Citations

Abstract Introduction Environmental exposure to food allergens (e.g. peanut antigens) through the skin or airways increases the risk of developing food allergy (e.g. peanut allergy). In contrast, oral antigen exposure (e.g., consumption of peanut products) early in life reduces the risk. The objective of this study was to investigate the immunologic mechanisms by which oral antigen exposure provides systemic immunotolerance and protection from food allergy development using murine models. Methods Naive mice were administered ovalbumin (OVA) through their drinking water to promote oral immunotolerance. Mice were subsequently exposed intranasally (i.n.) to OVA plus peanut flour, as an allergic adjuvant. CD4+ T cells from lung draining lymph nodes (dLNs) were assessed by single-cell RNA sequencing, MHC-II tetramer staining, and flow cytometry. Results Following i.n. exposure to OVA plus peanut flour, mice developed OVA-specific IgE (OVA-IgE) and showed symptoms of acute anaphylaxis upon intraperitoneal OVA challenge. Oral OVA feeding prior to i.n. OVA exposure reduced OVA-IgE and protected mice from anaphylaxis. Protection was associated with suppression of OVA-specific T follicular helper (Tfh) and germinal center B cells in dLNs. Tolerance was reversed by administration of anti-CTLA-4 blocking antibody but was not affected by deletion of Foxp3+Bcl6+ T follicular regulatory (Tfr) cells. Accordingly, conventional Foxp3+T regulatory (Treg) cells were not increased in dLNs of OVA-fed mice. Rather, protection was associated with the emergence of OVA-specific Foxp3-CTLA4+ cells expressing markers of stemness. Conclusion Oral antigen exposure suppresses the development of antigen-specific Tfh cells in remote dLNs thereby preventing systemic allergic sensitization. Systemic oral tolerance is dependent on CTLA-4 and unlikely requires conventional Foxp3+ Treg cells. Funding Source Supported by NIH F31 AI86242, R37 AI71106, and Mayo Clinic Graduate School of Biomedical Sciences. Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Abstract Introduction Mucosal immunization and respiratory IgA offer significant promise in protecting against airborne pathogens, including SARS-CoV-2. However, the conditions and mechanisms that lead to the robust induction of respiratory IgA responses following mucosal vaccination remain poorly understood. Methods In this project, we established SARS-CoV-2 breakthrough infection and mucosal adenovirus-based booster vaccination models. Using genetic mouse models and various in vivo and in vitro blockade or depletion approaches, we further investigated the mechanisms regulating respiratory IgA responses. Results Here we found that a mucosal adenovirus-based booster vaccination in mouse models revealed that respiratory booster immunization elicited markedly stronger and more durable respiratory IgA, T cell response, and protective immunity against SARS-CoV-2, supporting the promise of respiratory mucosal vaccination. Mechanistically, we identified that local CD4+ T cells support respiratory IgA production by promoting IgA+ B cell class switching, proliferation, and survival in situ. This T cell help depended on interactions between local Blimp-1+ T-bet+ Th1 cells and B cells, mediated through ICOS/CD40L/MHC-II engagement and IL-21 signaling. Furthermore, lung macrophages contributed to the mucosal IgA response via TGF-β production. Consequently, mucosal administration of SARS-CoV-2 Spike-encoding mRNA encapsulated in pulmonary surfactant (PS)- incorporated lipid nanoparticles designed to target lung macrophages elicited a stronger mucosal IgA response. Conclusion Our results uncover a local cellular network supporting enhanced mucosal IgA responses, with implications for the development of optimal mucosal immunization strategies against SARS-CoV-2 and other respiratory pathogens such as H5N1 influenza. Funding Source AI147394, AG069264, AI112844, HL170961, AI154598, AI176171 to J.S., AI67986, AI177467 to Hui.H; U54CA260582 to S.L.L; R01AI168684 to G. Z.; S.L.L was also supported by a fund provided by an anonymous private donor to The Ohio State University. Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Description Early oral route antigen exposure decreases the incidence of food allergy while environmental exposures through non-oral routes, such as the skin or airways, increase the risk. The goal of this study was to investigate the immunologic mechanisms of how oral allergen consumption protects from sensitization to the same allergens through environmental exposure routes. Naive mice were orally administered ovalbumin (OVA) in drinking water. Mice were subsequently exposed intranasally (i.n.) or epicutaneously (e.c.) to OVA plus peanut flour, as an adjuvant. OVA-specific CD4+ T cells from draining lymph nodes (dLNs) were assessed by MHC-II tetramer staining and flow cytometry. Following i.n. or e.c. exposure to OVA plus peanut flour, mice developed OVA-IgE and OVA-IgG1 and showed symptoms of acute anaphylaxis upon OVA challenge. Oral OVA feeding prior to i.n. or e.c. antigen exposure dramatically reduced OVA-IgE titers and protected mice from anaphylaxis. Protection was OVA-specific, as OVA-fed mice still developed peanut-IgE and anaphylaxis upon i.p. peanut challenge. Total OVA-specific CD4+ T and CD4+Foxp3+ T regulatory (Treg) cell numbers in dLNs were similar between control and OVA-fed mice. However, the majority of OVA-specific CD4+ T cells in control dLNs were T follicular helper (Tfh) cells, which were nearly abolished in OVA-fed mice. Oral route antigen exposure suppresses development of antigen-specific Tfh cells in remote dLNs and prevents systemic allergic sensitization. Funding Sources Supported by NIH R37 AI71106 and Mayo Clinic Graduate School of Biomedical Sciences. Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Abstract Description The immunological mechanism involved in regulation of chronic airway inflammation in allergic asthma is not fully understood. This study aimed to identify subpopulation(s) of T regulatory (Treg) cell responsible for controlling type 2 airway inflammation. Treg cells in the lung tissues of fungal allergen Alternaria-exposed mice expressed higher levels of Il2ra, Pdcd1, Ctla4, Il10 as compared to those in mediastinal lymph nodes. Mass cytometry analysis identified several populations of Treg cells including the one highly expressed CCR8, ST2, and GATA-3. CCR8+ST2+ Treg cells expanded after 4-week of Alternaria exposure and persisted in the lung tissues for additional 8 weeks without allergen re-exposure; their tissue residency was not affected by FTY720 treatment. Transcriptional analysis showed that CCR8+ST2+ Treg cells were enriched for Gata3 while minimally expressed type 2 cytokines. Moreover, CCR8+ST2+ Treg cells produced higher levels of IL-10 and amphiregulin compared to CCR8-ST2+ Treg cells and effectively suppressed proliferation of effector T cells. Severe airway inflammation and asthma-like pathology developed in Treg cell-deficient (Foxp3DTR) and Ccr8-/- mice underscored the functional significance of CCR8+ST2+ Treg cells to control airway inflammation in vivo. These findings suggest that heterogenous populations of Treg cells develop in the lung tissues during chronic airway inflammation and CCR8+ST2+ Treg subpopulation is likely to be highly suppressive. Funding Sources Supported by NIH grant R37 AI71106; Mayo Foundation; Mayo Graduate School of Biomedical Sciences Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Abstract Food allergy, such as peanut allergy can cause life-threatening anaphylaxis. While T follicular helper (Tfh) cells play a major role in production of allergen-specific IgE and IgG antibodies, the immunologic mechanisms that regulate the production of antibodies that mediate anaphylaxis are not well understood. While PD-1 is highly expressed on Tfh cells, our knowledge is limited regarding the role of the molecule in Tfh cells. Thus, the objective of this study was to investigate the roles of PD-1 in allergic immune responses using a mouse model of peanut allergy. Naïve wild-type mice were exposed to peanut flour intranasally (i.n.) and then challenged by intraperitoneal (i.p.) injection of peanut extract. The mice exposed i.n. to peanut flour produced peanut-specific IgE and IgG antibodies and developed anaphylaxis when challenged i.p. with peanut extract. PD-1 blockade with anti-PD-1 treatment Tfh and germinal center B cell numbers in mice exposed to peanut flour. Nonetheless, anti-PD-1 or anti-PD-L1 fully protected mice from anaphylaxis. Anti-PD-1 or genetic deficiency of PD-1 in CD4+ T cells reduced and increased levels of peanut-specific IgE and IgG, respectively. Anti-PD-1 promoted production of low-affinity IgE and IgG antibodies with no effect on high-affinity antibodies. By passive cutaneous anaphylaxis, peanut-specific antibodies generated in anti-PD1 treated mice failed to induce anaphylaxis, and plasma from these mice prevented anaphylaxis. Therefore, blockade of the PD-1 pathway is protective against allergic immune responses. The interaction between Tfh cells and B cells may play a pivotal role in controlling antibody quality and clinical manifestation of allergic diseases.

Abstract We investigated whether human IgM antibody, B7-DCXAb, which binds to B7-DC and enhances DCs' antigen uptake and retention, migration, and cytokine production, improves the efficacy of allergen immunotherapy. Mice were sensitized by intraperitoneal injection of OVA or house dust mite (HDM) antigens plus alum and then treated by intravenous B7-DCXAb injection intranasal or sublingual administration of antigen. OVA-sensitized mice developed anti-OVA IgE and IgG1, Th2-type splenocyte responses to OVA; marked airway eosinophilic inflammation was observed after intranasal OVA challenge. When these mice were treated by intravenous B7-DCXAb plus intranasal OVA, anti-OVA IgE and IgG1 decreased by 75~95% within 1 week; this decrease persisted for >8 weeks. Anti-OVA IgG2a decreased transiently. Splenocyte production of IL-4, IL-5, and IL-13 in response to OVA was abolished; no increases in IFN-g or IL-17 were noted. In OVA-challenged mice, airway eosinophilia and lung levels of IL-4, IL-5 and IL-13 were reduced by >90%. Immunotherapeutic effects were also observed with intravenous B7-DCXAb plus sublingual OVA, but not with intravenous B7-DCXAb alone. Furthermore, HDM-sensitized mice were treated successfully by intravenous B7-DCXAb plus intranasal HDM. B7-DCXAb injection plus mucosal administration of antigen effectively treats allergic responses and may provide novel strategy for allergen immunotherapy.

Abstract CD8+ T cells represent a relatively under studied cell type in allergic lung inflammation, despite their presence in the mucosal tissues. This is because CD8+ T cells are normally associated with a Th1 response as opposed to Th2 or Th17 responses, as are the most cases with allergic inflammation. However, recent papers have shown the production of Th2 and Th17 cytokines in these cells, yet knowledge of their physiological role in allergic inflammation is incomplete. The presence of IL-17 in asthma patients contributes to a severe, steroid resistant phenotype. Therefore, determining contributors of IL-17 and their physiological role is paramount to understanding the disease. Our preliminary data which suggests memory Tc17 cells are generated in CD4-/- mice when sensitized intranasally to ovalbumin with IL-33. We believe Tc17 cells are responsible for the inflammation because the inflamed lung tissue contained elevated IL-17, and stimulated T cells from inflamed tissue produced elevated levels of IL-17 as well. Additionally, we were unable to induce inflammation in Rag-/- mice or TCRβ-/- mice, which pinpoints the cell responsible to CD8+ T cells in CD4-/- mice. Using these mice to study an exaggerated phenotype will allow us to more easily detect the contributions that happen to a lesser degree in wild type animals. This work create a more complete picture of the disease environment and lead to therapies which can account for the contribution of this cell type.

Abstract We used an established model of asthma that involves intranasal administration of the fungal extract of Alternaria. Upon lung exposure to Alternaria, IL-33 was released by epithelial cells within one hour, followed by increased production of IL-5 in the lungs. We also found elevated serum levels of IL-5 in response to Alternaria exposure, which reached a peak at 6 hours post Alternaria administration. After prolonged exposure to Alternaria over 6 days, a significant increase in mature eosinophils was observed in bone marrow. In addition, the serum from mice challenged with Alternaria was able to stimulate bone marrow cells to undergo eosinophil maturation in vitro. Intraperitoneal administration of IL-5-blocking antibody in vivo decreased eosinophil maturation in bone marrow in response to intranasal Alternaria below intranasal PBS baseline levels. We have shown the source of IL-5 is by type 2 innate lymphoid cells directly through IL-5 reporter mice, and indirectly through IL-5 serum levels in CD127 knockout mice. Taken together, this study illustrates that the type 2 innate immune responses in the lungs leads to enhanced production of eosinophils in the bone marrow through the increase in lung and blood levels of IL-5. Thus, distant organs communicate during allergic inflammation by using cytokines as mediators. This concept will aid in the identification of novel therapeutic strategies to alleviate symptoms of asthma and allergic diseases.

Abstract Interleukin (IL)-1 family member cytokines are likely pivotal as interfaces between innate and adaptive immune responses. IL-33, an IL-1 family member, is implicated in airway Th2-type responses, including eosinophilia, increased mucus production and hyperresponsiveness. Our goal was to elucidate the immunological mechanisms involved in IL-33-mediated Th2-type responses in the lungs. Airway exposure of naïve BALB/c mice to IL-33 resulted in bronchoalveolar lavage (BAL) eosinophilia and increased lung levels of IL-5 and IL-13, but not IL-4 or IFNγ. Naïve Rag1-/- mice showed similar airway eosinophilia and cytokine responses as wild-type mice, suggesting a role for innate immune cells. By FACS analysis, IL-5-producing lung cells lacked CD3, B220 and other lineage markers (i.e. Lin-), but they expressed CD44 and CD25; markers for NK cells, mast cells, basophils and eosinophils were undetectable. FACS-sorted Lin-CD25+CD44+ lung cells expressed GATA-3 and the IL-33 receptor, T1/ST2, and responded to IL-33 by producing IL-5 and IL-13; morphologically, these lung cells resembled lymphocytes. In vitro blockade of CD25+ cells with anti-CD25 mAb (PC61) abrogated IL-33-induced Th2 cytokine production. Thus, mouse lungs contain a novel subset of innate immune cells that respond to IL-33. These airway cells may initiate early Th2-type immune responses and induce asthma and other allergic diseases.

Polyethyleneimine (PEI) induced immune responses were investigated in human bronchial epithelial (hBE) cells and mice. PEI rapidly induced ATP release from hBE cells and pretreatment with glutathione (GSH) blocked the response. PEI activated two conductive pathways, VDAC-1 and pannexin 1, which completely accounted for ATP efflux across the plasma membrane. Moreover, PEI increased intracellular Ca2+ concentration ([Ca2+]i), which was reduced by the pannexin 1 inhibitor, 10Panx (50 μM), the VDAC-1 inhibitor, DIDS (100 μM), and was nearly abolished by pretreatment with GSH (5 mM). The increase in [Ca2+]i involved Ca2+ uptake through two pathways, one blocked by oxidized ATP (oATP, 300 μM) and another that was blocked by the TRPV-1 antagonist A784168 (100 nM). PEI stimulation also increased IL-33 mRNA expression and protein secretion. In vivo experiments showed that acute (4.5 h) PEI exposure stimulated secretion of Th2 cytokines (IL-5 and IL-13) into bronchoalveolar lavage (BAL) fluid. Conjugation of PEI with ovalbumin also induced eosinophil recruitment and secretion of IL-5 and IL-13 into BAL fluid, which was inhibited in IL-33 receptor (ST2) deficient mice. In conclusion, PEI-induced oxidative stress stimulated type 2 immune responses by activating ATP-dependent Ca2+ uptake leading to IL-33 secretion, similar to allergens derived from Alternaria.