Resource and Technical Advance
Generating endogenous Myh11-driven Cre mice for sex-independent gene deletion in smooth muscle cells
Abstract
Specific and efficient smooth muscle cell (SMC)-targeted gene deletion is typically achieved by pairing SMMHC-CreERT2-Tg mice with mice carrying the loxP-flanked gene. However, the transgene, CreERT2, is not controlled by the endogenous Myh11 gene promoter, and the codon-modified iCreERT2 exhibits significant tamoxifen-independent leakage. Furthermore, because the Cre-bearing Bacterial Artificial Chromosome (BAC) is inserted onto the Y chromosome, the SMMHC-CreERT2-Tg mice strain can only exhibit gene deletions in male mice. Additionally, there is a lack of Myh11-driven constitutive Cre mice when tamoxifen usage is a concern. We used CRISPR/Cas9-mediated homologous recombination between a donor vector carrying the 1) CreNLSP2A or 2) CreERT2-P2A sequence and homologous arm surrounding the translation start site of the Myh11 gene to generate Cre knock-in mice. The P2A sequence enables the simultaneous translation of Cre and endogenous proteins. Using reporter mice, we assessed Cre-mediated recombination efficiency, specificity, tamoxifen-dependent controllability, and functionality in both sexes. Both constitutive (Myh11-CreNLSP2A) and inducible (Myh11-CreERT2-P2A) Cre mice demonstrated efficient, SMC-specific, sex-independent Cre recombinase activity without confounding endogenous gene expression. Combined with recently generated BAC transgenic Myh11-CreERT2-RAD mice and the Itga8-CreERT2 mouse models, our new models will help expand the research toolbox, facilitating unbiased and comprehensive research in SMCs and SMC-dependent cardiovascular diseases.
Authors
Yang Zhao, Guizhen Zhao, Ziyi Chang, Tianqing Zhu, Ying Zhao, Haocheng Lu, Chao Xue, Thomas L. Saunders, Yanhong Guo, Lin Chang, Y. Eugene Chen, Jifeng Zhang
Abstract
SARS-CoV-2 mRNA vaccination generates protective B cell responses targeting the SARS-CoV-2 spike glycoprotein. Whereas anti-spike memory B cell responses are long-lasting, the anti-spike humoral antibody response progressively wanes, making booster vaccinations necessary for maintaining protective immunity. Here we investigated qualitatively the plasmablast responses by measuring from single cells within hours of sampling the affinity of their secreted antibody for the SARS-CoV-2 spike receptor binding domain in cohorts of BNT162b2-vaccinated naive and COVID-19-recovered individuals. Using a unique droplet microfluidic and imaging approach, we analyzed >4,000 single IgG-secreting cells revealing high inter-individual variability in affinity for RBD with variations over 4 logs. High-affinity plasmablasts were induced by BNT162b2 vaccination against Hu-1 and Omicron RBD but disappeared quickly thereafter, whereas low-affinity plasmablasts represented >65% of the plasmablast response at all timepoints. Our droplet-based method thus proves efficient at fast and qualitative immune monitoring and should be helpful for optimization of vaccination protocols.
Authors
Matteo Broketa, Aurélien Sokal, Michael Mor, Pablo Canales-Herrerias, Angga Perima, Annalisa Meola, Ignacio Fernández, Bruno Iannascoli, Guilhem Chenon, Alexis Vandenberghe, Laetitia Languille, Marc Michel, Bertrand Godeau, Sebastien Gallien, Giovanna Melica, Marija Backovic, Felix A. Rey, Jean Baudry, Natalia T. Freund, Matthieu Mahevas, Pierre Bruhns
Abstract
While the development of different vaccines slowed the dissemination of SARS-CoV-2, the occurrence of breakthrough infections has continued to fuel the COVID-19 pandemic. To at least secure partial protection in majority of the population through one dose of a COVID-19 vaccine, delayed administration of boosters has been implemented in many countries. However, waning immunity and emergence of new variants of SARS-CoV-2 suggest that such measures may induce breakthrough infections due to intermittent lapses in protection. Optimizing vaccine dosing schedules to ensure prolonged continuity in protection could thus help control the pandemic. We developed a mechanistic model of immune response to vaccines as an in-silico tool for dosing schedule optimization. The model was calibrated with clinical datasets of acquired immunity to COVID-19 mRNA vaccines in healthy and immunocompromised subjects and showed robust validation by accurately predicting neutralizing antibody kinetics in response to multiple doses of COVID-19 mRNA vaccines. Importantly, by estimating population vulnerability to breakthrough infections, we predicted tailored vaccination dosing schedules to minimize breakthrough infections, especially for immunocompromised subjects. We identified that the optimal vaccination schedules vary from CDC-recommended dosing, suggesting that the model is a valuable tool to optimize vaccine efficacy outcomes during future outbreaks.
Authors
Prashant Dogra, Carmine Schiavone, Zhihui Wang, Javier Ruiz-Ramírez, Sergio Caserta, Daniela I. Staquicini, Christopher Markosian, Jin Wang, H. Dirk Sostman, Renata Pasqualini, Wadih Arap, Vittorio Cristini
Abstract
Trigeminal neuralgia (TN) is a classical neuralgic pain condition with distinct clinical characteristics. Modeling TN in rodents proves challenging. Recently, we found that a foramen in rodent skull base, the foramen lacerum, provides direct access to the trigeminal nerve root. Using this access, we developed FLIT (Foramen Lacerum Impingement of Trigeminal nerve root) model and observed distinct pain-like behaviors in rodents, including paroxysmal asymmetric facial grimaces, head tilt when eating, avoidance of solid chew, lack of wood chewing, etc. The FLIT model recapitulated key clinical features of TN, including lancinating pain-like behavior, and dental pain-like behavior. Importantly, when compared with a trigeminal neuropathic pain model (infraorbital nerve chronic constriction injury, IoN-CCI), the FLIT model was associated with significantly higher numbers of c-Fos positive cells in the primary somatosensory cortex (S1), unraveling robust cortical activation in the FLIT model. Using intravital two-photon calcium imaging, synchronized S1 neural dynamics were only present in the FLIT but not the IoN-CCI model, revealing differential implication of cortical activation in different pain models. Taken together, FLIT is a clinically relevant rodent model of TN which could facilitate pain research and therapeutics development.
Authors
Weihua Ding, Liuyue Yang, Qian Chen, Kun Hu, Yan Liu, Eric Bao, Changning Wang, Jianren Mao, Shiqian Shen
Abstract
Alloreactivity can drive autoimmune syndromes. After allogeneic hematopoietic stem cell transplantation (allo-HCT) chronic graft-versus-host disease (cGVHD), a B cell-mediated autoimmune-like syndrome, commonly occurs. Because donor-derived B cells continually develop under selective pressure from host alloantigens, aberrant B Cell Receptor (BCR)-activation and IgG production can emerge and contribute to cGVHD pathobiology. To better understand molecular programing of B cells under selective pressure of alloantigens, we performed scRNA-Seq analysis on high numbers of purified B cells from allo-HCT patients. An unsupervised analysis revealed 10 clusters, distinguishable by signature genes for maturation, activation and memory. We found striking transcriptional differences in the memory B cell compartment after allo-HCT compared to healthy or infected individuals. To identify intrinsic properties when B-cell tolerance is lost after allo-HCT, we then assessed clusters for differentially expressed genes (DEGs) between patients with vs. without autoimmune-like manifestations (Active cGVHD vs. No cGVHD, respectively). DEGs were found in Active cGVHD in both naive and BCR-activated clusters, suggesting functional diversity. Some DEGs were also differentially expressed across most clusters, suggesting common molecular programs that may promote B cell plasticity. Our study of human allo-HCT and cGVHD provides new understanding of B-cell memory in the face of chronic alloantigen stimulation.
Authors
Jonathan C. Poe, Jiyuan Fang, Dadong Zhang, Marissa R. Lee, Rachel A. DiCioccio, Hsuan Su, Xiaodi Qin, Jennifer Y. Zhang, Jonathan Visentin, Sonali J. Bracken, Vincent T. Ho, Kathy S. Wang, Jeremy J. Rose, Steven Z. Pavletic, Frances T. Hakim, Wei Jia, Amy N. Suthers, Itaevia M. Curry-Chisolm, Mitchell E. Horwitz, David A. Rizzieri, William C. McManigle, Nelson J. Chao, Adela R. Cardones, Jichun Xie, Kouros Owzar, Stefanie Sarantopoulos
Abstract
DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.
Authors
Amjad Horani, Deepesh Gupta, Jian Xu, Huihui Xu, Lis del C. Puga Molina, Celia M. Santi, Sruthi Ramagiri, Steven K. Brennan, Jiehong Pan, Jeffrey R. Koenitzer, Tao Huang, Rachael M. Hyland, Sean P. Gunsten, Shin-Cheng Tzeng, Jennifer M. Strahle, Pleasantine Mill, Moe R. Mahjoub, Susan K. Dutcher, Steven L. Brody
Abstract
The development and progression of endometriotic lesions are poorly understood, but immune cell dysfunction and inflammation are closely associated with the pathophysiology of endometriosis. There is a need for 3D in vitro models to permit the study of interactions between cell types and the microenvironment. To address this, we developed endometriotic spheroids (ES) to explore the role of epithelial-stromal interactions and model peritoneal invasion associated with lesion development. Using a non-adherent microwell culture system, spheroids were generated with immortalized endometriotic epithelial cells(12Z) combined with endometriotic stromal (iEc-ESC) or uterine stromal (iHUF) cell lines. Transcriptomic analysis found 4,522 differentially expressed genes in ES compared to spheroids containing uterine stromal cells. The top increased gene sets were inflammation-related pathways, and an overlap with baboon endometriotic lesions was highly significant. Finally, to mimic invasion of endometrial tissue into the peritoneum, a model was developed with human peritoneal mesothelial cells in an extracellular matrix. Invasion was increased in presence of estradiol or proinflammatory macrophages and suppressed by a progestin. Taken together, our results strongly support the concept that ES are an appropriate model for dissecting mechanisms that contribute to endometriotic lesion development.
Authors
Yong Song, Gregory W. Burns, Niraj R. Joshi, Ripla Arora, Ji-Yong Julie Kim, Asgerally T. Fazleabas
Abstract
Regular exercise leads to widespread salutary effects, and there is increasing recognition that exercise-stimulated circulating proteins can impart health benefits. Despite this, limited data exist regarding the plasma proteomic changes that occur in response to regular exercise. Here, we perform large-scale plasma proteomic profiling in 654 healthy human study participants before and after a supervised, 20-week endurance exercise training intervention. We identify hundreds of circulating proteins that are modulated, many of which are known to be secreted. We highlight proteins involved in angiogenesis, iron homeostasis, and the extracellular matrix, many of which are novel, including training-induced increases in fibroblast activation protein (FAP), a membrane-bound and circulating protein relevant in body-composition homeostasis. We relate protein changes to training-induced maximal oxygen uptake adaptations and validate our top findings in an external exercise cohort. Furthermore, we show that FAP is positively associated with survival in 3 separate, population-based cohorts.
Authors
Jeremy M. Robbins, Prashant Rao, Shuliang Deng, Michelle J. Keyes, Usman A. Tahir, Daniel H. Katz, Pierre M. Jean Beltran, François Marchildon, Jacob L. Barber, Bennet Peterson, Yan Gao, Adolfo Correa, James G. Wilson, J. Gustav Smith, Paul Cohen, Robert Ross, Claude Bouchard, Mark A. Sarzynski, Robert E. Gerszten
Abstract
Chronic lung allograft dysfunction (CLAD) is the leading cause of death in lung transplant recipients. CLAD is characterized clinically by a persistent decline in pulmonary function and histologically by the development of airway-centered fibrosis known as bronchiolitis obliterans. There are no approved therapies to treat CLAD, and the mechanisms underlying its development remain poorly understood. We performed single-cell RNA-Seq and spatial transcriptomic analysis of explanted tissues from human lung recipients with CLAD, and we performed independent validation studies to identify an important role of Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling in airway epithelial cells that contributes to airway-specific alloimmune injury. Specifically, we established that activation of JAK-STAT signaling leads to upregulation of major histocompatibility complex 1 (MHC-I) in airway basal cells, an important airway epithelial progenitor population, which leads to cytotoxic T cell–mediated basal cell death. This study provides mechanistic insight into the cell-to-cell interactions driving airway-centric alloimmune injury in CLAD, suggesting a potentially novel therapeutic strategy for CLAD prevention or treatment.
Authors
Aaditya Khatri, Jamie L. Todd, Fran L. Kelly, Andrew Nagler, Zhicheng Ji, Vaibhav Jain, Simon G. Gregory, Kent J. Weinhold, Scott M. Palmer
Abstract
Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants that is associated with an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. We describe an in vitro model of human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology. This model utilizes premature infant intestinal enteroids grown from surgically harvested intestinal tissue and co-cultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology by adding infant-derived microbiota. This model, named NEC-on-a-Chip, recapitulates the predominant features of NEC including significant upregulation of pro-inflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation, and disrupted epithelial barrier integrity. NEC-on-a-Chip provides an improved preclinical model of NEC that facilitates comprehensive analysis of the pathophysiology of NEC using precious clinical samples. This model is an advance towards a personalized medicine approach to test new therapeutics for this devastating disease.
Authors
Wyatt E. Lanik, Cliff J. Luke, Lila S. Nolan, Qingqing Gong, Lauren C. Frazer, Jamie M. Rimer, Sarah E. Gale, Raymond Luc, Shay S. Bidani, Carrie A. Sibbald, Angela N. Lewis, Belgacem Mihi, Pranjal Agrawal, Martin Goree, Marlie M. Maestas, Elise Hu, David G. Peters, Misty Good
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