Combined single-cell transcriptome and immune repertoire analysis reveals hepatic and renal immune injury by heat stroke
Zhang et al. report peripheral immune signatures of patients with heat stroke, identifying the NLRP3 inflammasome pathway as a potential intervention target for treatment. The cover art shows interactions between a complex vascular network and immune cells in the hot environment. Image credit: Ding Sun, Bin Wang, Yizhi Chen, and Min Zhang. The image was generated using the AI tool Yuanbao.
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Research Letter
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Abstract
Authors
Rimas V. Lukas, Ruochen Du, Harrshavasan Congivaram, Kathleen McCortney, Karan Dixit, Craig Horbinski, Margaret Schwartz, Raymond Lezon, Lauren Singer, Ditte Primdahl, Jigisha Thakkar, Amy B. Heimberger, Roger Stupp, Priya Kumthekar
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Research Articles
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Abstract
Glycosylation changes are pivotal in colorectal cancer (CRC) development. The role of bisecting GlcNAc, a specific N-glycosylation type catalyzed by glycosyltransferase MGAT3, in CRC progression remains elusive. Previous studies indicated that dietary interventions can be beneficial for patients with certain congenital disorders of glycosylation. However, the impact of dietary fatty acids, such as palmitic acid (PA), on glycosylation regulation remains largely unclear. Here, we observed markedly decreased levels of bisecting GlcNAc and MGAT3 in colonic tissues of CRC patients. Downregulation of bisecting GlcNAc in CRC cells increased cell proliferation, migration, and invasion, while decreasing apoptosis. Moreover, a PA-rich diet inhibited CRC carcinogenesis in azoxymethane/dextran sodium sulfate–induced CRC mice by elevating bisecting GlcNAc levels. However, in Mgat3fl/fl Villin-Cre mice the inhibitory effects of the PA-rich diet were abolished. Intact glycopeptide analysis revealed that PA enhanced the bisecting GlcNAc modification on desmoglein 2 (DSG2). Additionally, DSG2 was identified to inhibit CRC carcinogenesis through the EGFR/AKT signaling pathway. In conclusion, dietary PA suppresses CRC carcinogenesis by regulating bisecting GlcNAc modification on DSG2, providing a direct mechanistic link between dietary fatty acids and CRC.
Authors
Lei Lei, Juan Tang, Yuejiao Lv, Bingyi Jia, Wenqing Cai, Shuangshuang Sheng, Keying Li, Zhiwen Shi, Ning Fan, Zengqi Tan, Xiang Li, Feng Guan
×Abstract
Oxidative signaling is a central mechanism in alcohol-induced injury and has strong implications for blood-brain barrier (BBB) dysregulation and neuroinflammation. Here, by targeting oxidative signaling, we hypothesized an innovative approach to develop a clinically relevant therapeutic strategy for alleviating alcohol-mediated neurovascular damage. To accomplish this, we enhanced the endogenous activity of nuclear factor E2–related factor 2 (Nrf2) by treatment with a Nrf2 activator III TAT peptide (Nrf2 peptide [NP]) and investigated the neuroprotective role of Nrf2 in promoting antioxidant defense properties and reducing BBB damage and transmigration of leukocytes to the brain following alcohol ingestion. We administered the NP subcutaneously to alcohol-ingested mice and evaluated its therapeutic potential in alleviating alcohol-associated neurovascular impairments. We compared the results with those seen in animals treated with control peptide (random sequence with TAT). The studies showed that the NP treatment preserved the oxidant-antioxidant balance, downregulated ICAM-1 and its receptors, and mitigated BBB damage and leukocyte infiltration into the brain. We validated the effect of the NP in Nrf2-knockout (Nrf2−/−) mice. Thus, this study demonstrates that NP exerts neurovascular protective effects by regulating the oxidant-antioxidant balance, reducing oxidative stress–induced BBB disruption, and limiting transmigration of immune cells to the brain in a mouse model of alcohol ingestion.
Authors
Bibhuti Ballav Saikia, Saleena Alikunju, Yemin A. Poovanthodi, Zayan Kassim, P.M. Abdul Muneer
×Abstract
Nicotinamide adenine dinucleotide (NAD+) is essential for cellular metabolism, DNA repair, and stress responses. NAD+ is synthesized from nicotinamide, nicotinic acid (collectively termed niacin), and tryptophan. In humans, deficiencies in these nutrients result in pellagra, marked by dermatitis, diarrhea, and dementia. The dermatitis associated with pellagra typically manifests as photodermatosis in sun-exposed areas. This study examined the effects of NAD+ deficiency on skin homeostasis using epidermis-specific Nampt–conditional KO (Nampt-cKO) mice. These mice displayed substantial NAD+ depletion, reduced poly(ADP-ribose) polymerase (PARP) activity, and increased DNA damage. Consequently, Nampt-cKO mice developed spontaneous skin inflammation and epidermal hyperplasia. RNA-seq and IHC analyses demonstrated increased IL-36 cytokine expression, suggesting that DNA repair–related genomic stress triggers keratinocyte-driven IL-36 production, which promotes inflammation. Furthermore, reduced COL17A1 expression and elevated thymic stromal lymphopoietin (TSLP) levels were observed. NAD+ repletion by transdermal supplementation of nicotinamide mononucleotide (NMN) suppressed the rise of IL-36 levels and skin inflammation. These findings underscore the importance of Nampt-mediated NAD+ metabolism for epidermal stability and indicate that NAD+ depletion may contribute to IL-36–mediated skin inflammation, offering insights for therapeutic strategies in inflammatory skin disorders.
Authors
Taiki Seki, Jun-Dal Kim, Yasuhito Yahara, Hitoshi Uchida, Keisuke Yaku, Mariam Karim, Teruhiko Makino, Tadamichi Shimizu, Takashi Nakagawa
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Heat stroke (HS) is the most severe heat-related emergency, and its pathophysiology remains largely unknown, especially for exertional HS (EHS), which affects younger populations, athletes, and manual workers. Herein, we performed single-cell-transcriptomics, T cell receptor sequencing, and flow cytometry of PBMCs from 9 healthy control participants, 9 patients with heat exhaustion, and 9 patients with EHS to explore complex immunological responses associated with HS pathobiology. We showcased that granzyme-positive T cells and CD56dim NK cells with high cytotoxicity features and IL-1B+NLRP3+ monocytes with high inflammation and pyroptosis scores were enriched in HS, while the CD161+ T cells with innate immune-like, low cytotoxicity, and clonal expansion features were reduced in HS. Importantly, elevated granzyme-positive T and NK cells might interact with monocytes to induce pyroptosis of hepatic and renal cells and target organ injuries, and blocking the NLRP3 inflammasome pathway prior to the induction could alleviate organ injury in HS. This study offers deeper insights into the pathogenesis of HS, supporting the development of optimal treatment strategies.
Authors
Min Zhang, Bin Wang, Ding Sun, Xizhao Chen, Yena Zhou, Jin Yao, Liwen Du, Zehao Zhang, Hao Li, Zeyu Qu, Lu Chen, Qing Luo, Jie Zhang, Xinye Jin, Xiaowei Cheng, Jingxue Niu, Qinrui Xing, Xuezeng Tan, Tao Wang, Jie Liu, Lei Li, Qing Song, Xiangmei Chen, Yizhi Chen
×Abstract
Sarcomas are a heterogeneous group of cancers with few shared therapeutic targets. We show that PI3K signaling is frequently activated in sarcomas due to PTEN loss (in 30%–60%), representing a common therapeutic target. The PI3K pathway has lacked a downstream oncogenic transcription factor. We show TAZ and YAP are transcriptional coactivators regulated by PI3K and drive a transcriptome necessary for tumor growth in a PI3K-driven sarcoma mouse model. This PI3K/TAZ/YAP axis exists in parallel to the known PI3K/AKT/mTORC1 axis, providing a rationale for combination therapy targeting the TAZ/YAP-TEAD interaction and mTORC1. Combination therapy using IK-930 (TEAD inhibitor) and everolimus (mTORC1 inhibitor) synergistically diminished proliferation and anchorage-independent growth of PI3K-activated sarcoma cell lines at low, physiologically achievable doses. Furthermore, this combination therapy showed a synergistic effect in vivo, suggesting that an integrated view of PI3K and Hippo signaling can be leveraged therapeutically in PI3K-activated sarcomas.
Authors
Keith C. Garcia, Ali A. Khan, Krishnendu Ghosh, Souradip Sinha, Nicholas Scalora, Gillian DeWane, Colleen Fullenkamp, Nicole Merritt, Yuliia Drebot, Samuel Y. Yu, Mariah Leidinger, Michael D. Henry, Patrick J. Breheny, Michael S. Chimenti, Munir R. Tanas
×Abstract
Degradation of cellular waste from phagocytosis, endocytosis, and autophagy occurs through hydrolases that become activated during acidification of late endosomes and lysosomes (LELs). In our cross-sectional study, we showed diminished LEL acidification and the accumulation of surface-bound nucleosome on monocytes, dendritic cells, B cells, neutrophils, and T cells from patients with systemic lupus erythematosus (SLE). Diminished acidification and exocytosis of undegraded IgG-immune complexes were evident in active, but not inactive, disease. This was supported by our murine study in which LEL acidification was diminished, promoting exocytosis and the accumulation of cell surface IgG-immune complexes. Mechanistically, LEL dysfunction was induced by chronic PI3K activation in lupus-prone MRL/lpr mice. We also showed that on a non-autoimmune C57BL/6 background, deficiency in SHP-1 and inhibition of SHIP-1 activity were sufficient to recapitulate LEL dysfunction found in MRL/lpr mice. Non-acidic LELs were evident in the majority of patients and associated with SLEDAI arthritis, rash, and nephritis. The high frequency of LEL dysfunction in SLE suggests that it could serve as a biomarker identifying a specific disease endotype.
Authors
SunAh Kang, Andrew J. Monteith, Liubov Arbeeva, Karissa Grier, Shruti Saxena Beem, Anthony C. Trujillo, Xinyun Bi, Kai Sun, Rebecca E. Sadun, Mithu Maheswaranathan, Megan E.B. Clowse, Saira Z. Sheikh, Jennifer L. Rogers, Barbara J. Vilen
×Abstract
Liver macrophages are central in maintaining hepatic homeostasis and mediating immune responses during liver injury, including fibrosis. Macrophages may have proinflammatory or antiinflammatory properties, but which properties influence fibrosis remains unclear. To explore the role of macrophages in liver fibrosis, we performed single-cell RNA-seq in a mouse model of liver injury and found that macrophage diversity was increased. Marco was among the most significantly upregulated genes, and a population of Marcohi macrophages increased with injury and spatially segregated to nonfibrotic areas. The macrophage receptor with collagenous structure (MARCO) protein is a scavenger receptor expressed by specific subsets of macrophages, and its role in liver fibrosis is unclear. In vitro induction of Marco in bone marrow–derived macrophages decreased proinflammatory gene expression, increased antiinflammatory and antifibrotic gene expression, and enhanced phagocytosis, indicating a restorative phenotype. Adoptive transfer of MARCO+ macrophages in a mouse model of liver fibrosis reduced the expression of extracellular matrix–associated (ECM-associated) genes in hepatic stellate cells (HSCs) and reduced collagen deposition, which did not occur with the transfer of MARCO– macrophages. Therefore, MARCO+ macrophages have a tissue restorative role in the liver and attenuate fibrogenesis through interaction with HSCs, thereby providing a potential therapeutic pathway for liver fibrosis.
Authors
Sofia Jerez, Shawna A. Cooper, Usman Yaqoob, Maleeha F. Kalaiger, Abid A. Anwar, Mandy Wong, Bushra Arif, Luke C. Doskey, Maria Hernandez-Tejero, William A. Sherman, Ruben De Boeck, Ying Li, Moira B. Hilscher, Enis Kostallari, Nidhi Jalan-Sakrikar, Sheng Cao, Vijay H. Shah
×Abstract
Dysfunctional white adipose tissue contributes to the development of obesity-related morbidities, including insulin resistance, dyslipidemia, and other metabolic disorders. Adipose tissue macrophages (ATMs) accumulate in obesity and play both beneficial and harmful roles in the maintenance of adipose tissue homeostasis and function. Despite their importance, the molecules and mechanisms that regulate these diverse functions are not well understood. Lipid-associated macrophages (LAMs), the dominant subset of obesity-associated ATMs, accumulate in crown-like structures and are characterized by a metabolically activated and proinflammatory phenotype. We previously identified CD9 as a surface marker of LAMs. However, the contribution of CD9 to the activation and function of LAMs during obesity is unknown. Using a myeloid-specific CD9-KO model, we show that CD9 supports ATM-adipocyte adhesion and crown-like structure formation. Furthermore, CD9 promotes the expression of profibrotic and extracellular matrix remodeling genes. Loss of myeloid CD9 reduces adipose tissue fibrosis, increases visceral adipose tissue accumulation, and improves global metabolic outcomes during diet-induced obesity. These results identify CD9 as a causal regulator of pathogenic LAM functions, highlighting CD9 as a potential therapeutic target for treating obesity-associated metabolic disease.
Authors
Julia Chini, Nicole DeMarco, Dana V. Mitchell, Sam J. McCright, Kaitlyn M. Shen, Divyansi Pandey, Rachel L. Clement, Jessica Miller, Rajan Jain, Deanne M. Taylor, Mitchell A. Lazar, David A. Hill
×Abstract
Improper light focus on the retina, refractive error, is primarily caused by eye size differences and is the leading cause of vision loss worldwide. C-terminal variants in the Myelin regulatory factor (MYRF) gene, a retinal pigment epithelium–derived (RPE-derived) transcription factor, lead to isolated nanophthalmos characterized by a small, though structurally sound eye. However, other MYRF loss-of-function variants cause syndromic disease. To address this discrepancy, in vitro and animal studies were performed on a pathogenic C-terminal variant dG-MYRF (p.Gly1126fs30*, c.3376-1G>A). Human RPE cells or primary RPE transduced with dG-MYRF showed reduced target gene expression, with decreased steady-state levels of the C-terminal cleavage product, but normal cleavage and localization. A homozygous humanized MYRF C-terminal mouse model (MyrfhumdG/humdG) was embryonic lethal by E18.5, while WT (MyrfhumWT/humWT) mice were viable. Single-cell RNA-seq from E17.5 MyrfhumdG/humdG and KO RxCre;Myrffl/fl (E15.5 and P0) mice revealed shared differentially expressed genes, with decreased effect size in the MyrfhumdG/humdG eyes. These findings support dG-MYRF as a hypomorphic allele. Additionally, 2 MYRF splicing variants creating nonfunctional isoforms were found in families with isolated nanophthalmos. Overall, hypomorphic MYRF alleles underlie isolated nanophthalmos, supporting a tissue-specific threshold effect and highlighting unique roles for the MYRF C-terminus in the RPE.
Authors
Gabrielle M. Rozumek, Michelle L. Brinkmeier, Bin Guan, Su Qing Wang, Catherine Tower, Nina T. Yang, Rachel S. Lim, Dejuan Kong, Daniel Soden, Qitao Zhang, John Y.S. Han, Jason M.L. Miller, Lijin Dong, D. Ford Hannum, Sayoko E. Moroi, Julia E. Richards, Robert B. Hufnagel, Lev Prasov
×Abstract
Mutation of KRAS in endothelial cells (KRAS-ECs) leads to intracerebral hemorrhage (ICH) in brain arteriovenous malformation (bAVM), resulting in severe disabilities or even death. However, it is unclear what causes this hemorrhagic conversion of bAVMs. Here, using a locally established, clinically relevant sporadic bAVM mouse model, created by overexpressing mutant KRAS (KRASG12V) in brain ECs, we demonstrate that KRAS-ECs act as trigger for activation of microglia (MG) and infiltration of macrophages (Mϕ). Using a 3-dimensional immunostaining approach with cleared human and mouse bAVM tissues, we demonstrate an abundance of MG/Mϕ around the bAVM nidus. The presence of MG/Mϕ was correlated to the blood-brain barrier leakage in bAVM areas. Time-lapsed intravital imaging in Cx3cr1-gfp;Ccr2-rfp reporter mice demonstrated the dynamic activation of MG and infiltration of Mϕ toward mutant KRASG12V–modified dysplastic vessels. Importantly, a time-course analysis showed that these activated MG and infiltrated Mϕ are present around the bAVMs prior to hemorrhagic conversion, and controlled depletion of MG/Mϕ reduced ICH incidence in bAVMs. Inhibition of MG/Mϕ with long-term minocycline treatment attenuated the incidence of ICHs around bAVMs. Our study indicates that MG/Mϕ are involved in destabilization of KRASG12V-induced bAVM, leading to hemorrhagic conversion/ICH. Thus, modulation of MG/Mϕ may reduce ICH risk in patients with bAVM.
Authors
Hyejin Park, Jung-Eun Park, Bridger H. Freeman, Bosco Seong Kyu Yang, Shun-Ming Ting, Alexander K. Suh, Jude P.J. Savarraj, Shuning Huang, Jakob Körbelin, Huimahn Alex Choi, Sean P. Marrelli, Jaroslaw Aronowski, Peng Roc Chen, Eunhee Kim, Eun S. Park
×Abstract
Infectious diseases remain a global health challenge, driven by increasing antimicrobial resistance and the threat of emerging epidemics. Mycobacterium tuberculosis and Staphylococcus aureus are leading causes of mortality worldwide. Trained immunity — a form of innate immune memory — offers a promising approach to enhance pathogen clearance. Here, we demonstrate that IFN-γ induces trained immunity in human monocytes through a mechanism involving mTORC1 activation, glutaminolysis, and epigenetic remodeling. Macrophages derived from IFN-γ–trained monocytes exhibited increased glycolytic activity with enhanced cytokine and chemokine responses upon stimulation or infection. Crucially, trained macrophages had increased production of reactive oxygen species, which mediated enhanced bactericidal activity against methicillin-resistant S. aureus and M. tuberculosis. Furthermore, ATAC-sequencing analysis of IFN-γ–trained macrophages revealed increased chromatin accessibility in regions associated with host defense. Last, IFN-γ training restored impaired innate responses in macrophages from individuals homozygous for the TIRAP 180L polymorphism, a genetic variant associated with increased susceptibility to infection. These findings establish IFN-γ as a potent inducer of trained immunity in human monocytes and support its potential as a host-directed strategy to strengthen antimicrobial defenses, particularly in genetically susceptible individuals and high-risk clinical contexts.
Authors
Dearbhla M. Murphy, Isabella Batten, Aoife O’Farrell, Simon R. Carlile, Sinead A. O’Rourke, Chloe Court, Brenda Morris, Gina Leisching, Gráinne Jameson, Sarah A. Connolly, Adam H. Dyer, John P. McGrath, Emma McNally, Olivia Sandby-Thomas, Anjali Yennemadi, Conor M. Finlay, Clíona Ní Cheallaigh, Jean Dunne, Cilian Ó Maoldomhnaigh, Laura E. Gleeson, Aisling Dunne, Nollaig Bourke, Reinout van Crevel, Donal J. Cox, Niall Conlon, Arjun Raj, Rachel M. McLoughlin, Joseph Keane, Sharee A. Basdeo
×Abstract
Immune checkpoint inhibitors (ICIs) have reshaped the treatment landscape of several cancer types. However, their effectiveness remains limited to a subset of patients, in part due to insufficient preexisting antitumor immunity. In this study, we hypothesized that intracellular delivery of noncoding dsDNA encapsulated in lipid nanoparticles (DNA-LNPs), which have recently been demonstrated to activate both STING and absent in melanoma 2 (AIM2) pathways, could enhance antitumor immune responses and potentiate ICI therapy. Using multiple animal models of cancer, including hepatocellular carcinoma, acute myeloid leukemia, melanoma, and melanoma lung metastasis, we show that DNA-LNP treatment triggered strong cytokine induction and robust CD8+ T cell recruitment to the tumor microenvironment. This immune activation mediated potent CD8+ T cell–dependent antitumor effects and prolonged animal survival across multiple models. Notably, empty LNPs did not elicit potent cytokine elevation or antitumor effects, suggesting that these responses are triggered by the activation of cytosolic DNA-sensing pathways. Moreover, DNA-LNPs synergized with anti–PD-L1, substantially extending animal survival in both ICI-responsive and ICI-resistant tumor models. These findings position DNA-LNPs as a promising immunotherapy strategy, either alone or in combination with ICI therapies, to enhance antitumor immunity across diverse cancer types.
Authors
Seoyun Yum, Alba Rodríguez-Garcia, Joan Castellsagué, Marta Giménez-Alejandre, Guillem Colell, Salut Colell, Teresa Lobo-Jarne, Mark A. LaRue, Michael A. Minnier, Mustafa N. Yazicioglu, Rui Zhang, Xavier M. Anguela, Ali Nahvi, Matthew C. Walsh, Sean M. Armour, Sonia Guedan, Pedro J. Cejas
×Abstract
Modulation of miRNA expression in glomerular cells is associated with renal disease. Here, we investigated the role of miR-93-5p in mitigating glomerular damage in Alport syndrome and whether the disease-modifying activity of extracellular vesicles from human amniotic fluid stem cells (hAFSC-EVs) is mediated by their miR-93-5p cargo. We identified downregulation of miR-93-5p specifically in glomerular endothelial cells in Alport syndrome along disease progression. Silencing of miR-93-5p in hAFSC-EVs changed the transcriptomic and proteomic profile, regulating EV disease-modifying activity. Compared with naive hAFSC-EVs, silenced hAFSC-EVs did not rescue glomerular endothelial function in vitro and did not restore kidney function in vivo. We established that hAFSC-EVs regulate VEGFR1 and VEGFR2 signaling by miR-93-5p cargo transfer, highlighting that miR-93-5p can restore glomerular endothelial cell biology. Spatial transcriptomics analysis of hAFSC-EV–injected kidneys showed that these EVs can reverse pathways altered during disease progression by stimulating proregenerative processes, specifically in the glomerulus, by regulating miR-93-5p targets. Alteration of glomerular endothelial cell transcriptomics and miR-93-5p targets was also confirmed in biopsies of patients with Alport syndrome using spatial molecular imaging. We demonstrated the critical role of miR-93-5p in glomerular endothelial cells and the capability of hAFSC-EVs to regulate miR-93-5p and its targets in Alport syndrome.
Authors
Charmi Dedhia, Valentina Villani, Xiaogang Hou, Paolo Neviani, Geremy Clair, Mohammadreza Kasravi, Cristina Grange, Paolo Cravedi, Paola Aguiari, Velia Alcala, Giuseppe Orlando, Xue-Ying Song, Jonathan E. Zuckerman, Roger E. De Filippo, Stefano Da Sacco, Sargis Sedrakyan, Benedetta Bussolati, Laura Perin
×Abstract
Thyroid hormone signaling is an essential regulator of skeletal muscle development, function, and metabolism, yet the specific signaling pathways required for muscle regeneration are not yet defined. We used scRNA-seq and the FUCCI (fluorescent ubiquitination-based cell cycle indicator) reporter mouse model to examine how hypothyroidism impacts repair processes after cardiotoxin-induced injury in mice. During regeneration, and up to 2 months after injury, hypothyroid muscles displayed smaller myofibers and a shift to slower oxidative fiber types. scRNA-seq of tibialis anterior muscle during regeneration revealed that hypothyroidism reduced myogenic-lineage diversity. Cell cycle analysis confirmed delayed cell cycle progression at 5 and 14 days after injury, with skeletal muscle stem cells stalled at the G1/S transition, hindering differentiation. Transcriptomic data revealed altered nonmyogenic dynamics, including elevated activated fibro-adipogenic progenitors (FAPs) early in repair and persistent proinflammatory macrophages. Integrative regulon and ligand-receptor analysis further demonstrated that triiodothyronine acted through dual modes: a direct transcriptional control of myogenic cell cycle and oxidative programs and an indirect paracrine remodeling mediated by FAP and immune signaling networks. This study identified what we believe to be novel effects of hypothyroidism on myogenic heterogeneity and impaired tissue repair, offering insights into muscle-wasting mechanisms relevant to hypothyroidism-associated myopathy and sarcopenia.
Authors
Paola Aguiari, Valentina Villani, Yan-Yun Liu, Gianni Carraro, Gregory A. Brent, Laura Perin, Anna Milanesi
×Abstract
We hypothesized that bone marrow transplantation (BMT) using marrow extracted from the vertebral bodies (VBs) of an unrelated deceased lung transplant donor would be able to establish persistent hematopoiesis and generate immunity and tolerance. A teenager with severe combined immunodeficiency with lung failure due to recurrent pneumonias underwent lung transplantation in 2016 from a 1/8 HLA allele–matched unrelated donor, followed by BMT 4 months later using T cell/B cell–depleted, cryopreserved VB marrow. Rapid engraftment was followed by accelerating immune competence at 6 months, with independence from immunosuppression by 16 months. Donor T cell (>95%) and myeloid chimerism (7%–10%) has persisted for over 9 years. At 2 years after BMT, circulating T cells were hyporesponsive to host dendritic cells in vitro. T cell receptor clonotyping revealed the disappearance of host-reactive clones, and T cell RNA sequencing exhibited downmodulated signaling pathways for cytotoxicity/rejection, paired with upregulated immunomodulatory pathways, suggesting active suppression. In parallel, host monocytes upregulated certain signaling pathways, indicating active interactions between post-thymic donor T cells and host monocytes. In summary, for the first time to our knowledge, durable hematopoietic engraftment, immunity, and tolerance were demonstrable in a recipient of BMT obtained from a VB graft.
Authors
Paul Szabolcs, Xiaohua Chen, Marian G. Michaels, Memphis Hill, Evelyn Garchar, Zarreen Amin, Heather M. Stanczak, Shawna McIntyre, Aleksandra Petrovic, Dhivyaa Rajasundaram, Ansuman Chattopadhyay, Jonathan E. Spahr, Peter D. Wearden, Geoffrey Kurland
×Potentiation of fentanyl-induced respiratory depression by alcohol is not fully reversed by naloxone
Abstract
The high frequency of opioid overdose deaths often involves co-use of alcohol, which is reported in approximately 30% of fentanyl fatalities. Both substances depress respiratory function, and their combined effects can be lethal. The present study investigated physiological parameters of respiratory-depressant effects of fentanyl when coadministered with alcohol and their sensitivity to naloxone reversal using whole-body plethysmography in male and female Long-Evans rats. Administration of a high, sedative-like dose of alcohol alone or fentanyl alone resulted in no mortality, but fentanyl plus alcohol led to mortality rates of 42% and 33% in females and males, respectively. The fentanyl+alcohol combination reduced minute ventilation and increased apneic pauses compared with either drug alone. Lower, binge-like alcohol doses when combined with fentanyl also amplified respiratory depression. Pretreatment with naloxone did not fully restore normal respiration. Naloxone administered after fentanyl+alcohol transiently reversed the decrease in minute ventilation but did not reverse apneic pauses. Fentanyl-dependent rats were partially tolerant to fentanyl- and fentanyl+alcohol–induced respiratory depression, but alcohol-dependent rats exhibited sensitization to alcohol- and fentanyl+alcohol–induced apnea. These findings highlight physiological parameters of severe respiratory risks with fentanyl+alcohol co-use, which are inadequately reversed by naloxone, underscoring the need for targeted strategies to manage opioid+alcohol overdoses.
Authors
Emma V. Frye, Lyndsay E. Hastings, Aniah N. Matthews, Adriana Gregory-Flores, Janaina C.M. Vendruscolo, Lindsay A. Kryszak, Shelley N. Jackson, Aidan J. Hampson, Nora D. Volkow, Leandro F. Vendruscolo, Renata C.N. Marchette, George F. Koob
×Abstract
Latently infected cells persist in people living with HIV (PWH) despite suppressive antiretroviral therapy (ART) and evade immune clearance. “Shock and Kill” cure strategies are hampered by insufficient enhancement of targeted immune responses following latency reversal. We previously demonstrated that autologous Vδ2 T cells from PWH retain anti-HIV activity and can reduce CD4+ T cell reservoirs, although their use in cure approaches is limited due to their dual role as a viral reservoir. However, promising clinical data in oncology shows that their unique MHC-unrestricted antigen recognition affords potent on-target cytotoxicity in the absence of graft-versus-host disease when used as an allogeneic adoptive cell therapy modality. Here, we found expanded allogeneic Vδ2 T cells specifically eliminated HIV-infected CD4+ T cells and monocyte-derived macrophages (MDM), overcoming inherent resistance to killing by other cell types such as NK and CD8+ T cells. Notably, we demonstrated that allogeneic Vδ2 T cells recognized and eliminated the HIV-latent CD4+ T cell reservoir following latency reversal. Our study provides evidence for developing an allogeneic γδ T cell therapy for HIV cure and warrants preclinical investigation in combination approaches.
Authors
Brendan T. Mann, Marta Sanz, Alisha Chitrakar, Kayley Langlands, Marc Siegel, Natalia Soriano-Sarabia
×Abstract
Common variable immunodeficiency (CVID) is the most prevalent symptomatic primary antibody deficiency. For unclear reasons, inflammatory complications, like gastrointestinal (GI) disease, occur in ~50% of CVID cases, worsening morbidity and mortality. NFKB1 variants are among the most frequent genetic variants in CVID. While effect of NFKB1 variants is not well understood, we previously found frameshift heterozygous NFKB1 variants to increase cytokines, monocytes, and inflammatory complications in CVID. In this report, we used induced pluripotent stem cell–derived (iPSC-derived) monocytes (iMONOs) with CRISPR/Cas9-mediated gene editing to study a heterozygous NFKB1 frameshift found in a patient with CVID with severe GI disease. The heterozygous NFKB1 variant similarly reduced NFKB1 protein in CVID patient– and healthy donor–derived iMONOs, but elevated LPS-induced IL-1β release and expression of inflammatory genes, including IL1B, IL6, TNF, and neutrophil chemoattractants, only in CVID patient iMONOs. CVID patient iMONOs also had elevations of IL-12, CCL4, and CCL12 unaffected by presence or absence of the NFKB1 variant. TNF antagonism improved the patient’s GI disease, diminishing neutrophilic gastritis, circulating neutrophils, and the neutrophil chemoattractant CXCL1 in the blood. While the biology remains complex, our approach found heterozygous NFKB1 variant–induced inflammatory changes intensified in CVID iMONOs, corresponding with clinical response to TNF antagonism.
Authors
Kevin M. Hayes, Kai Boldt, Peter J. Schnorr, Pushpinder Bawa, Miranda L. Abyazi, Matthew S. Ware, Gavin Gyimesi, Marianne James, Huaibin M. Ko, Charlotte Cunningham-Rundles, Joseph P. Mizgerd, Gustavo Mostoslavsky, Darrell N. Kotton, Paul J. Maglione
×Abstract
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated chloride channel, cause cystic fibrosis (CF), the most common life-threatening inherited disorder among White individuals. Current CFTR correctors and potentiators, such as elexacaftor-tezacaftor-ivacaftor (ETI), only partially restore the function of the most prevalent mutant, F508del-CFTR, resulting in residual disease in people with CF. Here, we demonstrate that a mimetic peptide targeting the A-kinase–anchoring protein (AKAP) function of PI3Kγ (PI3Kγ MP), and driving localized cAMP elevation, enhances F508del-CFTR membrane localization, maximizing ETI efficacy in restoring chloride secretion. Mechanistically, PI3Kγ MP activates an AKAP-Lbc–anchored pool of PKD1, a known regulator of membrane trafficking. Consistently, PKD1 inhibition prevents PI3Kγ MP from enhancing the membrane expression of ETI-corrected F508del-CFTR. Overall, our findings reveal a regulatory pathway controlling CFTR membrane abundance via the AKAP function of PI3Kγ, which can be targeted to overcome the limitations of current CFTR modulator therapies.
Authors
Alessandra Murabito, Marco Mergiotti, Valeria Capurro, Alessia Loffreda, Mingchuan Li, Paola Peretto, Kai Ren, Andrea Raimondi, Carlo Tacchetti, Dario Diviani, Nicoletta Pedemonte, Emilio Hirsch, Alessandra Ghigo
×Abstract
The cardiac conduction system (CCS) develops asymmetrically along the body axes. In heterotaxy syndrome — resulting from aberrant left-right axis formation — atrial and atrioventricular conduction defects can cause life-threatening arrhythmias. However, the developmental mechanisms regulating the atrioventricular conduction system (AVCS) disposition and integrity remain unclear. To investigate the etiology of AVCS malformations in laterality defects, we analyzed CCS development and function in mouse mutants for Cryptic and Lefty1, which are key regulators of Pitx2 in the left-right axis formation. Cryptic–/– embryos exhibited bilateral sinoatrial nodes and an ectopic anterior AV node and bundle accompanied by reduced Pitx2 expression. In contrast, Lefty1–/– embryos showed a hypoplastic sinoatrial node and AV node–bundle dissociation with ectopic Pitx2 expression. Single-cell transcriptomic analysis of Pitx2–/– hearts revealed expansion of AV node and bundle populations, consistent with a repressive role of Pitx2 in AVCS specification. Genetic lineage tracing indicated that Pitx2-expressing cells from the left lateral plate mesoderm populate cranioventral cardiac regions, where AVCS development is suppressed. Together, these findings clarify how global left-right axis information is locally integrated to shape AVCS disposition and integrity, providing a mechanistic model for AVCS abnormalities in laterality-associated congenital heart disease.
Authors
Kunihiko Joo, Ryohei Matsuoka, Keiko Kitajima, Kenta Yashiro, Akira Shiose, Ryuji Tominaga, Michael M. Shen, Shinya Oki, Chikara Meno
×Abstract
Immunosuppression and metastasis are critical hallmarks of breast cancer, often linked to poor patient outcomes. The secreted cytokine chitinase-3–like 1 (CHI3L1) is frequently overexpressed in breast cancer samples and promotes an immunosuppressed tumor microenvironment. Notably, CHI3L1 expression is elevated in metastatic patient samples when compared with the matched primary breast tumor. To investigate its role in breast cancer metastasis, we generated an inducible genetically engineered mouse model that overexpresses CHI3L1 in the mammary epithelium. Ectopic expression of CHI3L1 in the polyomavirus middle T (PyMT) mouse model of breast cancer suppressed antitumor immune responses, accelerated mammary tumor onset, and enhanced lung metastasis. Mechanistically, elevated CHI3L1 expression in the mammary epithelium enhanced neutrophil recruitment, which subsequently degraded the extracellular matrix and increased the number of circulating tumor cells. These findings reveal a key mechanism driving metastatic dissemination and argue that therapeutically targeting Chi3l1 could enhance antitumor immunity and suppress metastasis.
Authors
Tarek Taifour, Adéline Massé, Yu Gu, Virginie Sanguin-Gendreau, Dongmei Zuo, Bin Xiao, Emilie Solymoss, Yunyun Shen, Hailey Proud, Sherif Samer Attalla, Vasilios Papavasiliou, Nancy U. Lin, Melissa E. Hughes, Kalie Smith, Chun Geun Lee, Suchitra Kamle, Josie Ursini-Siegel, Jack A. Elias, Peter M. Siegel, Rinath Jeselsohn, William J. Muller
×Abstract
Limb-girdle muscular dystrophy R2 (LGMD R2) is an autosomal recessive disorder caused by dysferlin deficiency, leading to progressive muscle weakness and wasting. The lack of reliable clinical biomarkers has limited disease monitoring and therapeutic evaluation. Here, we identified Disabled-2 (DAB2) as a molecular and clinical indicator of disease state in LGMD R2. Transcriptomic profiling revealed a significant upregulation of DAB2 in induced pluripotent stem cell–derived (iPSC-derived) myotubes from patients, a finding validated in muscle biopsies from 14 dysferlin-deficient individuals and in dysferlin-deficient Bla/J mice, where DAB2 levels increased with disease progression. Importantly, AAV-mediated expression of full-length dysferlin restored DAB2 levels, supporting its value as a dynamic readout of disease activity for both disease monitoring and therapeutic response. Given the established role of DAB2 in clathrin-mediated endocytosis, particularly in LDL receptor internalization and cholesterol homeostasis, and the pathological lipid accumulation reported in LGMD R2, we investigated its contribution to lipid dysregulation. High DAB2 expression paralleled lipid deposition in patient muscles, iPSC-derived myotubes, and mouse tissue, whereas siRNA-mediated DAB2 knockdown reduced lipid accumulation in LGMD R2 myotubes. Collectively, these findings suggest that DAB2 functions as a mechanistic link between dysferlin deficiency, altered lipid handling, and disease severity, and they highlight its potential as a prognostic marker and therapeutic response measure for LGMD R2.
Authors
Celine Bruge, Nathalie Bourg, Emilie Pellier, Quentin Miagoux, Manon Benabides, Noella Grossi, Hassan Hayat, Margot Jarrige, Helene Polveche, Valeria Agostini, Anthony Brureau, Stephane Vassilopoulos, Teresinha Evangelista, Gorka Fernández-Eulate, Tanya Stojkovic, Isabelle Richard, Xavier Nissan
×Abstract
Cachexia is a debilitating syndrome characterized by progressive skeletal muscle wasting, commonly affecting patients with cancer, particularly those with pancreatic cancer. Despite its clinical significance, the molecular mechanisms underlying cancer cachexia remain poorly understood. In this study, we utilized single-nucleus RNA-seq (snRNA-seq) and bulk RNA-seq, complemented by biochemical and histological analyses, to investigate molecular alterations in the skeletal muscle of the KPC mouse model of pancreatic cancer cachexia. Our findings demonstrated that KPC tumor growth induced myofiber-specific changes in the expression of genes involved in proteolytic pathways, mitochondrial biogenesis, and angiogenesis. Notably, tumor progression enhanced the activity of specific transcription factors that regulate the mTORC1 signaling pathway, along with genes involved in translational initiation and ribosome biogenesis. Skeletal muscle–specific, inducible inhibition of mTORC1 activity further exacerbated muscle loss in tumor-bearing mice, highlighting its protective role in maintaining muscle mass. Additionally, we uncovered new intercellular signaling networks within the skeletal muscle microenvironment during pancreatic cancer–induced cachexia. Our study reveals previously unrecognized molecular mechanisms that regulate skeletal muscle homeostasis, and it identifies potential therapeutic targets for the treatment of pancreatic cancer–associated cachexia.
Authors
Bowen Xu, Aniket S. Joshi, Meiricris Tomaz da Silva, Silin Liu, Ashok Kumar
×Abstract
Alveolar hemorrhage (AH) is a life-threatening condition with high mortality, yet the immunological mechanisms governing disease severity remain poorly defined. Here, we demonstrate a protective role for T cell–intrinsic β-catenin stabilization in AH using a transgenic mouse model (CAT-Tg) in which β-catenin is stabilized under the Lck promoter. We found β-catenin stabilization induced a distinct T cell phenotype marked by expansion of central effector memory cells (CD44+CD122+Eomes+T-bet+) and suppression of proinflammatory signaling, including reduced phosphorylation of STAT1, STAT3, and JAK1. Pristane-induced AH was attenuated in CAT-Tg mice, which exhibited reduced lung injury, decreased proteinuria, and diminished pulmonary proinflammatory cytokine production compared with WT controls. Protection was associated with a marked expansion of FOXP3+ Tregs. Mechanistically, β-catenin stabilization enhanced lung expression of amphiregulin and BATF, mediators of Treg stability and tissue repair. Adoptive transfer of CAT-Tg–derived Tregs into WT mice conferred superior protection against AH, reducing lung inflammation and proteinuria. Transcriptomic analyses revealed enrichment of tissue repair and immune homeostasis pathways, including PI3K-Akt, angiogenesis, and STAT5 signaling. Collectively, these findings identify β-catenin as a regulator of a protective amphiregulin/BATF/Treg axis, highlighting an immunomodulatory pathway with therapeutic potential for AH and inflammatory lung disease.
Authors
Fiona Mason, Hui Xiong, Ali Mobeen, Md Saddam Hossain, Sara Mahmudlu, Rosanne Trevail, Mikyal Mobeen, Li Chen, Sunny Lee, Tuncay Delibasi, Jyoti Misra Sen, Mobin Karimi
×Abstract
CD4 T cells are essential for immunity to M tuberculosis (Mtb), and emerging evidence indicates that IL-17–producing Th17 cells contribute to immunity to Mtb. While identifying protective T cell effector functions is important for TB vaccine design, T cell antigen specificity is also likely to be important. To identify antigens that induce protective immunity, we reasoned that, as in other pathogens, effective immune recognition drives sequence diversity in individual Mtb antigens. We previously identified Mtb genes under evolutionary diversifying selection pressure whose products we term Rare Variable Mtb Antigens (RVMA). Here, in 2 distinct human cohorts with recent exposure to TB, we found that RVMA preferentially induce CD4 T cells that express RoRγt and produce IL-17, in contrast to “classical” Mtb antigens that induce T cells that produce IFN-γ. Together with emerging evidence showing human Th17 responses are associated with prevention of progression to TB disease, our results suggest that RVMA can be valuable antigens in vaccines for those already infected with Mtb to amplify existing antigen-specific Th17 responses to prevent TB disease.
Authors
Paul Ogongo, Liya Wassie, Anthony Tran, Devin Columbus, Julia Huffaker, Lisa Sharling, Gregory Ouma, Samuel Gurrion Ouma, Kidist Bobosha, Cecilia S. Lindestam Arlehamn, Neel R. Gandhi, Sara C. Auld, Jyothi Rengarajan, Cheryl L. Day, Artur Queiroz, Mariana Araújo-Pereira, Eduardo Fukutani, Bruno B. Andrade, John D. Altman, Henry M. Blumberg, Joel D. Ernst, the TBRU ASTRa Study Group
×Abstract
The chronic inflammation of Crohn’s disease frequently leads to fibrosis and muscular hypertrophy of the intestinal wall. This often culminates in strictures, a serious condition lacking directed therapy. Severe pathological changes occur in the submucosa and muscularis propria intestinal wall layers of strictures, yet stricture-associated proteome changes in these layers is unexplored. We perform unbiased proteomics on submucosa and muscularis propria microdissected from transmural sections of strictured and nonstrictured ileum. Proteome changes in strictured submucosa reflected a transition from homeostasis to tissue remodeling, inflammation, and smooth muscle changes. Top submucosal features included reduced vascular components and lipid metabolism proteins accompanied by increased proteins with immune-, ECM-, or stress-related functions, including CTHRC1, TNC, IL-16, MZB1, and TXNDC5. In parallel, predominant changes in strictured muscularis propria included increased ECM (POSTN) and immune (mast cell CPA3) proteins alongside decreased proteins with lipid metabolic, mitochondrial, or key muscle functions. Finally, trends of differentially expressed proteins along nonstrictured submucosa suggest progressive profibrotic tissue remodeling and muscle expansion as proximity to strictures increases. The comprehensive proteome map presented here offers tissue-layer-resolved insight into the stricture microenvironment and potential drivers of fibrotic disease, providing a valuable resource to fuel biomarker and therapeutic target research.
Authors
Johannes Alfredsson, Carina Sihlbom Wallem, Maja Östling, Hanna de la Croix, Elinor Bexe-Lindskog, Mary Jo Wick
