Deleting the mitochondrial respiration negative regulator MCJ enhances the efficacy of CD8+ T cell adoptive therapies in pre-clinical studies.

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.

Respiratory viral infection promotes the awakening and outgrowth of dormant metastatic breast cancer cells in lungs.

Breast cancer is the second most common cancer globally. Most deaths from breast cancer are due to metastatic disease which often follows long periods of clinical dormancy1. Understanding the mechanisms that disrupt the quiescence of dormant disseminated cancer cells (DCC) is crucial for addressing metastatic progression. Infection with respiratory viruses (e.g. influenza or SARS-CoV-2) is common and triggers an inflammatory response locally and systemically2,3. Here we show that influenza virus infection leads to loss of the pro-dormancy mesenchymal phenotype in breast DCC in the lung, causing DCC proliferation within days of infection, and a greater than 100-fold expansion of carcinoma cells into metastatic lesions within two weeks. Such DCC phenotypic change and expansion is interleukin-6 (IL-6)-dependent. We further show that CD4 T cells are required for the maintenance of pulmonary metastatic burden post-influenza virus infection, in part through attenuation of CD8 cell responses in the lungs. Single-cell RNA-seq analyses reveal DCC-dependent impairment of T-cell activation in the lungs of infected mice. SARS-CoV-2 infected mice also showed increased breast DCC expansion in lungs post-infection. Expanding our findings to human observational data, we observed that cancer survivors contracting a SARS-CoV-2 infection have substantially increased risks of lung metastatic progression and cancer-related death compared to cancer survivors who did not. These discoveries underscore the significant impact of respiratory viral infections on the resurgence of metastatic cancer, offering novel insights into the interconnection between infectious diseases and cancer metastasis.

Manipulating mitochondrial electron flow enhances tumor immunogenicity.

Although tumor growth requires the mitochondrial electron transport chain (ETC), the relative contribution of complex I (CI) and complex II (CII), the gatekeepers for initiating electron flow, remains unclear. In this work, we report that the loss of CII, but not that of CI, reduces melanoma tumor growth by increasing antigen presentation and T cell-mediated killing. This is driven by succinate-mediated transcriptional and epigenetic activation of major histocompatibility complex-antigen processing and presentation (MHC-APP) genes independent of interferon signaling. Furthermore, knockout of methylation-controlled J protein (MCJ), to promote electron entry preferentially through CI, provides proof of concept of ETC rewiring to achieve antitumor responses without side effects associated with an overall reduction in mitochondrial respiration in noncancer cells. Our results may hold therapeutic potential for tumors that have reduced MHC-APP expression, a common mechanism of cancer immunoevasion.

In Vivo and In Vitro Expression of iC1, a Methylation-Controlled J Protein (MCJ) in Bovine Liver, and Response to In Vitro Bovine Fatty Liver Disease Model.

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.

Enhanced mitochondrial activity reshapes a gut microbiota profile that delays NASH progression.

BACKGROUND AND AIMS: Recent studies suggest that mitochondrial dysfunction promotes progression to NASH by aggravating the gut-liver status. However, the underlying mechanism remains unclear. Herein, we hypothesized that enhanced mitochondrial activity might reshape a specific microbiota signature that, when transferred to germ-free (GF) mice, could delay NASH progression. APPROACH AND RESULTS: Wild-type and methylation-controlled J protein knockout (MCJ-KO) mice were fed for 6 weeks with either control or a choline-deficient, L-amino acid-defined, high-fat diet (CDA-HFD). One mouse of each group acted as a donor of cecal microbiota to GF mice, who also underwent the CDA-HFD model for 3 weeks. Hepatic injury, intestinal barrier, gut microbiome, and the associated fecal metabolome were then studied. Following 6 weeks of CDA-HFD, the absence of methylation-controlled J protein, an inhibitor of mitochondrial complex I activity, reduced hepatic injury and improved gut-liver axis in an aggressive NASH dietary model. This effect was transferred to GF mice through cecal microbiota transplantation. We suggest that the specific microbiota profile of MCJ-KO, characterized by an increase in the fecal relative abundance of Dorea and Oscillospira genera and a reduction in AF12 , Allboaculum , and [ Ruminococcus ], exerted protective actions through enhancing short-chain fatty acids, nicotinamide adenine dinucleotide (NAD + ) metabolism, and sirtuin activity, subsequently increasing fatty acid oxidation in GF mice. Importantly, we identified Dorea genus as one of the main modulators of this microbiota-dependent protective phenotype. CONCLUSIONS: Overall, we provide evidence for the relevance of mitochondria-microbiota interplay during NASH and that targeting it could be a valuable therapeutic approach.

Interleukin-6-dependent epithelial fluidization initiates fibrotic lung remodeling.

Chronic disease results from the failure of tissues to maintain homeostasis. In the lung, coordinated repair of the epithelium is essential for preserving homeostasis. In animal models and human lung disease, airway epithelial cells mobilize in response to lung injury, resulting in the formation of airway-like cysts with persistent loss of functional cell types and parenchymal architecture. Using live-cell imaging of human lung epithelial cultures and mouse precision-cut lung slices, we demonstrated that distal airway epithelia are aberrantly fluidized both after injury and in fibrotic lung disease. Through transcriptomic profiling and pharmacologic stimulation of epithelial cultures, we identified interleukin-6 (IL-6) signaling as a driver of tissue fluidization. This signaling cascade occurred independently of canonical Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling but instead was dependent on a downstream SRC family kinase (SFK)-yes-associated protein (YAP) axis. Airway epithelial-fibroblast cocultures revealed that the fibrotic mesenchyme acts as a source of IL-6 family cytokines, which drive airway fluidization. Inhibition of the IL-6-SFK-YAP cascade was sufficient to prevent fluidization in both in vitro and ex vivo models. Last, we demonstrated a reduction in fibrotic lung remodeling in mice through genetic or pharmacologic targeting of IL-6-related signaling. Together, our findings illustrate the critical role of airway epithelial fluidization in coordinating the balance between homeostatic lung repair and fibrotic airspace remodeling.

GSK3ß Inhibition by Phosphorylation at Ser389 Controls Neuroinflammation.

The inhibition of Glycogen Synthase Kinase 3 ß (GSK3ß) by Ser9 phosphorylation affects many physiological processes, including the immune response. However, the consequences of GSK3ß inhibition by alternative Ser389 phosphorylation remain poorly characterized. Here we have examined neuroinflammation in GSK3ß Ser389 knock-in (KI) mice, in which the phosphorylation of Ser389 GSK3ß is impaired. The number of activated microglia/infiltrated macrophages, astrocytes, and infiltrated neutrophils was significantly higher in these animals compared to C57BL/6J wild-type (WT) counterparts, which suggests that the failure to inactivate GSK3ß by Ser389 phosphorylation results in sustained low-grade neuroinflammation. Moreover, glial cell activation and brain infiltration of immune cells in response to lipopolysaccharide (LPS) failed in GSK3ß Ser389 KI mice. Such effects were brain-specific, as peripheral immunity was not similarly affected. Additionally, phosphorylation of the IkB kinase complex (IKK) in response to LPS failed in GSK3ß Ser389 KI mice, while STAT3 phosphorylation was fully conserved, suggesting that the NF-κB signaling pathway is specifically affected by this GSK3ß regulatory pathway. Overall, our findings indicate that GSK3ß inactivation by Ser389 phosphorylation controls the brain inflammatory response, raising the need to evaluate its role in the progression of neuroinflammatory pathologies.

Mitochondrial bioenergetics boost macrophage activation, promoting liver regeneration in metabolically compromised animals.

BACKGROUND AND AIMS: Hepatic ischemia-reperfusion injury (IRI) is the leading cause of early posttransplantation organ failure as mitochondrial respiration and ATP production are affected. A shortage of donors has extended liver donor criteria, including aged or steatotic livers, which are more susceptible to IRI. Given the lack of an effective treatment and the extensive transplantation waitlist, we aimed at characterizing the effects of an accelerated mitochondrial activity by silencing methylation-controlled J protein (MCJ) in three preclinical models of IRI and liver regeneration, focusing on metabolically compromised animal models. APPROACH AND RESULTS: Wild-type (WT), MCJ knockout (KO), and Mcj silenced WT mice were subjected to 70% partial hepatectomy (Phx), prolonged IRI, and 70% Phx with IRI. Old and young mice with metabolic syndrome were also subjected to these procedures. Expression of MCJ, an endogenous negative regulator of mitochondrial respiration, increases in preclinical models of Phx with or without vascular occlusion and in donor livers. Mice lacking MCJ initiate liver regeneration 12 h faster than WT and show reduced ischemic injury and increased survival. MCJ knockdown enables a mitochondrial adaptation that restores the bioenergetic supply for enhanced regeneration and prevents cell death after IRI. Mechanistically, increased ATP secretion facilitates the early activation of Kupffer cells and production of TNF, IL-6, and heparin-binding EGF, accelerating the priming phase and the progression through G1 /S transition during liver regeneration. Therapeutic silencing of MCJ in 15-month-old mice and in mice fed a high-fat/high-fructose diet for 12 weeks improves mitochondrial respiration, reduces steatosis, and overcomes regenerative limitations. CONCLUSIONS: Boosting mitochondrial activity by silencing MCJ could pave the way for a protective approach after major liver resection or IRI, especially in metabolically compromised, IRI-susceptible organs.

IL-6 enhances CD4 cell motility by sustaining mitochondrial Ca2+ through the noncanonical STAT3 pathway.

Interleukin 6 (IL-6) is known to regulate the CD4 T cell function by inducing gene expression of a number of cytokines through activation of Stat3 transcription factor. Here, we reveal that IL-6 strengthens the mechanics of CD4 T cells. The presence of IL-6 during activation of mouse and human CD4 T cells enhances their motility (random walk and exploratory spread), resulting in an increase in travel distance and higher velocity. This is an intrinsic effect of IL-6 on CD4 T-cell fitness that involves an increase in mitochondrial Ca2+ Although Stat3 transcriptional activity is dispensable for this process, IL-6 uses mitochondrial Stat3 to enhance mitochondrial Ca2+-mediated motility of CD4 T cells. Thus, through a noncanonical pathway, IL-6 can improve competitive fitness of CD4 T cells by facilitating cell motility. These results could lead to alternative therapeutic strategies for inflammatory diseases in which IL-6 plays a pathogenic role.

Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance.

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.

Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury.

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.

Regulation of GSK3ß by Ser389 Phosphorylation During Neural Development.

GSK3ß is a constitutively active kinase that promotes cell death, which requires strict regulatory mechanisms. Although Akt-mediated phosphorylation at Ser9 is the default mechanism to inactivate GSK3ß, phosphorylation of GSK3ß at Ser389 by p38 MAPK has emerged as an alternative inhibitory pathway that provides cell protection and repair in response to DNA damage. Phosphorylation of Ser389 GSK3ß has been detected in adult brain, where it has been related to neuronal survival and behavior. However, the use of this pathway to regulate GSK3ß in the neonatal developing brain is unknown. In this study, we show that phosphorylation of GSK3ß at Ser389 in the brain is developmentally regulated, with the highest levels corresponding to the first 2 weeks of age. Moreover, we found that the phosphorylation of GSK3ß at Ser389 is the preferential mechanism for inactivating brain GSK3ß in 2-week-old mice. Importantly, we show that phospho-Ser389 GSK3ß expression is predominant in neuronal cell cultures from neonatal brain relative to other cell populations. However, phospho-Ser389 GSK3ß is triggered by DNA double-strand breaks in all developing neural cell types examined. Thus, the phosphorylation of GSK3ß on Ser389 could be a central regulatory mechanism to restrain GSK3ß during neurogenesis early in life.

Protective Intranasal Immunization Against Influenza Virus in Infant Mice Is Dependent on IL-6.

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.

Therapeutic efficacy of IL-17A neutralization with corticosteroid treatment in a model of antigen-driven mixed-granulocytic asthma.

Many mouse models of allergic asthma exhibit eosinophil-predominant cellularity rather than the mixed-granulocytic cytology in steroid-unresponsive severe disease. Therefore, we sought to implement a novel mouse model of antigen-driven, mixed-granulocytic, severe allergic asthma to determine biomarkers of the disease process and potential therapeutic targets. C57BL/6J wild-type, interleukin-6 knockout (IL-6-/-), and IL-6 receptor knockout (IL-6R-/-), mice were injected with an emulsion of complete Freund's adjuvant and house dust mite antigen (CFA/HDM) on day 1. Dexamethasone, a lymphocyte-depleting biological, or anti-IL-17A was administered during the intranasal HDM challenge on days 19-22. On day 23, the CFA/HDM model elicited mixed bronchoalveolar lavage (BAL) cellularity (typically 80% neutrophils and 10% eosinophils), airway hyperresponsiveness (AHR) to methacholine, diffusion impairment, lung damage, body weight loss, corticosteroid resistance, and elevated levels of serum amyloid A (SAA), pro-inflammatory cytokines, and T helper type 1/ T helper type 17 (Th1/Th17) cytokines compared with eosinophilic models of HDM-driven allergic airway disease. BAL cells in IL-6- or IL-6R-deficient mice were predominantly eosinophilic and associated with elevated T helper type 2 (Th2) and reduced Th1/Th17 cytokine production, along with an absence of SAA. Nevertheless, AHR remained in IL-6-deficient mice even when dexamethasone was administered. However, combined administration of anti-IL-17A and systemic corticosteroid significantly attenuated both overall and neutrophilic airway inflammation and also reduced AHR and body weight loss. Inhibition of IL-17A combined with systemic corticosteroid treatment during antigen-driven exacerbations may provide a novel therapeutic approach to prevent the pathological pulmonary and constitutional changes that greatly impact patients with the mixed-granulocytic endotype of severe asthma.

Silencing hepatic MCJ attenuates non-alcoholic fatty liver disease (NAFLD) by increasing mitochondrial fatty acid oxidation.

Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate ß-oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD.

miR-873-5p targets mitochondrial GNMT-Complex II interface contributing to non-alcoholic fatty liver disease.

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 ß-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.

Sonoporation as an Approach for siRNA delivery into T cells.

Delivery of small interfering RNAs (siRNAs) into primary T cells is quite challenging because they are non-proliferating cells and are difficult to transfect with non-viral approaches. Because sonoporation is independent of the proliferation status of cells and siRNA acts in the cell cytoplasm, we investigated whether sonoporation could be used to deliver siRNA into mouse and human T cells. Cells mixed with Definity microbubbles and siRNA were sonicated with a non-focused transducer of center frequency 2.20 MHz producing ultrasound at a 10% duty cycle, pulse repetition frequency of 2.20 kHz and spatial average temporal average ultrasound intensity of 1.29 W/cm2 for 5 s and then examined for siRNA fluorescence by flow cytometry analysis. These sonoporation conditions resulted in high-efficiency transfection of siRNA in mouse and human T cells. Further, the efficacy of siRNA delivery by sonoporation was illustrated by the successful visualization of decreased methylation-controlled J protein expression in mouse and human CD8 T cells via Western blot analysis. The results provide the first evidence that sonoporation is a novel approach to delivery of siRNA into fresh isolated mouse and human T cells in vitro, and might be used for in vivo studies in the future.

Glycolysis Induces MCJ Expression That Links T Cell Proliferation With Caspase-3 Activity and Death.

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.

A New Perspective: Mitochondrial Stat3 as a Regulator for Lymphocyte Function.

Stat3 as a transcription factor regulating gene expression in lymphocytes during the immune response is well known. However, since the pioneering studies discovering the presence of Stat3 in mitochondria and its role in regulating mitochondrial metabolism, only a few studies have investigated this non-conventional function of Stat3 in lymphocytes. From this perspective, we review what is known about Stat3 as a transcription factor and what is known and unknown about mitochondrial Stat3 (mitoStat3) in lymphocytes. We also provide a framework to consider how some of the functions previously assigned to Stat3 as regulator of gene transcription could be mediated by mitoStat3 in lymphocytes. The goal of this review is to stimulate interest for future studies investigating mitoStat3 in the immune response that could lead to the generation of alternative pharmacological inhibitors of mitoStat3 for the treatment of chronic inflammatory diseases.

Two Weeks of Variable Stress Increases Gamma-H2AX Levels in the Mouse Bed Nucleus of the Stria Terminalis.

Recent reports demonstrate that DNA damage is induced, and rapidly repaired, in circuits activated by experience. Moreover, stress hormones are known to slow DNA repair, suggesting that prolonged stress may result in persistent DNA damage. Prolonged stress is known to negatively impact physical and mental health; however, DNA damage as a factor in stress pathology has only begun to be explored. Histone H2A-X phosphorylated at serine 139 (γH2AX) is a marker of DNA double-strand breaks (DSB), a type of damage that may lead to cell death if unrepaired. We hypothesized that a 14-day period of variable stress exposure sufficient to alter anxiety-like behavior in male C57BL/6J mice would produce an increase in γH2AX levels in the bed nucleus of the stria terminalis (BNST), a region implicated in anxiety and stress regulation. We observed that 14 days of variable stress, but not a single stress exposure, was associated with increased levels of γH2AX 24 h after termination of the stress paradigm. Further investigation found that phosphorylation levels of a pair of kinases associated with the DNA damage response, glycogen synthase kinase 3 ß (GSK3ß) and p38 mitogen-activated protein kinase (MAPK) were also elevated following variable stress. Our results suggest that unrepaired DNA DSBs and/or repetitive attempted repair may represent an important component of the allostatic load that stress places on the brain.