Current state of biologics in treating eosinophilic esophagitis.

OBJECTIVE: Eosinophilic esophagitis (EoE) is a chronic, allergen-mediated, eosinophil-predominant, type 2 inflammatory disease that progresses to fibrostenosis of the esophagus if left untreated. This review focuses on biologics therapy in EoE. DATA SOURCES: Manuscripts on EoE treatments are identified on PubMed. STUDY SELECTIONS: Original research, randomized controlled trials, retrospective studies, meta-analyses, case series, and case reports of high relevance are selected and reviewed. RESULTS: Biologics have been used as investigational therapies for EoE in clinical studies over the years, based on earlier work that identified key cytokines and mediators of eosinophilic inflammation and, more recently, type 2 inflammation that underlie EoE pathogenesis. Dupilumab, a monoclonal antibody that targets the interleukin (IL)-4Rα chain, thereby interfering with IL-4 and IL-13 binding with the receptor, was recently approved by the Food and Drug Administration for EoE. Dupilumab improved clinical symptoms, endoscopic scores, histologic inflammation, and esophageal distensibility. Several clinical trials that target key cytokines such as IL-5, IL-13, and thymic stromal lymphopoietin in EoE are still ongoing. CONCLUSION: Topical corticosteroid, proton pump inhibitor therapy, elimination diet, and dilation are widely accepted treatment modalities for EoE. Dupilumab is the first Food and Drug Administration-approved therapy for EoE. Other studies evaluating biologics that target eosinophils, key cytokines, and inflammatory pathways in EoE are ongoing. Treatment algorithms are needed to position EoE therapies as they emerge.

LIGHT controls distinct homeostatic and inflammatory gene expression profiles in esophageal fibroblasts via differential HVEM and LTßR-mediated mechanisms.

Fibroblasts mediate tissue remodeling in eosinophilic esophagitis (EoE), a chronic allergen-driven inflammatory pathology. Diverse fibroblast subtypes with homeostasis-regulating or inflammatory profiles have been recognized in various tissues, but which mediators induce these alternate differentiation states remain largely unknown. We recently identified that TNFSF14/LIGHT promotes an inflammatory esophageal fibroblast in vitro. Herein we used esophageal biopsies and primary fibroblasts to investigate the role of the LIGHT receptors, herpes virus entry mediator (HVEM) and lymphotoxin-beta receptor (LTßR), and their downstream activated pathways, in EoE. In addition to promoting inflammatory gene expression, LIGHT down-regulated homeostatic factors including WNTs, BMPs and type 3 semaphorins. In vivo, WNT2B+ fibroblasts were decreased while ICAM-1+ and IL-34+ fibroblasts were expanded in EoE, suggesting that a LIGHT-driven gene signature was imprinted in EoE versus normal esophageal fibroblasts. HVEM and LTßR overexpression and deficiency experiments demonstrated that HVEM regulates a limited subset of LIGHT targets, whereas LTßR controls all transcriptional effects. Pharmacologic blockade of the non-canonical NIK/p100/p52-mediated NF-κB pathway potently silenced LIGHT's transcriptional effects, with a lesser role found for p65 canonical NF-κB. Collectively, our results show that LIGHT promotes differentiation of esophageal fibroblasts toward an inflammatory phenotype and represses homeostatic gene expression via a LTßR-NIK-p52 NF-κB dominant pathway.

Type 2 Immunity and Age Modify Gene Expression of Coronavirus-induced Disease 2019 Receptors in Eosinophilic Gastrointestinal Disorders.

ABSTRACT: Infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can lead to coronavirus-induced disease 2019 (COVID-19). The gastrointestinal (GI) tract is now an appreciated portal of infection. SARS-CoV-2 enters host cells via angiotensin-converting enzyme-2 (ACE2) and the serine protease TMPRSS2. Eosinophilic gastrointestinal disorders (EGIDs) are inflammatory conditions caused by chronic type 2 (T2) inflammation. the effects of the T2 atopic inflammatory milieu on SARS-COV-2 viral entry gene expression in the GI tract is poorly understood. We analyzed tissue ACE2 and TMPRSS2 gene expression in pediatric eosinophilic esophagitis (EoE), eosinophilic gastritis (EG), and in normal adult esophagi using publicly available RNA-sequencing datasets. Similar to findings evaluating the airway, there was no difference in tissue ACE2/TMPRSS2 expression in EoE or EG when compared with control non-EoE/EG esophagus/stomach. ACE2 gene expression was significantly lower in esophagi from children with or without EoE and from adults with EoE as compared with normal adult esophagi. Type 2 immunity and pediatric age could be protective for infection by SARS-CoV-2 in the gastrointestinal tract because of decreased expression of ACE2.

Antifibrotic Effects of the Thiazolidinediones in Eosinophilic Esophagitis Pathologic Remodeling: A Preclinical Evaluation.

INTRODUCTION: Eosinophilic esophagitis (EoE) is a T-helper 2 (Th2), eosinophilic disease associated with pathologic tissue remodeling that leads to end-organ dysfunction. During early-stage disease, inflammation and subepithelial fibrosis are coupled and reversible, but in late-stage or therapy-resistant disease, there can be uncoupling of these features with progressive esophageal rigidity and strictures contributing to clinical dysphagia and food impactions. No current pharmacotherapeutic interventions directly target esophageal fibrosis. Based on the ability of the thiazolidinediones (TZD) to regulate intestinal and hepatic fibrosis, we tested the antifibrotic effects of the TZDs, rosiglitazone and pioglitazone, in preclinical studies using primary human esophageal fibroblasts. METHODS: Primary fibroblasts isolated from normal or EoE esophagi were treated with transforming growth factor (TGF)-ß1 in the absence or presence of TZDs and, in some experiments, without or with budesonide and analyzed by quantitative real-time PCR and immunoblotting. Immunohistochemical analysis of human esophageal biopsies was performed. RESULTS: EoE esophageal biopsies and esophageal fibroblasts expressed higher levels of the TZD receptor, peroxisome proliferator-activated receptor-γ (PPAR-γ), than normal controls. PPAR-γ was inducible by the Th2 cytokine, interleukin 4 (IL-4). TZD significantly reduced TGF-ß1-induced myofibroblast and fibrotic gene and protein expression preferentially in EoE, but not normal esophageal fibroblasts. In esophageal fibroblasts, TGF-ß1 increased phosphorylated Smad2/3 and p38, but TZDs preferentially inhibited p38 phosphorylation, suggesting signaling pathway-specific effects. The TZDs were more potent than budesonide at decreasing collagen-1α1 expression. DISCUSSION: The TZDs preferentially exert antifibrotic effects in TGF-ß1-activated EoE fibroblasts and provide a preclinical foundation for further investigation of the potential of the TZDs in EoE pathologic remodeling.

New Developments in the Diagnosis and Treatment of Eosinophilic Esophagitis.

PURPOSE OF REVIEW: Eosinophilic esophagitis (EoE) is a chronic, allergen-driven, immune-mediated disease of the esophagus that progresses to esophageal fibrostenosis if left untreated. The aim of this review is to provide a concise update on recent clinically relevant advances in the development of diagnostic and therapeutic approaches for EoE. RECENT FINDINGS: Current diagnostic and disease monitoring protocols for EoE rely on repetitive endoscopic evaluations and esophageal tissue acquisition for histopathologic analysis. Recent advancements in EoE diagnosis include endoscopic functional lumen imaging probe (FLIP), transnasal endoscopy (TNE), and the emergence of non-invasive diagnostic tools including cytosponge, esophageal string test, and mucosal impedance probe. Biomarkers for EoE have not yet proven their clinical utility. No Food and Drug Administration (FDA)-approved drugs currently exist for the treatment of EoE. Topical corticosteroid, proton-pump inhibitors (PPI), elimination diet, and dilation are the current treatment modalities for confirmed EoE. Promising results from clinical trials are emerging for biologic agents that target the interleukin (IL)-13 and the IL-4/IL-13 receptor, specifically, RPC4046 and dupilumab, respectively. New diagnostic algorithms, non-invasive diagnostic strategies, and treatment modalities for EoE are emerging. Patients with EoE continue to require a multimodal and multi-disciplinary management approach.

Medical and dietary management of eosinophilic esophagitis.

OBJECTIVE: Eosinophilic esophagitis (EoE) is a disease of chronic, allergen-driven, T-helper 2 (Th2) immune-mediated inflammation that progresses to fibrostenosis of the esophagus if left untreated. There are currently no Food and Drug Administration (FDA)-approved drugs for the treatment of EoE. This review focuses on the medical and dietary management of EoE. DATA SOURCES: Manuscripts on EoE treatments were identified on PubMed. STUDY SELECTIONS: Original research, randomized control trials, retrospective studies, meta-analyses, case series, and on occasions, case reports of high relevance, were selected and reviewed. RESULTS: Current treatment strategies available to EoE patients center on monotherapy or combination therapy with dietary modification to exclude antigenic stimulation and topical corticosteroids to control Th2-mediated tissue inflammation and pathologic remodeling. Dilation as a rescue therapy for the narrowed, fibrostenotic, symptomatic esophagus can potentially be avoided with optimal medical and elimination diet therapies. The molecular mechanisms underlying EoE pathogenesis are being unraveled, from which targeted therapies can be developed and evaluated in preclinical and clinical studies. Current clinical research efforts focus on optimization of topical corticosteroid delivery, dosing, frequency, and duration of treatment, either alone or in combination with tailored elimination diet. Preliminary clinical trials with biologics targeting interleukin (IL)-5 and IL-13/IL-4 have been completed. CONCLUSION: Topical corticosteroid, elimination diet, and dilation are the current treatment modalities for confirmed EoE. The use of proton-pump inhibitors (PPI) is being suggested as a potential regimen to treat EoE, based on evolving understanding of PPI-responsive esophageal eosinophilia (PPI-REE). The complexity of EoE treatment regimens and frequent follow-ups require a multimodal, multi-disciplinary management approach to optimize patient care.

A rare localised nasal CD30+ primary cutaneous T-cell lymphoma following liver transplantation.

Cutaneous T-cell post-transplant lymphoproliferative disorder (PTLD) is a rare clinical presentation that can potentially turn aggressive in solid-organ transplant recipients if not detected and intervened on early. We encountered a rare case of rapidly worsening primary cutaneous CD30-positive, Epstein-Barr virus-negative anaplastic large cell lymphoma (ALCL) of T-cell origin, manifesting as an isolated nasal tip lesion in a 71-year-old man 4 years after orthotopic liver transplantation. Excisional biopsy with partial rhinectomy showed subepithelial diffuse infiltration of medium-to-large lymphoid cells having round-to-irregular nuclei, partially condensed chromatin and prominent nucleoli. Immunophenotypic studies revealed CD30-positive primary cutaneous ALCL. Positron emission tomography/CT imaging revealed a locally active disease, and radiation therapy was initiated with complete response. A high index of suspicion for PTLD when evaluating skin lesions in a post-transplant patient is paramount for its early recognition, prompt diagnosis and timely intervention while the window for curative therapy remains possible.

Tissue Remodeling in Chronic Eosinophilic Esophageal Inflammation: Parallels in Asthma and Therapeutic Perspectives.

Chronic eosinophilic inflammation is associated with tissue remodeling and fibrosis in a number of chronic T-helper 2 (Th2)-mediated diseases including eosinophilic esophagitis (EoE) and asthma. Chronic inflammation results in dysregulated tissue healing, leading to fibrosis and end organ dysfunction, manifesting clinically as irreversible airway obstruction in asthma and as esophageal rigidity, strictures, narrowing, dysmotility, dysphagia, and food impactions in EoE. Current therapies for EoE and asthma center on reducing inflammation-driven tissue remodeling and fibrosis with corticosteroids, coupled with symptomatic control and allergen avoidance. Additional control of Th2 inflammation can be achieved in select asthma patients with biologic therapies such as anti-IL-5 and anti-IL-13 antibodies, which have also been trialed in EoE. Recent molecular analysis suggests an emerging role for structural cell dysfunction, either inherited or acquired, in the pathogenesis and progression of EoE and asthma tissue remodeling. In addition, new data suggest that inflammation-independent end organ rigidity can alter structural cell function. Herein, we review emerging data and concepts for the pathogenesis of tissue remodeling and fibrosis primarily in EoE and relevant pathogenetic parallels in asthma, focusing additionally on emerging disease-specific therapies and the ability of these therapies to reduce tissue remodeling in subsets of patients.

Pulmonary complications of transcatheter arterial chemoembolization for hepatocellular carcinoma.

Transarterial chemoembolization (TACE) is an effective palliative intervention that is widely accepted for the management of hepatocellular carcinoma (HCC). Post-TACE pulmonary complications resulting in acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) are rare events. Pulmonary complications after TACE are thought to be related to chemical injury subsequent to the migration of the infused ethiodized oil or chemotherapeutic agent to the lung vasculature, facilitated by arteriovenous (AV) shunts within the hyper-vascular HCC. We review herein the literature on pulmonary complications related to TACE for HCC. Post-TACE pulmonary complications have included pulmonary oil embolism, interstitial pneumonitis, chemical pneumonitis, ALI, ARDS, lipoid pneumonia, acute eosinophilic and neutrophilic pneumonia, bilious pleuritis, pulmonary abscess, pulmonary tumor embolism, and possibly pulmonary metastasis with HCC. The risk factors associated with post-TACE pulmonary complications identified in the literature include large hyper-vascular HCC with AV shunts, large-volume Lipiodol infusion, and embolization via the right inferior phrenic artery. However, the absence of known risk factors is not a guarantee against serious complications. An astute awareness of the potential post-TACE pulmonary complications should expedite appropriate therapeutic interventions and increase potential for early recovery.

Sialyl residues modulate LPS-mediated signaling through the Toll-like receptor 4 complex.

We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFκB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation.

Proteinase-activated receptor 2 activation promotes an anti-inflammatory and alternatively activated phenotype in LPS-stimulated murine macrophages.

Proteinase-activated receptor 2 (PAR(2)), a 7-transmembrane G protein-coupled receptor, contributes to inflammation either positively or negatively in different experimental systems. Previously, we reported that concurrent activation of PAR(2) and TLRs in human lung and colonic epithelial cells resulted in a synergistic increase in NF-κB-mediated gene expression, but a down-regulation of IRF-3-mediated gene expression. In this study, the effect of PAR(2) activation on LPS-induced TLR4 signaling was examined in primary murine macrophages. The PAR(2) activation of wild-type macrophages enhanced LPS-induced expression of the anti-inflammatory cytokine, IL-10, while suppressing gene expression of pro-inflammatory cytokines, TNF-α, IL-6, and IL-12. Similar PAR(2)-mediated effects on LPS-stimulated IL-10 and IL-12 mRNA were also observed in vivo. In contrast, PAR 2-/- macrophages exhibited diminished LPS-induced IL-10 mRNA and protein expression and downstream STAT3 activation, but increased KC mRNA and protein. PAR(2) activation also enhanced both rIL-4- and LPS-induced secretion of IL-4 and IL-13, and mRNA expression of alternatively activated macrophage (AA-M) markers, e.g. arginase-1, mannose receptor, Ym-1. Thus, in the context of a potent inflammatory stimulus like LPS, PAR(2) activation acts to re-establish tissue homeostasis by dampening the production of inflammatory mediators and causing the differentiation of macrophages that may contribute to the development of a Th2 response.

The anti-tumor agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), induces IFN-beta-mediated antiviral activity in vitro and in vivo.

The 2009 outbreak of pandemic H1N1 influenza, increased drug resistance, and the significant delay in obtaining adequate numbers of vaccine doses have heightened awareness of the need to develop new antiviral drugs that can be used prophylactically or therapeutically. Previously, we showed that the experimental anti-tumor drug DMXAA potently induced IFN-ß but relatively low TNF-α expression in vitro. This study confirms these findings in vivo and demonstrates further that DMXAA induces potent antiviral activity in vitro and in vivo. In vitro, DMXAA protected RAW 264.7 macrophage-like cells from VSV-induced cytotoxicity and moreover, inhibited replication of influenza, including the Tamiflu®-resistant H1N1 influenza A/Br strain, in MDCK cells. In vivo, DMXAA protected WT C57BL/6J but not IFN-ß(-/-) mice from lethality induced by the mouse-adapted H1N1 PR8 influenza strain when administered before or after infection. Protection was accompanied by mitigation of weight loss, increased IFN-ß mRNA and protein levels in the lung, and significant inhibition of viral replication in vivo early after DMXAA treatment. Collectively, this study provides data to support the use of DMXAA as a novel antiviral agent.

Analysis of proteinase-activated receptor 2 and TLR4 signal transduction: a novel paradigm for receptor cooperativity.

Proteinase-activated receptor 2 (PAR2), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR2, often measured by Ca2+ release. PAR2 contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR2 in HEK293T cells resulted in concentration-dependent, PAR2 AP-inducible NF-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple Gi proteins or sequester intracellular Ca2+. Because previous studies described synergistic PAR2- and TLR4-mediated cytokine production, we hypothesized that PAR2 and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR2-induced NF-kappaB activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR2 in HEK293T cells led to a synergistic increase in AP-induced NF-kappaB signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR2 and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4(-/-) and PAR2(-/-) macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.

IFN regulatory factor-2 regulates macrophage apoptosis through a STAT1/3- and caspase-1-dependent mechanism.

IFN regulatory factor (IRF)-2(-/-) mice are significantly more resistant to LPS challenge than wild-type littermates, and this was correlated with increased numbers of apoptotic Kupffer cells. To assess the generality of this observation, and to understand the role of IRF-2 in apoptosis, responses of peritoneal macrophages from IRF-2(+/+) and IRF-2(-/-) mice to apoptotic stimuli, including the fungal metabolite, gliotoxin, were compared. IRF-2(-/-) macrophages exhibited a consistently higher incidence of apoptosis that failed to correlate with caspase-3/7 activity. Using microarray gene expression profiling of liver RNA samples derived from IRF-2(+/+) and IRF-2(-/-) mice treated with saline or LPS, we identified >40 genes that were significantly down-regulated in IRF-2(-/-) mice, including Stat3, which has been reported to regulate apoptosis. Compared with IRF-2(+/+) macrophages, STAT3alpha mRNA was up-regulated constitutively or after gliotoxin treatment of IRF-2(-/-) macrophages, whereas STAT3beta mRNA was down-regulated. Phospho-Y705-STAT3, phospho-S727-STAT1, and phospho-p38 protein levels were also significantly higher in IRF-2(-/-) than control macrophages. Activation of the STAT signaling pathway has been shown to elicit expression of CASP1 and apoptosis. IRF-2(-/-) macrophages exhibited increased basal and gliotoxin-induced caspase-1 mRNA expression and enhanced caspase-1 activity. Pharmacologic inhibition of STAT3 and caspase-1 abolished gliotoxin-induced apoptosis in IRF-2(-/-) macrophages. A novel IFN-stimulated response element, identified within the murine promoter of Casp1, was determined to be functional by EMSA and supershift analysis. Collectively, these data support the hypothesis that IRF-2 acts as a transcriptional repressor of Casp1, and that the absence of IRF-2 renders macrophages more sensitive to apoptotic stimuli in a caspase-1-dependent process.

Transcriptional regulation of lipopolysaccharide (LPS)-induced Toll-like receptor (TLR) expression in murine macrophages: role of interferon regulatory factors 1 (IRF-1) and 2 (IRF-2).

Activation of TLRs is most closely associated with induction of pro-inflammatory gene expression; however, expression of many other genes, including the TLR genes themselves, has also been shown to be modulated following TLR engagement. A large family of nuclear transcription factors, the interferon regulatory factors (IRFs), have been implicated in TLR signaling leading to pro-inflammatory gene expression. Given that IRF-1 and IRF-2 counter-regulate the transcriptional activity of many genes, we hypothesized that IRF-1 and IRF-2 might also regulate TLR gene expression following LPS stimulation of murine macrophages. mRNA derived from medium- or LPS-treated primary peritoneal macrophages was analyzed for TLR gene expression using quantitative real-time PCR. In wild-type macrophages, LPS up-regulated expression of TLRs 1-3 and 6-9 steady-state mRNA, while TLR4 mRNA was modestly down-regulated. IRF-2(-/-) macrophages responded to LPS with dysregulated expression of TLR3, TLR4, and TLR5 mRNA, whereas IRF-1 deficiency dampened LPS-induced mRNA expression for TLR3, TLR6, and TLR9. Functional studies revealed aberrant TLR3 signaling in IRF-2(-/-) macrophages. Collectively, these findings reveal an additional level of complexity associated with TLR transcriptional regulation and suggest that the trans-acting factors, IRF-1 and IRF-2, contribute to the innate immune response to infections by regulating TLR gene expression.