Neutrophil extracellular traps (NETs) are fibers of extracellular DNA released from neutrophils due to overwhelming phagocytic stimuli. The function of NETs is to trap and kill microbes to avoid spreading of potential pathogens. NETs are formed after encounter with various gram-positive and -negative bacteria but also in response to mediators causing sterile inflammation like interleukin-8 (IL-8), tumor necrosis factor (TNF), and phorbol myristate acetate (PMA). Here we show the formation of NETs (NETting) in response to monosodium urate (MSU) crystals as further model for sterile inflammation. We identified monocytes, neutrophils, and eosinophils as MSU phagocytosing cells. Basophils did not take up the crystals, instead they upregulated their activation marker CD203c after contact with MSU. Nevertheless, MSU crystals induced extracellular trap formation also in basophils, like in eosinophils and neutrophils, which phagocytose the crystals. In contrast, monocytes do not form NETs despite uptake of the MSU crystals. In contrast to the canonical stimuli like bacteria and PMA, MSU-induced NETosis was not abrogated by plasma. Our data show that MSU crystals induce extracellular DNA trap formation in all three granulocytes lineages (NETs, EETs, and BETs) but not in monocytes, and DNA externalization does not necessitate the uptake of the crystals.

In the presence of sodium, uric acid from purine metabolism precipitates as monosodium urate (MSU) needles and forms renal calculi or causes gouty arthritis in kidneys and joints, respectively. The latter is characterized by red, hot, and swollen arthritic joints. Here we report the in vitro effect of MSU crystals on blood granulocytes and analyze their contribution to granuloma formation and neutrophil extracellular traps (NETs) formation (NETosis) in synovial fluid of patients with gouty arthritis in vivo. We observed that MSU crystals induce NETosis in vitro in a reactive oxygen species (ROS)-dependent manner. Indeed, blocking ROS (e.g., the oxidative burst) by various anti-oxidants partially inhibited NETosis induced by MSU crystals. Analyses of synovial fluids and of tissue sections of patients suffering from gout revealed that NETs are also formed in vivo, especially during acute gouty flares and/or granuloma formation. Since prolonged exposure to NETs carries the risk for the development of chronic inflammation we also studied the opsonization of NETs, as a prerequisite for their clearance. The established dead cells’ opsonins C3b, galectin-9, and CRP decorated the residual dead cells’ corpses and opsonized these for disposal. Surprisingly, all three soluble pattern recognizing molecules spared the spread NET structures. We conclude that (i) MSU crystals are strong inducers of ROS-dependent NETosis and (ii) that the prolonged presence of NET-pathogen or NET-crystal aggregates observed in patients with systemic autoimmunity, especially in those with low serum DNase-1 activity, cannot be compensated by CRP, complement, and galectin-mediated phagocytic clearance.

Standard cancer treatments involve surgery, radiotherapy, chemotherapy, and immunotherapy. In clinical practice, the respective drugs are applied orally or intravenously leading to their systemic circulation in the whole organism. For chemotherapeutics or immune modulatory agents, severe side effects such as immune depression or autoimmunity can occur. At the same time the intratumoral drug doses are often too low for effective cancer therapy. Since monotherapies frequently cannot cure cancer, due to their synergistic effects multimodal therapy concepts are applied to enhance treatment efficacy. The targeted delivery of drugs to the tumor by employment of functionalized nanoparticles might be a promising solution to overcome these challenges. For multimodal therapy concepts and individualized patient care nanoparticle platforms can be functionalized with compounds from various therapeutic classes (e.g. radiosensitizers, phototoxic drugs, chemotherapeutics, immune modulators). Superparamagnetic iron oxide nanoparticles (SPIONs) as drug transporters can add further functionalities, such as guidance or heating by external magnetic fields (Magnetic Drug Targeting or Magnetic Hyperthermia), and imaging-controlled therapy (Magnetic Resonance Imaging).

Magnetic drug targeting (MDT) improves the integrity of healthy tissues and cells during treatment with cytotoxic drugs. An anticancer drug is bound to superparamagnetic iron oxide nanoparticles (SPION), injected into the vascular supply of the tumor and directed into the tumor by means of an external magnetic field. In this study, we investigated the impact of SPION, mitoxantrone (MTO) and SPION^MTO on cell viability in vitro and the nonspecific uptake of MTO into circulating leukocytes in vivo. MDT was compared with conventional chemotherapy. MTO uptake and the impact on cell viability were assessed by flow cytometry in a Jurkat cell culture. In order to analyze MTO loading of circulating leukocytes in vivo, we treated tumor-bearing rabbits with MDT and conventional chemotherapy. In vitro experiments showed a dose-dependent MTO uptake and reduction in the viability and proliferation of Jurkat cells. MTO and SPION^MTO showed similar cytotoxic activity. Non-loaded SPION did not have any effect on cell viability in the concentrations tested. Compared with systemic administration in vivo, MDT employing SPION^MTO significantly decreased the chemotherapeutic load in circulating leukocytes. We demonstrated that MDT spares the immune system in comparison with conventional chemotherapy.

The response of the immune system against dying and dead cells strongly depends on the cell death phenotype. Beside other forms of cell death, two clearly distinct populations, early apoptotic and secondary necrotic cells, have been shown to induce anti-inflammation/tolerance and inflammation/immune priming, respectively. Cytofluorometry is a powerful technique to detect morphological and phenotypical changes occurring during cell death. Here, we describe a new technique using AnnexinA5, propidiumiodide, DiIC1(5) and Hoechst 33342 to sub-classify populations of apoptotic and/or necrotic cells. The method allows the fast and reliable identification of several different phases and pathways of cell death by analysing the following cell death associated changes in a single tube: cellular granularity and shrinkage, phosphatidylserine exposure, ion selectivity of the plasma membrane, mitochondrial membrane potential, and DNA content. The clear characterisation of cell death is of major importance for instance in immunization studies, in experimental therapeutic settings, and in the exploration of cell-death associated diseases. It also enables the analysis of immunological properties of distinct populations of dying cells and the pathways involved in this process.

Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted increasing attention in many biomedical fields. In magnetic drug targeting SPIONs are injected into a tumour supplying artery and accumulated inside the tumour with a magnet. The effectiveness of this therapy is thus dependent on magnetic properties, stability and biocompatibility of the particles. A good knowledge of the effect of storage conditions on those parameters is of utmost importance for the translation of the therapy concept into the clinic and for reproducibility in preclinical studies. Here, core shell SPIONs with a hybrid coating consisting of lauric acid and albumin were stored at different temperatures from 4 to 45 °C over twelve weeks and periodically tested for their physicochemical properties over time. Surprisingly, even at the highest storage temperature we did not observe denaturation of the protein or colloidal instability. However, the saturation magnetisation decreased by maximally 28.8% with clear correlation to time and storage temperature. Furthermore, the biocompatibility was clearly affected, as cellular uptake of the SPIONs into human T-lymphoma cells was crucially dependent on the storage conditions. Taken together, the results show that the particle properties undergo significant changes over time depending on the way they are stored.