BACKGROUND:Non-specific esophagitis has been recently described in patients with dysphagia not fulfilling criteria for eosinophilic esophagitis (EoE). Whether it is a distinct entity and how dysphagia can develop in the absence of eosinophilic infiltration remains unknown. OBJECTIVE:In this study, we comprehensively characterized non-specific esophagitis aiming to elucidate eosinophil-independent fibrosis mechanisms. METHODS:We cross-sectionally and longitudinally analyzed treatment-naïve patients presenting with non-specific esophagitis defined by esophageal dysfunction and low-grade lymphocytic infiltration (< 30 lymphocytes/hpf) in the absence of tissue eosinophilia and reflux disease (GERD). We compared clinical, endoscopic, (immuno)-histological disease activity to EoE, lymphocytic esophagitis, and GERD. RNA sequencing was performed to investigate disease mechanisms. RESULTS:We identified 19 patients (6 males, median age 48 years), with a follow-up of 20 months (IQR 10-41). Clinical disease burden was considerable, contrasting mild endoscopic activity (EREFS 0, IQR 0-2). Immunostaining of esophageal biopsies did not reveal increased numbers of eosinophils or mast cells as contributors to the disease. Lymphocytic infiltration (15/hpf, IQR 9-24) was not increased compared to healthy controls (9/hpf, IQR 9-10) or GERD patients (13/hpf, IQR 9-23); 14 (73.6%) patients were treated with topical steroids (symptomatic response in 85.7%) and 6 patients (31.6%) underwent endoscopic dilation. Progression to EoE was observed in 1 patient, while esophageal eosinophilia (< 15 eos/hpf) developed in 3 patients. RNA sequencing (n = 10) revealed a distinct transcriptomic profile, with highly enriched fibrosis-related pathways. Immunostaining for E-Cadherin/Vimentin as a marker for epithelial-mesenchymal transition (EMT, n = 10) confirmed increased EMT compared to controls. CONCLUSION:Non-specific esophagitis appears to be a distinct non-eosinophilic phenotype with fibrotic traits. Our data suggest eosinophil-independent development of fibrosis.

Glycogen storage disease type Ib (GSD-Ib) engenders neutropenia and severe neutrophil dysfunction, leading to recurrent infections and inflammatory complications. Recent studies have identified intracellular accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P) as a key mechanism underlying neutrophil impairment and have suggested therapeutic benefits of sodium-glucose cotransporter 2 (SGLT2) inhibitors, which lower plasma levels of its precursor 1,5-AG. In this study, we performed a four-year longitudinal evaluation of empagliflozin therapy in a genetically confirmed GSD-Ib infant, contributing to the growing body of long-term data on empagliflozin treatment in GSD-Ib. Routine laboratory parameters and key neutrophil effector functions were assessed before and during treatment, as well as in two additional GSD-Ib patients with and without therapy. Empagliflozin therapy resulted in complete restoration of neutrophil function, including reactive oxygen species (ROS) production and bactericidal activity. Notably, neutrophil extracellular trap (NET) formation and neutrophil survival recovered to levels comparable to healthy donors. These functional improvements occurred in conjunction with reduced plasma 1,5-AG levels, supporting the concept that GSD-Ib neutrophils are sensitive to physiological 1,5-AG concentrations. Functional recovery and normalization of neutrophil survival observed in vitro, was paralleled by improvement of absolute neutrophil counts to low-normal levels in vivo. Clinically, treatment was associated with a substantial reduction of severe infections. Collectively, these findings further support that empagliflozin corrects neutrophil dysfunction in GSD-Ib and demonstrate its potential to improve long-term clinical outcome across the lifespan, from infancy through adulthood.

Changes in cell number during in vitro experiments and pharmacological screenings primarily depend on two factors: cell death and proliferation. The dynamics of these processes determine whether cell populations expand and accumulate or, conversely, decrease over time. Understanding the biological mechanisms governing these changes is crucial for deciphering the mode of action of any pharmacological or genetic treatment in fundamental research and pre-clinical trials. In this context, we introduce a robust and efficient flow cytometry-based methodology that enables comprehensive analysis of key cellular parameters that indicate changes in cell numbers. This approach encompasses the assessment of cell count along with critical maintenance parameters including proliferation, cell cycle dynamics, apoptosis, cell permeability, and mitochondrial depolarization. These parameters are intricately linked, offering a detailed view of the cellular state. The described methodology is versatile and adaptable for analyzing various cell types, whether at steady state or in response to treatments. To develop this workflow, we integrated and optimised multiple flow cytometry-based stainings such as annexin V, propidium iodide, bromodeoxyuridine, CellTrace Violet, and JC-1 into a unified protocol. This article offers a detailed, step-by-step guide to the entire method, covering aspects such as timing, sample preparation techniques, and the reagents used. Additionally, it includes examples of the data that can be obtained with this technique and illustrates its multiparametric visualization. Collectively, this methodology facilitates the rapid acquisition of up to eight different parameters from a single sample in one experiment.

The extent of mitochondrial heterogeneity and the presence of mitochondrial archetypes in cancer remain unknown. Mitochondria play a central role in the metabolic reprogramming that occurs in cancer cells. This process adjusts the activity of metabolic pathways to support growth, proliferation, and survival of cancer cells. Using a panel of colorectal cancer (CRC) cell lines, we revealed extensive differences in their mitochondrial composition, suggesting functional specialisation of these organelles. We differentiated bioenergetic and mitochondrial phenotypes, which point to different strategies used by CRC cells to maintain their sustainability. Moreover, the efficacy of various treatments targeting metabolic pathways was dependent on the respiration and glycolysis levels of cancer cells. Furthermore, we identified metabolites associated with both bioenergetic profiles and cell responses to treatments. The levels of these molecules can be used to predict the therapeutic efficacy of anti-cancer drugs and identify metabolic vulnerabilities of CRC. Our study indicates that the efficacy of CRC therapies is closely linked to mitochondrial status and cellular bioenergetics.

IgA antibodies have an important role in clearing mucosal pathogens. In this study, we have examined the contribution of IgA to the immune control of the gastrointestinal bacterial pathogens Helicobacter pylori and Citrobacter rodentium. Both bacteria trigger a strong local IgA response that results in bacterial IgA coating in mice and in gastritis patients. Class switching to IgA depends on Peyer's patches, T-cells, eosinophils, and eosinophil-derived TGF-(3 in both models. In the case of H. pylori, IgA secretion and bacterial coating also depend on a functional bacterial type IV secretion system, which drives the generation of Th17 cells and the IL-17dependent expression of the polymeric immunoglobulin receptor PIGR. IgA- /- mice are hypercolonized with C. rodentium in all examined tissues, suffer from more severe weight loss and develop more colitis. In contrast, H. pylori is controlled more efficiently in IgA- /- mice than their WT counterparts. The effects of IgA deficiency of the offspring can be compensated by maternal IgA delivered by WT foster mothers. We attribute the improved immune control observed in IgA- /- mice to IgA-mediated protection from complement killing, as H. pylori colonization is restored to wild type levels in a composite strain lacking both IgA and the central complement component C3. IgA antibodies can thus have protective or detrimental activities depending on the infectious agent.

Cancer immunoediting is a dynamic process of tumor-immune system interaction that plays a critical role in cancer development and progression. Recent studies have highlighted the importance of innate signaling pathways possessed by both cancer cells and immune cells in this process. The STING molecule, a pivotal innate immune signaling molecule, mediates DNA-triggered immune responses in both cancer cells and immune cells, modulating the anti-tumor immune response and shaping the efficacy of immunotherapy. Emerging evidence has shown that the activation of STING signaling has dual opposing effects in cancer progression, simultaneously provoking and restricting anti-tumor immunity, and participating in every phase of cancer immunoediting, including immune elimination, equilibrium, and escape. In this review, we elucidate the roles of STING in the process of cancer immunoediting and discuss the dichotomous effects of STING agonists in the cancer immunotherapy response or resistance. A profound understanding of the sophisticated roles of STING signaling pathway in cancer immunoediting would potentially inspire the development of novel cancer therapeutic approaches and overcome the undesirable protumor effects of STING activation.

Tumor-associated macrophages (TAMs) play a pivotal role in the tumor microenvironment (TME), actively contributing to the formation of an immunosuppressive niche that fosters tumor progression. Consequently, there has been a growing interest in targeting TAMs as a promising avenue for cancer therapy. Recent advances in the field of immunometabolism have shed light on the influence of metabolic adaptations on macrophage physiology in the context of cancer. Here, we discuss the key metabolic pathways that shape the phenotypic diversity of macrophages. We place special emphasis on how metabolic reprogramming impacts the activation status of TAMs and their functions within the TME. Additionally, we explore alterations in TAM metabolism and their effects on phagocytosis, production of cytokines/chemokines and interaction with cytotoxic T and NK immune cells. Moreover, we examine the application of nanomedical approaches to target TAMs and assess the clinical significance of modulating the metabolism of TAMs as a strategy to develop new anti-cancer therapies. Taken together, in this comprehensive review article focusing on TAMs, we provide invaluable insights for the development of effective immunotherapeutic strategies and the enhancement of clinical outcomes for cancer patients.

Recent advancements in intravital microscopy have enabled the study of cell death in vivo under various experimental conditions, such as infection and cancer. However, the limited throughput of this technology, together with a lack of openly accessible datasets, affects the development of algorithms for the automatic detection and characterization of cell death, which in turn require the integration of extensive and curated datasets. To address these needs, we present a curated dataset of microscopy videos depicting the death of neutrophils, eosinophils, and dendritic cells, acquired in the spleen and in the lymph node of mice under inflammatory conditions. The dataset provides time-lapse imaging data, along with coordinates in space and time of cell death events displaying apoptotic-like morphodynamics, and 3D reconstruction of the cell morphology at each time point. Altogether, these data will be pivotal for developing computer vision and bioimage analysis methods to advance cell death research.

Macrophage activation is tightly coupled to cellular metabolism: classically activated pro-inflammatory (M1) macrophages rely on glycolysis and a disrupted tricarboxylic acid cycle, whereas alternatively activated (M2) macrophages depend on oxidative phosphorylation (OXPHOS) and fatty acid oxidation. Although mitochondria are central to this metabolic plasticity, it remains unclear whether mitochondrial dysfunction itself can dictate macrophage polarization. Using macrophage-specific OPA1 knockout mice, we investigated how mitochondrial dysfunction influences macrophage metabolism and immune homeostasis. Loss of OPA1 caused severe impairment of OXPHOS, reduced mitochondrial membrane potential, and a compensatory glycolytic shift, driving M0 and M2 macrophages toward an M1-like bioenergetic state. Integrative metabolomic and transcriptomic analyses revealed strong priming of OPA1-deficient macrophages towards classical activation, including accumulation of M1-associated metabolites (lactate, succinate, itaconate) and upregulation of NF-κB–driven and other inflammatory gene programs, resulting in increased secretion of IL-6 and TNF even in the absence of stimulation. Functionally, this metabolic shift primed non-activated and M2 macrophages toward partial M1 polarization with enhanced bactericidal capacity, while simultaneously suppressing M2-associated processes such as proliferation, efferocytosis, and expression of Arg1, CD206, and RELMα. In vivo, OPA1ΔM mice displayed reduced peritoneal macrophage abundance, impaired self-renewal after IL-4 complex stimulation, and compensatory monocyte recruitment. The remaining macrophages exhibited increased MHCII and reduced RELMα expression, consistent with partial M1 skewing and loss of alternative activation. These local alterations were mirrored systemically: blood profiling revealed enhanced T-cell activation and sex-specific remodeling of immune composition during inflammation and aging. Collectively, these findings demonstrate that mitochondrial dysfunction serves as a cell-intrinsic cue that primes macrophages toward a glycolytic, pro-inflammatory phenotype while constraining their M2 properties and proliferative capacities. This dual metabolic and functional rewiring highlights mitochondrial integrity as a pivotal determinant of macrophage immunometabolic identity and reveals how its disruption can reshape both local and systemic immune homeostasis. ### Competing Interest Statement The authors have declared no competing interest. SNF, No. 310030_184816 (HUS)

Neutrophils are central mediators of the hyperinflammatory response in severe SARS-CoV-2 infection. We report elevated cytosolic levels of proliferating cell nuclear antigen (PCNA) in neutrophils from patients with severe and critical COVID-19, correlating with enhanced NADPH oxidase-dependent reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation. Using T2AA, a small-molecule inhibitor of the PCNA scaffold, we demonstrate potent suppression of NADPH oxidase activation and NET release, particularly in response to SARS-CoV-2 RNA. Mechanistically, we identify a previously unrecognized interaction between PCNA and the heterodimeric S100A8/S100A9 (calprotectin), predominantly enriched in CD16highCD62Llow neutrophils expanded during COVID-19. PCNA binds the dimeric S100A8/S100A9 complex mediated via S100A8 subunit with micromolar affinity, and this interaction is abrogated by tetramerization, suggesting regulation by intracellular calcium. Disruption of this complex by T2AA inhibited ROS production in an S100A8/S100A9-dependent manner, implicating calprotectin as a functional regulator of neutrophil activation. In a betacoronavirus mouse model, T2AA treatment attenuated lung inflammation, reduced NET and calprotectin levels, and shifted pulmonary neutrophils away from hyperactivated and immunosuppressive phenotypes, consistent with immune reprogramming toward resolution. These findings establish cytosolic PCNA as a central scaffold in neutrophil hyperactivation during COVID-19 and highlight its pharmacological disruption as a promising host-directed strategy to limit inflammation and prevent organ damage.

INTRODUCTION:Eosinophilic esophagitis (EoE) variants have been recently characterized as conditions with symptoms of esophageal dysfunction resembling EoE, but absence of significant esophageal eosinophilia. Their disease course and severity have yet to be determined. METHODS:Patients from 6 EoE centers with symptoms of esophageal dysfunction, but peak eosinophil counts of <15/hpf in esophageal biopsies and absence of gastroesophageal reflux disease with at least one follow-up visit were included. Clinical, (immuno)histological, and molecular features were determined and compared with EoE and healthy controls. RESULTS:We included 54 patients with EoE variants (EoE-like esophagitis 53.7%; lymphocytic esophagitis 13.0%; and nonspecific esophagitis 33.3%). In 8 EoE-like esophagitis patients, EoE developed after a median of 14 months (interquartile range 3.6-37.6). Such progression increased over time (17.6% year 1, 32.0% year 3, and 62.2% year 6). Sequential RNA sequencing analyses revealed only 7 genes associated with this progression (with TSG6 and ALOX15 among the top 3 upregulated genes) with upregulation of a previously attenuated Th2 pathway. Immunostaining confirmed the involvement of eosinophil-associated proteins (TSG6 and ALOX15) and revealed a significantly increased number of GATA3-positive cells during progression, indicating a Th1/Th2 switch. Transition from one EoE variant (baseline) to another variant (during follow-up) was seen in 35.2% (median observation time of 17.3 months). DISCUSSION:Transition of EoE variants to EoE suggests the presence of a disease spectrum. Few genes seem to be associated with the progression to EoE with upregulation of a previously attenuated Th2 signal. These genes, including GATA3 as a Th1/Th2 switch regulator, may represent potential therapeutic targets in early disease pathogenesis.

Atopic dermatitis (AD) is characterized by skin barrier dysfunction and immune dysregulation. Autophagy, which is important for the epidermal differentiation, is impaired in AD. The treatment with dupilumab, an interleukin (IL)-4/IL-13 receptor blocker, has been shown to reduce skin inflammation and restore the skin barrier. This study aimed to investigate the effect of dupilumab on the expression of key proteins involved in autophagy and lysosomal degradation. We performed immunofluorescence staining and microscopic analyses of skin specimens of AD patients, taken before and under (6–10 weeks) therapy with dupilumab, to investigate the expression of autophagy-related (ATG) 5 and ATG7 proteins, beclin-1, microtubule-associated protein light chain 3 (LC3B), sequestosome-1 (p62), lysosomal proteases (cathepsins B, D and L), serine protease inhibitors (SERPINB3, SERPINB4) as well as IL-33 and thymic stromal lymphopoietin (TSLP). The expression of LC3B and p62 as well as SERPINB3 and SERPINB4 was highly increased in untreated AD skin compared to non-lesional skin and normal skin and decreased upon dupilumab therapy. In contrast, the AD-associated increased expression of both ATG5 and ATG7 further increased under therapy. Before therapy, cathepsin D and L expression levels were significantly lower compared to normal skin, but increased following the initiation of dupilumab therapy. The increased expression of IL-33 and TSLP in the epidermis of AD patients correlated with that of LC3B and p62. Our study provides further evidence that autophagy is inhibited in lesional AD skin owing to lysosomal dysfunction. Upon dupilumab therapy, a restoration of dysregulated key players of autophagy is observed.

The role of mitochondria in steroidogenesis is well established. However, the specific effects of mitochondrial dysfunction on androgen synthesis are not fully understood. In this study, we investigate the effects of various mitochondrial and metabolic inhibitors in H295R adrenal cells and perform a comprehensive analysis of steroid and metabolite profiling. We report that mitochondrial complex I inhibition by rotenone shifts cells toward anaerobic metabolism with a concomitant hyperandrogenic phenotype characterized by rapid stimulation of dehydroepiandrosterone (DHEA, 2h) and slower accumulation of androstenedione and testosterone (24h). Screening of metabolic inhibitors confirmed DHEA stimulation, which included mitochondrial complex III and mitochondrial pyruvate carrier inhibition. Metabolomic studies revealed truncated tricarboxylic acid cycle with an inverse correlation between citric acid and DHEA production as a common metabolic marker of hyperandrogenic inhibitors. The current study sheds light on a direct interplay between energy metabolism and androgen biosynthesis that could be further explored to identify novel molecular targets for efficient treatment of androgen excess disorders.

Eosinophils, a type of granulocyte derived from myeloid precursors in the bone marrow, are distinguished by their cytoplasmic granules. They play crucial roles in immunoregulation, tissue homeostasis, and host defense, while also contributing to the pathogenesis of various inflammatory diseases. Although long non-coding RNAs (lncRNAs) are known to be involved in eosinophilic conditions, their specific expression and functions within eosinophils have not been thoroughly investigated, largely due to the reliance on tissue homogenates. In an effort to address this gap, we analyzed publicly available high-throughput RNA sequencing data to identify lncRNAs associated with eosinophilic conditions. Among the identified lncRNAs, ITGB2 antisense RNA 1 (ITGB2-AS1) was significantly downregulated in blood eosinophils from patients with hypereosinophilia. To further explore its role in eosinophil biology, we generated a stable ITGB2-AS1 knockdown in the HL-60 cell line. Interestingly, ITGB2-AS1 deficiency led to impaired eosinophil differentiation, as evidenced by a reduction in cytoplasmic granules and decreased expression of key eosinophil granule proteins, including eosinophil peroxidase (EPX) and major basic protein-1 (MBP-1). Additionally, ITGB2-AS1-deficient cells exhibited compromised eosinophil effector functions, with reduced degranulation and impaired production of reactive oxygen species (ROS). These findings suggest that ITGB2-AS1 plays a pivotal role in eosinophil differentiation and function, positioning it as a novel regulator in eosinophil biology.