Inka Fedke

Histone Hyperacetylation Is Associated with Amelioration of Experimental Colitis in Mice

Inhibitors of histone deacetylases (HDAC) are being studied for their antiproliferative effects in preclinical cancer trials. Recent studies suggest an anti-inflammatory role for this class of compounds. Because inflammatory bowel disease is associated with an increased risk of malignancies, agents with antiproliferative and anti-inflammatory properties would be of therapeutic interest. HDAC inhibitors from various classes were selected and evaluated for their in vitro capacity to suppress cytokine production and to induce apoptosis and histone acetylation. Valproic acid (VPA) and suberyolanilide hydroxamic acid (SAHA) were chosen for further studies in dextran sulfate sodium- and trinitrobenzene sulfonic acid-induced colitis in mice. In vitro, inhibition of HDAC resulted in a dose-dependent suppression of cytokine synthesis and apoptosis induction requiring higher concentrations of HDAC inhibitors for apoptosis induction compared with cytokine inhibition. Oral administration of either VPA or SAHA reduced disease severity in dextran sulfate sodium-induced colitis. The macroscopic and histologic reduction of disease severity was associated with a marked suppression of colonic proinflammatory cytokines. In parallel to the beneficial effect observed, a dose-dependent increase in histone 3 acetylation at the site of inflammation was shown under VPA treatment. Furthermore, SAHA as well as VPA treatment resulted in amelioration of trinitrobenzene sulfonic acid-induced colitis, which was associated with an increase of apoptosis of lamina propria lymphocytes. Inhibitors of HDAC reveal strong protective effects in different models of experimental colitis by inducing apoptosis and suppressing proinflammatory cytokines, thereby representing a promising class of compounds for clinical studies in human inflammatory bowel disease.

Leptin receptor expression on T lymphocytes modulates chronic intestinal inflammation in mice

Background: Leptin regulates appetite through the long isoform of its receptor in the hypothalamus. Although leptin regulates immune responses, it is still unknown whether a direct effect of leptin on lymphocytes is required. Aims: To clarify whether expression of leptin receptors on T lymphocytes modulates intestinal inflammation in mice. Methods: The model of colitis induced by transfer of CD4+CD45RBhigh (RBhigh) cells into scid mice was used. Wild-type (WT) or leptin receptor deficient (db/db) RBhigh cells were transferred into scid mice and development of colitis evaluated. Results: Leptin receptors were expressed on both RBhigh and RBlow cells. Intestinal lymphocytes of mice with colitis expressed high leptin levels compared with healthy controls whereas the opposite was true for serum leptin levels. Transfer of RBhigh cells from db/db mice induced delayed disease compared with transfer of WT cells. A high rate of apoptosis in lamina propria lymphocytes and reduced cytokine production were observed early on in scid mice receiving db/db RBhigh cells. These effects were not due to the high levels of glucocorticoids present in db/db mice as administration of corticosterone to WT mice failed to reproduce this phenomenon. High expression of peroxisome proliferator activated receptor γ was observed in the colon of recipients of db/db compared with WT cells. Freshly isolated db/db RBhigh cells produced low levels of interferon γ. Despite delayed onset of colitis, as disease progressed differences between mice receiving WT or db/db cells were no longer apparent. Conclusions: These results suggest that leptin affects the immune response, partly by acting on the long isoform of its receptor expressed on T lymphocytes.

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.

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.

Defining the role of T cell-derived leptin in the modulation of hepatic or intestinal inflammation in mice.

The role of leptin in the immune system has been well established. While adipocytes represent the major source, leptin production by lymphocytes, infiltrating at the site of inflammation, was recently demonstrated. However, the significance of this locally released leptin remains unresolved. In the present study, two models in which absence of leptin‐signalling is associated with protection were employed: the model of ConA‐induced hepatitis and the CD4+CD45Rbhigh transfer model of colitis. For the ConA model, scid mice were reconstituted with either WT or leptin‐deficient (ob/ob) CD4+ T cells. Eight weeks post transfer, ConA was injected and serum ALT, TNFα, leptin as well as liver mononuclear cell activation and histological signs of inflammation were evaluated. No difference between recipients of WT or ob/ob cells was observed for any of the parameters evaluated. In the second model, either WT or ob/ob CD4+CD45Rbhigh cells were transferred into scid mice. No histological differences were detected, although recipients of ob/ob cells showed higher weight loss compared to recipients of WT cells. Spontaneous production of IL‐6 from colon cultures obtained from recipients of ob/ob cells was reduced compared to recipients of WT cells, whereas stimulation of lamina propria lymphocytes with leptin resulted in a higher IFNγ release in recipients of ob/ob cells compared to recipients of WT cells. In conclusion, the present study provides evidence that T cell‐derived leptin does not play a major role in the regulation of the inflammatory process, indicating that the adipose tissue is the critical player in the immune‐modulating effects of leptin.

Development of intestinal inflammation in double IL-10- and leptin-deficient mice

Leptin‐deficient (ob/ob) mice are resistant in different models of autoimmunity and inflammation, suggesting that leptin regulates immunity and inflammation. To investigate whether leptin deficiency modulates the spontaneous intestinal inflammation observed in interleukin (IL)‐10‐deficient mice, double IL‐10‐ and leptin‐deficient [IL‐10 knockout (KO) ob/ob] mice were generated and compared with single IL‐10 KO mice for colitis severity. Body weight in IL‐10 KO ob/ob mice was significantly reduced compared with that of ob/ob mice. However, when compared with wild‐type or IL‐10 KO mice, IL‐10 KO ob/ob mice were still markedly obese. IL‐10 KO and IL‐10 KO ob/ob mice developed colitis with a comparable time‐course and severity in terms of macroscopic and histologic scores. Likewise, production of inter feron‐γ, IL‐6, and IL‐13 from colon cultures and splenocytes did not differ among these two groups. Conversely, rates of apoptosis were higher in lamina propria lymphocytes obtained from the colon of IL‐10 KO ob/ob compared with IL‐10 KO mice. In conclusion, although leptin deficiency has been associated with resistance in models of autoimmunity and inflammation induced by exogenous stimuli, leptin appears not to play a significant role in the spontaneous colitis of IL‐10 KO mice, although it modulates survival of intestinal lymphocytes.

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.