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Rothenberg, M. E.

Publications and source records attributed to Rothenberg, M. E..

11 recordsLinked to original sources

Esophageal epithelial cell-state transitions underlie the severity of pediatric eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a leading cause of chronic esophageal dysfunction driven by immune-mediated inflammation. Peak eosinophil count (PEC) in esophageal biopsies is routinely used to assess disease activity, and its associated molecular mechanisms have been well studied. However, PEC only partially captures overall disease severity, which is comprehensively captured by the Index of Severity for Eosinophilic Esophagitis (I-SEE). In contrast to PEC, the molecular and cellular programs associated with the I-SEE-defined disease severity, particularly in children, remain poorly understood. We integrated bulk transcriptomic profiling of pediatric esophageal biopsies with clinical severity metrics and a matched single-cell transcriptomic reference. Increasing severity was associated with a shift from type 2 inflammatory activation toward epithelial stress, cytoskeletal and junctional disruption, metabolic dysfunction, and extracellular matrix remodeling. Single-cell-informed analyses identified that proliferating and transitional epithelial cell states were strongly associated with higher I-SEE scores and exhibited impaired differentiation, heightened metabolic and oxidative stress responses, and structural remodeling programs not captured by bulk transcriptomic analyses alone. These findings reposition epithelial remodeling, rather than eosinophil burden alone, as a central molecular correlate of disease severity in pediatric EoE and provide a framework for improved disease stratification and therapeutic intervention.

immunology↗

Machine Learning Based Identification and Characterization of Mast Cells in Eosinophilic Esophagitis

BackgroundEosinophilic esophagitis (EoE) is diagnosed and monitored using esophageal eosinophil levels; however, EoE also exhibits a marked, understudied esophageal mastocytosis. ObjectiveUsing machine learning, we localized and characterized esophageal mast cells to decipher their potential role in disease pathology. MethodsEsophageal biopsy samples (EoE, control) were stained for mast cells by anti-tryptase and imaged using immunofluorescence; high-resolution whole tissue images were digitally assembled. Machine learning software was trained to identify, enumerate, and characterize mast cells, designated Mast Cell-Artificial Intelligence (MC-AI). ResultsMC-AI enumerated cell counts with high accuracy. During active EoE, epithelial mast cells increased and lamina propria (LP) mast cells decreased. In controls and EoE remission patients, papillae had the highest mast cell density and negatively correlated with epithelial mast cell density. Mast cell density in the epithelium and papillae correlated with the degree of epithelial eosinophilic inflammation, basal zone hyperplasia, and LP fibrosis. MC-AI detected greater mast cell degranulation in the epithelium, papillae, and LP in EoE patients compared with control individuals. Mast cells were localized further from the basement membrane in active EoE than EoE remission and controls individuals but were closer than eosinophils to the basement membrane in active EoE. ConclusionUsing MC-AI, we identified a distinct population of homeostatic esophageal papillae mast cells; during active EoE, this population decreases, undergoes degranulation, negatively correlates with epithelial mast cell levels, and significantly correlates with distinct histologic features. Overall, MC-AI provides a means to understand the potential involvement of mast cells in EoE and other disorders. Clinical ImplicationWe have developed a methodology for identifying, enumerating, and characterizing mast cells using artificial intelligence; this has been applied to decipher eosinophilic esophagitis and provides a platform approach for other diseases. Capsule SummaryA machine learning protocol for identifying mast cells, designated Mast Cell-Artificial Intelligence, readily identified spatially distinct and dynamic populations of mast cells in EoE, providing a platform to better understand this cell type in EoE and other diseases.

immunology↗

Aryl hydrocarbon receptor (AHR) and IL-13 signaling crosstalk in human keratinocytes and atopic dermatitis

Atopic dermatitis (AD) is an allergic skin disease mediated by skin barrier impairment and IL-13-driven immune response. Activation of the aryl hydrocarbon receptor (AHR) has shown promise in early clinical trials for AD, however, the mechanism by which AHR mediates this function is unknown. Herein, AHR signaling is shown to be dysregulated in AD patients using publicly available gene expression data from biopsies of patients with AD compared with controls. AHR target genes, CYP1A1, CYP1A2 and NFE2L2 were decreased in lesional skin compared with healthy control skin (p=0.001, p<1.0*10-10 and p=6.1*10-6 respectively). Single cell RNA sequencing studies demonstrated increased expression of AHR (p<1.0*10-4 and p=0.049) and decreased expression of CYP1A1 in lesional AD keratinocytes compared with healthy control keratinocytes (p=5.0*10-4 and p=0.21). AHR activation reversed IL-13-dependent gene expression of several key genes in AD pathogenesis, most notably the eosinophil chemoattractant, CCL26 (Eotaxin-3). There was substantial overlap between differentially expressed genes in keratinocytes of patients with atopic dermatitis and AHR-regulated genes. Mechanistically, there was evidence for direct transcriptional effects of AHR as its binding motifs were identified in the differentially expressed genes from lesional AD keratinocytes compared to control keratinocytes and AHR activation did not modify IL-13-dependent signal transducer and activator of transcription 6 (STAT6) translocation to the nucleus. Together these data imply that AHR modulates IL-13 downstream signaling in keratinocytes through genome-wide direct transcriptional regulatory effects.

immunology↗

Aryl Hydrocarbon Receptor Suppresses Eosinophilic Esophagitis Responses through OVOL1 and SPINK7

Eosinophilic esophagitis (EoE) is a type 2 allergic disease characterized by esophageal inflammation and epithelial cell dysfunction. Acquired loss of the anti-serine protease of kazal type 7 (SPINK7) in the squamous epithelium of the esophagus has a causal role in EoE pathogenesis. Yet there is a limited understanding of the factors that regulate its expression and responsiveness to inflammatory stimuli. Herein, we identified the transcription factor, ovo like transcriptional repressor 1 (OVOL1) as an esophageal selective gene product that regulates SPINK7 promoter activity. Overexpression of OVOL1 increased SPINK7 expression, whereas, its depletion decreased SPINK7 expression, impaired epithelial barrier and increased production of the pro-atopy cytokine thymic stromal lymphopoietin (TSLP). Mechanistically, ligands of AHR induced nuclear translocation of OVOL1 which in turn promoted epithelial cell differentiation, barrier function and SPINK7 expression. Interleukin (IL)-4 and IL-13 abolished AHR ligand-induced OVOL1 nuclear translocation. Stimulation with IL-13 abrogated the nuclear translocation of OVOL1 and promoted enhanced degradation of OVOL1 protein. This effect of IL-13 was dependent on the esophageal specific cysteine protease calpain-14. Translational studies demonstrated loss of OVOL1 protein expression in patients with EoE. In summary, AHR mediates its action via OVOL1-induced SPINK7 transcription, and IL-4 and IL-13 repress this pathway in EoE. As such, activation of the AHR pathway is a potential intervention strategy for reversing EoE. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/541192v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1e946a1org.highwire.dtl.DTLVardef@1c087forg.highwire.dtl.DTLVardef@71ecc8org.highwire.dtl.DTLVardef@17044da_HPS_FORMAT_FIGEXP M_FIG C_FIG The influence of the exposome on regulatory networks in EoE pathogenesis. AHR is activated and influenced by diet nutrients, environmental toxicants, microbiome composition, tryptophan metabolites, and drugs. When AHR is activated, it promotes translocation of OVOL1 to the nucleus, which in turn promotes expression of epithelial genes including SPINK7. SPINK7 expression promotes epithelial differentiation, barrier function, decreased proteolytic activity, and decreased TSLP production. IL-4 and IL-13 inhibit OVOL1 nuclear translocation and therefore, repress SPINK7 expression. IL-13-stimulated CAPN14 expression decreases OVOL1 protein expression and SPINK7 transcription.

immunology↗

EGIDExpress: An Interactive Shiny Web App to Visualize and Share Large Biological Datasets

Biomedical research on rare diseases faces challenges such as low availability of biological specimens to study and limited funding. Eosinophilic gastrointestinal diseases (EGIDs) are rare conditions associated with inappropriate inflammation and the accumulation of eosinophils in various segments of the gastrointestinal tract. We aimed to build a repository of large datasets related to EGIDs that would be easily browsable, interpretable, and accessible in order to facilitate data sharing and hypothesis generation. Using the R-code based package Shiny, we built a website that allows visualization of multiple types of datasets including microarray, RNAseq, protein array, single-cell RNAseq, and ChIPseq. Users can access EGIDExpress (https://egidexpress.research.cchmc.org/) to browse data on a per-gene basis and to generate graphic representation of the data. Additionally, users can download the processed data to initiate their own analyses. Within 34 months of launching EGIDExpress, over 2400 users from 37 countries and 37 states within the United States accessed the site. Overall, EGIDExpress is accelerating research on EGIDs and provides a prototypic platform for broad research data sharing.

bioinformatics↗

Remote immune processes revealed by immune-derived circulating cell-free DNA

Blood cell counts often fail to report on immune processes occurring in remote tissues. Here we use immune cell type-specific methylation patterns in circulating cell-free DNA (cfDNA) for studying human immune cell dynamics. We characterized cfDNA released from specific immune cell types in healthy individuals (N=242), cross sectionally and longitudinally. Immune cfDNA levels had no individual steady state as opposed to blood cell counts, suggesting that cfDNA concentration reflects adjustment of cell survival to maintain homeostatic cell numbers. We also observed selective elevation of immune-derived cfDNA upon perturbations of immune homeostasis. Following influenza vaccination (N=92), B-cell-derived cfDNA levels increased prior to elevated B-cell counts and predicted efficacy of antibody production. Patients with Eosinophilic Esophagitis (N=21) and B-cell lymphoma (N=27) showed selective elevation of eosinophil and B-cell cfDNA respectively, which were undetectable by cell counts in blood. Immune-derived cfDNA provides a novel biomarker for monitoring immune responses to physiological and pathological processes that are not accessible using conventional methods.

genomics↗

A Web Portal and Workbench for Biological Dissection of Single Cell COVID-19 Host Responses

Numerous studies have provided single-cell transcriptome profiles of host responses to SARS-CoV-2 infection. Critically lacking however is a datamine that allows users to compare and explore cell profiles to gain insights and develop new hypotheses. To accomplish this, we harmonized datasets from COVID-19 and other control condition blood, bronchoalveolar lavage, and tissue samples, and derived a compendium of gene signature modules per cell type, subtype, clinical condition, and compartment. We demonstrate approaches to probe these via a new interactive web portal (http://toppcell.cchmc.org/ COVID-19). As examples, we develop three hypotheses: (1) a multicellular signaling cascade among alternatively differentiated monocyte-derived macrophages whose tasks include T cell recruitment and activation; (2) novel platelet subtypes with drastically modulated expression of genes responsible for adhesion, coagulation and thrombosis; and (3) a multilineage cell activator network able to drive extrafollicular B maturation via an ensemble of genes strongly associated with risk for developing post-viral autoimmunity.

immunology↗

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East Respiratory Syndrome Coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice with a half-life of 8.6 hours in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

microbiology↗

Molecular mechanism of SARS-CoV-2 cell entry inhibition via TMPRSS2 by Camostat and Nafamostat mesylate

The entry of the coronavirus SARS-CoV-2 into human cells can be inhibited by the approved drugs camostat and nafamostat. Here we elucidate the molecular mechanism of these drugs by combining experiments and simulations. In vitro assays confirm the hypothesis that both drugs act by inhibiting the human protein TMPRSS2. As no experimental structure is available, we provide a model of the TMPRSS2 equilibrium structure and its fluctuations by relaxing an initial homology structure with extensive 280 microseconds of all-atom molecular dynamics (MD) and Markov modeling. We describe the binding mode of both drugs with TMPRSS2 in a Michaelis complex (MC) state preceding the formation of a long-lived covalent inhibitory state. We find that nafamostat to has a higher MC population, which in turn leads to the more frequent formation of the covalent complex and thus higher inhibition efficacy, as confirmed in vitro and consistent with previous virus cell entry assays. Our TMPRSS2-drug structures are made public to guide the design of more potent and specific inhibitors.

biophysics↗

Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV2-Priming Protease TMPRSS2

Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. The main cellular location where the SARS-CoV-2 S protein priming occurs remains debatable, therefore hampering the development of targeted treatments. Herein, we identified the human extracellular serine protease inhibitor (serpin) alpha 1 antitrypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease-inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. Our data support the key role of extracellular serine proteases in SARS-CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells. SummaryDelivery of extracellular serine protease inhibitors (serpins) such as A1AT has the capacity to reduce SARS-CoV-2 dissemination by binding and inhibiting extracellular proteases on the host cells, thus, inhibiting the first step in SARS-CoV-2 cell cycle (i.e. cell entry).

microbiology↗

Increasing Rates of Diagnosis, Substantial Co-occurrence, and Variable Treatment Patterns of Eosinophilic Gastritis, Gastroenteritis and Colitis Based on 10 Year Data Across a Multi-Center Consortium

Financial Support and AcknowledgementsSupport for this project was provided through a research training grant as part of the Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR) (U54 AI117804). CEGIR is part of the Rare Disease Clinical Research Network (RDCRN), an initiative of the Office of Rare Diseases Research (ORDR), NCATS, and is funded through collaboration between NIAID, NIDDK, and NCATS. CEGIR is also supported by patient advocacy groups including APFED CURED and EFC. This project also received support from NIH T32 DK007634 (CCR).\n\nAuthor DisclosersPatricia Fulkerson: Grant funding from the NIH; Consultant for Genentech, Inc; Research support from Knopp Biosciences, LLC.\n\nGary Falk: Research support from Shire, Celgene, Adare, Regeneron. Consulting for Shire\n\nJonathan M. Spergel: Consultant for Regeneron, DBV Technology, Kaleo; Grant funding from DBV Technology, Aimmune Therapeutics, Food Allergy Research Education; Royalties from UpToDate\n\nNirmala Gonsalves: Royalties from UpToDate; Advisory board for Allakos\n\nSandeep K Gupta: Consultant for Alkalos, Abbott, QOL, Receptos; research support from Shire\n\nGlenn Furuta: Founder of EnteroTrack; Consultant for Shire; Royalties from UpToDate\n\nMarc E. Rothenberg: Consultant for Pulm One, Spoon Guru, ClostraBio, Celgene, Shire, Astra Zeneca, GlaxoSmithKline, Allakos, Adare, Regeneron and Novartis and has an equity interest in the first four listed and Immune Pharmaceuticals, and royalties from reslizumab (Teva Pharmaceuticals), PEESSv2 (Mapi Research Trust) and UpToDate. M.E.R. is an inventor of patents owned by Cincinnati Childrens.\n\nEvan Dellon: Consultant for Adare, Allakos, Alivio, Banner, Celgen/Receptos, Enumeral, GSK, Regeneron, Shire; Research funding from Adare, Celegene/Receptos, Miraca, Meritage, Nutricia, Regeneron, Shire, Educational grant from Banner, Holoclara\n\nStudy HighlightsO_ST_ABSWhat is current knowledge?C_ST_ABSO_LIEosinophilic gastrointestinal disorders (EGIDs) include eosinophilic esophagitis (EoE), eosinophilic gastritis (EG), gastroenteritis (EGE), and colitis (EC).\nC_LIO_LINon-EoE EGIDs are rare with most studies limited to case reports or review of single center experiences.\nC_LIO_LIThere are no widely established guidelines for the diagnosis of EG, EGE, or EC.\nC_LI\n\nWhat is new here?O_LIIn this multicenter study, EG, EGE, and EC were all diagnosed with increasing frequency over the past decade.\nC_LIO_LIPresenting symptoms are non-specific and do not reliably distinguish between disorders.\nC_LIO_LIThere was no male predominance and the majority of subjects had atopy.\nC_LIO_LICo-occurrence of EG, EGE, and EC diagnoses is common, seen in 41% of patients.\nC_LIO_LIThere is substantial variability between centers in initial treatment approaches.\nC_LI

epidemiology↗