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Abstract
It is necessary for naïve CD8 T cells to be actively maintained in a quiescent metabolic state in order to respond robustly to infection while avoiding inappropriate activation during homeostasis. With age this quiescent state is lost and the CD8 T cell response to infection decreases. The factors regulating metabolic quiescence of CD8 T cells and how this regulation is lost during aging are not completely understood. Herein, we identify the transcription factor AFF3 as a regulator of metabolic quiescence in naïve CD8 T cells. While naïve AFF3 deficient CD8 T cells are more metabolically active prior to infection, they have reduced accumulation in response to viral infection, and this is correlated with a poor capacity to engage glycolysis. During aging in both murine and human CD8 T cells, AFF3 expression is decreased. In mice, this is associated with a loss of metabolic quiescence and reduced capacity to accumulate following infection. Our data highlight the role of metabolic regulation in CD8 T cell quiescence and identifies a transcription factor that may be a target to reinvigorate CD8 T cell responses during aging.
Authors
Molly E. Lumnitzer, Stefanie F. Valbon, Stephanie A. Condotta, Allison E. Norlander, Sheng Liu, Jun Wan, Martin J. Richer
Abstract
Spinal cord injury (SCI) leads to severe neurological and functional impairments, yet reliable biomarkers for assessing injury severity and predicting recovery remain limited. Cerebrospinal fluid (CSF) is in direct contact with the central nervous system and provides a valuable source for detecting molecular changes after SCI. Although exosomal microRNAs and proteins are increasingly recognized as mediators of intercellular communication, the role of human CSF exosomes in SCI has not been systematically investigated. To identify exosome-based biomarkers and potential therapeutic targets, we analyzed CSF and serum exosomes from patients with acute SCI using RNA sequencing and proteomic profiling. Weighted Gene Co-expression Network Analysis (WGCNA) identified six gene modules significantly associated with injury severity and neurological recovery at three months. Proteomic analysis revealed a five-protein panel that distinguished complete from incomplete SCI and a four-protein panel that predicted neurological improvement. Additionally, fifteen CSF-specific and nine serum-specific exosomal miRNAs were identified independent of injury severity. Among ten tested miRNAs associated with neurological recovery, seven regulated astrocyte proliferation, and six promoted neurite extension and synapse formation. Overall, this study provides a comprehensive characterization of CSF exosomal miRNAs and proteins in human SCI and identifies molecular signatures associated with injury severity and recovery.
Authors
Dallas L. Sheinberg, Haichao Wei, Joseph S. Withrow, Farshad Homayouni Moghadam, Chia-Chen Lu, Jyotirmoy Rakshit, Jennifer Zaragoza, John R. Williams, Wen Li, Jacques J. Morcos, Jia Qian Wu
Abstract
Polymorphonuclear neutrophils (PMNs) serve as frontline defenders against injury and infection, eliminating pathogens and initiating mucosal tissue repair. However, excessive PMN transepithelial migration (TEpM) contributes to chronic mucosal inflammatory disorders, including inflammatory bowel disease. PMN pro-inflammatory and pro-repair functions are regulated by incompletely defined signaling cascades involving kinases and phosphatases. Here, we determined how the protein tyrosine phosphatase CD45/PTPRC regulates PMN trafficking and effector functions in the gut. Pharmacologic inhibition of CD45 significantly reduced PMN colonic TEpM in vitro and in vivo and decreased intestinal PMN trafficking was observed in transgenic mice with PMN-specific deletion of CD45 (MRP8-Cre;Cd45fl/fl). Beyond limiting TEpM, CD45 depletion impaired key antimicrobial functions, including degranulation and phagocytosis, indicating broader effects on PMN effector activity. Importantly, recovery from dextran sodium sulfate (DSS)–induced colitis and biopsy-induced colonic wounding was delayed in MRP8-Cre;Cd45fl/fl mice, linking altered PMN function to defective mucosal healing. Mechanistically, CD45 depletion reduced surface expression of the β2 integrin CD11b/CD18 and inactivated the Src family kinase member Lyn. Together, data highlight an important CD45–CD11b–Lyn signaling axis that regulates PMN trafficking and effector functions in the intestine and identify CD45 as a promising target for modulating PMN function to promote mucosal tissue repair.
Authors
Jael Miranda, Dylan J. Fink, Zachary S. Wilson, Roland Hilgarth, Asma Nusrat, Charles A. Parkos, Jennifer C. Brazil
Abstract
Authors
Aneri Shethji, Theresa Hutchins, Anwesha Sanyal, Tianhao Liu, Wei Chen, Kathryn S. Torok
Abstract
Postnatal growth faltering is a pervasive problem among extremely preterm infants that is independently associated with adverse neurodevelopmental outcomes. We previously observed that preterm infants with poor postnatal growth have altered development of the intestinal microbiota relative to preterm infants with appropriate postnatal growth. Here, we used gnotobiotic mice to investigate whether these differences in microbiota development independently contribute to growth faltering. We found that colonization of neonatal mice with microbiotas from extremely preterm infants with poor growth reproduced postnatal growth impairment and induced a metabolic signature of enhanced lipolysis and fatty acid oxidation in the mice, characterized by elevated hepatic acylcarnitines and circulating ketones. In mice colonized at birth with microbiotas from infants with poor growth, postnatal treatment with microbiotas from infants with appropriate growth prevented growth impairment. These results indicate that altered development of the intestinal microbiota contributes to growth faltering in extremely preterm infants, and that microbiota modification can restore postnatal growth.
Authors
Kwai Tei Chan Poon, Se Hyang Han, Olga Ilkayeva, Michael J. Muehlbauer, Christopher B. Newgard, Charles M. Cotten, Patricia L. Ashley, Patrick C. Seed, John F. Rawls, Noelle E. Younge
Abstract
BACKGROUND. In vitro fertilization (IVF) culminates in embryo transfer into a hormonally primed endometrium, often via a programmed cycle (PC) regimen postulated to influence hypertensive disorders of pregnancy (HDP) risk. We thus generated a single-cell atlas of PC endometrium to define cell type-specific differences relative to natural cycle (NC) endometrium, and evaluated whether PC-associated modulation of the window of implantation (WOI) endometrium influences angiogenic balance in pregnancy. METHODS. Single-nucleus RNA-seq of prospectively collected PC and NC WOI endometrium. An independent prospective cohort of 548 singleton pregnancies was separately analyzed for maternal serum angiogenic markers (soluble fms-like tyrosine kinase-1; placental growth factor) and HDP incidence in PC- versus NC-conceived pregnancies, adjusting for clinical confounders and IVF use. RESULTS. Prominent transcriptomic differences were observed between PC (n = 7; 48,843 nuclei) and NC (n = 9; 44,230 nuclei) WOI endometrium, particularly in glandular epithelium (682 up- and 979 down-regulated genes; adjusted P < 0.05) and stromal fibroblasts (108 up- and 168 down-regulated). PC endometrium showed reduced uterine natural killer cell abundance, potentially from CXCL14 downregulation. Functional enrichment revealed downregulation of embryo implantation, angiogenesis, and extracellular matrix remodeling pathways in PC. Altered cell-cell signaling in decidualization, angiogenesis, and inflammatory response was also observed. Despite these WOI perturbations, PC-conceived pregnancies were not associated with early gestational angiogenic imbalance or increased HDP risk. CONCLUSION. PC endometrial preparation induced distinct cellular and signaling alterations in the WOI, but was not associated with subsequent development of angiogenic imbalance or HDP, thereby underscoring the resilience and adaptability of the early maternal-fetal interface. TRIAL REGISTRATION. ClinicalTrials.gov NCT03799107. FUNDING. ABOG/AAOGF; NICHD-R01-HD084380; NCTRI-P50-HD055764; NIAMS-P30-AR070155.
Authors
David Huang, Emily Flynn, Brittany R. Davidson, Juan C. Irwin, Mohammad Naser, Ana Laura Almonte, Jennifer Qin, Yue Song, Fleurdeliza B. Rabara, Rebecca Wong, Lydia B. Zablotska, Mitchell P. Rosen, Torsten Wittmann, Gabriela K. Fragiadakis, Alexis J. Combes, Marina Sirota, Marcelle I. Cedars, Linda C. Giudice
Abstract
Significant loss of pigmentation can increase visual disability, skin cancer risk, and psychosocial stress. Tyrosinase (TYR) catalyzes the first and rate-limiting step of melanin synthesis. Inhibitors of TYR are well established and are currently used in clinical settings; however, there is a dearth of direct activators of TYR. Here, using a human TYR construct, we developed high-throughput screening methods, in cell confirmatory assays employing 13C-tyrosine tracing, and computational analysis techniques, and identified ampyrone (4-aminoantipyrine) as a TYR activator. Ampyrone increased the in vitro catalytic activity of the human recombinant intra-melanosomal domain of TYR (hTYR) and its hypomorphic variant, Pro406Leu (P406L), a cause of oculocutaneous albinism type 1B (OCA1B). Moreover, ampyrone induced melanin synthesis in both wild-type and OCA1B human melanocytes, mouse OCA2 melanocytes, as well as 3-dimensional (3D) human skin cultures. Computational studies provided additional insight into the effects of direct TYR agonists on enzyme activity. Our results identified ampyrone as a lead candidate for TYR activation, potentially supporting the development of therapies for patients with genetic and acquired diseases of hypopigmentation.
Authors
Monika B. Dolinska, Yuhong A. Wang, Nathan P. Coussens, Vijay K. Kalaskar, Zuhal Eraslan, Samuel J. Grondin, Joseph Bonica, Sarah Toay, Matthew D. Hall, Min Shen, Matthew Boxer, Qiuying Chen, Steven S. Gross, Nabeel Attarwala, Yingyos Jittayasothorn, Ramakrisha P. Alur, Dhyanam Shukla, Robin Kee, Charles DeYoung, Cuilee Sha, David R. Adams, Stacie K. Loftus, Tiziana Cogliati, Yuri V. Sergeev, Jonathan H. Zippin, Brian P. Brooks
Abstract
Several genes guide inner ear development, and mutations in these genes can cause malformations that result in congenital hearing loss. However, the contribution of noncoding regulatory elements remains largely unclear. This study investigates the function of distal enhancer elements in the transcriptional regulation of GDF6, a gene implicated in cochlear development. Using mouse models with targeted deletions, human inner ear organoids, and CRISPR interference (CRISPRi), we identified a downstream regulatory interval harboring a developmental enhancer required to maintain GDF6 expression during otic epithelial maturation and cochlear morphogenesis. Deletion of this regulatory region or targeting of CRISPRi-based repressors to these regions resulted in decreased GDF6 expression, failure of otic-epithelium development, and prevention of hair cell-like differentiation, reflecting cochlear aplasia observed in patients with corresponding genomic deletions. These findings highlight the contribution of long-range regulatory elements to auditory development and illustrate how their disruption contributes to human deafness.
Authors
Mohammad Faraz Zafeer, Clemer Abad, Havva Ortabozkoyun, Memoona Ramzan, Guney Bademci, Maria C. Robayo, Duygu Duman, Rolen M. Quadros, Shengru Guo, Juan I. Young, Anthony J. Griswold, Channabasavaiah B. Gurumurthy, Derek M. Dykxhoorn, Katherina Walz, Mustafa Tekin
Abstract
Pulmonary arterial hypertension (PAH) is a progressive vascular syndrome characterized by aberrant signaling, severe pulmonary artery remodeling, and right ventricular (RV) failure, a major driver of morbidity and mortality. Dysregulation of the apelinergic pathway has been implicated in pulmonary vascular remodeling in PAH. Using a sugen-hypoxia rat model of PAH, we assessed the ability of a novel apelin analog, resistant to native peptidase degradation, to reverse the pathological hallmarks of PAH and RV dysfunction. Apelin analog therapy corrected the vascular lesions in the lungs and nearly normalized pulmonary arterial pressures. Early cardiorenal syndrome, RV dilation and dysfunction as well as RV cardiomyocyte and fibroblast activation induced by pressure overload, were also reversed by apelin analog treatment. Single-nucleus RNA sequencing of the lungs and RV revealed apelin-analog treatment activated several protective pathways, including rebalancing protective BMPR2 (bone morphogenetic protein receptor type 2) signaling to counteract excessive pathogenic TGFBR2 (transforming growth factor β receptor 2) activity in PAH. These findings highlight the therapeutic potential of exogenous apelin in reversing pulmonary vascular and cardiac pathologies in PAH and support further investigation to evaluate the clinical benefits of apelin analog treatment in patients with PAH and RV failure.
Authors
Jennie Vu, Pavel Zhabyeyev, Kemar J. Brown, Joshua M. Gorham, Daniel M. DeLaughter, Huachen Chen, Thilina U. Jayawardena, Ander Vergara, Maria Alexiou, Anjalee Wijewardane, Conrad Fischer, Charlotte Avet, Abby Ewasiuk, Faqi Wang, Mark C. Chappell, Yuri Kim, Michel Bouvier, John C. Vederas, Christine E. Seidman, Jonathan G. Seidman, Gavin Y. Oudit
Abstract
Circulating fatty acids (FA) are constitutively taken up by basolateral kidney proximal tubule transporters and are the preferred metabolic substrate. In many chronic kidney diseases, the damaged glomerular filtration barrier permits passage of albumin-bound FA, which are reabsorbed by apical FA transport protein-2 (FATP2). Bilateral FA uptake leads to lipotoxicity and progressive renal function decline, but the relative apical versus basolateral contribution and intracellular mechanisms are not established. Apical or bilateral (but not basolateral) palmitate incubation with human proximal tubule cells stimulated endoplasmic reticulum (ER) stress gene expression, ER stress pathway activation, and ER fragmentation. Apical or bilateral palmitate was associated with reduced lipid droplets, and decreased expression of ER-localized lipid droplet biogenesis transcripts. Inhibition of lipid droplet formation also precipitated ER stress, suggesting diminished sequestration of FA metabolites as the cause. Indeed, C16:0 ceramide was increased in bilateral palmitate-treated cells, and in kidneys from mice that phenocopy progressive diabetic kidney disease. Ceramide synthesis inhibition abrogated ER stress, and transfection with C16:0 ceramide decreased ER membrane fluidity and caused ER stress. We conclude that aberrant filtration and uptake of FA by apical FATP2 exceeded the capacity for lipid droplet incorporation, and led to cytotoxicity from ceramide-induced ER lipid bilayer stress.
Authors
Zhiyu Liu, Robert J. Gaivin, Shenaz Khan, Vincent Li, Amal Chaba, Fraser J. Moss, Usman Sabir, Takhar Kasumov, Tingwei Mu, Jeffrey R. Schelling
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