Caspase-11 drives macrophage hyperinflammation in models of Polg-related mitochondrial disease.

Mitochondrial diseases (MtD) represent a significant public health challenge due to their heterogenous clinical presentation, often severe and progressive symptoms, and the lack of effective therapies. Environmental exposures, such bacterial and viral infection, can further compromise mitochondrial function and exacerbate the progression of MtD. Infections in MtD patients more frequently progress to sepsis, pneumonia, and other detrimental inflammatory endpoints. However, the underlying immune alterations that enhance immunopathology in MtD remain unclear, constituting a key gap in knowledge that complicates treatment and increases mortality in this population. Here we employ in vitro and in vivo approaches to clarify the molecular and cellular basis for innate immune hyperactivity in models of polymerase gamma (Polg)-related MtD. We reveal that type I interferon (IFN-I)-mediated upregulation of caspase-11 and guanylate-binding proteins (GBPs) increase macrophage sensing of the opportunistic microbe Pseudomonas aeruginosa (PA) in Polg mutant mice. Furthermore, we show that excessive macrophage cytokine secretion and pyroptotic cell death contribute to lung inflammation and morbidity after infection with PA. Our work sheds new light on innate immune dysregulation in MtD and reveals potential targets for limiting infection- and inflammation-related complications in Polg-related MtD.

Primary oxidative phosphorylation defects lead to perturbations in the human B cell repertoire.

Introduction: The majority of studies on oxidative phosphorylation in immune cells have been performed in mouse models, necessitating human translation. To understand the impact of oxidative phosphorylation (OXPHOS) deficiency on human immunity, we studied children with primary mitochondrial disease (MtD). Methods: scRNAseq analysis of peripheral blood mononuclear cells was performed on matched children with MtD (N = 4) and controls (N = 4). To define B cell function we performed phage display immunoprecipitation sequencing on a cohort of children with MtD (N = 19) and controls (N = 16). Results: Via scRNAseq, we found marked reductions in select populations involved in the humoral immune response, especially antigen presenting cells, B cell and plasma populations, with sparing of T cell populations. MTRNR2L8, a marker of bioenergetic stress, was significantly elevated in populations that were most depleted. mir4485, a miRNA contained in the intron of MTRNR2L8, was co-expressed. Knockdown studies of mir4485 demonstrated its role in promoting survival by modulating apoptosis. To determine the functional consequences of our findings on humoral immunity, we studied the antiviral antibody repertoire in children with MtD and controls using phage display and immunoprecipitation sequencing. Despite similar viral exposomes, MtD displayed antiviral antibodies with less robust fold changes and limited polyclonality. Discussion: Overall, we show that children with MtD display perturbations in the B cell repertoire which may impact humoral immunity and the ability to clear viral infections.

Inflammatory and interferon gene expression signatures in patients with mitochondrial disease.

BACKGROUND: People with mitochondrial disease (MtD) are susceptible to metabolic decompensation and neurological symptom progression in response to an infection. Increasing evidence suggests that mitochondrial dysfunction may cause chronic inflammation, which may promote hyper-responsiveness to pathogens and neurodegeneration. We sought to examine transcriptional changes between MtD patients and healthy controls to identify common gene signatures of immune dysregulation in MtD. METHODS: We collected whole blood from a cohort of MtD patients and healthy controls and performed RNAseq to examine transcriptomic differences. We performed GSEA analyses to compare our findings against existing studies to identify commonly dysregulated pathways. RESULTS: Gene sets involved in inflammatory signaling, including type I interferons, interleukin-1ß and antiviral responses, are enriched in MtD patients compared to controls. Monocyte and dendritic cell gene clusters are also enriched in MtD patients, while T cell and B cell gene sets are negatively enriched. The enrichment of antiviral response corresponds with an independent set of MELAS patients, and two mouse models of mtDNA dysfunction. CONCLUSIONS: Through the convergence of our results, we demonstrate translational evidence of systemic peripheral inflammation arising from MtD, predominantly through antiviral response gene sets. This provides key evidence linking mitochondrial dysfunction to inflammation, which may contribute to the pathogenesis of primary MtD and other chronic inflammatory disorders associated with mitochondrial dysfunction.

Epitope-level profiling in children with mitochondrial disease reveals limitations in the antibacterial antibody repertoire.

INTRODUCTION: Immunometabolic studies in mice have suggested the importance of oxidative phosphorylation (OXPHOS) in humoral immunity. However, there are important distinctions between murine and human immunity. Furthermore, translational studies on the role of OXPHOS in humoral immunity are nearly absent from the biomedical literature. Children with primary OXPHOS deficiency (i.e., mitochondrial disease, MtD), are an important patient population for demonstrating the functional effects of this bioenergetic defect on humoral immunity. METHODS: To define whether OXPHOS deficiency extended to human B cells, we performed extracellular flux analysis on lymphoblastoid B cell lines from children with MtD and controls (N = 4/group). To expand the immune phenotype of B cell OXPHOS deficiency, we conducted a cross-sectional multiplex serology study of the antibacterial antibody repertoire in children with MtD (N = 16) and controls (N = 16) using phage display and immunoprecipitation sequencing (PhIPseq). The PhIPseq library contained >3000 peptides (i.e., epitopes) covering >40 genera and > 150 species of bacteria that infect humans. RESULTS: B cell lymphoblastoid cell lines from children with MtD displayed depressed baseline oxygen consumption, ATP production and reserve capacity, indicating that OXPHOS deficiency extended to these key cells in humoral immunity. Characterization of the bacterial exposome revealed comparable bacterial species between the two groups, mostly Streptococcus and Staphylococcus. The most common species of bacteria was S. pneumoniae. By interrogating the antibacterial antibody repertoire, we found that children with MtD had less robust antibody fold changes to common epitopes. Furthermore, we also found that children with MtD failed to show a direct relationship between the number of bacterial epitopes recognized and age, unlike controls. OXPHOS deficiency extends to B cells in children with MtD, leading to limitations in the antibacterial antibody repertoire. Furthermore, the timing of bacterial exposures was asynchronous, suggesting different periods of increased exposure or susceptibility. CONCLUSIONS: Overall, the antibacterial humoral response is distinctive in children with MtD, suggesting an important role for OXPHOS in B cell function.

Inflammatory and interferon gene expression signatures in patients with mitochondrial disease.

Background: People with mitochondrial disease (MtD) are susceptible to metabolic decompensation and neurological symptom progression in response to an infection. Increasing evidence suggests that mitochondrial dysfunction may cause chronic inflammation, which may promote hyperresponsiveness to pathogens and neurodegeneration. Methods: We collected whole blood from a cohort of MtD patients and healthy controls and performed RNAseq to examine transcriptomic differences. We performed GSEA analyses to compare our findings against existing studies to identify commonly dysregulated pathways. Results: Gene sets involved in inflammatory signaling, including type I interferons, interleukin-1ß and antiviral responses, are enriched in MtD patients compared to controls. Monocyte and dendritic cell gene clusters are also enriched in MtD patients, while T cell and B cell gene sets are negatively enriched. The enrichment of antiviral response corresponds with an independent set of MELAS patients, and two mouse models of mtDNA dysfunction. Conclusions: Through the convergence of our results, we demonstrate translational evidence of systemic peripheral inflammation arising from MtD, predominantly through antiviral response gene sets. This provides key evidence linking mitochondrial dysfunction to inflammation, which may contribute to the pathogenesis of primary MtD and other chronic inflammatory disorders associated with mitochondrial dysfunction.

Pyruvate dehydrogenase complex integrates the metabolome and epigenome in memory T cell differentiation in vitro.

Background: Modulation of metabolic flux through pyruvate dehydrogenase complex (PDC) plays an important role in T cell activation and differentiation. PDC sits at the transition between glycolysis and the tricarboxylic acid cycle and is a major producer of acetyl-CoA, marking it as a potential metabolic and epigenetic node. Methods: To understand the role of pyruvate dehydrogenase complex in T cell differentiation, we generated mice deficient in T cell pyruvate dehydrogenase E1A (Pdha) subunit using a CD4-cre recombinase-based strategy. To control for the contribution of exogenous metabolites in vivo, we conducted our T cell functional studies in vitro. T cells were differentiated into memory and effector T cells using standardized protocols. Cells were analyzed using stable isotopic tracing studies, metabolomics, RNAseq, ATACseq, ChIPseq and histone proteomics. Results: Herein, we show that genetic ablation of PDC activity in T cells (TPdh-/-) leads to marked perturbations in glycolysis, the tricarboxylic acid cycle, and OXPHOS. Due to depressed OXPHOS, TPdh-/-T cells became dependent upon substrate level phosphorylation via glycolysis. Due to the block of PDC activity, histone acetylation was reduced, as were most other types of post translational modifications. Transcriptional and functional profiling revealed abnormal CD8+ memory T cell differentiation in vitro. Conclusions: Collectively, our data indicate that PDC integrates the metabolome and epigenome in memory T cell differentiation. Targeting this metabolic and epigenetic node can have widespread ramifications on cellular function.

Adaptive immune responses to SARS-CoV-2 persist in the pharyngeal lymphoid tissue of children.

Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection.

Comprehensive profiling of the human viral exposome in households containing an at-risk child with mitochondrial disease during the 2020-2021 COVID-19 pandemic.

BACKGROUND: Viral infection is a major cause of morbidity in children with mitochondrial disease (MtD). As a result, families with children with MtD are highly adherent to risk mitigation behaviours (RMBs) advised by the Centers for Disease Control and Prevention during the COVID-19 pandemic that can modulate infection risk. METHODS: Deep serologic phenotyping of viral infections was performed via home-based sampling by combining SARS-CoV-2 serologic testing and phage display immunoprecipitation and sequencing. Samples were collected approximately 1 year apart (October 2020 to April 2021 and October 2021 to March 2022) on households containing a child with MtD. RESULTS: In contrast to our first collection in 2020-2021, SARS-CoV-2 antibody profiles for all participants in 2021-2022 were marked by greater isotype diversity and the appearance of neutralizing antibodies. Besides SARS-CoV-2, households (N = 15) were exposed to >38 different respiratory and gastrointestinal viruses during the study, averaging five viral infections per child with MtD. Regarding clinical outcomes, children with MtD (N = 17) experienced 34 episodes of illness resulting in 6 hospitalizations, with some children experiencing multiple episodes. Neurologic events following illness were recorded in five patients. Infections were identified via clinical testing in only seven cases. Viral exposome profiles were consistent with clinical testing and even identified infections not captured by clinical testing. CONCLUSIONS: Despite reported adherence to RMBs during the COVID-19 pandemic by families with a child with MtD, viral infection was pervasive. Not all infections resulted in illness in the child with MtD, suggesting that some were subclinical or asymptomatic. However, selected children with MtD did experience neurologic events. Our studies emphasize that viral infections are inexorable, emphasizing the need for further understanding of host-pathogen interactions through broad serologic surveillance.

Short report: Vaccine attitudes in the age of COVID-19 for a population of children with mitochondrial disease.

BACKGROUND: Children with developmental disabilities are vulnerable to morbidity associated with COVID-19. AIMS: To understand attitudes toward routine childhood vaccinations versus the COVID-19 vaccine in a population of families affected by mitochondrial disease (MtD), a form of developmental disability. METHODS AND PROCEDURES: An online survey was administered via several advocacy groups for children with MtD. OUTCOMES AND RESULT: Eighty-six percent of families reported being up to date with the childhood vaccine schedule and seventy percent reported that their affected child receives the annual flu shot. However, only fifty percent reported that the benefits of the COVID-19 vaccine outweighed the risk for their affected child. One quarter of families expressed concern that their child may become sick or deteriorate after the COVID-19 vaccine. In comparison to other routine childhood vaccines, families expressed less confidence in the COVID-19 vaccine. CONCLUSIONS AND IMPLICATIONS: Families affected by this population of developmental disabilities are more comfortable with the vaccines included in the routine childhood immunization schedule than with the newly introduced COVID-19 vaccine, even despite this group's vulnerability.

Leukocyte cytokine responses in adult patients with mitochondrial DNA defects.

Patients with oxidative phosphorylation (OxPhos) defects causing mitochondrial diseases appear particularly vulnerable to infections. Although OxPhos defects modulate cytokine production in vitro and in animal models, little is known about how circulating leukocytes of patients with inherited mitochondrial DNA (mtDNA) defects respond to acute immune challenges. In a small cohort of healthy controls (n = 21) and patients (n = 12) with either the m.3243A > G mutation or single, large-scale mtDNA deletions, we examined (i) cytokine responses (IL-6, TNF-α, IL-1ß) in response to acute lipopolysaccharide (LPS) exposure and (ii) sensitivity to the immunosuppressive effects of glucocorticoid signaling (dexamethasone) on cytokine production. In dose-response experiments to determine the half-maximal effective LPS concentration (EC50), relative to controls, leukocytes from patients with mtDNA deletions showed 74-79% lower responses for IL-6 and IL-1ß (pIL-6 = 0.031, pIL-1ß = 0.009). Moreover, whole blood from patients with mtDNA deletions (pIL-6 = 0.006), but not patients with the m.3243A > G mutation, showed greater sensitivity to the immunosuppressive effects of dexamethasone. Together, these ex vivo data provide preliminary evidence that some systemic OxPhos defects may compromise immune cytokine responses and increase the sensitivity to immune cytokine suppression by glucocorticoids. Further work in larger cohorts is needed to define the nature of immune dysregulation in patients with mitochondrial disease, and their potential implications for disease phenotypes. KEY MESSAGES: Little is known about leukocyte cytokine responses in patients with mitochondrial diseases. Leukocytes of patients with mtDNA deletions show blunted LPS sensitivity and cytokine responses. Leukocytes of patients with mtDNA deletions are more sensitive to glucocorticoid-mediated IL-6 suppression. Work in larger cohorts is needed to delineate potential immune alterations in mitochondrial diseases.

IFNγ regulates NAD+ metabolism to promote the respiratory burst in human monocytes.

Interferon γ (IFNγ) is an essential and pleiotropic activator of human monocytes, but little is known about the changes in cellular metabolism required for IFNγ-induced activation. We sought to elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. We found that IFNγ increased oxygen consumption rates (OCR) in monocytes, indicative of reactive oxygen species generation by both mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Transcriptional profiling revealed that this oxidative phenotype was driven by IFNγ-induced reprogramming of NAD+ metabolism, which is dependent on nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. Consistent with this pathway, monocytes from patients with gain-of-function mutations in STAT1 demonstrated higher-than-normal OCR, whereas chemical or genetic disruption of mitochondrial complex I (rotenone treatment or Leigh syndrome patient monocytes) or NADPH oxidase (diphenyleneiodonium treatment or chronic granulomatous disease [CGD] patient monocytes) reduced OCR. Interestingly, inhibition of NAMPT in healthy monocytes completely abrogated the IFNγ-induced oxygen consumption, comparable to levels observed in CGD monocytes. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for IFNγ activation of human monocytes.

Undiagnosed COVID-19 in households with a child with mitochondrial disease.

Background: The impact of the COVID-19 pandemic on medically fragile populations, who are at higher risk of severe illness and sequelae, has not been well characterized. Viral infection is a major cause of morbidity in children with mitochondrial disease (MtD), and the COVID-19 pandemic represents an opportunity to study this vulnerable population. Methods: A convenience sampling cross-sectional serology study was conducted (October 2020 to June 2021) in households (N = 20) containing a child with MtD (N = 22). Samples (N = 83) were collected in the home using a microsampling apparatus and shipped to investigators. Antibodies against SARS-CoV-2 nucleocapsid (IgG), spike protein (IgG, IgM, IgA), and receptor binding domain (IgG, IgM, IgA) were determined by enzyme linked immunosorbent assay. Results: While only 4.8% of participants were clinically diagnosed for SARS-CoV-2 infection, 75.9% of study participants were seropositive for SARS-CoV-2 antibodies. Most samples were IgM positive for spike or RBD (70%), indicating that infection was recent. This translated to all 20 families showing evidence of infection in at least one household member. For the children with MtD, 91% had antibodies against SARS-CoV-2 and had not experienced any adverse outcomes at the time of assessment. For children with recent infections (IgM+ only), serologic data suggest household members as a source. Conclusions: COVID-19 was highly prevalent and undiagnosed in households with a child with MtD through the 2020-2021 winter wave of the pandemic. In this first major wave, children with MtD tolerated SARS-CoV-2 infection well, potentially due to household adherence to CDC recommendations for risk mitigation.

Risk mitigation behaviors to prevent infection in the mitochondrial disease community during the COVID-19 pandemic.

BACKGROUND: A challenge during the COVID-19 pandemic has been widespread adherence to risk-reducing behaviors. Individuals with mitochondrial disease (MtD) are special population with an increased risk of morbidity associated with infection. PURPOSE: To measure risk mitigation behaviors (RMBs) in families affected by MtD and identify factors that may influence these behaviors. METHODS: An online questionnaire was distributed in April and June 2020. Individuals with MtD or their caregivers completed the survey. RESULTS: We received 529 eligible responses with n = 312 completing all questions for our multivariate regression model. The most common RMBs were increased hand washing (96%), social distancing (94%), and avoiding public gatherings (93%). Higher numbers of recent healthcare visits (b = 0.62, p < 0.05) and expressed fear of the MtD patient contracting COVID-19 (b = 0.92, p < 0.05) were associated with more RMBs. Living in a rural community (b = -0.99,p < 0.05) and a history of COVID-19 testing (b = -2.14,p < 0.01) were associated with fewer RMBs. CONCLUSIONS: Our results suggest that during the COVID-19 pandemic, families affected by MtD have near universal adherence to basic RMBs. This may be motivated by fear of the severe morbidity associated with infection in MtD. Patients with frequent healthcare visits may be sicker and therefore take more precautions. Living in a rural community may also impact these behaviors. People who practice fewer RMBs may be more likely to seek testing. Our findings may generalize to other chronic diseases.

B cell receptor isotypes differentially associate with cell signaling, kinetics, and outcome in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the B cell receptor (BCR) plays a critical role in disease development and progression, as indicated by the therapeutic efficacy of drugs blocking BCR signaling. However, the mechanism(s) underlying BCR responsiveness are not completely defined. Selective engagement of membrane IgM or IgD on CLL cells, each coexpressed by more than 90% of cases, leads to distinct signaling events. Since both IgM and IgD carry the same antigen-binding domains, the divergent actions of the receptors are attributed to differences in immunoglobulin (Ig) structure or the outcome of signal transduction. We showed that IgM, not IgD, level and organization associated with CLL-cell birth rate and the type and consequences of BCR signaling in humans and mice. The latter IgM-driven effects were abrogated when BCR signaling was inhibited. Collectively, these studies demonstrated a critical, selective role for IgM in BCR signaling and B cell fate decisions, possibly opening new avenues for CLL therapy.

PI3Kδ coordinates transcriptional, chromatin, and metabolic changes to promote effector CD8+ T cells at the expense of central memory.

Patients with activated phosphatidylinositol 3-kinase delta (PI3Kδ) syndrome (APDS) present with sinopulmonary infections, lymphadenopathy, and cytomegalvirus (CMV) and/or Epstein-Barr virus (EBV) viremia, yet why patients fail to clear certain chronic viral infections remains incompletely understood. Using patient samples and a mouse model (Pik3cdE1020K/+ mice), we demonstrate that, upon activation, Pik3cdE1020K/+ CD8+ T cells exhibit exaggerated features of effector populations both in vitro and after viral infection that are associated with increased Fas-mediated apoptosis due to sustained FoxO1 phosphorylation and Fasl derepression, enhanced mTORC1 and c-Myc signatures, metabolic perturbations, and an altered chromatin landscape. Conversely, Pik3cdE1020K/+ CD8+ cells fail to sustain expression of proteins critical for central memory, including TCF1. Strikingly, activated Pik3cdE1020K/+ CD8+ cells exhibit altered transcriptional and epigenetic circuits characterized by pronounced interleukin-2 (IL-2)/STAT5 signatures and heightened IL-2 responses that prevent differentiation to memory-like cells in IL-15. Our data position PI3Kδ as integrating multiple signaling nodes that promote CD8+ T cell effector differentiation, providing insight into phenotypes of patients with APDS.

Mitochondrial disease disrupts hepatic allostasis and lowers the threshold for immune-mediated liver toxicity.

OBJECTIVE: In individuals with mitochondrial disease, respiratory viral infection can result in metabolic decompensation with mitochondrial hepatopathy. Here, we used a mouse model of liver-specific Complex IV deficiency to study hepatic allostasis during respiratory viral infection. METHODS: Mice with hepatic cytochrome c oxidase deficiency (LivCox10-/-) were infected with aerosolized influenza, A/PR/8 (PR8), and euthanized on day five after infection following three days of symptoms. This time course is marked by a peak in inflammatory cytokines and mimics the timing of a common clinical scenario in which caregivers may first attempt to manage the illness at home before seeking medical attention. Metabolic decompensation and mitochondrial hepatopathy in mice were characterized by serum hepatic testing, histology, electron microscopy, biochemistry, metabolomics, and bioenergetic profiling. RESULTS: Following influenza infection, LivCox10-/- mice displayed marked liver disease including hepatitis, enlarged mitochondria with cristae loss, and hepatic steatosis. This pathophysiology was associated with viremia. Primary hepatocytes from LivCox10-/- mice cocultured with WT Kupffer cells in the presence of PR8 showed enhanced lipid accumulation. Treatment of hepatocytes with recombinant TNFα implicated Kupffer cell-derived TNFα as a precipitant of steatosis in LivCox10-/- mice. Eliminating Kupffer cells or blocking TNFα in vivo during influenza infection mitigated the steatosis and mitochondrial morphologic changes. CONCLUSIONS: Taken together, our data shift the narrative of metabolic decompensation in mitochondrial hepatopathy beyond the bioenergetic costs of infection to include an underlying susceptibility to immune-mediated damage. Moreover, our work suggests that immune modulation during metabolic decompensation in mitochondrial disease represents a future viable treatment strategy needing further exploration.

Chemical individuality in T cells: A Garrodian view of immunometabolism.

Metabolically quiescent T cells circulate throughout the body in search of antigen. Following engagement of their cognate receptors, T cells undergo metabolic reprogramming to support their activation, differentiation, and ultimately function. In the spirit of Sir Archibald Garrod, this metabolic reprogramming actually imparts a chemical individuality which confers advantage, while in others confers vulnerability, depending upon the milieu. Studying T cell immunometabolism in the context of inborn errors of metabolism allows one to define essential pathways of intermediary metabolism as well metabolic vulnerabilities and plasticity. Inborn errors of metabolism, a class of diseases first named by Garrod, have a long history of being informative for common physiologic and pathologic processes. This endeavor may be accomplished through the study of patients, animal models, and in vitro models of inborn errors of metabolism. In this review, the basics of intermediary metabolism and core metabolic pathways will be discussed, along with their relationship to T cell immunometabolism. Due to their pleiotropic nature, the reader will be specifically directed toward various inborn errors of metabolism which may be helpful for answering important questions about the role of metabolism in T cells.

AMP-activated protein kinase signaling regulated expression of urea cycle enzymes in response to changes in dietary protein intake.

Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although urea cycle enzyme levels rise in response to a high-protein (HP) diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either HP or low-protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptomic data. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated adenosine monophosphate-activated protein kinase (AMPK) as a possible regulator. Primary human hepatocytes, treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) an activator of AMPK, were used to test whether AMPK regulates expression of urea cycle genes. The abundance of carbamoylphosphate synthetase 1 and ornithine transcarbamylase mRNA increased in hepatocytes treated with AICAR, which supports a role for AMPK signaling in regulation of the urea cycle. Because AMPK is either a target of drugs used to treat type-2 diabetes, these drugs might increase the expression of urea cycle enzymes in patients with partial urea cycle disorders, which could be the basis of a new therapeutic approach.

Macrophage derived TNFα promotes hepatic reprogramming to Warburg-like metabolism.

During infection, hepatocytes must undergo a reprioritization of metabolism, termed metabolic reprogramming. Hepatic metabolic reprogramming in response to infection begins within hours of infection, suggesting a mechanism closely linked to pathogen recognition. Following injection with polyinosinic:polycytidylic acid, a mimic of viral infection, a robust hepatic innate immune response could be seen involving the TNFα pathway at 2 h. Repeated doses led to the adoption of Warburg-like metabolism in the liver as determined by in vivo metabolic imaging, expression analyses, and metabolomics. Hepatic macrophages, Kupffer cells, were able to induce Warburg-like metabolism in hepatocytes in vitro via TNFα. Eliminating macrophages in vivo or blocking TNFα in vitro or in vivo resulted in abrogation of the metabolic phenotype, establishing an immune-metabolic axis in hepatic metabolic reprogramming. Overall, we suggest that macrophages, as early sensors of pathogens, instruct hepatocytes via TNFα to undergo metabolic reprogramming to cope with challenges to homeostasis initiated by infection. This work not only addresses a key component of end-organ physiology, but also raises questions about the side effects of biologics in the treatment of inflammatory diseases. KEY MESSAGES: • Hepatocytes develop Warburg-like metabolism in vivo during viral infection. • Macrophage TNFα promotes expression of glycolytic enzymes in hepatocytes. • Blocking this immune-metabolic axis abrogates Warburg-like metabolism in the liver. • Implications for patients being treated for inflammatory diseases with biologics.

Mitochondrial Dysfunction and the Aging Immune System.

Mitochondria are ancient organelles that have co-evolved with their cellular hosts, developing a mutually beneficial arrangement. In addition to making energy, mitochondria are multifaceted, being involved in heat production, calcium storage, apoptosis, cell signaling, biosynthesis, and aging. Many of these mitochondrial functions decline with age, and are the basis for many diseases of aging. Despite the vast amount of research dedicated to this subject, the relationship between aging mitochondria and immune function is largely absent from the literature. In this review, three main issues facing the aging immune system are discussed: (1) inflamm-aging; (2) susceptibility to infection and (3) declining T-cell function. These issues are re-evaluated using the lens of mitochondrial dysfunction with aging. With the recent expansion of numerous profiling technologies, there has been a resurgence of interest in the role of metabolism in immunity, with mitochondria taking center stage. Building upon this recent accumulation of knowledge in immunometabolism, this review will advance the hypothesis that the decline in immunity and associated pathologies are partially related to the natural progression of mitochondrial dysfunction with aging.