The airway epithelial barrier is the primary defense line of the lungs against allergens, such as house dust mite (HDM). A disrupted airway epithelial barrier plays a major role in asthma development. Short-chain fatty acids (SCFAs) are bacterial fermentation products that are associated with prevention against allergic inflammation. In this study, we aim to elucidate the direct preventative effects of SCFAs, particularly butyrate, on the HDM-induced bronchial epithelial cell (BEC) activation and barrier disruption in-vitro. Immortalized human 16HBE cells were exposed to HDM to simulate allergen-induced BEC inflammation. Pre-exposure of 1-1000 µM acetate, propionate or butyrate were studied for their ability to prevent epithelial activation. Histone deacetylase activity of cells was measured after pre-incubation with 100 or 200 µM butyrate and combined butyrate and HDM exposure. In a transwell, air-liquid interface (ALI)-culture, the effects of 100 µM butyrate on barrier integrity (transepithelial resistance (TEER) and 4 kDa FITC-dextran permeability) and inflammation were assessed in presence and absence of HDM. To gain broader insight in the effects of butyrate, RNA sequencing (RNAseq) was performed using 16HBE cells exposed to HDM, butyrate or both. Pre-incubation with butyrate protected against the production of HDM-induced IL-8 (1-100 µM), CCL5 (1-100-1000 µM) and CCL20 (1-10-100 µM) by solid phase cultured 16HBE-cells, although 1000 µM butyrate increased IL-8 and CCL20 release. Pre-incubation with acetate and propionate showed similar effects. 100 µM butyrate did not suppress HDAC activity, while a dose of 200 µM butyrate did. In ALI-culture, HDM increased barrier permeability, and pre-incubation with 100 µM butyrate tended to prevent this. Additionally, HDM-induced CCL20 production was significantly decreased by butyrate. RNAseq revealed 100 µM butyrate to significantly affect biological processes of the cells linked to gene expression, mitochondrial function, cell division and cell signaling. These effects were observed both in absence and presence of HDM-exposure. Butyrate, at physiologically relevant doses, modulates gene expression of 16HBE cells, affecting amongst others post-transcriptional and mitochondrial pathways. In ALI-cultures, butyrate prevented epithelial activation (CCL20) and tended to prevent HDM-induced barrier defects. These findings highlight the relevance of SCFAs such as butyrate in maintaining bronchial epithelial cell homeostasis relevant for prevention and control of allergic airway disease.

Background:Immune fitness refers to the body's capacity to respond to health challenges, such as infections, by activating an appropriate immune response. The aim of the current study was to investigate the relationship between oral and gut microbiota community structure and immune fitness scores. Methods:Stool and saliva samples were collected to assess compositions of both oral and gut microbiota. Immune fitness was assessed with a single-item scale ranging from 0 (very poor) to 10 (excellent). Additionally, saliva samples were analyzed to measure the concentrations (pg/ml) of pro-inflammatory biomarkers interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α). Spearman's correlations were computed between microbiota abundance, immune fitness, and salivary biomarker levels. Bootstrapping was used to adjust for the relatively small sample size in the correlation analysis. Results:A total of 29 healthy participants (15 males; 14 females) enrolled in the study, with a mean age of 21.1 years old. Analysis of the salivary microbiota revealed significant negative correlations between self-reported immune fitness scores and the relative abundances of putative oral proinflammatory genera Selenomonas (r = -0.610), and Lachnospiraceae uncultured (r = -0.501). In the fecal microbiota, immune fitness scores showed a significant positive correlation with the relative abundance of putative beneficial butyrate-producing genus Lachnoclostridium (r = 0.513), and significant negative correlations with commensal gut bacterial genera Colidextribacter (r = -0.582) and Lachnospiraceae FCS020 group (r = -0.504). Conclusion:Self-reported immune fitness is associated with the oral and gut microbiota community. The findings demonstrate the importance of microbiota in immune function and support the use of self-assessment scales to evaluate immune fitness.

In this study, we analysed differences in the infant gut microbiome between breastfed and formula-fed infants using novel machine learning techniques. Breast milk, rich in bioactive agents, supports microbiota composition and immune development, while formulas aim to replicate its nutritional profile. We applied a methodology combining the DADA2 pipeline for 16S rRNA sequencing with the Recursive Ensemble Feature Selection (REFS) algorithm for biomarker discovery. We analysed three publicly available 16S rRNA datasets: PRJNA633365 (70 stool samples from China), PRJDB7295 (40 stool samples from the Philippines), and PRJNA562650 (40 stool samples from China). The discovery dataset (PRJNA633365) revealed 16 significant taxa out of 1,227, validated across the other two datasets. Next, we compared REFS performance with another feature selection algorithm, SelectKBest. Finally, we conducted a literature review to explore links between identified taxa and medical conditions. Additionally, we used MicrobiomeAnalyst to examine associations with diseases, diet, and lifestyle. Our results show differences in the bacterial composition between breastfed and formula-fed infants, and these findings were validated in two independent datasets. Future research should explore the functional roles of these taxa and consider regional and dietary variability to enhance understanding of microbiome dynamics and long-term health outcomes.

IntroductionHuman milk oligosaccharides (HMOs) are structurally diverse carbohydrates found in high concentrations in human milk, supporting infant immune development and gut microbiota colonization. Their composition is influenced by maternal Secretor (Se) status, determined by the FUT2 gene. While immunological HMO effects are increasingly recognized, the influence of an infant’s own Se status on immune responses remains understudied. This study explores how peripheral blood mononuclear cells (PBMCs) from Se-positive (Se+) and Se-negative (Se−) individuals respond to Se+ and Se− HMOs under bacterial and viral stimulation.MethodsPBMCs from 14 healthy adult donors, classified based on FUT2 expression, were exposed to 0.1% pooled Se+ or Se− HMOs, individual HMOs (2′-fucosyllactose and 3-fucosyllactose), and immune triggers (αCD3/CD28, LPS, and/or Poly I:C) for 24–48 h. Cytokine secretion (IFNγ, IL10, IL13, TNFα) was measured.ResultsBasal cytokine secretion did not differ between Se+ and Se− PBMCs. Upon stimulation, Se+ PBMCs secreted more IL10, particularly in response to Se+ or Se− HMOs. Individual HMOs did not replicate effects seen with pooled mixtures, highlighting the importance of HMO complexity. Under LPS stimulation, TNFα secretion was significantly reduced only with genotype-matched HMOs, suggesting Secretor-specific immune modulation.ConclusionThis is the first study showing that PBMC cytokine responses are shaped mainly by host Secretor status than HMO composition. Both genotype and HMO profile influence immune reactivity and should be considered in HMO research and personalized infant nutrition strategies.

Background/Objectives: This study evaluated the immunomodulatory effects of multiple A. bisporus strains, applying both in vitro and in vivo murine models and aiming to identify specific strains with immune modulatory properties. Methods: Sixteen A. bisporus strains were screened in a dendritic cell (DC)-CD4+ T cell co-culture system, using cells from female C57BL/6 mice, for their capacity to induce Th1- and Th17-associated cytokine production. Based on these cytokine profiles, three potent strains (MES01856, MES01706, MES01637), one non-responder strain (MES01515), and the commercially available A15 strain were selected for investigation in a murine model, an ovalbumin (OVA)-induced allergic food allergy. In these sensitized female C3H/HeOuJ mice, ear swelling, anaphylactic shock scores, OVA-specific IgE, and serum mouse mast cell protease-1 (mMCP-1) were measured following an allergen challenge. Additionally, IL-6, IL-10, and TNF-α secretion from bone marrow-derived dendritic cells (BMDCs) was measured after stimulation with these A. bisporus fresh ground extracts (FGEs). Results: In vitro, increases in cytokine production were obtained, with MES01856, MES01706, and MES01637 identified as the most potent inducers. In vivo, OVA sensitization elicited significant ear swelling and elevated shock scores in the mice. Dietary supplementation with MES01706 or MES01515 significantly reduced ear swelling, while the other strains resulted in non-significant changes. No significant changes were observed in shock scores, OVA-specific IgE, or mMCP-1. Conclusions: Multiple different A. bisporus strains elicit specific immunomodulating effects both in vitro and in in vivo murine models, identifying the MES01706 strain as one with the highest potential to beneficially affect immune function.

Placental inflammation and dysfunction play critical roles in adverse pregnancy outcomes, including preeclampsia and intrauterine growth restriction. Exposure to environmental factors, such as dietary toxins, infectious agents, and pollution, is thought to increase the risk of these outcomes. Despite the widespread use of HTR-8/SVneo and BeWo cells as trophoblast models, their comparative sensitivity to maternal exposome-related stimuli has not been systematically evaluated within the same experimental framework. we examined the responses of HTR-8/SVneo and BeWo trophoblast cell lines to mycotoxin deoxynivalenol (DON), an inflammatory cytokine cocktail (CC; TNF-α, IL-1β, IFN-γ), and particulate matter (PM2.5). Cells were seeded with either 0.2% or 1% penicillin/streptomycin (P/S) and exposed for 24 h. Cytokine secretion (IL-6 and IL-8), human chorionic gonadotropin (hCG) production, and mRNA and protein expression of junctional markers (ZO-1, OCLD, CLDN-3, CLDN-4, E-CAD) were analysed. DON significantly increased IL-6 secretion in both cell lines and elevated IL-8 levels in HTR-8/SVneo cells only. DON suppressed hCG production in both cell lines. CC exposure markedly elevated IL-6 and IL-8 levels, particularly in HTR-8/SVneo cells, without affecting hCG level. PM exposure did not significantly alter IL-6, IL-8, or hCG levels in either cell line. DON and CC altered the mRNA and protein expression of junctional markers in BeWo cells(ZO-1, OCLD, CLDN-3,CLDN-4), whereas HTR-8/SVneo cells showed more limited changes. Most cellular responses were consistent across both P/S concentrations. HTR-8/SVneo and BeWo cells exhibit differential sensitivity to DON, CC, and PM2.5, underscoring the importance of cell model selection in in vitro placental toxicology research. These results provide a comparative framework for interpreting trophoblast responses to maternal exposome-related stimuli and highlight the need for functional validation in future studies.

The gut microbiome is believed to play an important role in the development and onset of Parkinson’s disease (PD). While human studies report differences in gut microbiota between PD individuals and healthy controls, it is unclear whether preclinical animal models show similar patterns. We performed a systematic review and Bayesian regularised meta-analysis of preclinical PD studies that assessed both motor function and gut microbiota. Motor deficits were consistently observed across models, but gut bacterial diversity (α-diversity) and changes in key taxa (e.g. Akkermansia, Lactobacillus, Bifidobacterium) were inconsistent and poorly aligned with human data. In contrast, short-chain fatty acids (SCFAs) showed more reproducible changes and greater translatability to human findings. Chronic toxin-based models demonstrated the highest reproducibility. Overall, gut microbiota composition in animal PD models lacks consistency and human relevance, whereas SCFAs may offer a more reliable outcome. Finally, our study makes possible recommendations for reporting to improve future studies.

House dust mite (HDM) is frequently used in animal models of allergic airway diseases. HDM extracts differ in allergen concentration and endotoxin contamination, requiring model optimalization for each batch. This study aimed to refine and validate our murine model of HDM-driven acute allergic airway inflammation with a new batch of HDM extract and comparing different administration doses. We evaluated three HDM extracts, HDM1 (old batch), HDM2 and HDM3, containing varying Der p1 concentrations (33.7, 71.4 and 9.0 mg/g protein) and endotoxin levels (94.8, 53.9 and 24.7 EU/g protein). Male BALB/c mice were intranasally sensitized (day 0) with 1 µg (HDMlow) or 5 µg (HDMhigh), and challenged (day 7–11) with 10 µg (HDMlow) or 15 µg (HDMhigh) of the same HDM extract. Bronchoalveolar lavage fluid (BALF), lung tissue and serum were collected on day 14. High doses of all extracts significantly increased BALF total cell influx, including eosinophils and lymphocytes, and elevated lung Th2 and CD69 + Th2 cell frequencies. Eosinophils and lymphocytes induction varied across extracts (6–17 × 104 and 3–8 × 104 cells respectively). HDM3high tended (p < 0.1) to increase serum HDM-specific IgE. Upon ex-vivo HDM-restimulation of lung cells comparing HDM3 high with HDM2 high, only cells of HDM3high-treated mice showed increased IL-5 and IL-13 production. Overall, the high dose protocol most effectively induced HDM-driven acute allergic airway inflammation, based on BALF eosinophil and lymphocyte influx. Even though having low Der p1 and endotoxin levels, only HDM3high enhanced cytokine production upon ex-vivo HDM-restimulation, indicating the value of this functional assay to discriminate between HDM batches. House dust mite (HDM) extracts are widely used to induce allergic airway inflammation in mice, but differences in allergen and endotoxin content of these extracts might affect model outcomes. This study demonstrates that variations in HDM extract composition and dosing could lead to distinct inflammatory and immunological responses in mice. These findings highlight the importance of characterizing and optimizing each HDM extract batch to ensure reproducibility and comparability across experimental allergy studies and prevent the use of large groups of animals in suboptimal models.

The accelerating shift in biomedical research toward human-relevant, ethical, and innovative methodologies has propelled the adoption of Non-animal models (NAMs) as vital alternatives to traditional animal models. This review examines the different types of NAMs, including in vitro models such as 2D cell cultures, organoids, spheroids, organs-on-a-chip, and advanced in silico tools such as artificial intelligence (AI)-driven models, molecular docking, Physiologically Based Pharmacokinetic (PBPK) models, and Quantitative Structure-Activity Relationship (QSAR) approaches, in the context of their capacity to replace, reduce, and refine animal use. Drawing on historical and contemporary applications, we analyze the experimental utility, translational value, and limitations of each NAM against the benchmark of well-established animal models, emphasizing the ability of NAMs to capture human physiology, enhance throughput, and address ethical imperatives. Despite significant progress, NAMs face challenges in recapitulating systemic complexity and regulatory standardization. The review identifies current technological gaps and articulates future directions, with particular emphasis on multi-organ platforms, computational innovations, and data harmonization. Ultimately, continual advances in NAMs, coupled with evolving regulatory frameworks and ethical considerations, are redefining the standards and trajectory of preclinical biomedical research, paving the way toward safer, more predictive, and humane scientific practices.

Co-trimoxazole (CTX), a combination of sulfamethoxazole (SMZ) and trimethoprim (TMP), is used during pregnancies complicated by infections like Urinary Tract Infections (UTI) and Human Immunodeficiency Virus (HIV), despite potential fetal safety concerns. This study examines CTX’s effects on the placenta using both in vivo and in vitro models. Pregnant mice received CTX for 4 days during both early and late gestation. On gestational day 19, tissues were collected for analysis. Furthermore, in vitro, human BeWo placental cells were exposed to non-cytotoxic concentrations of CTX for 24 h. Gene expression was analyzed by bulk RNA sequencing with pathway analysis, and placental barrier function was assessed by measuring transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FITC-D) permeability. In vivo, CTX significantly reduced uterine weight and litter size (p < 0.05), indicating potential reproductive toxicity, which was supported by placental transcriptomic analysis. In vitro, bulk RNA-sequencing of CTX-treated BeWo cells exhibited differential expression of genes related to tight and adherens junctions, indicating impaired placental barrier integrity. This was accompanied by a significant reduction in TEER (p < 0.0001) and an increase in FITC-D permeability (p < 0.001). In addition, CTX activated the ERK1/2 (MAPK3) pathway, as indicated by increased ERK1/2 (p < 0.01) phosphorylation and downregulation of the ERK pathway negative regulators (DUSP genes). Additionally, IL-6 expression levels were reduced in the amniotic fluid (p < 0.01) and placenta of CTX-treated mice, and a similar reduction was observed in CTX-treated BeWo cells at both the transcript (p < 0.05) and protein (p < 0.001) levels. In conclusion, CTX may induce reproductive toxicity and compromise placental barrier integrity, warranting further investigation into its safety during pregnancy.

BACKGROUND:Early oral immunotherapy for food allergy (<3 years of age; eOIT) shows promise for inducing sustained unresponsiveness. However, concerns about its feasibility remain. Parental experiences with eOIT have not yet been studied, but understanding these is crucial for improving its clinical implications. OBJECTIVE:Assessing the feasibility of eOIT from a parental perspective. METHODS:This sequential explanatory mixed-methods study enrolled parents of children from the ORKA study, a prospective intervention study on eOIT for various allergens. Parents completed a baseline anxiety questionnaire, monthly feasibility questionnaires, and daily adherence diaries. After treatment, focus groups were conducted and analyzed using interpretative phenomenological analysis. RESULTS:Parents of 124 children with 189 treated food allergies were included. They reported little anxiety (mean STAI score 32.6 on a scale of 20-80) and high confidence in managing eOIT. Overall, feasibility was assessed positively (mean feasibility score 16.1-23.6 on a scale of 12-60). Adherence was between 96.7% (dose escalation) and 94.7% (maintenance dosing). The participant dropout rate was 9.7%. Focus groups with 10 participants revealed 5 themes: driven by hope, the hidden burden, navigating obstacles, guiding hands, and gratitude in the journey. Parents were motivated by the hope of tolerance development and desire to actively manage the allergy. However, eOIT was emotionally burdensome, especially during initiation and in maintaining adherence. Practical challenges included dose administration. Professional support and expectation management were perceived as essential. Regardless of clinical outcomes, parents viewed the effort as worthwhile. CONCLUSIONS:eOIT is feasible from a parental perspective, though emotionally and practically demanding. A family-centered approach with adequate education, practical guidance, and psychosocial support is recommended.

Post-acute sequelae of SARS-CoV-2 infection (PASC), also referred to as Long COVID, affects millions worldwide and is characterized by persistent fatigue, reduced immune fitness, and mood disturbances. The aim of the current study was to identify if immune fitness, mood, fatigue, and quality of life prior to SARS-CoV-2 infection could predict PASC fatigue severity. A retrospective cross-sectional survey was conducted among 299 Dutch PASC patients. Participants completed validated measures of immune fitness, fatigue (assessed with both the Fatigue Severity Scale and a single-item scale), mood (including stress, anxiety, depression, hostility, loneliness, and happiness) and quality of life for the three months prior to SARS-CoV-2 infection. The same assessments were made for the month before survey completion (i.e., during PASC). Correlational and regression analyses were conducted to identify possible predictors of PASC fatigue severity. Participants were predominantly female (90%): mean age 44.1 (SD 11.2) years. Both assessments of PASC fatigue did not correlate significantly with the prior SARS-CoV-2 assessments of immune fitness, fatigue, mood, and quality of life. The regression analyses revealed no significant predictors for PASC fatigue severity. In conclusion, immune fitness, fatigue, mood and quality of life prior to SARS-CoV-2 infection were not identified as independent predictors of PASC fatigue severity.

The airway epithelium forms the frontline interface between the external environment and the respiratory system and is constantly exposed to microbes and their constituents. Epidemiological and preclinical evidence increasingly highlights an important role for microbial factors in shaping long-term respiratory health by maintaining immune homeostasis and modulating exacerbations in chronic inflammatory diseases. Here, we argue that these durable effects are likely due to "imprinting" events at the epithelial interface. Emerging evidence indicates that microbes can functionally imprint the airway epithelium through metabolic and epigenetic reprogramming, thereby shaping subsequent responses to microbial and inflammatory stimuli. We propose a conceptual framework of epithelial imprinting comprised of four categories: differentiation, tolerance, priming and trained immunity. This framework provides an important foundation for the mechanistic dissection of epithelial memory in the airways and highlights novel therapeutic opportunities to harness microbial factors to modulate respiratory health.

Integrative systems microbiology increasingly relies on algorithmic approaches capable of extracting biologically meaningful patterns from heterogeneous and often high dimensional, low-sample-size (HDLSS) biological datasets. A major obstacle in this setting is the instability of inferred molecular signatures across cohorts, tissues, and measurement platforms. Here, we address this problem by formulating molecular system inference as a multi-dataset integration task and by applying the Matthews Correlation Coefficient–Recursive Ensemble Feature Selection (MCC-REFS) algorithm to jointly analyze five independent transcriptomic datasets spanning peripheral blood mononuclear cells, whole blood, plasma, and post-mortem tissues. We compared MCC-REFS with three commonly used feature-selection strategies, GRACES, SelectKBest, and Deep Neural Pursuit (DNP), in order to evaluate robustness, convergence, and cross-context reproducibility. MCC-REFS consistently converged on a compact seven-gene system (PPP2CB, SOCS3, ARG1, IL6R, ECHS1, FZD2, TRGV3/5) exhibiting higher stability indices and stronger classification performance than alternative methods. Generalization was assessed using an independent multi-layer perceptron classifier across validation cohorts with differing tissue origin and sequencing technologies, demonstrating preservation of discriminative structure. To support interpretation, we integrated functional, pharmacological, and interventional knowledge from DrugBank, DGIdb, and Open Targets, enabling the mapping of inferred gene systems onto pathways, known drug targets, and ongoing clinical investigations. Taken together, this work presents an algorithmic framework for multi-dataset and multi-omics integration in systems microbiology, illustrating how stable and interpretable molecular patterns can be identified from heterogeneous data, with Long COVID serving as a representative case study.

BACKGROUND:Prebiotics promote growth of beneficial gut bacteria, whereas antibiotics can disrupt the microbial balance, reduce diversity, and potentially cause gut dysbiosis. This investigation aimed to determine whether antibiotic use before, during, or after birth altered the risk of infant allergic disease outcomes within a maternal prebiotic dietary supplementation trial. METHODS:Maternal participants were allocated to consume daily prebiotics (galacto-oligosaccharides and fructo-oligosaccharides) or placebo (maltodextrin) powder from 18 to 20 weeks' gestation until 6 months postnatal. All infants had a family history of allergic disease. Maternal and infant antibiotic use was prospectively collected monthly throughout the trial intervention period, and infant allergic disease was assessed by 1 year of age. RESULTS:Overall, 83.1% (472/568) mothers and 20.8% (115/554) infants received antibiotics during the trial intervention period. Prebiotic supplementation modified the association between maternal intrapartum antibiotic exposure and infant allergic disease. Among women not consuming prebiotics, maternal intrapartum antibiotics were associated with an increased risk of infant allergen sensitisation (adjusted relative risk [aRR] 3.56, 95% CI 1.65-7.69), IgE-mediated food allergy (aRR 5.67, 95% CI 2.04-15.73), and medically diagnosed atopic eczema (aRR 6.42, 95% CI 2.43-16.95); such associations were not evident in women consuming prebiotics. Maternal and infant antibiotics administered during other intervention phases were not significantly associated with any infant allergic disease outcomes. CONCLUSION:Our exploratory investigation findings indicate that maternal prebiotics supplementation reduces the risk of infant allergen sensitisation, food allergy and atopic eczema associated with intrapartum antibiotic use. Future allergy prevention trials could further optimise maternal prebiotic interventions during the intrapartum period.

Prebiotic dietary supplementation has been shown to improve glucose homeostasis in type 2 diabetes patients. The aim of this analysis was to determine whether pre-pregnancy body mass index (BMI) modifies the effect of prebiotic supplementation from mid-pregnancy on reducing the risk of gestational diabetes mellitus (GDM). In a double-blinded, randomised controlled trial, pregnant women < 21 gestational weeks were randomly assigned (1:1) to consume daily prebiotics (14.2 g galacto-oligosaccharides and fructo-oligosaccharides) or placebo (8.7 g maltodextrin) powder. An effect modification analysis was performed to assess the heterogeneity of the effect of prebiotic supplementation in relation to pre-pregnancy BMI on GDM diagnosis. Between June 2016 and November 2021, 329 women were assigned to the prebiotic group (50.4

IntroductionA balanced microbiome is crucial for local and systemic immune regulation. Dietary fibers can support the intestinal microbiome, protecting the host from allergic diseases, including asthma. The effects of fibers depend on their type, dose, and disease context. Here, we investigated the preventative effects of four doses of fructooligosaccharides (FOS) in a murine model for house dust mite (HDM)-induced allergic asthma.MethodsBALB/c mice received a diet containing 1%, 2.5%, 5%, or 10% FOS (w/w) both prior to and during sensitization and challenges with HDM. Bronchoalveolar lavage fluid (BALF), lung tissue, serum, and cecum content were collected at the endpoint. Fecal microbiome composition was analyzed, and levels of short-chain fatty acids (SCFAs) were measured in cecum content, serum, and lung samples.ResultsHDM-allergic mice showed eosinophilic airway inflammation and increased pulmonary type 2 inflammation, while cecal SCFA levels were lower compared to sham mice. Serum acetate concentrations showed a similar decline (p = 0.092). The 10% FOS diet did not prevent allergic sensitization or eosinophilic airway inflammation; however, it significantly reduced the proportions of T helper 2 (Th2) cells and the Th2/Th1 ratio in the lungs, decreased concentrations of chemokine (C-C motif) ligand 2 (CCL22) and interleukin (IL-13) in the BALF, and inhibited IL-13 production upon ex vivo HDM restimulation of lung cells. The 2.5% and 5% FOS diets also decreased Th2 cell frequency in the lungs. High doses of FOS increased the abundance of fecal Prevotellaceae, while reducing fecal Oscillospiraceae and Lactobacillaceae. These microbial shifts were correlated with protective effects against type 2 inflammation. In HDM-allergic mice, fecal Prevotellaceae abundance correlated positively with serum acetate concentrations, which were correlated with type protective effects. In allergic mice, the 2.5% and 5% FOS doses were associated with increased abundance of fecal Muribaculaceae and Bacteroidaceae, respectively, along with elevated cecal SCFA concentrations. In addition, the 5% FOS dose increased the relative abundance of fecal Lachnospiraceae, which correlated negatively with serum acetate levels and type prevention.DiscussionDietary FOS modulated the gut microbiome and attenuated pulmonary type 2 immune responses in a dose-dependent manner. These findings underscore the importance of fiber dosing for precision nutrition strategies in allergy management.

Peanut allergy represents a major food-allergy burden, raising concerns about food processing and novel dietary products. Current diagnostics assess primarily allergic endpoints rather than immune mechanisms initiating and maintaining type 2 inflammation, particularly DC2-mediated Th2 polarization. Here, an in vitro autologous monocyte-derived dendritic cell (moDC)-T cell and B cell assay has been established to study immunomodulatory effects induced by unprocessed (P-D) and processed (P-DH (heated) or P-DHG (heated and glycated)) peanut proteins and emerging foods (protein concentrates or whole biomass), related to ex vivo DC2-T cell reactions. CD14 + monocytes, CD4 + T cells and CD19 + B cells were isolated from six peanut-allergic patients’ PBMCs. MoDCs generated with IL4/GM-CSF were exposed (48h) to type 2 polarizing cytokine (DC2) mix, or DC2 mix combined with the food samples. Next, DC2s were co-cultured with T cells (5d), followed by B cells incubation with DC2/T cell supernatant and food samples (10d). Supernatants and cells were analyzed for Th1/Th2/Th-regulatory (Treg) cells, IgE and IgG profiles. DC2 induced a strong Th2 phenotype and activity, P-D DC2 further enhanced IL13 secretion and %Tregs. P-DH DC2 favored Th2, whereas P-DHG DC2 increased IFN γ , with neither increasing %Treg. All increased CD40L + CD25 + memory Th2 cells. Wheat, whey and seaweed biomass had little effect, whereas algae DC2 showed distinct immunomodulatory, adjuvant-like activity. In conclusion, this autologous in vitro assay captures peanut-specific and generic Th2 responses and reactivity to food samples, supporting its use as additional tool to assess type 2-driving potential and allergenicity of emerging foods and processing methods in peanut-allergic patients. The current in vitro study was conducted in accordance with the Declaration of Helsinki and approved by the Medical Ethics Review Committee (METC) of the Erasmus MC (NL79534.078.21 MEC-2021-0905) and registrated at International Clinical Trials Registry Platform (NL-OMON51765). Immune cell illustrations were adapted (1, 2) with permission; permission conveyed through Copyright Clearance Center, Inc. The autologous moDC-T cell-B cell in vitro assay may be used to assess whether processing methods or new foods might have intrinsic capacity to affect type 2 inflammation in peanut-allergic patients.