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Abstract
TGF-β-activating integrins promote solid-organ fibrosis, suggesting their potential as disease markers. Chronic lung allograft dysfunction (CLAD), a progressive fibrotic complication that limits lung transplant survival, is driven by intragraft TGF-β activation. However, TGF-β-activating integrin expression patterns remain undefined in lung transplants. Single-cell RNA sequencing and proteomic analysis of tissue specimens from a mouse CLAD model and human explanted lungs with CLAD revealed high levels of the TGF-β-activating integrin αvβ6, primarily localized to fibrosis-associated Keratin 8+ alveolar transitional epithelial cells, which were greatly reduced in tolerant lungs. Molecular imaging with a newly optimized positron emission tomography radiotracer specific for αvβ6, [64Cu]Cu-DOTA-A20-K16R, showed significantly higher uptake in CLAD than in tolerant transplants. [64Cu]Cu-DOTA-A20-K16R activity was also reduced by experimental treatments that lowered αvβ6 expression and inhibited CLAD severity. Finally, [64Cu]Cu-DOTA-A20-K16R autoradiographic analysis of human explanted lungs with CLAD showed elevated activity that correlated with increased αvβ6 expression. Collectively, these findings illustrate the potential utility of αvβ6 molecular imaging for detecting CLAD pathogenesis.
Authors
Marlene Cano, Chandra S. Bathula, Fuyi Liao, Cristian Wieczorek Villas Boas, Yan Tao, Zhiyi Liu, Ricky Ebenezer, Victoria Davis, Allison Cannady, Dequan Zhou, Shunning Liang, Hanqiu Zhao, Derek E. Byers, Alexander S. Krupnick, Daniel Kreisel, Susan M. Lin, Allen Duong, Tereza Martinu, Zhiyu Dai, Buck E. Rogers, Andrew E. Gelman
Abstract
Inflammatory bowel diseases (IBD) including Crohn’s disease (CD) are characterized by impaired intestinal epithelial barrier (IEB) function. Alterations in the desmosomal cadherin desmoglein 2 (Dsg2) have been linked to loss of IEB and intestinal inflammation in IBD. We investigated the pathogenic impact of a cytoplasmatic-truncating mutation in the desmosomal cadherin desmoglein 2 gene (DSG2) in an IBD patient. Genetic analysis of a CD patient revealed a likely pathogenic DSG2 mutation leading to a truncated protein lacking part of the intracellular domain. Generation of an enterocyte-specific mouse model to recapitulate the human mutation resulted in a lethal intestinal barrier defect which was evident in homozygous but not in heterozygous mice. In vitro, barrier defects correlated with changes in Dsg2 mobility and upregulation of pore-forming tight junction protein claudin-2. Organoids derived from heterozygous Dsg2 truncated mice exhibited increased epithelial susceptibility to proinflammatory cytokines. Bulk RNAseq followed by gene ontology analysis in homozygous mice revealed a skewed IL17/IL23 response overlapping with transcriptional changes in patients with CD. Furthermore, 16S rRNA sequencing revealed alterations in microbiome composition in animals that carried the Dsg2 truncation. In summary, a patient-derived mutation in the DSG2 gene causing cytoplasmic truncation in homozygous state induced a lethal intestinal barrier breakdown in mice paralleled by altered gut microbiota and intestinal immune response.
Authors
Daniela Kugelmann, Alexander García-Ponce, Nadja Thielemann, Mariya Y. Radeva, Maresa Anders, Da Eun Kang, Christian Siadjeu, Natalie Burkard, Anja K.E. Horn, Jessica Neubauer, Matthias Hiermaier, Desalegn T. Egu, Christoph Schmitz, Oliver Kurzai, Katri S. Vuopala, Outi Kuismin, Nicolas Schlegel, Jens Waschke
Abstract
Essential thrombocythemia (ET) and myelofibrosis (MF) are myeloproliferative neoplasms (MPN) that can result from heterozygous or homozygous mutations in the calreticulin (CRT) gene (CALR). Mutant CRT binds to the thrombopoietin receptor to induce signaling that drives ET and MF. CRT is also a chaperone for the assembly of the highly polymorphic human leukocyte antigen (HLA) class I molecules. The effects of heterozygous versus homozygous MPN CALR mutations on the expression of HLA class I variants are unknown. To assess mutant CRT-induced changes to HLA class I expression, we generated CRISPR-based mutant CALR knock-in cell lines and CALR-knockout cell lines reconstituted with wild-type or MPN mutant CALR and also undertook measurements in MPN patient platelets and monocytes. Collectively, our findings indicate that heterozygous CALR mutations preserve HLA class I surface expression, whereas full loss of wild-type CRT results in marked, allele-dependent reductions in HLA class I expression, which are rescued by wild-type CRT but not MPN mutants. Reduced expression of CRT-dependent HLA class I in cells lacking a functional CRT induces natural killer (NK) cell activation. These studies identify specific HLA class I alleles and homozygous CALR mutations as prerequisites for low HLA class I expression in MPN.
Authors
Amanpreet Kaur, Grace Pagnucco, Fawziyah S. Khatri, Harini Desikan, Malathi Kandarpa, Matthew Cusick, Moshe Talpaz, Malini Raghavan
Abstract
Tuberous sclerosis complex (TSC) is an inherited tumor predisposition syndrome caused by pathogenic variants in TSC1 or TSC2, yet no animal model faithfully recapitulates the cardiac rhabdomyomas (CR) that are among its earliest manifestations. We generated a heterozygous TSC2-mutant Yucatan minipig carrying the recurrent pathogenic TSC2 p.Arg505* variant and characterized its cardiac phenotype using histopathology, immunohistochemistry, and RNA sequencing. TSC2-mutant minipigs consistently developed multiple CR that closely resembled human lesions, including enlarged glycogen-rich spider cells and activation of mechanistic target of rapamycin complex 1 (mTORC1) signaling. Comparative transcriptomic analyses demonstrated enrichment of a conserved TSC-associated gene expression program and activation of microphthalmia/transcription factor E (MITF/TFE)-regulated lysosomal pathways in both human and minipig tumors. Although other manifestations of TSC were not observed during the first year of life, this large-animal model faithfully reproduced the principal histopathologic and molecular features of human CR. These findings have established the TSC2-mutant Yucatan minipig as what we believe to be the first large-animal model of TSC-associated CR and provide a physiologically relevant platform for investigating tumor pathogenesis, mechanisms of spontaneous regression, and therapeutic strategies targeting mTORC1- and MITF/TFE-dependent signaling.
Authors
Elias Bou Farhat, Heng Du, Chrystalle Katte Carreon, Jad Halawi, Sophie Kudler, Magdalena Losko, Zachary T. Herbert, Adrienne L. Watson, Daniel Carlson, Dennis A. Webster, Derek M. Korpela, David M. Meredith, Elizabeth P. Henske, David J. Kwiatkowski
Abstract
Osteoarthritis (OA) is a heterogeneous disease in which synovial inflammation drives structural progression and pain, yet the molecular diversity of synovitis remains poorly defined. Here, we performed a multimodal characterization of synovial tissues from 48 dogs with naturally occurring OA, integrating histopathology, immunohistochemistry, transcriptomics, MRI, and soluble mediator profiling. This analysis identified three discrete synovial pathotypes, pauci-immune, diffuse myeloid, and lympho-myeloid, defined by distinct immune cell composition and transcriptional programs reflecting biologically distinct endotypes. The lympho-myeloid pathotype, characterized by ectopic lymphoid structures and adaptive immune enrichment, was independently associated with greater structural joint damage on MRI despite comparable clinical scores, demonstrating that tissue stratification captures heterogeneity invisible to clinical assessment alone. Composite soluble mediator signatures correlated with disease severity across pathotypes. Cross-species analyses integrating a large human synovial cohort revealed conserved pathotype architecture and shared gene networks, including tyrosine kinase signaling and B cell receptor activation, validating the translational relevance of the canine model. Together, these findings provide a framework for patient stratification and therapeutic target discovery in OA, with broad implications for precision medicine within a One Medicine perspective.
Authors
Anaëlle Drieu-Vincent, Julien De Lima, Floriane M. Etienne, Romain Guiho, Noémie Podevin, Richard Danger, Florence Lezin, Nicolas Gaigeard, Sophie Allain, Jérôme Abadie, Thomas Rousseau, Pierre Maitre, Olivier Gauthier, Jérôme Guicheux, Marion S. Fusellier, Marie-Astrid Boutet
Abstract
Alveolar microcysts, a hallmark of Idiopathic Pulmonary Fibrosis (IPF), are basal cell (BC)-lined structures that develop near terminal airways and correlate with poor clinical outcomes. Using primary-cell organoids and spatial transcriptomics, we identified striking epithelial similarities between respiratory bronchioles (RBs) and IPF alveolar microcysts, both containing AT0 (ABCA3+/SCGB3A2+), SCGB3A2+, and basaloid/BC populations, although basaloid/BCs were markedly expanded in IPF microcysts. A CEACAM6+ subset of normal RB epithelial cells, termed respiratory airway secretory cells (RASCs), consisted predominantly of SCGB3A2+ cells with only rare BCs (1–5%) that by RNA trajectory analysis suggested BCs arise from SCGB3A2+ progenitors. Accordingly, in organoid cultures nearly all RASC-derived colonies converted to BCs within 9 days, resembling colonies generated by purified BCs. BCs derived from either RASCs or normal alveolar type II (AT2) cells strongly expressed the collagenase matrix metalloproteinase-1 (MMP1), recapitulating the phenotype of BCs lining IPF microcysts. MMP1high BCs degraded type I collagen ex vivo and generated microcyst-like structures in vivo following xenotransplantation, both abolished by MMP activity inhibition or specific MMP1 deletion by sh-RNAi. Finally, a Frizzled-5 WNT agonist antibody reversed the MMP1high phenotype, identifying AT2- and RB-derived basal cell transdifferentiation as a previously unrecognized driver of alveolar destruction and microcyst formation in IPF and a potential therapeutic target.
Authors
Marylene Toigo, Kiana Garakani, Alexis N. Brumwell, Ying Wei, Tsung Che Ho, Paul J. Wolters, Michael A. Matthay, Stefano A. Iantorno, Vincent C. Auyeung, Harold A. Chapman, Claude Jourdan Le Saux
Abstract
In alcohol use disorder (AUD), duodenal macrophage number are reduced, but the underlying mechanisms and consequences for gut permeability remain unknown. Actively drinking AUD patients undergoing standardized inpatient detoxification and healthy controls were enrolled. Fasting blood samples and duodenal biopsies were analyzed using proteomics, histology, multiplex immunohistofluorescence, flow cytometry, and quantitative polymerase chain reaction. Circulating monocytes were differentiated ex vivo into macrophages to assess migration, differentiation, phagocytosis, and cytokine release following stimulation with bacterial products. Gut permeability was evaluated using urinary 51Chromium- ethylenediaminetetraacetic acid excretion, and plasma surrogate markers of microbial translocation. AUD patients showed markedly reduced duodenal macrophages despite increased circulating monocytes and tissue monocyte-attracting chemokines. AUD monocytes exhibited reduced CCR2 expression and downregulation of tissue proteins involved in adhesion and trans-endothelial migration, thereby decreasing mucosal infiltration. Early macrophage differentiation associated was impaired, with reduced CSF1, CSF2, IL-34, and CSF1 receptor expression. Despite preserved phagocytic capacity, residual tissue macrophages displayed a mixed immune phenotype with reduced expression of scavenger receptors involved in microbial responses and cytokine hyporesponsiveness to lipopolysaccharide and peptidoglycan. Reduced macrophage numbers correlated with increased intestinal permeability and markers of microbial translocation. These results link disruption of the duodenal monocyte-macrophage axis to intestinal barrier failure in AUD.
Authors
Yifan Hu, Jerome Ambroise, Luca Maccioni, Laure-Alix Clerbaux, Caroline Bouzin, Bernd Schnabl, Peter Stärkel
Abstract
Children with cystic fibrosis (CwCF) have impaired linear growth that correlates with poor clinical outcomes. Growth-stunted CwCF also exhibit a marked fecal dysbiosis, and lower serum levels of the gut microbiome-regulated growth factor IGF-1, relative to those without stunting. We hypothesized that the CF gut microbiome negatively impacts IGF-1 and skeletal growth in CwCF. To test this hypothesis, we compared bone phenotypes, IGF-1 and linear growth trajectories of C57BL/6 Cftrtm1Unc mutant mice (CF) to their wild-type littermates (WT) either colonized with (specific pathogen free, SPF) or without (germ-free, GF) normal microbiota. At 4 weeks of age, CF mice had reduced femur length and diminished serum IGF-1 compared to WT mice under SPF, but not GF, conditions. In contrast, by 14 weeks femur lengths and IGF-1 were similar in CF mice raised under both SPF and GF conditions. Accordingly, we observed significantly slower growth in CF mice under SPF, but not GF, conditions only from 1 to 4 weeks. CF mice also exhibited more pronounced relative taxonomic and functional dysbiosis at 4 compared with 14 weeks. These results suggest that gut microbiota play a critical role in early postnatal CF skeletal growth impairment, while microbe-independent mechanisms likely predominate in later life.
Authors
Xiaomeng You, Jisun Paik, Stacey Meeker, Kevin S. Mears, Christopher E. Pope, Hillary S. Hayden, Kelsi Penewit, Adam Waalkes, Sruthi Sridevan, Stephen J. Salipante, Lucas R. Hoffman, Julia F. Charles
Abstract
Basal-like double-negative prostate cancer (DNPC) and triple-negative breast cancer (TNBC) are aggressive tumors with few targeted treatment options, raising the question of whether a shared, druggable cell-surface antigen can be leveraged for effective immunotherapy. Here, we integrated analyses of independent bulk and single-cell transcriptomic datasets with mechanistic and preclinical therapeutic studies to evaluate CDH3 (P-cadherin) as a candidate target in basal-like disease. CDH3 expression was consistently enriched in basal-like subtypes across prostate and breast cancer cohorts and was preferentially expressed in basal epithelial tumor compartments. Mechanistically, CDH3 expression was governed by a YAP1–WNT5A/ROR2 signaling axis, linking basal subtype gene expression to non-canonical Wnt signaling. To exploit this target, we evaluated both a CDH3-directed antibody-drug conjugate (ADC) and CDH3-specific chimeric antigen receptor (CAR)-T cells. The ADC exhibited antigen-dependent internalization and cytotoxicity in vitro and reduced metastatic tumor burden in vivo. CDH3 CAR-T cells showed antigen-specific activation and tumor cell killing in vitro and improved tumor control and survival in metastatic prostate and breast cancer xenograft models, with additional benefit when combined with PD-1 blockade (nivolumab). Together, these findings establish CDH3 as a clinically relevant and therapeutically actionable target and support CDH3-directed ADC and CAR-T strategies for aggressive, basal-like DNPC and TNBC.
Authors
Guoqiang Liu, Shiyu Wang, Ziyu Zeng, Loan Duong, Jing Yang, Nipuni Barupala, Yun Zhao, Sharif Rahmy, Shan Feng, Xiaotian Chi, Ariana Arce, Linchuan Jia, Jun Wan, Liang Cheng, Xuemin Lu, Xin Lu
Abstract
Authors
Alejandra M. Marinelarena, Thao D. Pham, Jeffrey H. Becker, Apurba Majumder, Hidayatullah G. Munshi
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