Research Article
Abstract
Intestinal fibrosis, a severe complication of Crohn’s disease (CD), is characterized by excessive extracellular matrix (ECM) deposition and induces intestinal strictures, but there are no effective antifibrosis drugs available for clinical application. We performed single-cell RNA sequencing (scRNA-Seq) of fibrotic and nonfibrotic ileal tissues from patients with CD with intestinal obstruction. Analysis revealed mesenchymal stromal cells (MSCs) as the major producers of ECM and the increased infiltration of its subset FAP+ fibroblasts in fibrotic sites, which was confirmed by immunofluorescence and flow cytometry. Single-cell transcriptomic profiling of chronic dextran sulfate sodium salt murine colitis model revealed that CD81+Pi16– fibroblasts exhibited transcriptomic and functional similarities to human FAP+ fibroblasts. Consistently, FAP+ fibroblasts were identified as the key subtype with the highest level of ECM production in fibrotic intestines. Furthermore, specific knockout or pharmacological inhibition of TWIST1, which was highly expressed by FAP+ fibroblasts, could significantly ameliorate fibrosis in mice. In addition, TWIST1 expression was induced by CXCL9+ macrophages enriched in fibrotic tissues via IL-1β and TGF-β signal. These findings suggest the inhibition of TWIST1 as a promising strategy for CD fibrosis treatment.
Authors
Yao Zhang, Jiaxin Wang, Hongxiang Sun, Zhenzhen Xun, Zirui He, Yizhou Zhao, Jingjing Qi, Sishen Sun, Qidi Yang, Yubei Gu, Ling Zhang, Chunhua Zhou, Youqiong Ye, Ningbo Wu, Duowu Zou, Bing Su
Abstract
Patients affected by glioma frequently experience epileptic discharges; however, the causes of brain tumor–related epilepsy (BTRE) are still not completely understood. We investigated the mechanisms underlying BTRE by analyzing the effects of exosomes released by U87 glioma cells and by patient-derived glioma cells. Rat hippocampal neurons incubated for 24 hours with these exosomes exhibited increased spontaneous firing, while their resting membrane potential shifted positively by 10–15 mV. Voltage clamp recordings demonstrated that the activation of the Na+ current shifted toward more hyperpolarized voltages by 10–15 mV. To understand the factors inducing hyperexcitability, we focused on exosomal cytokines. Western blot and ELISAs showed that TNF-α was present inside glioma-derived exosomes. Remarkably, incubation with TNF-α fully mimicked the phenotype induced by exosomes, with neurons firing continuously, while their resting membrane potential shifted positively. Real-time PCR revealed that both exosomes and TNF-α induced overexpression of the voltage-gated Na+ channel Nav1.6, a low-threshold Na+ channel responsible for hyperexcitability. When neurons were preincubated with infliximab, a specific TNF-α inhibitor, the hyperexcitability induced by exosomes and TNF-α was drastically reduced. We propose that infliximab, an FDA-approved drug to treat rheumatoid arthritis, could ameliorate the conditions of glioma patients with BTRE.
Authors
Cesar Adolfo Sanchez Trivino, Renza Spelat, Federica Spada, Camilla D’Angelo, Ivana Manini, Irene Giulia Rolle, Tamara Ius, Pietro Parisse, Anna Menini, Daniela Cesselli, Miran Skrap, Fabrizia Cesca, Vincent Torre
Abstract
Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate, we administered the fibroblast-selective TGF-β1 signaling inhibitor epigallocatechin gallate (EGCG) to interstitial lung disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA-Seq on spare tissue. Biopsies from untreated patients showed higher fibroblast TGF-β1 signaling compared with nondisease donor or end-stage ILD tissues. In vivo, EGCG downregulated TGF-β1 signaling and several proinflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted frizzled-related protein 2 (sFRP2), an unrecognized TGF-β1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s) in situ. Using AEC2-fibroblast coculture organoids and precision-cut lung slices (PCLSs) from nondiseased donors, we found TGF-β1 signaling promotes a spread AEC2 KRT17+ basaloid state, whereupon sFRP2 then activates a mature cytokeratin 5+ (Krt5+) basal cell program. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin signaling were required for sFRP2-induced nuclear NFATc3 accumulation and KRT5 expression. These findings highlight stage-specific TGF-β1 signaling in ILD and the therapeutic potential of EGCG in reducing idiopathic pulmonary fibrosis–related (IPF-related) transcriptional changes and identify TGF-β1/noncanonical Wnt pathway crosstalk via sFRP2 as a mechanism for dysfunctional epithelial signaling in IPF/ILD.
Authors
Max L. Cohen, Alexis N. Brumwell, Tsung Che Ho, Kiana Garakani, Genevieve Montas, Darren Leong, Vivianne W. Ding, Jeffrey A. Golden, Binh N. Trinh, David M. Jablons, Michael A. Matthay, Kirk D. Jones, Paul J. Wolters, Ying Wei, Harold A. Chapman, Claude Jourdan Le Saux
Abstract
Soluble host factors in the upper respiratory tract can serve as the first line of defense against SARS-CoV-2 infection. In this study, we described the identification and function of a human airway trypsin–like protease (HAT), capable of reducing the infectivity of ancestral SARS-CoV-2. Further, in mouse models, HAT analogue expression was upregulated by SARS-CoV-2 infection. The antiviral activity of HAT functioned through the cleavage of the SARS-CoV-2 spike glycoprotein at R682. This cleavage resulted in inhibition of the attachment of ancestral spike proteins to host cells, which inhibited the cell-cell membrane fusion process. Importantly, exogenous addition of HAT notably reduced the infectivity of ancestral SARS-CoV-2 in vivo. However, HAT was ineffective against the Delta variant and most circulating Omicron variants, including the BQ.1.1 and XBB.1.5 subvariants. We demonstrate that the P681R mutation in Delta and P681H mutation in the Omicron variants, adjacent to the R682 cleavage site, contributed to HAT resistance. Our study reports what we believe to be a novel soluble defense factor against SARS-CoV-2 and resistance of its actions in the Delta and Omicron variants.
Authors
Wenyan Ren, Weiqi Hong, Jingyun Yang, Jun Zou, Li Chen, Yanan Zhou, Hong Lei, Aqu Alu, Haiying Que, Yanqiu Gong, Zhenfei Bi, Cai He, Minyang Fu, Dandan Peng, Yun Yang, Wenhai Yu, Cong Tang, Qing Huang, Mengli Yang, Bai Li, Jingmei Li, Junbin Wang, Xuelei Ma, Hongbo Hu, Wei Cheng, Haohao Dong, Jian Lei, Lu Chen, Xikun Zhou, Jiong Li, Wei Wang, Guangwen Lu, Guobo Shen, Li Yang, Jinliang Yang, Zhenling Wang, Guowen Jia, Zhaoming Su, Bin Shao, Hanpei Miao, Johnson Yiu-Nam Lau, Yuquan Wei, Kang Zhang, Lunzhi Dai, Shuaiyao Lu, Xiawei Wei
Abstract
Fibrosis represents the uncontrolled replacement of parenchymal tissue with extracellular matrix (ECM) produced by myofibroblasts. While genetic fate-tracing and single-cell RNA-Seq technologies have helped elucidate fibroblast heterogeneity and ontogeny beyond fibroblast to myofibroblast differentiation, newly identified fibroblast populations remain ill defined, with respect to both the molecular cues driving their differentiation and their subsequent role in fibrosis. Using an unbiased approach, we identified the metalloprotease ADAMTS12 as a fibroblast-specific gene that is strongly upregulated during active fibrogenesis in humans and mice. Functional in vivo KO studies in mice confirmed that Adamts12 was critical during fibrogenesis in both heart and kidney. Mechanistically, using a combination of spatial transcriptomics and expression of catalytically active or inactive ADAMTS12, we demonstrated that the active protease of ADAMTS12 shaped ECM composition and cleaved hemicentin 1 (HMCN1) to enable the activation and migration of a distinct injury-responsive fibroblast subset defined by aberrant high JAK/STAT signaling.
Authors
Konrad Hoeft, Lars Koch, Susanne Ziegler, Ling Zhang, Steffen Luetke, Maria C. Tanzer, Debashish Mohanta, David Schumacher, Felix Schreibing, Qingqing Long, Hyojin Kim, Barbara M. Klinkhammer, Carla Schikarski, Sidrah Maryam, Mathijs Baens, Juliane Hermann, Sarah Krieg, Fabian Peisker, Laura De Laporte, Gideon J.L. Schaefer, Sylvia Menzel, Joachim Jankowski, Benjamin D. Humphreys, Adam Wahida, Rebekka K. Schneider, Matthias Versele, Peter Boor, Matthias Mann, Gerhard Sengle, Sikander Hayat, Rafael Kramann
Abstract
CD8+ T cells destroy insulin-producing pancreatic β cells in type 1 diabetes through HLA class I–restricted presentation of self-antigens. Combinatorial peptide library screening was used to produce a preferred peptide recognition landscape for a patient-derived T cell receptor (TCR) that recognized the preproinsulin-derived (PPI-derived) peptide sequence LWMRLLPLL in the context of disease risk allele HLA A*24:02. Data were used to generate a strong superagonist peptide, enabling production of an autoimmune HLA A*24:02–peptide–TCR structure by crystal seeding. TCR binding to the PPI epitope was strongly focused on peptide residues Arg4 and Leu5, with more flexibility at other positions, allowing the TCR to strongly engage many peptides derived from pathogenic bacteria. We confirmed an epitope from Klebsiella that was recognized by PPI-reactive T cells from 3 of 3 HLA A*24:02+ patients. Remarkably, the same epitope selected T cells from 7 of 8 HLA A*24+ healthy donors that cross-reacted with PPI, leading to recognition and killing of HLA A*24:02+ cells expressing PPI. These data provide a mechanism by which molecular mimicry between pathogen and self-antigens could have resulted in the breaking of self-tolerance to initiate disease.
Authors
Garry Dolton, Anna Bulek, Aaron Wall, Hannah Thomas, Jade R. Hopkins, Cristina Rius, Sarah A.E. Galloway, Thomas Whalley, Li Rong Tan, Théo Morin, Nader Omidvar, Anna Fuller, Katie Topley, Md Samiul Hasan, Shikha Jain, Nirupa D’Souza, Thomas Hodges-Hoyland, the TIRID Consortium, Owen B. Spiller, Deborah Kronenberg-Versteeg, Barbara Szomolay, Hugo A. van den Berg, Lucy C. Jones, Mark Peakman, David K. Cole, Pierre J. Rizkallah, Andrew K. Sewell
Abstract
The widespread use of potent androgen receptor signaling inhibitors (ARSIs) has led to an increasing emergence of AR-independent castration-resistant prostate cancer (CRPC), typically driven by loss of AR expression, lineage plasticity, and transformation to prostate cancers (PCs) that exhibit phenotypes of neuroendocrine or basal-like cells. The anti-apoptotic protein BCL2 is upregulated in neuroendocrine cancers and may be a therapeutic target for this aggressive PC disease subset. There is an unmet clinical need, therefore, to clinically characterize BCL2 expression in metastatic CRPC (mCRPC), determine its association with AR expression, uncover its mechanisms of regulation, and evaluate BCL2 as a therapeutic target and/or biomarker with clinical utility. Here, using multiple PC biopsy cohorts and models, we demonstrate that BCL2 expression is enriched in AR-negative mCRPC, associating with shorter overall survival and resistance to ARSIs. Moreover, high BCL2 expression associates with lineage plasticity features and neuroendocrine marker positivity. We provide evidence that BCL2 expression is regulated by DNA methylation, associated with epithelial-mesenchymal transition, and increased by the neuronal transcription factor ASCL1. Finally, BCL2 inhibition had antitumor activity in some, but not all, BCL2-positive PC models, highlighting the need for combination strategies to enhance tumor cell apoptosis and enrich response.
Authors
Daniel Westaby, Juan M. Jiménez-Vacas, Ines Figueiredo, Jan Rekowski, Claire Pettinger, Bora Gurel, Arian Lundberg, Denisa Bogdan, Lorenzo Buroni, Antje Neeb, Ana Padilha, Joe Taylor, Wanting Zeng, Souvik Das, Emily Hobern, Ruth Riisnaes, Mateus Crespo, Susana Miranda, Ana Ferreira, Brian P. Hanratty, Daniel Nava Rodrigues, Claudia Bertan, George Seed, Maria de Los Dolores Fenor de La Maza, Christina Guo, Juliet Carmichael, Rafael Grochot, Khobe Chandran, Anastasia Stavridi, Andreas Varkaris, Nataly Stylianou, Brett G. Hollier, Nina Tunariu, Steven P. Balk, Suzanne Carreira, Wei Yuan, Peter S. Nelson, Eva Corey, Michael Haffner, Johann de Bono, Adam Sharp
Abstract
Maintaining protein homeostasis (proteostasis) requires precise control of protein folding and degradation. Failure to properly respond to stresses disrupts proteostasis, which is a hallmark of many diseases, including cataracts. Hibernators are natural cold-stress adaptors; however, little is known about how they keep a balanced proteome under conditions of drastic temperature shift. Intriguingly, we identified a reversible lens opacity phenotype in ground squirrels (GSs) associated with their hibernation-rewarming process. To understand this “cataract-reversing” phenomenon, we first established induced lens epithelial cells differentiated from GS-derived induced pluripotent stem cells, which helped us explore the molecular mechanism preventing the accumulation of protein aggregates in GS lenses. We discovered that the ubiquitin-proteasome system (UPS) played a vital role in minimizing the aggregation of the lens protein αA-crystallin (CRYAA) during rewarming. Such function was, for the first time to our knowledge, associated with an E3 ubiquitin ligase, RNF114, which appears to be one of the key mechanisms mediating the turnover and homeostasis of lens proteins. Leveraging this knowledge gained from hibernators, we engineered a deliverable RNF114 complex and successfully reduced lens opacity in rats with cold-induced cataracts and zebrafish with oxidative stress–related cataracts. These data provide new insights into the critical role of the UPS in maintaining proteostasis in cold and possibly other forms of stresses. The newly identified E3 ubiquitin ligase RNF114, related to CRYAA, offers a promising avenue for treating cataracts with protein aggregates.
Authors
Hao Yang, Xiyuan Ping, Jiayue Zhou, Hailaiti Ailifeire, Jing Wu, Francisco M. Nadal-Nicolás, Kiyoharu J. Miyagishima, Jing Bao, Yuxin Huang, Yilei Cui, Xin Xing, Shiqiang Wang, Ke Yao, Wei Li, Xingchao Shentu
Abstract
T cell–based immunotherapies are a promising therapeutic approach for multiple malignancies, but their efficacy is limited by tumor hypoxia arising from dysfunctional blood vessels. Here, we report that cell-intrinsic properties of a single vascular component, namely the pericyte, contribute to the control of tumor oxygenation, macrophage polarization, vessel inflammation, and T cell infiltration. Switching pericyte phenotype from a synthetic to a differentiated state reverses immune suppression and sensitizes tumors to adoptive T cell therapy, leading to regression of melanoma in mice. In melanoma patients, improved survival is correlated with enhanced pericyte maturity. Importantly, pericyte plasticity is regulated by signaling pathways converging on Rho kinase activity, with pericyte maturity being inducible by selective low-dose therapeutics that suppress pericyte MEK, AKT, or notch signaling. We also show that low-dose targeted anticancer therapy can durably change the tumor microenvironment without inducing adaptive resistance, creating a highly translatable pathway for redosing anticancer targeted therapies in combination with immunotherapy to improve outcome.
Authors
Zhi-Jie Li, Bo He, Alice Domenichini, Jiulia Satiaputra, Kira H. Wood, Devina D. Lakhiani, Abate A. Bashaw, Lisa M. Nilsson, Ji Li, Edward R. Bastow, Anna Johansson-Percival, Elena Denisenko, Alistair R.R. Forrest, Suraj Sakaram, Rafael Carretero, Günter J. Hämmerling, Jonas A. Nilsson, Gabriel Y.F. Lee, Ruth Ganss
Abstract
Neovascular age-related macular degeneration (nAMD) remains a major cause of visual impairment and puts considerable burden on patients and health care systems. l-DOPA–treated Parkinson’s disease (PD) patients have been shown to be partially protected from nAMD, but the mechanism remains unknown. Using murine models that combine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–induced (MPTP-induced) PD and laser-induced nAMD with standard PD treatment of l-DOPA/DOPA-decarboxylase inhibitor or specific dopamine receptor inhibitors, we here demonstrate that l-DOPA treatment–induced increase of dopamine-mediated dopamine receptor D2 (DRD2) signaling inhibits choroidal neovascularization independently of MPTP-associated nigrostriatal pathway lesion. Analyzing a retrospective cohort of more than 200,000 patients with nAMD receiving anti-VEGF treatment from the French nationwide insurance database, we show that DRD2 agonist–treated PD patients have a significantly delayed age of onset of nAMD and reduced need for anti-VEGF therapies, similar to the effects of the l-DOPA treatment. While providing a mechanistic explanation for an intriguing epidemiological observation, our findings suggest that systemic DRD2 agonists might constitute an adjuvant therapy to delay and reduce the need for anti-VEGF therapy in patients with nAMD.
Authors
Thibaud Mathis, Florian Baudin, Anne-Sophie Mariet, Sébastien Augustin, Marion Bricout, Lauriane Przegralek, Christophe Roubeix, Éric Benzenine, Guillaume Blot, Caroline Nous, Laurent Kodjikian, Martine Mauget-Faÿsse, José-Alain Sahel, Robin Plevin, Christina Zeitz, Cécile Delarasse, Xavier Guillonneau, Catherine Creuzot-Garcher, Catherine Quantin, Stéphane Hunot, Florian Sennlaub
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ISSN: 0021-9738 (print), 1558-8238 (online)