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Abstract
Heterozygous TBX4 variants are the second most common genetic cause of pediatric pulmonary hypertension (PH), yet mechanisms underlying TBX4-related lung disease remain poorly understood. This study developed a lung mesenchyme-specific Tbx4 loss-of-function (Tbx4cKO) mouse model that bypasses embryonic lethality to investigate this condition. Adult Tbx4cKO mice demonstrated significantly impaired pulmonary flow acceleration consistent with PH. Three-dimensional analysis of embryonic lungs revealed reduced lobe volumes and decreased distance between pleural edges and muscularized vessels. In adult Tbx4cKO lungs, we identified extensive vascular remodeling characterized by medial thickening and the extension of muscularized arteries into normally non-muscularized subpleural parenchymal zones. Contrary to previous reports suggesting vascular simplification, three-dimensional analysis demonstrated an elaborated pulmonary artery (PA) tree in addition to pathologic wall muscularization. Depletion of a single Tbx5 allele in addition to both Tbx4 alleles exacerbated histologic phenotypes with worsened right ventricular dilation. This model also demonstrated dysregulated airway smooth muscle patterning and prominent subpleural smooth muscle bands, similar to those in human TBX4 syndrome. We identify TBX4 as a critical regulator of smooth muscle differentiation and patterning across multiple lung compartments. Our model recapitulates key features of human TBX4 syndrome and identifies dysregulated smooth muscle differentiation as a potential future therapeutic target.
Authors
Lea C. Steffes, Kaylie A. Chiles, Sehar R. Masud, Aleen Rahman, Madeline Dawson, Csaba Galambos, Maya E. Kumar, Ripla Arora
Abstract
Human γδ T cells are a rare but functionally diverse lymphocyte subset critical for tumor surveillance and antimicrobial immunity. Although they express natural killer (NK) cell-associated receptors such as Killer-cell Immunoglobulin-like Receptors (KIRs), the relevance of KIR expression on γδ T cells remains largely unexplored. Using flow cytometry, ATAC-seq and RNA-seq, we identified KIR expression as a marker that distinguished two functionally and molecularly distinct γδ T cell subsets. KIR⁺ γδ T cells exhibited an advanced, memory-like differentiation state characterized by heightened cytotoxicity, stable epigenetic remodeling and a predominant IFNγ-producing profile. In contrast, KIR⁻ γδ T cells maintained a naïve-like phenotype and preferentially produced IL-17 upon polarization. Notably, KIR+ γδ T cells were consistently observed across individuals but were significantly enriched in cytomegalovirus (CMV)-seropositive donors, suggesting that chronic antigenic stimulation could promote the emergence of KIR⁺ effector γδ T cells. These findings reveal a functional dichotomy in human γδ T cells defined by KIR expression, linking IFNγ-driven cytotoxicity with KIR⁺ cells and IL-17 production with KIR⁻ cells. This insight advances our understanding of γδ T cell heterogeneity and has implications for viral immunity, immune memory and the development of γδ T cell-based immunotherapies.
Authors
Mahya Razmi, Yeganeh Almasi, Marilee Larrivée, Jonathan B. Angel, Alexandre Blais, Zakia Djaoud
Abstract
Survival after lung transplantation is limited by chronic, progressive graft failure, termed chronic lung allograft dysfunction (CLAD). Graft-resident mesenchymal cells (MCs) drive CLAD pathogenesis and exhibit stable dysregulated signaling, yet the transcriptomic and epigenomic drivers underlying this fibrogenic transformation remain elusive. We used single-cell multi-omic profiling to characterize gene expression and chromatin accessibility in MCs isolated from lavage fluid of lung transplant recipients with and without CLAD, collected early post-transplantation or after disease onset. MCs obtained after CLAD onset demonstrated a distinct transcriptomic signature compared with non-CLAD controls, enabling classification of disease status at the single-cell level with > 98% accuracy using signature genes. Chromatin accessibility analyses identified enrichment of CCAAT-enhancer-binding protein family transcription factors, specifically CEBPD, in CLAD MCs. Early post-transplant MCs showed minimal accessibility differences, suggesting that CEBPD-associated regulatory changes emerge over time. Integration analyses identified eight MC states and a CLAD-specific shift towards a fibrotic state. CEBPD, SOX4, and FOXP2 were identified as putative regulators of this state with substantial overlap in predicted targets. Targeting CEBPD reversed fibrotic phenotypes of CLAD MCs (decreased ECM expression, contractility, proliferation, and migration). Together, these data provide insights into transcriptomic and epigenomic changes in post-transplant MCs, nominating biomarkers and therapeutic targets.
Authors
Lu Lu, A. Patrick McLinden, Natalie M. Walker, Ragini Vittal, Yichen Wang, Fatemeh Fattahi, Stephen T. Russell, Michael P. Combs, Joshua D. Welch, Vibha N. Lama
Abstract
Pulmonary fibrosis is frequently accompanied by pulmonary hypertension, which can occur disproportionate to the extent of fibrosis, suggesting a fibrosis-independent vascular remodeling process. Here, we demonstrated that plasma growth differentiation factor 15 (GDF15) is elevated across diverse fibrotic lung disease subtypes and correlates with markers of elevated right heart pressures, but not pulmonary function indices, indicating a possible link to endothelial cell dysfunction. To investigate the import of endothelial GDF15 as a modifier of lung fibrosis pathogenesis, we generated endothelial cell-specific Gdf15 knockout mice, which showed protection from bleomycin-induced lung injury and fibrosis, with preserved lung function. RNA sequencing of human pulmonary microvascular endothelial cells revealed altered expression of barrier-regulatory genes in GDF15-deficient endothelial cells compared to controls. Functional studies confirmed that GDF15 knockdown attenuates thrombin-induced barrier disruption by reducing cytosolic Ca2+ responses. Together, these findings implicate endothelial GDF15 as a modifier of vascular permeability and Ca2+ signaling, and a contributor to lung injury and fibrosis.
Authors
Kristen Raffensperger, Marta Bueno, Brian J. Philips, Megan Miller, Máté Katona, Shuai Yuan, Adriana Estrada-Bernal, Byron Chuan, Pavan Suresh, Stephanie Taiclet, Scott Hahn, Yingze Zhang, Jonathan K. Alder, Seyed Mehdi Nouraie, Daniel J. Kass, Oliver Eickelberg, Adam C. Straub
Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease driven by aberrant fibroblast-to-myofibroblast differentiation, which requires metabolic reprogramming. Here, we identify alanine as an essential metabolite for myofibroblast differentiation. Transforming growth factor–β1 (TGF-β) increases intracellular alanine levels through enhanced synthesis and import in both normal and IPF lung fibroblasts. Alanine synthesis is primarily mediated by glutamate-pyruvate transaminase 2 (GPT2), whose expression is regulated by the glutamine–glutamate–α-ketoglutarate axis. Inhibition of GPT2 depletes alanine and suppresses TGF-β-induced α-SMA and COL1A1 expression, which are rescued by exogenous alanine. We also identify solute carrier family 38 member 2 (SLC38A2) as a transporter for both alanine and glutamine, upregulated by TGF-β or alanine deprivation. SLC38A2 and GPT2 form a coordinated regulatory axis sustaining intracellular alanine levels to support myofibroblast differentiation. Mechanistically, alanine deficiency impairs glycolytic flux and depletes tricarboxylic acid cycle intermediates, while alanine supplementation provides carbon and nitrogen for intracellular glutamate and proline biosynthesis, particularly under glutamine deprivation. Combined inhibition of alanine synthesis and uptake suppresses fibrogenic responses in fibroblasts and human precision-cut lung slices, highlighting dual metabolic targeting as a potential therapeutic strategy for fibrotic lung disease.
Authors
Fei Li, Niv Vigder, David R. Ziehr, Mari Kamiya, Hung N. Nguyen, Diana E. Ferreyra Faustino, Aseel H. Khalil, Hilaire C. Lam, Matthew L. Steinhauser, Edy Y. Kim, William M. Oldham
In Vivo Characterization of Candida Extracellular Vesicles Reveals Unique Infection Pathway Proteins
Abstract
Authors
Justin Massey, Robert Zarnowski, William Hartman Jr., Jeniel E. Nett, David R. Andes
Abstract
Fibroblast-like synoviocytes (FLSs) are crucial in driving synovial inflammation and joint damage in rheumatoid arthritis (RA). This study explored the functions and underlying mechanisms of GALNT1-mediated O-glycosylation, which is markedly upregulated in RA FLSs, in synovial aggression and subsequent experimental joint damage. Targeted suppression of GALNT1 effectively curtailed migration and invasion in RA FLSs and mitigated arthritis severity in a rat collagen-induced arthritis (CIA) model. Mechanistically, NEK9 was identified as a pivotal substrate and downstream effector of GALNT1, affecting the aggressive phenotype of RA FLSs. In vitro experiments further demonstrated that O-glycosylation of NEK9, mediated by GALNT1, promotes the pathogenic phenotype of RA FLSs by promoting cytoskeleton reorganization and restraining excessive endoplasmic reticulum (ER) stress activation. Our study provides mechanistic insights into the activation of RA FLSs and identifies GALNT1 as a potential therapeutic target for RA.
Authors
Yaoyao Zou, Haobo Lin, Jianling Su, Jieying Wang, Qin Zeng, Tianxiao Feng, Yunxia Lei, Jianda Ma, Hudan Pan, Hanshi Xu, Lie Dai, Yang Li
Abstract
Aortic dissection (AD) is a catastrophic vascular emergency with high mortality, and current pharmacologic interventions to prevent its progression are limited. Vascular smooth muscle cells (VSMCs) undergo a pathological phenotypic switch from a contractile to a synthetic state during AD, compromising aortic wall integrity; however, the underlying metabolic mechanisms remain poorly understood. In this study, we performed integrative transcriptomic analyses and identified glutaminase 1 (GLS1) as a key regulator of VSMC phenotypic switching in AD. GLS1 expression was significantly downregulated in VSMCs from both human AD aortic tissues and mouse models. Functionally, GLS1 deficiency promoted PDGF-BB–induced VSMC dedifferentiation in vitro. Smooth muscle cells specific Gls1 knockout (Gls1SMKO) mice exhibited aggravated AD upon BAPN treatment, whereas VSMCs specific GLS1 overexpression improved the contractile phenotype and reduced AD incidence. Mechanistically, GLS1 downregulation impaired glutamate metabolism, leading to reduced levels of glutathione and α-ketoglutarate. This metabolic disruption promoted reactive oxygen species accumulation and mitochondrial dysfunction, ultimately triggering VSMC phenotypic switching. Furthermore, we found that GLS1 transcription was repressed by retinoic acid receptor-α (RARα). Pharmacologic inhibition of RARα with AR7 restored GLS1 expression, ameliorated VSMC phenotypic switching, and conferred protection against AD. These findings reveal a critical role of GLS1-mediated glutamate metabolism in VSMC phenotypic switching and suggest a promising therapeutic strategy for AD.
Authors
Wei Xie, Chen Ning, Chen Lu, Dongjin Wang, Shuang Zhao, Tianyu Song, Hailong Cao
Abstract
Inflammatory bowel disease (IBD) is frequently accompanied by intestinal fibrosis, with non-response (NR) to long-term anti-tumor necrosis factor α (anti-TNFα) therapy occurring in approximately 23-46% of patients. Integrated analysis of single-cell and bulk RNA sequencing datasets revealed an expansion of IL11⁺ fibroblasts in inflamed intestine and their significant enrichment in non-responders. We further identified IL11⁺ fibroblasts as a central communication hub that engaged in extensive crosstalk with monocytes and may contribute to inflammatory amplification and fibrotic remodeling. Additionally, we employed machine learning approaches including least absolute shrinkage and selection operator (LASSO), support vector machines (SVM), and random forest (RF) to derive an IL11⁺ fibroblast-related gene signature effectively predicting NR to anti-TNFα in validation and test cohorts. IHC further confirmed the overexpression of IL-11 in non-responders. The signature genes we found are not only associated with immune and inflammatory responses but also with fibrosis, indicating a robust association between fibrosis and anti-TNFα treatment failure. In summary, this study highlights the important role of IL11⁺ fibroblasts in orchestrating both inflammation and fibrosis and provides an applicable model for predicting NR to anti-TNFα in IBD, thereby laying the foundation for precision medicine and targeted therapeutic strategies.
Authors
Wangyue Li, Wei Huang, Jiaxin Wang, Yiwen Tu, Qidi Yang, Yao Zhou, Zile Zhang, Haiming Zhuang, Yubei Gu, Duowu Zou, Yao Zhang
Abstract
Inhibiting the mammalian target of rapamycin (mTOR) during acute viral infection generates highly functional memory CD8 T cells. We investigated the effects of inhibiting mTOR by using rapamycin during the effector and contraction phases of the immune response to a DNA prime and Modified Vaccinia Ankara (MVA) boost SIV vaccination in rhesus macaques. Rapamycin administered either during MVA boosts alone (DMR) or during both primes and boosts (DRMR) reduced the contraction of effector CD8 T cells, resulting in higher frequencies of SIV-specific memory CD8 T cells with enhanced quality as indicated by expression of Bcl-2 and CD127. Additionally, rapamycin reduced the frequency of proliferating CCR5+ CD4 T cells in the blood following the MVA boost. Post SIV251 infection, rapamycin-treated macaques demonstrated marked expansion of SIV-specific CD8 T cells (reaching up to 50% in blood and 25% in gut). The heightened expansion of SIV-specific CD8 T cells in the DMR group was associated with markedly lower (2-logs compared to unvaccinated and 1-log compared to DM) peak viral load in the gut and set-point viremia, along with improved survival post infection. Thus, inhibiting the mTOR pathway during MVA boosts of a DNA/MVA vaccine enhances vaccine efficacy by improving memory CD4 and CD8 T cell function.
Authors
Shanmugalakshmi Sadagopal, Kasey Stokdyk, Suefen Kwa, Rahul Basu, Sailaja Gangadhara, Rafi Ahmed, Smita S. Iyer, Koichi Araki, Rama Rao Amara
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