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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.

BACKGROUND: Food allergy and acute anaphylaxis can be life-threatening. While T follicular helper (Tfh) cells play a pivotal role in the allergic immune responses, the immunologic mechanisms that regulate the production of antibodies that mediate anaphylaxis are not fully understood. OBJECTIVE: The aim of this study was to investigate the role of the inhibitory receptor programmed cell death protein 1 (PD-1), which is highly expressed on Tfh cells, in allergic immune responses using an animal model of peanut allergy and anaphylaxis. METHODS: Naïve wild-type mice were exposed to peanut flour intranasally and then challenged with peanut extract to induce systemic anaphylaxis. The roles of PD-1 were examined by blocking antibodies and using gene-deficient animals. A hapten model and passive cutaneous anaphylaxis were used to characterize allergen-specific antibodies. RESULTS: Treatment with anti-PD-1 enhanced development of Tfh cells and germinal center B cells in mice exposed to peanut flour. Nonetheless, anti-PD-1 or anti-PD-L1 fully protected mice from developing anaphylaxis. Anti-PD-1 treatment or genetic deficiency of PD-1 in CD4(+) T cells inhibited production of peanut-specific IgE and increased the levels of IgG. The passive cutaneous anaphylaxis showed that peanut-specific antibodies generated in anti-PD-1-treated animals prevented, rather than provoked, anaphylaxis when transferred to naïve animals. Anti-PD-1 promoted production of antibodies with low affinity for an antigen in the hapten model. CONCLUSION: Blockade of the PD-1/PD-L1 pathway is protective against allergic immune responses. The direct interaction between Tfh cells and B cells may play a pivotal role in controlling antibody quality and clinical manifestation of allergic diseases.

Alternaria alternata and house dust mite exposure evokes IL-33 secretion from the airway epithelium, which functions as an alarmin to stimulate type 2 immunity. Extracellular DNA (eDNA) is also an alarmin that intensifies inflammation in cystic fibrosis, chronic obstructive pulmonary disease, and asthma.We investigated the mechanisms underlying allergen-evoked DNA mobilization and release from the airway epithelium and determined the role of eDNA in type 2 immunity.Human bronchial epithelial (hBE) cells were used to characterize allergen-induced DNA mobilization and extracellular release using comet assays to measure DNA fragmentation, Qubit double-stranded DNA assays to measure DNA release, and DNA sequencing to determine eDNA composition. Mice were used to investigate the role of eDNA in type 2 immunity.Alternaria extract rapidly induces mitochondrial and nuclear DNA release from human bronchial epithelial cells, whereas house dust mite extract induces mitochondrial DNA release. Caspase-3 is responsible for nuclear DNA fragmentation and becomes activated after cleavage by furin. Analysis of secreted nuclear DNA showed disproportionally higher amounts of promotor and exon sequences and lower intron and intergenic regions compared to predictions of random DNA fragmentation. In mice, Alternaria-induced type 2 immune responses were blocked by pretreatment with a DNA scavenger. In caspase-3-deficient mice, Alternaria-induced DNA release was suppressed. Furthermore, intranasal administration of mouse genomic DNA with Alternaria amplified secretion of IL-5 and IL-13 into bronchoalveolar lavage fluid while DNA alone had no effect.These findings highlight a novel, allergen-induced mechanism of rapid DNA release that amplifies type 2 immunity in airways.

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.

Lung group 2 innate lymphoid cells (ILC2) determine the nature of the immune response to airway allergens. Some microbial products, including those stimulating interferons, block ILC2 activation, but whether this occurs following natural infections or causes durable ILC2 inhibition is unclear. We tested this using a model of physiological microbial exposures through cohousing laboratory and pet store mice. Laboratory mice cohoused for two weeks displayed an impaired ILC2 response and reduced lung eosinophilia toward intranasal allergens, while these responses were restored in mice cohoused at least two months. ILC2 inhibition at two weeks correlated with increased interferon receptor signaling, which waned by two months of cohousing. Re-induction of interferons in two-month cohoused mice blocked ILC2 activation. These findings suggest ILC2 respond dynamically to environmental cues and that microbial exposures do not dictate long-term desensitization of innate type-2 responses to allergens.

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.

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.

A human study, Learning Early About Peanut Allergy (LEAP), showed that early introduction of peanut products decreases the prevalence of peanut allergy among children. However, the immunologic mechanisms mediating the protective effects of consuming peanut products are not well understood.The objective was to develop a mouse model that simulates the LEAP study and investigate the underlying mechanisms for the study observations.Adult naive BALB/c mice were fed a commercial peanut butter product (Skippy) or buffer control and concomitantly exposed to peanut flour through the airway or skin to mimic environmental exposure. The animals were analyzed for anaphylactic reaction and by molecular and immunologic approaches.After exposure to peanut flour through the airway or skin, naive mice developed peanut allergy, as demonstrated by acute and systemic anaphylaxis in response to challenge with peanut extract. Ingestion of Skippy, however, nearly abolished the increase in peanut-specific IgE and IgG and protected animals from developing anaphylaxis. Skippy-fed mice showed reduced numbers of T follicular helper (Tfh) cells and germinal center B cells in their draining lymph nodes, and single-cell RNA sequencing revealed a CD4Ingestion of a peanut product protects mice from peanut allergy induced by environmental exposure to peanuts, and the CTLA-4 pathway, which regulates Tfh cell responses, likely plays a pivotal role in this protection.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are involved in type 2 immune responses in mucosal organs and are associated with various allergic diseases in humans. Studies are needed to understand the molecules and pathways that control ILC2s. OBJECTIVE: The aim of this study was to develop a mouse model that limits the innate type 2 immune response in the lung and to investigate the immunologic mechanisms involved in regulation of lung ILC2s. METHODS: Naïve BALB/c mice were administered various toll-like receptor (TLR) agonists and exposed intranasally (i.n.) to the fungal allergen Alternaria alternata. The mechanisms were investigated using gene knockout mice and cultures of lung cells and isolated lung ILC2s. RESULTS: Polyinosinic-polycytidylic acid [poly (I:C)] effectively inhibited innate type 2 response to A. alternata. Poly (I:C) promoted production of interferon (IFN)-α, -β, and -γ, and its inhibitory effects were dependent on the IFN-α/β receptor pathway. IFN-β was 100-times more potent than IFN-α at inhibiting type 2 cytokine production by lung ILC2s. Signal transducer and activator of transcription 5 (STAT5)-activating cytokines, including interleukin-2 (IL-2), IL-7, and thymic stromal lymphopoietin (TSLP), but not IL-33, promoted survival and proliferation of lung ILC2s in vitro, while IFN-β blocked these effects. Expression of the transcription factor GATA3, which is critical for differentiation and maintenance of ILC2s, was inhibited by IFN-β. CONCLUSION: IFN-β blocks the effects of STAT5-activating cytokines on lung ILC2s and inhibits their survival and effector functions. Administration of IFN-β may provide a new strategy to treat diseases involving ILC2s.

Early life respiratory insults, such as viral infections or hyperoxia, often increase asthma susceptibility later in life. The mechanisms underlying this increased susceptibility are not fully understood. IL-33 has been shown to be critically involved in allergic airway diseases. IL-33 expression in the neonatal lung can be increased by various respiratory insults associated with asthma development. Therefore, we investigated whether and how early life increases in IL-33 impact allergic airway responses later in life. Using a novel IL-33 transgenic mouse model, in which full-length IL-33 was inducible overexpressed in lung epithelial cells, we transiently upregulated lung IL-33 expression in neonatal mice for one week. After resting for 4–6 weeks, mice were intranasally exposed to a single-dose of recombinant IL-33 or the airborne allergen Alternaria. Alternatively, mice were exposed to Alternaria and ovalbumin multiple times for one month. We found that a transient increase in IL-33 expression during the neonatal period promoted IL-5 and IL-13 production when mice were later exposed to a single-dose of IL-33 or Alternaria in adulthood. However, increased IL-33 expression during the neonatal period did not affect airway inflammation, type 2 cytokine production, lung mucus production, or antigen-specific antibody responses when adult mice were exposed to Alternaria and ovalbumin multiple times. These results suggest that transient increased IL-33 expression early in life may have differential effects on allergic airway responses in later life, preferentially affecting allergen-induced acute type 2 cytokine production.

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.

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.

Group 2 innate lymphoid cells (ILC2s) reside in multiple organs in the body, where they play roles in immunity, tissue homeostasis, and metabolic regulation. However, little is known about the regulatory mechanisms of ILC2s in different organs. Here, we identified ILC2s in the mouse uterus and found that they express cell surface molecules, including the IL-33 receptor, ST2, that are roughly comparable to those expressed by lung ILC2s. Both in vivo and in vitro treatment with IL-33 induced type 2 cytokine production in uterine ILC2s, suggesting that they respond to IL-33 in a manner similar to ILC2s in other organs. Importantly, uterine ILC2s were nearly absent in ovariectomized mice and were increased in wild-type mice by estrogen administration, whereas lung ILC2s were unaffected by both ovariectomy and estrogen administration. Likewise, a marked reduction in uterine ILC2s was observed in mice deficient in estrogen receptor α or estrogen receptor β. Furthermore, uterine ILC2s highly expressed estrogen receptor α, and in vitro culture of isolated uterine ILC2s with 17β-estradiol modified expression of a number of genes. Finally, an increased prevalence in neonatal mortality was observed in litters from dams lacking the IL-33 receptor, ST2. Taken together, our findings indicate that unlike lung IL2Cs, uterine ILC2s are regulated by female sex hormones, which may specialize them for specific physiological functions.

Background Interleukin (IL)-33 is implicated in the pathophysiology of asthma and allergic diseases. However, our knowledge is limited regarding how IL-33 release is controlled. The transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a key role in antioxidant response regulation. Objective The goal of this project was to investigate the role of cellular oxidative stress in controlling IL-33 release in airway epithelium. Methods Complementary approaches were used that included human bronchial epithelial cells and mouse models of airway type-2 immunity that were exposed to fungus Alternaria extract. The clinically available Nrf2 activator 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me) was used to evaluate the role of Nrf2-induced antioxidant molecules. Results Human bronchial epithelial cells produced reactive oxygen species (ROS) when they were exposed to Alternaria extract. ROS scavengers, such as glutathione (GSH) and N-acetyl cysteine, prevented extracellular secretion of ATP and increases in intracellular calcium concentrations that precede IL-33 release. Administration of CDDO-Me to mice enhanced expression of a number of antioxidant molecules in the lungs and elevated lung levels of endogenous GSH. Importantly, CDDO-Me treatment reduced allergen-induced ATP secretion and IL-33 release by airway epithelial cells in vitro and protected mice from IL-33 release and asthma-like pathological changes in the lungs. Conclusions The balance between oxidative stress and antioxidant responses plays a key role in controlling IL-33 release in airway epithelium. The therapeutic potential of Nrf2 activators needs to be considered for asthma and allergic airway diseases.

Programmed cell death-1 (PD-1), a member of the CD28 family, is critical for regulation of immune cell activation and maintenance of peripheral tolerance. Blocking antibodies against PD-1 have been used to sustain activation of T cells in cancer immunotherapy. T follicular helper (Tfh) cells play important role in the differentiation and maturation of antigen-specific B cells. Tfh cells highly and constitutively express PD-1; however, our knowledge is limited regarding the role of PD-1 in Tfh cell function and antibody response. We addressed this question by using a Tfh-dependent mouse model of peanut allergy. When naïve BALB/c mice were exposed to peanut flour by inhalation for 4 weeks, they developed peanut allergy as demonstrated by increased plasma levels of peanut-specific IgE and IgG antibodies and manifestation of acute anaphylaxis upon challenge with peanut extract. To examine the roles of PD-1, we administered a PD-1 blocking antibody to the animals during their exposure to peanut flour. Mice administered anti-PD-1 did not develop anaphylaxis even when they were challenged with peanut extract. Anti-PD1 treatment increased the number of Tfh cells and germinal center B cells in draining lymph nodes, and it increased plasma levels of peanut-specific IgG antibodies. Importantly, PD-1 blockade promoted the accumulation of low-affinity antibodies while it maintained the levels of high-affinity antibodies. These findings suggest that quality of allergen-specific antibodies affects the severity of allergic immune responses. Understanding the mechanisms that explain how PD-1 regulates the Tfh-B interaction and affinity maturation of antibodies likely leads to a better understanding of pathophysiology of allergic diseases.

The ability of some microbes and their products to restrain the immune response to allergens has been extensively studied and is a key element of the hygiene hypothesis. However, it is unclear whether a modulated immune response relates to recent microbial exposure, or whether a diverse infectious history promotes sustained changes in reactivity to allergens. Thus, we explored how prior microbial experience in “dirty” mice affected innate immune responses to an airway fungal allergen. Dirty mice were generated by cohousing laboratory mice with pet store mice for at least 60 days to allow for physiological transmission of microbes. Despite changes induced in many immune cell populations, lung type 2 innate lymphoid cell (ILC2) numbers and phenotype were unaltered by cohousing. Short term (4.5 hour) production of the type 2 cytokines IL-5 and IL-13 in response to Alternaria alternata extract was consistently depressed in dirty mice relative to specific pathogen free (SPF) and germ-free animals. This correlated with diminished release of IL-33, an alarmin that activates ILC2, in dirty mice. Nevertheless, at a later timepoint (24 hours) or after repeated Alternaria exposure, the influx of eosinophils, neutrophils and T cells into the lungs was similar in dirty and SPF mice. Our data therefore indicate that a history of robust and diverse microbial exposure delays but ultimately does not restrain development of a type 2 response to airway allergens, in contrast to the established inhibitory effects of acute exposure to certain microbes and their products.

Innate lymphoid cells (ILCs) are a new family of immune cells that play important roles in innate immunity in mucosal tissues, and in the maintenance of tissue and metabolic homeostasis. Recently, group 2 ILCs (ILC2s) were found to promote the development and effector functions of Th2-type CD4+ T cells by interacting directly with T cells or by activating dendritic cells, suggesting a role for ILC2s in regulating adaptive immunity. However, our current knowledge on the role of ILCs in humoral immunity is limited. In this study, we found that ILC2s isolated from the lungs of naive BALB/c mice enhanced the proliferation of B1- as well as B2-type B cells and promoted the production of IgM, IgG1, IgA, and IgE by these cells in vitro. Soluble factors secreted by ILC2s were sufficient to enhance B cell Ig production. By using blocking Abs and ILC2s isolated from IL-5–deficient mice, we found that ILC2-derived IL-5 is critically involved in the enhanced production of IgM. Furthermore, when adoptively transferred to Il7r−/− mice, which lack ILC2s and mature T cells, lung ILC2s promoted the production of IgM Abs to a polysaccharide Ag, 4-hydroxy-3-nitrophenylacetyl Ficoll, within 7 d of airway exposure in vivo. These findings add to the growing body of literature regarding the regulatory functions of ILCs in adaptive immunity, and suggest that lung ILC2s promote B cell production of early Abs to a respiratory Ag even in the absence of T cells.

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.

Background Little is currently known regarding the immunologic mechanism(s) that initiate peanut allergy. Notably, peanut proteins have been detected in house dust, and their levels correlate with peanut allergy prevalence. Objective This study aimed to develop a new mouse model for peanut allergy and to investigate the immunologic mechanisms involved in peanut allergen sensitization. Methods To mimic environmental exposure, naive mice were exposed to peanut flour by inhalation for up to 4 weeks. We then analyzed serum levels of IgE antibody and challenged mice with peanut proteins. Immunological mechanisms involved in sensitization were analyzed using cytokine reporter mice, an adoptive cell transfer model, and gene knockout mice. Results When exposed to peanut flour by inhalation, both BALB/c and C57BL/6 mice developed peanut allergy, as demonstrated by the presence of peanut-specific IgE antibodies and manifestation of acute anaphylaxis on challenge. A large number of follicular helper T (Tfh) cells were also detected in draining lymph nodes of allergic mice. These cells produced IL-4 and IL-21, and they more robustly promoted peanut-specific IgE production than T h 2 cells did. Genetic depletion of Tfh cells decreased IgE antibody levels and protected mice from anaphylaxis, without affecting T h 2 cells. Furthermore, peanut flour exposure increased lung levels of IL-1α and IL-1β, and mice deficient in the receptor for these cytokines showed a significant decrease in Tfh cells compared with in wild-type mice. Conclusions Tfh cells play a key role in peanut allergy, and the IL-1 pathway is involved in the Tfh response to peanut allergen exposure.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by interstitial remodelling, leading to compromised lung function. Cellular senescence markers are detectable within IPF lung tissue and senescent cell deletion rejuvenates pulmonary health in aged mice. Whether and how senescent cells regulate IPF or if their removal may be an efficacious intervention strategy is unknown. Here we demonstrate elevated abundance of senescence biomarkers in IPF lung, with p16 expression increasing with disease severity. We show that the secretome of senescent fibroblasts, which are selectively killed by a senolytic cocktail, dasatinib plus quercetin (DQ), is fibrogenic. Leveraging the bleomycin-injury IPF model, we demonstrate that early-intervention suicide-gene-mediated senescent cell ablation improves pulmonary function and physical health, although lung fibrosis is visibly unaltered. DQ treatment replicates benefits of transgenic clearance. Thus, our findings establish that fibrotic lung disease is mediated, in part, by senescent cells, which can be targeted to improve health and function., Removal of senescent cells rejuvenates lungs of aged mice. Here the authors show that elimination of senescent cells using either genetic or pharmacological means improves lung function and physical health in a mouse model of idiopathic pulmonary fibrosis (IPF), suggesting potential therapy for treatment of human IPF.

Background Innate lymphoid cells (ILCs) play important roles in innate immunity and tissue remodeling via production of various cytokines and growth factors. Group 2 ILCs (ILC2s) were recently shown to mediate the immune pathology of asthma even without adaptive immunity. However, little is known about possible interactions between ILC2s and other immune cells. We sought to investigate the capacity of ILC2s to regulate effector functions of T cells. Methods We isolated ILC2s from the lungs of naive mice. We cultured CD4+ T cells with ILC2s in vitro and examined the functions of these cell types. The mechanisms were investigated using blocking antibodies and cells isolated from cytokine-deficient mice. For the in vivo study, we adoptively transferred ILC2s and CD4+ T cells into Il7ra−/− mice and subsequently exposed the mice to ovalbumin and a cysteine protease. Results Lung ILC2s enhanced CD4+ T-cell proliferation and promoted production of type 2 cytokines in vitro. The interaction between ILC2s and CD4+ T cells involved costimulatory molecule OX40L and cytokine IL-4, which was mainly derived from ILC2s. Adoptive transfer of both ILC2 and CD4+ T-cell populations, but not each population alone, into Il7ra−/− mice resulted in induction of a robust antigen-specific type 2 cytokine response and airway inflammation. Conclusion Lung ILC2s function to promote adaptive immunity in addition to their established roles in innate immunity. This novel function of ILC2s needs to be taken into account when considering the pathophysiology of asthma and other allergic airway diseases.

Although type 2 immune responses to environmental Ags are thought to play pivotal roles in asthma and allergic airway diseases, the immunological mechanisms that initiate the responses are largely unknown. Many allergens have biologic activities, including enzymatic activities and abilities to engage innate pattern-recognition receptors such as TLR4. In this article, we report that IL-33 and thymic stromal lymphopoietin were produced quickly in the lungs of naive mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and thymic stromal lymphopoietin sensitized naive animals to an innocuous airway Ag OVA, which resulted in production of type 2 cytokines and IgE Ab, and eosinophilic airway inflammation when mice were challenged with the same Ag. Importantly, upon exposure to proteases, uric acid (UA) was rapidly released into the airway lumen, and removal of this endogenous UA by uricase prevented type 2 immune responses. UA promoted secretion of IL-33 by airway epithelial cells in vitro, and administration of UA into the airways of naive animals induced extracellular release of IL-33, followed by both innate and adaptive type 2 immune responses in vivo. Finally, a potent UA synthesis inhibitor, febuxostat, mitigated asthma phenotypes that were caused by repeated exposure to natural airborne allergens. These findings provide mechanistic insights into the development of type 2 immunity to airborne allergens and recognize airway UA as a key player that regulates the process in respiratory mucosa.