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Non-alcoholic fatty liver (NAFLD) disease causes degeneration of the liver, affects about 25% of people globally, and has no approved treatment. Here, the authors show that the therapeutic siRNA-driven silencing of MCJ in the liver is an effective and safe treatment for NAFLD in multiple mouse models.

Objective: Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression. Methods: miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy. Results: We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid β-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment. Conclusion: GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment. Keywords: NASH, GNMT, Mitochondria, β-oxidation, Metabolism, microRNA

Background & Aims: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored. Methods: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury. Results: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics. Conclusions: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis. Lay summary: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.

Acetaminophen-induced liver injury is one of the most common causes of liver failure and has to be treated within hours of the overdose. Here Barbier-Torres et al. show that targeting MCJ, a mitochondrial negative regulator, even 24 h after the overdose protects liver from acetaminophen-induced damage.

An effective adaptive immune response requires rapid T cell proliferation, followed by equally robust cell death. These two processes are coordinately regulated to allow sufficient magnitude of response followed by its rapid resolution, while also providing the maintenance of T cell memory. Both aspects of this T cell response are characterized by profound changes in metabolism; glycolysis drives proliferation whereas oxidative phosphorylation supports the survival of memory T cells. While much is known about the separate aspects of T cell expansion and contraction, considerably less is understood regarding how these processes might be connected. We report a link between the induction of glycolysis in CD8+ T cells and upregulation of the inhibitor of complex I and oxidative phosphorylation, methylation-controlled J protein (MCJ). MCJ acts synergistically with glycolysis to promote caspase-3 activity. Effector CD8+ T cells from MCJ-deficient mice manifest reduced glycolysis and considerably less active caspase-3 compared to wild-type cells. Consistent with these observations, in non-glycolytic CD8+ T cells cultured in the presence of IL-15, MCJ expression is repressed by methylation, which parallels their reduced active caspase-3 and increased survival compared to glycolytic IL-2-cultured T cells. Elevated levels of MCJ are also observed in vivo in the highly proliferative and glycolytic subset of CD4-CD8- T cells in Fas-deficient lpr mice. This subset also manifests elevated levels of activated caspase-3 and rapid cell death. Collectively, these data demonstrate tight linkage of glycolysis, MCJ expression, and active caspase-3 that serves to prevent the accumulation and promote the timely death of highly proliferative CD8+ T cells.

Double stranded DNA breaks are generated during rearrangements of lymphocyte antigen receptors. Here the authors show that the DNA breaks induce phosphorylation of nuclear GSK3β at Ser389/Thr390, protecting the activated lymphocytes from necroptosis-mediated cell death.

IL-6 plays an important role in determining the fate of effector CD4 cells and the cytokines that these cells produce. Here we identify a novel molecular mechanism by which IL-6 regulates CD4 cell effector function. We show that IL-6-dependent signal facilitates the formation of mitochondrial respiratory chain supercomplexes to sustain high mitochondrial membrane potential late during activation of CD4 cells. Mitochondrial hyperpolarization caused by IL-6 is uncoupled from the production of ATP by oxidative phosphorylation. However, it is a mechanism to raise the levels of mitochondrial Ca2+ late during activation of CD4 cells. Increased levels of mitochondrial Ca2+ in the presence of IL-6 are used to prolong Il4 and Il21 expression in effector CD4 cells. Thus, the effect of IL-6 on mitochondrial membrane potential and mitochondrial Ca2+ is an alternative pathway by which IL-6 regulates effector function of CD4 cells and it could contribute to the pathogenesis of inflammatory diseases.

Abstract Mitochondrial respiration is essential for the survival and function of T cells used in adoptive cellular therapies. However, strategies that specifically enhance mitochondrial respiration to promote T cell function remain limited. Here, we investigate methylation-controlled J protein (MCJ), an endogenous negative regulator of mitochondrial complex I expressed in CD8 cells, as a target for improving the efficacy of adoptive T cell therapies. We demonstrate that MCJ inhibits mitochondrial respiration in murine CD8+ CAR-T cells and that deletion of MCJ increases their in vitro and in vivo efficacy against murine B cell leukaemia. Similarly, MCJ deletion in ovalbumin (OVA)-specific CD8+ T cells also increases their efficacy against established OVA-expressing melanoma tumors in vivo. Furthermore, we show for the first time that MCJ is expressed in human CD8 cells and that the level of MCJ expression correlates with the functional activity of CD8+ CAR-T cells. Silencing MCJ expression in human CD8 CAR-T cells increases their mitochondrial metabolism and enhances their anti-tumor activity. Thus, targeting MCJ may represent a potential therapeutic strategy to increase mitochondrial metabolism and improve the efficacy of adoptive T cell therapies.

Drug efflux through ABC transporters is a common mechanism leading to chemoresistance in cancer. Here, the authors show that mitochondrial respiration provides ATP to allow ABC transporters activity so mitochondrial respiration inhibition overcomes chemoresistance in preclinical cancer models.

Mitochondrial complex I inhibitor (iC1) is a methylation-controlled J protein (MCJ) that decreases cellular respiration by inhibiting oxidative phosphorylation. Recent rodent studies showed that loss or inhibition of iC1 was associated with preventing lipid accumulation. A common metabolic disorder of dairy cattle is a fatty liver disease (FLD), which often occurs during the periparturient period. In humans and rodents, iC1 is expressed in the liver and acts as a mitochondrial “brake”. However, iC1 expression in bovine liver and its possible role in FLD development have not yet been characterized. We hypothesized that iC1 is expressed in the bovine liver and that the expression of iC1 is correlated with FLD in periparturient dairy cattle. To test this hypothesis, we collected bovine liver tissue samples from an abattoir and isolated primary hepatic cells immediately following harvest. Utilizing an in vitro model of bovine FLD developed in our laboratory, we cultured primary hepatic cells in low-glucose DMEM supplemented with 10% FBS. The basal media was made to induce lipid accumulation and cytotoxicity in the primary liver cells with three treatments. To the basal media (control) we added 0.4 mM palmitate (treatment 1) or 20 ng/mL TNFα (treatment 2), or both 0.4 mM palmitate and 20 ng/mL TNFα (treatment 3). Consistent with our hypothesis, we present the novel characterization of iC1 expression in primary bovine liver cells cultured with or without the addition of lipotoxic factors made to emulate bovine FLD. We demonstrate both in situ and in vitro expression of iC1 in bovine liver and mRNA expression in hepatic cells and in the precipitates of conditioned media. The results of RT-qPCR, IHC, and western blot all demonstrated the expression of iC1 in bovine liver. In addition, we isolated precipitates of conditioned media further demonstrated iC1 expression by RT-qPCR. The transcript of iC1 tended to be more concentrated (4-fold; p > 0.05) in TNFα-treated conditioned media when compared with the control. Taken together, we present the novel finding that iC1 transcript and protein are expressed in liver tissue from dairy cattle, primary hepatic cells isolated from that liver tissue, and, finally, in the conditioned media derived from those cells. These novel findings and the prior findings on the role of iC1 in rodents and humans indicate that further investigation of the role of iC1 in the etiology and pathology of FLD in periparturient dairy cows is warranted.

Respiratory diseases adversely affect infants and are the focus of efforts to develop vaccinations and other modalities to prevent disease. The infant immune system differs from that of older children and adults in many ways that are as yet ill understood. We have used a C57BL/6 mouse model of infection with a laboratory- adapted strain of influenza (PR8) to delineate the importance of the cytokine IL-6 in the innate response to primary infection and in the development of protective immunity in adult mice. Herein, we used this same model in infant (14 days of age) mice to determine the effect of IL-6 deficiency. Infant wild type mice are more susceptible than older mice to infection, similar to the findings in humans. IL-6 is expressed in the lung in the early response to PR8 infection. While intramuscular immunization does not protect against lethal challenge, intranasal administration of heat inactivated virus is protective and correlates with expression of IL-6 in the lung, activation of lung CD8 cells, and development of an influenza-specific antibody response. In IL-6 deficient mice, this response is abrogated, and deficient mice are not protected against lethal challenge. These studies support the importance of the role of the tissue environment in infant immunity, and further suggest that IL-6 may be helpful in the generation of protective immune responses in infants.

The incidence and severity of chronic lung diseases is growing and affects between 100 and 150 million people worldwide and is associated with a significant rate of mortality. Unfortunately, the initial cause that triggers most chronic lung diseases remains unknown and current available therapies only ameliorate, but do not cure the disease. Thus, there is a need for identification of new targets and development of novel therapies especially for those most severely affected. IL-6, like other inflammatory cytokines, has been shown to be elevated in different lung diseases, but it was considered a byproduct of ongoing inflammation in the lung. However, recent studies support a dissociation of IL-6 from inflammation in the lung and suggest that this cytokine plays an active role in pathogenesis of asthma and, in all likelihood, COPD. IL-6 may therefore be a germane target for treatment of these and other chronic lung disease. Here, we provide an overview of the studies in mouse models and human patients that provide support for the involvement of IL-6 in lung diseases.