Issue published April 8, 2024
- Volume 9, Issue 7
- Previous Issue
Salas-Lucia et al. report that thyroid hormone cell transporter MCT8 is critical for early neurogenesis, mediating the bulk of the triiodothyronine (T3) uptake into developing human neural cells. The cover shows an immunofluorescence image of a neural rosette from an induced pluripotent stem cell–derived cerebral organoid stained to highlight dividing human neural precursor cells (green and red) and nuclei (blue). Images credit: Federico Salas-Lucia.
On the cover: Impaired T3 uptake and action in MCT8-deficient cerebral organoids underlie Allan-Herndon-Dudley syndrome
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Research Articles
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Abstract
Programmed cell death protein 1 (PD-1), a coinhibitory T cell checkpoint, is also expressed on macrophages in pathogen- or tumor-driven chronic inflammation. Increasing evidence underscores the importance of PD-1 on macrophages for dampening immune responses. However, the mechanism governing PD-1 expression in macrophages in chronic inflammation remains largely unknown. TGF-β1 is abundant within chronic inflammatory microenvironments. Here, based on public databases, significantly positive correlations between PDCD1 and TGFB1 gene expression were observed in most human tumors. Of note, among immune infiltrates, macrophages as the predominant infiltrate expressed higher PDCD1 and TGFBR1/TGFBR2 genes. MC38 colon cancer and Schistosoma japonicum infection were used as experimental models for chronic inflammation. PD-1hi macrophages from chronic inflammatory tissues displayed an immunoregulatory pattern and expressed a higher level of TGF-β receptors. Either TGF-β1–neutralizing antibody administration or macrophage-specific Tgfbr1 knockdown largely reduced PD-1 expression on macrophages in animal models. We further demonstrated that TGF-β1 directly induced PD-1 expression on macrophages. Mechanistically, TGF-β1–induced PD-1 expression on macrophages was dependent on SMAD3 and STAT3, which formed a complex at the Pdcd1 promoter. Collectively, our study shows that macrophages adapt to chronic inflammation through TGF-β1–triggered cooperative SMAD3/STAT3 signaling that induces PD-1 expression and modulates macrophage function.
Authors
Zhigang Lei, Rui Tang, Yu Wu, Chenxu Mao, Weijie Xue, Junyao Shen, Jiaojiao Yu, Xiaohong Wang, Xin Qi, Chuan Wei, Lei Xu, Jifeng Zhu, Yalin Li, Xiujun Zhang, Chunyan Ye, Xiaojun Chen, Xiaojun Yang, Sha Zhou, Chuan Su
×Abstract
In autoimmunity, FOXP3+ Tregs skew toward a proinflammatory, nonsuppressive phenotype and are, therefore, unable to control the exaggerated autoimmune response. This largely affects the success of autologous Treg therapy, which is currently under investigation for autoimmune diseases, including multiple sclerosis (MS). There is a need to ensure in vivo Treg stability before successful application of Treg therapy. Using genetic fate-mapping mice, we demonstrate that inflammatory, cytokine-expressing exFOXP3 T cells accumulate in the CNS during experimental autoimmune encephalomyelitis. In a human in vitro model, we discovered that interaction with inflamed blood-brain barrier endothelial cells (BBB-ECs) induces loss of function by Tregs. Transcriptome and cytokine analysis revealed that in vitro migrated Tregs have disrupted regenerative potential and a proinflammatory Th1/17 signature, and they upregulate the mTORC1 signaling pathway. In vitro treatment of migrated human Tregs with the clinically approved mTORC1 inhibitor rapamycin restored suppression. Finally, flow cytometric analysis indicated an enrichment of inflammatory, less-suppressive CD49d+ Tregs in the cerebrospinal fluid of people with MS. In summary, interaction with BBB-ECs is sufficient to affect Treg function, and transmigration triggers an additive proinflammatory phenotype switch. These insights help improve the efficacy of autologous Treg therapy of MS.
Authors
Paulien Baeten, Ibrahim Hamad, Cindy Hoeks, Michael Hiltensperger, Bart Van Wijmeersch, Veronica Popescu, Lilian Aly, Veerle Somers, Thomas Korn, Markus Kleinewietfeld, Niels Hellings, Bieke Broux
×Abstract
Myocardial ischemia/reperfusion (MI/R) injury is a major cause of adverse outcomes of revascularization following myocardial infarction. Anaerobic glycolysis during myocardial ischemia is well studied, but the role of aerobic glycolysis during the early phase of reperfusion is incompletely understood. Lactylation of Histone H3 (H3) is an epigenetic indicator of the glycolytic switch. Heat shock protein A12A (HSPA12A) is an atypic member of the HSP70 family. In the present study, we report that, during reperfusion following myocardial ischemia, HSPA12A was downregulated and aerobic glycolytic flux was decreased in cardiomyocytes. Notably, HSPA12A KO in mice exacerbated MI/R-induced aerobic glycolysis decrease, cardiomyocyte death, and cardiac dysfunction. Gain- and loss-of-function studies demonstrated that HSPA12A was required to support cardiomyocyte survival upon hypoxia/reoxygenation (H/R) challenge and that its protective effects were mediated by maintaining aerobic glycolytic homeostasis for H3 lactylation. Further analyses revealed that HSPA12A increased Smurf1-mediated Hif1α protein stability, thus increasing glycolytic gene expression to maintain appropriate aerobic glycolytic activity to sustain H3 lactylation during reperfusion and, ultimately, improving cardiomyocyte survival to attenuate MI/R injury.
Authors
Wansu Yu, Qiuyue Kong, Surong Jiang, Yunfan Li, Zhaohe Wang, Qian Mao, Xiaojin Zhang, Qianhui Liu, Pengjun Zhang, Yuehua Li, Chuanfu Li, Zhengnian Ding, Li Liu
×Abstract
Compromised vascular integrity facilitates extravasation of cancer cells and promotes metastatic dissemination. CD93 has emerged as a target for antiangiogenic therapy, but its importance for vascular integrity in metastatic cancers has not been evaluated. Here, we demonstrate that CD93 participates in maintaining the endothelial barrier and reducing metastatic dissemination. Primary melanoma growth was hampered in CD93–/– mice, but metastatic dissemination was increased and associated with disruption of adherens and tight junctions in tumor endothelial cells and elevated expression of matrix metalloprotease 9 at the metastatic site. CD93 directly interacted with vascular endothelial growth factor receptor 2 (VEGFR2) and its absence led to VEGF-induced hyperphosphorylation of VEGFR2 in endothelial cells. Antagonistic anti-VEGFR2 antibody therapy rescued endothelial barrier function and reduced the metastatic burden in CD93–/– mice to wild-type levels. These findings reveal a key role of CD93 in maintaining vascular integrity, which has implications for pathological angiogenesis and endothelial barrier function in metastatic cancer.
Authors
Kalyani Vemuri, Beatriz de Alves Pereira, Patricia Fuenzalida, Yelin Subashi, Stefano Barbera, Luuk van Hooren, Marie Hedlund, Fredrik Pontén, Cecilia Lindskog, Anna-Karin Olsson, Roberta Lugano, Anna Dimberg
×Abstract
BACKGROUND As Omicron is prompted to replicate in the upper airway, neutralizing antibodies (NAbs) delivered through inhalation might inhibit early-stage infection in the respiratory tract. Thus, elucidating the prophylactic efficacy of NAbs via nasal spray addresses an important clinical need.METHODS The applicable potential of a nasal spray cocktail containing 2 NAbs was characterized by testing its neutralizing potency, synergetic neutralizing mechanism, emergency protective and therapeutic efficacy in a hamster model, and pharmacokinetics/pharmacodynamic (PK/PD) in human nasal cavity.RESULTS The 2 NAbs displayed broad neutralizing efficacy against Omicron, and they could structurally compensate each other in blocking the Spike-ACE2 interaction. When administrated through the intranasal mucosal route, this cocktail demonstrated profound efficacy in the emergency prevention in hamsters challenged with authentic Omicron BA.1. The investigator-initiated trial in healthy volunteers confirmed the safety and the PK/PD of the NAb cocktail delivered via nasal spray. Nasal samples from the participants receiving 4 administrations over a course of 16 hours demonstrated potent neutralization against Omicron BA.5 in an ex vivo pseudovirus neutralization assay.CONCLUSION These results demonstrate that the NAb cocktail nasal spray provides a good basis for clinical prophylactic efficacy against Omicron infections.TRIAL REGISTRATION www.chictr.org.cn, ChiCTR2200066525.FUNDING The National Science and Technology Major Project (2017ZX10202203), the National Key Research and Development Program of China (2018YFA0507100), Guangzhou National Laboratory (SRPG22-015), Lingang Laboratory (LG202101-01-07), Science and Technology Commission of Shanghai Municipality (YDZX20213100001556), and the Emergency Project from the Science & Technology Commission of Chongqing (cstc2021jscx-fyzxX0001).
Authors
Xinghai Zhang, Feiyang Luo, Huajun Zhang, Hangtian Guo, Junhui Zhou, Tingting Li, Shaohong Chen, Shuyi Song, Meiying Shen, Yan Wu, Yan Gao, Xiaojian Han, Yingming Wang, Chao Hu, Xiaodong Zhao, Huilin Guo, Dazhi Zhang, Yuchi Lu, Wei Wang, Kai Wang, Ni Tang, Tengchuan Jin, Menglu Ding, Shuhui Luo, Cuicui Lin, Tingting Lu, Bingxia Lu, Yang Tian, Chengyong Yang, Guofeng Cheng, Haitao Yang, Aishun Jin, Xiaoyun Ji, Rui Gong, Sandra Chiu, Ailong Huang
×Abstract
BACKGROUND Diagnosis of PMM2-CDG, the most common congenital disorder of glycosylation (CDG), relies on measuring carbohydrate-deficient transferrin (CDT) and genetic testing. CDT tests have false negatives and may normalize with age. Site-specific changes in protein N-glycosylation have not been reported in sera in PMM2-CDG.METHODS Using multistep mass spectrometry–based N-glycoproteomics, we analyzed sera from 72 individuals to discover and validate glycopeptide alterations. We performed comprehensive tandem mass tag–based discovery experiments in well-characterized patients and controls. Next, we developed a method for rapid profiling of additional samples. Finally, targeted mass spectrometry was used for validation in an independent set of samples in a blinded fashion.RESULTS Of the 3,342 N-glycopeptides identified, patients exhibited decrease in complex-type N-glycans and increase in truncated, mannose-rich, and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man5GlcNAc2, which was not detected in controls, in 5 patients with normal CDT results, including 1 after liver transplant and 2 with a known genetic variant associated with mild disease, indicating greater sensitivity than CDT. It was detected by targeted analysis in 2 individuals with variants of uncertain significance in PMM2.CONCLUSION Complement C4–derived Man5GlcNAc2 glycopeptide could be a biomarker for accurate diagnosis and therapeutic monitoring of patients with PMM2-CDG and other CDGs.FUNDING U54NS115198 (Frontiers in Congenital Disorders of Glycosylation: NINDS; NCATS; Eunice Kennedy Shriver NICHD; Rare Disorders Consortium Disease Network); K08NS118119 (NINDS); Minnesota Partnership for Biotechnology and Medical Genomics; Rocket Fund; R01DK099551 (NIDDK); Mayo Clinic DERIVE Office; Mayo Clinic Center for Biomedical Discovery; IA/CRC/20/1/600002 (Center for Rare Disease Diagnosis, Research and Training; DBT/Wellcome Trust India Alliance)
Authors
Kishore Garapati, Rohit Budhraja, Mayank Saraswat, Jinyong Kim, Neha Joshi, Gunveen S. Sachdeva, Anu Jain, Anna N. Ligezka, Silvia Radenkovic, Madan Gopal Ramarajan, Savita Udainiya, Kimiyo Raymond, Miao He, Christina Lam, Austin Larson, Andrew C. Edmondson, Kyriakie Sarafoglou, Nicholas B. Larson, Hudson H. Freeze, Matthew J. Schultz, Tamas Kozicz, Eva Morava, Akhilesh Pandey
×Abstract
The roles of fibronectin leucine-rich transmembrane protein 2 (FLRT2) in physiological and pathological processes are not well known. Here, we identify a potentially novel function of FLRT2 in preventing endothelial cell senescence and vascular aging. We found that FLRT2 expression was lower in cultured senescent endothelial cells as well as in aged rat and human vascular tissues. FLRT2 mediated endothelial cell senescence via the mTOR complex 2, AKT, and p53 signaling pathway in human endothelial cells. We uncovered that FLRT2 directly associated with integrin subunit beta 4 (ITGB4) and thereby promoted ITGB4 phosphorylation, while inhibition of ITGB4 substantially mitigated the induction of senescence triggered by FLRT2 depletion. Importantly, FLRT2 silencing in mice promoted vascular aging, and overexpression of FLRT2 rescued a premature vascular aging phenotype. Therefore, we propose that FLRT2 could be targeted therapeutically to prevent senescence-associated vascular aging.
Authors
Hyun Jung Hwang, Donghee Kang, Jae-Ryong Kim, Joon Hyuk Choi, Ji-Kan Ryu, Allison B. Herman, Young-Gyu Ko, Heon Joo Park, Myriam Gorospe, Jae-Seon Lee
×Abstract
Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases facilitate viral entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleaved the S protein at multiple sites and abrogated viral entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with viral clearance and inflammation in COVID-19 patients.
Authors
Nathan G.F. Leborgne, Christelle Devisme, Nedim Kozarac, Inês Berenguer Veiga, Nadine Ebert, Aurélie Godel, Llorenç Grau-Roma, Melanie Scherer, Philippe Plattet, Volker Thiel, Gert Zimmer, Adriano Taddeo, Charaf Benarafa
×Abstract
Fibroblast growth factor 15/19 (FGF15/19, mouse/human ortholog) is expressed in the ileal enterocytes of the small intestine and released postprandially in response to bile acid absorption. Previous reports of FGF15–/– mice have limited our understanding of gut-specific FGF15’s role in metabolism. Therefore, we studied the role of endogenous gut-derived FGF15 in bile acid, cholesterol, glucose, and energy balance. We found that circulating levels of FGF19 were reduced in individuals with obesity and comorbidities, such as type 2 diabetes and metabolic dysfunction–associated fatty liver disease. Gene expression analysis of ileal FGF15-positive cells revealed differential expression during the obesogenic state. We fed standard chow or a high-fat metabolic dysfunction-associated steatohepatitis–inducing diet to control and intestine-derived FGF15-knockout (FGF15INT-KO) mice. Control and FGF15INT-KO mice gained similar body weight and adiposity and did not show genotype-specific differences in glucose, mixed meal, pyruvate, and glycerol tolerance. FGF15INT-KO mice had increased systemic bile acid levels but decreased cholesterol levels, pointing to a primary role for gut-derived FGF15 in regulating bile acid and cholesterol metabolism when exposed to obesogenic diet. These studies show that intestinal FGF15 plays a specific role in bile acid and cholesterol metabolism regulation but is not essential for energy and glucose balance.
Authors
Nadejda Bozadjieva-Kramer, Jae Hoon Shin, Ziru Li, Alan C. Rupp, Nicole Miller, Stace Kernodle, Nicolas Lanthier, Paulina Henry, Nikhil Seshadri, Andriy Myronovych, Ormond A. MacDougald, Robert W. O’Rourke, Rohit Kohli, Charles F. Burant, Amy E. Rothberg, Randy J. Seeley
×Abstract
Patients with mutations in the thyroid hormone (TH) cell transporter monocarboxylate transporter 8 (MCT8) gene develop severe neuropsychomotor retardation known as Allan-Herndon-Dudley syndrome (AHDS). It is assumed that this is caused by a reduction in TH signaling in the developing brain during both intrauterine and postnatal developmental stages, and treatment remains understandably challenging. Given species differences in brain TH transporters and the limitations of studies in mice, we generated cerebral organoids (COs) using human induced pluripotent stem cells (iPSCs) from MCT8-deficient patients. MCT8-deficient COs exhibited (i) altered early neurodevelopment, resulting in smaller neural rosettes with thinner cortical units, (ii) impaired triiodothyronine (T3) transport in developing neural cells, as assessed through deiodinase-3–mediated T3 catabolism, (iii) reduced expression of genes involved in cerebral cortex development, and (iv) reduced T3 inducibility of TH-regulated genes. In contrast, the TH analogs 3,5-diiodothyropropionic acid and 3,3′,5-triiodothyroacetic acid triggered normal responses (induction/repression of T3-responsive genes) in MCT8-deficient COs, constituting proof of concept that lack of T3 transport underlies the pathophysiology of AHDS and demonstrating the clinical potential for TH analogs to be used in treating patients with AHDS. MCT8-deficient COs represent a species-specific relevant preclinical model that can be utilized to screen drugs with potential benefits as personalized therapeutics for patients with AHDS.
Authors
Federico Salas-Lucia, Sergio Escamilla, Antonio C. Bianco, Alexandra Dumitrescu, Samuel Refetoff
×Abstract
IKK2/NF-κB pathway–mediated inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2/NF-κB pathway in medial calcification remains to be elucidated. In this study, we found that chronic kidney disease (CKD) induces inflammatory pathways through the local activation of the IKK2/NF-κB pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2/NF-κB pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NF-κB by SMC-specific IκBα deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2/NF-κB pathway induced cell death of VSMCs by reducing anti–cell death gene expression, whereas activation of NF-κB reduced CKD-dependent vascular cell death. In addition, increased calcification of extracellular vesicles through the inhibition of the IKK2/NF-κB pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death in vitro and in vivo. This study reveals that activation of the IKK2/NF-κB pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles.
Authors
Shinobu Miyazaki-Anzai, Masashi Masuda, Audrey L. Keenan, Yuji Shiozaki, Jose G. Miranda, Makoto Miyazaki
×Abstract
BACKGROUND Broadly neutralizing monoclonal antibodies (bNAbs) represent a promising strategy for HIV-1 immunoprophylaxis and treatment. 10E8VLS and VRC07-523LS are bNAbs that target the highly conserved membrane-proximal external region (MPER) and the CD4-binding site of the HIV-1 viral envelope glycoprotein, respectively.METHODS In this phase 1, open-label trial, we evaluated the safety and pharmacokinetics of 5 mg/kg 10E8VLS administered alone, or concurrently with 5 mg/kg VRC07-523LS, via s.c. injection to healthy non–HIV-infected individuals.RESULTS Eight participants received either 10E8VLS alone (n = 6) or 10E8VLS and VRC07-523LS in combination (n = 2). Five (n = 5 of 8, 62.5%) participants who received 10E8VLS experienced moderate local reactogenicity, and 1 participant (n = 1/8, 12.5%) experienced severe local reactogenicity. Further trial enrollment was stopped, and no participant received repeat dosing. All local reactogenicity resolved without sequelae. 10E8VLS retained its neutralizing capacity, and no functional anti-drug antibodies were detected; however, a serum t1/2 of 8.1 days was shorter than expected. Therefore, the trial was voluntarily stopped per sponsor decision (Vaccine Research Center, National Institute of Allergy and Infectious Diseases [NIAID], NIH). Mechanistic studies performed to investigate the underlying reason for the reactogenicity suggest that multiple mechanisms may have contributed, including antibody aggregation and upregulation of local inflammatory markers.CONCLUSION 10E8VLS resulted in unexpected reactogenicity and a shorter t1/2 in comparison with previously tested bNAbs. These studies may facilitate identification of nonreactogenic second-generation MPER-targeting bNAbs, which could be an effective strategy for HIV-1 immunoprophylaxis and treatment.TRIAL REGISTRATION Clinicaltrials.gov, accession no. NCT03565315.FUNDING Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH.
Authors
Seemal F. Awan, Amarendra Pegu, Larisa Strom, Cristina A. Carter, Cynthia S. Hendel, LaSonji A. Holman, Pamela J. Costner, Olga Trofymenko, Renunda Dyer, Ingelise J. Gordon, Ro Shauna S. Rothwell, Somia P. Hickman, Michelle Conan-Cibotti, Nicole A. Doria-Rose, Bob C. Lin, Sarah O’Connell, Sandeep R. Narpala, Cassandra G. Almasri, Cuiping Liu, Sungyoul Ko, Young D. Kwon, Aryan M. Namboodiri, Janardan P. Pandey, Frank J. Arnold, Kevin Carlton, Jason G. Gall, Peter D. Kwong, Edmund V. Capparelli, Robert T. Bailer, Adrian B. McDermott, Grace L. Chen, Richard A. Koup, John R. Mascola, Emily E. Coates, Julie E. Ledgerwood, Martin R. Gaudinski, the VRC 610 study team
×Abstract
Inflammatory lymphangiogenesis is intimately linked to immune regulation and tissue homeostasis. However, current evidence has suggested that classic lymphatic vessels are physiologically absent in intraocular structures. Here, we show that neolymphatic vessels were induced in the iris after corneal alkali injury (CAI) in a VEGFR3-dependent manner. Cre-loxP–based lineage tracing revealed that these lymphatic endothelial cells (LECs) originate from existing Prox1+ lymphatic vessels. Notably, the ablation of iridial lymphangiogenesis via conditional deletion of VEGFR3 alleviated the ocular inflammatory response and pathological T cell infiltration. Our findings demonstrate that iridial neolymphatics actively participate in pathological immune responses following injury and suggest intraocular lymphangiogenesis as a valuable therapeutic target for the treatment of ocular inflammation.
Authors
Zheng Liu, Keli Liu, Shunhua Shi, Xun Chen, Xinyu Gu, Weifa Wang, Keli Mao, Rukeye Yibulayi, Wanwen Wu, Lei Zeng, Weibin Zhou, Xiaofeng Lin, Feng Zhang, Bingsheng Lou
×Abstract
BACKGROUND Differentiating malignant from nonmalignant body fluids remains a clinical challenge because of the unsatisfying performance of conventional cytology. We aimed to improve the sensitivity and ubiquity of cancer cell detection by assaying universal cancer–only methylation (UCOM) markers in supernatant cell-free DNA (cfDNA).METHODS An observational prospective cohort including 1,321 nonmalignant and malignant body fluids of multiple cancers was used to develop and validate a cfDNA UCOM methylation diagnostic assay. All samples were divided into 2 portions for cytology and supernatant cfDNA methylation analysis.RESULTS The significant hypermethylation of a potentially novel UCOM marker, TAGMe, together with the formerly reported PCDHGB7, was identified in the cfDNA of malignant body fluid samples. The combined model, cell-free cancer-universal methylation (CUE), was developed and validated in a prospective multicancer cohort with markedly elevated sensitivity and specificity, and was further verified in a set containing additional types of malignant body fluids and metastases. In addition, it remained hypersensitive in detecting cancer cells in cytologically negative malignant samples.CONCLUSION cfDNA methylation markers are robust in detecting tumor cells and are applicable to diverse body fluids and tumor types, providing a feasible complement to current cytology-based diagnostic analyses.TRIAL REGISTRATION This study was registered at Chictr.org.cn (ChiCTR2200060532).FUNDING National Natural Science Foundation of China (32270645, 31872814, 32000505, 82170088), the National Key R&D Program of Ningxia Hui Autonomous region (2022BEG01003), Shanghai Municipal Key Clinical Specialty (shslczdzk02201), Science and Technology Commission of Shanghai Municipality (20DZ2261200, 20DZ2254400), and Major Special Projects of Basic Research of Shanghai Science and Technology Commission (18JC1411101).
Authors
Zhanrui Mao, Shihua Dong, Yu Yan, Chengyang Wang, Wei Li, Lu Wang, Chengchen Qian, Yuanlin Song, Lin Tong, Wenqiang Yu
×Abstract
Allelic heterogeneity (AH) has been noted in truncational TTN–associated (TTNtv-associated) dilated cardiomyopathy (DCM); i.e., mutations affecting A-band–encoding exons are pathogenic, but those affecting Z-disc–encoding exons are likely benign. The lack of an in vivo animal model that recapitulates AH hinders the deciphering of the underlying mechanism. Here, we explored zebrafish as a candidate vertebrate model by phenotyping a collection of zebrafish ttntv alleles. We noted that cardiac function and sarcomere structure were more severely disrupted in ttntv-A than in ttntv-Z homozygous embryos. Consistently, cardiomyopathy-like phenotypes were present in ttntv-A but not ttntv-Z adult heterozygous mutants. The phenotypes observed in ttntv-A alleles were recapitulated in null mutants with the full titin-encoding sequences removed. Defective autophagic flux, largely due to impaired autophagosome-lysosome fusion, was also noted only in ttntv-A but not in ttntv-Z models. Moreover, we found that genetic manipulation of ulk1a restored autophagy flux and rescued cardiac dysfunction in ttntv-A animals. Together, our findings presented adult zebrafish as an in vivo animal model for studying AH in TTNtv DCM, demonstrated TTN loss of function is sufficient to trigger ttntv DCM in zebrafish, and uncovered ulk1a as a potential therapeutic target gene for TTNtv DCM.
Authors
Ping Zhu, Jiarong Li, Feixiang Yan, Shahidul Islam, Xueying Lin, Xiaolei Xu
×Abstract
The central nervous system HIV reservoir is incompletely understood and is a major barrier to HIV cure. We profiled people with HIV (PWH) and uninfected controls through single-cell transcriptomic and T cell receptor (TCR) sequencing to understand the dynamics of HIV persistence in the CNS. In PWH on ART, we found that most participants had single cells containing HIV-1 RNA, which was found predominantly in CD4 central memory T cells, in both cerebrospinal fluid (CSF) and blood. HIV-1 RNA–containing cells were found more frequently in CSF than blood, indicating a higher burden of reservoir cells in the CNS than blood for some PWH. Most CD4 T cell clones containing infected cells were compartment specific, while some (22%) — including rare clones with members of the clone containing detectable HIV RNA in both blood and CSF — were found in both CSF and blood. These results suggest that infected T cells trafficked between tissue compartments and that maintenance and expansion of infected T cell clones contributed to the CNS reservoir in PWH on ART.
Authors
Meng Wang, Jennifer Yoon, Hailey Reisert, Bibhuprasad Das, Benjamin Orlinick, Jennifer Chiarella, Elias K. Halvas, John Mellors, Alina P.S. Pang, Lydia Aoun Barakat, Margaret Fikrig, Joshua Cyktor, Yuval Kluger, Serena Spudich, Michael J. Corley, Shelli F. Farhadian
×Abstract
The efficacy of chimeric antigen receptor T cell (CAR-T) therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28 EGFRvIII gliomas revealed impaired mitochondrial ATP production and a markedly hypoxic status compared with ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of the AMPK activator metformin and the mTOR inhibitor rapamycin (Met+Rap). Met+Rap–pretreated mouse CAR-T cells showed activated PPAR-γ coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap–pretreated CAR-T cells demonstrated persistent and effective antiglioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap–pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28 EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group, with fewer Ly6c+CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions under in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap–pretreated CAR-T cells in human trials.
Authors
Ryusuke Hatae, Keith Kyewalabye, Akane Yamamichi, Tiffany Chen, Su Phyu, Pavlina Chuntova, Takahide Nejo, Lauren S. Levine, Matthew H. Spitzer, Hideho Okada
×Abstract
To unravel the heterogeneity and molecular signature of effector memory Th2 cells (Tem2), we analyzed 23 individuals’ PBMCs of filaria-infected (Filaria+) and 24 healthy volunteers (Filaria–), with or without coincident house dust mite (HDM) allergic sensitization. Flow cytometry revealed 3 CD4+ Tem subsets — CCR4+CCR6+CRTH2– Tem17, CCR4+CCR6-CRTH2+ Tem2, and CCR6+CCR4+CRTH2+ Tem17.2 — markedly enriched in Filaria+ individuals. These subsets were sorted and analyzed by multiomic single-cell RNA immunoprofiling. SingleR-annotated Th2 cells from Tem2 and Tem17.2 cell subsets had features of pathogenic Th2 effector cells based on their transcriptional signatures, with downregulated CD27 and elevated expression levels of ITGA4, IL17RB, HPGDS, KLRB1, PTGDR2, IL9R, IL4, IL5, and IL13 genes. When the Filaria+ individuals were subdivided based on their allergic status, Tem2 cells in HDM+Filaria+ individuals showed an overall reduction in TCR diversity, suggesting the occurrence of antigen-driven clonal expansion. Moreover, HDM+Filaria+ individuals showed not only an expansion in the frequency of both Tem2 and Tem17.2 cell subsets, but also a change in their molecular program by overexpressing GATA3, IL17RB, CLRF2, and KLRB1, as well as increased antigen-induced IL-4, IL-5, and IL-13 production, suggesting that aeroallergens reshape the transcriptional and functional programming of Th2 cell subsets in human filarial infection toward a pathogenic immunophenotype.
Authors
Pedro H. Gazzinelli-Guimaraes, Brittany Dulek, Phillip Swanson, Justin Lack, Mario Roederer, Thomas B. Nutman
×Abstract
Prior studies showed that polyglutamine-expanded androgen receptor (AR) is aberrantly acetylated and that deacetylation of the mutant AR by overexpression of nicotinamide adenine dinucleotide–dependent (NAD+-dependent) sirtuin 1 is protective in cell models of spinal and bulbar muscular atrophy (SBMA). Based on these observations and reduced NAD+ in muscles of SBMA mouse models, we tested the therapeutic potential of NAD+ restoration in vivo by treating postsymptomatic transgenic SBMA mice with the NAD+ precursor nicotinamide riboside (NR). NR supplementation failed to alter disease progression and had no effect on increasing NAD+ or ATP content in muscle, despite producing a modest increase of NAD+ in the spinal cords of SBMA mice. Metabolomic and proteomic profiles of SBMA quadriceps muscles indicated alterations in several important energy-related pathways that use NAD+, in addition to the NAD+ salvage pathway, which is critical for NAD+ regeneration for use in cellular energy production. We also observed decreased mRNA levels of nicotinamide riboside kinase 2 (Nmrk2), which encodes a key kinase responsible for NR phosphorylation, allowing its use by the NAD+ salvage pathway. Together, these data suggest a model in which NAD+ levels are significantly decreased in muscles of an SBMA mouse model and intransigent to NR supplementation because of decreased levels of Nmrk2.
Authors
Danielle DeBartolo, Frederick J. Arnold, Yuhong Liu, Elana Molotsky, Hsin-Yao Tang, Diane E. Merry
×Abstract
BACKGROUND Intrinsic molecular subtypes define distinct biological breast cancers and can be used to further improve diagnosis and risk allocation.METHODS The Copenhagen Breast Cancer Genomics Study (CBCGS) prospectively included women diagnosed with breast cancer at Rigshospitalet from 2014 to 2021. Eligible patients were females with a primary invasive breast cancer (T1c, if N0M0; otherwise, any T, any N, or any M stage) and no prior malignancy. All patients underwent molecular profiling with the CIT256 and PAM50 molecular profile.RESULTS In the study period, 2,816 patients were included in the CBCGS. Molecular subtyping showed an increase in nonluminal (molecular-apocrine, luminal C, and Basal-like) as compared with luminal (luminal A, luminal B, and Normal-like) subtypes with increasing stage from I to IV. Across all stages, we found a significant difference in survival among subtypes; 91% of patients with LumA were alive at 5 years compared with 91% for LumB, 84% for LumC, 82% for mApo, and 80% for Basal-like. We identified 442 tumors (16%) that were discordant in subtype between CIT256 and IHC. Discordant subtype proved to be a risk factor of death among patients with IHC luminal breast cancer (hazard ratio [HR], 2.08; 95% CI, 1.51–2.86) in a multivariable Cox regression analysis. Discordance occurred more often among patients with N3, stage IV, or grade III disease.CONCLUSION Our findings indicate that molecular subtypes are a predominant classification for survival. Assessment is particularly crucial for patients with IHC luminal breast cancer with known high-risk factors, since they are at an increased risk of harboring an aggressive molecular subtype.
Authors
Tobias Berg, Maj-Britt Jensen, Alan Celik, Maj-Lis Talman, Maria Anna Misiakou, Ann Søegaard Knoop, Finn Cilius Nielsen, Bent Ejlertsen, Maria Rossing
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Abstract
Dysregulated lipid homeostasis is emerging as a potential cause of neurodegenerative disorders. However, evidence of errors in lipid homeostasis as a pathogenic mechanism of neurodegeneration remains limited. Here, we show that cerebellar neurodegeneration caused by Sorting Nexin 14 (SNX14) deficiency is associated with lipid homeostasis defects. Recent studies indicate that SNX14 is an inter-organelle lipid transfer protein that regulates lipid transport, lipid droplet (LD) biogenesis, and fatty acid desaturation, suggesting that human SNX14 deficiency belongs to an expanding class of cerebellar neurodegenerative disorders caused by altered cellular lipid homeostasis. To test this hypothesis, we generated a mouse model that recapitulates human SNX14 deficiency at a genetic and phenotypic level. We demonstrate that cerebellar Purkinje cells (PCs) are selectively vulnerable to SNX14 deficiency while forebrain regions preserve their neuronal content. Ultrastructure and lipidomic studies reveal widespread lipid storage and metabolism defects in SNX14 deficient mice. However, pre-degenerating SNX14 deficient cerebella show a unique accumulation of acylcarnitines and depletion of triglycerides. Furthermore, defects in LD content and telolysosome enlargement in pre-degenerating PCs, suggest lipotoxicity as a pathogenic mechanism of SNX14 deficiency. Our work shows a selective cerebellar vulnerability to altered lipid homeostasis and provides a mouse model for future therapeutic studies.
Authors
Yijing Zhou, Vanessa B. Sanchez, Peining Xu, Thomas Roule, Marco Flores-Mendez, Brianna Ciesielski, Donna Yoo, Hiab Teshome, Teresa Jimenez, Shibo Liu, Mike Henne, Tim O’Brien, Ye He, Clementina Mesaros, Naiara Akizu
Abstract
Background. Upper body obesity (UBO) results in insulin resistance with regards to free fatty acid (FFA) release; how this differs by fat depot and sex between UBO and lean adults is unknown. We tested the hypothesis that insulin suppression of FFA release from the splanchnic bed, leg fat and upper body non-splanchnic (UBNS) adipose tissue would be impaired in UBO. Methods. Fourteen UBO (7 men, 7 women) and 14 healthy, normal weight (7 men, 7 women) volunteers participated in studies that included femoral artery, femoral vein and hepatic vein catheterization. We then measured leg and splanchnic plasma flow as well as FFA kinetics (using isotopic tracers) under overnight fasting, low- and high-dose insulin infusion using the insulin clamp technique. Results. We found the expected insulin resistance in UBO; the most quantitatively important difference between UBO and lean adults was greater FFA release from UBNS adipose tissue when plasma insulin concentrations are in the post-prandial, physiological range. There were obesity, but not sex differences in the regulation of splanchnic FFA release and sex differences in the regulation of leg FFA release. Conclusion. Reversing the defects in insulin-regulated UBNS adipose tissue FFA release would have the greatest impact on systemic FFA abnormalities in UBO. Trial Registration: (not applicable) Funding: These studies were supported by grants DK45343 and DK40484 from the U.S. Public Health Service, and the Novo Nordic Foundation (grant numbers NNF18OC0031804 and NNF16OC0021406) and the Independent Research Fund Denmark (grant number 8020-00420B).
Authors
Søren Nielsen, Michael D. Jensen
Abstract
Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent anti-inflammatory and anti-tumor activity in several pre-clinical models and has proven both safe and efficacious in clinical trials for advanced gastro-intestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared to cyclophosphamide post-aHSCT (PTCy). Strikingly, we found Minnelide improved survival, weight loss and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC-matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score and colonic length. Notably, in addition to decreased donor T cell infiltration early post-HSCT, several regulatory cell populations including Tregs, ILC2s and MDSCs in the colon were increased which together may account for Minnelide’s GVHD suppression post-HSCT. Importantly, Minnelide GVHD prevention was accompanied by preservation of graft-versus-tumor (GVT) activity. As Minnelide possesses anti-AML activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for AML patients undergoing aHSCT.
Authors
Sabrina N. Copsel, Vanessa T. Garrido, Henry Barreras, Cameron S. Bader, Brent Pfeiffer, Beatriz Mateo-Victoriano, Dietlinde Wolf, Miguel Gallardo, Sophie Paczesny, Krishna V. Komanduri, Cara L. Benjamin, Alejandro Villarino, Ashok K. Saluja, Robert B. Levy
Intimomedial tears of the aorta heal by smooth muscle cell-mediated fibrosis without atherosclerosis
Abstract
Background. Disease of the aorta varies from atherosclerosis to aneurysms with complications including rupture, dissection, and poorly characterized limited tears. We studied limited tears without any mural hematoma, termed intimomedial tears to gain insight into aortic vulnerability to excessive wall stresses. Our premise is that minimal injuries in aortas with sufficient medial resilience to prevent tear progression correspond to initial mechanisms leading to complete structural failure in aortas with significantly compromised medial resilience. Methods. Intimomedial tears were macroscopically identified in 9 of 108 ascending aortas after surgery and analyzed by histology and immunofluorescence confocal microscopy. Results. Non-hemorrhagic, non atheromatous tears correlated with advanced aneurysmal disease and most lacked distinctive symptoms or radiological signs. Tears traversed the intima and part of the subjacent media, while the resultant defects were partially or completely filled with neointima characterized by differentiated smooth muscle cells, scattered leukocytes, dense fibrosis, and absent elastic laminae despite tropoelastin synthesis. Healed lesions contained organized fibrin at tear edges without evidence of plasma and erythrocyte extravasation or lipid accumulation. Conclusion. These findings suggest a multiphasic model of aortic wall failure in which primary lesions of intimomedial tears either heal if the media is sufficiently resilient or progress as dissection or rupture by medial delamination and tear completion, respectively. Moreover, mural incorporation of thrombus and cellular responses to injury, two historically important concepts in atheroma pathogenesis, contribute to vessel wall repair with adequate conduit function but even together are not sufficient to induce atherosclerosis. Funding. R01-HL146723, R01-HL168473, and Yale Department of Surgery.
Authors
Abdulrahman H.M. Hassab, David J. Hur, Prashanth Vallabhajosyula, George Tellides, Roland Assi
Abstract
Recent studies have uncovered that non-coding sequence variants may relate to Axenfeld-Rieger syndrome (ARS), a rare developmental anomaly with genetic heterogeneity. However, how these genomic regions are functionally and structurally associated with ARS is still unclear. In this study, we performed genome-wide linkage analysis and whole-genome sequencing in a Chinese ARS family and identified a heterozygous deletion of about 570 kb (termed LOH-1) in the intergenic sequence between PITX2 and FAM241A. Knockout of LOH-1 homologous sequences caused ARS phenotypes in mice. RNA-seq and RT-qPCR revealed a significant reduction in Pitx2 gene expression in LOH-1–/– mice, while Foxc1 expression remained unchanged. ChIP-seq and bioinformatics analysis identified a potential enhancer region (LOH-E1) within LOH-1. Deletion of LOH-E1 led to a significant downregulation of the PITX2 gene. Mechanistically, we found a sequence (hg38 chr4:111,399,594-111,399,691) which is on LOH-E1 could regulate PITX2 by binding to RAD21, a critical component of the cohesin complex. Knockdown of RAD21 resulted in reduced PITX2 expression. Collectively, our findings indicate that a potential enhancer sequence which is within LOH-1 may regulate PITX2 expression remotely through cohesin-mediated loop domains, leading to ARS when absent. 2
Authors
Yizheng Jiang, Yu Peng, Qi Tian, Zhe Cheng, Bei Feng, Junping Hu, Lu Xia, Hui Guo, Kun Xia, Liang Zhou, Zhengmao Hu
