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Abstract
Oxidative stress driven by malfunctioning respiratory complex I (RC-I) is a crucial pathogenic factor in liver ischemia/reperfusion (I/R) injury. This study investigates the role of alkaline ceramidase 3 (ACER3) and its unsaturated long-chain ceramide (CER) substrates in regulating liver I/R injury through RC-I. Our findings demonstrated that I/R upregulated ACER3/Acer3 and decreased unsaturated long-chain CER levels in human and mouse livers. Both global and hepatocyte-specific Acer3 ablation, as well as treatment with CER(d18:1/18:1), led to a significant increase of CER(d18:1/18:1) levels in the liver, which mitigated the I/R-induced hepatocyte damage and inflammation in mice. Mechanistically, Acer3 modulated CER(d18:1/18:1) levels in mitochondria-associated membranes and endoplasmic reticulum (ER), thereby influencing the transport of CER(d18:1/18:1) from ER to mitochondria. Acer3 ablation and CER(d18:1/18:1) treatment elevated CER(d18:1/18:1) in mitochondria, where CER(d18:1/18:1) bound to the RC-I subunit Ndufa6 to inactivate RC-I and reduced reactive oxygen species production in the I/R-injured mouse liver. These findings underscore the role of CER(d18:1/18:1)-Ndufa6 interaction in suppressing RC-I-mediated oxidative-stress-driven pathogenesis in liver I/R Injury.
Authors
Kai Wang, Leyi Liao, Hanbiao Liang, Pengxiang Huang, Qingping Li, Baoxiong Zhuang, Chen Xie, Xiangyue Mo, Xuesong Deng, Jieyuan Li, Yang Lei, Minghui Zeng, Cungui Mao, Ruijuan Xu, Cuiting Liu, Xianqiu Wu, Jie Zhou, Biao Wang, Yiyi Li, Chuanjiang Li
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
Abstract
Biliary atresia (BA) is a pediatric liver disease that often necessitates parenteral nutrition (PN) to support growth due to impaired liver function. While soy oil lipid emulsions (SLE) are commonly used in PN, they may contribute to cholestatic liver injury. In contrast, mixed oil lipid emulsions (MLE) show promise in preventing cholestasis in non-BA infants, potentially by restoring bile flow. However, their effectiveness in cases of complete bile duct obstruction, as seen in BA, remains uncertain. To explore the potential benefits of MLE in BA, we utilized a neonatal pig model of bile duct ligation (BDL). Pigs underwent either BDL or sham surgery and were subsequently fed either MLE or SLE via PN, or enterally with formula. The MLE-BDL pigs exhibited significantly greater weight gain compared to those fed SLE or formula enterally. Additionally, MLE-BDL pigs showed higher serum bile acid and gamma-glutamyl transferase concentrations compared to SLE-BDL pigs. However, no significant differences in liver injury, assessed by ductular reaction or fibrosis, were observed between MLE- and SLE-BDL pigs. Based on weight gain alone, MLE may be a superior lipid emulsion for use in neonates with obstructive cholestasis.
Authors
Greg Guthrie, Caitlin Vonderohe, Valeria Meléndez Hebib, Barbara Stoll, Douglas Burrin
Citation Information: JCI Insight. 2025. https://doi.org/10.1172/jci.insight.191359.
Abstract
The role of gamma-delta T (γδT) cells in immune responses to common allergens is poorly understood. Here, we utilized single-cell (sc) transcriptomic analysis of allergen-reactive γδT cells in humans to characterize the transcriptional landscapes and TCR repertoires in response to cockroach (CR) and mouse (MO) allergens. Using a novel Activation-Induced Marker (AIM) assay that allows detection of γδT cells combined with scRNA sequencing and TCR repertoire analysis, we identified both shared and allergen-specific γδT cell activation patterns and gene expression profiles. While CR extract activated both Vδ1 and Vδ2 subsets, MO extract primarily stimulated Vδ2 cells. Our analysis revealed allergen-specific clusters with distinct functional signatures, including enhanced inflammatory responses and cytotoxic effector functions in MO-specific γδT cells and natural killer cell-mediated immunity and IFNγ signaling in CR-specific populations. Comparison of allergic and non-allergic individuals highlighted differences in gene expression and TCR repertoires, including a higher IFNG expression in the CR-allergic compared to non-allergic cohorts, suggesting that phenotypic and functional differences are associated with γδT allergen responses. This study provides insights into the cellular and molecular heterogeneity and functionality of allergen-reactive γδT cells, offering a foundation for understanding their role in allergic diseases and potential therapeutic interventions
Authors
Kendall Kearns, Sloan A. Lewis, Esther Dawen Yu, Adam Abawi, Eric Wang, Synaida Maiche, Monalisa Mondal, Pandurangan Vijayanand, Grégory Seumois, Bjoern Peters, Alessandro Sette, Ricardo Da Silva Antunes
Abstract
Fibroblasts are central to pathogenesis of systemic sclerosis (SSc). However, studies of conventional explant fibroblast cultures incompletely reflect disease biology and treatment response. We isolated a second non-migratory “resident” population of fibroblasts from skin biopsies after outgrowth of explant “migratory” cells. These non-motile resident fibroblasts were compared with migratory cells from the same biopsy, using functional studies, bulk and scRNAseq, and localised in situ by multichannel immunofluorescence. Migratory and resident fibroblast populations in SSc showed distinct pro-fibrotic characteristics and gene expression for pathogenic pathways differing by stage and autoantibody subgroup. TGFβ signalling was highly active in migratory fibroblasts in early stage dcSSc. Conversely, resident fibroblasts had less upregulated TGFβ signalling, especially in late dcSSc. Increased chemokine expression was a hallmark of resident fibroblasts at all stages. In vitro studies confirmed differential response to TGFβ1 and CCL2 between migratory and resident cells. We suggest that migratory fibroblasts are especially important in early skin disease whereas non-migratory fibroblasts may have a regulatory role and contribute more to fibrosis in later stage disease. Thus, we have identified a pathogenic fibroblast population in SSc, not isolated by conventional explant culture, that could play an important role in fibrosis and be targeted therapeutically.
Authors
Kristina E.N. Clark, Shiwen Xu, Moustafa Attar, Voon H. Ong, Christopher D. Buckley, Christopher P. Denton
Abstract
Degenerative retinal disorders leading to irreversible photoreceptor death are a common cause of blindness. Optogenetic gene therapy aims to restore vision in affected individuals by introducing light sensitive opsins into the surviving neurons of the inner retina. While up until now the main focus of optogenetic therapy has been on terminally blind individuals, treating at stages where residual native vision is present could have several advantages. Yet, it is still unknown how residual native and optogenetic vision would interact if present at the same time. Using transgenic mice expressing the optogenetic tool ReaChR in ON-bipolar cells, we herein examine this interaction through electroretinography (ERG) and visually evoked potentials (VEP). We find that optogenetic responses show a peculiar ERG signature and are enhanced in retinas without photoreceptor loss. Conversely, native responses are dampened in the presence of ReaChR. Moreover, in VEP recordings we find that optogenetic responses reach the cortex asynchronous to the native response. These findings should be taken into consideration when planning future clinical trials and may direct future preclinical research to optimize optogenetic approaches for visual restoration. The identified ERG signatures moreover may serve to track treatment efficiency in clinical trials.
Authors
Eleonora Carpentiero, Steven Hughes, Jessica Rodgers, Nermina Xhaferri, Sumit Biswas, Michael J. Gilhooley, Mark W. Hankins, Moritz Lindner
Abstract
Bronchiolitis obliterans syndrome (BOS) is a progressive, fatal obstructive lung that occurs following lung transplant, where it is termed chronic lung allograft dysfunction BOS (CLAD-BOS), or as the primary manifestation of pulmonary chronic graft versus host disease (cGVHD-BOS) following allogeneic hematopoietic stem cell transplant. Disease pathogenesis is poorly understood, however chronic alloreactivity is common to both conditions, suggesting a shared pathophysiology. We performed single-cell RNA-Seq (scRNA-Seq) on explanted human lungs from 4 CLAD-BOS patients, 3 cGVHD-BOS patients, and 3 deceased controls to identify cell types, genes, and pathways enriched in BOS to better understand disease mechanisms. In both forms of BOS, we found an expanded population of CD8+ tissue resident memory T-cells (TRM), which was distinct to BOS compared to other chronic lung diseases. In addition, BOS samples expressed genes and pathways associated with macrophage chemotaxis and proliferation, including in non-immune cell populations. We also identify dysfunctional stromal cells in BOS, characterized by pro and anti-fibrotic gene programs. These data suggest substantial cellular and molecular overlap between CLAD- and cGVHD-BOS and therefore, common pathways for possible therapeutic intervention.
Authors
Patrick W. Mellors, Ana N. Nottingham, Bruno Casino Remondo, Maksim Shestov, Joseph D. Planer, Andrew R. Peterson, Yun Ying, Su Zhou, Jason D. Christie, Joshua M. Diamond, Edward Cantu, Maria C. Basil, Saar Gill
Abstract
We defined injury-induced transcriptome states in 4502 kidney transplant biopsies taken 1 day to 45 years post-transplant using genome-wide microarrays. Injury was measured by injury-induced gene sets and classifiers previously developed in transplants. In principal component analysis, PC1 correlated with both acute and chronic kidney injury and related inflammation, and PC2 with time post-transplant. PC3 was a novel dimension that correlated with epithelial remodeling pathways. Both PC1 and PC3 correlated with reduced survival, PC1 effects strongly increasing with time whereas PC3 effects being time-independent. In this model, we studied the expression of genes annotated in native kidneys in epithelial cells with failed repair: 12 “New” gene sets previously defined in single nucleus RNA sequencing of native kidneys with AKI (Genome Med.14(1):103). The “New4” gene sets reflecting epithelial-mesenchymal transition (EMT) correlated with injury PC1, lower eGFR, higher donor age, and future failure as strongly as any gene sets previously derived in transplants, independent of nephron segment of origin and graft rejection. These results suggest that there are two distinct dimensions in kidney transplant response to injury: PC1, AKI-induced changes, failed repair, and inflammation; and PC3, a response involving epithelial remodeling without inflammation. Increasing kidney age amplifies PC1 and particularly PC3.
Authors
Philip F. Halloran, Jessica Chang, Martina Mackova, Katelynn Madill-Thomsen, Enver Akalin, Tarek Alhamad, Sanjiv Anand, Miha Arnol, Rajendra Baliga, Mirosław Banasik, Christopher Blosser, Georg Böhmig, Daniel Brennan, Jonathan Bromberg, Klemens Budde, Andrzej Chamienia, Kevin Chow, Michał Ciszek, Declan de Freitas, Dominika Dęborska-Materkowska, Alicja Debska-Ślizień, Arjang Djamali, Leszek Domański, Magdalena Durlik, Gunilla Einecke, Farsad Eskandary, Richard Fatica, Iman Francis, Justyna Fryc, John Gill, Jagbir Gill, Maciej Glyda, Sita Gourishankar, Marta Gryczman, Gaurav Gupta, Petra Hruba, Peter Hughes, Arskarapurk Jittirat, Zeljka Jurekovic, Layla Kamal, Mahmoud Kamel, Sam Kant, Nika Kojc, Joanna Konopa, James Lan, Roslyn Mannon, Arthur Matas, Joanna Mazurkiewicz, Marius Miglinas, Thomas Mueller, Marek Myślak, Seth Narins, Beata Naumnik, Anita Patel, Agnieszka Perkowska-Ptasińska, Michael Picton, Grzegorz Piecha, Emilio Poggio, Silvie Rajnochová Bloudíčková, Thomas Schachtner, Soroush Shojai, Majid Sikosana, Janka Slatinská, Katarzyna Smykal-Jankowiak, Željka Veceric Haler, Ondrej Viklicky, Ksenija Vucur, Matthew R. Weir, Andrzej Wiecek, Zbigniew Włodarczyk, Harold Yang, Ziad Zaky, Patrick T. Gauthier, Christian Hinze
Abstract
Invasive aspergillosis is characterized by lung hemorrhage and release of extracellular heme, which promotes fungal growth. Heme can also mediate tissue injury directly, and both fungal growth and lung injury may induce hemorrhage. To assimilate these interdependent processes, we hypothesized that, during aspergillosis, heme mediates direct lung injury independent of fungal growth, leading to worse infection outcomes, and the scavenger protein, hemopexin, mitigates these effects. Mice with neutropenic aspergillosis were found to have a time-dependent increase in lung extracellular heme and a corresponding hemopexin induction. Hemopexin deficiency resulted in markedly increased lung injury, fungal growth, and lung hemorrhage. Using a computational model of the interactions of Aspergillus, heme, and the host, we predicted a critical role for heme-mediated generation of neutrophil-extracellular traps in this infection. We tested this prediction using a fungal strain unable to grow at body temperature, and found that extracellular heme and fungal exposure synergize to induce lung injury by promoting NET release, and disruption of NETs was sufficient to attenuate lung injury and fungal burden. These data implicate heme-mediated NETosis in both lung injury and fungal growth during aspergillosis, resulting in a detrimental positive feedback cycle that can be interrupted by scavenging heme or disrupting NETs.
Authors
Ganlin Qu, Henrique A.L. Ribeiro, Angelica L. Solomon, Luis Sordo Vieira, Yana Goddard, Nickolas G. Diodati, Arantxa V. Lazarte, Matthew Wheeler, Reinhard Laubenbacher, Borna Mehrad
Abstract
The dietary sodium-to-potassium ratio is positively correlated with blood pressure, and understanding this relationship is crucial for improving hypertension treatment. Moreover, few studies have examined these effects in both sexes. In this study, we aimed to investigate how supplementing (1.41% K+; HK) or depleting (DK) dietary potassium affects the development of salt-sensitive (SS) hypertension in male and female Dahl SS rats. Potassium supplementation attenuated blood pressure during 5 weeks of high salt (4% NaCl) diet in male but not in female rats. In contrast, potassium-deficient diet prevented the development of SS hypertension in both sexes, though this effect is unlikely to be protective. Both males and females on the DK diet were hypokalemic, had diminished heart rates, reduced weight gain, and females experienced high mortality. Males and females on the DK diet excreted less urinary sodium, but only females had elevated plasma sodium. Additionally, male and female DK-fed rats showed a decrease in multiple metabolites from the renin-angiotensin-aldosterone system. RNA-Sequencing of kidney cortical tissue revealed a number of genes that may underlie the sex-specific differences in phenotype. Analysis of renal ion channel and transporter expression revealed significant increases in basolateral inwardly rectifying potassium channels in DK rats. Male rats supplemented with potassium exhibited a decreased number and size of WNK4 puncta, whereas in potassium-supplemented females, there was no difference in puncta count and an increase in puncta size. Our data indicates there are sex-dependent differences in response to dietary potassium and that the distal nephron compensates for severe potassium deficiency.
Authors
Adrian Zietara, Lashodya V. Dissanayake, Melissa Lowe, Biyang Xu, Vladislav Levchenko, Vasundhara Kain, Ganesh V. Halade, Christine A. Klemens, Oleg Palygin, Alexander Staruschenko
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