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Sarcoidosis is a disease characterised by granuloma formation. There is an unmet need for new treatment strategies beyond corticosteroids. The NLRP3 inflammasome pathway is expressed in innate immune cells and senses danger signals to elicit inflammatory interleukin (IL)-1v; it has recently become a druggable target. This prompted us to test the role of the NLRP3 inflammasome and IL-1v pathway in granuloma formation and sarcoidosis. 19 sarcoid patients and 19 healthy volunteers were recruited into this pilot study. NLRP3 inflammasome activity was measured in bronchoalveolar lavage (BAL) cells and lung and skin biopsies using immunohistochemistry, Western blot, reverse-transcriptase PCR and ELISA. For in vivo experiments we used the trehalose 6,6'-dimycolate-granuloma mouse model and evaluated lung granuloma burden in miR-223 knockout and NLRP3 knockout mice, as well as the treatment effects of MCC950 and anti-IL-1v antibody therapy. We found strong upregulation of the NLRP3 inflammasome pathway, evidenced by expression of activated NLRP3 inflammasome components, including cleaved caspase-1 and IL-1v in lung granuloma, and increased IL-1v release of BAL cells from sarcoid patients compared to healthy volunteers (p=0.006). mRNA levels of miR-223, a micro-RNA downregulating NLRP3, were decreased and NLRP3 mRNA correspondingly increased in alveolar macrophages from sarcoid patients (p

Introduction: Sarcoidosis is an inflammatory lung disease characterized by granuloma formation. The NLRP3 inflammasome is crucially inolved in the production of biologically active IL-1v and IL-18. Objectives: We investigated the role of the NLRP3 inflammasome in granuloma formation in sarcoidosis and the effect of potential treatment strategies inhibiting the activation of this pathway. Methods: We analyzed BAL cells of 36 patients with sarcoidosis and 37 healthy volunteers (HV). NLRP3 inflammasome activity of AM was measured either by caspase-1p20 expression using Western Blot or IL-1v production (ELISA). Relative NLRP3/GAPDH and miRNA-223/U6 expression was detected by RT-PCR. NLRP3 inflammasome activation was induced by priming w LPS (1µg/ml) and subsequent activation with either ATP (1nM) or Nigericin (NIG 10µM) for 6h. We used the TDM-granuloma mouse model to evaluate lung granuloma burden in miR-223 KO and NLRP3 KO mice as well as to test the effects of the NLRP3 pathway inhibitor MCC950. Results: We found a significant increase in both spontaneous and LPS+NIGstimulated IL-1v production of AM derived from sarcoid patients compared to control AM ( p=0.01, p

Mitochondria form a highly dynamic network driven by opposing scission and fusion events. DRP1 is an essential modulator of mitochondrial fission and dynamics within mammalian cells. Its fission activity is regulated by posttranslational modifications such as activating phosphorylation at serine 616. DRP1 activity has recently been implicated as being dysregulated in numerous human disorders such as cancer and neurodegenerative diseases. Here we describe the development of a cellbased screening assay to detect DRP1 activation. We utilized this to undertake focused compound library screening and identified potent modulators that affected DRP1 activity including ICG-001, which is described as WNT/β-catenin signaling inhibitor. Our findings elucidate novel details about ICG-001’s mechanism of action (MOA) in mediating anti-proliferative activity. We show ICG-001 both inhibits mitochondrial fission and activates an early endoplasmic reticulum (ER) stress response to induce cell death in susceptible colorectal cancer cell lines.

The fibrillar peptide amyloid-beta (A beta) has a chief function in the pathogenesis of Alzheimer's disease. Interleukin 1 beta (IL-1 beta) is a key cytokine in the inflammatory response to A beta. Insoluble materials such as crystals activate the inflammasome formed by the cytoplasmic receptor NALP3, which results in the release of IL-1 beta. Here we identify the NALP3 inflammasome as a sensor of A beta in a process involving the phagocytosis of A beta and subsequent lysosomal damage and release of cathepsin B. Furthermore, the IL-1 beta pathway was essential for the microglial synthesis of proinflammatory and neurotoxic factors, and the inflammasome, caspase-1 and IL-1 beta were critical for the recruitment of microglia to exogenous A beta in the brain. Our findings suggest that activation of the NALP3 inflammasome is important for inflammation and tissue damage in Alzheimer's disease

Inhalation of silica crystals causes inflammation in the alveolar space. Prolonged exposure to silica can lead to the development of silicosis, an irreversible, fibrotic pulmonary disease. The mechanisms by which silica and other crystals activate immune cells are not well understood. Here we demonstrate that silica and aluminum salt crystals activated inflammasomes formed by the cytoplasmic receptor NALP3. NALP3 activation required phagocytosis of crystals, and this uptake subsequently led to lysosomal damage and rupture. 'Sterile' lysosomal damage (without crystals) also induced NALP3 activation, and inhibition of either phagosomal acidification or cathepsin B activity impaired NALP3 activation. Our results indicate that the NALP3 inflammasome senses lysosomal damage as an endogenous 'danger' signal