Studies of a mosaic patient with DBA and chimeric mice reveal erythroid cell-extrinsic contributions to erythropoiesis.

We follow a patient with Diamond-Blackfan anemia (DBA) mosaic for a pathogenic RPS19 haploinsufficiency mutation with persistent transfusion-dependent anemia. Her anemia remitted on eltrombopag (EPAG), but surprisingly, mosaicism was unchanged, suggesting that both mutant and normal cells responded. When EPAG was withheld, her anemia returned. In addition to expanding hematopoietic stem/progenitor cells, EPAG aggressively chelates iron. Because DBA anemia, at least in part, results from excessive intracellular heme leading to ferroptotic cell death, we hypothesized that the excess heme accumulating in ribosomal protein-deficient erythroid precursors inhibited the growth of adjacent genetically normal precursors, and that the efficacy of EPAG reflected its ability to chelate iron, limit heme synthesis, and thus limit toxicity in both mutant and normal cells. To test this, we studied Rpl11 haploinsufficient (DBA) mice and mice chimeric for the cytoplasmic heme export protein, FLVCR. Flvcr1-deleted mice have severe anemia, resembling DBA. Mice transplanted with ratios of DBA to wild-type marrow cells of 50:50 are anemic, like our DBA patient. In contrast, mice transplanted with Flvcr1-deleted (unable to export heme) and wild-type marrow cells at ratios of 50:50 or 80:20 have normal numbers of red cells. Additional studies suggest that heme exported from DBA erythroid cells might impede the nurse cell function of central macrophages of erythroblastic islands to impair the maturation of genetically normal coadherent erythroid cells. These findings have implications for the gene therapy of DBA and may provide insights into why del(5q) myelodysplastic syndrome patients are anemic despite being mosaic for chromosome 5q deletion and loss of RPS14.

Metabolic design in a mammalian model of extreme metabolism, the North American least shrew (Cryptotis parva).

Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ-specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the least shrew (Cryptotis parva). This was achieved through a combination of high-resolution 3D focused ion beam electron microscopy imaging and tandem mass tag mass spectrometry proteomics. We demonstrate that liver and kidney mitochondrial content are equivalent to the heart, permitting assessment of mitochondrial adaptations in different organs with similar metabolic demand. Muscle mitochondrial networks (cardiac and skeletal) are extensive, with a high incidence of nanotunnels - which collectively support the metabolism of large muscle cells. Mitochondrial networks were not detected in the liver and kidney as individual mitochondria are localized with sites of ATP consumption. This configuration is not observed in striated muscle, likely due to a homogeneous ATPase distribution and the structural requirements of contraction. These results demonstrate distinct, fundamental mitochondrial structural adaptations for similar metabolic demand that are dependent on the topology of energy utilization process in a mammalian model of extreme metabolism. KEY POINTS: Least shrews were studied to explore the relationship between metabolic function, mitochondrial morphology and protein content in different tissues. Liver and kidney mitochondrial content and enzymatic activity approaches that of the heart, indicating similar metabolic demand among tissues that contribute to basal and maximum metabolism. This allows an examination of mitochondrial structure and composition in tissues with similar maximum metabolic demands. Mitochondrial networks only occur in striated muscle. In contrast, the liver and kidney maintain individual mitochondria with limited reticulation. Muscle mitochondrial reticulation is the result of dense ATPase activity and cell-spanning myofibrils which require networking for adequate metabolic support. In contrast, liver and kidney ATPase activity is localized to the endoplasmic reticulum and basolateral membrane, respectively, generating a locally balanced energy conversion and utilization. Mitochondrial morphology is not driven by maximum metabolic demand, but by the cytosolic distribution of energy-utilizing systems set by the functions of the tissue.

GCN5L1 interacts with αTAT1 and RanBP2 to regulate hepatic α-tubulin acetylation and lysosome trafficking.

Although GCN5L1 (also known as BLOC1S1) facilitates mitochondrial protein acetylation and controls endosomal-lysosomal trafficking, the mechanisms underpinning these disparate effects are unclear. As microtubule acetylation modulates endosome-lysosome trafficking, we reasoned that exploring the role of GCN5L1 in this biology may enhance our understanding of GCN5L1-mediated protein acetylation. We show that α-tubulin acetylation is reduced in GCN5L1-knockout hepatocytes and restored by GCN5L1 reconstitution. Furthermore, GCN5L1 binds to the α-tubulin acetyltransferase αTAT1, and GCN5L1-mediated α-tubulin acetylation is dependent on αTAT1. Given that cytosolic GCN5L1 has been identified as a component of numerous multiprotein complexes, we explored whether novel interacting partners contribute to this regulation. We identify RanBP2 as a novel interacting partner of GCN5L1 and αTAT1. Genetic silencing of RanBP2 phenocopies GCN5L1 depletion by reducing α-tubulin acetylation, and we find that RanBP2 possesses a tubulin-binding domain, which recruits GCN5L1 to α-tubulin. Finally, we find that genetic depletion of GCN5L1 promotes perinuclear lysosome accumulation and histone deacetylase inhibition partially restores lysosomal positioning. We conclude that the interactions of GCN5L1, RanBP2 and αTAT1 function in concert to control α-tubulin acetylation and may contribute towards the regulation of cellular lysosome positioning. This article has an associated First Person interview with the first author of the paper.

Parkin targets NOD2 to regulate astrocyte endoplasmic reticulum stress and inflammation.

Loss of substantia nigra dopaminergic neurons results in Parkinson disease (PD). Degenerative PD usually presents in the seventh decade whereas genetic disorders, including mutations in PARK2, predispose to early onset PD. PARK2 encodes the parkin E3 ubiquitin ligase which confers pleotropic effects on mitochondrial and cellular fidelity and as a mediator of endoplasmic reticulum (ER) stress signaling. Although the majority of studies investigating ameliorative effects of parkin focus on dopaminergic neurons we found that astrocytes are enriched with parkin. Furthermore, astrocytes deficient in parkin display stress-induced elevation of nucleotide-oligomerization domain receptor 2 (NOD2), a cytosolic receptor integrating ER stress and inflammation. Given the neurotropic and immunomodulatory role of astrocytes we reasoned that parkin may regulate astrocyte ER stress and inflammation to control neuronal homeostasis. We show that, in response to ER stress, parkin knockdown astrocytes exhibit exaggerated ER stress, JNK activation and cytokine release, and reduced neurotropic factor expression. In coculture studied we demonstrate that dopaminergic SHSY5Y cells and primary neurons with the presence of parkin depleted astrocytes are more susceptible to ER stress and inflammation-induced apoptosis than wildtype astrocytes. Parkin interacted with, ubiquitylated and diminished NOD2 levels. Additionally, the genetic induction of parkin ameliorated inflammation in NOD2 expressing cells and knockdown of NOD2 in astrocytes suppressed inflammatory defects in parkin deficient astrocytes and concurrently blunted neuronal apoptosis. Collectively these data identify a role for parkin in modulating NOD2 as a regulatory node in astrocytic control of neuronal homeostasis.

The mitochondrial thiolase ACAT1 regulates monocyte/macrophage type I interferon via epigenetic control.

Lipid-derived acetyl-CoA is shown to be the major carbon source for histone acetylation. However, there is no direct evidence demonstrating lipid metabolic pathway contribututions to this process. Mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes the final step of ß-oxidation, the aerobic process catabolizing fatty acids (FA) into acetyl-CoA. To investigate this in the context of immunometabolism, we generated macrophage cell line lacking ACAT1. 13C-carbon tracing combined with mass spectrometry confirmed incorporation of FA-derived carbons into histone H3 and this incorporation was reduced in ACAT1 KO macrophage cells. RNA-seq identified a subset of genes downregulated in ACAT1 KO cells including STAT1/2 and interferon stimulated genes (ISGs). CHIP analysis demonstrated reduced acetyl-H3 binding to STAT1 promoter/enhancer regions. Increasing histone acetylation rescued STAT1/2 expression in ACAT1 KO cells. Concomitantly, ligand triggered IFNß release was blunted in ACAT1 KO cells and rescued by reconstitution of ACAT1. Furthermore, ACAT1 promotes FA-mediated histone acetylation in an acetylcarnitine shuttle-dependent manner. In patients with obesity, levels of ACAT1 and histone acetylation are abnormally elevated. Thus, our study identified a novel link between ACAT1 mediated FA metabolism and epigenetic modification on STAT1/2 that uncovers a regulatory role of lipid metabolism in innate immune signaling and opens novel avenues for interventions in human diseases such as obesity.

Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques.

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required. The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not been studied. To track the output and clonality of HDR-edited cells and to provide a comparison to lentivirally transduced HSCs in vivo, we developed a competitive rhesus macaque (RM) autologous transplantation model, co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV) and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decrease by 2 months following transplantation, with little improvement via p53 inhibition, in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonality was oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modification approaches on HSCs.

Propionate functions as a feeding state-dependent regulatory metabolite to counter proinflammatory signaling linked to nutrient load and obesity.

Generally, fasting and refeeding confer anti- and proinflammatory effects, respectively. In humans, these caloric-load interventions function, in part, via regulation of CD4+ T cell biology. However, mechanisms orchestrating this regulation remain incomplete. We employed integrative bioinformatics of RNA sequencing and high-performance liquid chromatography-mass spectrometry data to measure serum metabolites and gene expression of peripheral blood mononuclear cells isolated from fasting and refeeding in volunteers to identify nutrient-load metabolite-driven immunoregulation. Propionate, a short chain fatty acid (SCFA), and the SCFA-sensing G protein-coupled receptor 43 (ffar2) were coordinately and inversely regulated by fasting and refeeding. Propionate and free fatty acid receptor agonists decreased interferon-γ and interleukin-17 and significantly blunted histone deacetylase activity in CD4+ T cells. Furthermore, propionate blunted nuclear factor κB activity and diminished interleukin-6 release. In parallel, propionate reduced phosphorylation of canonical T helper 1 (TH1) and TH17 regulators, STAT1 and STAT3, respectively. Conversely, knockdown of free fatty acid receptors significantly attenuated the anti-inflammatory role of propionate. Interestingly, propionate recapitulated the blunting of CD4+ TH cell activation in primary cells from obese individuals, extending the role of this metabolite to a disease associated with low-grade inflammation. Together, these data identify a nutrient-load responsive SCFA-G protein-coupled receptor linked pathway to regulate CD4+ TH cell immune responsiveness.

Deep phenotyping of post-infectious myalgic encephalomyelitis/chronic fatigue syndrome.

Post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS) is a disabling disorder, yet the clinical phenotype is poorly defined, the pathophysiology is unknown, and no disease-modifying treatments are available. We used rigorous criteria to recruit PI-ME/CFS participants with matched controls to conduct deep phenotyping. Among the many physical and cognitive complaints, one defining feature of PI-ME/CFS was an alteration of effort preference, rather than physical or central fatigue, due to dysfunction of integrative brain regions potentially associated with central catechol pathway dysregulation, with consequences on autonomic functioning and physical conditioning. Immune profiling suggested chronic antigenic stimulation with increase in naïve and decrease in switched memory B-cells. Alterations in gene expression profiles of peripheral blood mononuclear cells and metabolic pathways were consistent with cellular phenotypic studies and demonstrated differences according to sex. Together these clinical abnormalities and biomarker differences provide unique insight into the underlying pathophysiology of PI-ME/CFS, which may guide future intervention.

Comparison of busulfan and total body irradiation conditioning on hematopoietic clonal dynamics following lentiviral gene transfer in rhesus macaques.

The clonal dynamics following hematopoietic stem progenitor cell (HSPC) transplantation with busulfan conditioning are of great interest to the development of HSPC gene therapies. Compared with total body irradiation (TBI), busulfan is less toxic and more clinically relevant. We used a genetic barcoded HSPC autologous transplantation model to investigate the impact of busulfan conditioning on hematopoietic reconstitution in rhesus macaques. Two animals received lower busulfan dose and demonstrated lower vector marking levels compared with the third animal given a higher busulfan dose, despite similar busulfan pharmacokinetic analysis. We observed uni-lineage clonal engraftment at 1 month post-transplant, replaced by multilineage clones by 2 to 3 months in all animals. The initial multilineage clones in the first two animals were replaced by a second multilineage wave at 9 months; this clonal pattern disappeared at 13 months in the first animal, though was maintained in the second animal. The third animal maintained stable multilineage clones from 3 months to the most recent time point. In addition, busulfan animals exhibit more rapid HSPC clonal mixing across bone marrow sites and less CD16+ NK-biased clonal expansion compared with TBI animals. Therefore, busulfan conditioning regimens can variably impact the marrow niche, resulting in differences in clonal patterns with implications for HSPC gene therapies.

MAVS Positively Regulates Mitochondrial Integrity and Metabolic Fitness in B Cells.

Activated B cells experience metabolic changes that require mitochondrial remodeling, in a process incompletely defined. In this study, we report that mitochondrial antiviral signaling protein (MAVS) is involved in BCR-initiated cellular proliferation and prolonged survival. MAVS is well known as a mitochondrial-tethered signaling adaptor with a central role in viral RNA-sensing pathways that induce type I IFN. The role of MAVS downstream of BCR stimulation was recognized in absence of IFN, indicative of a path for MAVS activation that is independent of viral infection. Mitochondria of BCR-activated MAVS-deficient mouse B cells exhibited a damaged phenotype including disrupted mitochondrial morphology, excess mitophagy, and the temporal progressive blunting of mitochondrial oxidative capacity with mitochondrial hyperpolarization and cell death. Costimulation of MAVS-deficient B cells with anti-CD40, in addition to BCR stimulation, partially corrected the mitochondrial structural defects and functionality. Our data reveal a (to our knowledge) previously unrecognized role of MAVS in controlling the metabolic fitness of B cells, most noticeable in the absence of costimulatory help.

N-arachidonylglycine is a caloric state-dependent circulating metabolite which regulates human CD4+T cell responsiveness.

Caloric deprivation interventions such as intermittent fasting and caloric restriction ameliorate metabolic and inflammatory disease. As a human model of caloric deprivation, a 24-h fast blunts innate and adaptive immune cell responsiveness relative to the refed state. Isolated serum at these time points confers these same immunomodulatory effects on transformed cell lines. To identify serum mediators orchestrating this, metabolomic and lipidomic analysis was performed on serum extracted after a 24-h fast and re-feeding. Bioinformatic integration with concurrent peripheral blood mononuclear cells RNA-seq analysis implicated key metabolite-sensing GPCRs in fasting-mediated immunomodulation. The putative GPR18 ligand N-arachidonylglycine (NAGly) was elevated during fasting and attenuated CD4+T cell responsiveness via GPR18 MTORC1 signaling. In parallel, NAGly reduced inflammatory Th1 and Th17 cytokines levels in CD4+T cells isolated from obese subjects, identifying a fasting-responsive metabolic intermediate that may contribute to the regulation of nutrient-level dependent inflammation associated with metabolic disease.

Boosting NAD preferentially blunts Th17 inflammation via arginine biosynthesis and redox control in healthy and psoriasis subjects.

To evaluate whether nicotinamide adenine dinucleotide-positive (NAD+) boosting modulates adaptive immunity, primary CD4+ T cells from healthy control and psoriasis subjects were exposed to vehicle or nicotinamide riboside (NR) supplementation. NR blunts interferon γ (IFNγ) and interleukin (IL)-17 secretion with greater effects on T helper (Th) 17 polarization. RNA sequencing (RNA-seq) analysis implicates NR blunting of sequestosome 1 (sqstm1/p62)-coupled oxidative stress. NR administration increases sqstm1 and reduces reactive oxygen species (ROS) levels. Furthermore, NR activates nuclear factor erythroid 2-related factor 2 (Nrf2), and genetic knockdown of nrf2 and the Nrf2-dependent gene, sqstm1, diminishes NR amelioratory effects. Metabolomics analysis identifies that NAD+ boosting increases arginine and fumarate biosynthesis, and genetic knockdown of argininosuccinate lyase ameliorates NR effects on IL-17 production. Hence NR via amino acid metabolites orchestrates Nrf2 activation, augments CD4+ T cell antioxidant defenses, and attenuates Th17 responsiveness. Oral NR supplementation in healthy volunteers similarly increases serum arginine, sqstm1, and antioxidant enzyme gene expression and blunts Th17 immune responsiveness, supporting evaluation of NAD+ boosting in CD4+ T cell-linked inflammation.

Impact of CRISPR/HDR-editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques.

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. The impact of HDR on true LT-HSCs clonal dynamics in a relevant large animal model has not previously been studied. To track the HDR-edited cells, autologous rhesus macaque (RM) CD34 + cells were electroporated with the gRNA/Cas9 ribonucleoprotein (RNP) and HDR cassette barcode library structure and reinfused into RMs following myeloablation. For competitive model animals, fractionated CD34 + cells were transduced with a barcoded GFP-expressing lentiviral vector (LV) and electroporated via HDR machinery, respectively. CD33 knockout (KO) neutrophils were prevalent early following engraftment and then rapidly decreased, resulting in less than 1% total editing efficiency. Interestingly, in competitive animals, a higher concentration of i53 mRNA result in a less steep reduction in CD33 KO cells, presented a modest decrease in HDR rate (0.1-0.2%) and total indels (1.5-6.5%). In contrast, the drop off of LV-transduced GFP + cells stabilized at 20% after 2 months. We next retrieved embedded barcodes and revealed that various clones contributed to early hematopoietic reconstitution, then after dominant clones appeared at steady state throughout the animals. In conclusion, CRISPR/HDR edited cells disappeared rapidly after the autologous transplantation in RM despite substantial gene editing outcome, whereas LV-transduced cells were relatively well maintained. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which can be mildly mitigated by the introduction of i53 mRNA.

Social Determinants modulate NK cell activity via obesity, LDL, and DUSP1 signaling.

Adverse social determinants of health (aSDoH) are associated with obesity and related comorbidities like diabetes, cardiovascular disease, and cancer. Obesity is also associated with natural killer cell (NK) dysregulation, suggesting a potential mechanistic link. Therefore, we measured NK phenotypes and function in a cohort of African-American (AA) women from resource-limited neighborhoods. Obesity was associated with reduced NK cytotoxicity and a shift towards a regulatory phenotype. In vitro, LDL promoted NK dysfunction, implicating hyperlipidemia as a mediator of obesity-related immune dysregulation. Dual specific phosphatase 1 (DUSP1) was induced by LDL and was upregulated in NK cells from subjects with obesity, implicating DUSP1 in obesity-mediated NK dysfunction. In vitro, DUSP1 repressed LAMP1/CD107a, depleting NK cells of functional lysosomes to prevent degranulation and cytokine secretion. Together, these data provide novel mechanistic links between aSDoH, obesity, and immune dysregulation that could be leveraged to improve outcomes in marginalized populations.

Ogfod1 deletion increases cardiac beta-alanine levels and protects mice against ischaemia- reperfusion injury.

AIMS: Prolyl hydroxylation is a post-translational modification that regulates protein stability, turnover, and activity. The proteins that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase family of proteins. 2-oxoglutarate- and iron-dependent oxygenase domain-containing protein 1 (Ogfod1), which hydroxylates a proline in ribosomal protein s23 is a newly described member of this family. The aims of this study were to investigate roles for Ogfod1 in the heart, and in the heart's response to stress. METHODS AND RESULTS: We isolated hearts from wild-type (WT) and Ogfod1 knockout (KO) mice and performed quantitative proteomics using tandem mass Tag labelling coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to identify protein changes. Ingenuity pathway analysis identified 'Urate Biosynthesis/Inosine 5'-phosphate Degradation' and 'Purine Nucleotides Degradation II (Aerobic)' as the most significantly enriched pathways. We performed metabolomics analysis and found that both purine and pyrimidine pathways were altered with the purine nucleotide inosine 5'-monophosphate showing a 3.5-fold enrichment in KO hearts (P = 0.011) and the pyrimidine catabolism product beta-alanine showing a 1.7-fold enrichment in KO hearts (P = 0.014). As changes in these pathways have been shown to contribute to cardioprotection, we subjected isolated perfused hearts to ischaemia and reperfusion (I/R). KO hearts showed a 41.4% decrease in infarct size and a 34% improvement in cardiac function compared to WT hearts. This protection was also evident in an in vivo I/R model. Additionally, our data show that treating isolated perfused WT hearts with carnosine, a metabolite of beta-alanine, improved protection in the context of I/R injury, whereas treating KO hearts with carnosine had no impact on recovery of function or infarct size. CONCLUSIONS: Taken together, these data show that Ogfod1 deletion alters the myocardial proteome and metabolome to confer protection against I/R injury.

Boosting NAD+ blunts TLR4-induced type I IFN in control and systemic lupus erythematosus monocytes.

BACKGROUNDFasting and NAD+-boosting compounds, including NAD+ precursor nicotinamide riboside (NR), confer antiinflammatory effects. However, the underlying mechanisms and therapeutic potential are incompletely defined.METHODSWe explored the underlying biology in myeloid cells from healthy volunteers following in vivo placebo or NR administration and subsequently tested the findings in vitro in monocytes extracted from patients with systemic lupus erythematosus (SLE).RESULTSRNA-Seq of unstimulated and LPS-activated monocytes implicated NR in the regulation of autophagy and type I IFN signaling. In primary monocytes, NR blunted LPS-induced IFN-ß production, and genetic or pharmacological disruption of autophagy phenocopied this effect. Given that NAD+ is a coenzyme in oxidoreductive reactions, metabolomics was performed and identified that NR increased the inosine level. Inosine supplementation similarly blunted autophagy and IFN-ß release. Finally, because SLE exhibits type I IFN dysregulation, we assessed the NR effect on monocytes from patients with SLE and found that NR reduced autophagy and IFN-ß release.CONCLUSIONWe conclude that NR, in an NAD+-dependent manner and in part via inosine signaling, mediated suppression of autophagy and attenuated type I IFN in myeloid cells, and we identified NR as a potential adjunct for SLE management.TRIAL REGISTRATIONClinicalTrials.gov registration numbers NCT02812238, NCT00001846, and NCT00001372.FUNDINGThis work was supported by the NHLBI and NIAMS Intramural Research divisions.

Integrated cell-free DNA and cytokine analysis uncovers distinct tissue injury and immune response patterns in solid organ transplant recipients with COVID-19.

COVID-19 pathogenesis is associated with an exuberant inflammatory response. However, the tissue injury pattern and immune response in solid-organ transplant recipients (SOTRs) taking immunosuppressive therapy have not been well characterized. Here, we perform both cfDNA and cytokine profiling on plasma samples to map tissue damage, including allograft injury and delineate underlying immunopathology. We identified injuries from multiple-tissue types, including hematopoietic cells, vascular endothelium, hepatocyte, adipocyte, pancreas, kidney, heart, and lung in SOTRs with COVID-19 that correlates with disease severity. SOTRs with COVID-19 have higher plasma levels of cytokines such as IFN-λ1, IFN-γ, IL-15, IL-18 IL-1RA, IL-6, MCP-2, and TNF-α as compared to healthy controls, and the levels of GM-CSF, IL-15, IL-6, IL-8, and IL-10 were associated with disease severity in SOTRs. Strikingly, IFN-λ and IP-10 were markedly increased in SOTRs compared to immunocompetent patients with COVID-19. Correlation analyses showed a strong association between monocyte-derived cfDNA and inflammatory cytokines/chemokines in SOTRs with COVID-19. Moreover, compared to other respiratory viral infections, COVID-19 induced pronounced injury in hematopoietic, vascular endothelial and endocrine tissues. Allograft injury, measured as donor-derived cfDNA was elevated in SOTRs with COVID-19, including allografts distant from the primary site of infection. Allograft injury correlated with inflammatory cytokines and cfDNA from immune cells. Furthermore, longitudinal analysis identified a gradual decrease of cfDNA and inflammatory cytokine levels in patients with a favorable outcome. Our findings highlight distinct tissue injury and cytokine features in SOTRs with COVID-19 that correlate with disease severity.

The Rho5 GTPase is necessary for oxidant-induced cell death in budding yeast.

In both animal and yeast cells, reactive oxygen species (ROS) are produced as byproducts of metabolism and upon exposure to diverse environmental stresses. Cellular defense systems operate to avoid molecular damage caused by ROS, but the redox balance is disturbed under excessive stress. Cells of the budding yeast Saccharomyces cerevisiae undergo apoptotic-like cell death upon exposure to hydrogen peroxide (H(2)O(2)). Here, we report that the Rho5 GTPase of budding yeast is necessary for H(2)O(2)-induced cell death, which accompanies ROS accumulation and DNA fragmentation. Unlike WT, a rho5 deletion mutant (rho5Delta) exhibits little cell death, whereas the constitutively active rho5(G12V) mutant exhibits excess ROS accumulation and increased cell death upon H(2)O(2) treatment. Consistent with a role in the oxidative stress response, Rho5 interacts with the thioredoxin reductase Trr1, a key component of the cytoplasmic thioredoxin antioxidant system, in a GTP-dependent manner. This interaction occurs on the vacuolar membrane before exposure to H(2)O(2) but also in the vacuolar lumen after H(2)O(2) treatment. Trr1 levels are elevated in rho5Delta cells but are elevated only slightly in WT and not in the rho5(G12V) cells after H(2)O(2) treatment. Taken together, these data suggest that Rho5 mediates H(2)O(2)-induced cell death by regulating the level of Trr1 or by excluding Trr1 from its cytoplasmic substrate.

Network Analysis and Transcriptome Profiling Identify Autophagic and Mitochondrial Dysfunctions in SARS-CoV-2 Infection.

Analyzing host cells' transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection will help delineate biological processes underlying viral pathogenesis. First, analysis of expression profiles of lung cell lines A549 and Calu3 revealed upregulation of antiviral interferon signaling genes in response to all three SARS-CoV-2, MERS-CoV, or influenza A virus (IAV) infections. However, perturbations in expression of genes involved in inflammatory, mitochondrial, and autophagy processes were specifically observed in SARS-CoV-2-infected cells. Next, a validation study in infected human nasopharyngeal samples also revealed perturbations in autophagy and mitochondrial processes. Specifically, mTOR expression, mitochondrial ribosomal, mitochondrial complex I, lysosome acidification, and mitochondrial fission promoting genes were concurrently downregulated in both infected cell lines and human samples. SARS-CoV-2 infection impeded autophagic flux either by upregulating GSK3B in lung cell lines or by downregulating autophagy genes, SNAP29, and lysosome acidification genes in human samples, contributing to increased viral replication. Therefore, drugs targeting lysosome acidification or autophagic flux could be tested as intervention strategies. Finally, age-stratified SARS-CoV-2-positive human data revealed impaired upregulation of chemokines, interferon-stimulated genes, and tripartite motif genes that are critical for antiviral signaling. Together, this analysis has revealed specific aspects of autophagic and mitochondrial function that are uniquely perturbed in SARS-CoV-2-infected host cells.

Reducing Fatty Acid Oxidation Improves Cancer-free Survival in a Mouse Model of Li-Fraumeni Syndrome.

Germline mutations of TP53, which cause the cancer predisposition disorder Li-Fraumeni syndrome (LFS), can increase mitochondrial activity as well as fatty acid ß-oxidation (FAO) in mice. Increased fatty acid metabolism can promote cancer malignancy, but its specific contribution to tumorigenesis in LFS remains unclear. To investigate this, we crossed LFS mice carrying the p53 R172H knock-in mutation (p53172H/H , homolog of the human TP53 R175H LFS mutation) with myoglobin-knockout (MB-/- ) mice known to have decreased FAO. MB-/- p53172H/H double-mutant mice also showed mildly reduced FAO in thymus, a common site of T lymphoma development in LFS mice, in association with an approximately 40% improvement in cancer-free survival time. RNA sequencing profiling revealed that the p53 R172H mutation promotes mitochondrial metabolism and ribosome biogenesis, both of which are suppressed by the disruption of MB. The activation of ribosomal protein S6, involved in protein translation and implicated in cancer promotion, was also inhibited in the absence of MB. To further confirm the role of FAO in lymphomagenesis, mitochondrial FAO enzyme, carnitine palmitoyltransferase 2 (CPT2), was specifically disrupted in T cells of p53172H/H mice using a Cre-loxP-mediated strategy. The heterozygous knockout of CPT2 resulted in thymus FAO haploinsufficiency and an approximately 30% improvement in survival time, paralleling the antiproliferative signaling observed with MB disruption. Thus, this study demonstrates that moderating FAO in LFS can suppress tumorigenesis and improve cancer-free survival with potential implications for cancer prevention. PREVENTION RELEVANCE: Mildly inhibiting the increased fatty acid oxidation observed in a mouse model of Li-Fraumeni syndrome, a cancer predisposition disorder caused by inherited mutations of TP53, dampens aberrant pro-tumorigenic cell signaling and improves the survival time of these mice, thereby revealing a potential strategy for cancer prevention in patients.