Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-α-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-α/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure.

Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside-in signaling pathway triggering NET formation is unknown. Here, we show that the receptor-interacting protein kinase (RIPK)-1-stabilizers necrostatin-1 or necrostatin-1s and the mixed lineage kinase domain-like (MLKL)-inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal-or PMAinduced NET formation in human and mouse neutrophils. These compounds do not affect PMA-or urate crystal-induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA-induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal-induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies. Additional supporting information may be found in the online version of this article at the publisher's web-site Keywords IntroductionNeutrophil extracellular trap (NET) formation was first described to enhance bacterial killing via the release of histones, ROS, and proteases [1,2], but also contributes to autoinflammatory Correspondence: Prof. Hans-Joachim Anders e-mail: hjanders@med.uni-muenchen.de and autoimmune disorders [3][4][5][6][7]. For example, monosodium urate (MSU) crystals trigger NET formation, which first drives massive inflammation and subsequently fosters the resolution of inflammation, explaining both gouty arthritis and tophusrelated immune energy [8]. MSU crystals, cytokines, and bacterial * These authors contributed equally to this work. Results and discussionPMA triggered death and NET formation of human neutrophils release around 50% of total DNA after 2 h (Supporting Information Fig. 1A). During these processes neutrophils release IL-1β (but not TNF-α) in time-dependent manner into the supernatant. However, pretreatment with IL-1R antagonist anakinra, TNF blocker etanercept or anti-TLR4 as well as lack of Fas (Fas lpr mice) had no effect on DNA release upon PMA or MSU stimulation as compared to their respective controls, which excludes their role in this process (Supporting Information Fig. 1B-F).In addition, neither zVAD-FMK nor ferrostatin-1 (Fer-1) had an effect (Fig. 1A), excluding caspase-mediated extrinsic apoptosis or pyroptosis, ferroptosis [12], respectively. In contrast, firstgeneration necrostatin (Nec)-1, Nec-1s, and the MLKL inhibitor necrosulfonamide (NSA) decreased overall cell death and NET formation as assessed by nuclear Sytox uptake, the release of DNA using PICO green dye, and chromatin release to induce NET structures. Nec-1 inhibits necroptosis via modulating RIPK1 and preventing RIPK3 and MLKL phosphorylation and necrosome formation [13][14][15][16]. Indeed, MSU-, LPS-, and PMA-induced cell death and NET formation of human neutrophils-induced RIPK3 expression and phospho...

BackgroundTo overcome the limitations of animal-based experiments, 3D culture models mimicking the tumor microenvironment in vivo are gaining attention. Herein, we investigated an alginate-based 3D scaffold for screening of 5-fluorouracil (5-FU) or/and curcumin on malignancy of colorectal cancer cells (CRC).MethodsThe potentiation effects of curcumin on 5-FU against proliferation and metastasis of HCT116 cell and its corresponding isogenic 5-FU-chemoresistant cells (HCT116R) were examined in a 3D-alginate tumor model.ResultsCRC cells encapsulated in alginate were able to proliferate in 3D-colonospheres in a vivo-like phenotype and invaded from alginate. During cultivation of cells in alginate, we could isolate 3 stages of cells, (1) alginate proliferating (2) invasive and (3) adherent cells. Tumor-promoting factors (CXCR4, MMP-9, NF-κB) were significantly increased in the proliferating and invasive compared to the adherent cells, however HCT116R cells overexpressed factors in comparison to the parental HCT116, suggesting an increase in malignancy behavior. In alginate, curcumin potentiated 5-FU-induced decreased capacity for proliferation, invasion and increased more sensitivity to 5-FU of HCT116R compared to the HCT116 cells. IC50 for HCT116 to 5-FU was 8nM, but co-treatment with 5 μM curcumin significantly reduced 5-FU concentrations in HCT116 and HCT116R cells (0.8nM, 0.1nM, respectively) and these effects were accompanied by down-regulation of NF-κB activation and NF-κB-regulated gene products.ConclusionsOur results demonstrate that the alginate provides an excellent tumor microenvironment and indicate that curcumin potentiates and chemosensitizes HCT116R cells to 5-FU-based chemotherapy that may be useful for the treatment of CRC and to overcome drug resistance.

Translation of the expanded (ggggcc)n repeat in C9orf72 patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) causes abundant poly-GA inclusions. To elucidate their role in pathogenesis, we generated transgenic mice expressing codon-modified (GA)149 conjugated with cyan fluorescent protein (CFP). Transgenic mice progressively developed poly-GA inclusions predominantly in motoneurons and interneurons of the spinal cord and brain stem and in deep cerebellar nuclei. Poly-GA co-aggregated with p62, Rad23b and the newly identified Mlf2, in both mouse and patient samples. Consistent with the expression pattern, 4-month-old transgenic mice showed abnormal gait and progressive balance impairment, but showed normal hippocampus-dependent learning and memory. Apart from microglia activation we detected phosphorylated TDP-43 but no neuronal loss. Thus, poly-GA triggers behavioral deficits through inflammation and protein sequestration that likely contribute to the prodromal symptoms and disease progression of C9orf72 patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-017-1711-0) contains supplementary material, which is available to authorized users.

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