Iron is essential for oxygen transport, energy metabolism, and immune regulation. Yet iron deficiency is the most common micronutrient disorder across all age groups, affecting nearly one quarter of the global population. Iron deficiency triggers nutritional immunity, a host defense mechanism that withholds and redistributes iron, contributing to increased morbidity and mortality. This review outlines normal iron physiology, distribution and absorption pathways and on the consequences of deficiency across body compartments, with particular attention to type 2-driven diseases. Beyond anemia, insufficient iron availability disrupts immune homeostasis by promoting type 2 inflammation, elevating IgE, and activating mast cells and eosinophils. Regulatory macrophages, the central hub of iron cycling, adopt an inflammatory, iron-sequestering state that reinforces malabsorption and redistribution. Epidemiology studies show higher iron-deficiency risk in allergic individuals; low maternal iron or early-life iron predisposes to eczema, wheeze, and asthma, while food-allergen elimination (notably cow's milk) further worsens anemia risk. Clinical evidence indicates that restoring iron status through diet, supplementation, or fortification lowers IgE levels, improves lung function, and alleviates symptoms of rhinitis, urticaria, and asthma. Iron may therefore represent a modifiable determinant of allergic disease development and severity. Integrating iron assessment and nutritional care into allergy management may reduce disease burden and slow the progression of allergic march.

Human bronchial organoids represent a highly advanced 3D cell culture model system that reflects complex features of the airway epithelium, including structure, developmental aspects, tissue-specific functions and heterogeneous cell-cell interactions. Thus, they serve as an ideal model to study physiological differentiation and activation processes of the bronchial epithelial cells, as well as pathophysiological mechanisms of lower airway diseases. We have established, refined and validated a series of protocols for the generation, perpetuation and characterization of human bronchial lung organoids based on somatic cells derived from surgical lung tissue samples as well as from primary bronchial epithelial cells which may be obtained from healthy and diseased donors. Such organoids are cultured in an extracellular matrix gel with a serum-free medium containing a precisely adjusted growth factor cocktail. This maintains the balance between the self-renewal and differentiation capacities of the local progenitor cells, allowing the long-term culture of organoids if specific splitting and dilution as well as freezing/thawing procedures are carried out regularly and appropriately. Here we provide these detailed protocols to enable researchers to apply the organoid technology and to generate highly comparable and complementary data. Furthermore, we present several examples for the detailed characterization and analysis of human bronchial organoids, such as gene-level expression analysis, covering single cell RNA sequencing, as well as imaging and metabolic activity-based assays.

Microglia, the brain's resident immune cells, constantly monitor their environment for signs of tissue damage or pathogens. Upon activation by stimuli like lipopolysaccharide (LPS), microglia undergo metabolic changes and release pro-inflammatory mediators. However, variations between human and rodent microglia, as well as differences between in vitro and in vivo conditions, likely influence microglial cellular functions and their responses to stimulation. In the present study, we compared several rodent and human model systems, including cell lines, primary cultures, induced pluripotent stem cell (iPSC)-derived cultures, and acutely isolated microglia, and revealed striking differences in LPS-induced metabolic changes and nitric oxide (NO) production. Using the murine microglial cell line BV-2, we demonstrated that NO was critical for restricting metabolism to glycolysis by blocking oxidative phosphorylation. In contrast, human iPSC-derived microglia and acutely isolated microglia from intraperitoneally injected rats maintained mitochondrial respiration upon LPS activation and did not show significant NO production and inducible nitric oxide synthase (iNOS) expression, respectively. Furthermore, we found that NO was not required for the increase in glycolysis rate or the release of pro-inflammatory cytokines upon LPS stimulation. Our results suggest that glycolysis is essential for microglial activation and cytokine production irrespective of NO production. However, the specific metabolic pathways involved may differ between species and experimental conditions. Understanding these differences is crucial for developing effective therapeutic strategies targeting microglial dysfunction in neurological diseases.

OBJECTIVE:Augmented mucosal expression of the transcription factor GATA3 has been implicated in the pathogenesis of ulcerative colitis (UC). Here, we evaluated the efficacy and safety of SB012, an enema formulation of the DNAzyme hgd40 that specifically inactivates GATA3 messenger RNA, for induction therapy in patients with active UC. DESIGN:In this randomized, double-blind, placebo-controlled, multicenter, phase 2a study, patients with moderately-to-severely active UC were randomized to either receive SB012 enema (225 mg hgd40) or placebo once daily for 4 weeks. The primary endpoint was change in the Total Mayo Score at week 4 compared to baseline values in the SB012 versus placebo group. RESULTS:Patients were randomized 2:1 to the SB012 (n = 13) or placebo (n = 7) group. The treatment difference between the SB012 and placebo group was not statistically significant at week 4 (P = .286). In patients not treated with glucocorticoids, the Total Mayo score in the SB012 group improved on day 28 by -2.2 (P = .027; 95%CI: -4.1 to -0.3) compared to placebo, whereas no improvement was seen in patients using corticosteroids. Further, the median Total Mayo Score in the SB012 group dropped significantly from 9.0 (Q1-Q3 6.5-10) at baseline to 7.0 (4.0-8.5) at week 4 (P = .004), while there were no significant changes in the placebo group. Endoscopic improvement was reached by 17% (2/12) and 57% (4/7) in the SB012 and 17% (1/6) and 50% (3/6) in the placebo group at weeks 4 (P = 1.0) and 8 (P = 1.0), respectively. SB012 application was well tolerated. CONCLUSION:Overall, topical application of the GATA3-specific DNAzyme formulation SB012 was well tolerated, but did not reach the defined primary endpoint of treatment difference at week 4 between the SB012 and placebo group in active moderate-to-severe UC patients, unless confounding by glucocorticoids was taken into account.

Background High-grade serous ovarian cancer (HGSOC) is commonly accompanied by malignant ascites, a clinically relevant tumor niche that promotes immune evasion, metastasis, and treatment resistance. Although natural killer (NK)-cell dysfunction has been described in ovarian cancer, the broader innate lymphoid landscape of ascites and the mechanisms linking ascites-derived signals to innate immune suppression remain insufficiently resolved.Methods We performed single-cell RNA sequencing of NK/innate lymphoid cells from ovarian cancer ascites to define cellular heterogeneity and differentiation states. Functional assays assessed NK-cell cytotoxicity, degranulation, and receptor expression following exposure to patient-derived ascites, with or without transforming growth factor-β (TGF-β) receptor inhibition. Proteomic profiling was used to characterize the soluble ascites milieu, and clinical associations were examined for innate lymphoid subsets.Results Single-cell analysis identified eight transcriptionally distinct NK/innate lymphoid states, including cytotoxic, precursor, early-like, tolerant/immunoregulatory, regulatory, proinflammatory, and innate lymphoid populations. Ovarian cancer ascites was characterized by depletion of cytotoxic and precursor NK-cell states together with enrichment of early-like, tolerant, regulatory, pro-inflammatory, and innate lymphoid cell (ILC) populations. Trajectory analysis indicated impaired maturation toward terminally differentiated cytotoxic NK cells. Notably, ascites contained an expanded population of programmed cell death protein 1 (PD-1)+ ILC2s, which were more abundant in patients with shorter progression-free survival. In functional assays, short-term exposure of healthy donor NK cells to ascites suppressed degranulation and tumor-cell killing, reduced expression of activating receptors including NKp30 and DNAM-1, increased inhibitory receptor expression, and shifted NK cells toward a CD56highCD16low phenotype. Proteomic profiling supported a soluble milieu consistent with type 2 immune skewing and NK-cell suppression. Importantly, TGF-β receptor inhibition partially restored NK-cell activation and function in the presence of ascites.Conclusions HGSOC ascites establishes a type 2-skewed immunoregulatory niche that coordinately drives NK cell dysfunction and PD-1+ ILC2 accumulation. The findings identify TGF-β-linked suppression and ascites-associated immune regulators as candidate immunotherapeutic vulnerabilities for restoring antitumor immunity in ovarian cancer.

Influenza A virus (IAV)-induced exacerbations are a major contributor to morbidity in chronic obstructive pulmonary disease (COPD), yet the epithelial mechanisms that govern these events remain unknown. We profiled the response to IAV infection of differentiated airway epithelial cells from healthy donors and individuals with COPD at single-cell resolution. The analysis revealed infection-driven shifts across multiple epithelial compartments and distinct alterations in cell-cell communication in COPD, associated with an increased CXCL11 expression. Functional assays demonstrated that CXCL11 augments mucus-associated gene and protein expression, particularly MUC5AC, increases mucus secretion and viscosity and is associated with reduction of virus-related immune pathways. This highlights CXCL11 as a contributor to both mucus hypersecretion and impaired antiviral epithelial responses in COPD exacerbations.

BACKGROUND:Pemphigus vulgaris (PV) is a CD4+ T-cell-dependent autoantibody-mediated blistering disease associated with human leucocyte antigen (HLA) class II molecules. IgG autoantibodies against the primary autoantigen desmoglein 3 (Dsg3), a desmosomal adhesion protein on epidermal keratinocytes, cause loss of epidermal cell adhesion. OBJECTIVES:To assess the clinical applicability of an innovative nanoparticle platform for the induction of immune tolerance exploiting the natural tolerance potential of liver sinusoidal endothelial cells. An open-label first-in-human study was conducted with TPM203, a mixture of four nanoparticle-coupled immunodominant Dsg3 T-cell peptides. METHODS:The efficacy and mechanism of action of TPM203 were first tested in a humanized HLA-DRB1*0402-transgenic PV mouse model. In the clinical phase I trial, TPM203 was administered intravenously in patients with PV with no-to-moderate disease activity in single ascending and multiple doses (three doses of TPM203 two weeks apart). Primary endpoints included safety and tolerability. As a secondary endpoint, pharmacokinetics were assessed. Exploratory endpoints comprised changes in Dsg3-specific and bulk T- and B-cell frequencies, anti-Dsg3 IgG levels and autoantibody-induced keratinocyte dissociation. The trial was registered with EudraCT (2019-001727-12). RESULTS:In the PV mouse model, two administrations of TPM203 significantly reduced anti-Dsg3 IgG. On the cellular level, TPM203 led to a significant decrease in CD4+ T cells in the spleen, accompanied by increased frequencies of regulatory T (Treg) cells. In the clinical trial, the 17 patients with PV enrolled across single- and multiple-dose groups did not experience any serious or severe adverse events, or treatment-related PV worsening. Pharmacokinetics confirmed rapid TPM203 clearance from the circulation. Significant TPM203-induced modulations in bulk lymphocyte subsets included an increase in Treg cells, and reductions in T helper 17.1 and CD27+ memory B cells, when dose groups were combined for analysis. Dsg3-specific T cells were found to be significantly reduced at week 8 following single administration of TPM203. Anti-Dsg3 IgG levels trended downward in the three lower single ascending dose groups, while IgG-induced keratinocyte-dissociating capacity was significantly reduced after multiple doses. CONCLUSIONS:Administered for the first time in humans, TPM203 was shown to be a safe and well-tolerated nanoparticle-based therapeutic approach with the potential to promote tolerance induction in PV, justifying further clinical development in this and other autoimmune diseases. An author video to accompany this article is available online.

Dietary patterns are major determinants of colorectal cancer risk, yet how nutritional cues are molecularly integrated to reprogram intestinal stem cell identity and fuel tumor initiation is not well understood. Here, we demonstrate that a Western-style diet (WSD) rapidly and reversibly reprograms intestinal stem cell identity. WSD suppresses canonical Lgr5 ⁺ stem cells while enhancing epithelial proliferation and stemness through activation of alternative stem cell states in Paneth and deep crypt secretory (DCS) cells in the small and large intestine, respectively. These diet-reprogrammed cells exhibit inflammatory and genotoxic stress and yet remain proliferative, suggesting increased susceptibility to tumor-initiating mutations. Mechanistically, WSD-induced remodeling is mediated by the gut microbiota, specifically through the expansion of enterotoxigenic Bacteroides fragilis (ETBF). ETBF and its secreted toxin fragilysin suppress Lgr5 ⁺ stem cells while directly promoting multipotency of c-Kit ⁺ DCS cells via Wnt signaling. Collectively, our findings identify diet-driven gut microbial shifts as a key regulator of stem cell plasticity, linking environmental exposure to epithelial reprogramming and colorectal cancer risk.

Human milk offers the best nutrition to the infant, which is crucial for the child's proper development and health status across the lifespan. Besides providing the substances optimally supplying the baby with energy and building materials, breast milk contains several immunometabolically active components. Those include molecules fully de novo synthesized by the mother, such as human milk oligosaccharides (HMO), and substances of nonhuman origin, transferred to the infant through mother's milk, such as dietary plant polyphenols. In this review, we outline the basic biology of HMO and polyphenols and deeply characterize their effects on the development of allergic disorders on the basis of available literature reporting data from in vitro, animal, and human studies. Further, we review the abundance of HMO and polyphenols, commonly present in mother's milk, and their mutual interactions in the context of the mechanisms underlying predisposition to, or protection against, the development of allergies. Finally, we discuss the potential of HMO and polyphenols in allergy prevention and therapy.

RATIONALE & AIM: The influence of processes of a preceding or ongoing allergic airway inflammation (AAI) on mechanisms of early differentiation and activation of progenitor cells within the bronchial epithelium is still poorly investigated. Therefore, the aim of the present study was to characterize early differentiation processes of airway cell progenitors after a preceding in vivo airway inflammation ex vivo using bronchial lung organoids. METHODS: Bronchial lung organoids were generated from mouse lungs affected by a preceding house dust mite-induced AAI and healthy control mice. At different time points of development such organoids were analyzed by flow cytometry, qPCR and single cell sequencing (scSeq). RESULTS: Organoids derived from control and AAI mice differed significantly in their morphological appearance with a spherical appearance in the first and a more budding-like shaped appearance in the latter group. The development of ciliated cells was significantly suppressed in AAI mice-derived organoids based on the lower presence of cell type-specific surface markers (flow cytometry) and mRNA expression levels (PCR analyses), e.g. of genes like Tuba1a and Foxj1. These changes were paralleled by an intensified differentiation into secretory cells as confirmed by higher expression levels of Scgb1a1 in organoids derived from AAI mice. Subsequent scSeq analyses confirmed these findings and further identified several secretory/club cell populations and their specific precursors based on distinct transcriptome profiles. These clusters significantly varied in their presence between the experimental groups. Further, cells within clusters characterized by the expression of basal cell markers such as Krt5, Krt14 and Krt17 were more abundant in the control group while cell clusters identified by other epithelial cell progenitor markers such as Pdpn, Trp63 and Itga6 appeared to be more prominent under AAI conditions. Thus, overall cell cluster composition of cells derived from organoids from AAI and control mice organoids differed significantly between the two conditions. CONCLUSIONS: A preceding or ongoing AAI has a long-lasting impact on early epithelial differentiation processes and thereby initiates continuous remodelling processes within the airway epithelium under such disease conditions. Most probably, epigenetic control mechanisms are involved at the level of early epithelial progenitor cells which, however, require further detailed investigations in the future.

Recent studies reveal a critical role of tumor cell-released extracellular vesicles (EVs) in pancreatic cancer (PC) progression. However, driver genes that direct EV function, the EV-recipient cells, and their cellular response to EV uptake remain to be identified. Therefore, we studied the role of Bcl-2-associated-anthanogene 6 (BAG6), a regulator of EV biogenesis for cancer progression. We used a Cre recombinase/LoxP-based reporter system in combination with single-cell RNA sequencing to monitor in vivo EV uptake and tumor microenvironment (TME) changes in mouse models for pancreatic ductal adenocarcinoma (PDAC) in a Bag6 pro- or deficient background. In vivo data were validated using mouse and human organoids and patient samples. Our data demonstrated that Bag6-deficient subcutaneous and orthotopic PDAC tumors accelerated tumor growth dependent on EV release. Mechanistically, this was attributed to mast cell (MC) activation via EV-associated IL33. Activated MCs promoted tumor cell proliferation and altered the composition of the TME affecting fibroblast polarization and immune cell infiltration. Tumor cell proliferation and fibroblast polarization were mediated via the MC secretome containing high levels of PDGF and CD73. Patients with high BAG6 gene expression and high protein plasma level have a longer overall survival indicating clinical relevance. The current study revealed a so far unknown tumor-suppressing activity of BAG6 in PDAC. Bag6-deficiency allowed the release of EV-associated IL33 which modulate the TME via MC activation promoting aggressive tumor growth. MC depletion using imatinib diminished tumor growth providing a scientific rationale to consider imatinib for patients stratified with low BAG6 expression and high MC infiltration.

BACKGROUND:IL-17A and TNF synergistically promote inflammation and tumorigenesis. Their interplay and impact on ovarian carcinoma (OC) progression are, however, poorly understood. We addressed this question focusing on mesothelial cells, whose interaction with tumor cells is known to play a pivotal role in transcoelomic metastasis formation.METHODS:Flow-cytometry and immunohistochemistry experiments were employed to identify cellular sources of IL-17A and TNF. Changes in transcriptomes and secretomes were determined by bulk and single cell RNA sequencing as well as affinity proteomics. Functional consequences were investigated by microscopic analyses and tumor cell adhesion assays. Potential clinical implications were assessed by immunohistochemistry and survival analyses.RESULTS:We identified Th17 cells as the main population of IL-17A- and TNF producers in ascites and detected their accumulation in early omental metastases. Both IL-17A and its receptor subunit IL-17RC were associated with short survival of OC patients, pointing to a role in clinical progression. IL-17A and TNF synergistically induced the reprogramming of mesothelial cells towards a pro-inflammatory mesenchymal phenotype, concomitantly with a loss of tight junctions and an impairment of mesothelial monolayer integrity, thereby promoting cancer cell adhesion. IL-17A and TNF synergistically induced the Th17-promoting cytokines IL-6 and IL-1β as well as the Th17-attracting chemokine CCL20 in mesothelial cells, indicating a reciprocal crosstalk that potentiates the tumor-promoting role of Th17 cells in OC.CONCLUSIONS:Our findings reveal a novel function for Th17 cells in the OC microenvironment, which entails the IL-17A/TNF-mediated induction of mesothelial-mesenchymal transition, disruption of mesothelial layer integrity and consequently promotion of OC cell adhesion. These effects are potentiated by a positive feedback loop between mesothelial and Th17 cells. Together with the observed clinical associations and accumulation of Th17 cells in omental micrometastases, our observations point to a potential role in early metastases formation and thus to new therapeutic options.

Aberrant type 2 inflammatory responses are the underlying cause of the pathophysiology of allergic asthma, allergic rhinitis, and other atopic diseases, with an alarming prevalence in relevant parts of the Western world. A bulk of evidence points out the important role of the DP2 receptor in these inflammation processes. A screening of different polyunsaturated fatty acids at a fluorescence resonance energy transfer-based DP2 receptor conformation sensor expressed in human embryonic kidney (HEK) cells revealed an agonistic effect of the prostaglandin (PG)-D2 precursor arachidonic acid on DP2 receptor activity of about 80% of the effect induced by PGD2. In a combination of experiments at the conformation sensor and using a bioluminescence resonance energy transfer-based G protein activation sensor expressed together with DP2 receptor wild type in HEK cells, we found that arachidonic acid acts as a direct activator of the DP2 receptor, but not the DP1 receptor, in a concentration range considered physiologically relevant. Pharmacological inhibition of cyclooxygenases and lipoxygenases as well as cytochrome P450 did not lead to a diminished arachidonic acid response on the DP2 receptor, confirming a direct action of arachidonic acid on the receptor.

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Impaired interaction of fibroblasts with pneumocytes contributes to the progression of chronic lung disease such as idiopathic pulmonary fibrosis (IPF). Mucin 5B (MUC5B) is associated with IPF. Here we analyzed the interaction of primary fibroblasts and alveolar type 2 (AT2) pneumocytes in the organoid model. Single-cell analysis, histology, and qRT-PCR revealed that fibroblasts expressing high levels of fibrosis markers regulate STAT3 signaling in AT2 cells, which is accompanied by cystic organoid growth and MUC5B expression. Cystic growth and MUC5B expression were also caused by the cytokine IL-6. The PI3K-Akt signaling pathway was activated in fibroblasts. The drug dasatinib prevented the formation of MUC5B-expressing cystic organoids. MUC5B associated with AT2 cells in samples obtained from IPF patients. Our model shows that fibrotic primary fibroblasts induce impaired differentiation of AT2 cells via STAT3 signaling pathways, as observed in IPF patients. It can be used for mechanistic studies and drug development.

BACKGROUND:Epigenetic modifications are known to mediate both beneficial and unfavorable effects of environmental exposures on the development and clinical course of asthma. On the molecular level, epigenetic mechanisms participate in multiple aspects of the emerging and ongoing asthma pathology. SUMMARY:Studies performed in the last several years expand our knowledge on the role of histone acetylation, a classical epigenetic mark, in the regulation of (patho)physiological processes of diverse cells playing a central role in asthma, including those belonging to the immune system (e.g., CD4+ T cells, macrophages) and lung structure (e.g., airway epithelial cells, pulmonary fibroblasts). Those studies demonstrate a number of specific histone acetylation-associated mechanisms and pathways underlying pathological processes characteristic for asthma, as well as report their modification modalities. KEY MESSAGES:Dietary modulation of histone acetylation levels in the immune system might protect against the development of asthma and other allergies. Interfering with the enzymes controlling the histone acetylation status of structural lung and (local) immune cells might provide future therapeutic options for asthmatics. Despite some methodological obstacles, analysis of the histone acetylation levels might improve asthma diagnostics.

Alveolar type 2 and club cells are part of the stem cell niche of the lung and their differentiation is required for pulmonary homeostasis and tissue regeneration. A disturbed crosstalk between fibroblasts and epithelial cells contributes to the loss of lung structure in chronic lung diseases. Therefore, it is important to understand how fibroblasts and lung epithelial cells interact during regeneration. Here, we analyzed the interaction of fibroblasts and the alveolar epithelium modeled in air-liquid interface cultures. Single-cell transcriptomics showed that cocultivation with fibroblasts leads to increased expression of type 2 markers in pneumocytes, activation of regulons associated with the maintenance of alveolar type 2 cells (e.g., Etv5), and transdifferentiation of club cells toward pneumocytes. This was accompanied by an intensified transepithelial barrier. Vice versa, the activation of NF-κB pathways and the CEBPB regulon and the expression of IL-6 and other differentiation factors (e.g., fibroblast growth factors) were increased in fibroblasts cocultured with epithelial cells. Recombinant IL-6 enhanced epithelial barrier formation. Therefore, in our coculture model, regulatory loops were identified by which lung epithelial cells mediate regeneration and differentiation of the alveolar epithelium in a cooperative manner with the mesenchymal compartment.