Single-cell RNA-sequencing of human eosinophils in allergic inflammation in the esophagus.

BACKGROUND: Eosinophils are elusive cells involved in allergic inflammation. Single-cell RNA-sequencing (scRNA-seq) is an emerging approach to deeply characterize cellular properties, heterogeneity, and functionality. OBJECTIVES: We sought to comprehensively characterize the transcriptome and biological functions of human eosinophils at a site of severe allergic inflammation in the esophagus (ie, eosinophilic esophagitis [EoE]). METHODS: We employed a gravity-based scRNA-seq methodology to sequence blood eosinophils from patients with EoE and control individuals compared to a reanalyzed public scRNA-seq dataset of human esophageal eosinophils of EoE patients. We used flow cytometry, immunostaining, and a stimulation assay to verify mRNA findings. RESULTS: In total, scRNA-seq was obtained from 586 eosinophils (188 from blood [n = 6 individuals] and 398 from esophagus [n = 6 individuals]). The esophageal eosinophils were composed of a population of activated eosinophils (enriched in 659 genes compared with peripheral blood-associated eosinophils) and a small population of eosinophils resembling peripheral blood eosinophils (enriched in 62 genes compared with esophageal eosinophils). Esophageal eosinophils expressed genes involved in sensing and responding to diverse stimuli, most notably IFN-γ, IL-10, histamine and leukotrienes, and succinate. Esophageal eosinophils were most distinguished from other esophageal populations by gene expression of the receptors CCR3, HRH4, SUCNR1, and VSTM1; transcription factors CEBPE, OLIG1, and OLIG2; protease PRSS33; and the hallmark eosinophil gene CLC. A web of bidirectional eosinophil interactions with other esophageal populations was derived. Comparing esophageal eosinophils and mast cells revealed that esophageal eosinophils expressed genes involved in DNAX-activation protein-12 (also known as TYROBP) interactions, IgG receptor-triggered events, immunoregulation, and IL-10 signaling. CONCLUSIONS: In EoE, esophageal eosinophils exist as 2 populations, a minority population resembling blood eosinophils and the other population characterized by high de novo transcription of diverse sensing receptors and inflammatory mediators readying them to potentially intersect with diverse cell types.

Amniotic Fluid Modifies Esophageal Epithelium Differentiation and Inflammatory Responses.

The interplay between genetic and environmental factors during pregnancy can predispose to inflammatory diseases postnatally, including eosinophilic esophagitis, a chronic allergic disease triggered by food. Herein, we examined the effects of amniotic fluid (AF) on esophageal epithelial differentiation and responsiveness to pro-allergic stimuli. Multiplex analysis of AF revealed the expression of 66 cytokines, whereas 5 cytokines including IL-4 and TSLP were not detected. Several pro-inflammatory cytokines including TNFa and IL-12 were highly expressed in the AF from women who underwent preterm birth, while EGF was the highest in term birth samples. Exposure of esophageal epithelial cells to AF resulted in transient phosphorylation of ERK1/2 and the transcription of early response genes, highlighting the direct impact of AF on esophageal epithelial cells. In a 3-dimensional spheroid model, AF modified the esophageal epithelial differentiation program and enhanced the transcription of IL-13-target genes, including CCL26 and CAPN14, which encodes for a major genetic susceptibility locus for eosinophilic esophagitis. Notably, CAPN14 exhibited upregulation in spheroids exposed to preterm but not term AF following differentiation. Collectively, our findings call attention to the role of AF as a potential mediator of the intrauterine environment that influences subsequent esophageal disorders.

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 covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) 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 h 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.

Broad transcriptional response of the human esophageal epithelium to proton pump inhibitors.

BACKGROUND: Proton pump inhibitors (PPIs) have been recognized as a primary treatment of eosinophilic esophagitis (EoE), an allergic inflammatory disease of the esophageal mucosa. The mechanisms underlying esophageal epithelial responses to PPIs remain poorly understood. OBJECTIVE: We hypothesized that PPIs can counteract IL-13-mediated esophageal epithelial responses that are germane for EoE pathogenesis. METHODS: Transcriptional responses of human esophageal cells to IL-13 and the PPIs omeprazole and esomeprazole were assessed by RT-PCR and RNA sequencing. Cytokine secretion was measured by multiplex analysis and ELISA. RESULTS: Human esophageal epithelial cells robustly responded to PPI stimulation by inducing a set of 479 core genes common between omeprazole and esomeprazole treatments. The transcriptional response to PPIs was partially mediated through the aryl hydrocarbon receptor signaling pathway, as the aryl hydrocarbon receptor antagonist GNF-351 modified approximately 200 genes, particularly those enriched in metabolic processes and regulation of cell death. PPI treatment reversed approximately 20% of the IL-13 transcriptome. Functional analysis of the PPI-responsive, upregulated genes revealed enrichment in metabolic and oxidation processes, and the unfolded protein response. In contrast, downregulated genes were overrepresented in functional terms related to cell division and cytoskeletal organization, which were also enriched for the genes in the EoE transcriptome reversed by PPIs. Furthermore, PPI treatment decreased the IL-13-induced proliferative response of esophageal epithelial cells. CONCLUSIONS: These results demonstrate broad effects of PPIs on esophageal epithelium, including their ability to curtail transcriptomic processes involved in cellular proliferation and IL-13-induced responses, and they highlight the importance of AHR signaling in mediating these responses.

Molecular mechanism of inhibiting the SARS-CoV-2 cell entry facilitator TMPRSS2 with camostat and nafamostat.

The entry of the coronavirus SARS-CoV-2 into human lung 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 that both drugs inhibit the human protein TMPRSS2, a SARS-Cov-2 spike protein activator. 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 330 microseconds of all-atom molecular dynamics (MD) and Markov modeling. Through Markov modeling, we describe the binding process of both drugs and a metabolic product of camostat (GBPA) to TMPRSS2, reaching a Michaelis complex (MC) state, which precedes the formation of a long-lived covalent inhibitory state. We find that nafamostat has a higher MC population than camostat and GBPA, suggesting that nafamostat is more readily available to form the stable covalent enzyme-substrate intermediate, effectively explaining its high potency. This model is backed by our in vitro experiments 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.

Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2-Priming Protease TMPRSS2.

BACKGROUND: 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. METHODS: We developed a cell-based assay to identify TMPRSS2 inhibitors. Inhibitory activity was established in SARS-CoV-2 viral load systems. RESULTS: We identified the human extracellular serine protease inhibitor (serpin) alpha 1 anti-trypsin (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. CONCLUSIONS: 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.

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 IC 50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC 50 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 EC 50 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 EC 50 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.

Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2-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. SUMMARY: Delivery 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).

Functional role of kallikrein 5 and proteinase-activated receptor 2 in eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, food antigen-driven, inflammatory disease of the esophagus and is associated with impaired barrier function. Evidence is emerging that loss of esophageal expression of the serine peptidase inhibitor, kazal type 7 (SPINK7), is an upstream event in EoE pathogenesis. Here, we provide evidence that loss of SPINK7 mediates its pro-EoE effects via kallikrein 5 (KLK5) and its substrate, protease-activated receptor 2 (PAR2). Overexpression of KLK5 in differentiated esophageal epithelial cells recapitulated the effect of SPINK7 gene silencing, including barrier impairment and loss of desmoglein-1 expression. Conversely, KLK5 deficiency attenuated allergen-induced esophageal protease activity, modified commensal microbiome composition, and attenuated eosinophilia in a murine model of EoE. Inhibition of PAR2 blunted the cytokine production associated with loss of SPINK7 in epithelial cells and attenuated the allergen-induced esophageal eosinophilia in vivo. Clinical samples substantiated dysregulated PAR2 expression in the esophagus of patients with EoE, and delivery of the clinically approved drug α1 antitrypsin (A1AT, a protease inhibitor) inhibited experimental EoE. These findings demonstrate a role for the balance between KLK5 and protease inhibitors in the esophagus and highlight EoE as a protease-mediated disease. We suggest that antagonizing KLK5 and/or PAR2 has potential to be therapeutic for EoE.