Abstract
We developed a 29-color spectral cytometry panel to enhance nonhuman primate (NHP) models for cross-reactive immunophenotyping. This panel is suitable for biosafety level 4 (BSL-4) viruses and can be used with both human and NHP samples in BSL-2 research settings. Tissues from humans, rhesus monkeys (RhMs), crab-eating macaques (CEMs), and green monkeys (GMs) were stained with a 29-color immunophenotyping panel requiring only two clone substitutions. Comparable staining was observed for all samples. Unbiased analysis showed acceptable overlap in T-cell phenotypes across samples, with differences in human and NHP B cells and granulocytes. In CEMs, most circulating CD8+ T cells were from effector memory cells, with significantly higher levels than in humans (p<0.0001), RhMs (p<0.05), and GMs (p<0.01). Analysis of samples from various anatomical sites revealed distinct location-specific phenotypes. In Nipah-virus-exposed animals, splenocytes showed a substantial increase in IgM+ B cells (p<0.0001) and a reduction in effector memory CD8+ T cells (p<0.0001) compared to unexposed controls. Lymph nodes from Ebola-virus-exposed animals showed a loss of CXCR3+CD8+ T cells vs unexposed controls. This panel may guide the development of additional multi-color panels in preclinical and clinical settings and potentially increase understanding of the pathogenesis of diseases caused by emerging and re-emerging viruses.
Authors
Andrew P. Platt, Bobbi Barr, Anthony Marketon, Rebecca Bernbaum, Deja F.P. Rivera, Vincent J. Munster, Daniel S. Chertow, Michael R. Holbrook, Scott M. Anthony, Bapi Pahar
Citation Information: JCI Insight. 2025. https://doi.org/10.1172/jci.insight.178689.
Abstract
Despite the accumulation of cisplatin in proximal tubules, direct visualization of the surrounding peritubular microcirculation, including its alteration in cisplatin-induced acute kidney injury (AKI), is lacking. Here, using fluorescence and cellular angiography through video-rate high-resolution intravital microscopy, progressive disturbance of peritubular microcirculation in cisplatin-induced AKI in mice was demonstrated. Fluorescence angiography revealed increasing perfusion defects, with a stepwise rise in time to peak (TTP), originating from capillaries surrounding S1 segments. Cellular angiography demonstrated a progressive decrease in the velocity and track length of individual erythrocytes during AKI progression, accompanied by a sequential decrease in the functional capillary ratio (FCR). Alterations in the perfusion area, TTP, and FCR preceded significant changes in blood urea nitrogen and cystatin C, suggesting the potential for early diagnosis. Although neutrophil infiltration near proximal tubules increased throughout the progression, it did not cause obstruction of the peritubular microcirculation. Depletion of neutrophils increased mortality due to systemic side effects, whereas functional inactivation of neutrophils using an anti-CD11b antibody improved peritubular microcirculation in cisplatin-induced AKI. This approach enables direct visualization and quantification of peritubular microcirculation and immune cell dynamics, providing insights into renal pathophysiology and potential therapeutic strategies.
Authors
Inwon Park, Seonghye Kim, Young Woo Um, Hee Eun Kim, Jae Hyuk Lee, Sejoong Kim, PILHAN KIM, You Hwan Jo
Citation Information: JCI Insight. 2025. https://doi.org/10.1172/jci.insight.183706.
Abstract
Adult stem cells decline in number and function in old age and identifying factors that can delay or revert age-associated adult stem cell dysfunction are vital for maintaining healthy lifespan. Here we show that Vitamin A, a micronutrient that is derived from diet and metabolized into retinoic acid, acts as an antioxidant and transcriptional regulator in muscle stem cells. We first show that obstruction of dietary Vitamin A in young animals drives mitochondrial and cell cycle dysfunction in muscle stem cells that mimics old age. Next, we pharmacologically targeted retinoic acid signaling in myoblasts and aged muscle stem cells ex vivo and in vivo and observed reductions in oxidative damage, enhanced mitochondrial function, and improved maintenance of quiescence through fatty acid oxidation. We next detected the receptor for vitamin A derived retinol, stimulated by retinoic acid 6 or Stra6, was diminished with muscle stem cell activation and in old age. To understand the relevance of Stra6 loss, we knocked down Stra6 and observed an accumulation of mitochondrial reactive oxygen species, as well as changes in mitochondrial morphology and respiration. These results demonstrate that Vitamin A regulates mitochondria and metabolism in muscle stem cells and highlight a unique mechanism connecting stem cell function with vitamin intake.
Authors
Paula M. Fraczek, Pamela Duran, Benjamin A. Yang, Valeria Ferre, Leanne Alawieh, Jesus A. Castor-Macias, Vivian T. Wong, Steve D. Guzman, Celeste Piotto, Klimentini Itsani, Jacqueline A Larouche, Carlos A. Aguilar
Citation Information: JCI Insight. 2025. https://doi.org/10.1172/jci.insight.179875.
Abstract
Skin inflammation in juvenile dermatomyositis (JDM) can signal disease onset or flare, and the persistence of cutaneous disease can prevent complete disease remission. The non-invasive study of cutaneous expression signatures through tape stripping (TS) holds the potential to reveal mechanisms underlying disease heterogeneity and organ-specific inflammation. The objectives of this study were to 1) define TS expression signatures in lesional and non-lesional JDM skin, 2) analyze TS signatures to identify JDM disease endotypes and 3) compare TS and blood signatures. While JDM lesional skin demonstrated interferon signaling as the top upregulated pathway, non-lesional skin uniquely highlighted pathways involved in metabolism, angiogenesis and calcium signaling. Both lesional and non-lesional skin shared inflammasome pathway dysregulation. Using unsupervised clustering of skin expression data, we identified a treatment-refractory JDM subgroup distinguished by upregulation of genes associated with mitochondrial dysfunction. The treatment-refractory JDM subgroup also demonstrated higher interferon, angiogenesis and innate immune expression scores in skin and blood, although scores were more pronounced in skin as compared to blood. Tape-stripping expression signatures in JDM provided insight into disease mechanisms and molecular subgroups. Skin, as compared to blood, transcriptional profiles served as more sensitive markers to classify disease subgroups and identify candidate treatment targets.
Authors
Jessica L. Turnier, Sarah M.H. Vandenbergen, Madison E. McClune, Christine Goudsmit, Sophia Matossian, Meredith Riebschleger, Nadine Saad, Jacqueline A. Madison, Smriti Mohan, Johann E. Gudjonsson, Lam C. Tsoi, Celine C. Berthier, J. Michelle Kahlenberg
Citation Information: JCI Insight. 2025. https://doi.org/10.1172/jci.insight.185159.
Abstract
Prader-Willi syndrome (PWS) is a multigenic disorder caused by the loss of seven contiguous paternally expressed genes. Mouse models with inactivation of all PWS genes are lethal. Knockout (KO) mouse models for each candidate gene have been generated, but they lack the functional interactions between PWS genes. Here, we revealed an interplay between Necdin and Magel2 “PWS” genes and generated a mouse model (named “Del Ndn-Magel2” mice) with a deletion including both genes. A subset of Del Ndn-Magel2 mice showed neonatal lethality. Behaviorally, surviving mutant mice exhibited sensory delays during infancy and alterations in social exploration at adulthood. Del Ndn-Magel2 mice had a lower body weight before weaning, persisting after weaning in males only, with reduced fat mass and improved glucose tolerance, and altered puberty. Adult mutant mice displayed increased ventilation and a persistent increase in apneas following a hypercapnic challenge. Transcriptomics analyses revealed a dysregulation of key circadian genes and alterations of genes associated with axonal function similar to PWS patients. At neuroanatomical levels, Del Ndn-Magel2 mice had an impaired maturation of oxytocin neurons and a disrupted development of melanocortin circuits. Together, these data indicate that the Del Ndn-Magel2 mouse is a pertinent and genetically relevant model of PWS
Authors
Pierre-Yves Barelle, Alicia Sicardi, Fabienne Schaller, Julie Buron, Denis Becquet, Felix Omnes, Françoise Watrin, Marie-Sophie Alifrangis, Catarina Santos, Clément Menuet, Anne-Marie François-Bellan, Emilie Caron, Jessica Klucznik, Vincent Prevot, Sebastien G. Bouret, Françoise Muscatelli
Citation Information: JCI Insight. 2025. https://doi.org/10.1172/jci.insight.190105.
Abstract
The use of genetically engineered tools, including combinations of Cre-LoxP and Flp-FRT systems, enable the interrogation of complex biology. Steroidogenic factor-1 (SF-1) is expressed in the ventromedial hypothalamic nucleus (VMH). Development of genetic tools, such as mice expressing Flp recombinase (Flp) in SF-1 neurons (Sf-1-Flp), will be useful for future studies that unravel the complex physiology regulated by the VMH. Here, we developed and characterized Sf-1-Flp mice and demonstrated its utility. Flp sequence was inserted into Sf-1 locus with P2A. This insertion did not affect Sf-1 mRNA expression levels and Sf-1-Flp mice do not have any visible phenotypes. They are fertile and metabolically comparable to wild-type littermate mice. Optogenetic stimulation using adeno-associated virus (AAV)-bearing Flp-dependent channelrhodopsin-2 (ChR2) increased blood glucose and skeletal muscle PGC-1α in Sf-1-Flp mice. This was similar to SF-1 neuronal activation using Sf-1-BAC-Cre and AAV-bearing Cre-dependent ChR2. Finally, we generated Sf-1-Flp mice that lack β2-adrenergic receptors (Adrβ2) only in skeletal muscle with a combination of Cre/LoxP technology (Sf-1-Flp::SKM∆Adrβ2). Optogenetic stimulation of SF-1 neurons failed to increase skeletal muscle PGC-1α in Sf-1-Flp::SKM∆Adrβ2 mice, suggesting that Adrβ2 in skeletal muscle is required for augmented skeletal muscle PGC-1α by SF-1 neuronal activation. Our data demonstrate that Sf-1-Flp mice are useful for interrogating complex physiology.
Authors
Marco Galvan, Mina Fujitani, Samuel R. Heaselgrave, Shreya Thomas, Bandy Chen, Jenny J. Lee, Steven C. Wyler, Joel K. Elmquist, Teppei Fujikawa
Citation Information: JCI Insight. 2025. https://doi.org/10.1172/jci.insight.187025.
Abstract
The functional plasticity of tumor-infiltrating B (TIL-B) cells spans from anti-tumor responses to non-canonical immune suppression. Yet, how tumor microenvironment (TME) influences TIL-B development is still underappreciated. Our current study integrated single cell transcriptomics and BCR (B cell receptor) sequencing to profile TIL-B phenotypes and clonalities in hepatocellular carcinoma (HCC). Using trajectory and gene regulatory network analysis, we were able to characterize plasma cells, memory and naïve B cells within the HCC TME and further revealed a downregulation of BCR-signaling genes in plasma cells and a subset of inflammatory TNF+ memory B cells. Within the TME, non-switch memory B cell subset acquires an age-associated B cell phenotype (TBET+, CD11c+) and expressed higher levels of PD-L1, CD25 and granzyme B. We further demonstrated that the presence of HCC tumor cells could confer suppressive functions on peripheral blood B cells which in turn, dampen T cell co-stimulation. To the best of our knowledge, these findings represent novel mechanisms of non-canonical immune suppression in HCC. While previous studies identified atypical memory B cells in chronic hepatitis and across several solid cancer types, we further highlighted their potential role as regulatory B cells (Bregs) within both the TME and peripheral blood of HCC patients.
Authors
Shi Yong Neo, Timothy Wai Ho Shuen, Shruti Khare, Joni Chong, Maichan Lau, Niranjan Shirgaonkar, Levene Chua, Junzhe Zhao, Keene Lee, Charmaine Tan, Rebecca Ba, Janice Lim, Joelle Chua, Hui Shi Cheong, Hui Min Chai, Chung Yip Chan, Alexander Yaw Fui Chung, Peng Chung Cheow, Prema Raj Jeyaraj, Jin Yao Teo, Ye Xin Koh, Aik Yong Chok, Pierce Kah Hoe Chow, Brian Goh, Wei Keat Wan, Wei Qiang Leow, Tracy Jie Zhen Loh, Po Yin Tang, Jayanthi Karunanithi, Nye Thane Ngo, Tony Kiat Hon Lim, Shengli Xu, Ramanuj Dasgupta, Han Chong Toh, Kong-Peng Lam
Abstract
Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) dysfunction, leading to neurodegeneration, is the pathological hallmark of primary open-angle glaucoma (POAG). Impaired axonal transport is an early and critical feature of glaucomatous neurodegeneration. However, a robust mouse model that accurately replicates these human POAG features has been lacking. We report the development and characterization of a novel Cre-inducible mouse model expressing a DsRed-tagged Y437H mutant of human myocilin (Tg.CreMYOCY437H). A single intravitreal injection of HAd5-Cre induced selective MYOC expression in the TM, causing TM dysfunction, reducing the outflow facility, and progressively elevating IOP in Tg.CreMYOCY437H mice. Sustained IOP elevation resulted in significant loss of retinal ganglion cells (RGCs) and progressive axonal degeneration in Cre-induced Tg.CreMYOCY437H mice. Notably, impaired anterograde axonal transport was observed at the optic nerve head before RGC degeneration, independent of age, indicating that impaired axonal transport contributes to RGC degeneration in Tg.CreMYOCY437H mice. In contrast, axonal transport remained intact in ocular hypertensive mice injected with microbeads, despite significant RGC loss. Our findings indicate that Cre-inducible Tg.CreMYOCY437H mice replicate all glaucoma phenotypes, providing an ideal model for studying early events of TM dysfunction and neuronal loss in POAG.
Authors
Balasankara Reddy Kaipa, Ramesh Kasetti, Yogapriya Sundaresan, Linya Li, Sam Yacoub, J. Cameron Millar, William Cho, Dorota Skowronska-Krawczyk, Prabhavathi Maddineni, Krzysztof Palczewski, Gulab S. Zode
Citation Information: JCI Insight. 2025. https://doi.org/10.1172/jci.insight.174264.
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a drug resistant and lethal cancer. Identification of the genes that consistently show altered expression across patients’ cohorts can expose effective therapeutic targets and strategies. To identify such genes, we separately analyzed five human PDAC microarray datasets. We defined genes as ‘consistent’ if upregulated or downregulated in ≥ 4 datasets (adjusted P<0.05). The genes were subsequently queried in additional datasets, including single-cell RNA-sequencing data, and we analyzed their pathway enrichment, tissue-specificity, essentiality for cell viability, association with cancer features e.g., tumor subtype, proliferation, metastasis and poor survival outcome. We identified 2,010 consistently upregulated and 1,928 downregulated genes of which >50%, to our knowledge, were uncharacterized in PDAC. These genes spanned multiple processes, including cell cycle, immunity, transport, metabolism, signaling and transcriptional/epigenetic regulation – cell cycle and glycolysis being the most altered. Several upregulated genes correlated with cancer features, and their suppression impaired PDAC cell viability in prior CRISPR/Cas9 and RNA interference screens. Further, the upregulated genes predicted sensitivity to bromodomain and extraterminal (epigenetic) protein inhibition, which, in combination with gemcitabine, disrupted amino acid metabolism and in vivo tumor growth. Our results highlight genes for further studies in the quest for PDAC mechanisms, therapeutic targets and biomarkers.
Authors
Zeribe C. Nwosu, Heather Giza, Maya Nassif, Verodia Charlestin, Rosa E. Menjivar, Daeho Kim, Samantha B. Kemp, Peter Sajjakulnukit, Anthony Andren, Li Zhang, William K.M. Lai, Ian Loveless, Nina G. Steele, Jiantao Hu, Biao Hu, Shaomeng Wang, Marina Pasca di Magliano, Costas A. Lyssiotis
Citation Information: JCI Insight. 2024. https://doi.org/10.1172/jci.insight.187328.
Abstract
Cerebral endothelial cell (EC) injury and blood-brain barrier (BBB) permeability contribute to neuronal injury in acute neurological disease states. Preclinical experiments have used animal models to study this phenomenon, yet the response of human cerebral ECs to BBB disruption remains unclear. In our Phase 1 clinical trial (NCT04528680), we used low-intensity pulsed ultrasound with microbubbles (LIPU/MB) to induce transient BBB disruption of peri-tumoral brain in patients with recurrent glioblastoma. We found radiographic evidence that BBB integrity was mostly restored within 1-hour of this procedure. Using single-cell RNA sequencing and transmission electron microscopy, we analyzed the acute response of human brain ECs to ultrasound-mediated BBB disruption. Our analysis revealed distinct EC gene expression changes after LIPU/MB, particularly in genes related to neurovascular barrier function and structure, including changes to genes involved in the basement membrane, EC cytoskeleton, and junction complexes, as well as caveolar transcytosis and various solute transporters. Ultrastructural analysis showed that LIPU/MB led to a decrease in luminal caveolae, the emergence of cytoplasmic vacuoles, and the disruption of the basement membrane and tight junctions, among other things. These findings suggested that acute BBB disruption by LIPU/MB led to specific transcriptional and ultrastructural changes and could represent a conserved mechanism of BBB repair after neurovascular injury in humans.
Authors
Andrew Gould, Yu Luan, Ye Hou, Farida V. Korobova, Li Chen, Victor A. Arrieta, Christina Amidei, Rachel Ward, Cristal Gomez, Brandyn Castro, Karl Habashy, Daniel Zhang, Mark Youngblood, Crismita Dmello, John Bebawy, Guillaume Bouchoux, Roger Stupp, Michael Canney, Feng Yue, M. Luisa Iruela-Arispe, Adam M. Sonabend
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