Macrophages Modulate Hepatic Injury Involving NLRP3 Inflammasome: The Example of Efavirenz.

Drug-induced liver injury (DILI) constitutes a clinical challenge due to the incomplete characterization of the mechanisms involved and potential risk factors. Efavirenz, an anti-HIV drug, induces deleterious actions in hepatocytes that could underlie induction of the NLRP3 inflammasome, an important regulator of inflammatory responses during liver injury. We assessed the potential of efavirenz to modulate the inflammatory and fibrogenic responses of major liver cell types involved in DILI. The effects of efavirenz were evaluated both in vitro and in vivo. Efavirenz triggered inflammation in hepatocytes, in a process that involved NF-κB and the NLRP3 inflammasome, and activated hepatic stellate cells (HSCs), thereby enhancing expression of inflammatory and fibrogenic markers. The NLRP3 inflammasome was not altered in efavirenz-treated macrophages, but these cells polarized towards the anti-inflammatory M2 phenotype and displayed upregulated anti-inflammatory mediators. Conversely, no evidence of damage was observed in efavirenz-treated animals, except when macrophages were depleted, which resulted in the in vivo manifestation of the deleterious effects detected in hepatocytes and HSCs. Efavirenz elicits a cell-specific activation of the NLRP3 inflammasome in hepatocytes and HSCs, but macrophages appear to counteract efavirenz-induced liver injury. Our results highlight the dynamic nature of the interaction among liver cell populations and emphasize the potential of targeting macrophage polarization as a strategy to treat NLRP3 inflammasome-induced liver injury.

Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis.

BACKGROUND & AIMS: Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. METHODS: We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). RESULTS: We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1ß secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. CONCLUSIONS: These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. LAY SUMMARY: Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.

Rilpivirine attenuates liver fibrosis through selective STAT1-mediated apoptosis in hepatic stellate cells.

OBJECTIVE: Liver fibrosis constitutes a major health problem worldwide due to its rapidly increasing prevalence and the lack of specific and effective treatments. Growing evidence suggests that signalling through cytokine-activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways regulates liver fibrosis and regeneration. Rilpivirine (RPV) is a widely used anti-HIV drug not reported to produce hepatotoxicity. We aimed to describe the potential hepatoprotective effects of RPV in different models of chronic liver injury, focusing on JAK-STAT signalling regulation. DESIGN: The effects of RPV on hepatic steatosis, inflammation and fibrogenesis were studied in a nutritional mouse model of non-alcoholic fatty liver disease, carbon tetrachloride-induced fibrosis and bile duct ligation-induced fibrosis. Primary human hepatic stellate cells (hHSC) and human cell lines LX-2 and Hep3B were used to investigate the underlying molecular mechanisms. RESULTS: RPV exerted a clear anti-inflammatory and antifibrotic effect in all the in vivo models of liver injury employed, and enhanced STAT3-dependent proliferation in hepatocytes and apoptosis in HSC through selective STAT1 activation. These results were reproduced in vitro; RPV undermined STAT3 activation and triggered STAT1-mediated pathways and apoptosis in HSC. Interestingly, this selective pro-apoptotic effect completely disappeared when STAT1 was silenced. Conditioned medium experiments showed that HSC apoptosis activated STAT3 in hepatocytes in an interleukin-6-dependent mechanism. CONCLUSION: RPV ameliorates liver fibrosis through selective STAT1-dependent induction of apoptosis in HSC, which exert paracrinal effects in hepatocytes, thus promoting liver regeneration. RPV's actions may represent an effective strategy to treat chronic liver diseases of different aetiologies and help identify novel therapeutic targets.

The interaction between RUNX2 and core binding factor beta as a potential therapeutic target in canine osteosarcoma.

Osteosarcoma remains the most common primary bone tumour in dogs with half of affected dogs unable to survive 1 year beyond diagnosis. New therapeutic options are needed to improve outcomes for this disease. Recent investigations into potential therapeutic targets have focused on cell surface molecules with little clear therapeutic benefit. Transcription factors and protein interactions represent underdeveloped areas of therapeutic drug development. We have utilized allosteric inhibitors of the core binding factor transcriptional complex, comprised of core binding factor beta (CBFß) and RUNX2, in four canine osteosarcoma cell lines Active inhibitor compounds demonstrate anti-tumour activities with concentrations demonstrated to be achievable in vivo while an inactive, structural analogue has no activity. We show that CBFß inhibitors are capable of inducing apoptosis, inhibiting clonogenic cell growth, altering cell cycle progression and impeding migration and invasion in a cell line-dependent manner. These effects coincide with a reduced interaction between RUNX2 and CBFß and alterations in expression of RUNX2 target genes. We also show that addition of CBFß inhibitors to the commonly used cytotoxic chemotherapeutic drugs doxorubicin and carboplatin leads to additive and/or synergistic anti-proliferative effects in canine osteosarcoma cell lines. Taken together, we have identified the interaction between components of the core binding factor transcriptional complex, RUNX2 and CBFß, as a potential novel therapeutic target in canine osteosarcoma and provide justification for further investigations into the anti-tumour activities we describe here.

Saxitoxin Poisoning in Green Turtles (Chelonia mydas) Linked to Scavenging on Mass Mortality of Caribbean Sharpnose Puffer Fish (Canthigaster rostrata-Tetraodontidae).

Fish within the family Tetraodontidae are potential sources of both endogenous tetrodotoxins (TTXs) and dietary derived saxitoxins (STXs). Ingestion of fish tissues containing these toxins by other vertebrates can lead to severe illness and death. The Caribbean sharpnose puffer (Canthigaster rostrata) is a widespread tetraodontid species within the western Atlantic. Mass settlement of juveniles into foraging habitats have been associated with large-scale puffer fish mortality events. In 2013, 2014, and 2017, puffer mortality events on the southern Caribbean coast of Costa Rica were also associated with strandings of green turtles (Chelonia mydas) found to have fed on C. rostrata. Stranded sea turtles were found dead without apparent cause or alive with severe neurological signs that resolved during short periods of captivity. Puffer fish and turtle organ samples were analyzed for both TTXs and STXs. Concentrations of TTXs were extremely low in the fish (0.5-0.7 µg/g) and undetectable in turtle stomach contents. However, concentrations of STXs in whole fish (16.6-47.5 µg STX-eq/g) exceeded the 0.8 µg STX-eq/g human seafood safety threshold for STXs by orders of magnitude. Saxitoxins were also detected in samples of stomach contents (ingested fish), brain, lung, kidney, and serum from three affected turtles. Study results indicate that saxitoxicosis resulting from opportunistic foraging on C. rostrata during fish mortality events may be a significant factor in episodic stranding of green sea turtles in this region.

A genetically engineered microRNA-34a prodrug demonstrates anti-tumor activity in a canine model of osteosarcoma.

Osteosarcoma (OSA) represents the most common primary bone tumor in humans and pet dogs. Little progress has been made with regard to viable treatment options in the past three decades and patients presenting with metastatic disease continue to have a poor prognosis. Recent mouse studies have suggested that microRNA-34a (miR-34a) may have anti-tumor activities in human OSA models. Due to the conservation of microRNA across species, we hypothesized that a bioengineered miR-34a prodrug (tRNA/miR-34a) would have similar effects in canine OSA, providing a valuable preclinical model for development of this therapeutic modality. Using a panel of canine OSA cell lines, we found that tRNA/miR-34a reduced viability, clonogenic growth, and migration and invasion while increasing tumor cell apoptosis. Furthermore, canine OSA cells successfully process the tRNA/miR-34a into mature miR-34a which reduces expression of target proteins such as platelet derived growth factor receptor alpha (PDGFRα), Notch1 and vascular endothelial growth factor (VEGF). Additionally, our subcutaneous OSA xenograft model demonstrated in vivo tumor growth delay, increased necrosis and apoptosis by tRNA/miR-34a, and decreased cellular proliferation ability. Taken together, these data support that this novel microRNA-based therapy may possess clinical utility in a spontaneously-occurring large animal model of OSA, which can then serve to inform the clinical development of this therapy for human OSA patients.

Role of p62/SQSTM1 beyond autophagy: a lesson learned from drug-induced toxicity in vitro.

BACKGROUND AND PURPOSE: SQSTM1/p62 is a multifunctional, stress-induced, scaffold protein involved in multiple cellular processes including autophagic clearance, regulation of inflammatory responses and redox homeostasis. Its altered function has been associated with different human pathologies, such as neurodegenerative, metabolic and bone diseases (down-regulation), and cancerogenesis (up-regulation). However, its role in the off-target effects of clinically used drugs is still not understood. EXPERIMENTAL APPROACH: We evaluated the expression of p62 in cultured Hep3B cells and their derived ρ° cells (lacking mitochondria), along with markers of autophagy and mitochondrial dysfunction. The effects of efavirenz were compared with those of known pharmacological stressors, rotenone, thapsigargin and CCCP, and we also used transient silencing with siRNA and p62 overexpression. Western blotting, quantRT-PCR and fluorescence microscopy were used to assay these effects and their underlying mechanisms. KEY RESULTS: In Hep3B cells, efavirenz augmented p62 protein content, an effect not observed in the corresponding ρ° cells. p62 up-regulation followed enhanced SQSTM1 expression mediated through the transcription factor CHOP/DDIT3, while other well-known regulators (NF-kB and Nrf2) were not involved. Inhibition of autophagy with 3MA or with transient silencing of Atg5 did not affect SQSTM1 expression in efavirenz-treated cells while p62 overexpression ameliorated the deleterious effect of efavirenz on cell viability. CONCLUSION AND IMPLICATIONS: In our model, p62 exerted a specific, autophagy-independent role and protected against efavirenz-induced mitochondrial ROS generation and activation of the NLRP3 inflammasome. These findings add to the multifunctional nature of p62 and may help to understand the off-target effects of clinically useful drugs.

Lon protease: a novel mitochondrial matrix protein in the interconnection between drug-induced mitochondrial dysfunction and endoplasmic reticulum stress.

BACKGROUND AND PURPOSE: Mitochondria-associated membranes (MAMs) are specific endoplasmic reticulum (ER) domains that enable it to interact directly with mitochondria and mediate metabolic flow and Ca2+ transfer. A growing list of proteins have been identified as MAMs components, but how they are recruited and function during complex cell stress situations is still not understood, while the participation of mitochondrial matrix proteins is largely unrecognized. EXPERIMENTAL APPROACH: This work compares mitochondrial/ER contact during combined ER stress/mitochondrial dysfunction using a model of human hepatoma cells (Hep3B cell line) treated for 24 h with classic pharmacological inducers of ER stress (thapsigargin), mitochondrial dysfunction (carbonyl cyanide m-chlorophenyl hydrazone or rotenone) or both (the antiretroviral drug efavirenz used at clinically relevant concentrations). KEY RESULTS: Markers of mitochondrial dynamics (dynamin-related protein 1, optic atrophy 1 and mitofusin 2) were expressed differently with these stimuli, pointing to a specificity of combined ER/mitochondrial stress. Lon, a matrix protease involved in protein and mtDNA quality control, was up-regulated at mRNA and protein levels under all conditions. However, only efavirenz decreased the mitochondrial content of Lon while increasing its extramitochondrial presence and its localization to MAMs. This latter effect resulted in an enhanced mitochondria/ER interaction, as shown by co-immunoprecipitation experiments of MAMs protein partners and confocal microscopy imaging. CONCLUSION AND IMPLICATIONS: A specific dual drug-induced mitochondria-ER effect enhances the MAMs content of Lon and its extramitochondrial expression. This is the first report of this phenomenon and suggests a novel MAMs-linked function of Lon protease.

Inflammasomes in Liver Fibrosis.

Cell death and inflammation are two central elements in the development of liver fibrosis. Inflammasomes are intracellular multiprotein complexes expressed in both hepatocytes and nonparenchymal cells in the liver that are key regulators of inflammation and cell fate. They respond to cellular danger signals by activating caspase 1, releasing the proinflammatory cytokines IL-1ß and IL-18, as well as initiating a novel pathway of programmed cell death termed "pyroptosis." These processes can initiate and perpetuate an abnormal wound-healing response with the principle cellular target being the activation of hepatic stellate cells. From the various inflammasomes, the NLRP3 inflammasome has been increasingly implicated in the pathogenesis of chronic inflammatory liver diseases, including nonalcoholic steatohepatitis, a disease process that is soaring and has evolved as a primary cause of liver fibrosis and need for liver transplantation. In this review, the authors highlight the growing evidence for both indirect and direct effects of inflammasomes in triggering liver fibrosis as well as potential novel targets for antifibrotic therapies.

The purine analogues abacavir and didanosine increase acetaminophen-induced hepatotoxicity by enhancing mitochondrial dysfunction.

BACKGROUND: NRTIs are essential components of HIV therapy with well-documented, long-term mitochondrial toxicity in hepatic cells, but whose acute effects on mitochondria are unclear. As acetaminophen-induced hepatotoxicity also involves mitochondrial interference, we hypothesized that it would be exacerbated in the context of ART. METHODS: We evaluated the acute effects of clinically relevant concentrations of the most widely used NRTIs, alone or combined with acetaminophen, on mitochondrial function and cellular viability. RESULTS: The purine analogues abacavir and didanosine produced an immediate and concentration-dependent inhibition of oxygen consumption and complex I and III activity. This inhibition was accompanied by an undermining of mitochondrial function, with increased production of reactive oxygen species and reduction of mitochondrial membrane potential and intracellular ATP levels. However, this interference did not compromise cell survival. Co-administration with concentrations of acetaminophen below those considered hepatotoxic exacerbated the deleterious effects of both compounds on mitochondrial function and compromised cellular viability, showing a clear correlation with diminished glutathione levels. CONCLUSIONS: The simultaneous presence of purine analogues and low concentrations of acetaminophen significantly potentiates mitochondrial dysfunction, increasing the risk of liver injury. This new mechanism is relevant given the liver's susceptibility to mitochondrial dysfunction-related toxicity and the tendency of the HIV infection to increase oxidative stress.

Involvement of nitric oxide in the mitochondrial action of efavirenz: a differential effect on neurons and glial cells.

The anti-human immunodeficiency virus (HIV) drug efavirenz (EFV) alters mitochondrial function in cultured neurons and glial cells. Nitric oxide (NO) is a mediator of mitochondrial dysfunction associated with HIV central nervous system symptoms. We show that EFV promotes inducible nitric oxide synthase (iNOS) expression in cultured glial cells and generated NO undermines their mitochondrial function, as inhibition of NOS partially reverses this effect. EFV inhibits mitochondrial Complex I in both neurons and glia; however, when the latter cells are treated for longer periods, other mitochondrial complexes are also affected in accordance with the increased NO production. These findings shed light on the mechanisms responsible for the frequent EFV-associated neurotoxicity.

Brucella ceti infection in dolphins from the Western Mediterranean sea.

BACKGROUND: Brucella ceti infections have been increasingly reported in cetaceans. Brucellosis in these animals is associated with meningoencephalitis, abortion, discospondylitis', subcutaneous abscesses, endometritis and other pathological conditions B. ceti infections have been frequently described in dolphins from both, the Atlantic and Pacific Oceans. In the Mediterranean Sea, only two reports have been made: one from the Italian Tyrrhenian Sea and the other from the Adriatic Sea. RESULTS: We describe the clinical and pathological features of three cases of B. ceti infections in three dolphins stranded in the Mediterranean Catalonian coast. One striped dolphin had neurobrucellosis, showing lethargy, incoordination and lateral swimming due to meningoencephalitis, A B. ceti infected bottlenose dolphin had discospondylitis, and another striped dolphin did not show clinical signs or lesions related to Brucella infection. A detailed characterization of the three B. ceti isolates was performed by bacteriological, molecular, protein and fatty acid analyses. CONCLUSIONS: All the B. ceti strains originating from Mediterranean dolphins cluster together in a distinct phylogenetic clade, close to that formed by B. ceti isolates from dolphins inhabiting the Atlantic Ocean. Our study confirms the severity of pathological signs in stranded dolphins and the relevance of B. ceti as a pathogen in the Mediterranean Sea.

Lack of mitochondrial toxicity of darunavir, raltegravir and rilpivirine in neurons and hepatocytes: a comparison with efavirenz.

OBJECTIVES: Growing evidence associates the non-nucleoside reverse transcriptase inhibitor efavirenz with several adverse events. Newer antiretrovirals, such as the integrase inhibitor raltegravir, the non-nucleoside reverse transcriptase inhibitor rilpivirine and the protease inhibitor darunavir, claim to have a better toxicological profile than efavirenz while producing similar levels of efficacy and virological suppression. The objective of this study was to determine the in vitro toxicological profile of these three new antiretrovirals by evaluating their effects on the mitochondrial and cellular parameters altered by efavirenz in hepatocytes and neurons. METHODS: Hep3B cells and primary rat neurons were treated with clinically relevant concentrations of efavirenz, darunavir, rilpivirine or raltegravir. Parameters of mitochondrial function, cytotoxicity and oxidative and endoplasmic reticulum stress were assessed using standard cell biology techniques. RESULTS: None of the new compounds altered the mitochondrial function of hepatic cells or neurons, while efavirenz decreased mitochondrial membrane potential and enhanced superoxide production in both cell types, effects that are known to significantly compromise the functioning of mitochondria, cell viability and, ultimately, cell number. Of the four drugs assayed, efavirenz was the only one to alter the protein expression of LC3-II, an indicator of autophagy, and CHOP, a marker of endoplasmic reticulum stress and the unfolded protein response. CONCLUSIONS: Darunavir, rilpivirine and raltegravir do not induce toxic effects on Hep3B cells and primary rat neurons, which suggests a safer hepatic and neurological profile than that of efavirenz.

Neuronal bioenergetics and acute mitochondrial dysfunction: a clue to understanding the central nervous system side effects of efavirenz.

BACKGROUND: Neurological pathogenesis is associated with mitochondrial dysfunction and differences in neuronal/glial handling of oxygen and glucose. The main side effects attributed to efavirenz involve the CNS, but the underlying mechanisms are unclear. METHODS: Human cell lines and rat primary cultures of neurons and astrocytes were treated with clinically relevant efavirenz concentration. RESULTS: Efavirenz alters mitochondrial respiration, enhances reactive oxygen species generation, undermines mitochondrial membrane potential, and reduces adenosine triphosphate (ATP) levels in a concentration-dependent fashion in both neurons and glial cells. However, it activates adenosine monophosphate-activated protein kinase only in glial cells, upregulating glycolysis and increasing intracellular ATP levels, which do not occur in neurons. To reproduce the conditions that often exist in human immunodeficiency virus-related neuroinflammatory disorders, the effects of efavirenz were evaluated in the presence of exogenous nitric oxide, an inflammatory mediator and mitochondrial inhibitor. The combination potentiated the effects on mitochondrial parameters in both neurons and glial cells, but ATP generation and lactate production were enhanced only in glial cells. CONCLUSIONS: Efavirenz affects the bioenergetics of neurons through a mechanism involving acute mitochondrial inhibition, an action exacerbated in neuroinflammatory conditions. A similar scenario of glial cells survival and degeneration of neurons with signs of mitochondrial dysfunction and oxidative stress has been associated with neurocognitive disorders.

ER stress in human hepatic cells treated with Efavirenz: mitochondria again.

BACKGROUND & AIMS: ER stress is associated with a growing number of liver diseases, including drug-induced hepatotoxicity. The non-nucleoside analogue reverse transcriptase inhibitor Efavirenz, a cornerstone of the multidrug strategy employed to treat HIV1 infection, has been related to the development of various adverse events, including metabolic disturbances and hepatic toxicity, the mechanisms of which remain elusive. Recent evidence has pinpointed a specific mitochondrial effect of Efavirenz in human hepatic cells. This study assesses the induction of ER stress by Efavirenz in the same model and the implication of mitochondria in this process. METHODS: Primary human hepatocytes and Hep3B were treated with clinically relevant concentrations of Efavirenz and parameters of ER stress were studied using standard cell biology techniques. RESULTS: ER stress markers, including CHOP and GRP78 expression (both protein and mRNA), phosphorylation of eIF2α, and presence of the spliced form of XBP1 were upregulated. Efavirenz also enhanced cytosolic Ca(2+) content and induced morphological changes in the ER suggestive of ER stress. This response was greatly attenuated in cells with altered mitochondrial function (Rho°). The effects of Efavirenz on the ER, and particularly in regard to the mitochondrial involvement, differed from those elicited by a standard pharmacological ER stressor. CONCLUSIONS: This newly discovered mechanism of cellular insult involving ER stress and UPR response may help comprehend the hepatic toxicity that has been associated with the widespread and life-long use of Efavirenz. In addition, the specificity of the actions of Efavirenz observed expands our knowledge of the mechanisms that trigger ER stress and shed some light on the mitochondria/ER interplay in drug-induced hepatic challenge.

The game of futsal as an adaptive process.

Some researchers have described team sports as complex, open, and hierarchical systems. This study aimed to investigate and describe how the game of futsal could be characterized as a dynamic adaptive process. One game, which included participation by two amateur teams, was analyzed by examining players' individual (space occupied, skills with and without ball) and collective actions (attacks and defenses). Data were collected through time-continuum notation, and were analyzed through frequencies and clustering, using trend analysis and multiple comparisons, and Ward's minimum variance method with Euclidean distance, respectively. Results revealed four attack patterns for each team, with four defense patterns for one (Blue), and seven for the other (Red), and they showed within-pattern variability. All were performed in an unpredictable manner, with no absolute correspondence between attacks and defenses. The futsal game as an adaptive process was characterized by changing intra- and inter-patterns.

Reemplazo total de articulación temporo mandibular por anquilosis / Total joint replacement for ankylosed temporomandibular

Introducción: La anquilosis de la Articulación Temporo Mandibular (ATM) condiciona la incapacidad parcial o total para abrir la boca, por lo que el paciente no puede masticar, higienizarse la boca, ni hablar adecuadamente. Objetivo: Evaluar el tratamiento quirúrgico mediante el reemplazo total de la articulación temporo mandibular con prótesis de titanio. Material y Método: Se reseca la articulación temporo mandibular afectada incluyendo una parte de la rama ascendente de la mandíbula para implantar en su lugar una prótesis de titanio molibdeno y cromo cobalto. Resultado: La satisfacción y felicidad que manifiesta el paciente al poder hablar y comer adecuadamente reflejan que esta forma de tratamiento es por demás satisfactoria. Conclusiones: El reemplazo total de la ATM con una prótesis integral es de fácil manejo y con muy buenos resultados.