Muscular dystrophies are a group of genetic neuromuscular disorders that involve severe muscle wasting. Transforming growth factor β-activated kinase 1 (TAK1) is an important signaling protein that regulates cell survival, growth, and inflammation. TAK1 has been recently found to promote myofiber growth in the skeletal muscle of adult mice. However, the role of TAK1 in muscle diseases remains poorly understood. In the present study, we have investigated how TAK1 affects the progression of dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD). TAK1 is highly activated in the dystrophic muscle of mdx mice during the peak necrotic phase. While targeted inducible inactivation of TAK1 inhibits myofiber injury in young mdx mice, it results in reduced muscle mass and contractile function. TAK1 inactivation also causes loss of muscle mass in adult mdx mice. By contrast, forced activation of TAK1 through overexpression of TAK1 and TAB1 induces myofiber growth without having any deleterious effect on muscle histopathology. Collectively, our results suggest that TAK1 is a positive regulator of skeletal muscle mass and targeted regulation of TAK1 can suppress myonecrosis and ameliorate disease progression in DMD.
Anirban Roy, Tatiana E. Koike, Aniket S. Joshi, Meiricris Tomaz da Silva, Kavya Mathukumalli, Mingfu Wu, Ashok Kumar
Leber congenital amaurosis (LCA) is a group of inherited retinal diseases (IRDs) characterized by the early onset and rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy (U-ExM), we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localize at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient for lebercilin exhibit early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, AAV-based LCA5 gene augmentation partially restores the bulge region, preserves OS axoneme structure and membrane disc formation, and results in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.
Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman
BACKGROUND. Currently, no laboratory tests exist to stratify for the risk of developing sinusoidal obstruction syndrome (SOS), an early endothelial complication after hematopoietic cell transplantation (HCT). Risk biomarkers of SOS have not been verified in a prospective cohort accounting for differences between practices across institutions. Herein, we aimed to define risk groups for SOS occurrence using three proteins: L-Ficolin, Hyaluronic Acid (HA), and Stimulation-2 (ST2). METHODS. Between 2017 to 2021, we prospectively accrued 80 pediatric patients across 4 US centers. Biomarkers were tested by ELISA blind to patient groupings and associated with SOS incidence at day 35 post-HCT, and overall survival (OS) at day 100 post-HCT. Cutpoints were identified using retrospective cohorts and applied to the prospective cohort. RESULTS. Combination of the three biomarkers measured at day 3 post-HCT in the prospective cohort provided 80% (95%CI, 55-100%) sensitivity and 73% (95%CI, 62-83%) specificity for risk of SOS occurrence. Patients with low L-Ficolin were 9 times (95%CI 3-32) more likely to develop SOS, while patients with high HA and ST2 were 6.5 (95%CI 1.9-22.0) and 5.5 (95%CI 2.3-13.1) times more likely to develop SOS. These three markers also predicted worse day 100 OS [L-Ficolin: HR, 10.0 (95%CI 2.2-45.1), P=0.0002; HA: HR, 4.1 (95%CI 1.0-16.4), P=0.031; ST2: HR, 3.9 (95%CI 0.9-16.4), P=0.04]. CONCLUSION. L-Ficolin, HA, and ST2 levels measured as early as three days post-HCT improved risk stratification for SOS occurrence and OS and may guide risk-adapted preemptive therapy. TRIAL REGISTRATION. ClinicalTrials.gov NCT03132337. FUNDING. NICHD P50HD090215, R01HD074587, NCI R01CA168814 and NHLBI K24HL156896.
Yan Han, Alan Bidgoli, Brittany P. DePriest, Alejandra Méndez, Khadijeh Bijangi-Vishehsaraei, Evelio D. Perez-Albuerne, Robert A. Krance, Jamie Renbarger, Jodi L. Skiles, Sung W. Choi, Hao Liu, Sophie Paczesny
Neutrophilic inflammation characterizes several respiratory viral infections including COVID-19-related ARDS, although its contribution to disease pathogenesis remains poorly understood. Blood and airway immune cells from 52 severe COVID-19 subjects were phenotyped by flow cytometry. Samples and clinical data were collected at two separate time points to assess changes during ICU stay. Blockade of type I interferon and IFIT3 signaling was performed in vitro to determine their contribution to viral clearance in A2 neutrophils. We identified two neutrophil subpopulations (A1 and A2) in the airway compartment, where loss of the A2 subset correlated with increased viral burden and reduced 30-days survival. A2 neutrophils showcased a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation with consequent reduced viral catabolism, providing the first discrete mechanism of type I interferon signaling in neutrophils. The identification of this novel neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.
Camilla Margaroli, Timothy R. Fram, Nirmal S. Sharma, Siddharth B. Patel, Jennifer Tipper, Sarah W. Robison, Derek W. Russell, Seth D. Fortmann, Mudassir Meraj Banday, Yixel M. Soto-Vazquez, Tarek Abdalla, Sawanan Saitornuang, Matthew C. Madison, Sixto M. Leal Jr., Kevin S. Harrod, Nathaniel B. Erdmann, Amit Gaggar
Familial exudative vitreoretinopathy (FEVR) is a complex hereditary eye disorder characterized by incomplete development of the retinal vasculature, thereby affecting retinal angiogenesis. But the genetic factors contributing to its development or pathogenesis remain elusive. In a Chinese FEVR family with 19 members, by utilizing whole exome sequencing, we identified a candidate disease-causing DNA variant in sorting nexin 31 (SNX31) (c.963delG; p. Trp321Cys), which results in a frameshift mutation. Herein we studied the biochemical mechanism of this mutation and uncovered that it is deficient in β1-integrin binding and integrin stability. The SNX31 c.963delG point mutation mouse model (SNX31m/m) was constructed using CRISPR/Cas9 technology. At 2-4 months of age, SNX31m/m mice showed fundus phenotypes similar to FEVR-like changes, including vascular leakage and retinal atrophy. Moreover, we found that VEGF and apoptotic pathways were involved in these ocular phenotypes. At present, the FEVR-like mouse model is mainly constructed by intravitreal injection, and we are the first to construct it by gene knockout. Hence our study extended FEVR mutation spectrum to include SNX31. Meanwhile, these findings expanded our understanding of the molecular pathogenesis of FEVR and may facilitate the development of methods for the diagnosis and prevention of FEVR patients.
Ningda Xu, Yi Cai, Jiarui Li, Tianchang Tao, Caifei Liu, Yan Shen, Xiaoxin Li, Leiliang Zhang, Mingwei Zhao, Xuan Shi, Jing Li, Lvzhen Huang
ASXL1 (Additional sex combs-like 1) plays key roles in epigenetic regulation of early developmental gene expression. De novo truncating mutations in ASXL1 cause Bohring-Opitz syndrome (BOS, OMIM #605039), a rare neurodevelopmental condition characterized by severe intellectual disabilities, characteristic facial features, hypertrichosis, increased risk of Wilms tumor, and variable congenital anomalies including heart defects and severe skeletal defects giving rise to a typical ‘BOS posture’. These BOS-causing ASXL1 variants are also high-prevalence somatic driver mutations in acute myeloid leukemia (AML). We use primary cells from BOS individuals (n = 18) and controls (n = 49) to dissect gene regulatory changes caused by ASXL1 mutations using comprehensive multi-omics assays for chromatin accessibility (ATAC-seq), DNA methylation, histone methylation binding, and transcriptome in peripheral blood and skin fibroblasts. Our data shows that regardless of cell type, ASXL1 mutations drive strong cross-tissue effects that disrupt multiple layers of the epigenome. The data showed a broad activation of canonical Wnt signaling at the transcriptional and protein levels and upregulation of VANGL2, a planar cell polarity pathway protein that acts through non-canonical Wnt signaling to direct tissue patterning and cell migration. This multi-omics approach identifies the core impact of ASXL1 mutations and therapeutic targets for BOS and myeloid leukemias.
Isabella Lin, Angela Wei, Zain Awamleh, Meghna Singh, Aileen Ning, Analeyla Herrera, Bianca E. Russell, Rosanna Weksberg, Valerie A. Arboleda
Viral illnesses like SARS-CoV-2 have pathologic effects on non-respiratory organs in the absence of direct viral infection. We injected mice with cocktails of rodent equivalents of human cytokine storms resulting from SARS-CoV-2 / COVID-19 or Rhinovirus common cold infection. At low doses, COVID-19 cocktails induced glomerular injury and albuminuria in Zhx2 hypomorph and Zhx2+/+ mice to mimic COVID-19 related proteinuria. Common Cold cocktail induced albuminuria selectively in Zhx2 hypomorph mice to model relapse of Minimal Change Disease (MCD), that improved after depletion of TNF-α or sIL-4Rα or IL-6. The Zhx2 hypomorph state increased cell membrane to nuclear migration of podocyte ZHX proteins in vivo (both cocktails) and lowered pSTAT6 activation (COVID-19 cocktail) in vitro. At higher doses, COVID-19 cocktails induced acute heart injury, myocarditis, pericarditis, acute liver injury and acute kidney injury, and high mortality in Zhx2+/+ mice, whereas Zhx2 hypomorph mice were relatively protected, due in part to early asynchronous activation of STAT5 and STAT6 pathways in these organs. Dual depletion of cytokine combinations of TNF-α with IL-2 or IL-13 or IL-4 in Zhx2+/+ mice reduced multiorgan injury and eliminated mortality. Using genome sequencing and CRISPR-Cas9, an insertion upstream of ZHX2 was identified as a cause of the human ZHX2 hypomorph state.
Maria Del Nogal Avila, Ranjan Das, Joubert B. Kharlyngdoh, Eduardo Molina-Jijon, Hector Donoro-Blazquez, Stéphanie Gambut, Michael R. Crowley, David K. Crossman, Rasheed A. Gbadagesin, Sunveer S. Chugh, Sunjeet S. Chugh, Carmen Avila-Casado, Camille Macé, Lionel C. Clement, Sumant S. Chugh
To improve our limited understanding of the pathogenesis of thoracic aortic aneurysm (TAA) leading to acute aortic dissection, single-cell RNA sequencing (scRNA-seq) was employed to profile disease-relevant transcriptomic changes of aortic cell populations in a well-characterized mouse model of the most commonly diagnosed form of Marfan syndrome (MFS). As result, two discrete sub-populations of aortic cells (SMC3 and EC4) were identified only in the aorta of Fbn1mgR/mgR mice. SMC3 highly express genes related to extracellular matrix formation and nitric oxide signaling, whereas EC4 transcriptional profile is enriched in SMC, fibroblast, and immune cell-related genes. Trajectory analysis predicted close phenotypic modulation between SMC3 and EC4, which were therefore analyzed together as a discrete MFS-modulated (MFSmod) sub-population. In situ hybridizations of diagnostic transcripts located MFSmod cells to the intima of Fbn1mgR/mgR aortas. Reference-based dataset integration revealed transcriptomic similarity between MFSmod and an SMC-derived cell cluster modulated in human TAA. Consistent with angiotensin II type I receptor (At1r) contribution to TAA development, MFSmod cells were absent in the aorta of Fbn1mgR/mgR mice treated with the At1r antagonist losartan. Altogether, our findings indicate that a discrete dynamic alteration of aortic cell identity is associated with dissecting TAA in MFS mice and increased risk of aortic dissection in MFS patients.
Yifei Sun, Keiichi Asano, Lauriane Sedes, Anna Cantalupo, Jens Hansen, Ravi Iyengar, Martin J. Walsh, Francesco Ramirez
Rationale. RNA binding protein 47 (RBM47) is required for embryonic endoderm development but a role in adult intestine is unknown. Objective. We studied intestine-specific Rbm47 knockout mice (Rbm47-IKO) following intestinal injury and made crosses into Apcmin/+ mice to examine alterations in intestinal proliferation, response to injury and tumorigenesis. We also interrogated human colorectal polyps and colon carcinoma tissue. Findings. Rbm47-IKO mice exhibit increased proliferation, abnormal villus morphology and cellularity, with corresponding changes in Rbm47-IKO organoids. Rbm47-IKO mice adapt to radiation injury and are protected against chemical-induced colitis, with Rbm47-IKO intestine showing upregulation of antioxidant and Wnt signaling pathways as well as stem cell and developmental genes. Furthermore, Rbm47-IKO mice are protected against colitis-associated cancer. By contrast, aged Rbm47-IKO mice develop spontaneous polyposis and Rbm47-IKO, Apcmin/+ mice manifest an increased intestinal polyp burden. RBM47 mRNA was decreased in human colorectal cancer versus paired normal tissue along with alternative splicing of TJP1 mRNA. Public databases revealed stage-specific reduction in RBM47 expression in colorectal cancer, associated independently with decreased overall survival. Conclusions. These findings implicate RBM47 as a cell-intrinsic modifier of intestinal growth, inflammatory and tumorigenic pathways.
Saeed Soleymanjahi, Valerie Blanc, Elizabeth A. Molitor, David M. Alvarado, Yan Xie, Vered Gazit, Jeffrey W. Brown, Kathleen Byrnes, Ta-Chiang Liu, Jason C. Mills, Matthew A. Ciorba, Deborah C. Rubin, Nicholas O. Davidson
GM3 synthase deficiency (GM3SD) is an infantile-onset epileptic encephalopathy syndrome caused by biallelic loss-of-function mutations in ST3GAL5. Loss of ST3GAL5 activity in humans results in systemic ganglioside deficiency and severe neurological impairment. No disease-modifying treatment is currently available. Certain recombinant adeno-associated viruses (rAAVs) are capable of crossing the blood-brain barrier to induce widespread, long-term gene expression in the central nervous system (CNS), and represent a promising therapeutic strategy. Here, we show that a first-generation rAAV-ST3GAL5 replacement vector employing a ubiquitous promoter restored tissue ST3GAL5 expression and normalized cerebral gangliosides in patient-derived iPSC neurons and brain tissue from St3gal5 knock-out mice, but caused fatal hepatotoxicity when administered systemically. In contrast, a second-generation vector optimized for CNS-restricted ST3GAL5 expression, administered by either intracerebroventricular or intravenous route at postnatal day 1, allowed for safe and effective rescue of lethality and behavior impairment in symptomatic GM3SD mice up to a year. These results support further clinical development of ST3GAL5 gene therapy.
Huiya Yang, Robert Brown, Dan Wang, Kevin A. Strauss, Guangping Gao
Sorbitol dehydrogenase (SORD) deficiency has been identified as the most frequent autosomal recessive form of hereditary neuropathy. Loss of SORD causes high sorbitol levels in tissues due to the inability to convert sorbitol to fructose in the two-step polyol pathway, leading to degenerative neuropathy. The underlying mechanisms of sorbitol-induced degeneration have not been fully elucidated, and no current FDA-approved therapeutic options are available to reduce sorbitol levels in the nervous system. Here, in a Drosophila model of SORD deficiency, we showed synaptic degeneration in the brain, neurotransmission defect, locomotor impairment, and structural abnormalities in the neuromuscular junctions. In addition, we found reduced ATP production in the brain and reactive oxygen species accumulation in the central nervous system (CNS) and muscle, indicating mitochondrial dysfunction. Applied Therapeutics, Inc has developed a CNS-penetrant next-generation aldose reductase inhibitor (ARI), AT-007 (govorestat), which inhibits the conversion of glucose to sorbitol. AT-007 significantly reduced sorbitol levels in patient-derived fibroblasts, iPSC-derived motor neurons, and Drosophila brains. AT-007 feeding in Sord-deficient Drosophila mitigated synaptic degeneration and significantly improved synaptic transduction, locomotor activity, and mitochondrial function. Moreover, AT-007 treatment significantly reduced ROS accumulation in Drosophila CNS, muscle, and patient-derived fibroblasts. These findings uncover the molecular and cellular pathophysiology of SORD neuropathy and provide a potential treatment strategy for patients with SORD deficiency.
Yi Zhu, Amanda G. Lobato, Adriana P. Rebelo, Tijana Canic, Natalie Ortiz Vega, Xianzun Tao, Sheyum Syed, Christopher Yanick, Mario Saporta, Michael Shy, Riccardo Perfetti, Shoshana Shendelman, Stephan L. Zuchner, R. Grace Zhai
The prevalence of obesity and type 2 diabetes is growing at an alarming rate, including among pregnant women. Low-calorie sweeteners (LCS) have increasingly been used as an alternative to sugar to deliver a sweet taste without the excessive caloric load. However, there is little evidence regarding their biological effects, particularly during development. Here, we used a mouse model of maternal LCS consumption to explore the impact of perinatal LCS exposure on the development of neural systems involved in metabolic regulations. We report that adult male, but not female, offspring from both aspartame- and rebaudioside A-exposed dams displayed increased adiposity and developed glucose intolerance. Moreover, maternal LCS consumption reorganized hypothalamic melanocortin circuits and disrupted parasympathetic innervation of pancreatic islets in male offspring. We then identified phenylacetylglycine (PAG) as a unique metabolite that is upregulated in the milk of LCS-fed dams and the serum of their pups. Furthermore, maternal PAG treatment recapitulates some of the key metabolic and neurodevelopmental abnormalities associated with maternal LCS consumption. Together, our data indicate that maternal LCS consumption has enduring consequences on the offspring's metabolism and neural development and that these effects are likely to be mediated through the gut microbial co-metabolite PAG.
Soyoung Park, Amine M. Belfoul, Marialetizia Rastelli, Alice Jang, Magali Monnoye, Hosung Bae, Anna Kamitakahara, Patrick Giavalisco, Shan Sun, Pierre-Yves Barelle, Jasmine Plows, Cholsoon Jang, Anthony Fodor, Michael I. Goran, Sebastien G. Bouret
Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1, an inwardly rectifying potassium channel that regulates membrane hyperpolarization, contributes to vasodilation in resistance arteries. First, we show phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data has implied PS may compete with PIP2 binding on Kir2.1. We found 83.33% of Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate PS blocks PIP2 activation of Kir2.1, and addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggests PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and demonstrates the intracellular lipid localization within endothelium is an important determinant of vascular function.
Claire A. Ruddiman, Richard G. Peckham, Melissa A. Luse, Yen-Lin Chen, Maniselvan Kuppusamy, Bruce A. Corliss, Jordan Hall, Chien-Jung Lin, Shayn M. Peirce, Swapnil K. Sonkusare, Robert P. Mecham, Jessica E. Wagenseil, Brant E. Isakson
Synovial Fibroblasts (SFs) are key pathogenic drivers in Rheumatoid arthritis (RA). Their in vivo activation by TNF is sufficient to orchestrate full arthritic pathogenesis in animal models and TNF blockade proved efficacious for a high percentage of RA patients albeit co-inducing rare but serious side effects. Aiming to find new potent therapeutics, we applied the L1000CDS2 search engine, in order to repurpose drugs that could reverse the pathogenic expression signature of arthritogenic human TNF transgenic (hTNFtg) SFs. We identified a neuroleptic drug, namely Amisulpride, which reduced SFs’ inflammatory potential while decreasing the clinical score of hTNFtg polyarthritis. Notably, we found that Amisulpride function is neither through its known targets Dopamine receptors 2 and 3 and Serotonin Receptor 7, nor through TNF-TNFRI binding inhibition. Through a click chemistry approach, novel potential targets of Amisulpride were identified, which were further validated to repress hTNFtg SFs’ inflammatory potential ex vivo (Ascc3 and Sec62), while phosphoproteomics analysis revealed that treatment altered important fibroblast activation pathways, such as adhesion. Thus, Amisulpride could prove beneficial to patients suffering from RA and the often-accompanying comorbid dysthymia, reducing SF pathogenicity along with its anti-depressive activity, serving further as a “lead” compound for the development of novel therapeutics against fibroblast activation.
Dimitra Papadopoulou, Fani Roumelioti, Christos Tzaferis, Panagiotis Chouvardas, Anna-Kathrine Pedersen, Filippos Charalampous, Eleni Christodoulou-Vafeiadou, Lydia Ntari, Niki Karagianni, Maria C. Denis, Jesper V. Olsen, Alexis Ν. Matralis, George Kollias
Although the expression of Mex3 RNA binding family member B (MEX3B) is upregulated in human nasal epithelial cells (HENCs) predominately in the eosinophilic chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) subtype, its functions as an RNA binding protein in airway epithelial cells remain unknown. Here, we revealed the role of MEX3B based on different subtypes of CRS, and demonstrated that MEX3B decreased TGF-β receptor III (TGFBR3) mRNA level by binding to its 3’ UTR and reducing its stability in HNECs. TGF-βR3 was found to be a TGF-β2 specific coreceptor in HNECs. Knocking down or overexpressing MEX3B promoted or inhibited TGF-β2-induced phosphorylation of Smad2 in HNECs, respectively. TGF-βR3 and p-Smad2 levels were downregulated in CRSwNP compared with controls and CRS without nasal polyps (CRSsNP), with a more prominent downregulation in the eosinophilic CRSwNP. TGF-β2 promoted collagen production in HNECs. Collagen abundance decreased and edema scores increased in CRSwNP compared to control, again more prominently in the eosinophilic type. Collagen expression in eosinophilic CRSwNP was negatively correlated with MEX3B but positively correlated with TGF-βR3. These results suggest that MEX3B inhibits tissue fibrosis in eosinophilic CRSwNP by downregulating epithelial cell TGFBR3 expression; consequently, MEX3B might be a valuable therapeutic target against eosinophilic CRSwNP.
Jin-Xin Liu, Chen Ao-Nan, Qihong Yu, Ke-Tai Shi, Yi-Bo Liu, Cui-Lian Guo, Zhe-Zheng Wang, Yin Yao, Li Pan, Xiang Lu, Kai Xu, Heng Wang, Ming Zeng, Chaohong Liu, Robert P. Schleimer, Ning Wu, Bo Liao, Zheng Liu
Vascular smooth muscle-derived Sca1+ adventitial progenitor (AdvSca1-SM) cells are tissue resident, multipotent stem cells that contribute to progression of vascular remodeling and fibrosis. Upon acute vascular injury, AdvSca1-SM cells differentiate into myofibroblasts and are embedded in perivascular collagen and extracellular matrix. While the phenotypic properties of AdvSca1-SM-derived myofibroblasts have been defined, the underlying epigenetic regulators driving the AdvSca1-SM-to-myofibroblast transition are unclear. We show that the chromatin remodeler, Smarca4/Brg1, facilitates AdvSca1-SM myofibroblast differentiation. Brg1 mRNA and protein was upregulated in AdvSca1-SM cells after acute vascular injury and pharmacological inhibition of Brg1 by the small molecule PFI-3 attenuated perivascular fibrosis and adventitial expansion. TGF-β1 stimulation of AdvSca1-SM cells in vitro reduced expression of stemness genes while inducing expression of myofibroblast genes that was associated with enhanced contractility; PFI blocked TGF-β1-induced phenotypic transition. Similarly, genetic knockdown of Brg1 in vivo reduced adventitial remodeling and fibrosis and reversed AdvSca1-SM-to-myofibroblast transition in vitro. Mechanistically, TGF-β1 promoted redistribution of Brg1 from distal intergenic sites of stemness genes and recruitment to promoter regions of myofibroblast-related genes, which was blocked by PFI-3. These data shed insight into epigenetic regulation of resident vascular progenitor cell differentiation and support that manipulating the AdvSca1-SM phenotype will provide important anti-fibrotic clinical benefit.
Austin J. Jolly, Sizhao Lu, Allison M. Dubner, Keith A. Strand, Marie F. Mutryn, Aaron Pilotti-Riley, Etienne P. Danis, Raphael A. Nemenoff, Karen S. Moulton, Mark W. Majesky, Mary C.M. Weiser-Evans
Invariant Natural Killer T (iNKT) cells act at the interface between lipid metabolism and immunity, due to their restriction to lipid antigens presented on CD1d by antigen presenting cells (APC). How foreign lipid antigens are delivered to APC remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), Isoglobotrihexosylceramide (iGb3) and OCH, a sphingosine-truncated analogue of αGalCer, with very-low-density (VLDL) and/or low-density (LDL) lipoproteins in vitro and in vivo. We demonstrate LDL receptor (LDLR)-mediated uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a novel mechanism of lipid antigen delivery to APCs, and provides further insight in the immunological capacities of circulating lipoproteins.
Suzanne E. Engelen, Francesca A. Ververs, Angela Markovska, B. Christoffer Lagerholm, Jordan M. Kraaijenhof, Laura I.E. Yousif, Yasemin-Xiomara Zurke, Can M.C. Gulersonmez, Sander Kooijman, Michael Goddard, Robert J. van Eijkeren, Peter J. Jervis, Gurdyal S. Besra, Saskia Haitjema, Folkert W. Asselbergs, Eric Kalkhoven, Hidde L. Ploegh, Marianne Boes, Vincenzo Cerundolo, G. Kees Hovingh, Mariolina Salio, Edwin C.A. Stigter, Patrick C.N. Rensen, Claudia Monaco, Henk S. Schipper
Loss of function mutations in the DNA methyltransferase 3A (DNMT3A) are seen in a large number of AML patients with normal cytogenetics and are frequently associated with poor prognosis. DNMT3A mutations are an early pre-leukemic event, which when combined with other genetic lesions result in full blown leukemia. Here, we show that loss of Dnmt3a in HSC/Ps results in myeloproliferation, which is associated with hyperactivation of the PI3Kinase pathway. PI3Kα/β or the PI3Kα/δ inhibitor treatment partially corrects myeloproliferation, although the partial rescue is more efficient in response to the PI3Kα/β inhibitor treatment. In vivo RNA-seq analysis on drug treated Dnmt3a–/– HSC/Ps showed a reduction in the expression of genes associated with chemokines, inflammation, cell attachment and extracellular matrix compared to controls. Remarkably, drug treated leukemic mice showed a reversal in the enhanced fetal liver HSC like gene signature observed in vehicle treated Dnmt3a–/– LSK cells as well as a reduction in the expression of genes involved in regulating actin cytoskeleton-based functions including the RHO/RAC GTPases. In a human PDX model bearing DNMT3A mutant AML, PI3Kα/β inhibitor treatment prolonged their survival and rescued the leukemic burden. Our results identify a new target for treating DNMT3A mutation driven myeloid malignancies.
Lakshmi Reddy Palam, Baskar Ramdas, Katelyn M. Pickerell, Santhosh Kumar Pasupuleti, Rahul Kanumuri, Annamaria Cesarano, Megan Szymanski, Bryce M. Selman, Utpal P. Davé, George Sandusky, Fabiana Perna, Sophie Paczesny, Reuben Kapur
Several pre-clinical studies have demonstrated that certain cytotoxic drugs enhance metastasis, but the importance of host responses triggered by chemotherapy in regulating cancer metastasis has not been fully explored. Here, we showed that multi-dose Gemcitabine (GEM) treatment promoted breast cancer lung metastasis in a transgenic spontaneous breast cancer model. GEM treatment significantly increased accumulation of CCR2+ macrophages and monocytes in the lungs of tumor-bearing as well as tumor-free mice. These changes were largely caused by chemotherapy induced reactive myelopoiesis that is biased toward monocyte development. Mechanistically, enhanced production of mitochondrial ROS was observed in GEM treated BM LSK cells and monocytes. Treatment with the mitochondrial targeted antioxidant abrogated GEM induced hyper-differentiation of BM progenitors. In addition, GEM treatment induced up-regulation of host cell derived CCL2, and knockout CCR2 signaling abrogated the pro-metastatic host response induced by chemotherapy. Furthermore, chemotherapy treatment resulted in the upregulation of coagulation factor X (FX) in lung interstitial macrophages. Targeting activated FX (FXa) using FXa inhibitor or F10 gene knockdown reduced pro-metastatic effect of chemotherapy. Together, these studies suggest a novel mechanism for chemotherapy induced metastasis via the host response induced accumulation of monocytes/macrophages and interplay between coagulation and inflammation in the lungs.
Caijun Wu, Qian Zhong, Rejeena Shrestha, Jingzhi Wang, Xiaoling Hu, Hong Li, Eric C. Rouchka, Jun Yan, Chuanlin Ding
BACKGROUND. Due to their immunoregulatory and tissue regenerative features, mesenchymal stromal cells (MSCs) are a promising novel tool for the management of ulcerative proctitis (UP). Here we report on a phase IIa clinical study to evaluate the impact of local MSC therapy in UP. METHODS. Thirteen refractory UP patients, with endoscopic Mayo score (EMS) 2 or 3, were included. Seven patients received 20-40 x 106 allogeneic MSCs (cohort 1), while six patients received 40-80 x 106 MSCs (cohort 2). Adverse events (AEs) were assessed at baseline and week 2, 6, 12, and 24. Clinical, endoscopic, and biochemical parameters were assessed at baseline, week 2 and 6. Furthermore, we evaluated the engraftment of MSCs, presence of donor-specific human leukocyte antigen (HLA) antibodies (DSAs), and we determined the impact of MSC therapy on the local immune compartment. RESULTS. No serious AEs were observed. The clinical Mayo score was significantly improved at week 2 and 6, and the EMS was significantly improved at week 6, compared to baseline. At week 6, donor MSCs were still detectable in rectum biopsies of 4/9 patients and DSAs against both HLA-class I and -class II were found. Mass cytometry showed a reduction of activated CD8+ T cells and CD16+ monocytes and an enrichment in mononuclear phagocytes and natural killer cells in biopsies after local MSC therapy. CONCLUSION. Local administration of allogeneic MSCs is safe, tolerable, and feasible for treatment of refractory UP and shows encouraging signs of clinical efficacy and modulation of local immune responses. This sets the stage for larger clinical trials. TRIAL REGISTRATION. clinicaltrialsregister.eu, EudraCT: 2017-003524-75, Dutch Trial register: NTR7205. FUNDING. ECCO grant 2020.
Laura F. Ouboter, Marieke C. Barnhoorn, Hein W. Verspaget, Leonie Plug, Emma S. Pool, Karoly Szuhai, Lukas J.A.C. Hawinkels, Melissa van Pel, Jaap Jan Zwaginga, Dave Roelen, Frits Koning, M. Fernanda Pascutti, Andrea van der Meulen - de Jong