Yuki I. Kawamura

Elimination of Colonic Patches with Lymphotoxin β Receptor-Ig Prevents Th2 Cell-Type Colitis

Past studies have shown that colonic patches, which are the gut-associated lymphoreticular tissues (GALT) in the colon, become much more pronounced in hapten-induced murine colitis, and this was associated with Th2-type T cell responses. To address the role of GALT in colonic inflammation, experimental colitis was induced in mice either lacking organized GALT or with altered GALT structures. Trinitrobenzene sulfonic acid was used to induce colitis in mice given lymphotoxin-β receptor-Ig fusion protein (LTβR-Ig) in utero, a treatment that blocked the formation of both Peyer’s and colonic patches. Mice deficient in colonic patches developed focal acute ulcers with Th1-type responses, whereas lesions in normal mice were of a diffuse mucosal type with both Th1- and Th2-type cytokine production. We next determined whether LTβR-Ig could be used to treat colitis in normal or Th2-dominant, IFN-γ gene knockout (IFN-γ−/−) mice. Four weekly treatments with LTβR-Ig resulted in deletion of Peyer’s and colonic patches with significant decreases in numbers of dendritic cells. This pretreatment protected IFN-γ−/− mice from trinitrobenzene sulfonic acid-induced colitis; however, in normal mice this weekly treatment was less protective. In these mice hypertrophy of colonic patches was seen after induction of colitis. We conclude that Th2-type colitis is dependent upon the presence of colonic patches. The effect of LTβR-Ig was mediated through prevention of colonic patch hypertrophy in the absence of IFN-γ. Thus, LTβR-Ig may offer a possible treatment for the Th2-dominant form of colitis.

The epigenetic regulator Uhrf1 facilitates the proliferation and maturation of colonic regulatory T cells

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 (Uhrf1fl/flCd4-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, Uhrf1fl/flCd4-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.

Cholera toxin activates dendritic cells through dependence on GM1-ganglioside which is mediated by NF-κB translocation

Cholera toxin (CT) is a potent adjuvant; however, the mechanism for its ability to enhance mucosal immunity has not been fully elucidated. We report here that CT exerts its adjuvant propertiesby signaling through the GM1 ganglioside receptor. When ganglioside‐defective mice were given the antigen (Ag) ovalbumin (OVA) with CT by the oral route, CT failed to support either OVA‐specific antibody or CD4+ T cell responses. In vitro treatment of murine bone marrow‐derived dendritic cells (DC) with CT induced full maturation as evidenced by up‐regulation of the costimulatory molecules, as well as by an enhanced ability to effectively present OVA for Ag‐specific T cell responses. On the other hand, ganglioside‐defective DC failed to differentiate to full function as Ag‐presenting cells in response to CT. Since ganglioside‐defective DC showed a mature phenotype after stimulation with lipopolysaccharide (LPS), the effects of CT on DC was independent of signal transduction through adjuvant receptor for LPS, the Toll‐like receptor 4. Furthermore, CT also induced nuclear translocation of nuclear factor (NF)‐κB in DC in a GM1‐dependent fashion. These results highlight gangliosides expressed by DC for recognition of the non‐self protein bacterial enterotoxin, which employ a unique signaling pathway to induce both innate and adaptive immunity.

Introduction of Sda Carbohydrate Antigen in Gastrointestinal Cancer Cells Eliminates Selectin Ligands and Inhibits Metastasis

The Sd(a) blood group carbohydrate structure is expressed in the normal gastrointestinal mucosa. We reported previously that the expression of Sd(a) carbohydrate structures and beta1,4-N-acetylgalactosaminyltransferase (beta1,4GalNAcT) activity responsible for Sd(a) synthesis were remarkably decreased in cancer lesions of the gastrointestinal tract. In this study, we found that Sd(a) antigen was expressed mainly in chief cells of normal stomach but not in cancer tissue by immunohistologic staining. In separated gastric mucosal cells, the Sd(a) glycolipids and beta1,4GalNAcT activity were concentrated in a fraction that contained chief cells as a major population. We cloned the cDNA encoding the glycosyltransferase that catalyzes the synthesis of Sd(a) (Sd(a)-beta1,4GalNAcT). Introduction of this cloned cDNA into KATO III gastric or HT29 colonic cancer cell lines, which originally expressed the E-selectin ligands, sialyl Lewis(x) and sialyl Lewis(a), resulted in a marked increase in cell-surface expression of Sd(a) along with the concomitant total loss of both sialyl Lewis(x) and sialyl Lewis(a). Both KATO III and HT29 cells transfected with the Sd(a)-beta1,4GalNAcT gene showed significantly decreased adhesion to activated human umbilical vein endothelial cells when compared with mock-transfected cells. Sd(a) determinants showed no direct binding to Siglec-3, -5, -7, and -9. These Sd(a)-beta1,4GalNAcT-transfected cells showed strikingly reduced metastatic potential in vivo when compared with mock-transfected cells. In summary, forced expression of Sd(a) carbohydrate determinant caused remarkable elimination of carbohydrate ligands for selectin and reduced metastasis of human gastrointestinal tract cancer cells.

Interleukin-13 Damages Intestinal Mucosa via TWEAK and Fn14 in Mice—A Pathway Associated With Ulcerative Colitis

BACKGROUND & AIMS TWEAK, a member of the tumor necrosis factor (TNF) superfamily, promotes intestinal epithelial cell injury and signals through the receptor Fn14 following irradiation-induced tissue damage and during development of colitis in mice. Interleukin (IL)-13, an effector of tissue damage in similar models, has been associated with the pathogenesis of ulcerative colitis (UC). We investigated interactions between TWEAK and IL-13 following mucosal damage in mice. METHODS We compared patterns of gene expression in intestinal tissues from wild-type and TWEAK knockout mice following γ-irradiation. Intestinal explants from these mice were used to detect cell damage induced by IL-13 and TNF-α. Levels of messenger RNA for IL-13, TWEAK, and Fn14 were measured in mucosal samples from patients with UC. RESULTS Based on gene expression analysis, TWEAK mediates γ-irradiation-induced epithelial cell cycle arrest and apoptosis. However, TWEAK alone did not induce damage or apoptosis of primary intestinal epithelial cells. On the other hand, exogenous IL-13 activated caspase-3 in naïve intestinal explants; this process required TWEAK, Fn14, and secretion of endogenous TNF-α which was mediated by ADAM17. Conversely, activation of caspase by exogenous TNF-α required IL-13, TWEAK, and Fn14. In mucosa from patients with UC, messenger RNA levels of IL-13, TWEAK, and Fn14 increased with level of disease severity. CONCLUSIONS IL-13-induced damage of intestinal epithelial cells requires TWEAK, its receptor (Fn14), and TNF-α. IL-13, TNF-α, TWEAK, and Fn14 could perpetuate and aggravate intestinal inflammation in patients with UC.

Inhibition of CCL1-CCR8 Interaction Prevents Aggregation of Macrophages and Development of Peritoneal Adhesions

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.

Predominant T helper type 2-inflammatory responses promote murine colon cancers

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‐γ) 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‐γ−/− 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‐γ−/− 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‐γ−/− mice showed distinct nuclear expression of β‐catenin, in contrast to the strong membrane staining seen in tumors of IL‐4−/− mice. In conclusion, colonic inflammation associated with Th2‐donimant cytokine responses enhanced the formation of malignant neoplasms.

Microbiota-derived lactate accelerates colon epithelial cell turnover in starvation-refed mice

Oral food intake influences the morphology and function of intestinal epithelial cells and maintains gastrointestinal cell turnover. However, how exactly these processes are regulated, particularly in the large intestine, remains unclear. Here we identify microbiota-derived lactate as a major factor inducing enterocyte hyperproliferation in starvation-refed mice. Using bromodeoxyuridine staining, we show that colonic epithelial cell turnover arrests during a 12- to 36-h period of starvation and increases 12-24 h after refeeding. Enhanced epithelial cell proliferation depends on the increase in live Lactobacillus murinus, lactate production and dietary fibre content. In the model of colon tumorigenesis, mice exposed to a carcinogen during refeeding develop more aberrant crypt foci than mice fed ad libitum. Furthermore, starvation after carcinogen exposure greatly reduced the incidence of aberrant crypt foci. Our results indicate that the content of food used for refeeding as well as the timing of carcinogen exposure influence the incidence of colon tumorigenesis in mice.

Dose-Dependent Differential Regulation of Cytokine Secretion from Macrophages by Fractalkine

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.

A Second Generation of Double Mutant Cholera Toxin Adjuvants: Enhanced Immunity without Intracellular Trafficking

Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Th1-type cytokine responses by OVA-specific CD4+ T cells, dmCT E112K/KDEV exhibited significantly lower Th1-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity.