Gong et al. report that repressor element 1–silencing transcription factor (REST) activates ferroptosis and that renal tubular epithelial cell–specific knockout of REST alleviates murine acute kidney injury (AKI) and attenuates AKI-to–chronic kidney disease transition by inhibiting ferroptosis. The cover image shows a transmission electron micrograph of mouse kidney following ischemia/reperfusion injury.
BACKGROUND. Antibody-based therapies for respiratory viruses are of increasing importance. The INSIGHT006 trial administered anti-influenza hyperimmune intravenous immunoglobulin (Flu-IVIG) to patients hospitalised with influenza. Flu-IVIG treatment improved outcomes in patients with influenza B but showed no benefit for influenza A. METHODS. To probe potential mechanisms of Flu-IVIG utility, sera collected from patients hospitalised with influenza A or B viruses (IAV or IBV) were analysed for antibody isotype/subclass and Fc-gamma receptor (FcgR) binding by ELISA, bead-based multiplex and NK cell activation assays. RESULTS. Influenza-specific FcgR binding antibodies were elevated in Flu-IVIG infused IBV- and IAV-infected patients. In IBV-infected participants (n = 62), increased IgG3 and FcgR binding were associated with more favourable outcomes. Flu-IVIG therapy also improved the odds of a more favourable outcome in patients with low levels of anti-IBV Fc-functional antibody. Higher FcgR binding antibody was associated with less favourable outcomes in IAV-infected patients (n = 50), and Flu-IVIG worsened the odds of a favourable outcome in participants with low levels of anti-IAV Fc-functional antibody. CONCLUSION. These detailed serological analyses provide insights into antibody features and mechanisms required for a successful humoral response against influenza, suggesting that IBV-specific, but not IAV-specific, antibodies with Fc-mediated functions may assist in improving influenza outcome. This work will inform development of improved influenza immunotherapies. TRIAL REGISTRATION. ClinicalTrials.gov NCT02287467 FUNDING SOURCES. Funding for this research was provided by Subcontract 13XS134 under Leidos Biomedical Research Prime Contract HHSN261200800001E and HHSN261201500003I, NCI/NIAID.
Hillary A. Vanderven, Deborah N. Wentworth, Win Min Han, Heidi Peck, Ian G. Barr, Richard T. Davey, Jr., John H. Beigel, Dominic E. Dwyer, Mamta K. Jain, Brian Angus, Christian T. Brandt, Analia Mykietiuk, Matthew G. Law, James D. Neaton, Stephen J. Kent
Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multi-system disease. The wide range of manifestations observed in mitochondrial disease patients results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify non-random distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA sequencing, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profile the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and healthy control donors. Utilizing the retina as a model for complex multi-lineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm-derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm-derived lineage, namely the vasculature of the choroid, was near homoplasmic for the wildtype allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the non-random nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.
Nathaniel K. Mullin, Andrew P. Voigt, Miles J. Flamme-Wiese, Xiuying Liu, Megan J. Riker, Katayoun Varzavand, Edwin M. Stone, Budd A. Tucker, Robert F. Mullins
Specific and efficient smooth muscle cell (SMC)-targeted gene deletion is typically achieved by pairing SMMHC-CreERT2-Tg mice with mice carrying the loxP-flanked gene. However, the transgene, CreERT2, is not controlled by the endogenous Myh11 gene promoter, and the codon-modified iCreERT2 exhibits significant tamoxifen-independent leakage. Furthermore, because the Cre-bearing Bacterial Artificial Chromosome (BAC) is inserted onto the Y chromosome, the SMMHC-CreERT2-Tg mice strain can only exhibit gene deletions in male mice. Additionally, there is a lack of Myh11-driven constitutive Cre mice when tamoxifen usage is a concern. We used CRISPR/Cas9-mediated homologous recombination between a donor vector carrying the 1) CreNLSP2A or 2) CreERT2-P2A sequence and homologous arm surrounding the translation start site of the Myh11 gene to generate Cre knock-in mice. The P2A sequence enables the simultaneous translation of Cre and endogenous proteins. Using reporter mice, we assessed Cre-mediated recombination efficiency, specificity, tamoxifen-dependent controllability, and functionality in both sexes. Both constitutive (Myh11-CreNLSP2A) and inducible (Myh11-CreERT2-P2A) Cre mice demonstrated efficient, SMC-specific, sex-independent Cre recombinase activity without confounding endogenous gene expression. Combined with recently generated BAC transgenic Myh11-CreERT2-RAD mice and the Itga8-CreERT2 mouse models, our new models will help expand the research toolbox, facilitating unbiased and comprehensive research in SMCs and SMC-dependent cardiovascular diseases.
Yang Zhao, Guizhen Zhao, Ziyi Chang, Tianqing Zhu, Ying Zhao, Haocheng Lu, Chao Xue, Thomas L. Saunders, Yanhong Guo, Lin Chang, Y. Eugene Chen, Jifeng Zhang
Fatty acid binding protein 4 (FABP4) is a lipid chaperone secreted from adipocytes upon stimulation of lipolysis. Circulating FABP4 levels strongly correlate with obesity and metabolic pathologies in experimental models and humans. While adipocytes have been presumed to be the major source of hormonal FABP4, this question has not been addressed definitively in vivo. We generated mice with Fabp4 deletion in cells known to express the gene; adipocytes (Adipo-KO), endothelial cells (Endo-KO), myeloid cells (Myeloid-KO), and the whole body (Total-KO) to examine the contribution of these cell types to basal and stimulated plasma FABP4 levels. Unexpectedly, baseline plasma FABP4 was not significantly reduced in Adipo-KO mice, whereas Endo-KO mice showed ~87% reduction versus wildtype controls. In contrast, Adipo-KO mice exhibited ~62% decreased induction of FABP4 responses to lipolysis, while Endo-KO mice showed only mildly decreased induction, indicating that adipocytes are the main source of increases in FABP4 during lipolysis. We did not detect any myeloid contribution to circulating FABP4. Surprisingly, despite the nearly intact induction of FABP4, Endo-KO mice showed blunted lipolysis-induced insulin secretion, identical to Total-KO mice. We conclude that the endothelium is the major source of baseline hormonal FABP4 and is required for the insulin response to lipolysis.
Karen E. Inouye, Kacey J. Prentice, Alexandra Lee, Zeqiu B. Wang, Carla Dominguez-Gonzalez, Mu Xian Chen, Jillian K. Riveros, M. Furkan Burak, Grace Y. Lee, Gokhan S. Hotamisligil
During alveolar repair, alveolar type 2 (AT2) epithelial cell progenitors rapidly proliferate and differentiate into flat type 1 alveolar epithelial cells. Failure of normal alveolar repair mechanisms can lead to loss of alveolar structure (emphysema) or development of fibrosis, depending on the type and severity of injury. To test if β1-containing integrins are required during repair following acute injury, we administered E. coli lipopolysaccharide (LPS) by intratracheal injection to mice with a post-developmental deletion of β1 integrin in AT2 cells. While control mice recovered from LPS injury without structural abnormalities, β1-deficient mice had more severe inflammation and developed emphysema. In addition, recovering alveoli were repopulated with an abundance of rounded epithelial cells co-expressing type 2, type 1, and mixed intermediate cell state markers, with few mature type 1 cells. β1-deficient AT2 cells showed persistently increased proliferation after injury, which was blocked by inhibiting NF-kB activation in these cells. Lineage tracing experiments revealed that β1-deficient AT2 cells failed to differentiate into mature type 1 alveolar epithelial cells. Together, these findings demonstrate that functional alveolar repair after injury with terminal alveolar epithelial differentiation requires β1-containing integrins.
Jennifer M.S. Sucre, Fabian Bock, Nicholas M. Negretti, John T. Benjamin, Peter M. Gulleman, Xinyu Dong, Kimberly T. Ferguson, Christopher S. Jetter, Wei Han, Yang Liu, Seunghyi Kook, Jason J. Gokey, Susan H. Guttentag, Jonathan A. Kropski, Timothy S. Blackwell, Roy Zent, Erin J. Plosa
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