Basic Research Poster Abstracts

Stem cell factor (SCF) binds to the receptor c-Kit expressed on several myeloid and lymphoid cell populations, including Type 2 innate lymphoid cells (ILC2). However, the importance of the SCF/c-Kit interaction with ILC2 has not been completely understood. In this work, we observed that whole-body KO of SCF using Kitl fl/fl-UBCCreERT mice diminished airway allergy by decreasing the expression of IL-13 and significantly reduced total numbers of ILC2 in the lung when compared with Cre- (control) mice. In addition, we observed a systemic effect where the bone marrow and fibroblast grown from control allergic mice showed significantly increased expression of SCF248 isoform but not SCF220 and an increased number of ILC progenitors (ILCp) in the bone marrow. In contrast, as expected, no upregulation of SCF was detected in the Cre+ SCF KO allergic mice. Furthermore, no difference in the numbers of ILCp was observed, suggesting that SCF expression, specifically the SCF248 isoform, is systemically upregulated during a chronic allergic response. Bone marrow fibroblast from naïve mice stimulated in vitro with murine recombinant IL-13 upregulated the expression of SCF428. This work suggests that IL-13 upregulation during chronic airway allergy generated a systemic allergic microenvironment through the overexpression of SCF248 isoform in bone marrow, allowing the persistent differentiation of ILCp-ILC2 to maintain the Th2 phenotype in the lung and possibly other organs like the gut.

IL-9 is a cytokine that signals for anti-helminth and allergic responses and erythroid differentiation. IL-9 is mainly produced by T cells, mast cells, and ILC2. IL-9 production in T cells is positively regulated by several cytokines such as IL-4, TGFb1, and IL-1b, which activates transcriptional activators such as Pu.1, IRF4 and Batf for IL-9 gene expression. Here, we report that a transcription factor that plays a negative role in IL-9 expression in T cells. Moreover, the expression of this transcription factor is controlled by the RARa-retinoic acid axis, allowing it to mediate the negative function of RA on IL-9 expression. This transcription factor is one of the many genes that are rapidly up-regulated by RA. The gene has multiple RARa binding sites in the regulatory/super enhancer regions that recruit nuclear coactivator SRC3 (NCOA3) and histone epigenetic modifications in an RA-dependent manner. In high RA environments, RARa induces the expression of this transcription factor via retinoic acid receptor binding motifs. Increased expression of this gene suppressed the transcriptional activity of the IL-9 gene. We also found that T cells that are deficient in the expression of this factor become hyperpolarized to Th9 cells. Thus, our results establish a novel negative regulator of IL-9 gene expression and Th9 differentiation.

Innate lymphoid cells (ILCs) originate from embryonic hematopoietic tissues during fetal development but later develop from hematopoietic progenitors in the bone marrow. We report here that the expression of Basic Leucine Zipper ATF-Like Transcription Factor (BATF) is induced during ILC development in the bone marrow at the -lymphoid progenitor (LP) stage in response to the cytokine IL-7 in a STAT3-dependent manner and is required for production of committed ILC progenitors in the marrow and functional ILCs in the periphery. As the mechanism, the up-regulated BATF induces the expression of Nfil3 and Id2, two transcription factors necessary for the development of fetal and adult bone marrow ILC progenitors. Moreover, Batf-/- ILC progenitors fail to generate mature ILCs and NK cells in the periphery and lymphoid-tissue inducer (LTi) cells in the fetal liver and adult gut tissues. In sum, BATF plays an essential role in establishing a competent ILC defense system in the body.

Background: Eosinophilic Esophagitis (EoE) is a chronic inflammatory disease of the esophagus driven by Type-2 inflammatory responses and characterized by esophageal eosinophilia and epithelial remodeling. Corroborative clinical and discovery-based studies have demonstrated a central role for the Type 2-cytokine Interleukin-13 (IL-13) in the underlying esophageal inflammatory and epithelial remodeling responses. However, the IL-13 responsive pathways that drive the chronic inflammatory versus esophageal epithelial remodeling remains unclear.

Objective: We sought to investigate the role of IL-13-STAT3-SFRP1 signaling axis in driving esophageal epithelial proliferation and epithelial remodeling in EoE.

Methods: We performed bulk RNA sequencing analysis, western blot, RT-qPCR and histological analyses on esophageal epithelial keratinocytes and primary esophageal cells derived from patients with EoE. We utilized the STAT3 degrader (SD-36) and pharmacological agonist and antagonist of SFRP1 to define the role of STAT3 and SFRP1 in esophageal epithelial proliferation and gene expression. Finally, we employed the in vivo model, Krt5-rtTA x tetO-IL-13Tg model of esophageal remodeling to define the role of STAT3 in esophageal epithelial proliferation and remodeling.

Results: We identify an enrichment of STAT3 target genes in the EoE transcriptome and IL-13-stimulated EPC2-ALI cells. We demonstrate that IL-13 stimulation of EPC2-ALI cells and primary esophageal cells induces phosphorylation of STAT3 (Y705 and S727) and STAT6 (Y641). We show that STAT3 is activated by IL-13 and is required for IL-13-induced esophageal epithelial proliferation, and that SFRP1 is a novel candidate gene which is involved in cell proliferation and is a STAT3 target. We show that IL-13-induced mRNA expression of ANO1 and TP63 is enhanced with recombinant SFRP1 protein and attenuated by SFRP1 pharmacologic inhibition. Further, we demonstrate that IL-13 stimulation abrogates SFRP1 protein, and this effect in is rescued with STAT3 degradation. We also demonstrate that Krt5-rtTA x tetO-IL-13Tg mice which received Doxycycline exhibit esophageal remodeling (BZH and DIS) and increased basal epithelial cell proliferation.

Conclusions: These data identify a functional role of IL-13-STAT3-SFRP1 signaling axis in driving esophageal epithelial proliferation and BZH in EoE.

Background: Food allergy diagnosis and management causes a number of social and emotional challenge for individuals with food allergies and caregivers. This has led to increasing interest in the development and usage of approaches to accurately predict food allergy diagnosis, severity of food allergic reactions and treatment outcomes. However, the utility of these approaches is somewhat conflicting.

Objective: To develop and utilize a murine model that mimics the natural course of food allergy

diagnosis and treatment in humans and to identify biomarkers that predict reactivity during food

challenge and responsiveness during oral immunotherapy and how these outcomes are modified by genetics.

Methods: Sub-cutaneous (s.c.) sensitized Intestinal IL-9Tg (Intl IL-9Tg) mice and Intl IL-9Tg mice backcrossed onto the IL-4RαY709F (IL-9TgY709F) background received single exposure of egg antigen (OVA), underwent oral food challenge (OFC) and oral immunotherapy (OIT). Food allergy severity, mast cell activation and OVA-specific IgE levels were examined to determine the predictability of these outcomes in determining reactivity and treatment outcomes.

Results: We show that s.c. sensitization and a single oral allergen-challenge of egg antigen to BALB/c, Intl IL-9Tg and IL-9TgY709F mice induced a food allergic reaction. We show that enhanced IL-4Rα-signaling altered the symptoms of the first real-world allergic reaction, decreased the cumulative antigen dose and increased severity of reaction during OFC and altered side effect frequency and OIT outcomes. Analysis of the biomarkers during first real-world allergic reaction revealed that only the severity of the initial reaction significantly correlated with the cumulative reactive antigen dose during OFC.

Conclusion: Collectively, these data indicate that SNPs in IL-9TgY709F can alter clinical symptoms of food allergic reactions, severity, and reactive dose during OFC and OIT and that severity of first allergic reaction can predict the likelihood of reaction during an oral food challenge in mice with IL-9TgY709F gain of function.

Background: Mast cells (MCs) are considered the main effectors in allergic reactions and well known for their contribution to the pathogenesis of immune disorders. In vitro models to study MCs function include isolation of mast cells from tissues or differentiation from hematopoietic progenitors. The drawbacks of such techniques are low number of cells, donor-dependent heterogeneity, and the lack of a continuous cell source. Immortalized MC lines are available, but these cells display several phenotypic and functional alterations.

Objectives: To overcome the limitations we used human induced pluripotent stem cells (iPSC) to develop in vitro model of MC differentiation.

Methods: We developed a five-step protocol for the generation of MCs from human iPSCs. Human iPSCs were exposed to bFGF inducing embryoid body formation (EB) and mast cell media I supplemented with IL-3, FLT-3, IL-6 and SCF to promote myeloid cells-forming complex (MCFC). Hemopoietic cells derived from the MCFC were harvested weekly for twelve weeks and mast cell lineage populations were characterized by flow Cytometry and scRNA and IgE-degranulation analysis.

Results: Flow cytometry phenotyping of hemopoietic cells derived from the MCFC revealed an initial increase in CD34+ hematopoietic progenitors within Weeks 1-3 followed by an increase in CD34- CD45RA- hematopoietic cells. Among CD34- population we identified low granular cell populations (SSClow) characterized by cKit+ FcERI-7-integrinhigh CD203c+ , cKit- FcERI+ 7-integrinMid CD203c-/+ cells and c-KIt+ FcERI+ cells which are consistent with early basophil and mast cells progenitors. Analyses of SSChigh cells identified cKit+ FcERI- and cKit+ FcERI+ supporting mature basophils and mature mast cells. scRNA seq analysis of the cells harvested at week 4 of the MCFC culture revealed the presence of hemopoietic progenitors such as CMP/GMPs (6.6%), GMP-derived myeloid cell (13.9%), neutrophils (1.7%), basophils (2.1%), eosinophils (5.9), MC precursors (20.1%) and mature MC population (6.6%). Culturing MC precursor populations in Mast cell maturation media led to MC maturation as evidence by high expression of high affinity IgE receptor, metachromatic granules, presence of MC granule proteins (Tryptase and Chymase) and activation following FcεRI crosslinking.

Conclusion: This novel human iPSC-based approach provides a sustainable and homogeneous source for prolonged and highly productive generation of phenotypically mature, functional MCs. This system will be a useful model for studying mechanisms controlling early mast cells development and maturation, which may have considerable therapeutic implications.

Candida albicans is a fungus that colonizes the gastrointestinal (GI) tract of most healthy humans. Stable colonization is thought to be a prerequisite for C. albicans pathogenesis since infections are almost always seeded by indigenous populations in the GI tract following disruption to the bacterial microbiota (typically from broad-spectrum antibiotics). Nevertheless, very little is known about the mechanisms by which bacteria control C. albicans persistence in the GI tract. We recently identified a strain of C. albicans (CHN1) that more stably colonizes the murine GI tract than the canonical laboratory strain SC5314 in the presence of a bacterial microbiota. CHN1 colonization significantly alters the lactic acid bacteria (LAB) community, including prolonged depletion of Lactobacillus johnsonii and outgrowth of Enterococcus faecalis. This suggests that interactions with LAB are central to C. albicans colonization of the GI tract. Using RNA-seq to compare baseline global gene expression in CHN1 and SC5314, we found that CHN1 expressed significantly lower levels of many hyphal growth-associated genes relative to SC5314, despite being able to undergo hyphal transformation under inducing conditions. CHN1 also had significantly higher expression of CDC21 (a putative thymidylate synthase), ACF2 (an endo-1,3- glucanase), PGA19 (a GPI-anchored protein involved in macrophage interaction), and MNN13 (an -1,3-mannosyltransferase involved in cell wall protein glycosylation), as well as many genes with unknown functions. We predict that these genes mediate fungal interactions with the bacterial microbiota, and that their differential regulation may determine the capacity for C. albicans to persist in the GI tract alongside a bacterial microbiota.

Lactobacillus johnsonii, a gram-positive GRAS (generally regarded as safe) microorganism, is a well-known probiotic bacterium. Its health-promoting activities, like inhibition and exclusion of pathogens, immunomodulation, and enhancement of epithelial barrier function, have been documented in strains isolated from various ecological niches, ranging from food to vertebrate hosts. The optimal performance of any probiotic strain depends heavily on its ability to adapt to and stably colonize the gastrointestinal tract. Though the probiotic activities of L. johnsonii are being studied, it is important to address the gap in our knowledge regarding the host adaptability of these strains. To determine whether specific genes in L. johnsonii drive host adaptation, we compared the genomes of 25 strains isolated from different vertebrate hosts. Phylogenetic classification of these strains separates them into host-specific-phylogenetic lineages, with the human probiotic strains being the only exception to this. Several of the host-specific genes identified in this study fall under carbohydrate metabolism and transport pathway, and cell-wall/membrane/envelope biogenesis pathways. Notably, several genes of the defense mechanism pathway, such as toxin and antitoxin components, endonuclease, and ABC-type multidrug transport system were highly strain-specific, and not found in other strains even of the same clade. The results from this comparative genomic analysis provide insight into L. johnsonii host-adaptation and highlights putative targets for enhancing probiotic functions.

The use of broad-spectrum antibiotics and/or yeast colonization drives dysbiosis of the microbiome which can alter systemic immunity and favor the development of mucosal Type 2 immunity to aeroallergens. Our objective was to determine how dysbiosis might enhance the manifestation of food allergies and identify significant elements of the response. We utilized a well-characterized murine (Balb/c) model of food allergies (chicken egg ovalbumin, OVA) and found that mice with a conventional microbiome developed a low-level allergic response where IL-13 and mast cell proteases -1, -4 and -6 were upregulated. In genetically identical mice with a dysbiotic MB and Candida albicans colonization, a highly polarized Type 2 allergic response developed in the GI tract with more robust induction of IL-4, IL-13, IL-5, a number of chemokines, in addition to high expression of mast cell proteases and increased incidence of diarrhea. The loss of a specific Lachnospiraceae OTU in the dysbiotic mice was the prominent difference in the microbiome. Germ-free (GF) mice did not develop this same augmented Type 2 response. Conventionalization of GF mice resulted in Akkermansia muciniphila outgrowth and more variability in the allergic response between individual mice. Moreover, genetically identical conventional mice from different rooms in our vivarium had distinct microbiomes and developed different levels of the Type 2 response. Thus, our data recapitulate the heterogeneity in anaphylactic reactions seen in patients that have circulating levels of food allergen-reactive IgE and support the concept that alterations in the MB can be one factor underlying this heterogeneity.

Candida albicans is a yeast that is a member of the gastrointestinal (GI) tract of most healthy individuals. C. albicans infections are almost always seeded by indigenous yeast populations in the GI tract following disruption to the bacterial microbiota (typically from broad-spectrum antibiotics). Thus, stable colonization is a prerequisite for C. albicans overgrowth and pathogenesis. Nonetheless, very little is known about the mechanisms by which bacteria control C. albicans persistence in the GI tract. Our objective was to study the effect of a C. albicans overgrowth on microbiome reassembly after broad-spectrum antibiotics in a murine model. We utilized two strains of C. albicans from genomically distinct clades as a tool to model differences in colonization dynamics of C. albicans. Mice inoculated with SC5314 progressively cleared this strain over time, whereas CHN1-inoculated mice maintained significantly higher levels of colonization. Notably, these differences in colonization were associated with how the two strains interact with, and differentially modify, the host microbiome after antibiotic perturbation. The bacterial community structure of SC5314-colonized mice progressively became more similar to the pre-antibiotic structure over time; however, in CHN1-inoculated mice, the bacterial community structure never recovered to its pre-antibiotic state. This dysbiosis accompanying stable C. albicans colonization was also evident at phyla-level taxonomic changes. The indigenous bacterial microbiome of the GI tract, including Lactobacillus spp., is vital in promoting colonization resistance against C. albicans. After cessation of Amoxicillin treatment in SC5314-inoculated mice, Lactobacillus johnsonii levels quickly recovered to pre-antibiotic levels, correlating with the diminished levels of SC5314 in the GI tract. In contrast, stable colonization by strain CHN1 accompanied suppression of Lactobacillus johnsonii levels and elevated Enterococcus faecalis levels, a known causative agent of common infections. Altogether, these data elucidate the contribution

of the indigenous bacterial microbiota in C. albicans colonization, and how stable yeast colonization in an antibiotic-disrupted community can significantly alter the bacterial recolonization process.

Background: A mucosal adjuvant that induces mucosal as well systemic immunity could be more effective in combating respiratory and gut pathogens. However, this type of adjuvant is very challenging to develop due to the limited understanding of mucosal environment. In our previous studies, we have shown that an intranasal oil-in-water based nanoemulsion (NE) adjuvant interacts with epithelial cells and induces dendritic cell-specific retinaldehyde dehydrogenase (RALDH) activity in immunized animals. RALDH converts vitamin A into retinoic acid (RA). It appeared that the RA then orchestrated T cell homing towards the mucosal sites as well as IgA production. We sought to investigate the role of RA signaling in underlying this effect.

Methods: We used CD11c cre-dnRARα animals to study role of RA signaling in DCs and its impact on T cell immunity. These animals express dominant negative form of retinoic acid receptor α (RARα) under CD11c, rendering RA signaling in DCs. To study effect of these DCs, we intranasally immunized CD11c Cre-dnRARα and wt littermate controls with ova-albumin (Ova) in combination to NE. We studied DCs and T cells activation, T cells cytokine production as well as IgG production post immunization.

Results: We have shown NE-ova immunization produces Th1/Th17 biased immune response in immunized animals and increases antigen specific IgG and broncho alveolar lavage (BAL) IgA. Immunization of CD11c Cre-dnRARα animals with NE-ova had significant reduction in DCs activation. These animals also had significantly reduced T cell activation and cytokines production (both Th1/Th17 phenotype). Of interest the CD11c Cre-dnRARa animals still made some antibody responses. This data suggested NE induces Th1/Th17 cytokines in immunized animals via RARα pathway.

Conclusion: RARα signaling appears to play a major role in DCs activation. Further, RA signaling in DCs leads to compromised cellular and mucosal immune responses.

Background: IgE-mast cell (MC)-derived mediators, including histamine, drive many severe food-related anaphylactic reactions. These mediators cause vasodilatation, leading to fluid extravasation and possible cardiovascular collapse. We previously revealed a synergistic interaction between IL-4 and histamine in vascular endothelial (VE) dysfunction and exacerbation of the severity of IgE-mediated reactions.

Objectives: To define the molecular pathways involved in IL-4 enhancement of histamine-induced VE dysfunction and severity of IgE-mediated reactions.

Methods: We performed RNAseq analyses on murine vascular endothelial cells. We stimulated lentivral transduced shRNA scrambled and STAT3 human vascular endothelial cell line (EAHY926) (VEWT and VESTAT3) with IL-4 and histamine and performed RNAseq, vascular endothelial barrier function, western blot and cell signaling analyses for pSRC, intracellular calcium and Rho-GTPase activity. We performed passive anaphylaxis employing Cdh5creSTAT3flox mice to define the role of IL-4-STAT3 signaling in IgE-mediated reactions.

Results: Herein, we show that prior exposure of IL-4 amplified histamine-induced VE dysfunction including histamine-induced intracellular calcium flux, phosphorylation of src, and modulating kinetic of histamine-induced Rho-GTPase activity and this was associated with E-Cadherin degradation and barrier dysfunction. IL-4 enhancement of histamine-induced barrier function was sensitive to Cycloheximide (CHX) inhibition indicating a requirement for de novo protein synthesis and transcription. RNA-seq analysis revealed that IL-4 dysregulated 83 genes in VE cells. Transcription factor motif analyses revealed that 46 / 83 genes possessed a putative STAT3 motif, including cell-cell adhesion signaling genes and Guanylate-binding proteins (such as Thbs1, Gbp4, and Gbp6). Consistent with this, IL-4 stimulation in EA.hy926 cells induced phosphorylation on STAT3Y705 (required for nuclear translocation) and STAT3S727 and this was associated with barrier dyfunction. In vivo, conditional deletion of STAT3 in VE cells protects mice from IL-4 amplification of Histamine-induced anaphylaxis via diminution of the histamine response.

Conclusions: These data suggest a novel role of the IL4/ STAT3 signaling axis in priming VE cells, thus enhancing IgE- and histamine-induced anaphylaxis

Background: While aluminum adjuvant (alum)-based vaccines are commonly administered to neonates, a major gap in knowledge exists in understanding their immune effects specific in early life. The immature infant immune system is Th2-biased and is dependent on environmental cues to mature and shift towards a Th1/Th2 balance. Thus, the induction of strong Th2 immunity with alum vaccines in early life may perturb this balance, leading to the predisposition towards development of Th2-polarized immune responses to subsequent allergen exposure.

Methods: Mice were immunized with the hepatitis B-alum vaccine (HB) as neonates, infants or adults. Following HB-alum immunization, mice were exposed to ovalbumin (ova) intranasally weekly for a total of 4 exposures. Lymphocytes from the spleen and lymph nodes were harvested to characterize the ova-specific cellular immune response, and mice were challenged with ova to determine induction of allergic reactivity.

Results: Immune responses to HB-alum vaccine were more strongly Th2 polarized when given earlier in life. The effects on Th2 skewing were not limited to HB, as early life immunization with HB-alum also led to the development of Th2 polarized immune response to subsequent unadjuvanted ova exposure. Increased induction of Th2 polarized immune responses to ova also correlated with allergic reactivity, suggesting that early life alum immunization predisposed for sensitization following allergen exposure. Interestingly, mice that were immunized with HB-alum in adulthood generated ova-specific responses more consistent with tolerance and were not sensitized following ova exposure.

Conclusions: Immunization with alum in early life induces stronger and more long-lived Th2 immune responses and predisposes mice to develop Th2-biased immunity to subsequent allergen exposure likely through the induction of trained innate immunity that prolongs the Th2 bias of the immature infant immune system and may be a factor that predisposes towards allergic sensitization. Because vaccines are crucial elements of health in infancy, furthering our understanding of early life immunization will be critical moving forward in the design of vaccines for infants to reduce bystander immune events triggered by alum immunization.

Introduction

Food allergy is a common and potentially fatal disease caused by the loss of immunological tolerance to food antigens. Circumventing CD103+ dendritic cell (DC)-mediated tolerance, which is dependent the generation of α4β7-expressing regulatory T cells (Tregs), allergen-specific T cells coordinate an inflammatory immune response ultimately leading to oral food challenge (OFC)-induced mast cell activation. Our laboratory has developed a biodegradable allergen-encapsulating nanoparticle (aeNP) platform that can efficiently deliver masked antigen to tolerance-inducing immune populations, efficiently restoring the dysregulated immune response in an antigen-specific manner. This nanoparticle platform is utilized in phase 2 clinical trials for celiac disease (clinicaltrials.gov identifier: NCT04530123) and peanut allergy (clinicaltrials.gov identifier: NCT04950504). Here we demonstrated that aeNPs reduce the clinical and immunological effects of food allergy and provide preliminary evidence suggesting potential mechanisms responsible for these effects.

Methods

aeNPs containing ovalbumin (OVA; PLG(OVA) NPs) were generated using a water-in-oil-in-water double emulsion protocol to meet size (~500 nm diameter) and zeta potential (-40 to -60 mV) specifications. OVA-allergic mice were generated using Balb/cJ mice sensitized with two doses of i.p. OVA adsorbed onto aluminum hydroxide at days 0 and 14, followed by intravenous administration of 2.5 mg of PLG(OVA) NPs or control phosphate buffered saline (PBS) via tail vein injection at days 28 and 42. Mice were administered 7 intragastric challenges of OVA via oral gavage between days 45 and 59. Outcomes analysis included OFC-induced clinical anaphylaxis scores, anaphylactic temperature drops, and quantification of serum mast cell protease 1 (MCPT-1). Single cells were isolated from the spleen, mesenteric lymph nodes (mLNs), Peyer’s patches (PPs), and small intestine lamina propria (SILP) and assessed using flow cytometry and ex vivo recall assays.

Results and Discussion

Therapeutically delivered PLG(OVA) NPs protect recipients from OFC-induced anaphylaxis as demonstrated by a significant reduction in clinical anaphylaxis scores and a decrease in OFC-induced temperature drops. No differences in OFC-induced clinical scores or temperature changes were observed between PLG(OVA) NP-treated mice and naïve unsensitized, untreated mice. Additionally, PLG(OVA) NP treatment eliminated the incidence of OFC-induced diarrhea. These results indicate that aeNPs confer robust therapeutic suppression of allergic reactivity and offer a novel and efficacious approach to food allergen-specific immunotherapy. Early efforts to determine the mechanisms through which aeNPs bestow desensitization implicate changes in the phenotypes of gut-homing T cells. Cy5.5-tagged PLG(OVA) NPs directly associate with CD103+ dendritic cells (DCs) to a greater degree in OVA-sensitized mice compared to PBS-sensitized mice; these cells have an intrinsic ability to induce Treg differentiation, leading us to investigate how aeNPs may alter T cell populations. T cell phenotype experiments revealed that CD4+ and CD8+ T cells in the spleen and mLN more frequently express the integrin α4β7, a gut-homing marker, compared to PBS-treated controls. Preliminary data also shows that PLG(OVA) NPs induced a trend towards increased α4β7 expression among splenic and mLN regulatory T cells, as well as CD4+, CD8+, and regulatory T cell populations in the PPs. Finally, early results from recall assays involving mLN and PP lymphocytes demonstrate a trend towards reductions in the Th2 cytokines IL-4, IL-5, IL-6, and IL-13 without increases in the Th1/17 cytokines IL-17 and IFN-γ. In addition to their effects on T cell phenotype, PLG(OVA) NP administration results in a trend towards reduced mast cell frequency in the SILP. A similar trend is observed in serum levels of the mast cell degranulation marker MCPT-1 one hour after the final OFC. Collectively, these results support the hypothesis that aeNP-mediated desensitization proceeds through an increase in gut-homing T cell populations, and possibly gut-homing Treg populations, as well as a possible reduction in mast cell activity following allergen exposure.

Despite the great potential of oral immunotherapy (OIT) for food allergy treatment, the side effects and risk of relapse after treatment cessation still remain. Dysbiosis of the intestinal commensal microbiota have been implicated in the development of food allergy especially for infants, yet it is unknown how to precisely exploit the gut microbiota and the derived metabolites in the small intestine as adjunct therapy for OIT. Here we demonstrate a “small intestine-retentive” inulin gel loaded with allergen (inulin gel/allergen) that can remodel the ileal microbiota and immune homeostasis in situ, achieving robust OIT efficacy with sustained tolerance in multiple food allergy models.