Oral administration of egg ovalbumin allergen induces dysregulation of tryptophan metabolism in sensitized BALB/c mice.

Food allergy (FA), triggered by specific dietary allergens, has emerged as a substantial global concern for food safety and public health. While studies have elucidated changes in immune cells and cytokines associated with allergen exposure, a comprehensive analysis of the host's metabolic features and the interaction between metabolites and the gut microbiota has not been conducted. In this study, egg allergen ovalbumin (OVA) was administered by the oral route to sensitized BALB/c mice to faithfully replicate key aspects of human FA, including severe allergic diarrhea, mast cell infiltration, and elevated levels of serum IgE, mMCPT-1, and Th2 cell hallmark cytokines (such as IL-4, IL-5, and IL-13). Furthermore, the untargeted and targeted metabolomic analyses indicated that FA in mice precipitated a substantial decrease in the tryptophan metabolites indole-3-acrylic acid (IA) and indole-3-lactic acid (ILA). The integration of shotgun metagenome and metabolome data further unveiled that the dysregulation of indole metabolism is related to a decline in the abundance of beneficial bacteria such as Lactobacillus and Bifidobacterium. Additionally, disruption of the tryptophan indole derivative pathway compromises the maintenance of intestinal mucosal function through the AHR signaling pathway, manifested by decreased expression of Reg3g and IL22. Taken together, this study demonstrated that the anaphylaxis triggered by oral ingestion of food allergens can lead to disruptions in tryptophan metabolism, consequently impairing intestinal immune homeostasis.

Co-exposure to polystyrene microplastics and di-(2-ethylhexyl) phthalate aggravates allergic asthma through the TRPA1-p38 MAPK pathway.

Increasing attention has been paid to the potential impact of microplastics (MPs) pollution on human health. MPs and phthalates coexist in the environment, however, the effects of exposure to MPs alone or to a combination of di-(2-ethylhexyl) phthalate (DEHP) and MPs on allergic asthma are unclear. This study investigates the effects of exposure to polystyrene microplastics (PS-MPs) or co-exposure with DEHP, on allergic asthma, and the underlying molecular mechanisms. We established an allergic asthma model using ovalbumin, and mice were exposed to PS-MPs (5 mg/kg bw/day) alone, or combined with DEHP (0.5, 5 mg/kg bw/day), for 28 days. The results showed that in the presence of ovalbumin (OVA) sensitization, exposure to PS-MPs alone slightly affected airway inflammation, and airway hyperresponsiveness, while co-exposure to PS-MPs and DEHP caused more significant damage. Co-exposure also induced more oxidative stress and Th2 immune responses, and activation of the TRPA1 and p38 MAPK pathways. The aggravation of asthmatic symptoms induced by co-exposure to PS-MPs and DEHP were inhibited by blocking TRPA1 ion channel or p38 MAPK pathway. The results demonstrated that co-exposure to PS-MPs and DEHP exacerbates allergic asthma, by exacerbating oxidative stress and inflammatory responses, and activating the TRPA1-p38 MAPK pathway.

Factor VIII trafficking to CD4+ T cells shapes its immunogenicity and requires several types of antigen-presenting cells.

Despite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.

Inulin has a beneficial effect by modulating the intestinal microbiome in a BALB/c mouse model.

Food allergy is an important health problem that affects human quality of life and socioeconomic development, and its treatment requires improvement. Intestinal flora dysbiosis is closely associated with food allergies. A sensitised mouse model was established by the intraperitoneal injection of ovalbumin (OVA). The mice were randomly divided into four groups: control, model, high-dose (H), and low-dose (L) inulin. The mice were administered water containing different concentrations of inulin four weeks before the OVA injection. Body weight changes were monitored. After the last OVA injection, the mice were scored for allergic reactions. The levels of total immunoglobulin E (IgE) and diamine oxidase (DAO) in the serum and secretory IgA (sIgA) in the small intestinal mucus were measured, and 16S rRNA sequencing of the faecal flora was performed to evaluate microbial parameters. The intestinal flora biomarkers, correlations between them, and biochemical indicators were analysed. Inulin treatment had no effect on the body weight of OVA-sensitised mice but attenuated allergic reactions and intestinal injury in mice. Compared with the control group, the model group had significantly higher levels of serum DAO and IgE and significantly lower levels of intestinal mucus IgA. IgA levels in the intestinal mucus of mice treated with inulin prior to OVA sensitisation were higher than those in non-inulin-treated OVA-sensitised mice. Furthermore, analysis of operational taxonomic units showed that inulin treatment decreased the abundance of Alloprevotella, Rikenellaceae RC9, Eubacterium siraeum, and Eubacterium xylanophilum, and increased the abundance of Blautia and Lachnospiraceae. Serum DAO levels were positively associated with Eubacterium siraeum, Alloprevotella, Eubacterium xylanophilum, and Odoribacter and negatively associated with Blautia, Tyzzerella, Alistipes, Desulfovibrionaceae, and Ruminococcaceae UCG005. In addition, IgE levels were positively associated with Eubacterium siraeum, Alloprevotella, Eubacterium xylanophilum, Odoribacter, and Citrobacter and negatively associated with Blautia, unclassified Ruminococcaceae, and Alistipes. IgA exhibited significant positive correlation with Blautia, norank_f_Eubacterium coprostanoligenes, Alistipes, norank Desulfovibrionaceae, Muribaculum, and Ruminococcaceae U C G 005 and significant negative correlation with Eubacterim siraeum, Eubacterium xylanophilum, Odoribacter, and Citrobacter. Inulin exerts a protective effect against food allergies in mice, which is partially mediated by alterations in the gut microbiota.

Frequency of allergic reactions in egg allergic patients after receiving the yellow fever vaccine

Background: Immunization with live attenuated viral yellow fever vaccine (YFV) grants effective immunity in most cases, and is recommended and prioritized for residents and travelers of endemic countries. YFV is seldom administered to egg-allergic patients (EAP) since it is cultivated in embryonated chicken eggs and may contain residual egg proteins, being a problem for egg-allergic residents and travelers of endemic countries. Objective: Describe the frequency of allergic reactions after YFV administration in confirmed EAP from an allergy outpatient center in Bogotá, Colombia. Methods: An observational, retrospective, cross-sectional, and descriptive study was conducted from January 2017 to December 2019. EAP whose allergy was confirmed with a positive Skin Prick Test (SPT) and/or egg protein–specific IgE levels who hadn’t received the YFV were included. Every patient had an SPT, severe EAP, and an additional Intradermal Test (IDT) done with the vaccine. If the vaccine SPT and IDT were negative, the YFV was administered as a single dose; if either were positive, the YFV was administered in graded doses. Statistical analysis was done in Stata16MP. Results: Seventy one patients were included, 24 (33.8%) of those had a history of egg anaphylaxis. All patients had negative YFV SPTs, and two of the five YVF IDTs were positive. Two patients, with previous egg-anaphylaxis, presented allergic reactions to the vaccine. Conclusions: YFV did not trigger allergic reactions in EAP without history of egg-anaphylaxis. With further research, safe single-dose vaccination to this population could be considered; however, patients with previous egg-anaphylaxis should be evaluated by an allergist before vaccination (AU)

Engineered Nanoparticles inside a Microparticle Oral System for Enhanced Mucosal and Systemic Immunity.

Antigen delivery through an oral route requires overcoming multiple challenges, including gastrointestinal enzymes, mucus, and epithelial tight junctions. Although each barrier has a crucial role in determining the final efficiency of the oral vaccination, transcytosis of antigens through follicle-associated epithelium (FAE) represents a major challenge. Most of the research is focused on delivering an antigen to the M-cell for FAE transcytosis because M-cells can easily transport the antigen from the luminal site. However, the fact is that the M-cell population is less than 1% of the total gastrointestinal cells, and most of the oral vaccines have failed to show any effect in clinical trials. To challenge the current dogma of M-cell targeting, in this study, we designed a novel tandem peptide with a FAE-targeting peptide at the front position and a cell-penetrating peptide at the back position. The tandem peptide was attached to a smart delivery system, which overcomes the enzymatic barrier and the mucosal barrier. The result showed that the engineered system could target the FAE (enterocytes and M-cells) and successfully penetrate the enterocytes to reach the dendritic cells located at the subepithelium dome. There was successful maturation and activation of dendritic cells in vitro confirmed by a significant increase in maturation markers such as CD40, CD86, presentation marker MHC I, and proinflammatory cytokines (TNF-α, IL-6, and IL-10). The in vivo results showed a high production of CD4+ T-lymphocytes (helper T-cell) and a significantly higher production of CD8+ T-lymphocytes (killer T-cell). Finally, the production of mucosal immunity (IgA) in the trachea, intestine, and fecal extracts and systemic immunity (IgG, IgG1, and IgG2a) was successfully confirmed. To the best of our knowledge, this is the first study that designed a novel tandem peptide to target the FAE, which includes M-cells and enterocytes rather than M-cell targeting and showed that a significant induction of both the mucosal and systemic immune response was achieved compared to M-cell targeting.

Measuring the Manipulation of T Helper Immune Responses by Schistosoma mansoni.

Schistosoma mansoni uses different mechanisms to escape its host's immunity. Understanding the ability of memory T cells to withstand this pathogen's manipulation is important for the development of effective vaccines against this immunomodulatory pathogen. In this study, ovalbumin (OVA) transgenic S. mansoni is used as a tool to investigate whether fully differentiated Th1, Th2 and Th17 cells are able to withstand pathogen manipulation. Naïve T cells from OT-II T cell receptor transgenic mice with a specificity for OVA were differentiated into Th1, Th2, and Th17 polarised memory cells in vitro. These cells were adoptively transferred into recipient mice to investigate whether these polarised immune memory T cells are resilient in the face of pathogen-mediated manipulation. After transferring memory cells, mice were challenged with OVA-transduced S. mansoni eggs as well as wild-type controls. The in vitro differentiated Th1, Th2 and Th17 memory cells continued to produce the same cytokines when challenged by OVA-expressing S. mansoni eggs as to these they produced when transferred in vivo, suggesting that the Th phenotypes of the memory T cells remains unaltered in the face of stimulation by S. mansoni. The ability of memory T cells to remain resilient to manipulation by the parasite suggests that vaccines might be able to produce immune memory responses able to withstand S. mansoni immune manipulation and hence protect the host from infection.

Eupatilin Suppresses OVA-Induced Asthma by Inhibiting NF-κB and MAPK and Activating Nrf2 Signaling Pathways in Mice.

To investigate the effect of eupatilin in asthma treatment, we evaluated its therapeutic effect and related signal transduction in OVA-induced asthmatic mice and LPS-stimulated RAW264.7 cells. The BALF was tested for changes in lung inflammatory cells. Th2 cytokines in the BALF and OVA-IgE in the serum were measured by ELISA. H&E and PAS staining were used to evaluate histopathological changes in mouse lungs. The key proteins NF-κB, MAPK, and Nrf2 in lung tissues were quantitatively analyzed by Western blotting. Finally, we evaluated the effect of eupatilin on cytokines and related protein expression in LPS-stimulated RAW 264.7 cells in vitro. In OVA-induced asthmatic mice, eupatilin reduced the numbers of inflammatory cells, especially neutrophils and eosinophils. Eupatilin also decreased the levels of IL-5, IL-13 in the BALF and OVA-IgE in the serum. Furthermore, eupatilin inhibited the activation of NF-κB and MAPK pathways and increased the expression of Nrf2 in OVA-induced asthmatic mice. In vitro, eupatilin significantly reduced LPS-stimulated NO, IL-6, and ROS production. Additionally, the NF-κB, MAPK, and Nrf2 protein expression in LPS-stimulated RAW264.7 cells was consistent with that in OVA-induced asthmatic lung tissues. In summary, eupatilin attenuated OVA-induced asthma by regulating NF-κB, MAPK, and Nrf2 signaling pathways. These results suggest the utility of eupatilin as an anti-inflammatory drug for asthma treatment.

CCR3 gene knockout in bone marrow cells ameliorates combined allergic rhinitis and asthma syndrome (CARAS) by reducing airway inflammatory cell infiltration and Th2 cytokines expression in mice model.

The present study aims to investigate the effects of CCR3 gene knockout in bone marrow cells (CCR3-KO) on the mouse model of combined allergic rhinitis and asthma syndrome (CARAS). It was found that CCR3-KO significantly reduced eosinophil (EOS) migration into the nasal (NALF) and bronchoalveolar (BALF) cavities of mice, and decreased Th2 cytokines (such as, IL-4, IL-5 and IL-13) levels in nasal mucosa and lung tissues. In addition, histological analysis showed that the damage degree of nasal mucosa structure in ovalbumin (OVA) modulated CCR3-KO mice was significantly less than that in OVA modulated Wild type (WT) mice, with reduced inflammatory cell infiltration and nasal mucus secretion. The infiltration of inflammatory cells in lung tissue was significantly reduced, and the proliferation of lung smooth muscle layer and extracellular matrix (ECM) production were decreased. Symptom analysis showed that CCR3-KO can reduced allergic rhinitis (AR) signals as nose scratching and sneezing. It was also found CCR3-KO reduce OVA-induced weight loss. The results showed that CCR3-KO could reduce the symptoms of allergic inflammation in CARAS mice by reducing airway inflammatory cell infiltration and down-regulating the expression of Th2 cytokines, and CCR3 gene could be used as a target gene for the treatment of CARAS.

Small Molecule Calcium Channel Activator Potentiates Adjuvant Activity.

There remains an unmet need for reliable fully synthetic adjuvants that increase lasting protective immune responses from vaccines. We previously reported a high-throughput screening for small molecules that extended nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) activation after a Toll-like receptor 4 (TLR4) ligand, lipopolysaccharide (LPS), stimulation using a human myeloid reporter cell line. We identified compounds with a conserved aminothiazole scaffold including 2D216 [N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide], which increased murine antigen-specific antibody responses when used as a co-adjuvant with LPS. Here, we examined the mechanism of action in human cells. Although 2D216 activated the major mitogen-activated protein kinases, it did not interact with common kinases and phosphatases and did not stimulate many of the pattern recognition receptors (PRRs). Instead, the mechanism of action was linked to intracellular Ca2+ elevation via Ca2+ channel(s) at the plasma membrane and nuclear translocation of the nuclear factor of activated T-cells (NFAT) as supported by RNA-seq data, analysis by reporter cells, Ca2+ flux assays, and immunoblots. Interestingly, 2D216 had minimal, if any, activity on Jurkat T cells but induced cytokine production and surface expression of costimulatory molecules on cells with antigen-presenting functions. A small series of analogs of 2D216 were tested for the ability to enhance a TLR4 ligand-stimulated autologous mixed lymphocyte reaction (MLR). In the MLR, 2E151, N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-((4-propylpiperidin-1-yl)sulfonyl)benzamide, was more potent than 2D216. These results indicate that a small molecule that is not a direct PRR agonist can act as a co-adjuvant to an approved adjuvant to enhance human immune responses via a complementary mechanism of action.

Dietary Exposure to Flame Retardant Tris (2-Butoxyethyl) Phosphate Altered Neurobehavior and Neuroinflammatory Responses in a Mouse Model of Allergic Asthma.

Tris (2-butoxyethyl) phosphate (TBEP) is an organophosphate flame retardant and used as a plasticizer in various household products such as plastics, floor polish, varnish, textiles, furniture, and electronic equipment. However, little is known about the effects of TBEP on the brain and behavior. We aimed to examine the effects of dietary exposure of TBEP on memory functions, their-related genes, and inflammatory molecular markers in the brain of allergic asthmatic mouse models. C3H/HeJSlc male mice were given diet containing TBEP (0.02 (TBEP-L), 0.2 (TBEP-M), or 2 (TBEP-H) µg/kg/day) and ovalbumin (OVA) intratracheally every other week from 5 to 11 weeks old. A novel object recognition test was conducted in each mouse at 11 weeks old. The hippocampi were collected to detect neurological, glia, and immunological molecular markers using the real-time RT-PCR method and immunohistochemical analyses. Mast cells and microglia were examined by toluidine blue staining and ionized calcium-binding adapter molecule (Iba)-1 immunoreactivity, respectively. Impaired discrimination ability was observed in TBEP-H-exposed mice with or without allergen. The mRNA expression levels of N-methyl-D aspartate receptor subunits Nr1 and Nr2b, inflammatory molecular markers tumor necrosis factor-α oxidative stress marker heme oxygenase 1, microglia marker Iba1, and astrocyte marker glial fibrillary acidic protein were significantly increased in TBEP-H-exposed mice with or without allergen. Microglia and mast cells activation were remarkable in TBEP-H-exposed allergic asthmatic mice. Our results indicate that chronic exposure to TBEP with or without allergen impaired object recognition ability accompanied with alteration of molecular expression of neuronal and glial markers and inflammatory markers in the hippocampus of mice. Neuron-glia-mast cells interaction may play a role in TBEP-induced neurobehavioral toxicity.

Ligustrazine Inhibits Lung Phosphodiesterase Activity in a Rat Model of Allergic Asthma.

OBJECTIVES: This study sought to examine whether ligustrazine was capable of inhibiting phosphodiesterase (PDE) activity and improving lung function in a rat model of asthma. METHODS: Rats were initially sensitized using ovalbumin (OVA) and then were challenged daily with aerosolized OVA beginning 14 days later (30 min/day) to generate a rat model of asthma. Changes in airway function following methacholine (MCh) injection were evaluated by monitoring lung resistance (R L) and dynamic lung compliance (C dyn) values using an AniRes2005 analytic system. In addition, serum IgE was measured via ELISA, while PDE expression was evaluated via qPCR and western blotting. Key Findings. Ligustrazine significantly impaired allergen-induced lung hyperresponsivity and inflammation in this asthma model system. Ligustrazine treatment was also associated with reduced expression of PDEs including PDE4 in the lungs of these rats. CONCLUSIONS: Ligustrazine suppresses airway inflammation and bronchial hyperresponsivity in this rat model system, and these changes are associated with decreased PDE expression at the protein and mRNA levels.

The mixture of siRNAs targeted to IL-4 and IL-13 genes effectively reduces the airway hyperreactivity and allergic inflammation in a mouse model of asthma.

Bronchial asthma (BA) is one of the most common chronic inflammatory disease of airways. There are huge experimental data indicating that Th2-cytokines IL-4 and IL-13 play a key role in BA pathogenesis. They are implicated in the IgE synthesis, eosinophil infiltration to the lungs and in the development of airway hyperreactivity (AHR), that makes these cytokines the promising targets. Neutralization of IL-4 and IL-13 or its common receptor chain (IL-4Rα) by monoclonal antibodies substantially reduce asthma symptoms. RNA interference provides a novel method for regulation of gene expression by siRNA molecules. In this study we evaluated whether the siRNA targeted to IL-4 and IL-13 reduce BA symptoms in mice model. Experimental BA was induced in BALB/c mice by sensitization to ovalbumin allergen followed by intranasal challenge. The intranasal delivery of siRNAs targeted to IL-4 and IL-13 inhibited the lung expression of these cytokines by more than 50% that led to the attenuation of AHR and pulmonary inflammation; the quantity of eosinophils in lungs which are one of the major inflammatory cells involved in allergic asthma pathogenesis decreased by more than 50% after siRNA treatment. These data support the possibility of a dual IL-4 and IL-13 inhibition by locally delivered siRNAs which in turn leads to the suppression of allergen-induced pulmonary inflammation and AHR.

MicroRNA-335-5p alleviates inflammatory response, airway fibrosis, and autophagy in childhood asthma through targeted regulation of autophagy related 5.

Childhood asthma is the most universal chronic disease, with significant cases reported. Despite the current progress in treatment, prognosis remains poor and the existing drugs cause serious side effects. This investigation explored the mechanisms and use of miR-335-5p on childhood asthma therapy. MiR-335-5p and ATG5 expression was analyzed in clinical plasma samples through RT-qPCR. Airway smooth muscle cells (ASMCs) were cultured, and transfected with miR-335-5p mimic, miR-335-5p inhibitor, and pcDNA3.1-ATG5, or co-transfected with miR-335-5p mimic + pcDNA3.1-ATG5. Asthma cell models were constructed through TGF-ß1, and animal models through ovalbumin (OVA). Monocyte-macrophage infiltration in bronchoalveolar lavage fluid (BALF) was determined by May-Grunwald-Giemsa staining, and collagen in lung tissue was assessed via Masson staining. Relationship between miR-335-5p and ATG5 was detected by dual-luciferase assay. Cell proliferation was detected by MTT assay. MiR-335-5p and ATG5 RNA expression was determined by RT-qPCR. Collagen I, collagen III, α-SMA, ATG5, LC3I/II, Beclin-1, and p62 protein expression levels in ASMCs were detected by western blot. MiR-335-5p expression was low, but ATG5 expression was high in childhood asthma. Versus OVA+ mimic NC group, the number of eosinophil and collagen in OVA+ miR-335-5p mimic group were reduced. In contrast to TGF-ß1 + mimic NC group, TGF-ß1 + miR-335-5p mimic group reduced inflammatory, airway fibrosis, and autophagy in ASMCs. ATG5 was miR-335-5p target. Overexpressing ATG5 significantly reversed the inhibitory effects of miR-335-5p on inflammatory response, fibrosis, and autophagy in ASMCs. Overall, the study concludes that MiR-335-5p alleviate inflammatory response, airway fibrosis, and autophagy in childhood asthma through targeted regulation of ATG5.

HuR-Targeted Inhibition Impairs Th2 Proinflammatory Responses in Asthmatic CD4+ T Cells.

RNA-binding protein HuR (ELAVL1) is a master regulator of gene expression in human pathophysiology. Its dysregulation plays an important role in many diseases. We hypothesized that HuR plays an important role in Th2 inflammation in asthma in both mouse and human. To address this, we used a model of airway inflammation in a T cell-specific knockout mouse model, distal lck-Cre HuRfl/fl, as well as small molecule inhibitors in human peripheral blood-derived CD4+ T cells. Peripheral CD4+ T cells were isolated from 26 healthy control subjects and 45 asthmatics (36 type 2 high and 9 non-type 2 high, determined by blood eosinophil levels and fraction of exhaled NO). Our mouse data showed conditional ablation of HuR in T cell-abrogated Th2 differentiation, cytokine production, and lung inflammation. Studies using human T cells showed that HuR protein levels in CD4+ T cells were significantly higher in asthmatics compared with healthy control subjects. The expression and secretion of Th2 cytokines were significantly higher in asthmatics compared with control subjects. AMP-activated protein kinase activator treatment reduced the expression of several cytokines in both type 2 high and non-type 2 high asthma groups. However, the effects of CMLD-2 (a HuR-specific inhibitor) were more specific to endotype-defining cytokines in type 2 high asthmatics. Taken together, these data suggest that HuR plays a permissive role in both allergen and non-allergen-driven airway inflammation by regulating key genes, and that interfering with its function may be a novel method of asthma treatment.

Pollutants enhance IgE sensitization in the gut via local alteration of vitamin D-metabolizing enzymes.

Mechanisms linking ingested pollutants to increased incidence of allergy are poorly understood. We report that mice exposed to low doses of cadmium develop higher IgE responses following oral allergen sensitization and more severe allergic symptoms upon allergen challenge. The environmentally relevant doses of this pollutant also induced oxidative/inflammatory responses in the gut of SPF, but not germ-free mice. Interestingly, the increased IgE responses correlated with stimulation of the vitamin D3-metabolizing enzymes CYP27B1 and CYP24A1 in the gut and increased luminal levels of oxidized vitamin D3 metabolites that are not ligands of the vitamin D receptor. Inhibition of CYP27B1 and CYP24A1 via oral administration of pharmacological inhibitors reduced IgE responses induced in mice orally exposed to cadmium. Our findings identify local alteration of vitamin D signaling as a new mechanism for induction of IgE responses by environmental pollutants. They also identify vitamin D3-metabolizing enzymes as therapeutic targets for the treatment of allergy.

Sphagneticola trilobata (L.) Pruski-derived kaurenoic acid prevents ovalbumin-induced asthma in mice: Effect on Th2 cytokines, STAT6/GATA-3 signaling, NFκB/Nrf2 redox sensitive pathways, and regulatory T cell phenotype markers.

ETHNOPHARMACOLOGICAL RELEVANCE: Sphagneticola trilobata (L.) Pruski is used in traditional medicine in Brazil for inflammatory diseases treatment including asthma. The diterpene kaurenoic acid (KA) is one of its active compounds, but whether KA activity could explain the traditional use of S. trilobata in asthma is unknown. AIM: Investigate KA effect and mechanisms in asthma. METHODS: Experimental asthma was induced by ovalbumin immunization and challenge in male Swiss mice. KA (0.1-10 mg/kg, gavage) was administered 1 h before the ovalbumin challenge. Total leukocytes, eosinophil, and mast cell were counted in bronchoalveolar lavage fluid (BALF), and lung histopathology was performed. Lung mRNA expression of Th2 and regulatory T cells markers, and BALF type 2 cytokine production were quantitated. NFκB activation and oxidative stress-related components in pulmonary tissue were measured. RESULTS: KA inhibited the migration of total leukocytes and eosinophils to BALF, reduced lung histopathology (inflammatory cells and mast cells), mRNA expression of IL-33/ST2, STAT6/GATA-3 and NFκB activation in the lung, and reduced IL-33, IL-4, IL-5 production in the BALF. KA also reduced the mRNA expression of iNOS and gp91phox, and superoxide anion production accompanied by the induction of Nrf2, HO-1 and NQO1 mRNA expression, thus, exerting an antioxidant effect. Finally, KA induced nTreg-like and Tr1-like, but not Th3-like markers of suppressive T cell phenotypes in the lung tissue. CONCLUSION: KA prevents antigen-induced asthma by down-regulating Th2 and NFκB/cytokine-related pathways, and up-regulating Nrf2 and regulatory T cells' markers. Thus, explaining the ethnopharmacological use of S. trilobata for the treatment of lung inflammatory diseases.

Ncf1 Governs Immune Niches in the Lung to Mediate Pulmonary Inflammation in Mice.

Neutrophil cytosolic factor 1 (Ncf1) is a major genetic factor associated with autoimmune diseases and has been identified as a key player in autoimmune mediated inflammation. We addressed the role of Ncf1 in an antigen-induced pulmonary inflammation model, and found that the Ncf1m1j mutation, causing a deficient reactive oxygen species response, alleviated disease. The Ncf1m1j mutation was associated with a reduced inflammatory cell infiltration in airways, but had limited effect on mucus secretion, antibody production and lung fibrosis. The disease remission in the Ncf1 mutated mice was reversed when functional Ncf1 was transgenically expressed in alveolar macrophages, suggesting that the cellular inflammation was depended on functional Ncf1 in alveolar macrophages. By determining cytokine and chemokine profiles in lung and serum, we found that Ncf1 deficiency allowed an increased expression of Th1 cytokines, including TNF-α, IFN-γ and IL-12. Since also epithelial cytokines were found to be regulated by Ncf1, we tested the effect of Ncf1 in IL-33 and IL-25 induced lung inflammation models. Mice with the Ncf1m1j mutation showed less sensitivity to IL-33, but not IL-25, induced lung inflammation, in a macrophage independent manner. The mice with deficient Ncf1 showed a reduced eosinophil infiltration and group 2 innate lymphoid cell (ILC2) activation. The production of IFN-γ in CD4+ T cells was increased, whereas IL-5 and IL-13 in ILC2 were decreased. Importantly, anti-IFN-γ antibody treatment of Ncf1 deficient mice increased eosinophil infiltration and rescued ILC2 activation in the lung. We conclude that Ncf1 deficiency enhances Th1 response, deactivates ILC2, and protects against pulmonitis.

Assessment of Allergen-Responsive Regulatory T Cells in Experimental Asthma Induced in Different Mouse Strains.

BACKGROUND: Regulatory T cells (Tregs) are important in regulating responses to innocuous antigens, such as allergens, by controlling the Th2 response, a mechanism that appears to be compromised in atopic asthmatic individuals. Different isogenic mouse strains also have distinct immunological responses and susceptibility to the experimental protocols used to develop lung allergic inflammation. In this work, we investigated the differences in the frequency of Treg cell subtypes among A/J, BALB/c, and C57BL/6, under normal conditions and following induction of allergic asthma with ovalbumin (OVA). METHODS: Subcutaneous sensitization followed by 4 consecutive intranasal OVA challenges induced asthma characteristic changes such as airway hyperreactivity, inflammation, and production of Th2 cytokines (IL-4, IL-13, IL-5, and IL-33) in the lungs of only A/J and BALB/c but not C57BL/6 strain and evaluated by invasive whole-body plethysmography, flow cytometry, and ELISA, respectively. RESULTS: A/J strain naturally showed a higher frequency of CD4+IL-10+ T cells in the lungs of naïve mice compared to the other strains, accompanied by higher frequencies of CD4+IL-4+ T cells. C57BL/6 mice did not develop lung inflammation and presented higher frequency of CD4+CD25+Foxp3+ Treg cells in the bronchoalveolar lavage fluid (BALF) after the allergen challenge. In in vitro settings, allergen-specific stimulation of mediastinal LN (mLN) cells from OVA-challenged animals induced higher frequency of CD4+IL-10+ Treg cells from A/J strain and CD4+CD25+Foxp3+ from C57BL/6. CONCLUSIONS: The observed differences in the frequencies of Treg cell subtypes associated with the susceptibility of the animals to experimental asthma suggest that CD4+CD25+Foxp3+ and IL-10-producing CD4+ Treg cells may play different roles in asthma control. Similar to asthmatic individuals, the lack of an efficient regulatory response and susceptibility to the development of experimental asthma in A/J mice further suggests that this strain could be preferably chosen in experimental models of allergic asthma.

Pattern-Recognition Receptor Agonist-Containing Immunopotentiator CVC1302 Boosts High-Affinity Long-Lasting Humoral Immunity.

Ideally, a vaccine should provide life-long protection following a single administered dose. In our previous study, the immunopotentiator CVC1302, which contains pattern- recognition receptor (PRR) agonists, was demonstrated to prolong the lifetime of the humoral immune response induced by killed foot-and-mouth disease virus (FMDV) vaccine. To elucidate the mechanism by which CVC1302 induces long-term humoral immunity, we used 4-hydroxy-3-nitrophenylacetyl (NP)-OVA as a pattern antigen and administered it to mice along with CVC1302, emulsified together with Marcol 52 mineral oil (NP-CVC1302). From the results of NP-specific antibody levels, we found that CVC1302 could induce not only higher levels of NP-specific antibodies but also high-affinity NP-specific antibody levels. To detect the resulting NP-specific immune cells, samples were taken from the injection sites, draining lymph nodes (LNs), and bone marrow of mice injected with NP-CVC1302. The results of these experiments show that, compared with mice injected with NP alone, those injected with NP-CVC1302 had higher percentages of NP+ antigen-presenting cells (APCs) at the injection sites and draining LNs, higher percentages of follicular helper T cells (TFH), germinal center (GC) B cells, and NP+ plasma-blasts in the draining LNs, as well as higher percentages of NP+ long-lived plasma cells (LLPCs) in the bone marrow. Additionally, we observed that the inclusion of CVC1302 in the immunization prolonged the lifetime of LLPCs in the bone marrow by improving the transcription expression of anti-apoptotic transcription factors such as Mcl-1, Bcl-2, BAFF, BCMA, Bax, and IRF-4. This research provides a blueprint for designing new generations of immunopotentiators.