Thorsten Stroh

Histone deacetylases: novel targets for prevention of colitis-associated cancer in mice

Objective: Inhibition of histone deacetylases, well known for its antiproliferative efficacy in vivo, was recently shown to ameliorate inflammation in experimental colitis. Since inflammatory bowel disease is associated with an increased risk of developing colon cancer, here the combined anti-inflammatory and proapoptotic efficacy of histone deacetylase inhibitors was studied in mouse models. Methods: The novel histone deacetylase inhibitor ITF2357 was compared with suberoylanilide hydroxamic acid in models of experimental colitis. Effects on tumour growth were studied after treatment of mice with azoxymethane and dextran sulphate sodium, and in interleukin 10 (IL10) knockout mice, respectively. Possible underlying mechanisms involving apoptosis and nuclear factor (NF)-κB activation were addressed by flow cytometry and western blot. Results: In dextran sulphate sodium- and trinitrobenzene sulphonic acid-induced colitis, treatment with ITF2357 was superior to suberoylanilide hydroxamic acid as shown by macroscopic and histological amelioration of inflammation, by reduced production of interferon γ (IFNγ) and by increased production of IL10. In both models of inflammation-mediated tumourigenesis, inhibition of histone deacetylases resulted in a significant suppression of tumour growth in terms of size and number, along with reduced signs of inflammation. As for potential mechanisms of ITF2357 action, increased acetylation of histone 3, reduced production of IFNγ and enhanced apoptosis in lamina propria mononuclear cells were found to accompany a histone deacetylase-dependent activation of NF-κB. Conclusions: These results indicate that inhibition of histone deactylases can attenuate inflammation-mediated tumour growth, which is paralled by an inhibition of NF-κB. Thus histone deacetylase inhibitors provide a promising strategy that combines anti-inflammatory and proapoptotic modes of action.

Leptin: A Critical Regulator of CD4+ T-cell Polarization in Vitro and in Vivo

Besides being mandatory in the metabolic system, adipokines like leptin directly affect immunity. Leptin was found to be necessary in T helper 1 (Th1)-dependent inflammatory processes, whereas effects on Th2 cells are rarely understood. Here, we focused on leptin in T-helper cell polarization and in Th2-mediated intestinal inflammation in vivo. The induction of cytokine-producing Th1 or Th2 cells from naive CD4(+) T cells under polarizing conditions in vitro was generally decreased in cells from leptin-deficient ob/ob mice compared with wild-type mice. To explore the in vivo relevance of leptin in Th2-mediated inflammation, the model of oxazolone-induced colitis was employed in wild-type, ob/ob, and leptin-reconstituted ob/ob mice. Ob/ob mice were protected, whereas wild-type and leptin-reconstituted ob/ob mice developed colitis. The disease severity went in parallel with local production of the Th2 cytokine IL-13. A possible explanation for the protection of ob/ob mice in Th1- as well as in Th2-dependent inflammation is provided by a decreased expression of the key transcription factors for Th1 and Th2 polarization, T-bet and GATA-3, in naive ob/ob T cells. In conclusion, these results support the regulatory function of the adipokine leptin within T-cell polarization and thus in the acquired immune system and support the concept that there is a close interaction with the endocrine system.

Resistance of Janus Kinase-2 Dependent Leptin Signaling in Natural Killer (NK) Cells: A Novel Mechanism of NK Cell Dysfunction in Diet-Induced Obesity

Leptin acts not only as an anorexigenic hormone but also regulates cell-mediated immunity via leptin receptors (Ob-R) expressed on T and B lymphocytes. However, the impact of leptin on natural killer (NK) cells is currently elusive. We evaluated leptin effects on NK cells in relation to the body weight in rats using in vivo and in vitro approaches. Leptin was injected iv in male lean and diet-induced obese Lewis and F344 rats. NK cell numbers were analyzed in blood and spleen by fluorescence activated cell sorting and immunohistochemistry, and the activity of NK cells was measured by chromium release assay. Ob-R expression was investigated by confocal laser scanning and quantitative RT-PCR. To compare leptin-dependent intracellular signaling under basal and leptin- and tumor cell (MADB106)-stimulated conditions, intracellular target proteins of NK cells were evaluated by Western blotting. Number and distribution pattern of splenic NK cells were significantly different in lean and obese animals. Leptin administration resulted in a 4-fold higher stimulation of the NK activity in lean than obese animals. This was not due to a decreased expression of Ob-R because quantitative RT-PCR revealed significantly higher Ob-Rb mRNA levels in NK cells from obese rats. In contrast, postreceptor signaling is differentially abrogated in obese animals with significantly lower activation of postreceptor signaling components (Janus kinase-2p, protein kinase B pT308, AMPalphapT172) after an in vivo leptin challenge. In conclusion, the results for the first time assign leptin a central role as a modulator of NK cell number and activity only in lean but not obese subjects. The differential role of leptin has important implications for the influence of body weight in the response to systemic inflammations and in the immunological defense of cancer.

Toll-like receptor expression and response to specific stimulation in adipocytes and preadipocytes : On the role of fat in inflammation

Abstract:  Data in this study indicate that both adipocytes and preadipocytes express abroad set of TLRs and they also respond to specific stimulation by cytokine production. The may be of relevance to Crohns disease and a suggests a closer link between adipose tissue and innate immunity.

Adipokines from local fat cells shape the macrophage compartment of the creeping fat in Crohn's disease

Objective The creeping fat in Crohns disease (CD) is infiltrated by macrophages; local adipokine levels are increased. This study aimed to link these observations to define a role for macrophages in the pathology of human CD. Methods Human peripheral blood CD14 cells were polarised in vitro into M1 and M2 macrophages. The effects on adipokine receptors, phenotypic surface markers, cytokines and chemokines were assessed after treatment with leptin and adiponectin. Immunohistochemistry visualised macrophage subtypes in samples of mesenteric fat tissue from patients with CD. The chemotactic potential of secreted macrophage products was determined by T cell migration and chemokine production in vitro. Results Although both adipokines altered the phenotype and function of M1 and M2 macrophages, M2 macrophages were more susceptible. M1 responded to leptin by increased cytokine production, but the stronger effect was seen in M2 macrophages with high expression of interleukin (IL)-10, IL-6 and tumour necrosis factor α. Adiponectin exerted similar effects and led to upregulated mannose receptor expression by M2 macrophages. Large macrophage numbers within the mesenteric fat tissue of patients with CD comprise a unique infiltration predominantly of M2 macrophages, leading to an IL-10-rich environment. While leptin increased the potency of both subtypes to attract CD3 T cells, adiponectin only affected M2 macrophages. Conclusion The adipocyte-dependent microenvironment within the creeping fat of patients with CD modulates the local macrophage compartment to a preference for the M2 subtype. The findings in this study with human cells suggest a protective role for the mesenteric fat in CD in terms of an enveloping barrier with the potential to limit intestinal inflammation.

Combined pulse electroporation--a novel strategy for highly efficient transfection of human and mouse cells.

The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect.

A hepatoprotective Lindera obtusiloba extract suppresses growth and attenuates insulin like growth factor-1 receptor signaling and NF-kappaB activity in human liver cancer cell lines

BackgroundIn traditional Chinese and Korean medicine, an aqueous extract derived from wood and bark of the Japanese spice bush Lindera obtusiloba (L.obtusiloba) is applied to treat inflammations and chronic liver diseases including hepatocellular carcinoma. We previously demonstrated anti-fibrotic effects of L.obtusiloba extract in hepatic stellate cells. Thus, we here consequently examine anti-neoplastic effects of L.obtusiloba extract on human hepatocellular carcinoma (HCC) cell lines and the signaling pathways involved.MethodsFour human HCC cell lines representing diverse stages of differentiation were treated with L.obtusiloba extract, standardized according to its known suppressive effects on proliferation and TGF-β-expression. Beside measurement of proliferation, invasion and apoptosis, effects on signal transduction and NF-κB-activity were determined.ResultsL.obtusiloba extract inhibited proliferation and induced apoptosis in all HCC cell lines and provoked a reduced basal and IGF-1-induced activation of the IGF-1R signaling cascade and a reduced transcriptional NF-κB-activity, particularly in the poorly differentiated SK-Hep1 cells. Pointing to anti-angiogenic effects, L.obtusiloba extract attenuated the basal and IGF-1-induced expression of hypoxia inducible factor-1α, vascular endothelial growth factor, peroxisome proliferator-activated receptor-γ, cyclooxygenase-2 and inducible nitric oxide synthase.ConclusionsThe traditional application of the extract is confirmed by our experimental data. Due to its potential to inhibit critical receptor tyrosine kinases involved in HCC progression via the IGF-1 signaling pathway and NF-κB, the standardized L.obtusiloba extract should be further analysed for its active compounds and explored as (complementary) treatment option for HCC.

Histone deacetylase 5 regulates the inflammatory response of macrophages

Modifying the chromatin structure and interacting with non‐histone proteins, histone deacetylases (HDAC) are involved in vital cellular processes at different levels. We here specifically investigated the direct effects of HDAC5 in macrophage activation in response to bacterial or cytokine stimuli. Using murine and human macrophage cell lines, we studied the expression profile and the immunological function of HDAC5 at transcription and protein level in over‐expression as well as RNA interference experiments. Toll‐like receptor‐mediated stimulation of murine RAW264.7 cells significantly reduced HDAC5 mRNA within 7 hrs but presented baseline levels after 24 hrs, a mechanism that was also found for Interferon‐γ treatment. If treated with lipopolysaccharide, RAW264.7 cells transfected for over‐expression only of full‐length but not of mutant HDAC5, significantly elevated secretion of tumour necrosis factor α and of the monocyte chemotactic protein‐1. These effects were accompanied by increased nuclear factor‐κB activity. Accordingly, knock down of HDAC5‐mRNA expression using specific siRNA significantly reduced the production of these cytokines in RAW264.7 or human U937 cells. Taken together, our results suggest a strong regulatory function of HDAC5 in the pro‐inflammatory response of macrophages.

Extraluminal factors contributing to inflammatory bowel disease.

Many identified and yet unknown factors contribute to the pathogenesis of inflammatory bowel disease (IBD). The genome-wide association studies clearly support the earlier developed concept that IBD occurs in genetically predisposed individuals who are exposed to distinct environmental factors, which together result in dysregulation of the mucosal immune system. Thus, the majority of previous studies have focused on the immune response within the intestinal wall. The present review aims to emphasize the contribution of three extraluminal structures to this inflammatory process, namely the mesenteric fat tissue, the lymphatics and the microvasculature. Broadening our view across the intestinal wall will not only facilitate our understanding of the disease, but will also us to identify future therapeutic targets.

1094 The Impact of HDAC Inhibition On CD4+ T Cells in Chronic Intestinal Inflammation

group, consisting of 10 wild-type mice. We induced a mild colitis through administration of 1.5% Dextran Sodium Sulphate (DSS) in their drinking water for 7 days. On day 8 we sacrificed the mice and scored blood loss and stool consistency. The average of the scores for weight loss, blood loss and stool consistency was defined as the disease activity index. A pathologist examined the degree of inflammation by scoring influx of inflammatory cells, crypt loss, ulceration, fibrosis, edema and the area involved. Neutrophils, macrophages and T-cells were stained using standard immunohistochemical techniques and counted. Results: The PI3Kγ-kinase-dead mice developed a significantly milder colitis than the control group. On day 8 they had gained 1.1% ± 0.9% weight, whereas the wild-type mice had lost 1.4% ± 0.6% weight, P=0.027. Their disease activity index was lower than control mice (0.63 ± 0.14, versus 1.63 ± 0.24, P=0.002). They also scored better on the histology-parameters (0.8 ± 0.1, versus 1.1 ± 0.1, P=0.02). Influx of T-cells and macrophages was significantly reduced in the PI3Kγ-kinase-dead mice (7.9 ± 0.6 versus 10.5 ± 0.9 positive cells per intercrypt area, P=0.02, and 8.7 ± 0.9, versus 11.1 ± 0.8 positive cells per intercrypt area, P=0.03, respectively). Conclusions: These data show that mice lacking functional PI3Kγ develop less severe colitis upon induction with DSS. These results suggest that the reduced influx of inflammatory cells could play an important role in this outcome and that inhibition of PI3Kγ might be a valuable target in the treatment of IBD.