Keita Saeki

Smad2 and Smad3 Inversely Regulate TGF-β Autoinduction in Clostridium butyricum-Activated Dendritic Cells

Colonization with a mixture of Clostridium species has been shown to induce accumulation of induced regulatory T (iTreg) cells in the colon. Transforming growth factor-β (TGF-β) is an essential factor for iTreg cell induction; however, the relationship between Clostridium species and TGF-β remains to be clarified. Here we demonstrated that a gram-positive probiotic bacterial strain, Clostridium butyricum (C. butyricum), promoted iTreg cell generation in the intestine through induction of TGF-β1 from lamina propria dendritic cells (LPDCs). C. butyricum-mediated TGF-β1 induction was mainly Toll-like receptor 2 (TLR2) dependent, and the ERK-AP-1 kinase pathway played an important role. In addition, the autocrine TGF-β-Smad3 transcription factor signal was necessary for robust TGF-β expression in DCs, whereas Smad2 negatively regulated TGF-β expression. Smad2-deficient DCs expressed higher concentrations of TGF-β and were tolerogenic for colitis models. This study reveals a novel mechanism of TGF-β induction by Clostridia through a cooperation between TLR2-AP-1 and TGF-β-Smad signaling pathways.

C-C motif chemokine receptor 9 positive macrophages activate hepatic stellate cells and promote liver fibrosis in mice†‡

Chemokine receptors mediate migration of immune cells into the liver, thereby promoting liver inflammation. C‐C motif chemokine receptor (CCR) 9+ macrophages are crucial in the pathogenesis of acute liver inflammation, but the role and underlying mechanisms of this macrophage subset in chronic liver injury and subsequent liver fibrosis are not fully understood. We confirmed that tumor necrosis factor alpha (TNF‐α)‐producing CCR9+ macrophages accumulated during the initiation of carbon tetrachloride (CCl4)‐induced liver injury, and CCR9 deficiency attenuated the degree of liver damage. Accumulation of CCR9+ macrophages persisted prominently during the process of liver fibrosis induced by repetitive CCl4 or thioacetamide (TAA)/leptin administration. Increased CCR9 expression was also found on activated hepatic stellate cells (HSCs). Importantly, experimental liver fibrosis was significantly ameliorated in CCR9−/− mice compared with wild‐type (WT) mice, assessed by α‐smooth muscle actin (α‐SMA) immunostain, Sirius red staining, and messenger RNA (mRNA) expression levels of α‐SMA, collagen 1α1, transforming growth factor (TGF)‐β1, and tissue inhibitor of metalloproteinase (TIMP)‐1. Accumulated CD11b+ macrophages in CCl4‐treated WT mice showed marked increases in TNF, NO synthase‐2, and TGF‐β1 mRNA expression compared with CCR9−/− mice, implying proinflammatory and profibrogenic properties. Hepatic CD11b+ macrophages from CCl4‐treated WT mice (i.e., CCR9+ macrophages), but not CD8+ T lymphocytes or non‐CD11b+ cells, significantly activated HSCs in vitro compared with those from CCR9−/− mice. TNF‐α or TGF‐β1 antagonism attenuated CCR9+ macrophage‐induced HSC activation. Furthermore, C‐C motif chemokine ligand (CCL) 25 mediated migration and, to a lesser extent, activation of HSCs in vitro. Conclusion: Accumulated CD11b+ macrophages are critical for activating HSCs through the CCR9/CCL25 axis and therefore promote liver fibrosis. CCR9 antagonism might be a novel therapeutic target for liver fibrosis. (HEPATOLOGY 2013;)

CCL2-induced migration and SOCS3-mediated activation of macrophages are involved in cerulein-induced pancreatitis in mice

BACKGROUND & AIMS Acute pancreatitis is a common inflammatory disease mediated by damage to acinar cells and subsequent pancreatic inflammation with recruitment of leukocytes. We investigated the pathologic roles of innate immune cells, especially macrophages, in cerulein- and L-arginine-induced acute pancreatitis in mice. METHODS Acute pancreatitis was induced by sequential peritoneal administration of cerulein to mice. We determined serum concentrations of amylase and lipase, pancreatic pathology, and features of infiltrating mononuclear cells. We performed parabiosis surgery to assess the hemodynamics of pancreatic macrophages. RESULTS Almost all types of immune cells, except for CD11b(high)CD11c(-) cells, were detected in the pancreas of healthy mice. However, activated CD11b(high)CD11c(-) cells, including Gr-1(low) macrophages and Gr-1(high) cells (granulocytes and myeloid-derived suppressor cells), were detected in damaged pancreas after cerulein administration. CCL2(-/-) mice given cerulein injections developed significantly less severe pancreatitis, with less infiltration of CD11b(high)CD11c(-)Gr-1(low) macrophages, but comparable infiltration of myeloid-derived suppressor cells, compared with cerulein-injected wild-type mice. Parabiosis and bone marrow analyses of these mice revealed that the CD11b(high)CD11c(-)Gr-1(low) macrophages had moved out of the bone marrow. Furthermore, mice with macrophage-specific deletion of suppressor of cytokine signaling 3 given injections of cerulein developed less severe pancreatitis and Gr-1(low) macrophage produced less tumor necrosis factor-α than wild-type mice given cerulein, although the absolute number of CD11b(high)CD11c(-)Gr-1(low) macrophages was comparable between strains. Induction of acute pancreatitis by L-arginine required induction of macrophage migration by CCL2, via the receptor CCR2. CONCLUSIONS Cerulein induction of pancreatitis in mice involves migration of CD11b(high)CD11c(-)Gr-1(low) macrophage from the bone marrow (mediated by CCL2 via CCR2) and suppressor of cytokine signaling 3-dependent activation of macrophage. These findings might lead to new therapeutic strategies for acute pancreatitis.

Transforming growth factor β and Ras/MEK/ERK signaling regulate the expression level of a novel tumor suppressor lefty

Objectives The objectives of the present study were (i) to identify a novel tumor suppressor gene whose expression level was regulated by transforming growth factor (TGF-&bgr;) and (ii) to evaluate the effect of Ras/MEK/ERK signaling on TGF-&bgr;–dependent Lefty up-regulation. Methods Human pancreatic cancer cell lines were used. The effect of Ras/MEK/ERK pathway on TGF-&bgr;–mediated Lefty up-regulation was tested by adding K-ras small interfering RNA, MEK inhibitor U0126, or extracellular signal–regulated kinase (ERK) inhibitor LY294002. Results Transforming growth factor &bgr; upregulated Lefty messenger RNA levels within 6 of the 7 cell lines. Lefty exerts an antagonistic effect against the tumor-promoting molecule, Nodal, as recombinant Lefty suppressed Nodal-mediated proliferation. Interestingly, inhibition of the Ras/MEK/ERK pathway dramatically enhanced TGF-mediated Lefty up-regulation, suggesting that Ras/MEK/ERK signaling suppresses TGF-&bgr;–Lefty pathway. Conclusions Our data suggest that Lefty is a novel TGF-&bgr; target molecule that mediates growth inhibition of pancreatic cancer cells. In addition, activation of the Ras/MEK/ERK pathway serves as a mechanism by which pancreatic cancer escapes from growth inhibition by the TGF-&bgr;–Lefty axis. The results imply a novel therapeutic strategy for pancreatic cancer, that is, combination treatment with Ras/MEK/ERK inhibitors and TGF-&bgr;.

Hoxa9 Transduction Induces Hematopoietic Stem and Progenitor Cell Activity through Direct Down-Regulation of Geminin Protein

Hoxb4, a 3′-located Hox gene, enhances hematopoietic stem cell (HSC) activity, while a subset of 5′-located Hox genes is involved in hematopoiesis and leukemogenesis, and some of them are common translocation partners for Nucleoporin 98 (Nup98) in patients with leukemia. Although these Hox gene derivatives are believed to act as transcription regulators, the molecular involvement of the Hox gene derivatives in hematopoiesis and leukemogenesis remains largely elusive. Since we previously showed that Hoxb4 forms a complex with a Roc1-Ddb1-Cul4a ubiquitin ligase core component and functions as an E3 ubiquitin ligase activator for Geminin, we here examined the E3 ubiquitin ligase activities of the 5′-located Hox genes, Hoxa9 and Hoxc13, and Nup98-Hoxa9. Hoxa9 formed a similar complex with the Roc1-Ddb1-Cul4a component to induce ubiquitination of Geminin, but the others did not. Retroviral transduction-mediated overexpression or siRNA-mediated knock-down of Hoxa9 respectively down-regulated or up-regulated Geminin in hematopoietic cells. And Hoxa9 transduction-induced repopulating and clonogenic activities were suppressed by Geminin supertransduction. These findings suggest that Hoxa9 and Hoxb4 differ from Hoxc13 and Nup98-Hoxa9 in their molecular role in hematopoiesis, and that Hoxa9 induces the activity of HSCs and hematopoietic progenitors at least in part through direct down-regulation of Geminin.

CCR2 knockout exacerbates cerulein-induced chronic pancreatitis with hyperglycemia via decreased GLP-1 receptor expression and insulin secretion.

Glucagon-like peptide-1 (GLP-1) promotes insulin release; however, the relationship between the GLP-1 signal and chronic pancreatitis is not well understood. Here we focus on chemokine (C-C motif) ligand 2 (CCL2) and its receptor (CCR2) axis, which regulates various immune cells, including macrophages, to clarify the mechanism of GLP-1-mediated insulin secretion in chronic pancreatitis in mice. One and multiple series of repetitive cerulein administrations were used to induce acute and chronic cerulein pancreatitis, respectively. Acute cerulein-administered CCR2-knockout (KO) mice showed suppressed infiltration of CD11b(+)Gr-1(low) macrophages and pancreatic inflammation and significantly upregulated insulin secretion compared with paired wild-type (WT) mice. However, chronic cerulein-administered CCR2-KO mice showed significantly increased infiltration of CD11b(+)/Gr-1(-) and CD11b(+)/Gr-1(high) cells, but not CD11b(+)/Gr-1(low) cells, in pancreas with severe inflammation and significantly decreased insulin secretion compared with their WT counterparts. Furthermore, although serum GLP-1 levels in chronic cerulein-administered WT and CCR2-KO mice were comparably upregulated after cerulein administrations, GLP-1 receptor levels in pancreases of chronic cerulein-administered CCR2-KO mice were significantly lower than in paired WT mice. Nevertheless, a significantly higher hyperglycemia level in chronic cerulein-administered CCR2-KO mice was markedly restored by treatment with a GLP-1 analog to a level comparable to the paired WT mice. Collectively, the CCR2/CCL2 axis-mediated CD11b(+)-cell migration to the pancreas is critically involved in chronic pancreatitis-mediated hyperglycemia through the modulation of GLP-1 receptor expression and insulin secretion.

MyD88-dependent interleukin-10 production from regulatory CD11b+Gr-1high cells suppresses development of acute cerulein pancreatitis in mice

We explored the role of the MyD88 signaling pathway. This pathway mediates the recognition of pathogen-associated molecular patterns and damage-associated molecular patterns via Toll-like receptors (TLRs) and/or IL-1/IL-18 via each cytokine receptor in a murine model of acute pancreatitis induced by cerulein administration. Our analysis revealed that: various TLRs and MyD88 molecules were constitutively expressed in the pancreas of cerulein-treated and untreated wild-type (WT) mice. MyD88⁻/⁻ mice administered cerulein developed severe pancreatitis as compared with MyD88⁺/⁺ WT mice. The number of IL-10-expressing CD11b⁺Gr-1(high) cells in cerulein-administered MyD88⁻/⁻ mice was significantly decreased. This was in accordance with a reciprocal increase in the infiltration of CD4⁺ T cells as compared with that in control MyD88⁺/⁺ mice. WT mice pretreated with antibiotics and administered cerulein developed milder pancreatitis as compared with control cerulein-administered mice without antibiotic treatment. The MyD88 signaling pathway contributes to the induction of regulatory IL-10-producing macrophages/myeloid-derived suppressor cells, possibly in response to non-bacterial components in the damaged pancreas. These results provide a new concept for therapeutic strategies against acute pancreatitis.

Scmh1 Has E3 Ubiquitin Ligase Activity for Geminin and Histone H2A and Regulates Geminin Stability Directly or Indirectly via Transcriptional Repression of Hoxa9 and Hoxb4

ABSTRACT Polycomb-group (PcG) complex 1 acts as an E3 ubiquitin ligase both for histone H2A to silence transcription and for geminin to regulate its stability. Scmh1 is a substoichiometric component of PcG complex 1 that provides the complex with an interaction domain for geminin. Scmh1 is unstable and regulated through the ubiquitin-proteasome system, but its molecular roles are unknown, so we generated Scmh1-deficient mice to elucidate its function. Loss of Scmh1 caused derepression of Hoxb4 and Hoxa9, direct targets of PcG complex 1-mediated transcriptional silencing in hematopoietic cells. Double knockdown of Hoxb4 and Hoxa9 or transduction of a dominant-negative Hoxb4N→A mutant caused geminin accumulation. Age-related transcriptional downregulation of derepressed Hoxa9 also leads to geminin accumulation. Transduction of Scmh1 lacking a geminin-binding domain restored derepressed expression of Hoxb4 and Hoxa9 but did not downregulate geminin like full-length Scmh1. Each of Hoxb4 and Hoxa9 can form a complex with Roc1-Ddb1-Cul4a to act as an E3 ubiquitin ligase for geminin. We suggest that geminin dysregulation may be restored by derepressed Hoxb4 and Hoxa9 in Scmh1-deficient mice. These findings suggest that PcG and a subset of Hox genes compose a homeostatic regulatory system for determining expression level of geminin.

A novel JAK-STAT inhibitor, 2-[(3-Carbamoyl-2-thienyl)amino]-2-oxoethyl(2,6-dichlorophenyl)acetate, suppresses helper T cell differentiation in vitro and collagen-induced arthritis in vivo.

Th17 cells, which have been implicated in autoimmune diseases including rheumatoid arthritis (RA), require the JAK-STAT3 pathway for their differentiation and functions. Recently, JAK inhibitors have been developed as a therapeutic drug for RA. However, the current JAK inhibitors are not optimized to STAT3 compared with other STATs. In this study, we found a new lead compound of a small molecule JAK-STAT inhibitor, 2-[(3-Carbamoyl-2-thienyl)amino]-2-oxoethyl (2,6-dichlorophenyl)acetate, which inhibits STAT3 as efficiently as other STATs. This compound, named JI069, was selected by STAT3 reporter assay in combination with an in silico docking model. JI069 inhibited gp130 signaling by inducing dissociation between gp130 and JAK1. In HEK293T cells and primary T cells, JI069 suppressed STAT3 activation as efficiently as other STATs, including STAT1, STAT5, and STAT6. JI069 effectively suppressed Th1, Th2, and Th17 differentiation while strongly promoted iTreg differentiation. JI069 suppressed symptoms of the collagen-induced arthritis (CIA) model in mice, and inhibited the cytokine production from T cells as well as the STAT3 phosphorylation of synovial cells. These data suggest that JI069 is a new type of JAK inhibitor which has potential for the treatment of immunological disorders.

Ampullary Adenoma Treated by Endoscopic Double-Snare Retracting Papillectomy.

We report herein improved methods for the safe and successful completion of endoscopic papillectomy (EP). Between January 2008 and November 2011, 12 patients underwent double-snare retracting papillectomy for the treatment of lesions of the major duodenal papilla. The main outcomes were en bloc resection rates, pathological findings, and adverse events. All of the patients (mean age, 60.1 years; range, 38 to 80 years) were diagnosed with ampullary adenoma by endoscopic forceps biopsies prior to endoscopic snare papillectomy. En bloc resection by double-snare retracting papillectomy was successfully performed for all lesions (median size, 12.3 mm), comprising six tubular adenomas, one tubulovillous adenoma, three cases of epithelial atypia, one hamartomatous polyp, and one case of duodenitis with regenerative change. Significant hemorrhage and pancreatitis were observed in one case after EP. Adenoma recurrence occurred in three patients during follow-up (median, 28.5 months) at a mean interval of 2 months postoperatively (range, 1 to 3 months). No serious adverse events were observed. Double-snare retracting papillectomy is effective and feasible for treating lesions of the major duodenal papilla. Further treatment experience, including a single-arm phase II study, needs to be accumulated before conducting a randomized controlled study.