HPV16 activates the AIM2 inflammasome in keratinocytes.

Human papillomaviruses (HPV) are double-stranded DNA viruses, which selectively infect keratinocytes in stratified epithelia. After an initial infection, many patients clear HPV. In some patients, however, HPV persist, and dysfunctional innate immune responses to HPV infection could be involved in the ineffective clearing of these viruses. In this study, the mechanisms of HPV-induced immune responses in keratinocytes were investigated. Binding of viral DNA leads to AIM2 inflammasome activation and IL-1ß release, while IFI16 activation results in IFN-ß release. Using immunohistochemistry, AIM2 and IFI16-two recently identified sensors for cytosolic DNA-were also detected in HPV positive skin lesions. CISH stainings further confirmed the presence of cytosolic HPV16 DNA in biopsy samples. Moreover, active IL-1ß and cleaved caspase-1 were detected in HPV infected skin, suggesting inflammasome activation by viral DNA. In subsequent functional studies, HPV16 DNA triggered IL-1ß and IL-18 release via the AIM2 inflammasome in normal human keratinocytes. Although HPV DNA did not induce IFN-ß in keratinocytes, IFN-ß secretion was observed when AIM2 was blocked. Meanwhile, blocking of IFI16 increased HPV16 DNA-induced IL-1ß, but not IL-18, secretion. These findings suggest crosstalk between IFI16 and AIM2 in the immune response to HPV DNA. In sum, novel aspects concerning HPV-induced innate immune responses were identified. Eventually, understanding the mechanisms of HPV-induced inflammasome activation could lead to the development of novel strategies for the prevention and treatment of HPV infections.

Botulinum toxin A for the treatment of keloids.

INTRODUCTION: Keloids are the result of excessive scar tissue formation. Besides their poor aesthetic appearance, keloids can be associated with severe clinical symptoms such as pain, itching, and rigidity. Unfortunately, most therapeutic approaches remain clinically unsatisfactory. Recently, injections with botulinum toxin A (BTA) were proposed for the treatment of established keloids in a clinical trial. In this study, we aimed to verify the effects of intralesional BTA for the treatment of therapy-resistant keloids using objective measurements. In addition, the underlying molecular mechanisms were investigated using cultured keloid-derived fibroblasts. MATERIALS AND METHODS: Four patients received BTA (doses varying from 70 to 140 Speywood units per session) injected directly into their keloids every 2 months for up to 6 months. Differences in height and volume were evaluated clinically and measured with a 3-D optical profiling system. Keloid-derived fibroblasts were treated with different concentrations of BTA, and expression of collagen (COL)1A1, COL1A2, COL3A1, TGF-ß1, TGF-ß2, TGF-ß3, fibronectin-1, laminin-ß2, and α-SMA was determined by real-time quantitative PCR. MTT and BrdU assays were used to analyze the effects of BTA on fibroblast proliferation and metabolism. RESULTS: Intralesional administration of BTA did not result in regression of keloid tissue. No differences in expression of ECM markers, collagen synthesis, or TGF-ß could be observed after BTA treatment of keloid fibroblasts. In addition, cell proliferation and metabolism of keloid fibroblasts was not affected by BTA treatment. CONCLUSION: The suggested clinical efficiency of intralesional BTA for the therapy of existent keloids could not be confirmed in this study. Based on our data, the potential mechanisms of action of BTA on keloid-derived fibroblasts remain unclear.

Honey bee (Apis mellifera) venom induces AIM2 inflammasome activation in human keratinocytes.

BACKGROUND: Following allergen exposure, cytokines and other pro-inflammatory signals play an important role in the immunological cascade leading to allergic sensitization. Inflammasomes sense exogenous and endogenous danger signals and trigger IL-1ß and IL-18 activation which in turn shape Th2 responses. Honey bee venom (BV) allergies are very common; however, the local inflammatory cascade leading to the initiation of allergic sensitization is poorly understood. In this study, the local inflammatory cascades in skin after exposure to BV were investigated. METHODS: The mechanisms of inflammasome activation in human skin and in cultured keratinocytes upon BV exposure were analyzed by ELISA, Western blot, flow cytometry, siRNA techniques, and immunofluorescence. RESULTS: In an ex vivo bee sting model, BV induced IL-1ß release suggesting the activation of inflammasomes. Indeed, in cultured keratinocytes, the BV component melittin triggered IL-1ß and IL-18 release via the AIM2 inflammasome. AIM2 is a cytosolic DNA receptor, and mitochondrial as well as genomic DNA was detected in the cytosol of melittin-treated keratinocytes as triggers of inflammasome activation. As a mechanism, melittin mediated destruction of mitochondrial membranes leading to the leakage of mitochondrial DNA into the cytosolic compartment. CONCLUSION: These data suggest that upon BV exposure, keratinocytes are involved in an innate immune response by the activation of the AIM2 inflammasome and subsequent IL-1ß and IL-18 release triggered by endogenous DNA. As IL-1ß and IL-18 are involved in Th2- and IgE-mediated immune reactions, these results could add to the understanding of the role of the tissue microenvironment to subsequent allergic responses.

MIC-1 (a multifunctional modulator of dendritic cell phenotype and function) is produced by decidual stromal cells and trophoblasts.

BACKGROUND: Macrophage inhibitory cytokine-1 (MIC-1) is a multifunctional cytokine produced in high amounts by placental tissue. Inhibiting trophoblast invasion and suppressing inflammation through inhibition of macrophage activation, MIC-1 is thought to provide pleiotropic functions in the establishment and maintenance of pregnancy. So far, little is known about the decidual cell subsets producing MIC-1 and the effect of this cytokine on dendritic cells (DCs), which are known to play a distinct role in the development of pro-fetal tolerance in pregnancy. METHODS: To identify the decidual cell types expressing and secreting MIC-1, immunohistochemical staining, PCR experiments, western blot analysis and ELISAs were performed. Immature DCs (iDCs) were generated from peripheral blood-derived monocytes and differentiated in the presence of MIC-1 or dexamethasone (Dex) for control. Migratory and proliferative activity of DCs after MIC-1 exposure was investigated by migration and proliferation assay. Cytokine secretion after MIC-1 exposure was tested in isolated uNK cells, isolated CD14+ monocytes, monocyte-derived iDCs and mature DCs. Subsequently, the phenotype of DCs was studied using FACS analysis. To test the T-cell stimulatory capacity of pre-incubated DCs, mixed lymphocyte reaction was applied. Finally, the expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) after the exposure of MIC-1 to maturing DCs was analysed by western blot. RESULTS: Immunohistochemical staining, PCR and western blot experiments demonstrated that MIC-1 is mainly expressed by trophoblast cells and decidual stromal cells. Analysis of the MIC-1 secretion of decidual cell types by ELISA again characterized trophoblast and stromal cells as main producers. The migratory activity of iDCs was significantly induced by MIC-1. No changes in proliferative activity of DCs were observed after MIC-1 pre-incubation. The secretion of pro- or anti-inflammatory cytokines was not affected significantly by MIC-1. Studying the phenotype of DCs after MIC-1 exposure by FACS analysis, we observed that MIC-1 suppresses the expression of typical maturation molecules such as CD25 and CD83 as well as of CD86 during cytokine-induced DC maturation similar to Dex. In addition, T-cell stimulatory capacity of DCs was significantly reduced after MIC-1 exposure. MIC-1 was also able to increase slightly the expression of IDO (a key immunomodulatory enzyme promoting periphereal tolerance) in maturing DCs. CONCLUSIONS: We have identified MIC-1 as a novel factor (secreted by decidual cells in early pregnancy) that could promote the increase of a tolerogenic subtype of DC in decidua.

Expression of transketolase-like 1 protein (TKTL1) in human endometrial cancer.

BACKGROUND: Malignant tumors metabolize glucose to lactate even in the presence of oxygen (aerobic glycolysis). The metabolic switch from oxidative glycolysis to non-oxidative fermentation of glucose and proteins performed by the tumor cells seems to be associated with TKTL1 and pAkt overexpression. Therefore the aim of the present study was to investigate the expression of TKTL1 and pAkt in human specimens of endometrial cancer as compared to benign endometrium. Additionally, expression of the glucose transporter GLUT1 was also investigated as aerobic glycolysis is associated with an increased need for glucose. MATERIALS AND METHODS: Levels of TKTL1, pAkt, and GLUT1 expression were immunhistochemically evaluated on paraffin embedded biopsy material from 10 benign and 41 malignant endometrial tissue samples. TKTL1 mRNA levels in the endometrial cancer cell lines Ishikawa and HEC-1A were evaluated by RT-PCR. RESULTS: Expression of TKTL1, GLUT1 and pAKT was significantly increased in endometrial carcinomas as compared to benign endometrial tissue. There was a significantly weaker TKTL1 expression in highly differentiated G1 tumors. In the human endometrial cancer cell lines Ishikawa and HEC-1A, TKTL1 mRNA was clearly detectable. CONCLUSION: The levels of TKTL1, GLUT1 and pAKT expression point to the glycolytic phenotype of malignant endometrial tissue. Given the pronounced TKTL1 expression across all different subtypes of endometrial cancer, this protein could serve as a target for future cancer treatments.

Effect of isomalt consumption on faecal microflora and colonic metabolism in healthy volunteers.

Due to its low digestibility in the small intestine, a major fraction of the polyol isomalt reaches the colon. However, little is known about effects on the intestinal microflora. During two 4-week periods in a double-blind, placebo-controlled, cross-over design, nineteen healthy volunteers consumed a controlled basal diet enriched with either 30 g isomalt or 30 g sucrose daily. Stools were collected at the end of each test phase and various microbiological and luminal markers were analysed. Fermentation characteristics of isomalt were also investigated in vitro. Microbiological analyses of faecal samples indicated a shift of the gut flora towards an increase of bifidobacteria following consumption of the isomalt diet compared with the sucrose diet (P<0.05). During the isomalt phase, the activity of bacterial beta-glucosidase decreased (P<0.05) whereas beta-glucuronidase, sulfatase, nitroreductase and urease remained unchanged. Faecal polyamines were not different between test periods with the exception of cadaverine, which showed a trend towards a lower concentration following isomalt (P=0.055). Faecal SCFA, lactate, bile acids, neutral sterols, N, NH3, phenol and p-cresol were not affected by isomalt consumption. In vitro, isomalt was metabolized in several bifidobacteria strains and yielded high butyrate concentrations. Isomalt, which is used widely as a low-glycaemic and low-energy sweetener, has to be considered a prebiotic carbohydrate that might contribute to a healthy luminal environment of the colonic mucosa.