TLR9 independent interferon α production by neutrophils on NETosis in response to circulating chromatin, a key lupus autoantigen.

OBJECTIVES: Interferon (IFN) α is a key immunoregulatory cytokine secreted by activated plasmacytoid dendritic cells (PDC) that constitute less than 1% of leucocytes. IFNα plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). Nevertheless, the natural IFNα inducers in SLE as well as the different IFNα secreting cell types are only partially characterised. METHODS: Chromatin was purified from calf thymus. Human peripheral blood mononuclear cells (PBMC), neutrophils and mouse bone marrow neutrophils were purified and cultured with different stimuli. IFNα production was estimated by flow cytometry, ELISA and a bioassay, and gene expression by quantitative real time PCR. Neutrophil activation and NETosis were analysed by flow cytometry, ELISA and confocal microscopy. RESULTS: Neutrophils produced a bioactive IFNα on stimulation with purified chromatin. IFNα secretion was observed with steady state neutrophils purified from 56 independent healthy individuals and autoimmune patients in response to free chromatin and not chromatin containing immune complexes. Chromatin induced IFNα secretion occurred independently of Toll-like receptor 9 (TLR9). Neutrophil priming by granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor or IFNα was not necessary but PBMC sustained IFNα secretion by neutrophils. PDC were 27 times more efficient than neutrophils but blood neutrophils were 100 times more frequent than PDC. Finally, neutrophil activation by chromatin was associated with NETosis and DNA sensor upregulation. CONCLUSIONS: Neutrophils have the capability of producing IFNα on selective triggering, and we identified a natural lupus stimulus involved, unveiling a new mechanism involved in SLE. Neutrophils represent another important source of IFNα and important targets for future therapies aimed at influencing IFNα levels.

Primary blood neutrophils express a functional cell surface Toll-like receptor 9.

Polymorphonuclear leukocytes (PMNs) represent one of the first lines of defense against pathogens. TLR9 is normally expressed in endosomes/lysosomes where it is activated by pathogen-derived DNA. Here we show that freshly isolated human and mouse primary PMNs express TLR9 at the cell surface ex vivo. Moreover, surface TLR9 expression is upregulated upon activation of PMNs with different stimuli and not only TLR9 agonists. Importantly, surface TLR9 is processed, active, and functional. TLR9 ligands, oligo-nucleotides containing unmethylated CpG motifs, indeed bind to surface TLR9 and binding was strongly observed at the cell surface of human cells expressing surface TLR9 and at the surface of WT but not TLR9-deficient mouse PMNs. Finally, CpG oligonucleotides cross-linked onto a solid phase and having no access to intracellular TLR9 are able to trigger cell surface TLR9 and induce neutrophil activation, even when endosomal acidification is inhibited. This is the first demonstration of a functional TLR9 expressed at the cell surface of human primary cells. This pathway may be triggered when pathogen-derived TLR9 ligands cannot reach the endosome, offering a rescue mechanism for neutrophil activation.

The immunosuppressive tumour network: myeloid-derived suppressor cells, regulatory T cells and natural killer T cells.

Myeloid-derived suppressor cells (MDSC) and regulatory T (Treg) cells are major components of the immune suppressive tumour microenvironment (TME). Both cell types expand systematically in preclinical tumour models and promote T-cell dysfunction that in turn favours tumour progression. Clinical reports show a positive correlation between elevated levels of both suppressors and tumour burden. Recent studies further revealed that MDSCs can modulate the de novo development and induction of Treg cells. The overlapping target cell population of Treg cells and MDSCs is indicative for the importance and flexibility of immune suppression under pathological conditions. It also suggests the existence of common pathways that can be used for clinical interventions aiming to manipulate the TME. Elimination or reprogramming of the immune suppressive TME is one of the major current challenges in immunotherapy of cancer. Interestingly, recent findings suggest that natural killer T (NKT) cells can acquire the ability to convert immunosuppressive MDSCs into immunity-promoting antigen-presenting cells. Here we will review the cross-talk between MDSCs and other immune cells, focusing on Treg cells and NKT cells. We will consider its impact on basic and applied cancer research and discuss how targeting MDSCs may pave the way for future immunocombination therapies.

Immunocombination therapy for high-risk neuroblastoma.

Neuroblastoma (NBL) is an aggressive malignancy of the sympathetic nervous system. Advanced-stage NBLs prove fatal in approximately 50% of patients within 5 years. Therefore, new treatment modalities are urgently needed. Immunotherapy is a treatment modality that can be combined with established forms of treatment. Administration of monoclonal antibodies or dendritic cell-based therapies alone can lead to favorable clinical outcomes in individual cancer patients; for example patients with melanoma, lymphoma and NBL. However, clinical benefit is still limited to a minority of patients, and further improvements are clearly needed. In this article, we review the most commonly used approaches to treat patients with NBL and highlight the prerequisites and opportunities of cell-based immunotherapy, involving both innate and adaptive immune-effector cells. Furthermore, we discuss the potential of the combined application of immunotherapy and novel tumor-targeted therapies for the treatment of both cancer in general and NBL in particular.

Simvastatin reduces tumor cell adhesion to human peritoneal mesothelial cells by decreased expression of VCAM-1 and ß1 integrin.

Peritoneal carcinomatosis describes cancer metastasis onto the surface of the peritoneum. It is frequently caused by ovarian and colorectal cancer. Once a tumor has penetrated the peritoneum, cancer cells disseminate into the abdominal cavity. Additionally, surgery can account for the spread of free tumor cells. Their subsequent adhesion to mesothelial cells (HMCs) initiates peritoneal carcinomatosis. Therefore, this study analyzed the effect of simvastatin on tumor cell adherence. HMCs were isolated from human greater omentum. Fluorescence-labeled tumor cells (SKOV-3, OvCar-29, OAW42, FraWü; ovarian/HT29; colorectal) were incubated on confluent mesothelial monolayers with 10 µM simvastatin for 48 h. Adhesion was quantified using a fluorescence reader. Expression of the adhesion molecules VCAM-1, ICAM-1 and ß1 integrin chain under the influence of simvastatin 0.1-100 µM for 24-72 h was analyzed using flow cytometry. Simvastatin significantly reduced the adhesion of all ovarian cancer cells and HT29 to HMCs (P≤0.001). Concomitantly simvastatin decreased the expression of VCAM-1 on HMCs. ICAM-1 and ß1 integrin chain expression on ovarian cancer cells was also clearly reduced. By contrast, the expression of the analyzed adhesion molecules on HT29 cells remained unchanged. Simvastatin clearly inhibits tumor cell adhesion to HMCs. In the case of ovarian cancer cell lines it appears to be mediated by decreased expression of both VCAM-1 on HMCs and the integrin α4ß1 on tumor cells. As an example of adhesion molecule down-regulating drugs, simvastatin may provide a novel therapeutic approach to the prevention of peritoneal carcinomatosis.

Phagocytosis of dying tumor cells by human peritoneal mesothelial cells.

Peritoneal carcinomatosis is an advanced form of metastatic disease characterized by cancer cell dissemination onto the peritoneum. It is commonly observed in ovarian and colorectal cancers and is associated with poor patient survival. Novel therapies consist of cytoreductive surgery in combination with intraperitoneal chemotherapy, aiming at tumor cell death induction. The resulting dying tumor cells are considered to be eliminated by professional as well as semi-professional phagocytes. In the present study, we have identified a hitherto unknown type of 'amateur' phagocyte in this environment: human peritoneal mesothelial cells (HMCs). We demonstrate that HMCs engulf corpses of dying ovarian and colorectal cancer cells, as well as other types of apoptotic cells. Flow cytometric, confocal and electron microscopical analyses revealed that HMCs ingest dying cell fragments in a dose- and time-dependent manner and the internalized material subsequently traffics into late phagolysosomes. Regarding the mechanisms of prey cell recognition, our results show that HMCs engulf apoptotic corpses in a serum-dependent and -independent fashion and quantitative real-time PCR (qRT-PCR) analyses revealed that diverse opsonin receptor systems orchestrating dying cell clearance are expressed in HMCs at high levels. Our data strongly suggest that HMCs contribute to dying cell removal in the peritoneum, and future studies will elucidate in what manner this influences tumor cell dissemination and the antitumor immune response.

Nucleosome-induced neutrophil activation occurs independently of TLR9 and endosomal acidification: implications for systemic lupus erythematosus.

The nucleosome is a major autoantigen known to activate PMN in systemic lupus erythematosus (SLE). TLR9 recognizes bacterial and even mammalian DNA under certain circumstances. Nevertheless, the role of TLR9 in SLE development is still unclear. Since nucleosomes are composed of DNA, we investigated whether TLR9 is required for nucleosome-induced PMN activation. Isolated neutrophils were cultured with nucleosomes, plasma from lupus patients and other stimuli in the presence/absence of various inhibitors. Cells were analyzed by flow cytometry, ELISA and confocal microscopy. We found that nucleosomes circulating in lupus plasma induce the secretion of pro-inflammatory cytokines by PMN. Nucleosomes activate human PMN independently of unmethylated CpG sequences in nucleosomal DNA, leading to IL-8/IL-6/TNF secretion and CD11b up-regulation. Nucleosomes accumulate in the cytoplasm of PMN upon endocytosis, induce TLR9 up-regulation and act synergistically with TLR9 ligands. Nucleosome-induced activation was not inhibited by polymyxin B (PB), chloroquine (CQ), ammonium chloride (AC) or a TLR9 antagonist. Moreover, both PMN isolated from WT and TLR9-KO mice were activated by nucleosomes, as detected by MIP-2 secretion and CD11b up-regulation. Activation occurred therefore independently of endotoxins, endosomal acidification, TLR9 and CpG motifs. TLR9 may thus be differently required in the triggering of nucleosome-induced innate immunity and anti-nucleosome B-cell autoimmunity.

Nph, DC and chromatin-mediated pathogenesis in systemic lupus erythematosus.

Nucleosomes (Nuc) are key lupus autoantigen and have been shown to activate several types of immune cells as well as the complement system, resulting in inflammation. The elevated concentrations and the immunostimulatory capacities of circulating Nuc may favour the break of peripheral tolerance in genetically predisposed individuals. Nevertheless, in most cases the signalling pathways involved are not elucidated yet, although Tir8/Sigirr deficiency has been recently shown to enhance cell activation upon exposure to Nuc-containing immune complexes.

Abeta42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology.

We have generated a novel transgenic mouse model on a C57BL/6J genetic background that coexpresses KM670/671NL mutated amyloid precursor protein and L166P mutated presenilin 1 under the control of a neuron-specific Thy1 promoter element (APPPS1 mice). Cerebral amyloidosis starts at 6-8 weeks and the ratio of human amyloid (A)beta42 to Abeta40 is 1.5 and 5 in pre-depositing and amyloid-depositing mice, respectively. Consistent with this ratio, extensive congophilic parenchymal amyloid but minimal amyloid angiopathy is observed. Amyloid-associated pathologies include dystrophic synaptic boutons, hyperphosphorylated tau-positive neuritic structures and robust gliosis, with neocortical microglia number increasing threefold from 1 to 8 months of age. Global neocortical neuron loss is not apparent up to 8 months of age, but local neuron loss in the dentate gyrus is observed. Because of the early onset of amyloid lesions, the defined genetic background of the model and the facile breeding characteristics, APPPS1 mice are well suited for studying therapeutic strategies and the pathomechanism of amyloidosis by cross-breeding to other genetically engineered mouse models.

Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use.

The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.