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Abstract
Fibroblasts in the lung mesenchyme produce growth factors and extracellular matrix components that guide formation of distal airspaces during the saccular stage of lung development. Inflammation in preterm infants disrupts this process, leading to bronchopulmonary dysplasia (BPD). To examine how mesenchymal inflammation contributes to BPD pathogenesis, we developed a transgenic mouse model (“IKKβTbx4”) in which expression of activated human IκB kinase beta (IKKβ), an upstream activator of NF-κB, was induced in Tbx4 lung enhancer-positive mesenchymal cells during the saccular stage of lung development (postnatal day 0 [PN0] - PN5). Saccular stage IKKβTbx4 mice exhibited a BPD-like phenotype with interstitial thickening and reduced distal airspaces at PN5, progressing to emphysematous enlargement of the distal lung at 2 mo of age. Mesenchymal NF-κB activity upregulated the chemokines CCL2 and CCL7, recruiting CCR2pos monocyte-derived macrophages to the lung. Recruited macrophages disrupted the elastin scaffold and impaired microvascular organization with reductions in CAP2 endothelial cells (aCaps) and pericytes. Blocking CCR2-dependent monocyte recruitment with a small molecule CCR2 antagonist rescued the abnormal lung phenotype. These findings identify mesenchyme-macrophage crosstalk as a mechanism by which inflammation disrupts saccular stage lung development, suggesting a role for this signaling axis in BPD pathogenesis.
Authors
Benjamin C. Crawford, Jessica Chauviere Lee, Bertha C. Elias, Shivangi Dave, Riet van der Meer, Wei Han, Alexandria L. Sharkey, David S. Nichols, Charles Shissias, Lauren Pate, Hayden Tan, Dawn C. Newcomb, Wei Shi, Lawrence S. Prince, Erin J. Plosa, Bradley W. Richmond, Timothy S. Blackwell, Susan H. Guttentag, John T. Benjamin
Abstract
Systemic lupus erythematosus (SLE) is a heterogeneous systemic autoimmune disease, yet the molecular basis underlying this variability remains incompletely understood. We profiled the plasma proteome in 260 SLE patients and 86 healthy volunteers (HVs) using the SomaScan v4.1 platform, quantifying 7,288 analytes corresponding to 6,595 unique proteins. We identified 215 proteins that were robustly differentially abundant between SLE patients and HVs in both discovery (n=207 SLE, n=45 HVs) and validation sets (n=53 SLE, n=41 HVs). Within-cases analyses identified 421 proteins associated with disease activity. Network-based clustering delineated correlated protein modules, including an interferon-associated module and a renal-associated module. Autoantibody-stratified analyses further uncovered distinct proteomic endotypes: positivity for antibodies targeting RNA-binding proteins (anti-Sm, anti-Ro-60, anti-RNP68, anti-RNP-A) was associated with increased interferon-stimulated protein levels (e.g., MX1, ISG15, CXCL10), independent of disease activity. Anti-Sm, anti-RNP-A and anti-Ro52 antibodies were associated with reduced plasma levels of their respective autoantigens. Anti-dsDNA antibodies were associated with elevated levels of CD40 ligand (CD40LG) and the neutrophil protease proteinase-3. Moreover, we identified an association between CD40LG and disease activity specific to the anti-dsDNA positive subgroup. Together, these data define plasma protein signatures of SLE and disease activity, highlight autoantibody-specific molecular phenotypes, and provide a basis for precision medicine.
Authors
Geoffrey H. D. Leung, Charlotte Bottomley, Norzawani Buang, Robert T. Maughan, Benjamin J. Whittle, Boroumand Zeidaabadi, Yun-Ju Huang, Tabitha Turner-Stokes, Marie Condon, Liz Lightstone, Tom Cairns, Marina Botto, Matthew C. Pickering, James E. Peters
Abstract
Vascular tortuosity (VT) is a critical biomarker of disease progression and decision to treat ischemic retinal disorders, particularly retinopathy of prematurity (ROP). The murine oxygen-induced retinopathy model is the most widely-used model of ischemic retinopathy. Although VT has been described in OIR, its temporal dynamics have not been systematically defined. In this study, a semi-automated artificial intelligence (AI)-based pipeline was used to quantify VT throughout OIR. Retinal flat mounts from age-matched normoxic and OIR mice (postnatal days [P]10-P56) underwent vessel segmentation using a generative adversarial network (GAN), and VT was quantified as a cumulative tortuosity index (CTI) with the iROP-Assist algorithm. Concurrently, standard OIR outcomes of neovascularization (NV) and vaso-obliteration (VO) were quantified using OIRseg.org. NV peaked at P17 and resolved by P23, while VO regressed over a similar interval. VT peaked with NV at P17 but remained elevated through P56. These temporal changes mirror both the development of VT and its persistence after NV regression observed clinically in ROP. Collectively, these findings establish VT as a durable, quantifiable phenotype in OIR and expand the model’s utility beyond neovascular endpoints, providing a translational platform for investigating VT pathogenesis and evaluating the effects of therapeutic agents on vascular tortuosity.
Authors
Kyle V. Marra, Tomoya Murakami, Jimmy S. Chen, Edith Aguilar, Jacob I. Robinson, Maxwell Prenner, Richard Daneman, Martin Friedlander, Eric Nudleman
Abstract
Spreading depolarizations (SDs) are propagating waves of near-complete breakdown of transmembrane ion gradients that occur during acute ischemic stroke and worsen outcome by driving calcium overload and glutamate release in neurons and astrocytes. The plasmalemmal sodium-calcium exchanger (NCX) plays a key role in such changes, in that the complex ionic disequilibrium during ischemia induces reverse-mode activity of NCX, leading to cellular calcium overload in exchange for sodium. However, the cell type-specific roles of NCX in neurons and astrocytes during SDs remain unclear. Here, we used ion and glutamate reporters in an in vivo stroke model in mice carrying inducible, cell-specific deletions of NCX isoform-1. Neuronal NCX1 deletion reduced neuronal and astrocytic calcium transients, increased neuronal sodium transients, decreased extracellular glutamate levels, and raised SD initiation threshold. In contrast, astrocytic NCX1 deletion increased sodium transients in both neurons and astrocytes, and increased neuronal calcium as well as extracellular glutamate levels. A computational model of ischemia confirmed that these effects are consistent with reverse-mode NCX1 activity. Together, these findings indicate opposing roles of reverse-mode NCX1 during ischemia. Neuronal NCX1 promotes SD susceptibility, calcium overload and glutamate release, whereas astrocytic NCX1 exerts protective effects by attenuating glutamate elevation and neuronal calcium accumulation.
Authors
Somayyeh Hamzei Taj, Pawan Kumar Thapaliya, Cordula Rakers, Niklas J. Gerkau, Christine R. Rose, Ghanim Ullah, Gabor C. Petzold
Abstract
The sodium-dependent multivitamin transporter, encoded by SLC5A6, mediates cellular uptake of biotin and pantothenic acid, essential cofactors for energy metabolism. We identified two families with SLC5A6 mutations presenting with early-onset dilated cardiomyopathy (DCM). To investigate the link between vitamin deficiency and cardiomyopathy, we generated a cardiac-specific SLC5A6 knockout (Slc5a6cKO) mouse model and evaluated the impact of vitamin supplementation. Slc5a6cKO mice developed progressive cardiac dysfunction, culminating in cardiac pathology and premature death at 26 weeks; earlier stages exhibited cardiomyocyte hypertrophy, fibrosis, impaired Coenzyme A synthesis, and metabolic imbalance, indicating progression toward cardiomyopathy. Cardiac magnetic resonance imaging and ECG confirmed progressive functional decline. Proteomic analysis revealed early mitochondrial metabolic disruption and extracellular matrix protein upregulation at 8 weeks, preceding overt cardiac dysfunction. Strikingly, vitamin supplementation from preconception onwards prevented the cardiac phenotype, preserving cardiac structure, function, morphology and survival. This paralleled the clinical outcome in one patient who received early vitamin treatment, compared to another who required a heart transplant without vitamin treatment. This study establishes a direct link between SLC5A6-mediated vitamin transport, mitochondrial function, and cardiac health. It highlights how vitamin deficiency contributes to cardiomyopathy pathogenesis and supports early vitamin supplementation as a potential therapeutic strategy for metabolic cardiomyopathies.
Authors
Millie O. Fullerton, Lauren C. Phillips, Rachael E. Redgrave, Luke Spray, Vincent Haufroid, George Merces, Scott T. Kerridge, Gavin D. Richardson, Nathalie Mercier, Dominique Roland, Rebecca Crossley, Andrew D.H. Morgan, Joseph P. Dewulf, John Burn, Simon D. Bamforth, Helen M. Phillips
Abstract
Virally suppressed people with HIV (PWH) remain at risk for developing comorbidities due to chronic inflammation with one potential contributor being the HIV reservoir. Associations between the CD4-reservoir and inflammation have been extensively characterized, while the role the monocyte-reservoir is poorly understood despite evidence that inflammatory monocytes play a role in HIV-associated comorbidities. Additionally, most studies focus on a single cellular reservoir, while it is highly likely that these reservoirs are interdependent. In a cohort of 164 PWH, we used the intact proviral DNA assay to quantify cell-specific reservoirs, applied unsupervised clustering to identify reservoir phenotypes, and then determined if reservoir phenotypes were associated with distinct immune signatures compared to people without HIV. Five unique reservoir clusters emerged driven primarily by variability in the monocyte reservoir, and each associated with a distinct immune landscape. These included profiles characterized by systemic inflammation, leukocyte–vascular activation, T cell activation with vascular and neuronal injury, enhanced CD8 activation and NK cell recovery, and altered monocyte survival, activation, and migration. This multidimensional approach provides a framework to identify reservoir-immune profiles that may explain heterogeneity in inflammation despite viral suppression and may inform strategies to mitigate HIV-associated comorbidities.
Authors
Ruoyu Wang, Aparna B. Bhattacharyya, Lily Pohlenz, Erin N. Shirk, Hayley S. Romero, Katherine Haas, Jennifer M. Coughlin, Raha M. Dastgheyb, Leah H. Rubin, Rebecca T. Veenhuis
Abstract
Idiopathic Pulmonary Fibrosis (IPF) is a fatal, aging-related disease characterized by persistent lung fibroblast activation, progressive lung scarring and several vascular abnormalities. We have previously demonstrated that aging-associated vascular dysfunction drives maladaptive endothelial responses to injury and exacerbates lung fibrosis via secretion of pro-fibrotic endothelial-derived factors. However, regulatory mechanisms governing endothelial dysfunction during progressive lung fibrosis remain poorly understood. Here, using preclinical mouse models of progressive lung fibrosis as well as human IPF lungs, we demonstrate that miR-205-5p is overexpressed in lung ECs from fibrotic lungs, and coordinates gene expression programs implicated in endothelial dysfunction and progressive fibrosis. Mechanistically, miR-205-5p induces senescence in lung ECs, mirroring the senescent phenotype of IPF lung ECs. Consistently, conditioned medium derived from lung ECs overexpressing miR-205-5p promotes lung fibroblast activation. Importantly, miR-205-5p inhibition in IPF lung ECs attenuates endothelial senescence and limits paracrine fibroblast activation. Finally, inhibition of miR-205-5p in vivo preserves the pulmonary vascular network and attenuates lung fibrosis progression in aged mice challenged with bleomycin. Collectively, our findings support a novel connection between lung endothelial miR-205-5p, endothelial senescence and pro-fibrotic alteration of the endothelial secretome, and highlight miR-205-5p inhibition as a potential therapeutic intervention for pulmonary fibrosis.
Authors
Giuseppe Muscato, Benjamin B. Roos, Sharonda Harris, Xiaoyu Tracy Cai, Gina Civettini, Enrico Sciacca, Ahmed Raslan, Alessandra Castaldi, Sharon Elliot, Marilyn K. Glassberg, Carlo Vancheri, Daniel J. Tschumperlin, Giovanni Ligresti, Nunzia Caporarello
Abstract
Identifying mechanisms of kidney disease commonly involves comparing diseased samples to healthy reference tissues; however, the effects of variability in tissue procurement, storage, and donor characteristics remain underexplored. In this study, we systematically evaluated three reference tissue types—tumor nephrectomy (TN), pre-transplant biopsies from living donors (LD), and percutaneous biopsies from healthy control volunteers (HC)—to determine their impact on differential gene expression across three diabetic kidney disease (DKD) states. We observed distinct injury markers, cell state proportions, and gene signatures associated with procurement method, sex, and donor age. Adjustment for these confounding factors significantly influenced pathway analysis results. Specifically, correcting for age and sex eliminated significant enrichment of interferon gamma response in the diabetes mellitus–resilient (DM-R) versus HC comparison. Processes related to biological aging were enriched in older reference tissues, potentially confounding disease-specific interpretations. Importantly, tumor necrosis factor signaling via nuclear factor-κB remained enriched in LD and TN samples relative to HC, even after accounting for confounders. These results underscore the critical importance of selecting appropriate control tissues and rigorously adjusting for confounding variables to reliably discern the molecular mechanisms underlying kidney diseases.
Authors
Rajasree Menon, Paul L. Kimmel, Edgar A. Otto, Lalita Subramanian, Christopher L. O'Connor, Bradley Godfrey, Cathy Smith, Fadhl Alakwaa, Celine C. Berthier, Minnie M. Sarwal, E. Steve Woodle, Laura Pyle, Ye Ji Choi, Patricia Ladd, John R. Sedor, Sylvia E. Rosas, Sushrut S. Waikar, Abhijit S. Naik, Ricardo Melo Ferreira, Michael T. Eadon, Markus Bitzer, Petter Bjornstad, Jeffrey B. Hodgin, Matthias Kretzler
Abstract
HLA-E-restricted HIV-specific T cells offer exciting possibilities for immunotherapy. However, HLA-E binding peptides are rare. A recent study showed that in HLA-B*57:01 people living with HIV (PLWH), the peptide that dominates the T cell response, KAFSPEVIPMF (KF11), also stimulates HLA-E-restricted T cells, even though direct binding of this peptide to HLA-E could not be demonstrated. We therefore changed position 2 alanine for methionine in the peptide (referred to as KMF11) which greatly enhanced binding to HLA-E. This enabled the generation of stabilised HLA-E-KMF11 tetramers which were used to select and then grow specific T cell clones from T cells of HLA-B*57:01 negative blood donors primed with this peptide in vitro. Approximately 20% of these T cell clones reacted with HLA-E positive cells presenting the native KF11 peptide. Furthermore, these T cells inhibited replication of HIV-1 NL4-3 in CD4 T cells in vitro. Therefore, this native peptide can be presented by HLA-E to CD8 T cells, although priming in vivo may depend on cross reactivities to classical MHC Ia types. Nevertheless, such T cells could be exploitable for immunotherapy given the conservation of this HIV1 peptide epitope and the non-polymorphism in HLA-E.
Authors
Hong Sun, Hongbing Yang, Max N. Quastel, Simon Brackenridge, Wanlin He, Anna E. Kliszczak, Margarida Rei, Persephone Borrow, Geraldine M. Gillespie, Andrew J. McMichael
Abstract
Biallelic loss-of-function variants in the adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including the autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the ‘druggable genome’, in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans-Golgi network, suggesting they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.
Authors
Marvin Ziegler, Cedric Günter, Julian E. Alecu, Xutong Xue, Hyo-Min Kim, Afshin Saffari, Alexandra K. Davies, Mustafa Sahin, Darius Ebrahimi-Fakhari
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