Doyle, Alfred D.

ABSTRACT Background Eosinophilic esophagitis is associated with epithelial barrier dysfunction. Epidemiologic studies suggest environmental factors promote disease pathogenesis. The common household detergent sodium dodecyl sulfate (SDS) induces epithelial barrier dysfunction and eosinophilic inflammation in mice. We hypothesized that acute SDS exposure would compromise the esophageal mucosal barrier in humans. Methods Healthy adults brushed with 2 g of an SDS‐containing toothpaste. Esophageal impedance was measured continuously pre‐/post‐toothbrushing to assess the effect of toothpaste on epithelial barrier function. SDS was measured in saliva using a methylene blue anionic substances assay. Participants completed 1‐h esophageal string tests (ESTs) pre‐/post‐toothbrushing and protein isolates from EST eluates from the proximal and distal esophagus were analyzed by tandem mass tag mass spectrometry. Results Ten of twelve subjects completed the clinical study. SDS in the initial expectorate measured immediately after toothbrushing varied markedly (median: 321.40 μg/mL; range: 169.90–1243.00 μg/mL) and was detectable in saliva in 80% of subjects (median: 9.55 μg/mL; range: 0–123.40 μg/mL) at 60 min post toothbrushing. Esophageal mucosal impedance decreased within 30 min of toothbrushing ( p < 0.01). Toothbrushing increased salivary viscosity and proteins associated with mucosal defense in EST eluates. The tight junction pathway was downregulated while the salivary secretion and complement and coagulation cascades pathways were upregulated in the proximal esophagus. Conclusion In healthy individuals, SDS remained detectable in saliva for a prolonged period after toothbrushing at concentrations previously shown to disrupt esophageal epithelial barrier function in vitro. Toothbrushing with an SDS‐containing toothpaste was associated with reduced esophageal mucosal impedance, altered salivary properties, and activated mucosal immune responses.

Background Eosinophilic oesophagitis (EoE) is a food allergen-induced inflammatory disorder characterised by interleukin (IL)-13-mediated oesophageal inflammation and epithelial basal cell hyperplasia (BCH). The role of mitochondria in EoE pathogenesis remains elusive. Design Prompted by single cell transcriptomics data, we interrogated the role of mitochondria in EoE pathobiology using patient biopsies, EoE-mouse models and oesophageal epithelial cells grown in monolayer and three-dimensional (3D) organoid cultures treated with EoE-relevant cytokines. 3D organoids and EoE-bearing mice were treated with omeprazole—a proton-pump inhibitor used as first-line EoE therapy. We performed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference in mouse organoids to identify the key mitochondrial regulatory genes whose depletion may lead to BCH. We analysed mitochondrial membrane potential, mass and superoxide production by flow cytometry, cellular oxygen consumption by respirometry, mitochondrial structures and perturbation of cellular energy homeostasis by immunoblotting. Results Mitochondrial dysfunction appeared to be a hallmark of EoE-related BCH where mitochondrial structural damage was associated with impaired oxidative respiratory capacity, elevation of mitochondrial superoxide and decreased adenosine triphosphate (ATP) production, as corroborated by activation of the adenosine monophosphate (AMP) -activated protein kinase and suppression of mammalian target-of-rapamycin signalling. Depletion of PGC1A, the master regulator of mitochondria biogenesis, recapitulated EoE-related BCH, suggesting that mitochondrial dysfunction drives BCH. Further, omeprazole alleviated mitochondrial damage and dysfunction in EoE-related BCH modelled in mice and patient-derived organoids. Conclusion Mitochondrial dysfunction is tightly linked to perturbation of redox homeostasis in EoE-related BCH, which is promoted by IL-13 and reversible with omeprazole treatment.

Abstract Eosinophil effector functions have been hypothesized to be mediated in part by the release of proteins stored in the secondary granules of mature cells of this leukocyte lineage. Major basic protein-1 (MBP-1) and eosinophil peroxidase (EPO) comprise the majority of protein in these granules. No role has been speculated for these proteins in eosinophilopoiesis and/or the accumulation of mature cells in circulation. Methods: Eosinophilopoiesis and eosinophil accumulation in the periphery were assessed in single and double knockout (MBP-1-/- and/or EPO-/-) mice. Bone marrow and peripheral blood leukocytes were assessed by IHC, FACS and in vitro cell culture assays. Mice were subjected to an acute OVA protocol assessing induced pulmonary pathologies in each of the strains of granule protein knockout mice. Results: We demonstrate that, unlike the single deficiency of MBP-1 or EPO, the absence of both granule proteins resulted in the loss of peripheral blood eosinophils. IHC assessments of bone marrow and spleen demonstrate that eosinophil lineage commitment occurs in these mice. However, these assessments and FACS studies demonstrated a blockade in the maturation of eosinophil-lineage committed cells. This blockade is not rescued by ex vivo culture or by bone marrow engraftment into wild type recipient mice. Similar to other eosinophil-less mouse models, MBP-1-/-/EPO-/- mice also fail to develop pulmonary inflammation in an OVA protocol.

INTRODUCTION: We aimed to assess the diagnostic utility of eosinophil peroxidase (EPX) staining on Cytosponge (CS) samples in eosinophilic esophagitis (EoE). METHODS: Esophageal biopsy (BX) samples from adult subjects with EoE were assessed using peak eosinophils per high-power field (eos/hpf), EPX, and the EoE histologic scoring system. EPX staining and eos/hpf were compared (BX vs CS). RESULTS: CS EPX positivity correlated with eos/hpf (CS [ r = 0.82, P < 0.0001]; BX [ r = 0.65, P < 0.0001]) and EoE histologic scoring system (grade [ r = 0.62, P < 0.00001]; stage [ r = 0.61, P < 0.0001]). CS EPX identified subjects with active EoE (area under the curve = 0.86, P < 0.0001). DISCUSSION: The correlation of CS EPX with eosinophilic inflammation and histologic disease severity supports its diagnostic utility in EoE.

AbstractBackgroundEosinophilic esophagitis (EoE) is a chronic allergic disease associated with type 2 inflammation and epithelial barrier dysfunction. The etiology is unknown, however, genetic heritability studies suggest environmental factors play a key role in pathogenesis. Detergents, such as sodium dodecyl sulfate (SDS), are common ingredients in household products such as dish soap and toothpaste. We hypothesized detergent exposure decreases epithelial barrier function and induces esophageal inflammation.MethodsImmortalized esophageal epithelial cells (EPC2) were cultured in air‐liquid interface (ALI) and exposed to SDS. Barrier function/activity was assessed by transepithelial electrical resistance (TEER), FITC‐dextran flux, and RT‐PCR. Additionally, SDS‐treated mouse esophageal organoids were evaluated for morphology. To investigate the effects of SDS in vivo, mice were treated with 0.5% SDS in drinking water for 14 days. Esophagi were assessed by gross morphology, histopathology, protein expression, and bulk RNA sequencing.ResultsWhen EPC2 cells were exposed to SDS (5 μg/ml) for 96 h, TEER decreased (p = 0.03), and FITC‐dextran flux increased (p = 0.0002). mRNA expression of IL‐33 increased 4.5‐fold (p = 0.02) at 6 h and DSG1 decreased (p < 0.0001) by 72 h. Disrupted epithelial integrity was noted in SDS‐treated esophageal organoids. When mice were exposed to SDS, they showed increased esophageal width, chemokine, and metalloprotease levels. Mice treated with SDS also showed increased IL‐33 protein expression, basal zone hyperplasia, CD4+ cell infiltration, and esophageal eosinophilia. RNA sequencing revealed upregulation of immune response pathway genes.ConclusionExposure to SDS decreases esophageal barrier integrity, stimulates IL‐33 production, and promotes epithelial hyperplasia and tissue eosinophilia. Detergents may be a key environmental trigger in EoE pathogenesis.

Background Diagnosis of non-esophageal eosinophilic gastrointestinal disorders requires quantification of tissue eosinophils. Our objective was to evaluate eosinophil peroxidase (EPX) immunohistochemistry (IHC) as a method for histologic diagnosis of eosinophilic gastritis (EG) and eosinophilic duodenitis (EoD). Methods We performed a retrospective analysis of biopsies from pediatric EG/EoD cases and controls. Subjects with EG or EoD had ≥30 eosinophils per high power field (eos/hpf) in ≥5 hpf in the stomach and/or ≥3 hpf in the duodenum, respectively. Controls had no histopathologic diagnosis recorded. Tissue eosinophil counts were assessed by hematoxylin & eosin stains. EPX stains were assessed using a unique histopathologic scoring system. Slides were digitized and EPX+ staining area/mm 2 was quantified by image analysis. Results Twenty-six EG/EoD cases and 40 controls were analyzed. EPX scores and EPX/mm 2 levels were markedly elevated in EG/EoD (p ≤ 0.0001). Eosinophil density (eos/mm 2 ) correlated strongly with EPX scores and EPX/mm 2 levels in the stomach (r ≥ 0.77) and moderately with EPX scores and EPX/mm 2 levels in the duodenum (r ≥ 0.52); (p < 0.0001). EPX quantification identified EG/EoD subjects with high diagnostic accuracy (EPX score: AUC = 1 for EG and EoD; EPX/mm 2 : AUC = 0.98 (95%CI 0.96-1) for EG, AUC = 0.91 (95%CI 0.81-1) for EoD). Conclusion EPX-based assessment of eosinophilic inflammation may facilitate automated histologic diagnosis.

Abstract Eosinophils and the release of cationic granule proteins have long been implicated in the development of the type 2–induced pathologies linked with respiratory inflammation. Paradoxically, the ablation of the two genes encoding the most abundant of these granule proteins, major basic protein-1 (MBP-1) and eosinophil peroxidase (EPX), results in a near collapse of eosinophilopoiesis. The specificity of this lineage ablation and the magnitude of the induced eosinopenia provide a unique opportunity to clarify the importance of eosinophils in acute and chronic inflammatory settings, as well as to identify potential mechanism(s) of action linked with pulmonary eosinophils in those settings. Specifically, we examined these issues by assessing the induced immune responses and pathologies occurring in MBP-1−/−/EPX−/− mice after 1) ovalbumin sensitization/provocation in an acute allergen-challenge protocol, and 2) crossing MBP-1−/−/EPX−/− mice with a double-transgenic model of chronic type 2 inflammation (i.e., I5/hE2). Acute allergen challenge and constitutive cytokine/chemokine expression each induced the accumulation of pulmonary eosinophils in wild-type controls that was abolished in the absence of MBP-1 and EPX (i.e., MBP-1−/−/EPX−/− mice). The expression of MBP-1 and EPX was also required for induced lung expression of IL-4/IL-13 in each setting and, in turn, the induced pulmonary remodeling events and lung dysfunction. In summary, MBP-1−/−/EPX−/− mice provide yet another definitive example of the immunoregulatory role of pulmonary eosinophils. These results highlight the utility of this unique strain of eosinophil-deficient mice as part of in vivo model studies investigating the roles of eosinophils in health and disease settings.

Abstract Rationale The release of eosinophil granule proteins in the lungs of patients with asthma has been dogmatically linked with lung remodeling and airway hyperresponsiveness. However, the demonstrated inability of established mouse models to display the eosinophil degranulation occurring in human subjects has prevented a definitive in vivo test of this hypothesis. Objectives To demonstrate in vivo causative links between induced pulmonary histopathologies/lung dysfunction and eosinophil degranulation. Methods A transgenic mouse model of chronic T-helper cell type 2–driven inflammation overexpressing IL-5 from T cells and human eotaxin 2 in the lung (I5/hE2) was used to test the hypothesis that chronic histopathologies and the development of airway hyperresponsiveness occur as a consequence of extensive eosinophil degranulation in the lung parenchyma. Measurement and Main Results Studies targeting specific inflammatory pathways in I5/hE2 mice surprisingly showed that eosinophil-dependent immunoregulative events and not the release of individual secondary granule proteins are the central contributors to T-helper cell type 2–induced pulmonary remodeling and lung dysfunction. Specifically, our studies highlighted a significant role for eosinophil-dependent IL-13 expression. In contrast, extensive degranulation leading to the release of major basic protein-1 or eosinophil peroxidase was not causatively linked to many of the induced pulmonary histopathologies. However, these studies did define a previously unappreciated link between the release of eosinophil peroxidase (but not major basic protein-1) and observed levels of induced airway mucin. Conclusions These data suggest that improvements observed in patients with asthma responding to therapeutic strategies ablating eosinophils may occur as a consequence of targeting immunoregulatory mechanisms and not by simply eliminating the destructive activities of these purportedly end-stage effector cells.

AbstractBackgroundEosinophils are hallmark cells of allergic Th2 respiratory inflammation. However, the relative importance of eosinophil activation and the induction of effector functions such as the expression ofIL‐13 to allergic Th2 pulmonary disease remain to be defined.MethodsWild‐type or cytokine‐deficient (IL‐13−/−orIL‐4−/−) eosinophils treated with cytokines (GM‐CSF,IL‐4,IL‐33) were adoptively transferred into eosinophil‐deficient recipient mice subjected to allergen provocation using established models of respiratory inflammation. Allergen‐induced pulmonary changes were assessed.ResultsIn contrast to the transfer of untreated blood eosinophils to the lungs of recipient eosinophil deficient mice, which induced no immune/inflammatory changes either in the lung or in the lung draining lymph nodes (LDLN), pretreatment of blood eosinophils withGM‐CSFprior to transfer elicited trafficking of these eosinophils toLDLN. In turn, theseLDLNeosinophils elicited the accumulation of dendritic cells andCD4+T cells to these sameLDLNs without inducing pulmonary inflammation. However, exposure of eosinophils toGM‐CSF,IL‐4, andIL‐33 prior to transfer induced not only immune events in theLDLN, but also allergen‐mediated increases in airway Th2 cytokine/chemokine levels, the subsequent accumulation ofCD4+T cells as well as alternatively activated (M2) macrophages, and the induction of pulmonary histopathologies. Significantly, this allergic respiratory inflammation was dependent on eosinophil‐derivedIL‐13, whereasIL‐4 expression by eosinophils had no significant role.ConclusionThe data demonstrate the differential activation of eosinophils as a function of cytokine exposure and suggest that eosinophil‐specificIL‐13 expression by activated cells is a necessary component of the subsequent allergic Th2 pulmonary pathologies.