Immunometabolic features of natural killer cells are associated with infection outcomes in critical illness.

Immunosuppression increases the risk of nosocomial infection in patients with chronic critical illness. This exploratory study aimed to determine the immunometabolic signature associated with nosocomial infection during chronic critical illness. We prospectively recruited patients who were admitted to the respiratory care center and who had received mechanical ventilator support for more than 10 days in the intensive care unit. The study subjects were followed for the occurrence of nosocomial infection until 6 weeks after admission, hospital discharge, or death. The cytokine levels in the plasma samples were measured. Single-cell immunometabolic regulome profiling by mass cytometry, which analyzed 16 metabolic regulators in 21 immune subsets, was performed to identify immunometabolic features associated with the risk of nosocomial infection. During the study period, 37 patients were enrolled, and 16 patients (43.2%) developed nosocomial infection. Unsupervised immunologic clustering using multidimensional scaling and logistic regression analyses revealed that expression of nuclear respiratory factor 1 (NRF1) and carnitine palmitoyltransferase 1a (CPT1a), key regulators of mitochondrial biogenesis and fatty acid transport, respectively, in natural killer (NK) cells was significantly associated with nosocomial infection. Downregulated NRF1 and upregulated CPT1a were found in all subsets of NK cells from patients who developed a nosocomial infection. The risk of nosocomial infection is significantly correlated with the predictive score developed by selecting NK cell-specific features using an elastic net algorithm. Findings were further examined in an independent cohort of COVID-19-infected patients, and the results confirm that COVID-19-related mortality is significantly associated with mitochondria biogenesis and fatty acid oxidation pathways in NK cells. In conclusion, this study uncovers that NK cell-specific immunometabolic features are significantly associated with the occurrence and fatal outcomes of infection in critically ill population, and provides mechanistic insights into NK cell-specific immunity against microbial invasion in critical illness.

The role of antipsychotics and other drugs on the development and progression of neuroleptic malignant syndrome.

Neuroleptic malignant syndrome (NMS) is a rare but serious and sometimes fatal complication in patients taking antipsychotic drugs, and its underlying mechanism still remains unclear. The pharmacotherapy for psychotic disorders is complicated and often involves a combination of two or more drugs, including drugs other than antipsychotics. In the present study, we used the Japanese Adverse Drug Event Report (JADER) database to broadly investigate the drugs associated with NMS, following their related pathways, as well as the drug-drug interactions (DDIs) in NMS. All analyses were performed using data from the JADER database from April 2004 to May 2022. Single-drug signals were evaluated using the reporting odds ratio (ROR) and proportional reporting ratio (PRR), and drug pathways were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG). DDIs were evaluated using the Ω shrinkage measure and Chi-square statistics models. All drugs associated with 20 or more NMS cases in the JADER database exhibited signals for NMS, including non-antipsychotics. Pathways associated with the drugs included the dopaminergic or serotonergic synapses related to antipsychotics. DDIs leading to NMS were confirmed for several drug combinations exhibiting single-drug signals. This study confirmed the significant association of various drugs, including non-psychotics, with NMS and suggested that various pathways related to these drugs may be involved in the progression of NMS. In addition, several combinations of these drugs were found to interact (DDI), increasing the risk of NMS, which suggests that appropriate caution should be taken when administering these drugs.

Analysis of Appendicitis Cases in the Japanese Adverse Drug Event Report (JADER) Database.

Appendicitis is one of the most common abdominal surgical emergencies worldwide; however, its causes remain poorly understood. The Japanese Adverse Drug Event Report (JADER) database is a spontaneous reporting system (SRS) that can be utilized to analyze the safety signals of adverse events. In this study, we investigated the association between drug use and the onset of appendicitis using the JADER database. We first used the reporting odds ratio (ROR) as the signal and found signals for appendicitis, perforated appendicitis, and complicated appendicitis for 23, 9, and 1 drug, respectively. To investigate the level of hazard over time in drug-associated appendicitis, the Weibull shape parameter ß was calculated using a Weibull plot, which revealed drug-dependent patterns for changes in the risk of appendicitis over time for the eight drugs. Furthermore, logistic regression analysis was performed to account for the influence of age, sex, and primary disease, and a significant association was detected between two drugs and appendicitis. Several types of drugs, such as antitumor, antirheumatic, and anti-inflammatory drugs, were included in our analyses; however, only clozapine, which is used for patients with schizophrenia, was commonly identified in these analyses. The resulting data suggest that certain drugs may be associated with appendicitis and may require adequate attention.

Single cell profiling of γδ hepatosplenic T-cell lymphoma unravels tumor cell heterogeneity associated with disease progression.

PURPOSE: Hepatosplenic T-cell lymphoma (HSTCL), mostly derived from γδ T cells, is a rare but very aggressive lymphoma with poor outcomes. In this study, we generated the first single cell landscape for this rare disease and characterized the molecular pathogenesis underlying the disease progression. METHODS: We performed paired single cell RNA-seq and T cell receptor (TCR) sequencing on biopsies from a HSTCL patient pre- and post- chemotherapy treatments. Following by a series of bioinformatics analysis, we investigated the gene expression profile of γδ HSTCS as well as its tumor microenvironment (TME). RESULTS: We characterized the unique gene expressing signatures of malignant γδ T cells with a set of marker genes were newly identified in HSTCL (AREG, PLEKHA5, VCAM1 etc.). Although the malignant γδ T cells were expanded from a single TCR clonotype, they evolved into two transcriptionally distinct tumor subtypes during the disease progression. The Tumor_1 subtype was dominant in pre-treatment samples with highly aggressive phenotypes. While the Tumor_2 had relative mild cancer hallmark signatures but expressed genes associated with tumor survival signal and drug resistance (IL32, TOX2, AIF1, AKAP12, CD38 etc.), and eventually became the main tumor subtype post-treatment. We further dissected the tumor microenvironment and discovered the dynamically rewiring cell-cell interaction networks during the treatment. The tumor cells had reduced communications with the microenvironment post-treatment. CONCLUSIONS: Our study reveals heterogenous and dynamic tumor and microenvironment underlying pathogenesis of HSTCL and may contribute to identify novel targets for diagnosis and treatment of HSTCL in the future.

Circulating N-formylmethionine and metabolic shift in critical illness: a multicohort metabolomics study.

BACKGROUND: Cell stress promotes degradation of mitochondria which release danger-associated molecular patterns that are catabolized to N-formylmethionine. We hypothesized that in critically ill adults, the response to N-formylmethionine is associated with increases in metabolomic shift-related metabolites and increases in 28-day mortality. METHODS: We performed metabolomics analyses on plasma from the 428-subject Correction of Vitamin D Deficiency in Critically Ill Patients trial (VITdAL-ICU) cohort and the 90-subject Brigham and Women's Hospital Registry of Critical Illness (RoCI) cohort. In the VITdAL-ICU cohort, we analyzed 983 metabolites at Intensive Care Unit (ICU) admission, day 3, and 7. In the RoCI cohort, we analyzed 411 metabolites at ICU admission. The association between N-formylmethionine and mortality was determined by adjusted logistic regression. The relationship between individual metabolites and N-formylmethionine abundance was assessed with false discovery rate correction via linear regression, linear mixed-effects, and Gaussian graphical models. RESULTS: Patients with the top quartile of N-formylmethionine abundance at ICU admission had a significantly higher adjusted odds of 28-day mortality in the VITdAL-ICU (OR, 2.4; 95%CI 1.5-4.0; P = 0.001) and RoCI cohorts (OR, 5.1; 95%CI 1.4-18.7; P = 0.015). Adjusted linear regression shows that with increases in N-formylmethionine abundance at ICU admission, 55 metabolites have significant differences common to both the VITdAL-ICU and RoCI cohorts. With increased N-formylmethionine abundance, both cohorts had elevations in individual short-chain acylcarnitine, branched chain amino acid, kynurenine pathway, and pentose phosphate pathway metabolites. CONCLUSIONS: The results indicate that circulating N-formylmethionine promotes a metabolic shift with heightened mortality that involves incomplete mitochondrial fatty acid oxidation, increased branched chain amino acid metabolism, and activation of the pentose phosphate pathway.

Conditional deletion of myeloid-specific mitofusin 2 but not mitofusin 1 promotes kidney fibrosis.

Macrophages exert critical functions during kidney injury, inflammation, and tissue repair or fibrosis. Mitochondrial structural and functional aberrations due to an imbalance in mitochondrial fusion/fission processes are implicated in the pathogenesis of chronic kidney disease. Therefore, we investigated macrophage-specific functions of mitochondrial fusion proteins, mitofusin (MFN)1 and MFN2, in modulating macrophage mitochondrial dynamics, biogenesis, oxidative stress, polarization, and fibrotic response. MFN1 and MFN2 were found to be suppressed in mice after adenine diet-induced chronic kidney disease, in transforming growth factor-beta 1-treated bone marrow-derived macrophages, and in THP-1-derived human macrophages (a human leukemic cell line). However, abrogating Mfn2 but not Mfn1 in myeloid-lineage cells resulted in greater macrophage recruitment into the kidney during fibrosis and the macrophage-derived fibrotic response associated with collagen deposition culminating in worsening kidney function. Myeloid-specific Mfn1 /Mfn2 double knockout mice also showed increased adenine-induced fibrosis. Mfn2-deficient bone marrow-derived macrophages displayed enhanced polarization towards the profibrotic/M2 phenotype and impaired mitochondrial biogenesis. Macrophages in the kidney of Mfn2-deficient and double knockout but not Mfn1-deficient mice exhibited greater mitochondrial mass, size, oxidative stress and lower mitophagy under fibrotic conditions than the macrophages in the kidney of wild-type mice. Thus, downregulation of MFN2 but not MFN1 lead to macrophage polarization towards a profibrotic phenotype to promote kidney fibrosis through a mechanism involving suppression of macrophage mitophagy and dysfunctional mitochondrial dynamics.

Association of plasma mitochondrial DNA with COPD severity and progression in the SPIROMICS cohort.

BACKGROUND: There is a lack of mechanism-driven, clinically relevant biomarkers in chronic obstructive pulmonary disease (COPD). Mitochondrial dysfunction, a proposed disease mechanism in COPD, is associated with the release of mitochondrial DNA (mtDNA), but plasma cell-free mtDNA has not been previously examined prospectively for associations with clinical COPD measures. METHODS: P-mtDNA, defined as copy number of mitochondrially-encoded NADH dehydrogenase-1 (MT-ND1) gene, was measured by real-time quantitative PCR in 700 plasma samples from participants enrolled in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) cohort. Associations between p-mtDNA and clinical disease parameters were examined, adjusting for age, sex, smoking status, and for informative loss to follow-up. RESULTS: P-mtDNA levels were higher in participants with mild or moderate COPD, compared to smokers without airflow obstruction, and to participants with severe COPD. Baseline increased p-mtDNA levels were associated with better CAT scores in female smokers without airflow obstruction and female participants with mild or moderate COPD on 1-year follow-up, but worse 6MWD in females with severe COPD. Higher p-mtDNA levels were associated with better 6MWD in male participants with severe COPD. These associations were no longer significant after adjusting for informative loss to follow-up. CONCLUSION: In this study, p-mtDNA levels associated with baseline COPD status but not future changes in clinical COPD measures after accounting for informative loss to follow-up. To better characterize mitochondrial dysfunction as a potential COPD endotype, these results should be confirmed and validated in future studies. TRIAL REGISTRATION:  ClinicalTrials.gov NCT01969344 (SPIROMICS).

Cell-free DNA in human ex vivo lung perfusate as a potential biomarker to predict the risk of primary graft dysfunction in lung transplantation.

BACKGROUND: Cell-free DNA (cfDNA), such as mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA), are known to be released from injured cells and as such have been explored as biomarkers for tissue injury in different clinical settings. Ex vivo lung perfusion (EVLP) has been developed as an effective technique for marginal donor lung functional assessment. We hypothesized that the level of cfDNA in EVLP perfusate may reflect tissue injury and thus can be developed as a biomarker to quantify the degree of donor lung injury or predict the development of primary graft dysfunction (PGD) after lung transplantation (LTx). METHODS: The perfusate from 62 donor lungs transplanted at our institution between May 2010 and December 2015 was sampled for cfDNA at 1 and 4 hours of perfusion. Sequences of genes encoding nicotinamide adenine dinucleotide dehydrogenase 1 (NADH-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to represent mtDNA and nuDNA, respectively. Levels were quantified by real-time polymerase chain reaction and correlated with clinical outcome after LTx. RESULTS: In our entire cohort, 14 patients developed PGD grade 3 (PGD3) within 72 hours after LTx. The non-PGD group included 48 patients (PGD0-1). Concentrations of mtDNA in the perfusate of the PGD3 group were significantly higher than those in non-PGD group at 1 hour of EVLP (1874 ± 844 vs 1259 ± 885 copies/µL; P = .011). The perfusate of the PGD3 group had significantly higher levels of nuDNA compared with the non-PGD group at both 1 hour (1498 ± 1895 vs 675 ± 391 copies/µL; P = .008) and 4 hours (4521 ± 5810 vs 1764 ± 1494 copies/µL; P = .001). In donation after cardiac death (DCD) cases, mtDNA levels were significantly higher in the PGD3 group compared with the non-PGD group at 1 hour of EVLP (2060 ± 997 vs 1184 ± 782 copies/µL; P = .040), and the levels of nuDNA were significantly higher in the PGD3 group compared with the non-PGD group at both 1 hour (1021 ± 495 vs 606 ± 305 copies/µL; P = .041) and 4 hours (2358 ± 1028 vs 1185 ± 967 copies/µL; P = .006). In donation after brain death (DBD) cases, cfDNA scores did not show a significant difference. CONCLUSIONS: We found that the amount of cfDNA, especially nuDNA, in EVLP perfusate was higher in the severe PGD group (PGD3) compared with the non-PGD group. This proof-of-concept study supports the concept that the analysis of cfDNA levels in EVLP perfusate can help estimate the damage to donor lungs before implantation. Larger studies are needed to validate this concept.

Multi-kinase framework promotes proliferation and invasion of lung adenocarcinoma through activation of dynamin-related protein 1.

Recent studies revealed the role of dynamin-related protein 1 (DRP1), encoded by the DNM1L gene, in regulating the growth of cancer cells of various origins. However, the regulation, function, and clinical significance of DRP1 remain undetermined in lung adenocarcinoma. Our study shows that the expression and activation of DRP1 are significantly correlated with proliferation and disease extent, as well as an increased risk of postoperative recurrence in stage I to stage IIIA lung adenocarcinoma. Loss of DRP1 in lung adenocarcinoma cell lines leads to an altered mitochondrial morphology, fewer copies of mitochondrial DNA, decreased respiratory complexes, and impaired oxidative phosphorylation. Additionally, the proliferation and invasion are both suppressed in DRP1-depleted lung adenocarcinoma cell lines. Our data further revealed that DRP1 activation through serine 616 phosphorylation is regulated by ERK/AKT and CDK2 in lung adenocarcinoma cell lines. Collectively, we propose the multikinase framework in activating DRP1 in lung adenocarcinoma to promote the malignant properties. Biomarkers related to mitochondrial reprogramming, such as DRP1, can be used to evaluate the risk of postoperative recurrence in early-stage lung adenocarcinoma.

Cell-free DNA (cfDNA) and Exosome Profiling from a Year-Long Human Spaceflight Reveals Circulating Biomarkers.

Liquid biopsies based on cell-free DNA (cfDNA) or exosomes provide a noninvasive approach to monitor human health and disease but have not been utilized for astronauts. Here, we profile cfDNA characteristics, including fragment size, cellular deconvolution, and nucleosome positioning, in an astronaut during a year-long mission on the International Space Station, compared to his identical twin on Earth and healthy donors. We observed a significant increase in the proportion of cell-free mitochondrial DNA (cf-mtDNA) inflight, and analysis of post-flight exosomes in plasma revealed a 30-fold increase in circulating exosomes and patient-specific protein cargo (including brain-derived peptides) after the year-long mission. This longitudinal analysis of astronaut cfDNA during spaceflight and the exosome profiles highlights their utility for astronaut health monitoring, as well as cf-mtDNA levels as a potential biomarker for physiological stress or immune system responses related to microgravity, radiation exposure, and the other unique environmental conditions of spaceflight.

Upregulation of iNOS Protects Cyclic Mechanical Stretch-Induced Cell Death in Rat Aorta Smooth Muscle Cells.

Aortic dissection and aneurysm are associated with abnormal hemodynamic loads originating from hypertension. Our previous study demonstrated that cyclic mechanical stretch (CMS, mimicked hypertension) caused the death of rat aortic smooth muscle cells (RASMCs) in a mitogen activated-protein kinases (MAPKs)-dependent manner. The current study investigated the effects of inducible nitric oxide synthase (iNOS) on CMS-induced RASMC death. cDNA microarrays for CMS-treated RASMCs showed that iNOS expression levels were increased in response to CMS. Real-time polymerase chain reaction (PCR) analysis demonstrated that this increase was p38 MAPK (p38)-dependent. NO production was also increased. This increase could be inhibited by p38 and iNOS inhibitors. Thus, CMS-induced iNOS synthesized NO. CMS-induced cell death in RASMCs was increased by the iNOS inhibitor but abrogated by the long-acting NO donor DETA-NONOate. Increased iNOS expression was confirmed in the abdominal aortic constriction mouse model. Signal transducers and activators of transcription 1 (STAT1) was activated in stretched RASMCs, and iNOS expression and NO production were inhibited by the STAT1 inhibitor nifuroxazide. Our findings suggest that RASMCs were protected by iNOS from CMS-stimulated cell death through the STAT1 and p38 signal pathways independently.

Association of urine mitochondrial DNA with clinical measures of COPD in the SPIROMICS cohort.

BACKGROUNDMitochondrial dysfunction, a proposed mechanism of chronic obstructive pulmonary disease (COPD) pathogenesis, is associated with the leakage of mitochondrial DNA (mtDNA), which may be detected extracellularly in various bodily fluids. Despite evidence for the increased prevalence of chronic kidney disease in COPD subjects and for mitochondrial dysfunction in the kidneys of murine COPD models, whether urine mtDNA (u-mtDNA) associates with measures of disease severity in COPD is unknown.METHODSCell-free u-mtDNA, defined as copy number of mitochondrially encoded NADH dehydrogenase-1 (MTND1) gene, was measured by quantitative PCR and normalized to urine creatinine in cell-free urine samples from participants in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) cohort. Urine albumin/creatinine ratios (UACR) were measured in the same samples. Associations between u-mtDNA, UACR, and clinical disease parameters - including FEV1 % predicted, clinical measures of exercise tolerance, respiratory symptom burden, and chest CT measures of lung structure - were examined.RESULTSU-mtDNA and UACR levels were measured in never smokers (n = 64), smokers without airflow obstruction (n = 109), participants with mild/moderate COPD (n = 142), and participants with severe COPD (n = 168). U-mtDNA was associated with increased respiratory symptom burden, especially among smokers without COPD. Significant sex differences in u-mtDNA levels were observed, with females having higher u-mtDNA levels across all study subgroups. U-mtDNA associated with worse spirometry and CT emphysema in males only and with worse respiratory symptoms in females only. Similar associations were not found with UACR.CONCLUSIONU-mtDNA levels may help to identify distinct clinical phenotypes and underlying pathobiological differences in males versus females with COPD.TRIAL REGISTRATIONThis study has been registered at ClinicalTrials.gov ( NCT01969344).FUNDINGUS NIH, National Heart, Lung and Blood Institute, supplemented by contributions made through the Foundation for the NIH and the COPD Foundation from AstraZeneca/MedImmune, Bayer, Bellerophon Therapeutics, Boehringer-Ingelheim Pharmaceuticals Inc., Chiesi Farmaceutici S.p.A., Forest Research Institute Inc., GlaxoSmithKline, Grifols Therapeutics Inc., Ikaria Inc., Novartis Pharmaceuticals Corporation, Nycomed GmbH, ProterixBio, Regeneron Pharmaceuticals Inc., Sanofi, Sunovion, Takeda Pharmaceutical Company, and Theravance Biopharma and Mylan.

Apolipoprotein E and Periostin Are Potential Biomarkers of Nasal Mucosal Inflammation. A Parallel Approach of In Vitro and In Vivo Secretomes.

No previously suggested biomarkers of nasal mucosal inflammation have been practically applied in clinical fields, and nasal epithelium-derived secreted proteins as biomarkers have not specifically been investigated. The goal of this study was to identify secreted proteins that dynamically change during the differentiation from basal cells to fully differentiated cells and examine whether nasal epithelium-derived proteins can be used as biomarkers of nasal mucosal inflammation, such as chronic rhinosinusitis. To achieve this goal, we analyzed two secretomes using the isobaric tag for relative and absolute quantification technique. From in vitro secretomes, we identified the proteins altered in apical secretions of primary human nasal epithelial cells according to the degree of differentiation; from in vivo secretomes, we identified the increased proteins in nasal lavage fluids obtained from patients 2 weeks after endoscopic sinus surgery for chronic sinusitis. We then used a parallel approach to identify specific biomarkers of nasal mucosal inflammation; first, we selected apolipoprotein E as a nasal epithelial cell-derived biomarker through screening proteins that were upregulated in both in vitro and in vivo secretomes, and verified highly secreted apolipoprotein E in nasal lavage fluids of the patients by Western blotting. Next, we selected periostin as an inflammatory mediator-inducible biomarker from in vivo secretomes, the secretion of which was not induced under in vitro culture conditions. We demonstrated that those two nasal epithelium-derived proteins are possible biomarkers of nasal mucosal inflammation.

Mitophagy-dependent macrophage reprogramming protects against kidney fibrosis.

Mitophagy, by maintaining mitochondrial quality control, plays a key role in maintaining kidney function and is impaired in pathologic states. Macrophages are well known for their pathogenic role in kidney fibrosis. Here, we report that PINK1/Parkin-mediated mitophagy in macrophages is compromised in experimental and human kidney fibrosis. We demonstrate downregulation of mitophagy regulators mitofusin-2 (MFN2) and Parkin downstream of PINK1 in kidney fibrosis. Loss of either Pink1 or Prkn promoted renal extracellular matrix accumulation and frequency of profibrotic/M2 macrophages. Pink1-/- or Prkn-/- BM-derived macrophages (BMDMs) showed enhanced expression of rictor. Mitochondria from TGF-ß1-treated Pink1-/- BMDMs exhibited increased superoxide levels, along with reduced respiration and ATP production. In addition, mitophagy in macrophages involves PINK1-mediated phosphorylation of downstream MFN2, MFN2-facilitated recruitment of Parkin to damaged mitochondria, and macrophage-specific deletion of Mfn2 aggravates kidney fibrosis. Moreover, mitophagy regulators were downregulated in human CKD kidney and TGF-ß1-treated human renal macrophages, whereas Mdivi1 treatment suppressed mitophagy mediators and promoted fibrotic response. Taken together, our study is the first to our knowledge to demonstrate that macrophage mitophagy plays a protective role against kidney fibrosis via regulating the PINK1/MFN2/Parkin-mediated pathway.

Heteroplasmy concordance between mitochondrial DNA and RNA.

Mitochondrial DNA (mtDNA) heteroplasmies are associated with various diseases but the transmission of heteroplasmy from mtDNA to mitochondrial RNA (mtRNA) remains unclear. We compared heteroplasmies in mtRNA from 446 human B-lymphoblastoid cell lines to their corresponding mtDNA using deep sequencing data from two independent studies. We observed 2786 heteroplasmies presenting in both DNA and RNA at 1% frequency cutoff. Among them, the frequencies of 2427 (87.1%) heteroplasmies were highly consistent (less than 5% frequency difference) between DNA and RNA. To validate these frequency consistencies, we isolated DNA and RNA simultaneously from GM12282 cell line used in those two sequencing studies, and resequenced its heteroplasmy sites. Interestingly, we also observed the rapid changes of heteroplasmy frequencies during 4 weeks of the cell culture: the frequencies at Day 14 increased by >25% than those at Day 0. However, the heteroplasmy frequencies from the same time point were highly consistent. In summary, our analysis on public data together with in vitro study indicates that the heteroplasmies in DNA can be transcribed into RNA with high fidelity. Meanwhile, the observed rapid-changing heteroplasmy frequency can potentially disturb cell functions, which could be an overlooked confounding factor in cell line related studies.