Iijima, Koji

Abstract The objective of this study was to test the hypothesis that the increasing global incidence of allergy and atopy are due in part to improved hygiene and decreased microbial exposure. We have adopted a novel mouse model of normalized microbial exposure to test the impact of immune experience on subsequent responses to airway allergens. In this model, specific pathogen free (SPF) B6 mice are cohoused with mice from pet stores and become “dirty” – many commensals and pathogens are transmitted through cohousing and influence the immune cell populations systemically and in the lungs. We treated mice intranasally with a single dose of A. alternata fungal extract (Alt) and assessed production of type 2 cytokines. IL5 and IL13 levels in the lungs and bronchoalveolar lavage fluid were dramatically elevated by Alt in SPF mice but were not significantly increased by Alt in dirty mice. Type 2 innate lymphoid cells (ILC2) are the cells responsible for IL5 and IL13 after acute Alt treatment, and interestingly the number of lung ILC2 was unaltered by cohousing and their activation status appeared similar in both housing conditions. We treated mice with recombinant IL33, the alarmin released by lung epithelial cells in response to allergens, and the results suggest an impaired response to IL33 by dirty lung ILC2. In a repeated Alt exposure model, ILC2 cells expanded in SPF lungs and recruited eosinophils, neutrophils, and T cells. Most dirty mouse lungs contained these cell populations at steady state but were only modestly recruited with repeated Alt exposure. Lung function experiments are in progress. This study suggests that increased microbial exposure leads to more type-2 associated immune cells in the lungs, however responses to airway allergens are dampened.

Abstract Peanut allergies are life-long diseases that cause severe anaphylactic responses to affected individuals. Oral ingestion of peanut products prevents infants from developing peanut allergy. However, little information is currently available regarding the immunologic mechanisms for this protection. Therefore, the goal of this project was to establish an oral tolerance model to peanut allergy in mice and to determine the immunologic mechanisms. Naïve BALB/c mice that were exposed intranasally (i.n.) to peanut flour (PNf) developed peanut allergy as demonstrated by increased serum levels of IgE antibody as well as acute anaphylactic response when challenged with peanut extract. When mice were fed peanut butter (PNB) prior to i.n PNf exposure, they were protected from developing peanut allergy; these mice showed decreased serum levels of IgE antibody and lacked anaphylactic response. T follicular helper (Tfh) cells and germinal center (GC) B cells were decreased in lung draining lymph nodes of mice fed PNB. Furthermore, scRNSseq revealed 2 distinct CD4+ T cell subpopulations that are increased in those protected mice. CTLA-4, a checkpoint molecule, was highly expressed in one of these subpopulations. Systemic administration of a CTLA-4 blocking antibody to mice fed PNB increased the numbers of Tfh cells and GC B cells and increased serum levels of IgE antibody. Finally, anti-CTLA-4 reversed the clinical efficacy of oral PNB administration, resulting in manifestation of acute anaphylaxis when challenged with peanut extract. Thus, oral administration of peanut product(s) prevents development of peanut allergy in mice. CTLA4+CD4+ T cells likely play a pivotal role in this process by regulating the development of Tfh cells.

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.

Abstract The impact of microbes on restraining the immune response to allergens has been extensively studied and is a key element of the hygiene hypothesis. Lung type 2 innate lymphoid cell responses to airway allergens can be inhibited by administration of a number of microbial products; however, it is unclear whether such an effect would be observed with natural infections and how sustained any observed inhibitory effects would be. To answer these questions, we used a murine model of physiological microbial exposures through cohousing SPF laboratory mice with pet store mice and examined the acute type 2 response to intranasally delivered fungal allergen extract Alternaria alternata. We found laboratory mice cohoused with pet store mice for two weeks display a suppressed ILC2 response to A. alternata which resulted in reduced eosinophilia. By comparison, mice cohoused for at least two months had ILC2 and eosinophil responses similar to SPF mice despite dramatic changes to the composition of the immune cell populations in the lungs. Lung ILC2 in two-month cohoused mice were still sensitive to subsequent inflammatory cues, as administration of poly(I:C) was able to suppress ILC2 activation and eosinophilia equally well in SPF and two-month cohoused animals. These findings suggest ILC2 dynamically respond to their environment and are not easily desensitized long-term.

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.

Abstract Peanut allergy is a growing public concern; however, little is known about the immunological mechanism(s) that initiate the disease process. Our knowledge is also limited regarding the role of group 2 innate lymphoid cells (ILC2s) in regulating humoral immunity. To fill these major gaps in our knowledge, we investigated the immunological mechanisms involved in peanut allergen sensitization by using mouse models. To mimic environmental exposure in humans, naive BALB/c mice were exposed to peanut flour by inhalation without any exogenous adjuvants. When exposed to peanut flour, naive mice developed T follicular helper (Tfh) cells in their lung draining lymph nodes and produced IgE Abs to peanuts. Mice deficient in IL-13 showed decreased numbers of Tfh cells and germinal center B cells and produced significantly fewer IgE Abs. IL-13 was necessary and sufficient for induction of CD11c+ MHC class IIhi dendritic cells that are implicated in Tfh cell development. Importantly, lung ILC2s served as a predominant early source of IL-13 when naive mice were exposed to peanut flour. Furthermore, mice that are deficient in lung ILC2s by bone marrow transfer from Rorasg/sg mice or by genetic manipulation produced significantly fewer IgE Abs to peanuts compared with control mice. These findings suggest lung ILC2s that serve as a rapid source of IL-13 upon allergen exposure play a major role in Tfh cell development, IgE Ab production, and initiation of peanut allergy.

Abstract IL-33, an IL-1 family cytokine, is constitutively expressed in mucosal tissues and other organs in healthy humans and animals, and expression levels increase in inflammatory conditions. Although IL-33–mediated promotion of type 2 immune responses has been well established, a gap in our knowledge regarding the functional diversity of this pleiotropic cytokine remains. To address this gap, we developed a new IL-33 transgenic mouse model in which overexpression of full-length IL-33 is induced in lung epithelial cells under conditional control. In adult mice, an ∼3-fold increase in the steady-state IL-33 levels produced no pathologic effects in the lungs. When exposed to airborne allergens, adult transgenic mice released more IL-33 extracellularly and exhibited robust type 2 immune responses. In neonatal transgenic mice, up to postnatal day 14, a similar increase in steady-state IL-33 levels resulted in increased mortality, enlarged alveolar spaces resembling bronchopulmonary dysplasia, and altered expression of genes associated with tissue morphogenesis. Processed 25-kDa IL-33 protein was detected in bronchoalveolar lavage fluids without any exogenous stimuli, and pathologic changes were abolished in mice deficient in the IL-33 receptor ST2. These findings suggest that adult lungs are relatively resistant to IL-33 overexpression unless they encounter environmental insults, whereas developing lungs are highly susceptible, with IL-33 overexpression resulting in detrimental and pathologic outcomes.