Iijima, Koji

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)

Exposure of airway epithelia to environmental allergens or nanoparticles invokes a novel genome-protective physiological response to oxidative stress. Fragments of double-stranded genomic DNA released into the airway lumen originate from repetitive, transcriptionally active, and fragile sites within the genome. Exporting DNA with high repetitive content potentially aids cell survival through the preservation of genome integrity and fidelity during the repair process.

Abstract Description Rationale Mice are used for models of human diseases. The immune cell composition of specific-pathogen-free (SPF) mice closely resembles that of newborn humans. Adult humans have been exposed to a multitude of pathogens throughout their lives. In this project, we sought to create a mouse model resembling humans by co-housing SPF mice to pet store mice. Methods Male and Female SPF B6 mice were co-housed with pet store mice. Multiplexed PCR was used to assess pathogen transmission. After 2 months, lungs, small intestines, and colons were analyzed by high-dimensional mass cytometry and multiplexed cytokine assay. Results Co-housed male mice frequently succumbed to infection. After two months, co-housed males still exhibited signs of active infection across all organs examined including profound myeloid and T effector cell infiltration. In female mice, infection was less fatal and active sickness resolved by two months. Across tissues, T cells exhibited both memory and effector phenotypes. Increases in monocytes and granulocytes were observed in lungs and colons while CD8+ T cells increased in the small intestine. Importantly, innate lymphoid cells were not displaced but underwent phenotypic changes. Conclusion Asynchronous systemic exposure to multiple pathogens in mice results in dramatic alterations to the immune landscape in a sex- and tissue-dependent manner. When used in disease models, these mice may provide new insight beyond those obtained previously with SPF mice. Funding Sources •NIAID R37 AL71106 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)

Allergens from the fungus Alternaria alternata promote secretion of alarmins from the airway epithelium including ATP, IL-33 and both genomic and mitochondrial DNA fragments, which produce type 2 immune responses in airways. Alternaria -induced oxidative stress activates ATP release by opening apical voltage-dependent anion channels (VDAC-1) and stimulating exocytosis of ATP. This investigation demonstrated that treatment of human bronchial epithelial (hBE) cells with a selective peptide antagonist or small molecule inhibitor (FPS-ZM 1 ) of the receptor for advanced glycation end products (RAGE), reduced Alternaria -evoked ATP secretion, decreasing the initial rate by ~40%. Furthermore, the Alternaria -induced increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) was inhibited by ~75% following pretreatment with RAGE antagonist peptide. Intranasal administration of Alternaria extract (50 μg) produced a significant increase in IL-33 secretion into bronchoalveolar lavage (BAL) fluid, which was inhibited in RAGE-deficient ( Ager -/- ) mice. Moreover, Alternaria -induced increases in IL-5 and IL-13 secretion into BAL fluid were also significantly reduced in Ager -/- mice. These findings support the conclusion that Alternaria -dependent ATP release and Ca 2+ mobilization was dependent in part on RAGE activation and that RAGE antagonists have the potential of reducing type 2 immune responses in airways following Alternaria allergen exposure. This study was supported by grants from the NIH (R01A1128729 & 1R01AI169530) to HK and SMO and by the Minnesota Agricultural Experiment station grant (AES0016134) to SMO. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Objective To evaluate eosinophil peroxidase (EPX) as a biomarker for tissue levels of eosinophilia, cytokines, and chemokines within chronic rhinosinusitis (CRS). Methods Twenty‐eight subjects undergoing sinonasal surgery were prospectively enrolled. Ethmoid tissue was analyzed with an in‐house EPX immunoassay and a 48‐plex cytokine‐chemokine array. Clinical severity was assessed using SNOT‐22 and Lund‐Mackay scores. Subjects were grouped as follows: controls, polyp status (CRS with [CRSwNP] and without nasal polyps [CRSsNP]), tissue eosinophilia (eosinophilic CRS [eCRS], non‐eosinophilic CRS [neCRS]), or combinations thereof (eCRSwNP, eCRSsNP, neCRSsNP). eCRS was defined as >10 eosinophils per high power field (HPF). Subjects without CRS or asthma were enrolled as controls. Results EPX was elevated in CRSwNP compared to control ( p = 0.007), in eCRS compared to neCRS ( p = 0.002), and in eCRSwNP along with eCRSsNP compared to neCRSsNP ( p = 0.023, p = 0.015, respectively). eCRS displayed elevated IL‐5 compared to neCRS ( p = 0.005). No significant differences in EPX or IL‐5 were observed between eCRSwNP and eCRSsNP. IL‐5 was elevated in eCRSwNP ( p = 0.019) compared neCRSsNP. Area under the receiver operator characteristic curve was 0.938 (95% CI, 0.835–1.00) for EPX and tissue eosinophilia, with an optimal cut‐point of 470 ng/mL being 100% specific and 81.25% sensitive for tissue eosinophilia. Linear regression revealed a strong correlation between EPX and IL‐5 (R 2 = 0.64, p < 0.001). Comparing EPX and IL‐5, only EPX displayed significant correlation with SNOT‐22 ( p = 0.04) and Lund‐Mackay score ( p = 0.004). Conclusion EPX is associated with tissue eosinophilia in CRS patients regardless of polyp status. EPX correlates with IL‐5 and could be potentially considered a biomarker for anti‐IL‐5 therapies. Level of Evidence 3 Laryngoscope , 134:69–78, 2024

Exposure of human bronchial epithelial cells (hBECs) and mouse airways to allergens from the fungus Alternaria alternata triggers the release of alarmin signaling molecules including ATP, IL-33 and DNA fragments. The goal of this study was to determine the mechanisms that regulate release of genomic DNA fragments and how extracellular DNA (eDNA) contributes to type 2 immunity. Treatment of hBECs with Alternaria extract induces oxidative stress, which leads to ATP release and uptake of Ca2+ following activation of P2X receptors. DNA release was dependent on a sustained increase in intracellular [Ca2+], which stimulates cleavage of caspase 3 by the proprotein convertase enzyme furin, resulting in activation of caspase 3 and ultimately nuclear DNA fragmentation as shown in comet assays. Some of the fragmented DNA was released into the media under conditions where barrier function of the epithelium was maintained, and nuclear and plasma membranes remained intact. Alternaria-induced DNA release was detected within 30 minutes following exposure, reached its peak at 4 hours and ceased after 12 hours. Pretreatment of hBECs with a calpain inhibitor resulted in continued DNA release and loss of cell viability by 24 hours. This result suggested that calpain activity contributed to termination of Alternaria-evoked DNA release. In mice, intranasal administration of Alternaria extract produced release of DNA fragments into bronchoalveolar lavage (BAL) fluid over a similar time course as observed in hBECs. In caspase 3 deficient mice however, DNA release into BAL fluid was significantly reduced. Furthermore, Alternaria-induced type 2 immune responses were suppressed following pretreatment with a DNA scavenger. Conversely, intranasal administration of mouse genomic DNA with Alternaria amplified IL-5 and IL-13 secretion into BAL fluid while DNA alone had no effect. These findings indicate that DNA release from the airway epithelium is a regulated process involving noncanonical activation of caspase 3 to produce DNA fragmentation and calpain activity, which appears to be essential for the termination of DNA release. Moreover in airways, eDNA appears to function as a secondary alarmin that acts on immune cells to enhance type 2 immunity. This work was supported by grants from the NIH (AI128729 and AI169530) to HK and SMO. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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 &gt;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 &gt;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.