NLRP3 inflammasome activation is required for fibrosis development in NAFLD

NLR inflammasomes, caspase 1 activation platforms critical for processing key pro-inflammatory cytokines, have been implicated in the development of nonalcoholic fatty liver disease (NAFLD). As the direct role of the NLRP3 inflammasome remains unclear, we tested effects of persistent NLRP3 activation as a contributor to NAFLD development and, in particular, as a modulator of progression from benign hepatic steatosis to steatohepatitis during diet-induced NAFLD. Gain of function tamoxifen-inducible Nlrp3 knock-in mice allowing for in vivo temporal control of NLRP3 activation and loss of function Nlrp3 knockout mice were placed on short-term choline-deficient amino acid-defined (CDAA) diet, to induce isolated hepatic steatosis or long-term CDAA exposure, to induce severe steatohepatitis and fibrosis, respectively. Expression of NLRP3 associated proteins was assessed in liver biopsies of a well-characterized group of patients with the full spectrum of NAFLD. Nlrp3−/− mice were protected from long-term feeding CDAA-induced hepatomegaly, liver injury, and infiltration of activated macrophages. More importantly, Nlrp3−/− mice showed marked protection from CDAA-induced liver fibrosis. After 4 weeks on CDAA diet, wild-type (WT) animals showed isolated hepatic steatosis while Nlrp3 knock-in mice showed severe liver inflammation, with increased infiltration of activated macrophages and early signs of liver fibrosis. In the liver samples of patients with NAFLD, inflammasome components were significantly increased in those patients with nonalcoholic steatohepatitis (NASH) when compared to those with non-NASH NAFLD with mRNA levels of pro-IL1 beta correlated to levels of COL1A1. Our study uncovers a crucial role for the NLRP3 inflammasome in the development of NAFLD. These findings may lead to novel therapeutic strategies aimed at halting the progression of hepatic steatosis to the more severe forms of this disease.Fil: Wree, Alexander. University of California at San Diego; Estados Unidos. University Hospital Essen; AlemaniaFil: McGeough, Matthew D.. University of California at San Diego; Estados UnidosFil: Peña, Carla A.. University of California at San Diego; Estados UnidosFil: Schlattjan, Martin. University Hospital Essen; AlemaniaFil: Li, Hongying. University of California at San Diego; Estados UnidosFil: Inzaugarat, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Messer, Karen. University of California at San Diego; Estados UnidosFil: Canbay, Ali. University Hospital Essen; AlemaniaFil: Hoffman, Hal M.. University of California at San Diego; Estados Unidos. Ludwig Institute of Cancer Research; Estados UnidosFil: Feldstein, Ariel E.. University of California at San Diego; Estados Unido

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis

Interleukin‐18 signaling promotes activation of hepatic stellate cells in mouse liver fibrosis

Background and Aims: Nucleotide‐binding oligomerization domain‐like receptor‐family pyrin domain‐containing 3 (NLRP3) inflammasome activation has been shown to result in liver fibrosis. Mechanisms and downstream signaling remain incompletely understood. Here, we studied the role of IL‐18 in hepatic stellate cells (HSCs), and its impact on liver fibrosis. Approach and Results: We observed significantly increased serum levels of IL‐18 (128.4 pg/ml vs. 74.9 pg/ml) and IL‐18 binding protein (BP; 46.50 ng/ml vs. 15.35 ng/ml) in patients with liver cirrhosis compared with healthy controls. Single cell RNA sequencing data showed that an immunoregulatory subset of murine HSCs highly expresses Il18 and Il18r1. Treatment of cultured primary murine HSC with recombinant mouse IL‐18 accelerated their transdifferentiation into myofibroblasts. In vivo, IL‐18 receptor‐deficient mice had reduced liver fibrosis in a model of fibrosis induced by HSC‐specific NLRP3 overactivation. Whole liver RNA sequencing analysis from a murine model of severe NASH‐induced fibrosis by feeding a choline‐deficient, L‐amino acid‐defined, high fat diet showed that genes related to IL‐18 and its downstream signaling were significantly upregulated, and Il18−/− mice receiving this diet for 10 weeks showed protection from fibrotic changes with decreased number of alpha smooth muscle actin‐positive cells and collagen deposition. HSC activation triggered by NLRP3 inflammasome activation was abrogated when IL‐18 signaling was blocked by its naturally occurring antagonist IL‐18BP. Accordingly, we observed that the severe inflammatory phenotype associated with myeloid cell‐specific NLRP3 gain‐of‐function was rescued by IL‐18BP. Conclusions: Our study highlights the role of IL‐18 in the development of liver fibrosis by its direct effect on HSC activation identifying IL‐18 as a target to treat liver fibrosis

Atomic Layer Deposition of Molybdenum and Tungsten Oxide Thin Films Using Heteroleptic Imido-Amidinato Precursors : Process Development, Film Characterization, and Gas Sensing Properties

Heteroleptic bis(tert-butylimido)bis(N,N'-diisopropylacetamidinato) compounds of molybdenum and tungsten are introduced as precursors for atomic layer deposition of tungsten and molybdenum oxide thin films using ozone as the oxygen source. Both precursors have similar thermal properties but exhibit different growth behaviors. With the molybdenum precursor, high growth rates up to 2 angstrom/cycle at 300 degrees C and extremely uniform films are obtained, although the surface reactions are not completely saturative. The corresponding tungsten precursor enables saturative film growth with a lower growth rate of 0.45 angstrom/cycle at 300 degrees C. Highly pure films of both metal oxides are deposited, and their phase as well as stoichiometry can be tuned by changing the deposition conditions. The WO films the crystallize as gamma-WO3 at 300 degrees C and above, whereas films deposited at lower temperatures are amorphous. Molybdenum oxide can be deposited as either amorphous (= 325 degrees C) films. MoOr films are further characterized by synchrotron photoemission spectroscopy and temperature-dependent resistivity measurements. A suboxide MoOx film deposited at 275 degrees C is demonstrated to serve as an efficient hydrogen gas sensor at a low operating temperature of 120 degrees C.Peer reviewe

Are Metastases from Metastases Clinical Relevant? Computer Modelling of Cancer Spread in a Case of Hepatocellular Carcinoma

Background: Metastasis formation remains an enigmatic process and one of the main questions recently asked is whether metastases are able to generate further metastases. Different models have been proposed to answer this question; however, their clinical significance remains unclear. Therefore a computer model was developed that permits comparison of the different models quantitatively with clinical data and that additionally predicts the outcome of treatment interventions. Methods: The computer model is based on discrete events simulation approach. On the basis of a case from an untreated patient with hepatocellular carcinoma and its multiple metastases in the liver, it was evaluated whether metastases are able to metastasise and in particular if late disseminated tumour cells are still capable to form metastases. Additionally, the resection of the primary tumour was simulated. The simulation results were compared with clinical data. Results: The simulation results reveal that the number of metastases varies significantly between scenarios where metastases metastasise and scenarios where they do not. In contrast, the total tumour mass is nearly unaffected by the two different modes of metastasis formation. Furthermore, the results provide evidence that metastasis formation is an early event and that late disseminated tumour cells are still capable of forming metastases. Simulations also allow estimating how the resection of the primary tumour delays the patient’s death. Conclusion: The simulation results indicate that for this particular case of a hepatocellular carcinoma late metastases, i.e.

The neurobiology of Etruscan shrew active touch

The Etruscan shrew, Suncus etruscus, is not only the smallest terrestrial mammal, but also one of the fastest and most tactile hunters described to date. The shrew's skeletal muscle consists entirely of fast-twitch types and lacks slow fibres. Etruscan shrews detect, overwhelm, and kill insect prey in large numbers in darkness. The cricket prey is exquisitely mechanosensitive and fast-moving, and is as big as the shrew itself. Experiments with prey replica show that shape cues are both necessary and sufficient for evoking attacks. Shrew attacks are whisker guided by motion- and size-invariant Gestalt-like prey representations. Shrews often attack their prey prior to any signs of evasive manoeuvres. Shrews whisk at frequencies of approximately 14 Hz and can react with latencies as short as 25–30 ms to prey movement. The speed of attacks suggests that shrews identify and classify prey with a single touch. Large parts of the shrew's brain respond to vibrissal touch, which is represented in at least four cortical areas comprising collectively about a third of the cortical volume. Etruscan shrews can enter a torpid state and reduce their body temperature; we observed that cortical response latencies become two to three times longer when body temperature drops from 36°C to 24°C, suggesting that endothermy contributes to the animal's high-speed sensorimotor performance. We argue that small size, high-speed behaviour and extreme dependence on touch are not coincidental, but reflect an evolutionary strategy, in which the metabolic costs of small body size are outweighed by the advantages of being a short-range high-speed touch and kill predator

Cytoarchitectonic and chemoarchitectonic characterization of the prefrontal cortical areas in the mouse

This study describes cytoarchitectonic criteria to define the prefrontal cortical areas in the mouse brain (C57BL/6 strain). Currently, well-illustrated mouse brain stereotaxic atlases are available, which, however, do not provide a description of the distinctive cytoarchitectonic characteristics of individual prefrontal areas. Such a description is of importance for stereological, neuronal tracing, and physiological, molecular and neuroimaging studies in which a precise parcellation of the prefrontal cortex (PFC) is required. The present study describes and illustrates: the medial prefrontal areas, i.e., the infralimbic, prelimbic, dorsal and ventral anterior cingulate and Fr2 area; areas of the lateral PFC, i.e., the dorsal agranular insular cortical areas and areas of the ventral PFC, i.e., the lateral, ventrolateral, ventral and medial orbital areas. Each cytoarchitectonically defined boundary is corroborated by one or more chemoarchitectonic stainings, i.e., acetylcholine esterase, SMI32, SMI311, dopamine, parvalbumin, calbindin and myelin staining

NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

Background & Aims NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. Methods We fed foz/foz and wild-type mice an atherogenic diet for 16\ua0weeks, gavaged MCC950 or vehicle until 24\ua0weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6\ua0weeks and determined the effects on liver fibrosis. Results In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24\ua0weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. Conclusion MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. Lay summary Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis