CD8+ T cell-mediated rejection of allogenic human-induced pluripotent stem cell-derived cardiomyocyte sheets in human PBMC-transferred NOG MHC double knockout mice.

BACKGROUND: Transplantation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has emerged as a promising therapy to treat end-stage heart failure. However, the immunogenicity of hiPS-CMs in transplanted patients has not been fully elucidated. Thus, in vivo models are required to estimate immune responses against hiPS-CMs in transplant recipients. METHODS: We transferred human peripheral blood mononuclear cells (hPBMCs) into NOD/Shi-scid IL-2rgnull (NOG) MHC class I/II double knockout (NOG-ΔMHC) mice, which were reported to accept hPBMCs without xenogeneic-graft-versus-host disease (xeno-GVHD). Then, hiPS-CM sheets generated from the hiPS cell line 201B7 harboring a luciferase transgene were transplanted into the subcutaneous space of NOG-ΔMHC mice. Graft survival was monitored by bioluminescent images using a Xenogen In Vivo Imaging System. RESULTS: The human immune cells were engrafted for more than 3 months in NOG-ΔMHC mice without lethal xeno-GVHD. The hiPS-CMs expressed a moderate level of human leukocyte antigen (HLA)-class I, but not HLA-class II, molecules even after interferon-gamma (IFN-γ) stimulation. Consistently, the allogenic IFN-γ-treated hiPS-CMs induced weak CD8+ but not CD4+ T cell responses in vitro. hiPS-CM sheets disappeared approximately 17 to 24 days after transplantation in hPBMC-transferred NOG-ΔMHC mice, and CD8+ T cell depletion significantly prolonged graft survival, similar to what was observed following tacrolimus treatment. CONCLUSIONS: hiPS-CMs are less immunogenic in vitro but induce sufficient CD8+ T cell-mediated immune responses for graft rejection in vivo.

Interleukin-11-expressing fibroblasts have a unique gene signature correlated with poor prognosis of colorectal cancer.

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

Inhibition of Importin ß1 Augments the Anticancer Effect of Agonistic Anti-Death Receptor 5 Antibody in TRAIL-resistant Tumor Cells.

TNF-related apoptosis-inducing ligand (TRAIL) and an agonistic antibody against the death-inducing TRAIL receptor 5, DR5, are thought to selectively induce tumor cell death and therefore, have gained attention as potential therapeutics currently under investigation in several clinical trials. However, some tumor cells are resistant to TRAIL/DR5-induced cell death, even though they express DR5. Previously, we reported that DR5 is transported into the nucleus by importin ß1, and knockdown of importin ß1 upregulates cell surface expression of DR5 resulting in increased TRAIL sensitivity in vitro Here, we examined the impact of importin ß1 knockdown on agonistic anti-human DR5 (hDR5) antibody therapy. Drug-inducible importin ß1 knockdown sensitizes HeLa cells to TRAIL-induced cell death in vitro, and exerts an antitumor effect when combined with agonistic anti-hDR5 antibody administration in vivo Therapeutic importin ß1 knockdown, administered via the atelocollagen delivery system, as well as treatment with the importin ß inhibitor, importazole, induced regression and/or eradication of two human TRAIL-resistant tumor cells when combined with agonistic anti-hDR5 antibody treatment. Thus, these findings suggest that the inhibition of importin ß1 would be useful to improve the therapeutic effects of agonistic anti-hDR5 antibody against TRAIL-resistant cancers.

Diagnostic performance of real-time tissue elastography in chronic hepatitis C patients with sustained virological response.

BACKGROUND AND AIMS: Real-time tissue elastography is a non-invasive method for measuring liver elasticity. However, there are no reports evaluating the value of real-time tissue elastography for liver fibrosis in hepatitis C virus-infected patients with sustained virological response. The aim of this study is to clarify the diagnostic performance of real-time tissue elastography in patients with sustained virological response. METHODS: In this prospective study, we enrolled 425 chronic hepatitis C patients who underwent liver biopsy: 118 patients with sustained virological response (45.8% women) and 307 patients with hepatitis C virus (51.1% women). The post-sustained virological response biopsy was performed 5.9 ± 1.8 years after the therapy. Liver fibrosis index measurements as assessed using real-time tissue elastography were performed on the same day of biopsy. RESULTS: The respective mean liver fibrosis index values for fibrosis stages F0, F1, F2, F3, and F4 were 2.82 ± 0.33, 2.90 ± 0.51, 3.06 ± 0.58, 3.65 ± 0.24, and 3.83 ± 0.65, respectively, in patients with sustained virological response. The diagnostic accuracies expressed as areas under the receiver operating characteristic curves in patients with sustained virological response were 0.776 for the diagnosis of significant fibrosis (≥F2), 0.885 for severe fibrosis (≥F3), and 0.860 for cirrhosis (F4), respectively. The optimum cut-off values liver fibrosis index were 3.14 for ≥F2, 3.24 for ≥F3, and 3.30 for F4 in patients with sustained virological response. CONCLUSION: Real-time tissue elastography is an acceptable method for predicting the severity of fibrosis in hepatitis C virus patients with sustained virological response.

Predictive ability of shear wave elastography for pruritus in chronic hepatitis C patients with sustained virological response.

BACKGROUND AND AIMS: Pruritus is one of the complications with chronic liver disease and markedly worsens quality of life. However, the current status of pruritus in chronic hepatitis C patients who have achieved a sustained virological response (SVR) has not been clarified sufficiently. The aim of this study was to investigate the predictors of pruritus in post-SVR patients treated with direct-acting antivirals (DAA). PATIENTS AND METHODS: In this retrospective study, we enrolled 110 hepatitis C patients with SVR who underwent serial shear wave elastography before DAA therapy and at the end of treatment. The severity of pruritus was evaluated using Kawashima's pruritus scores and a visual analog scale. RESULTS: The prevalence of pruritus before treatment and after SVR was 28.2 and 25.5%. Multivariate logistic regression analysis confirmed that a history of hepatocellular carcinoma [odds ratio (OR): 9.72; 95% confidence interval (CI): 2.05-46.15; P=0.004], high γ-glutamyl transpeptidase levels at baseline (OR: 5.77; 95% CI: 1.83-18.21; P=0.003), low serum albumin at the end of treatment (OR: 4.85; 95% CI: 1.31-17.99; P=0.018), and high liver stiffness measurement assessed by shear wave elastography at the end of treatment (OR: 3.16; 95% CI: 1.19-11.01; P=0.024) were significant independent factors associated with pruritus in patients who had achieved an SVR following DAA therapy. CONCLUSIONS: In chronic hepatitis C patients with SVR after DAA therapy, the incidence of pruritus is not uncommon. Liver stiffness measurement is useful for predicting the incidence of pruritus. Thus, even if SVR is achieved, patients with higher liver stiffness at the end of treatment must be monitored carefully for pruritus.

Skeletal muscle fat deposition is associated with hepatocellular carcinoma development in patients with chronic liver disease.

OBJECTIVES: The effect of skeletal muscle fat deposition on the prognosis of patients with chronic liver disease remains unclear. Skeletal muscle fat deposition can be estimated by attenuation of skeletal muscle in Hounsfield units (HU) on computed tomography (CT). The aim of this retrospective cohort study was to investigate the association between skeletal muscle fat deposition assessed by skeletal muscle attenuation (SMA), and hepatocellular carcinoma (HCC). METHODS: We enrolled 288 patients with chronic liver disease (139 men, 149 women; mean age 67.5 ± 10.4 y; hepatitis C virus, 239; hepatitis B virus, 17; without viral infection, 32; chronic hepatitis, 227; and cirrhosis, 61) who underwent liver biopsy and CT scanning between January 2013 and February 2017. The patients were divided into two groups based on SMA levels, with the cutoff value of 31 HU. We analyzed the effect of SMA on HCC development. RESULTS: During the study follow-up period (median, 2.50 y; range, 0.5-4.7 y), HCC was identified in 19 patients (7%). The cumulative incidence of HCC in patients with lower SMA (<31 HU) was significantly higher than in patients with SMA ≥31 HU (P = 0.007). Cox proportional hazards regression analysis confirmed cirrhosis (hazard ratio [HR], 6.626; 95% confidence interval [CI], 2.57-17.12; P <0.001) and lower SMA (HR, 3.502; 95% CI, 1.25-9.83; P = 0.017) as significant independent factors associated with HCC development in patients with chronic liver disease. CONCLUSIONS: Patients with cirrhosis and skeletal muscle fat deposition assessed by SMA had a higher risk for developing HCC.

Liver stiffness reduction correlates with histological characteristics of hepatitis C patients with sustained virological response.

BACKGROUND & AIMS: We investigated the correlation between histological characteristics and changes in liver stiffness (LS) in patients with sustained virological response (SVR) using acoustic radiation force impulse (ARFI) elastography. METHODS: In this prospective study, we enrolled 176 hepatitis C patients with SVR who underwent ARFI elastography and liver biopsy before antiviral treatment, and serial ARFI elastography at the end of treatment (EOT) and at 24 weeks after the EOT. To compare the long-term changes in LS in patients with SVR using ARFI elastography, another group of 140 patients who had undergone paired biopsy after achieving SVR was included. RESULTS: Mean LS values were 1.60±0.63 m/s, 1.48±0.56 m/s and 1.37±0.62 m/s at baseline, EOT and 24 weeks after EOT, respectively, P<.001. Higher inflammatory activity at baseline was associated with an improvement in LS at the EOT, with an odds ratio of 1.940. Significant fibrosis at baseline was associated with an improvement in LS at 24 weeks after the EOT, with an odds ratio of 2.617. Among patients in the paired biopsy group with baseline fibrosis stage identical to the ARFI group, LS values at 24 weeks after the EOT did not show any difference with values at 5 years after EOT. CONCLUSIONS: Pre-treatment histological characteristics influence LS reduction after SVR is achieved.

Induction of Colonic Regulatory T Cells by Mesalamine by Activating the Aryl Hydrocarbon Receptor.

BACKGROUND & AIMS: Mesalamine is a first-line drug for treatment of inflammatory bowel diseases (IBD). However, its mechanisms are not fully understood. CD4+ Foxp3+ regulatory T cells (Tregs) play a potential role in suppressing IBD. This study determined whether the anti-inflammatory activity of mesalamine is related to Treg induction in the colon. METHODS: We examined the frequencies of Tregs in the colons of wild-type mice, mice deficient for aryl hydrocarbon receptor (AhR-/- mice), and bone marrow-chimeric mice lacking AhR in hematopoietic cells (BM-AhR-/- mice), following oral treatment with mesalamine. We also examined the effects of mesalamine on transforming growth factor (TGF)-ß expression in the colon. RESULTS: Treatment of wild-type mice with mesalamine increased the accumulation of Tregs in the colon and up-regulated the AhR target gene Cyp1A1, but this effect was not observed in AhR-/- or BM-AhR-/- mice. In addition, mesalamine promoted in vitro differentiation of naive T cells to Tregs, concomitant with AhR activation. Mice treated with mesalamine exhibited increased levels of the active form of TGF-ß in the colon in an AhR-dependent manner and blockade of TGF-ß signaling suppressed induction of Tregs by mesalamine in the colon. Furthermore, mice pretreated with mesalamine acquired resistance to dextran sodium sulfate-induced colitis. CONCLUSIONS: We propose a novel anti-inflammatory mechanism of mesalamine for colitis: induction of Tregs in the colon via the AhR pathway, followed by TGF-ß activation.

IFN-γ is required for cytotoxic T cell-dependent cancer genome immunoediting.

Genetic evolution that occurs during cancer progression enables tumour heterogeneity, thereby fostering tumour adaptation, therapeutic resistance and metastatic potential. Immune responses are known to select (immunoedit) tumour cells displaying immunoevasive properties. Here we address the role of IFN-γ in mediating the immunoediting process. We observe that, in several mouse tumour models such as HA-expressing 4T1 mammary carcinoma cells, OVA-expressing EG7 lymphoma cells and CMS5 MCA-induced fibrosarcoma cells naturally expressing mutated extracellular signal-regulated kinase (ERK) antigen, the action of antigen-specific cytotoxic T cell (CTL) in vivo results in the emergence of resistant cancer cell clones only in the presence of IFN-γ within the tumour microenvironment. Moreover, we show that exposure of tumours to IFN-γ-producing antigen-specific CTLs in vivo results in copy-number alterations (CNAs) associated with DNA damage response and modulation of DNA editing/repair gene expression. These results suggest that enhanced genetic instability might be one of the mechanisms by which CTLs and IFN-γ immunoedits tumours, altering their immune resistance as a result of genetic evolution.

Liver stiffness measurement predicts hepatocellular carcinoma development in patients treated with direct-acting antivirals.

BACKGROUND AND AIM: Predictive factors for hepatocarcinogenesis following eradication of hepatitis C virus by direct-acting antivirals (DAAs) are unknown. The aim of the study was to investigate the relationships between liver stiffness (LS) using acoustic radiation force impulse (ARFI) erastograghy and the development of hepatocellular carcinoma (HCC) in patients who achieved sustained virological response (SVR) treated with DAA. METHODS: In this prospective study, we enrolled 263 hepatitis C patients with SVR who underwent ARFI before DAA treatment. Thirty patients had previous HCC. RESULTS: The median LS value according to ARFI measurements was 1.34 m/s (range: 0.67-4.35). During the follow-up period (median: 18.1 months), development of HCC occurred in 19 patients (7.2%; HCC occurrence in 7 patients and HCC recurrence in 12 patients). By multivariate Cox regression analysis, HCC history (hazard ratio [HR]: 10.634; 95% confidence interval [CI]: 4.13-27.37; P = 0.001), older age (HR: 4.638; 95% CI: 1.63-13.61; P = 0.004) and higher total bilirubin levels (HR: 4.189; 95% CI: 1.66-10.60; P = 0.002) were independent predictors for the development of HCC, and higher LS value (≥1.73 m/s) at baseline was an independent predictor for HCC occurrence (HR: 8.350; 95% CI: 1.62-43.09; P = 0.011). The cumulative recurrence of HCC was statistically similar according to the degree of LS in patients who were previously treated for HCC. CONCLUSION: The LS value at baseline is useful for predicting HCC occurrence. Thus, even if SVR is achieved, patients with higher LS at baseline must be followed carefully for HCC occurrence.

Critical Contribution of Nuclear Factor Erythroid 2-related Factor 2 (NRF2) to Electrophile-induced Interleukin-11 Production.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a crucial role in protection of cells from electrophile-induced toxicity through up-regulating phase II detoxifying enzymes and phase III transporters. We previously reported that oxidative stress induces up-regulation of interleukin-11 (IL-11), a member of the IL-6 family that ameliorates acetaminophen-induced liver toxicity. However, a role for IL-11 in protection of cells from electrophile-induced toxicity remains unclear. Here we show that an environmental electrophile, 1,2-naphthoquinone (1,2-NQ), but not 15d-prostaglandin J2 (PGJ2) or tert-butylhydroxyquinone (tBHQ), induced IL-11 production. Consistent with a crucial role for prolonged ERK activation in H2O2-induced IL-11 production, 1,2-NQ, but not 15d-PGJ2 or tBHQ, elicited prolonged ERK activation. Conversely, inhibition of the ERK pathway by a MEK inhibitor completely blocked 1,2-NQ-induced IL-11 production at both protein and mRNA levels, further substantiating an intimate cross-talk between ERK activation and 1,2-NQ-induced IL-11 production. Promoter analysis of the Il11 gene revealed that two AP-1 sites were essential for 1,2-NQ-induced promoter activities. Among various members of the AP-1 family, Fra-1 was up-regulated by 1,2-NQ, and its up-regulation was blocked by a MEK inhibitor. Although NRF2 was not required for H2O2-induced IL11 up-regulation, NRF2 was essential for 1,2-NQ-induced IL11 up-regulation by increasing Fra-1 proteins possibly through promoting mRNA translation of FOSL1 Finally, intraperitoneal administration of 1,2-NQ induced body weight loss in wild-type mice, which was further exacerbated in Il11ra1-/- mice compared with Il11ra1+/- mice. Together, both Fra-1 and NRF2 play crucial roles in IL-11 production that protects cells from 1,2-NQ intestinal toxicity.

Depletion of myeloid cells exacerbates hepatitis and induces an aberrant increase in histone H3 in mouse serum.

Tissue-resident macrophages and bone marrow (BM)-derived monocytes play a crucial role in the maintenance of tissue homeostasis; however, their contribution to recovery from acute tissue injury is not fully understood. To address this issue, we generated an acute murine liver injury model using hepatocyte-specific Cflar-deficient (CflarHep-low ) mice. Cellular FLICE-inhibitory protein expression was down-regulated in Cflar-deficient hepatocytes, which thereby increased susceptibility of hepatocytes to death receptor-induced apoptosis. CflarHep-low mice developed acute hepatitis and recovered with clearance of apoptotic hepatocytes at 24 hours after injection of low doses of tumor necrosis factor α (TNFα), which could not induce hepatitis in wild-type (WT) mice. Depletion of Kupffer cells (KCs) by clodronate liposomes did not impair clearance of dying hepatocytes or exacerbate hepatitis in CflarHep-low mice. To elucidate the roles of BM-derived monocytes and neutrophils in clearance of apoptotic hepatocytes, we examined the effect of depletion of these cells on TNFα-induced hepatitis in CflarHep-low mice. We reconstituted CflarHep-low mice with BM cells from transgenic mice in which human diphtheria toxin receptor (DTR) was expressed under control of the lysozyme M (LysM) promoter. TNFα-induced infiltration of myeloid cells, including monocytes and neutrophils, was completely ablated in LysM-DTR BM-reconstituted CflarHep-low mice pretreated with diphtheria toxin, whereas KCs remained present in the livers. Under these experimental conditions, LysM-DTR BM-reconstituted CflarHep-low mice rapidly developed severe hepatitis and succumbed within several hours of TNFα injection. We found that serum interleukin-6 (IL-6), TNFα, and histone H3 were aberrantly increased in LysM-DTR BM-reconstituted, but not in WT BM-reconstituted, CflarHep-low mice following TNFα injection. CONCLUSION: These findings indicate an unexpected role of myeloid cells in decreasing serum IL-6, TNFα, and histone H3 levels via the suppression of TNFα-induced hepatocyte apoptosis. (Hepatology 2017;65:237-252).

Anagliptin, a potent dipeptidyl peptidase IV inhibitor: its single-crystal structure and enzyme interactions.

The single-crystal structure of anagliptin, N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide, was determined. Two independent molecules were held together by intermolecular hydrogen bonds, and the absolute configuration of the 2-cyanopyrrolidine ring delivered from l-prolinamide was confirmed to be S. The interactions of anagliptin with DPP-4 were clarified by the co-crystal structure solved at 2.85 Å resolution. Based on the structure determined by X-ray crystallography, the potency and selectivity of anagliptin were discussed, and an SAR study using anagliptin derivatives was performed.

Effect of polyphenols on 3-hydroxy-3-methylglutaryl-coenzyme A lyase activity in human hepatoma HepG2 cell extracts.

When carbohydrate metabolism is impaired, fatty acid metabolism is activated. Excess acetyl-coenzyme A (CoA) is generated from fatty acids by ß-oxidation and is used for the formation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) and subsequently for acetoacetate. High levels of secreted ketone bodies (acetoacetate and 3ß-hydroxybutyrate) lower the pH of blood and urine, resulting in ketoacidosis. HMG-CoA lyase in hepatic cells is a rate-limiting enzyme catalyzing the cleavage of HMG-CoA to acetoacetate, and thus inhibition of this enzyme results in reduced acetoacetate production, in other words, impaired ketoacidosis. Inhibition of HMG-CoA lyase activity possibly prevents ketoacidosis and should be the therapeutic target. Polyphenols are common and abundant dietary constituents with beneficial effects on human health. We examined the inhibitory effects of dietary polyphenols on HMG-CoA lyase activity in cellular extracts of human hepatoma HepG2 cells. Of the nine representative dietary polyphenols tested, (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), and gallic acid (GA) effectively inhibited HMG-CoA lyase activity. Lineweaver-Burk analysis revealed that EGC and EGCG are likely to be mixed-type noncompetitive inhibitors. Pyrogallol with the gallyl structure also inhibited HMG-CoA lyase activity, suggesting that the gallyl moiety of polyphenols is important for the inhibition of HMG-CoA lyase activity.

TIM-4 has dual function in the induction and effector phases of murine arthritis.

T cell Ig and mucin domain (TIM)-4 is involved in immune regulation. However, the pathological function of TIM-4 has not been understood and remains to be clarified in various disease models. In this study, DBA/1 mice were treated with anti-TIM-4 mAb during the induction or effector phase of collagen-induced arthritis (CIA). Anti-TIM-4 treatment in the induction phase exacerbated the development of CIA. In vitro experiments suggest that CD4 T cells bind to TIM-4 on APCs, which induces inhibitory effect to CD4 T cells. In contrast, therapeutic treatment with anti-TIM-4 mAb just before or after the onset or even at later stage of CIA significantly suppressed the development and progression by reducing proinflammatory cytokines in the ankle joints without affecting T or B cell responses. Consistently, clinical arthritis scores of collagen Ab-induced arthritis, which is not mediated by T or B cells, were significantly reduced in anti-TIM-4-treated mice with a concomitant decrease of proinflammatory cytokines in the joints. In vitro, macrophages secreted proinflammatory cytokines in response to TIM-4-Ig protein and LPS, which were reduced by the anti-TIM-4 mAb. The anti-TIM-4 mAb also inhibited the differentiation and bone-resorbing activity of osteoclasts. These results indicate that TIM-4 has two distinct functions depending on the stage of arthritis. The therapeutic effect of anti-TIM-4 mAb on arthritis is mediated by the inhibition of proinflammatory cytokine production by inflammatory cells, osteoclast differentiation, and bone resorption, suggesting that TIM-4 might be an appropriate target for the therapeutic treatment of arthritis.

Immunomodulation of dendritic cells differentiated in the presence of nicotine with lipopolysaccharide from Porphyromonas gingivalis.

Tobacco smoking is a significant risk factor for periodontal diseases. Nicotine, one of the most studied constituents in cigarette smoke, is thought to modify immune responses. Dendritic cells (DCs), which are key mediators between innate and adaptive immunity, stimulate naive T cells to differentiate to effector T-cell subsets that may be actively involved in the immunopathogenesis of periodontal diseases. In this study, we evaluated the effects of nicotine and lipopolysaccharide (LPS) from Porphyromonas gingivalis, alone and in combination, on the functions of human monocyte-derived DCs to elucidate the mechanism of tissue destruction of smoking-associated periodontal diseases. P. gingivalis LPS-stimulated DCs differentiated with nicotine (NiDCs) induced lower T-cell proliferation and human leukocyte antigen (HLA)-DR expression, but elevated expression of programmed cell death ligand 1. Additionally, NiDCs impaired interferon-γ production but maintained interleukin (IL)-5 and IL-10 production in co-cultured T cells. Furthermore, NiDCs produced lower levels of proinflammatory cytokines compared with DCs differentiated in the absence of nicotine. Interestingly, NiDCs preferentially produced the T helper 2 (Th2)-type chemokines macrophage chemotactic protein-1 and macrophage-derived chemokine. These results suggest that the presence of nicotine during differentiation of DCs modulates the immunoregulatory functions of P. gingivalis LPS-stimulated DCs.

Nicotine modulates the immunological function of dendritic cells through peroxisome proliferator-activated receptor-γ upregulation.

We examined the effects of nicotine on differentiation and function of monocyte-derived human dendritic cells (DCs). NiDCs, which were the DCs differentiated in the presence of nicotine, showed lower levels of CD1a. Secretion of IL-12 and TNF-α by lipopolysaccharide (LPS)-stimulated NiDCs was significantly suppressed compared to monocyte-derived DCs grown without nicotine. NiDCs displayed a diminished capacity to induce allogeneic T cell proliferation with a reduced production of IFN-γ, and maintained/enhanced LPS-mediated expression of coinhibitory molecules. Interestingly, NiDCs enhanced the expression of nuclear receptor peroxisome proliferator-activated receptors γ (PPAR γ), which has immunomodulatory properties. Expression of PPAR γ and PPAR γ-target genes was significantly inhibited by pretreatment with d-tubocurarine, antagonist of non-selective nicotinic acetylcholine receptors (nAChR). In addition, reduction of Th1 responses was inhibited after blocking nAChR-mediated signal. These data suggest the effect of nicotine on altering DC immunogenicity by impeding Th1 immunity is partially mediated by upregulation of PPAR γ.

Interleukin-11 links oxidative stress and compensatory proliferation.

Apoptotic cells can stimulate the compensatory proliferation of surrounding cells to maintain tissue homeostasis. Although oxidative stress is associated with apoptosis and necrosis, whether it contributes to compensatory proliferation is unknown. Here, we showed that interleukin-11 (IL-11), a member of the IL-6 family of proinflammatory cytokines, was produced by cells in an oxidative stress-dependent manner. IL-11 production depended on the activation in dying cells of extracellular signal-regulated kinase 2, which in turn caused the phosphorylation and accumulation of the transcription factor Fra-1 by preventing its proteasome-dependent degradation. Fra-1 was subsequently recruited to the Il11 promoter and activated gene transcription. Upon acute liver injury in mice, IL-11 was mainly produced by hepatocytes in response to reactive oxygen species that were presumably released from dying hepatocytes. IL-11 that was secreted by the dying cells then induced the phosphorylation of the transcription factor STAT3 in adjacent healthy hepatocytes, which resulted in their compensatory proliferation. Furthermore, an IL-11 receptor (IL-11R) agonist enhanced the proliferation of hepatocytes and ameliorated oxidative stress upon acetaminophen-induced liver injury. Conversely, the effects of acetaminophen were exacerbated in mice deficient in the IL-11R α subunit. Together, these results suggest that IL-11 provides a functional link between oxidative stress and compensatory proliferation.

Nicotine up-regulates IL-8 expression in human gingival epithelial cells following stimulation with IL-1ß or P. gingivalis lipopolysaccharide via nicotinic acetylcholine receptor signalling.

OBJECTIVE: Cigarette smoking is an important risk factor for periodontal disease. The aim of this study is to evaluate the effect of nicotine, a major component of cigarette smoke, on interleukin-8 (IL-8) production and cellular signalling via nicotinic acetylcholine receptors (nAChRs) in human gingival epithelial cells (HGECs). DESIGN: Messenger RNA (mRNA) expression of nAChR subunits in three different HGEC lines (epi 4, Tfx and E6E7) was assessed using reverse transcription-polymerase chain reaction (RT-PCR). HGECs were stimulated by 1×10(-3)M nicotine in the presence or absence of IL-1ß or Porphyromonas gingivalis lipopolysaccharide (LPS). IL-8 production was then examined using real-time PCR and enzyme-linked immunosorbent assay. Nicotine-mediated signalling in the epi 4 cell line was also evaluated by Western blotting. RESULTS: HGECs expressed several nAChR subunits. Nicotine increased the secretion of IL-8 from HGECs that were cultured in the presence of IL-1ß or P. gingivalis LPS and also induced the phosphorylation of extracellular signal-regulated kinase (ERK) in epi 4. Pretreatment with non-selective nAChR antagonist or intracellular calcium chelator reduced the nicotine-induced phosphorylation of ERK. Furthermore, nicotine-induced IL-8 secretion was decreased by pretreatment with non-selective nAChR antagonist, ERK1/2 inhibitor or intracellular calcium chelator. CONCLUSION: These findings indicate that nicotine increases IL-8 production in gingival epithelial cells via ERK phosphorylation following Ca(2+) signalling after nAChR activation.

Importin ß1 protein-mediated nuclear localization of death receptor 5 (DR5) limits DR5/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cell death of human tumor cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/death receptor 5 (DR5)-mediated cell death plays an important role in the elimination of tumor cells and transformed cells. Recently, recombinant TRAIL and agonistic anti-DR5 monoclonal antibodies have been developed and applied to cancer therapy. However, depending on the type of cancer, the sensitivity to TRAIL has been reportedly different, and some tumor cells are resistant to TRAIL-mediated apoptosis. Using confocal microscopy, we found that large amounts of DR5 were localized in the nucleus in HeLa and HepG2 cells. Moreover, these tumor cells were resistant to TRAIL, whereas DU145 cells, which do not have nuclear DR5, were highly sensitive to TRAIL. By means of immunoprecipitation and Western blot analysis, we found that DR5 and importin ß1 were physically associated, suggesting that the nuclear DR5 was transported through the nuclear import pathway mediated by importin ß1. Two functional nuclear localization signals were identified in DR5, the mutation of which abrogated the nuclear localization of DR5 in HeLa cells. Moreover, the nuclear transport of DR5 was also prevented by the knockdown of importin ß1 using siRNA, resulting in the up-regulation of DR5 expression on the cell surface and an increased sensitivity of HeLa and HepG2 cells to TRAIL. Taken together, our findings suggest that the importin ß1-mediated nuclear localization of DR5 limits the DR5/TRAIL-induced cell death of human tumor cells and thus can be a novel target to improve cancer therapy with recombinant TRAIL and anti-DR5 antibodies.