CD137 Signaling Regulates Acute Colitis via RALDH2-Expressing CD11b-CD103+ DCs.

CD137, a potent costimulatory receptor for CD8+ T cells, is expressed in various non-T cells, but little is known about its regulatory functions in these cells. In this study, we show that CD137 signaling, specifically in intestinal CD11b-CD103+ dendritic cells (DCs), restricts acute colitis progression. Mechanistically, CD137 engagement activates TAK1 and subsequently stimulates the AMPK-PGC-1α axis to enhance expression of the Aldh1a2 gene encoding the retinoic acid (RA) metabolizing enzyme RALDH2. RA can act on CD11b+CD103- DCs and induce SOCS3 expression, which, in turn, suppresses p38MAPK activation and interleukin-23 (IL-23) production. Administration of RA in DC-specific CD137-/- mice represses IL-23-producing CD11b+CD103- DCs and TH17 cells, indicating that RA is a major inhibitory effector molecule against intestinal CD11b+CD103- DCs. Additionally, the therapeutic effect of the anti-CD137 antibody is abrogated in DC-specific CD137-/- mice. Taken together, our results define a mechanism of paracrine immunoregulation operating between adjacent DC subsets in the intestine.

Netrin-1-Induced Stem Cell Bioactivity Contributes to the Regeneration of Injured Tissues via the Lipid Raft-Dependent Integrin α6ß4 Signaling Pathway.

Netrin-1 (Ntn-1) is a multifunctional neuronal signaling molecule; however, its physiological significance, which improves the tissue-regeneration capacity of stem cells, has not been characterized. In the present study, we investigate the mechanism by which Ntn-1 promotes the proliferation of hUCB-MSCs with regard to the regeneration of injured tissues. We found that Ntn-1 induces the proliferation of hUCB-MSCs mainly via Inα6ß4 coupled with c-Src. Ntn-1 induced the recruitment of NADPH oxidases and Rac1 into membrane lipid rafts to facilitate ROS production. The Inα6ß4 signaling of Ntn-1 through ROS production is uniquely mediated by the activation of SP1 for cell cycle progression and the transcriptional occupancy of SP1 on the VEGF promoter. Moreover, Ntn-1 has the ability to induce the F-actin reorganization of hUCB-MSCs via the Inα6ß4 signaling pathway. In an in vivo model, transplantation of hUCB-MSCs pre-treated with Ntn-1 enhanced the skin wound healing process, where relatively more angiogenesis was detected. The potential effect of Ntn-1 on angiogenesis is further verified by the mouse hindlimb ischemia model, where the pre-activation of hUCB-MSCs with Ntn-1 significantly improved vascular regeneration. These results demonstrate that Ntn-1 plays an important role in the tissue regeneration process of hUCB-MSC via the lipid raft-mediated Inα6ß4 signaling pathway.

Vibrio vulnificus VvpE Stimulates IL-1ß Production by the Hypomethylation of the IL-1ß Promoter and NF-κB Activation via Lipid Raft-Dependent ANXA2 Recruitment and Reactive Oxygen Species Signaling in Intestinal Epithelial Cells.

An inflammatory response is a hallmark of necrosis evoked by bacterial pathogens. Vibrio vulnificus, VvpE, is an elastase that is responsible for tissue necrosis and inflammation; however, the molecular mechanism by which it regulates host cell death has not been characterized. In the present study, we investigate the cellular mechanism of VvpE with regard to host cell death and the inflammatory response of human intestinal epithelial (INT-407) cells. The recombinant protein (r)VvpE (50 pg/ml) caused cytotoxicity mainly via necrosis coupled with IL-1ß production. The necrotic cell death induced by rVvpE is highly susceptible to the knockdown of annexin A (ANXA)2 and the sequestration of membrane cholesterol. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into membrane lipid rafts coupled with ANXA2 to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of redox-sensitive transcription factor NF-κB. The silencing of NF-κB inhibited IL-1ß production during necrosis. rVvpE induced hypomethylation and region-specific transcriptional occupancy by NF-κB in the IL-1ß promoter and has the ability to induce pyroptosis via NOD-, LRR-, and pyrin domain-containing 3 inflammasome. In a mouse model of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus negated the proinflammatory responses and maintained the physiological levels of the proliferation and migration of enterocytes. These results demonstrate that VvpE induces the hypomethylation of the IL-1ß promoter and the transcriptional regulation of NF-κB through lipid raft-dependent ANXA2 recruitment and ROS signaling to promote IL-1ß production in intestinal epithelial cells.

Silencing of MUC8 by siRNA increases P2Y2-induced airway inflammation.

Mucin hypersecretion and overproduction are frequent manifestations of respiratory disease. Determining the physiological function of airway mucin is presently considered more important than identifying the relevant signaling pathways. The lack of a full-length human mucin 8 (MUC8) cDNA sequence has hindered the generation of a Muc8 knockout mouse line. Thus, the precise physiological functions of MUC8 are unclear. Herein, we investigated the function of MUC8 using a small-interfering RNA (siRNA)-mediated genetic silencing approach in human airway epithelial cells. Herein, intracellular IL-1α production was stimulated by an ATP/P2Y2 complex. While ATP/P2Y2 increased IL-1α secretion in a time-dependent manner, treatment with P2Y2-specific siRNA significantly decreased IL-1α secretion. Moreover, ATP increased P2Y2-mediated upregulation of MUC8 expression; however, IL-1α significantly decreased the extent to which ATP/P2Y2 upregulated MUC8 expression. Interestingly, treatment with MUC8-specific siRNA decreased the production of anti-inflammatory cytokines (TGF-ß and IL-1 receptor antagonist) and increased the production of inflammatory cytokines (IL-1α and IL-6) in our system. In addition, siRNA-mediated knockdown of MUC8 expression dramatically increased the secretion of inflammatory chemokines and resulted in an approximately threefold decrease in cell chemotaxis. We propose that MUC8 may function as an anti-inflammatory mucin that participates in inflammatory response by attracting immune cells/cytokines to the site of inflammation. Our results provide new insight into the physiological function of MUC8 and enhance our understanding of mucin overproduction during airway inflammation.

Multivessel versus single vessel spasm, as assessed by the intracoronary acetylcholine provocation test, in Korean patients.

1. Coronary artery spasm (CAS) is known to be a major cause of myocardial ischaemia. Multivessel coronary spasm (MVS) in particular is likely to induce more severe and prolonged myocardial ischaemia than single vessel spasm (SVS). 2. In the present study, a total of 1082 consecutive patients without significant coronary artery disease who underwent an acetylcholine (ACh) provocation test between March 2004 and April 2009 were investigated. Patients were divided into three groups: an MVS group (n = 275), an SVS group (n = 376) and a non-CAS group (n = 431). Differences in clinical and angiographic characteristics following the ACh provocation test were evaluated between the MVS, SVS and non-CAS groups. 3. At baseline, patients in the MVS group had the highest prevalence of peripheral artery disease (PAD), hyperlipidaemia, smoking and old age, as well as the highest triglyceride levels. Calcium channel blockers were most frequently prescribed in MVS patients before the ACh test. During the ACh test, the highest prevalence of chest pain, ischaemic electrocardiogram changes, baseline spasms and diffuse and severe spasms were observed in the MVS group. The response rate to lower ACh doses that induce CAS was also higher in the MVS group. Multivariate analysis showed that the presence of PAD (odds ratio (OR) 2.0; P = 0.006) and baseline spasm (OR 1.4; P = 0.045) were independent predictors of ACh-induced MVS. 4. In conclusion, ischaemic symptoms, diffuse and severe spasm and baseline spasm were more frequently associated with MVS patients, suggesting more intensive medical therapies and close clinical follow up would be required for this patient group.

Allogeneic renal graft rejection in a rat model: in vivo MR imaging of the homing trait of macrophages.

PURPOSE: To evaluate the feasibility of MR imaging to depict the in vivo recruitment of superparamagnetic iron oxide (SPIO)-labeled macrophages and to aid diagnosis of graft rejection in kidney transplantation. MATERIALS AND METHODS: This study was approved by the institution's committee on animal research. Eighteen male Lewis rats received a kidney transplant; 12 had an F344 rat donor and six had a Lewis rat donor. Peritoneal macrophages were harvested from thioglycollate-treated Lewis rats, cultured, and labeled with SPIO. After resuspension of macrophages in a concentration of 1 x 10(7) cells per milliliter of Hanks balanced salt solution, 5 x 10(6) of SPIO-labeled macrophages was administered through the tail vein 2 or 5 days after transplantation in each group. The transplanted kidneys were imaged on a 4.7-T MR imager 24 hours after macrophage administration. The Wilcoxon signed rank test was performed for evaluating the differences between the relative signal intensity (SI) before and after SPIO-labeled macrophage administration. RESULTS: A low-SI zone was predominantly noted in the medulla of the transplanted kidneys, and the relative SI decreased significantly from 1.40 to 0.53 (P < .001) in the allogeneic transplants following SPIO-labeled macrophage administration 5 days after the allogeneic transplantation. In the syngeneic group, the lower-SI zone was not noted in the grafts. At histopathologic examination, the lower-SI zone corresponded to the distribution of the SPIO-labeled macrophages. CONCLUSION: This study demonstrates that the homing of intravenously administered SPIO-labeled macrophages can be monitored in the allograft rejection model on in vivo MR images.

[Comparative quantification of plasma hnRNP B1 mRNA in non-small cell lung cancer patients by real-time PCR].

BACKGROUND: Circulating cell-free nucleic acids are known to be a noninvasive diagnostic tool for cancer detection. Heterogeneous nuclear ribonucleoprotein (hnRNP) B1, a nuclear core complex, is overexpressed in early stage lung cancer. We intended to evaluate the usefulness of plasma hnRNP B1 mRNA in differentiating non-small cell lung cancer (NSCLC) from other benign lung diseases, especially pulmonary tuberculosis, which is highly prevalent in Korea and often difficult to distinguish from lung cancer. METHODS: Plasma RNA was extracted from 30 patients with NSCLC, 30 patients with benign lung diseases including pulmonary tuberculosis, and 10 healthy controls. Plasma hnRNP B1 mRNA was measured by TaqMan Gene Expression Assay (Applied Biosystems, USA), and pre-developed beta-actin (ACTB) mRNA was used for normalization. We analyzed the relative gene expression data using the delta-delta Ct method. RESULTS: Plasma hnRPN B1 mRNA was measurable in 93.3% (28/30) of NSCLC patients. Normalized 2-DeltaDeltaCt of plasma hnRPN B1 mRNA was 62.2 (95%Cl, 6.4-210.1) in NSCLC patients and 2.7 (95%Cl, 0.5-13.6) in benign lung disease patients (P<0.001, Mann-Whitney U test). CONCLUSIONS: Plasma hnRNP B1 mRNA was significantly increased in patients with lung cancer compared with that in patients with other benign lung diseases. Plasma hnRNP B1 mRNA may be useful as a potential marker for the detection of NSCLC.

[Treatment outcome of multidrug resistance related mRNA expression and c-jun-N-terminal kinase activity in patients with acute myeloid leukemia].

BACKGROUND: The multidrug resistance (mdr1), multidrug resistance associated protein (mrp1), and glutathione-s-transferase (gst) pi genes have been associated with treatment failure in acute myeloid leukemia (AML). c-jun N-terminal kinase (JNK) activity is increased in response to chemotherapeutic agent. METHODS: To investigate the significance of multidrug resistance (mdr) parameters and JNK activity, bone marrow or peripheral blood cells from 52 patients with AML were analyzed. RT-PCR was performed for mdr1, mrp1, and gst pi gene expression. JNK expression and activity were measured using an immunoe- nzymatic kinase assay and a western blot method. RESULTS: High level expression of mdr1, mrp1, and gst pi mRNA was observed in 38.5%, 48.1% and 54.3% of AML cases, respectively. The remission rate was significantly low in cases with an older age (>55 yr), a high WBC count, poor chromosomal abnormalities, a high level expression of mdr1 and mrp1. The WBC count and mdr1 mRNA expression were independent predictors for the outcome to induction chemotherapy. There was a shorter duration of overall survival in the patients with an older age, a high WBC count, chromosome aberrations, high level expressions of mdr1 and mrp1 mRNA, and JNK activation. The patient's age, WBC count and chromosomal abnormalities were independent predictors for overall survivals. The majority (28/30) of AML cases did not show any levels of JNK activation except for two cases, which were associated with an extremely high WBC count, chromosomal aberration, high level expressions of mdr1, mrp1 and gst pi mRNA, and treatment resistance. CONCLUSIONS: These data indicate the influences of mdr1 and mrp1 mRNA expression on the clinical outcome of AML to induction chemotherapy. But it will be necessary to investigate further whether blast cells of AML resistant to chemotherapy retain the capacity to activate JNK, and relate to MDR parameters.