Abstract
Pulmonary arterial hypertension (PAH) is a rare and incurable disease characterized by progressive narrowing of pulmonary arteries (PA), resulting in right ventricular (RV) hypertrophy, RV failure, and eventually death. Orai1 inhibition has emerged as promising therapeutic approach to mitigate PAH. In this study, we investigated the efficacy of a clinically applicable selective Orai1 inhibitor, CM5480, and its effects when combined with standard PAH therapies in a preclinical PAH model. In male and female monocrotaline PAH-rats, CM5480 monotherapy improved hemodynamics, PA, and RV remodeling, as confirmed by RV catheterization, echocardiography, histology, and unbiased RNA-Seq. Standard PAH therapies, ambrisentan or sildenafil, achieved modest improvements in experimental PAH. In contrast, combination therapies with CM5480 yielded significantly greater benefits in reducing PA remodeling and improving cardiac function compared with monotherapies. Furthermore, in vitro experiments showed that Orai1 knockdown reduced pulmonary endothelial cell dysfunction in PAH and that the Orai1 pathway is independent of standard PAH-targeted pathways in PA smooth muscle cells (PASMCs). Finally, we found enhanced Orai1 expression/function in PASMCs and pulmonary vein SMCs from patients with pulmonary veno-occlusive disease. These findings suggest that Orai1 inhibition represents a potentially novel and complementary therapeutic strategy for PAH by acting at pulmonary vascular and RV levels.
Authors
Anaïs Saint-Martin Willer, Grégoire Ruffenach, Bastien Masson, Kristelle El Jekmek, Angèle Boët, Rui Adão, Mathieu Gourmelon, Antoine Beauvais, Jessica Sabourin, Mary Dutheil, Maria-Rosa Ghigna, Laurent Tesson, Séverine Ménoret, Ignacio Anegon, Fabrice Bauer, Vincent de Montpréville, Sudarshan Hebbar, Carmen Brás-Silva, Kenneth Stauderman, Marc Humbert, Olaf Mercier, David Montani, Véronique Capuano, Fabrice Antigny
Abstract
Prolonged and dysregulated neutrophilic inflammation causes tissue damage in chronic inflammatory diseases, including antibody-mediated glomerulonephritis (AGN). An increase in glycolysis, supported by enhanced glucose uptake, is a hallmark of hyperneutrophilic inflammation. Neutrophils upregulate glucose transporter 1–mediated (Glut1-mediated) glucose incorporation for renal antimicrobial activities. However, little is known about the role of neutrophil-specific Glut1 function in the pathogenesis of AGN. Using a well-vetted mouse model of AGN, we show that neutrophils upregulate Glut1 expression and function in the nephritic kidney. We demonstrate that Glut1 function in the hematopoietic cells during the early stage of the disease is necessary for kidney pathology. Most importantly, neutrophil-intrinsic Glut1 function is critical for AGN. While neutrophil-specific Glut1 ablation diminished the expression of tissue-damaging effector molecules in both the early and late stages, renal cytokines’ and chemokines’ production were compromised only in the late stage of the disease. Consequently, Glut1 inhibitor treatment ameliorated renal pathology in AGN mice. These data identify a Glut1-driven inflammatory circuit in neutrophils, which is amenable to therapeutic targeting in AGN.
Authors
Hossein Rahimi, Wonseok Choi, Doureradjou Peroumal, Shuxia Wang, Partha S. Biswas
Abstract
Elexacaftor/tezacaftor/ivacaftor (ETI) cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has led to rapid and substantial improvements in cystic fibrosis (CF) airway disease. Underlying molecular and cellular mechanisms, long-term efficacy, and ability to reverse airway epithelial remodeling in established disease remain unclear. Longitudinal nasal brushes from an adult CF cohort were used to evaluate gene expression, cellular composition, stem cell function, and microbiome changes at baseline and at 6 months and 2 years after ETI. The baseline to 6 month span showed a massive downregulation of extensive neutrophilic inflammatory gene expression programs that correlated with increased pulmonary function and decreased sinusitis. Primary airway epithelial stem cell cultures from matched donor samples showed partially improved differentiation and barrier capacity at 6 months. Although clinical outcomes remained stable during the 6 month to 2 year span, transcriptional changes revealed a resurgence of baseline inflammatory programs. The time course of gene expression was consistent with ongoing normalization of epithelial remodeling. Relative abundance of Pseudomonas also decreased during the time course. These data suggest that ETI rectifies inflammation, epithelial remodeling, and bacterial infection in the airways, but resurgence of inflammatory gene expression may indicate ongoing inflammation, potentially presaging disease progression with long-term therapy.
Authors
Eszter K. Vladar, Austin E. Gillen, Sangya Yadav, Mikayla R. Murphree, David Baraghoshi, J. Kirk Harris, Elmar Pruesse, Sierra S. Niemiec, Alexandra W.M. Wilson, Katherine B. Hisert, Stephen M. Humphries, Matthew Strand, David A. Lynch, Max A. Seibold, Daniel M. Beswick, Jennifer L. Taylor-Cousar
Abstract
Cardiac hypertrophy is a common adaptation to cardiovascular stress and often a prelude to heart failure. We examined how S-palmitoylation of the small GTPase, Ras-related C3 botulinum toxin substrate 1 (Rac1), impacts cardiomyocyte stress signaling. Mutation of the Cys-178 palmitoylation site impaired activation of Rac1 when overexpressed in cardiomyocytes. Cardiomyocyte-specific Rac1 conditional knockin (Rac1cKI) mice expressing a Rac1C178S mutant protein exhibited normal cardiac structure and function but developed more severe cardiac hypertrophy in response to angiotensin II (AngII) infusion, cardiomyocyte-specific overexpression of AngII type 1 receptor (AT1R), and cardiac pressure overload. Moreover, pressure overload and AT1R overexpression evoked cardiac failure phenotypes in Rac1cKI mice not observed in controls. Mechanistically, Rac1cKI hearts and cardiomyocytes genetically resistant to Rac1 S-palmitoylation had a profound increase in protein kinase A (PKA) substrate phosphorylation in response to acute β-adrenergic stimulation, as did Rac1cKI hearts subjected to chronic AngII treatment, AT1R overexpression, or pressure overload that correlates with more advanced heart failure phenotypes. This was not associated with increased PKA enzymatic activity, suggesting potential deficits in phosphatase activity at PKA-regulated phospho-sites. Taken together, this study suggests Rac1 S-palmitoylation dampens adrenergic drive and PKA-dependent modulation of the phospho-proteome in response to cardiovascular stress, revealing essential functions for S-acylated Rac1 in cardiac adaptation.
Authors
James P. Teuber, Rachel E. Scissors, Arasakumar Subramani, Nageswara Madamanchi, Matthew J. Brody
Abstract
The intestinal mucosal epithelium forms a barrier between luminal contents and the body. MicroRNAs (miRNAs) regulate mucosal homeostasis by controlling inflammatory responses and structural integrity. Here, we discovered a protective role for miR147 in intestinal inflammation using a miR147tdTomato reporter mouse. miR147 was enriched in the intestines, with the highest expression in the colonic epithelial cells at the luminal surface, with prominent expression in differentiated enterocytes. Mice with general or intestinal epithelial deletion of miR147 showed increased intestinal inflammation and diminished mucosal healing during colitis. RNA sequencing of miR147-deficient cells showed dysregulated immune signaling, with upregulated proinflammatory cytokine pathways and reduced type I interferon responses and revealed Ndufa4 as a likely miR147 target. Ndufa4, a mitochondrial protein regulating energy metabolism and inflammation, was elevated at the crypt base, inversely correlating with miR147. Mice lacking the miR147 binding site in Ndufa4’s 3′-UTR phenocopied miR147-deficient mice during colitis. Spatial and single-cell transcriptomic analyses in murine and human colons showed mutually exclusive miR147 and Ndufa4 expression, consistent with a regulatory relationship in epithelial differentiation and metabolism. These findings underscore miR147’s role in intestinal homeostasis and mucosal healing, suggesting it as a therapeutic target for inflammatory bowel disease.
Authors
Agnieszka K. Czopik, Arash Dabiri, Chia-Hao Tung, Victoria Vaughn, Xiangsheng Huang, Jinlian Wang, Hui Li, Nicolas F. Moreno, Natalia V. Piwko, Katherine Figarella, Hongfang Liu, Zhongming Zhao, Xiaoyi Yuan, Holger K. Eltzschig
Abstract
Wiedemann-Steiner syndrome (WDSTS) is a rare genetic cause of intellectual disability that is primarily caused by heterozygous loss-of-function variants in the gene encoding the histone lysine methyltransferase 2A (KMT2A). Prior studies have shown successful postnatal amelioration of disease phenotypes for Rett, Rubinstein-Taybi, and Kabuki syndromes, which are related Mendelian disorders of the epigenetic machinery. To explore whether the neurological phenotype in WDSTS is treatable in utero, we created a mouse model carrying a loss-of-function variant placed between 2 loxP sites. Kmt2a+/LSL mice demonstrated core features of WDSTS including growth retardation, craniofacial abnormalities, and hypertrichosis as well as hippocampal memory defects. The neurological phenotypes were rescued upon restoration of KMT2A in utero following breeding to a nestin-Cre. Together, our data provide a mouse model to explore the potential therapeutic window in WDSTS. Our work suggests that WDSTS has a window of opportunity extending at least until the midpoint of in utero development, making WDSTS an ideal candidate for future therapeutic strategies.
Authors
Tinna Reynisdottir, Kimberley J. Anderson, Katrin Möller, Stefán Pétursson, Andrew Brinn, Katheryn P. Franklin, Juan Ouyang, Asbjorg O. Snorradottir, Cathleen M. Lutz, Aamir R. Zuberi, Valerie B. DeLeon, Hans T. Bjornsson
Abstract
Dravet syndrome (DS) is an early-onset epilepsy caused by loss-of-function mutations in the SCN1A gene, which encodes Nav1.1 channels that preferentially regulate activity of inhibitory neurons early in development. DS is associated with a high incidence of sudden unexpected death in epilepsy (SUDEP) by a mechanism that may involve respiratory failure. Evidence also shows that loss of Scn1a impaired activity of neurons in the retrotrapezoid nucleus (RTN) that regulate breathing in response to CO2/H+, suggesting breathing problems precede seizures and serve as a biomarker of SUDEP. Consistent with this, we showed that Scn1a+/– mice exhibited a blunted ventilatory response to CO2/H+ prior to overt seizure activity that worsened with disease progression. Later in development, some Scn1a+/– mice also showed a blunted ventilatory response to hypoxia. Importantly, the severity of respiratory problems correlated with mortality. We also found that pharmacological activation of Nav1.1 rescued activity deficits of RTN neurons in Scn1a+/– mice. We conclude that disordered breathing may be an early biomarker of SUDEP in DS, and at the cellular level, loss of Scn1a disrupts RTN neurons by mechanisms involving disinhibition and pharmacological activation of Nav1.1 to reestablish inhibitory control of RTN neurons rescues activity deficits.
Authors
Brenda M. Milla, Eliandra N. da Silva, Cleyton R. Sobrinho, Monica L. Strain, Daniel K. Mulkey
Abstract
Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of survival motor neuron (SMN) protein. Several therapeutic approaches boosting SMN are approved for human patients, delivering remarkable improvements in lifespan and symptoms. However, emerging phenotypes, including neurodevelopmental comorbidities, are being reported in some treated patients with SMA, indicative of alterations in brain development. Here, using a mouse model of severe SMA, we revealed an underlying neurodevelopmental phenotype in SMA where prenatal SMN-dependent defects in translation drove disruptions in nonmotile primary cilia across the central nervous system (CNS). Low levels of SMN caused widespread perturbations in translation at E14.5 targeting genes associated with primary cilia. The density of primary cilia in vivo, as well as cilial length in vitro, was significantly decreased in prenatal SMA mice. Proteomic analysis revealed downstream perturbations in primary cilia-regulated signaling pathways, including Wnt signaling. Cell proliferation was concomitantly reduced in the hippocampus of SMA mice. Prenatal transplacental therapeutic intervention with SMN-restoring risdiplam rescued primary cilia defects in SMA mouse embryos. Thus, SMN protein is required for normal cellular and molecular development of primary cilia in the CNS. Early, systemic treatment with SMN-restoring therapies can successfully target neurodevelopmental comorbidities in SMA.
Authors
Federica Genovese, Yu-Ting Huang, Anna A.L. Motyl, Martina Paganin, Gaurav Sharma, Ilaria Signoria, Deborah Donzel, Nicole C.H. Lai, Marie Pronot, Rachel A. Kline, Helena Chaytow, Kimberley J. Morris, Kiterie M.E. Faller, Thomas M. Wishart, Ewout J.N. Groen, Michael A. Cousin, Gabriella Viero, Thomas H. Gillingwater
Abstract
In vitro studies have implicated orphan receptor GPRC5B in β cell survival, proliferation, and insulin secretion, but its relevance for glucose homeostasis in vivo is largely unknown. Using tamoxifen-inducible, β cell–specific GPRC5B-KO mice (Ins-G5b–KOs), we show here that loss of GPRC5B does not affect β cell function in the lean state but results in strongly reduced insulin secretion and disturbed glucose tolerance in mice subjected to high-fat diet for 16 weeks. Flow cytometry and single-cell expression analyses in islets from obese mice show a reduced β cell abundance and a less mature β cell phenotype in Ins-G5b–KOs. Expression of β cell–specific transcription factor MafA is reduced both on the RNA and protein level, as are transcripts of MafA target genes. Mechanistically, we show that phosphorylation of cAMP response element-binding protein (CREB), a major regulator of MafA expression, is reduced in islets of obese Ins-G5b–KOs, and we show that this phenotype precedes the downregulation of MafA and MafA target genes. Taken together, GPRC5B helps to maintain mature β cell function in obesity through cAMP/CREB-dependent regulation of MafA expression.
Authors
Tianpeng Wang, Remy Bonnavion, Janett Piesker, Stefan Günther, Nina Wettschureck
Bcl6 expression is associated with a distinct immune landscape and spatial transcriptome in COVID-19
Abstract
The regulation of follicular (F) and germinal center (GC) immune reactivity in human lymph nodes (LNs), particularly during the acute stages of viral infection, remains poorly understood. We have analyzed lung-draining lymph nodes (LD-LNs) from COVID-19 autopsies using multiplex imaging and spatial transcriptomics to examine the immune landscape with respect to follicular immune reactivity. We identified 3 groups of donors based on the Bcl6 prevalence of their reactive follicles (RFs): RF-Bcl6no/lo, RF-Bcl6int, and RF-Bcl6hi. A distinct B/Tfh immune landscape, associated with increased prevalence of proliferating B cell and Tfh cell subsets, was found in RF-Bcl6hi LD-LNs. The comparison between LD-LNs and subdiaphragmatic (SD) LNs from the same donor revealed a divergent Bcl6 expression between the 2 anatomical sites. LD-LN Bcl6 expression was also associated with a distinct spatial transcriptomic profile. TH1-associated genes/pathways (e.g., CXCR3, STAT5, TNF signaling) were significantly upregulated in RF-Bcl6no/lo tissues, while the RF-Bcl6hi tissues exhibited significant upregulation of GC-promoting genes/pathways (e.g., CXCL13, B-cell receptor signaling). Our findings reveal a heterogeneous F/GC landscape in COVID-19 LD-LNs, highlighting specific molecular targets and pathways that could regulate human F/GC immune dynamics during acute viral infections.
Authors
Cloé Brenna, Bernat Bramon Mora, Kalliopi Ioannidou, Julien Bodelet, Mia L. Siebmanns, Simon Burgermeister, Spiros Georgakis, Michail Orfanakis, Yannick D. Muller, Nazanin Sédille, Matthew J. Feinstein, Jon W. Lomasney, Oliver Y. Chén, Giuseppe Pantaleo, Sabina Berezowska, Laurence de Leval, Raphael Gottardo, Constantinos Petrovas
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