Physiological microbial exposure has a temporally limited inhibitory effect on lung ILC2 responses to allergens in mice

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.

Peroxiredoxin Asp f3 Is Essential for Aspergillus fumigatus To Overcome Iron Limitation during Infection

Asp f3 is one of the most abundant proteins in the pathogenic mold Aspergillus fumigatus . It has an enigmatic multifaceted role as a fungal allergen, virulence factor, reactive oxygen species (ROS) scavenger, and vaccine candidate.

Novel Mouse Models of Fungal Asthma

Alternaria alternata is a ubiquitous fungus and a major allergen associated with the development of asthma. Inhalation of intact spores is the primary cause of human exposure to fungal allergen. However, allergen-rich cultured fungal filtrates are oftentimes used in the current models of fungal sensitization that do not fully reflect real-life exposures. Thus, establishing novel spore exposure models is imperative. In this study, we established novel fungal exposure models of both adult and neonate to live spores. We examined pathophysiological changes in the spore models as compared to the non-exposure controls and also to the conventional filtrate models. While both Alternaria filtrate- and spore-exposed adult BALB/c mice developed elevated airway hyperresponsiveness (AHR), filtrates induced a greater IgE mediated response and higher broncholavage eosinophils than spores. In contrast, the mice exposed to Alternaria spores had higher numbers of neutrophils. Both exposures induced comparable levels of lung tissue inflammation and mucous cell metaplasia (MCM). In the neonatal model, exposure to Alternaria spores resulted in a significant increase of AHR in both adult and neonatal mice. Increased levels of IgE in both neonatal and adult mice exposed to spores was associated with increased eosinophilia in the treatment groups. Adult demonstrated increased numbers of lymphocytes that was paralleled by increased IgG1 production. Both adults and neonates demonstrated similarly increased eosinophilia, IgE, tissue inflammation and MCM.

Polymorphism of Aspergillus Fumigatus Major Allergen Genes Associating with Their Isolated Sites Affects Their IgE Epitope Structures

The circumstances in which organisms live induce polymorphism in their genes, including fungal allergen genes, leading to altered structures and functions of proteins, related to their pathogenicity. Major allergen genes of Aspergillus fumigatus, Asp f 1, Asp f 2, and Asp f 3, were examined in 59 strains [environment and animal/human-body origin] to determine their nucleotide sequences, and then categorized. The location and number of IgE epitopes on the allergen molecules were predicted using a computer software. The Asp f 1 gene was classified into two groups (f1-1 and f1-2). One of the groups possessed one-nucleotide mutation point with one amino-acid substitution. The mutated Asp f 2 gene accompanying 6-amino acid substitution was classified into 7 groups (f2-1 to f2-7). Six of the groups possessed a newborn IgE epitope. The Asp f 3 gene contained two mutations, resulted in three groups (f3-1 to f3-3) without any amino-acid substitutions. Category E, consisting of groups f1-1, f2-5, and f3-2, was specific to an environmental origin. Our findings suggest that nucleotide mutation of the fungal allergen genes, associated with the origin of the fungus, modifies the structure of proteins, and affects their pathogenic properties, such as the localization of IgE epitopes.

Role of PAR1 in Food Allergies

Background and Hypothesis: The prevalence of food allergies continues to rise. In a mouse model, food allergy to peanuts develops in flaky tail mice with skin barrier mutations and exposure to peanut (PNE) and Alternaria Alternata (fungal allergen, Alt) on the skin. In the skin, keratinocytes respond to proteases in allergens through protease activating receptor 1 (PAR1). Blocking PAR1 decreased the severity of viral induced inflammation in mice. Whether PAR1 has a major role in food allergies has not been investigated. We tested the hypothesis that blocking PAR1 would halt the development of food allergy to peanuts in neonatal mice. Project Methods: In our studies, pups were injected intradermally (i.d.) with a PAR1 antagonist and then treated with PNE/Alt. In another group, pups received i.d. injections of a PAR1 agonist and then treated with PNE only. Control groups received allergens only. Pups were treated and skin sensitized 5 times every 3-4 days. Forty-eight hours after the last treatment, pups were challenged with PNE through oral gavage, and temperatures were recorded every 15-30 minutes for 3 hours. Skin, ileum, and jejunum samples were collected and used for qPCR to determine the expression of inflammatory mediators. Plasma serum was used for analysis of anti-PNE specific antibodies by ELISA. Results: PAR1 antagonist blocked anaphylaxis in allergic mice sensitized with PNE and Alt. PAR1 agonist is sufficient to induce anaphylaxis in mice sensitized with PNE only. Conclusion and Potential Impact: This study demonstrates that PAR1 is involved in the development of food allergies, where blocking the receptor blocked food allergies in neonatal mice. The signaling mechanisms and activators of PAR1 need further studies, using PAR1 deficient mice. This novel pathway may lead to therapies to stop the development of food allergies.

Identification and biochemical characterization of Asp t 36, a new fungal allergen from Aspergillus terreus

Aspergillus terreus is an allergenic fungus, in addition to causing infections in both humans and plants. However, the allergens in this fungus are still unknown, limiting the development of diagnostic and therapeutic strategies. We used a proteomic approach to search for allergens, identifying 16 allergens based on two-dimensional immunoblotting with A. terreus susceptible patient sera. We further characterized triose-phosphate isomerase (Asp t 36), one of the dominant IgE (IgE)-reactive proteins. The gene was cloned and expressed in Escherichia coli. Phylogenetic analysis showed Asp t 36 to be highly conserved with close similarity to the triose-phosphate isomerase protein sequence from Dermatophagoides farinae, an allergenic dust mite. We identified four immunodominant epitopes using synthetic peptides, and mapped them on a homology-based model of the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure, rendering it an IgE-binding hotspot. Biophysical analysis indicated that Asp t 36 shows similar secondary structure content and temperature sensitivity with other reported triose-phosphate isomerase allergens. In vivo studies using a murine model displayed that the recombinant Asp t 36 was able to stimulate airway inflammation, as demonstrated by an influx of eosinophils, goblet cell hyperplasia, elevated serum Igs, and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus, and define a new fungal allergen more broadly. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.

RNA-binding protein RBM3 negatively regulates innate lymphoid cells (ILCs) and lung inflammation

Innate lymphoid cells (ILCs) promote lung inflammation through cytokine production in diseases such as asthma. RNA-binding proteins (RBPs) are critical post-transcriptional regulators of cellular function, including inflammatory responses, though the role of RBPs in innate lymphoid cells is unknown. Here, we demonstrate that RNA-binding motif 3 (RBM3) is one of the most highly expressed RBPs in Thy1.2 + lung ILCs after fungal allergen challenge and is further induced by epithelial cytokines TSLP and IL-33 in both human and mouse ILCs. Single (rbm3-/-) and double (rbm3-/-rag2-/-) knockout mice exposed via the airway to the asthma-associated fungal allergen Alternaria alternata displayed increases in eosinophilic lung inflammation and ILC activation compared to control mice. In addition to increased Th2 cytokine production, rbm3-/- ILCs produced elevated IL-17A. The negative regulation by RBM3 in ILC responses was direct as purified rbm3-/- ILCs were hyperinflammatory in vitro and in vivo after stimulation with IL-33. Transcriptomic analysis by RNA-sequencing of rbm3-/- lung ILCs showed increased type 2 and 17 cytokines as well as global expression differences in critical cytokines, receptors, transcription factors, and survival transcripts compared with WT ILCs. Intriguingly, these transcript changes did not correlate with the presence of AU-rich elements (AREs), which RBM3 is known to bind. Thus, regulation of ILC responses by RNA-binding proteins offers novel mechanistic insight into lung ILC biology and ILC-driven inflammatory diseases.

RNA-binding protein RBM3 negatively regulates innate lymphoid cells (ILCs) and lung inflammation

Innate lymphoid cells (ILCs) promote lung inflammation through cytokine production in diseases such as asthma. RNA-binding proteins (RBPs) are critical post-transcriptional regulators of cellular function, including inflammatory responses, though the role of RBPs in innate lymphoid cells is unknown. Here, we demonstrate that RNA-binding motif 3 (RBM3) is one of the most highly expressed RBPs in Thy1.2+ lung ILCs after fungal allergen challenge and is further induced by epithelial cytokines TSLP and IL-33 in both human and mouse ILCs. Single (rbm3−/−) and double (rbm3−/−rag2−/−) knockout mice exposed via the airway to the asthma-associated fungal allergen Alternaria alternata displayed increases in eosinophilic lung inflammation and ILC activation compared to control mice. In addition to increased Th2 cytokine production, rbm3−/− ILCs produced elevated IL-17A. The negative regulation by RBM3 in ILC responses was direct as purified rbm3−/− ILCs were hyperinflammatory in vitro and in vivo after stimulation with IL-33. Transcriptomic analysis by RNA-sequencing of rbm3−/− lung ILCs showed increased type 2 and 17 cytokines as well as global expression differences in critical cytokines, receptors, transcription factors, and survival transcripts compared with WT ILCs. Intriguingly, these transcript changes did not correlate with the presence of AU-rich elements (AREs), which RBM3 is known to bind. Thus, regulation of ILC responses by RNA-binding proteins offers novel mechanistic insight into lung ILC biology and ILC-driven inflammatory diseases.