Neurodegeneration mediates neurological disability in inflammatory demyelinating diseases of the CNS. The role of innate immune cells in mediating this damage has remained controversial with evidence for destructive and protective effects. This has complicated efforts to develop treatment. The time sequence and dynamic evolution of the opposing functions are especially unclear. Given limits of in vivo monitoring in human diseases such as multiple sclerosis (MS), animal models are warranted to investigate the association and timing of innate immune activation with neurodegeneration. Using noninvasive in vivo retinal imaging of experimental autoimmune encephalitis (EAE) in CX3CR1GFP/+–knock-in mice followed by transcriptional profiling, we are able to show 2 distinct waves separated by a marked reduction in the number of innate immune cells and change in cell morphology. The first wave is characterized by an inflammatory phagocytic phenotype preceding the onset of EAE, whereas the second wave is characterized by a regulatory, antiinflammatory phenotype during the chronic stage. Additionally, the magnitude of the first wave is associated with neuronal loss. Two transcripts identified — growth arrest–specific protein 6 (GAS6) and suppressor of cytokine signaling 3 (SOCS3) — might be promising targets for enhancing protective effects of microglia in the chronic phase after initial injury.
Andrés Cruz-Herranz, Frederike C. Oertel, Kicheol Kim, Ester Cantó, Garrett Timmons, Jung H. Sin, Michael Devereux, Nicholas Baker, Brady Michel, Ryan D. Schubert, Lakshmisahithi Rani, Christian Cordano, Sergio E. Baranzini, Ari J. Green
Existing patient-derived-xenograft (PDX) mouse models of solid tumors lack a fully tumor-donor matched, syngeneic, and functional immune system. We developed such a model by engrafting lymphopenic recipient mice with a fresh, undisrupted piece of solid tumor, whereby tumor-infiltrating lymphocytes (TILs) persisted in the recipient mice for several weeks. Successful tumor engraftment was achieved in eighty-three to eighty-nine percent of tumor-infiltrating-lymphocytes-PDX (TIL-PDX) mice, and these were seen to harbor exhausted immuno-effector as well as functional immuno-regulatory cells persisting for at least six months post-engraftment. Combined treatment with interleukin-15 (IL-15) stimulation and immune checkpoint inhibition (ICI) resulted in complete or partial tumor response in this model. Further, depletion of Cytotoxic T-lymphocytes (CTLs) and/or Natural Killer (NK) cells before combined immunotherapy revealed that both cell types were required for maximal tumor regression. Our novel TIL-PDX model provides a valuable resource for powerful mechanistic and therapeutic studies in solid tumors.
Duy T. Le, Tridu R. Huynh, Bryan M. Burt, George Van Buren, Shawn A. Abeynaike, Cristina Zalfa, Rana Nikzad, Farrah Kheradmand, John J. Tyner, Silke Paust
Recent advances in high-throughput T cell receptor (TCR) sequencing have allowed for new insights into the human TCR repertoire. However, methods for capturing antigen-specific repertoires remain an area of development. Here, we describe a potentially novel approach that utilizes both a biological and statistical enrichment to define putatively antigen-specific complementarity-determining region 3 (CDR3) repertoires in unselected individuals. The biological enrichment entails fluorescence-activated cell sorting of in vitro antigen-activated memory CD4+ T cells, followed by TCRβ sequencing. The resulting TCRβ sequences are then filtered by selecting those that are statistically enriched when compared to their frequency in the autologous resting T cell compartment. Applying this method to define putatively peanut protein-specific repertoires in 27 peanut-allergic individuals resulted in a library of 7345 unique CDR3β amino acid sequences that had similar characteristics to other validated antigen-specific repertoires in terms of homology and diversity. In-depth analysis of these CDR3βs revealed 36 public sequences that demonstrated high levels of convergent recombination. In a network analysis, the public CDR3βs unveiled themselves as core sequences with more edges than their private counterparts. This method has the potential to be applied to a wide range of T cell-mediated disorders, and to yield new biomarkers and biological insights.
Neal P. Smith, Bert Ruiter, Yamini V. Virkud, Ang A. Tu, Brinda Monian, James J. Moon, J. Christopher Love, Wayne G. Shreffler
There is an emerging need for accurate and rapid identification of bacteria in the human body to achieve diverse biomedical objectives. Copper homeostasis is vital for the survival of bacterial species owing to the roles of the metal as a nutrient, respiratory enzyme cofactor, and a toxin. Here, we report the development of a copper-64–labeled bacterial metal chelator, yersiniabactin, to exploit a highly conserved metal acquisition pathway for noninvasive and selective imaging of bacteria. Compared with traditional techniques used to manufacture probes, our strategy simplifies the process considerably by combining the function of metal attachment and cell recognition to the same molecule. We demonstrate, for the first time to our knowledge, how a copper-64 PET probe can be used to identify specific bacterial populations, monitor antibiotic treatment outcomes, and track bacteria in diverse niches in vivo.
Nabil A. Siddiqui, Hailey A. Houson, Nitin S. Kamble, Jose R. Blanco, Robert E. O’Donnell, Daniel J. Hassett, Suzanne E. Lapi, Nalinikanth Kotagiri
Single cell sequencing studies have characterized the transcriptomic signature of cell types within the kidney. However, the spatial distribution of acute kidney injury (AKI) is regional and affects cells heterogeneously. We first optimized coordination of spatial transcriptomics and single nuclear sequencing datasets, mapping 30 dominant cell types to a human nephrectomy. The predicted cell type spots corresponded with the underlying histopathology. To study the implications of AKI on transcript expression, we then characterized the spatial transcriptomic signature of two murine AKI models: ischemia reperfusion injury (IRI) and cecal ligation puncture (CLP). Localized regions of reduced overall expression were associated with injury pathways. Using single cell sequencing, we deconvoluted the signature of each spatial transcriptomic spot, identifying patterns of colocalization between immune and epithelial cells. Neutrophils infiltrated the renal medulla in the ischemia model. Atf3 was identified as a chemotactic factor in S3 proximal tubules. In the CLP model, infiltrating macrophages dominated the outer cortical signature and Mdk was identified as a corresponding chemotactic factor. The regional distribution of these immune cells was validated with multiplexed CO-Detection by inDEXing (CODEX) immunofluorescence. Spatial transcriptomic sequencing complements single cell sequencing by uncovering mechanisms driving immune cell infiltration and detection of relevant cell subpopulations.
Ricardo Melo Ferreira, Angela R. Sabo, Seth Winfree, Kimberly S. Collins, Danielle Janosevic, Connor J. Gulbronson, Ying-Hua Cheng, Lauren Casbon, Daria Barwinska, Michael J. Ferkowicz, Xiaoling Xuei, Chi Zhang, Kenneth W. Dunn, Katherine J. Kelly, Timothy A. Sutton, Takashi Hato, Pierre C. Dagher, Tarek M. El-Achkar, Michael T. Eadon
Gut microbe–derived metabolites influence human physiology and disease. However, establishing mechanistic links between gut microbial metabolites and disease pathogenesis in animal models remains challenging. The major route of absorption for microbe-derived small molecules is venous drainage via the portal vein to the liver. In the event of presystemic hepatic metabolism, the route of metabolite administration becomes critical. To our knowledge, we describe here a novel portal vein cannulation technique using a s.c. implanted osmotic pump to achieve continuous portal vein infusion in mice. We first administered the microbial metabolite trimethylamine (TMA) over 4 weeks, during which increased peripheral plasma levels of TMA and its host liver-derived cometabolite, trimethylamine-N-oxide, were observed when compared with a vehicle control. Next, 4-hydroxyphenylacetic acid (4-HPAA), a microbial metabolite that undergoes extensive presystemic hepatic metabolism, was administered intraportally to examine effects on hepatic gene expression. As expected, hepatic levels of 4-HPAA were elevated when compared with the control group while peripheral plasma 4-HPAA levels remained the same. Moreover, significant changes in the hepatic transcriptome were revealed by an unbiased RNA-Seq approach. Collectively, to our knowledge this work describes a novel method for administering gut microbe–derived metabolites via the portal vein, mimicking their physiologic delivery in vivo.
Danny Orabi, Lucas J. Osborn, Kevin Fung, William Massey, Anthony J. Horak III, Federico Aucejo, Ibrahim Choucair, Beckey DeLucia, Zeneng Wang, Jan Claesen, J. Mark Brown
Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have been used extensively to model inherited heart diseases, but hiPSC-CM models of ischemic heart disease are lacking. Here our objective was to generate an hiPSC-CM model of ischemic heart disease. To this end, hiPSCs were differentiated to functional hiPSC-CMs and then purified using either a simulated ischemia media or by using magnetic antibody-based purification targeting the non-myocyte population for depletion from the cell population. Flow cytometry analysis confirmed that each purification approach generated hiPSC-CM cultures of >94% cTnT+ cells. Following purification hiPSC-CMs were re-plated as confluent syncytial monolayers for electrophysiological phenotype analysis and protein expression by Western blotting. Metabolic selected hiPSC-CM monolayers’ phenotype recapitulated many of the functional and structural hallmarks of ischemic cardiomyocytes, including: elevated diastolic calcium, diminished calcium transient amplitude, prolonged action potential duration, depolarized resting membrane potential, hypersensitivity to chemotherapy induced cardiotoxicity, depolarized mitochondrial membrane potential, depressed SERCA2a expression, reduced maximal oxygen consumption rate and abnormal response to β1-adrenergic receptor stimulation. These findings indicate that metabolic selection of hiPSC-CMs generates cell populations with phenotype like what is well known to occur in the setting of ischemic heart failure, and thus provides a novel opportunity for study of human ischemic heart disease.
Justin Davis, Ahmad Chouman, Jeffery Creech, Andre Monteiro da Rocha, Daniela Ponce-Balbuena, Eric N. Jimenez Vazquez, Ruthann Nichols, Andrey Lozhkin, Nageswara R. Madamanchi, Katherine F. Campbell, Todd J. Herron
The recently proposed glymphatic pathway for solute transport and waste clearance from the brain has been the focus of intense debate. By exploiting an isotopically enriched MRI tracer, H217O, we directly imaged glymphatic water transport in the rat brain in vivo for the first time. Our results reveal glymphatic transport that is dramatically faster and more extensive than previously thought and unlikely to be explained by diffusion alone. Moreover, we confirm the critical role of aquaporin-4 channels in glymphatic transport.
Mohammed S. Alshuhri, Lindsay Gallagher, Lorraine M. Work, William M. Holmes
Endothelial cells are important in the maintenance of healthy blood vessels and in the development of vascular diseases. However, the origin and dynamics of endothelial precursors and remodeling at the single-cell level have been difficult to study in vivo due to technical limitations. We aimed to develop a direct visual approach to track the fate and function of single endothelial cells over several days-weeks in the same vascular bed in vivo using multiphoton microscopy (MPM) of transgenic Cdh5-Confetti mice and the kidney glomerulus as a model. Individual cells of the vascular endothelial lineage were identified and tracked due to their unique color combination, based on the random expression of cyan/green/yellow/red fluorescent proteins. Experimental hypertension, hyperglycemia, and laser-induced endothelial cell ablation rapidly increased the number of new glomerular endothelial cells that appeared in clusters of the same color, suggesting clonal cell remodeling by local precursors at the vascular pole. Furthermore, intravital MPM allowed the detection of distinct structural and functional alterations of proliferating endothelial cells. No circulating Cdh5-Confetti+ cells were found in the renal cortex. The heart, lung, and kidneys showed more significant clonal endothelial cell expansion compared to the brain, pancreas, liver and spleen. Serial MPM of Cdh5-Confetti mice in vivo is a powerful new technical advance to study endothelial remodeling and repair in the kidney and other organs under physiological and disease conditions.
Dorinne Desposito, Ina Maria Schiessl, Georgina Gyarmati, Anne Riquier-Brison, Audrey Izuhara, Hiroyuki Kadoya, Balint Der, Urvi Nikhil Shroff, Young-Kwon Hong, Janos Peti-Peterdi
Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy–based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.
Tony Chour, Lei Tian, Edward Lau, Dilip Thomas, Ilanit Itzhaki, Olfat Malak, Joe Z. Zhang, Xulei Qin, Mirwais Wardak, Yonggang Liu, Mark Chandy, Katelyn E. Black, Maggie P.Y. Lam, Evgenios Neofytou, Joseph C. Wu
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