We are interested in defining the role of IL-13–induced transcriptional programs in esophageal epithelial proliferation in EoE. To do this, we employ a combinatory approach involving RNA sequencing and bioinformatics, cellular and molecular technologies (western blot, reverse transcriptase quantitative PCR), histologic assessment of esophageal biopsies from healthy control and patients with EoE, primary immortalized esophageal cells and in vivo mouse model systems.

Utilizing these experimental systems, we have identified 82 genes that were differentially expressed in biopsies of patients with EoE and IL-13–stimulated esophageal epithelial cells grown at the air-liquid interface (EPC2-ALI). Computational analysis revealed that these differentially expressed genes (DEGs) were enriched for putative STAT3 targets.

Employing esophageal epithelial keratinocytes and a primary esophageal culture system derived from patient biopsies, we show that IL-13 induces esophageal epithelial STAT3 phosphorylation and activation (pSTAT3-Y705 and pSTAT3-S727) and esophageal epithelial proliferation. Notably, using Genetic (shRNA) and pharmacologic (proteolysis-targeting chimera degrader) approaches we showed that STAT3 was required for IL-13–induced esophageal epithelial proliferation and expression of EoE proliferative genes.

Interestingly, in silico analyses identified the putative STAT3 target secreted frizzled-related protein 1 (SFRP1) as a DEG associated with the IL-13–induced esophageal epithelial proliferative network. SFRP1 is a secreted glycoprotein that is a member of the secreted frizzled-related receptor family, which act as soluble modulators of Wnt signaling. SFRP1 acts as a competitive antagonist binding to the Wnt ligand through the netrin domain and preventing Wnt binding to its receptor negatively regulating β-catenin–induced pro-survival and pro-proliferation transcriptional programs.

Agonism and antagonism of SFRP1 experiments revealed that SFRP1 plays a key regulatory role in IL-13–induced STAT3-dependent esophageal proliferation. Strikingly, we showed that SFRP1 was expressed predominantly in esophageal epithelial cells in active EoE compared with EoE in remission or normal control samples and that SFRP1+ esophageal epithelial cells were enriched for the core EoE proinflammatory transcriptome (CCL26, ALOX15, TNFAIP6, POSTN, and ANO1).

These studies identify SFRP1 as a key regulator of IL-13–induced and STAT3-dependent esophageal proliferation and BZH in EoE and identify an SFRP1+ esophageal epithelial cell population in esophageal inflammation and remodeling in EoE.

We are currently pursuing studies defining the molecular mechanism by which SFRP1 regulates STAT3-dependent esophageal proliferation and BZH in EoE.

PMID: 37652141 

We have had long interest in immune inhibitory receptors originating from studies performed by Takai and Kubagawa in the late 1990s who independently identified two gene products with 98% sequence identity (originally designated p91 and PIRB) termed Paired immunoglobulin-like receptor B (PIR-B).

PIR-B is an immunoreceptor tyrosine-based inhibitory motif (ITIM) containing type I transmembrane glycoprotein expressed predominantly on myeloid cells, B cells, and granulocytes. Activation of PIR-B via major histocompatibility class I molecules in cis and trans fashion, and cell wall components of certain gram-negative and gram-positive bacteria, induces PIR-B ITIM domain engagement and activation of the intracellular phosphatases Src-homology region 2 domain-containing phosphatase (SHP)-1 and SHP-2. SHP-1 dephosphorylates downstream signaling molecules including p65 and extracellular signal-regulated kinase (ERK)1/2, resulting in the inhibition of downstream nuclear factor-κB– and mitogen-activated protein kinase–signaling pathways and inhibition of B-cell receptor, Toll-like receptor, and chemokine-receptor signaling.

In the mid-2000’s, we examined the role of the inhibitory receptor PIR-B in the regulation of macrophage function in innate intestinal immunity. We showed that PIR-B restrains innate-immune–induced proinflammatory cytokine (IL1β, IL6, and TNFα) production by macrophages and limits acute intestinal inflammation and epithelial cell injury.

Recently, we observed PIR-B expression on CD4+ T cells. Employing PIR-B-deficient (Pirb-/-) mice and using the Il10-/- spontaneous, αCD3-mediated, and CD4+CD45RBhi T-cell transfer model of colitis, we showed that loss of PIR-B expression protected mice from the development of CD4+ T-cell–dependent colitis. Notably, disease protection was associated with significantly reduced frequency of tissue resident memory (TRM) CD4+IL17A+ T cells. This observation was important as clinical and discovery-based studies have provided corroborative evidence identifying a central role for pathogenic TRM CD4+ IL17A cells in the exacerbation of the clinical manifestations of IBD.

Performing adoptive transfer experiments, we showed that PIR-B expression on CD4+ T cells conferred a competitive advantage for T-cell survival and TRM CD4+ T-cell development. Our studies showed that Pirb-/- naïve CD4+ T cells have decreased capacity to differentiate into Th17 cells, impaired cell-cycle entry into G1 and S phases, and enhanced cell death. Mechanistic analysis has shown that PIR-B acted as a rheostat, controlling mammalian target of rapamycin complex 1 (mTORC1) signaling in CD4+ T cells and limiting CD4+ IL17A+ T-cell outgrowth.

The human homologue of PIR-B is the leukocyte immunoglobulin-like receptor (LIR) family member, LILRB3. We were able to demonstrate that LILRB3 was expressed by human CD4+ IL17A+ cells, and a relationship between LILRB3+ CD4+ IL17A+ function and the IBD phenotype LILRB3 expression on CD4+ IL17A+ cells was associated with mucosal injury and a proinflammatory Th17 signature in a treatment-naïve endoscopically severe pediatric CD population. Collectively, these data suggest an intrinsic role for PIR-B in the regulation of the outgrowth and maintenance of TRM CD4+ IL17A+ T cells and the development of T-cell–dependent colitis.

We are currently pursuing studies defining the role of immune inhibitory receptors in the regulation of pathogenic TRM CD4+ IL17A cell function and the exacerbation of the clinical manifestations of IBD.

PMCID: PMC8531983

A food-induced anaphylactic reaction encompasses a variety of symptoms that can affect one or more target organs, including those of the gastrointestinal, cutaneous, respiratory, and cardiovascular systems. In human subjects compromise of either the cardiovascular or respiratory system defines a severe reaction and it is postulated that basophil- and mast cell (MC)–derived mediators, through inducing pulmonary venous vasodilatation and fluid extravasation, cause the respiratory and cardiovascular collapse that leads to the severe, life-threatening anaphylactic phenotype.

Clinical studies have reported increased levels of the cytokine IL-4, and mast cell derived mediator histamine in the sera of human patients with severe anaphylaxis suggesting a role for these molecules in the regulation of anaphylaxis severity. Consistent with this, we have previously demonstrated that IL-4 can interact with vasoactive mediators such as histamine to increase hemoconcentration and the severity of anaphylaxis. The gap in knowledge is the cellular target of these IL-4–mediated effects and the underlying IL-4 receptor α chain (IL-4Rα)–dependent signaling processes involved in the amplification of histamine-induced vascular endothelial (VE) barrier dysfunction and fluid extravasation in IgE-mediated reactions is not yet fully understood.

Employing both in vitro and in vivo model systems, we are defining the relationship between IL-4 and histamine in IgE-mediated VE leak and hypovolemic shock.

So far, we have identified that IL-4 amplifies histamine-induced hypovolemic shock in mice through VE IL-4Rα chain–dependent process. Notably, IL-4 and histamine stimulated activation of ABL1 kinase activity in VE cells and VE barrier dysfunction was inhibited by pharmacologic and genetic ablation of ABL1 activity. Importantly, using both passive and active models of food-induced anaphylaxis, we showed that blockade of ABL kinase activity using the inhibitor imatinib protected the mice from the severe IgE-mediated anaphylactic phenotype after allergen exposure.

These studies implicate an important contribution by the IL-4Rα/ABL1 signaling pathway in the VE compartment in the severity of IgE- and histamine-induced anaphylaxis.

The laboratory is currently studying the IL-4Rα chain–dependent signaling processes that alter vascular endothelial function and drive severe IgE-mediated allergic reactions.

PMID: 29157947

Translocation of dietary antigens across the SI epithelium is thought to occur through microfold cell–mediated transcytosis, transepithelial dendrites, goblet cell antigen passages (GAPs), and paracellular leak. Recent data suggests that dysregulation of the mechanisms of dietary antigen sampling and presentation at the gastrointestinal epithelium to hematopoietic compartment predisposes to aberrant adaptive immune responses.

The gastrointestinal (GI) epithelial barrier comprises a single cell layer of polarized intestinal epithelial cells (IEC) that forms a physical barrier between the gut lumen (containing food, bacteria, fungi, viruses, environmental particulates, carcinogens and toxins), and the internal systems of the body. The GI epithelial barrier acts as a selective filter, limiting the uptake of pathogens and harmful substances while permitting the absorption of critical dietary components (proteins, carbohydrates, nutrients and electrolytes) that are essential for cellular growth and survival. To regulate these critical processes the intestinal epithelium consists of a functionally diverse array of epithelial cell types (i.e. enterocytes, goblet cells (GCs), neuroendocrine cells, tuft cells, Paneth cells and microfold (M) cells), which act cooperatively to promote the development and maintenance of physical and chemical barriers, host defense and nutrient absorption.

In healthy subjects microfold cell–mediated transcytosis and GAP-mediated transcellular transport seem to be the primary route of dietary antigen sampling. These processes are thought to be important in inducing systemic immunologic non-responsiveness, termed oral tolerance. However, the contribution of these processes to food allergen translocation across the allergic intestinal epithelium and induction of a food-induced IgE-mediated reaction remained unclear.

The Hogan Lab is interested in defining the processes involved in the translocation of dietary allergens across the intestinal epithelial surface to the subepithelial immune compartment in food allergic reactions.

These studies have identified that secretory intestinal epithelial cells (GC’s, enteroendocrine cells, and Paneth cells) in the small intestine (SI) of mice with food allergy act as conduits to permit dietary allergens to transport across the intestinal epithelium to underlying allergic immune cells such as mast cells. We showed that SI intestinal secretory epithelial cell antigen passages (secretory antigen passages [SAPs]) that comprise of villi and cryptic goblet cells (GCs), enteroendocrine cells, and Paneth cells; are induced by the cytokine IL-13 in a CD38/cyclic adenosine diphosphate ribose (cADPR)–dependent manner; and are conserved in human subjects. Our in vivo analyses reveal that SAPs are integral for initial translocation of food allergens across the SI epithelium to underlying MCs, which leads to induction of a food-induced anaphylactic reaction.

The laboratory is currently studying the role of SAPs in regulating the interaction between tolerogenic induction mechanisms and tolerogenic maintenance and/or reinforcement mechanisms in sustaining unresponsiveness to food antigens through life.

PMCID: PMC6779525