suppressed the expressions of E-cadherin and β-catenin at the cell–cell contact site. We quantified the phosphorylation levels of key proteins of MAPK signaling pathway and identified phosphorylation of p38. Additionally, HSP27, a downstream molecule of p38, was accelerated in GSTO2-transfected cells, unlike in mock-transfected cells. When GSTO2-transfected cells were treated with a p38 inhibitor, the expression of β-catenin and the membrane localization of E-cadherin was recovered. We next examined GSTO2 expression in 61 ESCC tissues using quantitative reverse transcription polymerase chain reaction and immunostaining. The results showed that GSTO2 mRNA and protein were significantly reduced in ESCC compared with normal tissues. When human ESCC cell lines were treated with 5-aza-2′-deoxycytidine, a DNA-methyltransferase inhibitor, GSTO2 transcription was induced, suggesting that aberrant hypermethylation is the cause of the down-regulated expression. Our results indicate that GSTO2 expression inhibits the membrane localization of E-cadherin, probably by modulation of the p38 signaling pathway. Down-regulation of GSTO2 by DNA hypermethylation contributes to the growth and progression of ESCC.

Jaw1/LRMP is a type II integral membrane protein that is localized at the endoplasmic reticulum (ER) and outer nuclear membrane. We previously reported that a function of Jaw1 is to maintain the nuclear shape as a KASH protein via its carboxyl terminal region, a component of linker of nucleoskeleton and cytoskeleton complex in the oligomeric state. Although the oligomerization of some KASH proteins via the cytosolic regions serves to stabilize protein-protein interactions, the issue of how the oligomerization of Jaw1 is regulated is not completely understood. Therefore, we focused on three distinct regions on the cytosolic face of Jaw1: the N-terminal region, the coiled-coil domain and the stem region, in terms of oligomerization. A co-immunoprecipitation assay showed that its coiled-coil domain is a candidate for the oligomerization site. Furthermore, our data indicated that the N-terminal region prevents the aberrant oligomerization of Jaw1 as an intrinsically disordered region (IDR). Importantly, the ectopic expression of an N-terminal region deleted mutant caused the formation of organized smooth ER (OSER), structures such as nuclear karmellae and whorls, in B16F10 cells. Furthermore, this OSER interfered with the localization of the oligomer and interactors such as the type III inositol 1,4,5-triphosphate receptor (IP3R3) and SUN2. In summary, the N-terminal region of Jaw1 inhibits the formation of OSER as an IDR to maintain the homeostatic localization of interactors on the ER membrane.

Background & Aim E-cadherin is a member of the cadherin family and plays a major role in cell–cell adhesion of normal epithelium. Decreased or lack of E-cadherin expression has been reported in breast, colon and bladder cancer, and the disruption of the E-cadherin-mediated cell–cell adhesion is considered the first crucial step of metastasis. The esophageal stratified epithelial cells have many spines that intermingle with each other through desmosomes, in which E-cadherins play an important role for cell–cell adhesion. However, the significance of E-cadherin expression in the progression of esophageal squamous cell carcinoma (ESCC) still remains to be clarified. In the present study, we investigated the relation between E-cadherin expression in tumors and the clinicopathological features of ESCC. Method E-cadherin expression was evaluated in ESCC specimens of 61 patients who underwent surgery from April 2008 to December 2015 at the National Center for Global Health and Medicine by immunohistochemical staining. Distribution of E-cadherin in cancerous tissues was classified as Preserved (expression in cell membrane alone) or Disturbed (expression in cytoplasm or decreased/lack of expression of E-cadherin). Significance of the distribution pattern was analyzed by comparison to clinicopathological features. Results Thirty-seven of 61 (61%) cases revealed Preserved expression pattern while Disturbed distribution was observed in 24 of 61 (39%) cases. Compared to Preserved pattern, Disturbed expression of E-cadherin in tumors was significantly associated with advanced TNM stages (P=0.0462). Conclusion E-cadherin was expressed in most cases of ESCC; however, its distribution was not restricted in membrane but in the cytoplasm of tumors. Disturbed expression of E-cadherin in tumors correlated with advanced stage of ESCC, suggesting that the disruption of the E-cadherin-mediated cell–cell adhesion may play an important role in the progression of ESCC.

Esophageal squamous cell carcinoma (ESCC) is associated with the accumulation of genetic and epigenetic changes in the background mucosa. Dysregulated DNA methylation is known to lead to the inactivation of tumor suppressor genes and the activation of oncogenes. To identify the genes whose expression is perturbed by abnormal DNA methylation in ESCC, integrative transcriptomics by serial analysis of gene expression (SAGE) and methylome sequencing by methyl-DNA immunoprecipitation (MeDIP) analysis were performed. We found 159 genes with significantly decreased expression in ESCC compared to that in noncancerous esophageal mucosa. MeDIP-seq analysis identified hypermethylation in the promoter region of 56 of these genes. Using surgically resected tissues of 40 cases, we confirmed that the paired-like homeodomain 1 (PITX1) gene was hypermethylated in ESCC compared to that in normal tissues (P < 0.0001) by pyrosequencing. PITX1 overexpression in ESCC cell lines inhibited cell growth and colony formation, whereas PITX1 knockdown accelerated cell growth. A PITX1-transfected ESCC cell line, KYSE30, formed smaller tumors in nude mice than in mock-transfected cells. Hypermethylation of PITX1 was associated with tumor depth (P = 0.0011) and advanced tumor stage (P = 0.0052) and predicted poor survival in ESCC (hazard ratio, 0.1538; 95% confidence interval, 0.03159–0.7488; P = 0.0169). In this study, we found a novel tumor suppressor gene of ESCC, PITX1, which is silenced by DNA hypermethylation. Downregulation of PITX1 contributes to the growth and progression of ESCC. Hypermethylation of the PITX1 in ESCC correlated with tumor progression and advanced stage cancer, and may predict a poor prognosis.

Obesity is associated with risk of adenocarcinoma in the proximal stomach. We aimed to identify the links between dietary fat and gastric premalignant lesions. C57BL/6 mice were fed high fat diet (HFD), and gastric mucosa was histologically analysed. Morphological changes were also analysed using an electron microscope. Transcriptome analysis of purified parietal cells was performed, and non-parietal gastric corpus epithelial cells were subjected to single-cell gene-expression profiling. Composition of gastric contents of HFD-fed mice was compared with that of the HFD itself. Lipotoxicity of free fatty acids (FFA) was examined in primary culture and organoid culture of mouse gastric epithelial cells in vitro, as well as in vivo, feeding FFA-rich diets. During ~8–20 weeks of HFD feeding, the parietal cells of the stomach displayed mitochondrial damage, and a total of 23% of the mice developed macroscopically distinct metaplastic lesions in the gastric corpus mucosa. Transcriptome analysis of parietal cells indicated that feeding HFD enhanced pathways related to cell death. Histological analysis and gene-expression profiling indicated that the lesions were similar to previously reported precancerous lesions identified as spasmolytic polypeptide-expressing metaplasia. FFAs, including linoleic acid with refluxed bile acids were detected in the stomachs of the HFD-fed mice. In vitro, FFAs impaired mitochondrial function and decreased the viability of parietal cells. In vivo, linoleic acid-rich diet, but not stearic acid-rich diet induced parietal-cell loss and metaplastic changes in mice. Dietary lipids induce parietal-cell damage and may lead to the development of precancerous metaplasia.

Glycans conserved across species, Early embryogenesis, Embryonic stem cells, Human embryogenesis, Essential signals

Summary Nutritional status potentially influences immune responses; however, how nutritional signals regulate cellular dynamics and functionality remains obscure. Herein, we report that temporary fasting drastically reduces the number of lymphocytes by ∼50% in Peyer’s patches (PPs), the inductive site of the gut immune response. Subsequent refeeding seemingly restored the number of lymphocytes, but whose cellular composition was conspicuously altered. A large portion of germinal center and IgA+ B cells were lost via apoptosis during fasting. Meanwhile, naive B cells migrated from PPs to the bone marrow during fasting and then back to PPs during refeeding when stromal cells sensed nutritional signals and upregulated CXCL13 expression to recruit naive B cells. Furthermore, temporal fasting before oral immunization with ovalbumin abolished the induction of antigen-specific IgA, failed to induce oral tolerance, and eventually exacerbated food antigen-induced diarrhea. Thus, nutritional signals are critical in maintaining gut immune homeostasis.

ObjectiveThe clinical significance of polymorphisms in the interleukin-28B gene encoding interferon (IFN)-λ3, which has antiviral effects, is known in chronic HCV but not in HBV infection. Thus, we measured IFN-λ3 levels in patients with HBV and investigated its clinical significance and association with nucleos(t)ide (NUC) analogue administration.DesignSerum IFN-λ3 level was measured in 254 patients with HBV with varying clinical conditions using our own high sensitivity method. The resulting values were compared with various clinical variables. In addition, cell lines originating from various organs were cultured with NUCs, and the production of IFN-λ3 was evaluated.ResultsHigher serum IFN-λ3 levels were detected in the patients treated with nucleotide analogues (adefovir or tenofovir) compared with those treated with nucleoside analogues (lamivudine or entecavir). There were no other differences in the clinical background between the two groups. A rise in the serum IFN-λ3 levels was observed during additional administration of the nucleotide analogues. In vitro experiments showed that the nucleotide analogues directly and dose-dependently induced IFN-λ3 production only in colon cancer cells. Furthermore, the supernatant from cultured adefovir-treated colon cancer cells significantly induced IFN-stimulated genes (ISGs) and inhibited hepatitis B surface antigen (HBsAg) production in hepatoma cells, as compared with the supernatant from entecavir-treated cells.ConclusionsWe discovered that the nucleotide analogues show an additional pharmacological effect by inducing IFN-λ3 production, which further induces ISGs and results in a reduction of HBsAg production. These findings provide novel insights for HBV treatment and suggest IFN-λ3 induction as a possible target.

ESET/SETDB1, one of the major histone methyltransferases, catalyzes histone 3 lysine 9 (H3K9) trimethylation. ESET is critical for suppressing expression of retroviral elements in embryonic stem cells; however, its role in the immune system is not known. We found that thymocyte-specific deletion of ESET caused impaired T cell development, with CD8 lineage cells being most severely affected. Increased apoptosis of CD8 single-positive cells was observed, and TCR-induced ERK activation was severely inhibited in ESET−/− thymocytes. Genome-wide comprehensive analysis of mRNA expression and H3K9 trimethylation revealed that ESET regulates expression of numerous genes in thymocytes. Among them, FcγRIIB, whose signaling can inhibit ERK activation, was strongly and ectopically expressed in ESET−/− thymocytes. Indeed, genetic depletion of FcγRIIB in ESET−/− thymocytes rescued impaired ERK activation and partially restored defective positive selection in ESET−/− mice. Therefore, impaired T cell development in ESET−/− mice is partly due to the aberrant expression of FcγRIIB. Collectively, to our knowledge, we identify ESET as the first trimethylated H3K9 histone methyltransferase playing a crucial role in T cell development.

Lung cancer is one of the most common malignant diseases and the leading cause of cancer death globally. The clinical, pathologic, and genomic characteristics of lung cancer are very diverse. Non-small cell lung cancer (NSCLC) is the most frequent type of lung cancers and can be histologically classified into lung adenocarcinoma and squamous cell carcinoma (SCC). Lung adenocarcinoma is generally located in alveolar or endobronchial, whereas LSCC frequently arises in bronchi and bronchiole. Molecular-targeted drugs have been developed for lung adenocarcinoma; however, there is currently no definite clinical implication for lung SCC (LSCC). Therefore, to increase the cure and survival rates of lung cancers, developing type-specific diagnostic/prognostic markers and treatment methods might improve this situation. One of the major risk factors of LSCC is smoking, which causes aberrant DNA methylation, abnormal gene expression, and eventually malignant transformation. We previously identified glutathione S-transferase omega 2 (GSTO2) as one of DNA methylation target genes in esophageal SCC (Otsubo T, Oncotarget, 8, 84434-84448, 2017). We reported that GSTO2 was exclusively expressed in nethermost basal cells which are believed to function as stem cells in normal esophageal mucosa. Furthermore, GSTO2 overexpression in esophageal SCC cells significantly inhibited cell growth in vitro and in vivo. (Terayama M, Carcinogenesis, in press). In present study, we investigated the expression and function of GSTO2 in normal lung and LSCC. In normal bronchi and bronchiole containing a variety of epithelial cell populations such as ciliated cells, non-ciliated, columnar Clara cells, and basal cells, GSTO2 was expressed in cytokeratin 5/6-positive airway basal cells. In contrast, all 94 LSCC specimens examined in this study showed no GSTO2 expression. To clarify the significance of GSTO2 silencing LSCC, we restored GSTO2 expression in LK-2 LSCC cells. Overexpression of GSTO2 significantly inhibited cell growth compared with the mock-transfected LK-2 cells. In colony formation assay, GSTO2-transfected cells failed to form colonies, whereas mock-transfected cells formed colonies efficiently. When human LSCC cell lines (LK-2 and EBC-1) were treated with 5-aza-2'-deoxycytidine (5-aza-dC), a DNA-methyltransferase inhibitor, the GSTO2 transcription was induced, indicating that aberrant hypermethylation of the GSTO2 in LSCC was likely the cause of the downregulated expression. Considering its expression in basal cells which are proposed to be the precursor cells of LSCC, the silencing of GSTO2 by DNA hypermethylation may contribute malignant transformation of LSCC. Citation Format: Ryusuke Sumiya, Masayoshi Terayama, Teruki Hagiwara, Keigo Sekihara, Satoshi Nagasaka, Kazuhiko Yamada, Norihiro Kokudo, Yuki I. Kawamura. Glutathione S transferase omega 2 (GSTO2) is a novel tumor suppressor gene of human lung squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4716.

Surgery for esophageal carcinoma is known to be associated with high morbidity. Recent studies have reported a correlation of nutritional and inflammatory parameters with postoperative course. This study aims to clarify the risk factors for operative morbidity after resection of esophageal carcinoma. Consecutive patients who underwent esophagectomy for esophageal squamous cell carcinoma at our institute were included (n = 102; 89 males and 13 females; mean age: 67.3 years). Clinicopathological characteristics, presence or absence of sarcopenia, and modified Glasgow prognostic score were assessed, and their correlation with postoperative complications was investigated using univariate and multivariate analyses. Sarcopenia was defined using a combination of muscle mass area and body mass index. Of the included 102 patients, 45 (44.1%) exhibited sarcopenia (sarcopenia group), while 57 (55.9%) did not (non-sarcopenia group). No significant difference was observed between the groups regarding surgical procedures and tumor stage; furthermore, there was no mortality. Twenty-six patients developed respiratory complications (including 20 cases of pneumonia). On univariate analysis, sarcopenia, modified Glasgow prognostic score, and American Society of Anesthesiologists physical status were found to be significantly associated with the development of postoperative respiratory complications. On multivariate analysis, sarcopenia was found to be an independent risk factor for postoperative respiratory complications after esophagectomy. We believe that identifying patients at risk and providing preoperative nutritional support as well as physical therapy aimed at strengthening of body muscles may help reduce the incidence of postoperative respiratory complications in such patients.

In the original publication of the article, Fig. 1 was published incorrectly.

We previously conducted transcriptome analysis of a paired specimen of normal and esophageal squamous cell carcinoma (ESCC) tissues and found that mRNA expression of cystatin A (CSTA), a member of the cystatin superfamily, was perturbed in tumors compared with that in the background mucosa. However, little is known about the significance of CSTA expression in ESCC. The mRNA expression of CSTA was evaluated by qRT-PCR using 28 paired frozen samples of tumor and nontumor mucosae. The protein expression of CSTA was evaluated by the immunostaining of formalin-fixed, paraffin-embedded sections of ESCC samples from 59 patients who underwent surgery, and its relationship with clinical features was analyzed. The mRNA expression of CSTA was significantly decreased in ESCC compared with that in matched normal mucosa (P

Our previous differential transcriptome analysis between a paired specimen of normal and esophageal squamous cell carcinoma (ESCC) tissues found aberrant expression of kallikrein-related peptidase 13 (KLK13) in tumors. In this study, we evaluated the expression of KLK13 in many ESCC cases in relation with clinical features, and the prognosis. Eighty-eight ESCC cases were subjected to immunohistological staining for KLK13 and classified into KLK13-negative and KLK13-positive groups. Difference of clinical features and the prognosis between the groups was analyzed. In normal esophageal mucosa, KLK13 expression was evident but limited in the stratum granulosum in all cases. By contrast, only 27 of 88 ESCC samples showed KLK13 expression, whereas the remaining 61 tumors showed no KLK13 expression. The KLK13-positive group was significantly associated with pT classification (deeper tumor invasions; P = 0.0282), pN classification (lymph node metastasis; P = 0.0163), and advanced TNM stage (P = 0.0198). In KLK13-positive samples, KLK13-expressing cells often expressed Ki67, a proliferation marker, unlike normal mucosa, in which Ki67-expressing cells were limited to the basal layer and did not express KLK13. Compared with patients with KLK13-negative group, KLK13-positive group showed poorer postoperative prognosis. Relatively high levels of KLK13 expression in ESCC were associated with cell proliferation and correlated with tumor progression, advanced cancer stage, and poor prognosis.

Periplakin (PPL), a member of the plakin family of proteins that localizes to desmosomes and intermediate filaments, is downregulated in human esophageal squamous cell carcinoma (ESCC). Little is known, however, about the molecular mechanism underlying the regulation of PPL expression and the contribution of PPL loss to the malignant property of the cancer is unclear. We demonstrated that PPL mRNA expression was significantly reduced in ESCC tissues compared with that in normal tissues. Therefore, we hypothesized that CpG hypermethylation is the cause of the downregulation of PPL. Bisulfite-pyrosequencing of 17 cases demonstrated that the frequency of PPL methylation was higher in ESCC tissues than in normal tissues. When human ESCC cell lines were treated with 5-aza-2′-deoxycytidine (5-aza-dC), a DNA-methyltransferase inhibitor, PPL transcription was induced. Human KYSE270 ESCC cells do not stratify under ordinary culture conditions and rarely produce desmosomes; however, the forced expression of PPL promoted cell stratification. PPL induction also promoted adhesion to extracellular matrix but delayed cell migration. The abundance of desmosome-like structures was greatly increased in PPL transfectant as determined by transmission electron microscopy. Very low expression of another desmosome protein EVPL in ESCC, even in PPL transfectant, also supported the significant role of PPL in desmosome formation and cell stratification. Our results first indicate that the downregulation of PPL mediated by DNA hypermethylation, which may play an important role in the loss of ESCC stratification and likely in metastatic phenotype.

Scientific Data, 4, ISSN:2052-4463

Fasting-refeeding in mice induces transient hyperproliferation of colonic epithelial cells, which is dependent on the lactate produced as a metabolite of commensal bacteria. We attempted to manipulate colonic epithelial cell turnover with intermittent fasting to prompt recovery from acute colitis. Acute colitis was induced in C57BL/6 mice by administration of dextran sulfate sodium in the drinking water for 5 days. From day 6, mice were fasted for 36 h and refed normal bait, glucose powder, or lactylated high-amylose starch. On day 9, colon tissues were subjected to analysis of histology and cytokine expression. The effect of lactate on the proliferation of colonocytes was assessed by enema in vivo and primary culture in vitro. Intermittent fasting resulted in restored colonic crypts and less expression of interleukin-1β and interleukin-17 in the colon than in mice fed ad libitum. Administration of lactate in the colon at refeeding time by enema or by feeding lactylated high-amylose starch increased the number of regenerating crypts. Addition of lactate but not butyrate or acetate supported colony formation of colonocytes in vitro. In conclusion, intermittent fasting in the resolution phase of acute colitis resulted in better recovery of epithelial cells and reduced inflammation.

Background Our previous report showed that periplakin (PPL), a member of the plakin family of proteins, was expressed in all the normal esophageal squamous cells, except the Ki67 + basal cell layer; however, PPL-negative cells were observed in esophageal squamous cell carcinoma (ESCC) tissue samples and the proportion of PPL-positive areas in tumors showed considerable variation. In this study, we analyzed the relationships between PPL expression in tumors and the clinicopathological features of ESCC. Methods PPL expression was evaluated by immunostaining ESCC samples from 70 patients who underwent surgery and we classi ed the samples into PPL-negative and PPL-positive groups based on the proportion of PPL-positive areas in tumors, and the relationships among PPL expression, clinical features, and the ESCC prognosis were analyzed. ESCC cell line KYSE270 cells were stably transfected with the PPL expression vector and their growth was assessed by colony formation assay and subcutaneous xenografts in athymic mice. Results As found in our previous study, decreased PPL staining intensity was observed in all of the cancer tissues analyzed, compared with that observed in paired normal esophageal mucosae; however, the PPL-positive group (the percentage of PPL-positive tumor cells was > 20% on immunostaining) exhibited positive associations with the pT classi cation (larger primary tumor) (P = 0.029), pN classi cation (lymph node metastasis) (P = 0.0462), and advanced stages of cancer (P = 0.0253). High PPL expression was observed in all of 26 lymph node metastases analyzed. Furthermore, patients with PPL-positive tumors showed poor postoperative prognosis compared to those with PPL-negative ones. Forced expression of PPL in the KYSE270 ESCC cell line induced a strati ed structure and colony formation. In addition, PPL-transfected cells formed larger tumors in nude mice than mock-transfected cells, suggesting that relatively high PPL expression in tumors may facilitate cell-cell adhesion by desmosome formation and eventual cell growth in vivo. Conclusion PPL expression was generally reduced in ESCC compared with paired non-cancer tissue; however, relatively high levels of PPL expression in tumors correlated with tumor progression, lymph node metastasis, advanced stage cancer, and a poor prognosis. Disclosure All authors have declared no conflicts of interest.

We recently found that chemokine-driven peritoneal cell aggregation is the primary mechanism of postoperative adhesion in a mouse model. To investigate this in humans, paired samples of peritoneal lavage fluid were obtained from seven patients immediately after incision (preoperative) and before closure (postoperative), and were assayed for the presence of 27 cytokines and chemokines using multiplex beads assay. As a result, IL-6 and CCL5 showed the most striking increase during operation. Recombinant CCL5 or lavage fluid induced chemotaxis of human peripheral blood mononuclear cells. We propose that CCL5 is possibly involved in the mechanism of postoperative adhesion in humans.

Background & Aims Epithelial cells that cover the intestinal mucosal surface maintain immune homeostasis and tolerance in the gastrointestinal tract. However, little is known about the molecular mechanisms that regulate epithelial immune functions. Epithelial cells are distinct in that they are highly polarized; this polarity is, at least in part, established by the epithelium-specific polarized sorting factor adaptor protein (AP)-1B. We investigated the role of AP-1B–mediated protein sorting in the maintenance of gastrointestinal immune homeostasis. Methods The role of AP-1B in intestinal immunity was examined in AP-1B–deficient mice ( Ap1m2 −/− ) by monitoring their phenotypes, intestinal morphology, and epithelial barrier functions. AP-1B–mediated protein sorting was examined in polarized epithelial cells from AP-1B knockdown and Ap1m2 −/− mice. Results Ap1m2 −/− mice developed spontaneous chronic colitis, characterized by accumulation of interleukin-17A–producing, T-helper 17 cells. Deficiency of AP-1B caused epithelial immune dysfunction, such as reduced expression of antimicrobial proteins and impaired secretion of immunoglobulin A. These defects promoted intestinal dysbiosis and increased bacterial translocation within the mucosa. Importantly, AP-1B deficiency led to mistargeting of a subset of basolateral cytokine receptors to the apical plasma membrane in a polarized epithelial cell line and in colonic epithelial cells from mice. AP1M2 expression was reduced significantly in colonic epithelium samples from patients with Crohn's disease. Conclusions AP-1B is required for proper localization of a subset of cytokine receptors in polarized epithelial cells, which allows them to respond to cytokine signals from underlying lamina propria cells. The AP-1B–mediated protein sorting machinery is required for maintenance of immune homeostasis and prevention of excessive inflammation.

Background Alcohol drinking and smoking are substantial risk factors of esophageal squamous cell carcinoma (ESCC) and are supposed to induce genetic mutations and epigenetic disorders, including aberrant DNA methylation. Previously, we have conducted transcriptome and methylome analyses of a paired specimen of ESCC and adjacent non-cancerous tissues and found that both gene expression and promotor methylation of PRSS27 were perturbed in ESCC. PRSS27 was a trypsin-like serine protease (also known as marapsin) and expressed in normal esophagus; however, little is known about the significance of PRSS27 expression in ESCC. In this study, we evaluated the expression of PRSS27 in many ESCC cases in relation with clinical features and the prognosis. Methods ESCC tissue specimens were obtained from 80 patients who had undergone esophagectomy between April 2008 and December 2016 in our hospital and were subjected to immunostaining for PRSS27. ESCC cases were classified into PRSS27-negative and PRSS27-positive groups and difference of clinical features and the prognosis between the groups was analyzed. Results The mRNA expression of PRSS27 was significantly decreased in ESCC compared with those in matched normal mucosa (P Conclusion PRSS27 gene and protein expression was both downregulated in ESCC; its functional significance in relation to malignancy is underinvestigation. Disclosure All authors have declared no conflicts of interest.

Background Epigenetic changes such as DNA methylation are involved in the onset of ESCC. Previously, we have conducted transcriptome and methylome analyses using a paired specimen of ESCC and adjacent non-cancerous tissues and found promotor hypomethylation of NT5E with altered gene expression level in ESCC. NT5E has been known to be an extracellular nucleoside metabolism-related enzyme; however, the significance of its expression in ESCC has never been clarified. In this study, we aimed to evaluate the ESCC-related alteration of the expression of NT5E in using many cases of ESCC. Methods Formalin-fixed, paraffin-embedded sections of surgical specimens from 67 ESCC patients who had undergone esophagectomy or endoscopic submucosal dissection between April 2008 and December 2015 in our hospital were immunostained using anti-NT5E antibody, and classified into NT5E-negative and NE5E-positive groups. To evaluate the mRNA expression of NT5E, qRT-PCR was performed using paired normal and ESCC frozen tissue samples from 24 patients. Results Compared with matched normal mucosa, cancer tissues showed significantly higher NT5E mRNA expression (P Conclusion NT5E gene and protein expression was both upregulated in ESCC; its functional significance in relation to malignancy is underinvestigation. Disclosure All authors have declared no conflicts of interest.

Cancer-associated changes in cell surface carbohydrates, including incomplete synthesis of normal carbohydrate epitopes, strongly affect malignant and metastatic potential. Here, we report that compensating for the cancer-associated loss of a single glycosyltransferase, β1,4 N -acetylgalactosaminyltransferese T2, dramatically decreased cell surface expression of both E-selectin ligands (sialyl Lewis x and sialyl Lewis a ). This modification was associated with elevated expression of the Sd a carbohydrate determinant, which is expressed in normal gastrointestinal mucosa and is strikingly downregulated in cancer tissues. Loss of E-selectin ligands resulted in decreased adhesion of cancer cells to activated human endothelial cells in vitro and eventually suppressed metastatic potential in vivo .

Although expression of the fractalkine (CX3CL1, FKN) is enhanced in inflamed tissues, it is detected at steady state in various organs such as the intestine, and its receptor CX3CR1 is highly expressed in resident-type dendritic cells and macrophages. We hypothesized that FKN might regulate the inflammatory responses of these cells. Therefore, murine macrophages were pretreated with FKN and then stimulated with LPS. We found that macrophages pretreated with 0.03 nM FKN but not with 3 nM FKN secreted 50% less TNF-α than did cells treated with LPS alone. Cells treated with 0.03 nM FKN and LPS also showed reduced phosphorylation of ERK1/2 and reduced NF-κB p50 subunit. Interestingly, the p65 subunit of NF-κB was translocated to the nuclei but redistributed to the cytoplasm in the early phase by forming a complex with peroxisome proliferator-activated receptor (PPAR) γ. Exogenous 15-deoxy-Δ(12,14)-prostaglandin J2, a natural ligand for PPAR-γ, also induced redistribution of p65 with decreased TNF-α secretion after LPS challenge. Pretreatment with 0.03 nM but not 3 nM FKN increased the cellular levels of 15-deoxy-Δ(12,14)-prostaglandin J2 as well as mRNA of PPAR-γ. Requirement of PPAR-γ for the effect of 0.03 nM FKN was confirmed by small interfering RNA of PPAR-γ. In contrast, pretreatment with 3 nM FKN induced higher levels of IL-23 compared with cells pretreated with 0.03 nM FKN and produced TNF-α in a CX3CR1-dependent manner. These dose-dependent differential effects of FKN establish its novel role in immune homeostasis and inflammation.

Peritoneal adhesions are a significant complication of surgery and visceral inflammation; however, the mechanism has not been fully elucidated. The aim of this study was to clarify the mechanism of peritoneal adhesions by focusing on the cell trafficking and immune system in the peritoneal cavity. We investigated the specific recruitment of peritoneal macrophages (PMφ) and their expression of chemokine receptors in murine models of postoperative and postinflammatory peritoneal adhesions. PMφ aggregated at the site of injured peritoneum in these murine models of peritoneal adhesions. The chemokine receptor CCR8 was up-regulated in the aggregating PMφ when compared with naive PMφ. The up-regulation of CCR8 was also observed in PMφ, but not in bone marrow-derived Mφ, treated with inflammatory stimulants including bacterial components and cytokines. Importantly, CCL1, the ligand for CCR8, a product of both PMφ and peritoneal mesothelial cells (PMCs) following inflammatory stimulation, was a potent enhancer of CCR8 expression. Cell aggregation involving PMφ and PMCs was induced in vitro in the presence of CCL1. CCL1 also up-regulated mRNA levels of plasminogen activator inhibitor-1 in both PMφ and PMCs. CCR8 gene-deficient mice or mice treated with anti-CCL1-neutralizing Ab exhibited significantly reduced postoperational peritoneal adhesion. Our study now establishes a unique autocrine activation system in PMφ and the mechanism for recruitment of PMφ together with PMCs via CCL1/CCR8, as immune responses of peritoneal cavity, which triggers peritoneal adhesions.

Group 2 innate lymphoid cells (ILC2s) produce a significant amount of interleukin-5 (IL-5), which supports eosinophil responses in various tissues; they also produce IL-13, which induces mucus production and contributes to tissue repair or fibrosis. The ILC2s are activated by alarmins, such as IL-33 released from epithelia, macrophages and natural killer T (NKT) cells in response to infection and allergen exposure, leading to epithelial injury. We examined gene expression in lung ILC2s and found that ILC2s expressed Ifngr1, the receptor for interferon-γ (IFN-γ). Interferon-γ severely inhibited IL-5 and IL-13 production by lung and kidney ILC2s. To evaluate the effects in vivo, we used α-galactosylceramide (α-GalCer) to induce NKT cells to produce IL-33 and IFN-γ. Intraperitoneal injection of α-GalCer in mice induced NKT cell activation resulting in IL-5 and IL-13 production by ILC2s. Administration of anti-IFN-γ together with α-GalCer significantly enhanced the production of IL-5 and IL-13 by ILC2s in lung and kidney. Conversely, cytokine production from ILC2s was markedly suppressed after injection of exogenous IL-33 in Il33(-/-) mice pre-treated with α-GalCer. Hence, IFN-γ induced or already present in tissues can impact downstream pleiotropic functions mediated by ILC2s, such as inflammation and tissue repair.

Background: Prognosis of Kawasaki disease and its main complications, coronary artery aneurysm (CAA) and myocardial ischemia were remarkably improved by intravenous immunoglobulin (IVIG) and other additional anti-inflammatory and anti-coagulation therapy. In contrast, rupture of coronary artery aneurysm is a rare, and still definitely fatal complication. Though approximately 10 cases were reported with sporadic occurrence in every three to four years in Japan, there is no patient whose life was saved. Case report: We experienced a 5-year-old boy who had already developed left CAA of 9 mm in diameter on the 7th day from onset of fever. Despite of repeated IVIG, left CAA was rapidly expanding day by day and finally the diameter became 18mm on the 12th day. Early in the next morning, sudden cardiac arrest was noted, but resuscitation was not effective. Autopsy revealed cardiac tamponade caused by rupture of very fragile wall of huge left CAA. Discussions: After this experience, we have tried to carry patients into the intensive care unit with deep sedation, if patient’s CAA is expanding more than 10mm within 2 weeks in acute stage of Kawasaki disease. Furthermore, we also recommend use of antihypertensive drugs such as calcium channel blocker and/or beta-blockers. We also have advised to colleagues in other hospitals to try in the same way when they asked for the management of similar cases. At least three patients with huge aneurysm were survived with this method in recent reports. If patient has strong inflammation sustaining with expanding huge aneurysm, plasma exchange and all possible anti-inflammatory agents including steroid or infliximab under the percutaneous cardiopulmonary support and surgeons' stand-by until inflammation will disappear. Following repair or platy of coronary aneurysm and coronary artery bypass surgery may be considered, but possibility and results are unknown. Conclusions: Because of its rarity, it is difficult to detect exact indication of deep sedation or intensive care, however, earlier and more cautious management will be safer for ‘super-giant’ aneurysm. Concerning to the sustaining inflammation with huge aneurysm, we would like to hear advices for this strategy from cardiac surgeons and pediatric intensivists.

Long interspersed element-1 (L1) is a transposable element that can move within the genome, potentially leading to genome diversity and modified gene function. Although L1 activity in somatic cells is normally suppressed through DNA methylation, some L1s are activated in tumors including colorectal carcinoma. However, how L1-retrotransposition (L1-RTP) is involved in gastrointestinal disorders remains to be elucidated. We hypothesized that L1-RTP in somatic cells might contribute to colitis-associated cancer (CAC). To address this, we employed an experimental model of CAC using transgenic L1-reporter mice carrying a human L1-EGFP reporter gene. Mice were subjected to repeated cycles of colitis induced by administration of dextran sodium sulfate (DSS) in drinking water with injection of carcinogen azoxymethane (AOM). L1-RTP levels were measured by a quantitative polymerase chain reaction targeting the newly inserted reporter EGFP in various tissues and cell types, including samples obtained by laser microdissection and cell sorting with flow cytometry. DNA methylation levels of the human L1 promoter were analyzed by bisulfite pyrosequencing. AOM+DSS-treated mice exhibited significantly higher levels of L1-RTP in whole colon tissue during the acute phase of colitis when compared with control naive mice. L1-RTP levels in whole colon tissue were positively correlated with the histological severity of colitis and degree of neutrophil infiltration into the lamina propria (LP), but not with tumor development in the colon. L1-RTP was enriched in LP mesenchymal cells rather than epithelial cells (ECs), myeloid, or lymphoid cells. DNA methylation levels of the human L1 promoter region showed a negative correlation with L1-RTP levels. L1-RTP was absent from most tumors found in 22-week-old mice. In conclusion, we demonstrated that L1-RTP was induced in the mouse CAC mucosa in accordance with the acute inflammatory response; however, retrotransposition appears not to have direct relevance to colitis-induced cancer initiation.

Colon cancer is one of the most serious complications of inflammatory bowel diseases, especially ulcerative colitis (UC). Previous studies have shown that characteristic immunological event during inflammation in UC is the expression of T helper-type 2 (Th2) cell-derived cytokines. In this study, we investigated the influence of a predominant Th2-type cytokine response in colitis on carcinogen-induced colon tumors. Wild type (WT), interferon gamma (IFN-gamma) gene deficient (-/-) [Th2 dominant] or interleukin (IL)-4(-/-) [Th1-dominant] mice of BALB/c background were used in this study. To compare tumor formation, mice were given the carcinogen azoxymethane (AOM) and intrarectal administration of trinitrobenzene sulfonic acid (TNBS), to induce colitis. Thirty-three weeks after initial treatment, the total colon was examined. When IFN-gamma(-/-) mice were treated with AOM and TNBS, significantly higher number of tumors were seen (8.4 +/- 1.7) than in WT (3.3 +/- 2.9) or IL-4(-/-) (3.1 +/- 3.4) mice, which received identical treatments. A separate set of experiment, using less doses of AOM and TNBS also showed the higher frequency of tumor formation in IFN-gamma(-/-) mice than in IL-4(-/-) mice. Histologically, the tumors were well- or moderately-differentiated adenocarcinomas. No invasion into the submucosal or serosal layers of the intestine was seen. In immunohistological staining, some tumors in IFN-gamma(-/-) mice showed distinct nuclear expression of beta-catenin, in contrast to the strong membrane staining seen in tumors of IL-4(-/-) mice. In conclusion, colonic inflammation associated with Th2-dominant cytokine responses enhanced the formation of malignant neoplasms.

Background & Aims: Activins belong to the transforming growth factor-β superfamily. Recent studies have shown that activin and its natural antagonist, follistatin, are involved in tissue repair and inflammatory processes. The aim of this study was to determine whether neutralization of activins with follistatin would have an in vivo anti-inflammatory effect in several murine models of colitis. Methods: We assessed activin levels in the colitis induced by intracolonic administration of trinitrobenzene sulfonic acid (TNBS). We subsequently tested the effects of an intraperitoneal injection of follistatin before or after induction of TNBS colitis. We also examined the established colitis induced by oral dextran sulfate sodium (DSS) as well as the spontaneous colitis that develops in interleukin (IL)-10 gene-deficient (IL-10−/−) mice. Results: Levels of activin transcripts in the colon during the acute phase of TNBS colitis were up-regulated. Epithelial cells, infiltrating macrophages (Mϕ), and endothelial cells produced excess activin βA. Pretreatment with follistatin increased the survival rate of mice with TNBS colitis from 33% to 82% and decreased the plasma levels of IL-6 and amyloid A. Administration of follistatin also reduced the histologic score and tissue myeloperoxidase activity in established TNBS and DSS colitis and reduced the severity of the colitis in IL-10−/− mice. Based on results obtained from 3 mouse models and from in vitro experiments, follistatin promoted the proliferation of colonic epithelial cells. Conclusions: Neutralization of activins by follistatin promoted epithelial cell division and tissue repair, clearly suggesting a treatment modality for intestinal inflammation.

We have analyzed the gastrointestinal inflammation which develops following adoptive transfer of IL-4 gene knockout (IL-4(-/-)) CD4(+)CD45RB(Hi) (RB(Hi)) T cells to severe combined immunodeficient (SCID) or to T cell-deficient, T cell receptor beta and delta double knockout (TCR(-/-)) mice. Transfer of IL-4(-/-) RB(Hi) T cells induced a similar type of colitis to that seen in SCID or TCR(-/-) recipients of wild-type (wt) RB(Hi) T cells as reported previously. Interestingly, transfer of both wt and IL-4(-/-) RB(Hi) T cells to TCR(-/-) but not to SCID mice induced inflammation in the gastric mucosa. Notably, TCR(-/-) recipients of IL-4(-/-) RB(Hi) T cells developed a more severe gastritis with erosion, apoptosis of the antral epithelium, and massive infiltration of macrophages. This gastritis was partially dependent on the indigenous microflora. Recipients of both wt and IL-4(-/-) RB(Hi) T cells developed duodenitis with multinuclear giant cells, expansion of mucosal macrophages, and dendritic cells. Full B cell responses were reconstituted in TCR(-/-) recipients of RB(Hi) T cells; however, anti-gastric autoantibodies were not detected. We have now developed and characterized a novel model of chronic gastroduodenitis in mice, which will help in our understanding of the mechanisms involved in chronic inflammation in the upper gastrointestinal tract of humans.

Background & Aims: T helper (Th) 1 and Th2 cell subsets significantly influence the pathological features of inflammation in the gastrointestinal tract in a distinct manner. It is now established that the transfer of CD4 + CD45RB Hi (RB Hi ) T cells to either severe combined immunodeficient (SCID) or recombinase activation gene 2–deficient (RAG −/− ) mice results in a severe granulomatous hypertrophic colitis mediated by Th1 cells. We have modified this approach to address the role of Th2 cells. Methods: RB Hi T cells from wild-type (Wt) mice or mice genetically predisposed to Th2 responses (interferon-γ–defective [IFN-γ −/− ]) with or without B cells were transferred to T cell receptor (TCR)-β and δ-chain–defective (TCR −/− ) or SCID mice. Results: Transfer of Wt RB Hi T cells induced wasting disease with severe colitis in the TCR −/− mice. In contrast, IFN-γ −/− RB Hi T cells induced severe weight loss and hypoalbuminemia without significant inflammation in the colon. The small intestine of these mice exhibited villus atrophy, a decrease in brush-border enzymes, reduced enterocyte proliferation, and an increased number of goblet cells. The presence of B cells was necessary for these changes, because SCID recipients required cotransfer of B cells, together with IFN-γ −/− RB Hi T cells for ileal lesions to develop. Treatment of TCR −/− recipients of IFN-γ −/− RB Hi T cells with anti–IL-4 mAb abrogated both the wasting disease and the villus atrophy. Conclusions: Dysregulated Th2 cells cause atrophic changes and goblet cell transformation in the small intestinal epithelium and wasting disease mediated by excess interleukin-4 and B cells. GASTROENTEROLOGY 2003;124:672-682

Aim To clarify the roles of TWEAK and its receptor Fn14 in 5-fluorouracil (5-FU)-induced diarrhea. Methods Diarrhea was induced in wild-type (WT), Fn14 knockout (KO), and IL-13 receptor (IL-13R)α1 KO BALB/c mice using a single injection of 5-FU. Histological analysis, cytokine analysis, and flow cytometry was performed on ileal tissues and cells. Murine colon carcinoma-bearing mice were co-treated with an anti-TWEAK antibody and 5-FU. Embryonic fibroblast response to cytokines was also analyzed. Results 5-FU induced high Fn14 expression in epithelial cells. The severity of 5-FU-induced diarrhea was lower in Fn14 KO mice compared with WT mice. Administration of anti-TWEAK antibody reduced 5-FU-induced diarrhea without affecting the antitumor effects of 5-FU in vivo. 5-FU-induced expression of IL-13, IL-17A, TNF-α, and IFN-γ in the ileum was Fn14 dependent. The severity of 5-FU-induced diarrhea was lower in IL-13Rα1 KO mice, indicating major role for IL-13 signaling via IL-13Rα1 in pathogenesis. We found that IL-13Rα2, an IL-13 neutralizing/cell protective receptor, was strongly induced by IL-33 in vitro and in vivo. IL-13Rα2 was upregulated in the ileum of 5-FU-treated Fn14 KO mice. Thus, the deletion of Fn14 upregulated IL-13Rα2 expression, which reduced IL-13 expression and activity. Conclusion Disruption of the TWEAK/Fn14 pathway affects several interconnected pathways, including those associated with IL-13, IL-33, and IL-13Rα2, to attenuate 5-FU-induced intestinal side effects.

Tumor necrosis factor (TNF)-α is a major effector in various inflammatory conditions. TNF-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily that promotes inflammatory tissue damage through its receptor, FGF-inducible molecule 14 (Fn14). Since both TWEAK and TNF-α have been shown to mediate pathological responses through inter-dependent or independent pathways by in vitro, the potential interplay of these pathways was investigated in a mouse colitis model. Acute colitis was induced by rectal injection of trinitrobenzene sulfonic acid (TNBS), with administration of control IgG, TNF receptor (TNFR)-Ig chimeric protein, anti-TWEAK monoclonal antibody, or the combination of TNFR-Ig and anti-TWEAK antibody. On day 4, disease severity was evaluated and gene expression profiling was analyzed using whole colon tissue. NF-κB activation was investigated with Western blot. Levels of transcript of TWEAK, Fn14 and NF-κB-related molecules were measured in purified colon epithelial cells (ECs). As a result, activation of the canonical (p50/RelA), but not noncanonical (p100/RelB)-mediated pathway was the hallmark of inflammatory responses in this model. Inflammation induced upregulation of Fn14 only in ECs but not in other cell types. Combination treatment of TNFR-Ig and anti-TWEAK antibody synergistically reduced disease severity in comparison with the control antibody or single agent treatment. Gene expression profile of the colon indicated downregulation of canonical NF-κB pathway with combination treatment. In conclusion, synergistic activation of canonical NF-κB by TWEAK and TNF-α is critical for the induction of inflammatory tissue damage in acute inflammation.

Intestinal regulatory T cells (Treg cells) are necessary for the suppression of excessive immune responses to commensal bacteria. However, the molecular machinery that controls the homeostasis of intestinal Treg cells has remained largely unknown. Here we report that colonization of germ-free mice with gut microbiota upregulated expression of the DNA-methylation adaptor Uhrf1 in Treg cells. Mice with T cell-specific deficiency in Uhrf1 (Uhrf1(fl/fl)Cd4-Cre mice) showed defective proliferation and functional maturation of colonic Treg cells. Uhrf1 deficiency resulted in derepression of the gene (Cdkn1a) that encodes the cyclin-dependent kinase inhibitor p21 due to hypomethylation of its promoter region, which resulted in cell-cycle arrest of Treg cells. As a consequence, Uhrf1(fl/fl)Cd4-Cre mice spontaneously developed severe colitis. Thus, Uhrf1-dependent epigenetic silencing of Cdkn1a was required for the maintenance of gut immunological homeostasis. This mechanism enforces symbiotic host-microbe interactions without an inflammatory response.