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In the past, the genetically diabetic-obese diabetes/diabetes (db/db) and obese/obese (ob/ob) mouse strains were used to investigate mechanisms of diabetes-impaired wound healing. Here we determined patterns of skin repair in genetically normal C57Bl/6J mice that were fed using a high fat diet (HFD) to induce a diabetes-obesity syndrome. Wound closure was markedly delayed in HFD-fed mice compared to mice which had received a standard chow diet (CD). Impaired wound tissue of HFD mice showed a marked prolongation of wound inflammation. Expression of vascular endothelial growth factor (VEGF) was delayed and associated with the disturbed formation of wound margin epithelia and an impaired angiogenesis in the reduced granulation tissue. Normal wound contraction was retarded and disordered. Wound disorders in obese C57Bl/6J mice were paralleled by a prominent degradation of the inhibitor of NFκB (IκB-α) in the absence of an Akt activation. By contrast to impaired wound conditions in ob/ob mice, late wounds of HFD mice did not develop a chronic inflammatory state and were epithelialized after 11 days of repair. Thus, only genetically obese and diabetic ob/ob mice finally developed chronic wounds and therefore represent a better suited experimental model to investigate diabetes-induced wound healing disorders.
IL-18 is an important mediator involved in chronic inflammatory conditions such as cutaneous lupus erythematosus, psoriasis and chronic eczema. An imbalance between IL-18 and its endogenous antagonist IL-18 binding protein (BP) may account for increased IL-18 activity. IL-27 is a cytokine with dual function displaying pro- and anti-inflammatory properties. Here we provide evidence for a yet not described anti-inflammatory mode of action on skin resident cells. Human keratinocytes and surprisingly also fibroblasts (which do not produce any IL-18) show a robust, dose-dependent and highly inducible mRNA expression and secretion of IL-18BP upon IL-27 stimulation. Other IL-12 family members failed to induce IL-18BP. The production of IL-18BP peaked between 48–72 h after stimulation and was sustained for up to 96 h. Investigation of the signalling pathway showed that IL-27 activates STAT1 in human keratinocytes and that a proximal GAS site at the IL-18BP promoter is of importance for the functional activity of IL-27. The data are in support of a significant anti-inflammatory effect of IL-27 on skin resident cells. An important novel property of IL-27 in skin pathobiology may be to counter-regulate IL-18 activities by acting on keratinocytes and importantly also on dermal fibroblasts.
Simultaneous and dose dependent melanoma cytotoxic and immune stimulatory activity of betulin
(2015)
Conventional cytostatic cancer treatments rarely result in the complete eradication of tumor cells. Therefore, new therapeutic strategies focus on antagonizing the immunosuppressive activity of established tumors. In particular, recent studies of antigen-loaded dendritic cells (DCs) eliciting a specific antitumor immune response has raised the hopes of achieving the complete elimination of tumor tissue. Genistein, fingolimod and betulin have already been described as active compounds in different types of cancer. Herein, we applied an integrated screening approach to characterize both their cytostatic and their immune-modulating properties side-by-side. As will be described in detail, our data confirmed that all three compounds exerted proapoptotic and antiproliferative activity in different B16 melanoma cell lines to a given extent, as revealed by an MTT assay, CFSE and DAPI staining. However, while genistein and fingolimod also affected the survival of primary bone marrow (BM) derived DCs of C57BL/6 mice, betulin exhibited a lower cytotoxicity for BMDCs in comparison to the melanoma cells. Moreover, we could show for the first time, that only betulin caused a simultaneous, highly specific immune-stimulating activity, as measured by the IL-12p70 release of Toll-like receptor 4-stimulated BMDCs by ELISA, which was due to increased IL-12p35 mRNA expression. Interestingly, the activation of DCs resulted in enhanced T lymphocyte stimulation, indicated by increased IL-2 and IFN-γ production of cytotoxic T cells in spleen cell co-culture assays which led to a decreased viability of B16 cells in an antigen specific model system. This may overcome the immunosuppressive environment of a tumor and destroy tumor cells more effectively in vivo if the immune response is specific targeted against the tumor tissue by antigen-loaded dendritic cells. In summary, cytostatic agents, such as betulin, that simultaneously exhibit immune stimulatory activity may serve as lead compounds and hold great promise as a novel approach for an integrated cancer therapy.
Interleukin (IL)-22 is a STAT3-activating cytokine displaying characteristic AU-rich elements (ARE) in the 3'-untranslated region (3'-UTR) of its mRNA. This architecture suggests gene regulation by modulation of mRNA stability. Since related cytokines undergo post-transcriptional regulation by ARE-binding tristetraprolin (TTP), the role of this destabilizing protein in IL-22 production was investigated. Herein, we demonstrate that TTP-deficient mice display augmented serum IL-22. Likewise, IL-22 mRNA was enhanced in TTP-deficient splenocytes and isolated primary T cells. A pivotal role for TTP is underscored by an extended IL-22 mRNA half-life detectable in TTP-deficient T cells. Luciferase-reporter assays performed in human Jurkat T cells proved the destabilizing potential of the human IL-22-3'-UTR. Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3'-UTR. Transcript destabilization by TTP was nullified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity. Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126. Altogether, data indicate that TTP directly controls IL-22 production, a process counteracted by MEK1/2. The TTP-dependent regulatory pathway described herein likely contributes to the role of IL-22 in inflammation and cancer and may evolve as novel target for pharmacological IL-22 modulation.
Because of its highly bioactive properties sphingosine 1-phosphate (S1P) is an attractive target for the treatment of several diseases. Since the expression of sphingosine kinases as well as S1P receptors was demonstrated in the kidney, questions about the physiological and pathophysiological functions of S1P in this organ have been raised. In this review, we summarize the current state of knowledge about S1P-mediated functions in the kidney. A special focus is put on S1P modulated signal transduction in renal glomerular and tubular cells and consequences for the development and treatment of several kidney diseases, diabetic nephropathy, glomerulonephritis, ischemia-reperfusion injury, as well as for Wilms tumor progression.
Interleukin (IL)-18, formerly known as interferon (IFN)-γ-inducing factor, is a crucial mediator of host defence and inflammation. Control of IL-18 bioactivity by its endogenous antagonist IL-18 binding protein (IL-18BP) is a major objective of immunoregulation. IL-18BP is strongly up-regulated by IFN-γ, thereby establishing a negative feedback mechanism detectable in cell culture and in vivo. Here we sought to investigate in D.L. Dexter (DLD) colon carcinoma cells molecular mechanisms of IL-18BP induction under the influence of IFN-γ. Mutational analysis revealed that a proximal γ-activated sequence (GAS) at the IL-18BP promoter is of pivotal importance for expression by IFN-γ-activated cells. Use of siRNA underscored the essential role of the signal transducer and activator of transcription (STAT)-1 in this process. Indeed, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis proved STAT1 binding to this particular GAS site. Maximal expression of IL-18BP was dependent on de novo protein synthesis but unaffected by silencing of interferon regulatory factor-1. Altogether, data presented herein indicate that direct action of STAT1 on the IL-18BP promoter at the proximal GAS element is key to IL-18BP expression by IFN-γ-stimulated DLD-1 colon carcinoma cells.
The widely varying therapeutic response of patients with inflammatory bowel disease (IBD) continues to raise questions regarding the unclarified heterogeneity of pathological mechanisms promoting disease progression. While biomarkers for the differentiation of Crohn’s disease (CD) versus ulcerative colitis (UC) have been suggested, specific markers for a CD subclassification in ileal CD versus colonic CD are still rare. Since an altered signature of the tryptophan metabolism is associated with chronic inflammatory disease, we sought to characterize potential biomarkers by focusing on the downstream enzymes and metabolites of kynurenine metabolism. Using immunohistochemical stainings, we analyzed and compared the mucosal tryptophan immune metabolism in bioptic samples from patients with active inflammation due to UC or CD versus healthy controls. Localization-specific quantification of immune cell infiltration, tryptophan-metabolizing enzyme expression and mucosal tryptophan downstream metabolite levels was performed. We found generally increased immune cell infiltrates in the tissue of all patients with IBD. However, in patients with CD, significant differences were found between regulatory T cell and neutrophil granulocyte infiltration in the ileum compared with the colon. Furthermore, we observed decreased kynurenine levels as well as strong kynureninase (KYNU) expression specifically in patients with ileal CD. Correspondingly, significantly elevated levels of the kynurenine metabolite 3-hydroxyanthranilic acid were detected in the ileal CD samples. Highlighting the heterogeneity of the different phenotypes of CD, we identified KYNU as a potential mucosal biomarker allowing the localization-specific differentiation of ileal CD versus colonic CD.
Sphingosine‐1‐phosphate lyase 1 (S1P lyase or SGPL1) is an essential sphingosine‐1‐phosphate‐degrading enzyme. Its manipulation favors onset and progression of colorectal cancer and others in vivo. Thus, SGPL1 is an important modulator of cancer initiation. However, in established cancer, the impact of retrospective SGPL1 modulation is elusive. Herein, we analyzed how SGPL1 siRNA affects malignancy of the human colorectal cancer cells DLD‐1 and found that in parallel to the reduction of SGPL1 expression levels, migration, invasion, and differentiation status changed. Diminished SGPL1 expression was accompanied with reduced cell migration and cell invasion in scratch assays and transwell assays, whereas metabolic activity and proliferation was not altered. Decreased migration was attended by increased cell–cell‐adhesion through upregulation of E‐cadherin and formation of cadherin‐actin complexes. Spreading cell islets showed lower vimentin abundance in border cells. Furthermore, SGPL1 siRNA treatment induced expression of epithelial cell differentiation markers, such as intestinal alkaline phosphatase and cytokeratin 20. Hence, interference with SGPL1 expression augmented a partial redifferentiation of colorectal cancer cells toward normal colon epithelial cells. Our investigation showed that SGPL1 siRNA influenced tumorigenic activity of established colorectal cancer cells. We therefore suggest SGPL1 as a target for lowering malignant potential of already existing cancer.
IL-22 is an immunoregulatory cytokine displaying pathological functions in models of autoimmunity like experimental psoriasis. Understanding molecular mechanisms driving IL-22, together with knowledge on the capacity of current immunosuppressive drugs to target this process, may open an avenue to novel therapeutic options. Here, we sought to characterize regulation of human IL22 gene expression with focus on the established model of Jurkat T cells. Moreover, effects of the prototypic immunosuppressant cyclosporin A (CsA) were investigated. We report that IL-22 induction by TPA/A23187 (T/A) or αCD3 is inhibited by CsA or related FK506. Similar data were obtained with peripheral blood mononuclear cells or purified CD3(+) T cells. IL22 promoter analysis (-1074 to +156 bp) revealed a role of an NF-AT (-95/-91 nt) and a CREB (-194/-190 nt) binding site for gene induction. Indeed, binding of CREB and NF-ATc2, but not c-Rel, under the influence of T/A to those elements could be proven by ChIP. Because CsA has the capability to impair IκB kinase (IKK) complex activation, the IKKα/β inhibitor IKKVII was evaluated. IKKVII likewise reduced IL-22 induction in Jurkat cells and peripheral blood mononuclear cells. Interestingly, transfection of Jurkat cells with siRNA directed against IKKα impaired IL22 gene expression. Data presented suggest that NF-AT, CREB, and IKKα contribute to rapid IL22 gene induction. In particular the crucial role of NF-AT detected herein may form the basis of direct action of CsA on IL-22 expression by T cells, which may contribute to therapeutic efficacy of the drug in autoimmunity.