BACKGROUND:Expression of CD25, the IL-2 receptor alpha chain, on human mast cells is primarily associated with aberrant mast cells in clonal mast cell disorders. However, the regulation of CD25 expression in normal, mature tissue-resident mast cells remains poorly understood. OBJECTIVE:IL-33 is a key modulator of immune responses, including in lung inflammatory conditions. Because mast cells are prominent IL-33 receptor-expressing cells, we investigated the effect of IL-33 on CD25 expression in purified human lung mast cells (HLMCs). METHODS:Purified HLMCs were stimulated with IL-33 and the transcriptional responses measured by RNA sequencing. The expression of the IL-2 receptor subunits CD25 (IL2RA), CD122 (IL2RB), and CD132 (IL2RG) was quantified by real-time quantitative PCR and flow cytometry. IL2RA expression was further examined in publicly available single-cell RNA sequencing datasets, and in situ CD25 protein expression on HLMCs was assessed in human lung tissue by immunofluorescence staining. RESULTS:IL-33 robustly induced the expression of CD25 and CD132 in HLMCs without a corresponding upregulation of CD122, resulting in absent IL-2-mediated signaling despite enhanced IL-2 binding via CD25. Single-cell RNA sequencing data identified IL2RA+ mast cells as a distinct subpopulation with enriched IL-33 response signatures and upregulation of genes linked to immune signaling and inflammatory pathways. CD25-positive HLMCs were also detected in situ, displaying substantial heterogeneity in expression levels and spatial distribution. CONCLUSIONS:CD25 expression in HLMCs is upregulated by IL-33 and is dynamically regulated in human lung tissues.

Background Chronic obstructive pulmonary disease (COPD) in never-smokers may have other clinical characteristics than tobacco smoking-related COPD.Research question What are the risk factors, biomarkers, respiratory symptoms and health status in never-smoking individuals with COPD?Study design and methods We investigated never-smokers with COPD (n=154, mean age 60 years) from the population-based Swedish CArdioPulmonary bioImage Study (SCAPIS), and compared them with four control groups: never-smokers with normal lung function (n=281), current smokers with normal lung function (n=97), ex-smokers with COPD (n=103) and current smokers with COPD (n=55). COPD was defined as forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) less than the lower limit of normal (LNN) after bronchodilation. We examined fractional exhaled nitric oxide (FeNO), blood biomarkers, respiratory symptoms, health status, medical history and living conditions.Results The never-smoker COPD group reported more respiratory symptoms and worse health status than never-smokers with normal lung function, but fewer symptoms, milder airflow limitation and better health status compared with ex-smokers and smokers with COPD. Never-smokers with COPD had more self-reported asthma. Moreover, never-smokers with COPD had higher Immunoglobulin E sensitisations to a mix of aeroallergens, higher geometrical mean FeNO levels and blood eosinophil counts than never-smokers with normal lung function. When participants with self-reported asthma were excluded, never-smokers with COPD still had more wheeze, cough and higher FeNO.Conclusion Never-smokers with COPD had more respiratory symptoms and elevated markers of type-2 inflammation, suggesting they might represent a distinct clinical phenotype which may differ from smoking-related COPD. They may therefore need to be treated and followed differently.Trial registration number NCT03049202.

BACKGROUND:Seasonal allergic rhinitis (SAR) is viewed as a type 2 (T2) disease driven by the IL-5/IL-5 receptor α (IL-5Rα) and IL-4/IL-13/IL-4 receptor (IL-4R) pathways and the upstream epithelial alarmins IL-33 and TSLP. However, the dynamics and expression patterns of T2-immunity in the nasal mucosa during sustained allergen exposure remain poorly understood. AIM/OBJECTIVE:To investigate whether traditional T2-cytokine and alarmin expressions persist during sustained, well-developed eosinophilic inflammation in patients with SAR. METHODS:Twenty patients were examined late in and outside the birch pollen season. Symptoms, blood, nasal secretions, and mucosal biopsies were evaluated. Analyses included multiplex immunohistochemistry, in situ hybridisation, and computerized image analysis to quantify immune cells, cytokine/alarmin-expression, and their receptors, focusing on IL-5Rα. RESULTS:Late in the season, patients presented increased symptoms, blood/tissue eosinophilia, and elevated basophils, mast cells, and T helper 2 cells, consistent with a T2-phenotype. Unexpectedly, cells expressing IL-4 and IL-5 mRNA were undetectable. Surprisingly, IL-5Rα was abundant on epithelial cells, but not on mucosal eosinophils. Few scattered IL-13HIGH cells were identified. IL-33 expression remained robust but was lower compared with post-seasonal observations. CONCLUSIONS AND SIGNIFICANCE:Sustained eosinophilic inflammation in SAR may be maintained by non-T2-pathways in established allergic disease, potentially informing future treatment strategies.

Rationale Information is missing on the tissue cell expression patterns of interleukin 33 (IL-33) and splice variants of the IL-33 receptor ST2 in normal and chronic obstructive pulmonary disease (COPD) lungs.Objectives To characterize the expression patterns of IL-33, the soluble ST2 (sST2) and membrane-bound ST2 (ST2L) splice variants in the poorly studied small airway and distal lung compartments in COPD and controls.Methods Surgically excised lung tissue was collected from 38 COPD patients and 21 non-COPD controls. Lung compartment expression of IL-33 and ST2 and key expressing cell types were assessed histologically by combined in situ hybridization and multiplex immunohistochemistry. Expression dynamics of IL-33, ST2L, and sST2 were explored by spatially resolved single-cell analysis.Measurements and Main Results COPD lungs displayed increased IL-33 mRNA and IL-33 mRNA/protein ratios, suggesting increased IL-33 turnover. Total ST2/IL1RL1 mRNA levels were upregulated in COPD lungs. Mast cells constituted the major ST2-expressing immune cell population in controls and displayed a microenvironmental-specific upregulation of both ST2L and sST2 in COPD. In control alveolar regions, ST2Lhigh sST2high mast cells were present alongside IL-33-expressing general capillary (gCap) and sST2moderate ST2Llow aerocyte endothelial subsets. In COPD, patchy alveolar regions displayed markedly elevated capillary sST2 and numbers of ST2L+ and IL-33+ gCaps.Conclusions By unraveling the expression patterns of IL-33 and the biologically opposing ST2L and sST2 splice variants in control and COPD lungs, the present study provides novel insights into IL-33-mediated immunity in the distal lung, information that has bearing on treatment strategies targeting this pathway in lung diseases.

Background: Histological examination of mucosal tissue in inflammatory bowel diseases (IBD) is a sensitive tool to measure disease activity, and histological remission is emerging as a potentially important treatment target. There are several existing histopathological indices, but they often encompass caveats such as not primarily having been designed to measure the degree of inflammation, encompassing subjective components with poor intra- and interindividual reproducibility, and requiring expert pathologists who are scarce, thus resulting in extended response times. Aim: To construct a new computerized, automated index to objectively measure histological disease activity in the ileal and colonic mucosa, applicable to both Crohn's disease (CD) and ulcerative colitis (UC). Materials and methods: Ileocolonic biopsies were collected from control subjects and patients with CD or UC. A group of CD patients was sampled before and after 12 weeks of anti-TNFα therapy. Another group of CD and UC patients functioned as a small validation cohort. Epithelial cells, neutrophils, macrophages, and T cells were immunohistochemically stained, followed by digitalization of the color signal and computerized delineation of the epithelial and lamina propria compartments. The various immune cell types within the epithelium and the lamina propria, respectively, were enumerated, and the numbers were compared between control subjects and patients with CD or UC. Results: The numbers of neutrophils and macrophages in the epithelium, and neutrophils in the lamina propria, showed the highest sensitivity and specificity for distinguishing control-subject tissues from CD and UC tissues. These three parameters were thus chosen to construct a new index, named QiC3 1.0, that could separate tissues from control subjects and patients with CD or UC with high precision. It performed equally well in a small validation cohort of patients. The QiC3 index correlated well with previously described histopathological indices, fecal calprotectin, and endoscopic scores in UC, but showed worse correlation with endoscopic scores in CD and symptomatic scores. When applying the new index to tissues from CD patients before and after therapy, it showed good responsiveness, demonstrating a distinct amelioration in the microscopic inflammatory status that corresponded well to improvements in histopathological scores. Conclusion: We describe a new quantitative, computerized, automated, non-subjective, and response-sensitive immunohistological index (QiC3) for measuring disease activity in ileal and colonic mucosal biopsies, suitable for both CD and UC. ### Competing Interest Statement Erik Hertervig has served as consultant for Abbvie, Merck, Sharp & Dohme, and Takeda. Jonas S Erjefalt is the founder and CEO of Medetect AB. Jan Marsal has served as consultant for AbbVie, BMS, Ferring, Galapagos, Lilly, Takeda, Tillotts. Jan Marsal has received investigator-initiated study grants from AbbVie, Ferring, Pfizer, and Takeda. The other authors have no financial conflicts of interest. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was performed in accordance with the declaration of Helsinki and was approved by the regional ethics committees in Lund and Linkoping, Sweden (Dnr. 2011/60 and 2011-201-31, respectively). Written informed consent was obtained from all subjects before they were included. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. Hedlund Foundation Julin Foundation Bengt Ihres Foundation, https://ror.org/04v0hex10 Swedish Society for Medical Research, https://ror.org/0433fd238 Royal Physiographic Society of Lund, https://ror.org/05rnj0j91 Skåne University Hospital, https://ror.org/02z31g829 Swedish Society of Medicine, https://ror.org/016ks4x90 investigator-initiated study grant from AbbVie Healthcare Region of Southern Sweden Swedish government (ALFSKANE-539811)

Eosinophils are increasingly recognized as adaptable immune cells that exhibit diverse phenotypes and effector functions across different tissues and disease states. While they can induce pathology through degranulation and cytotoxic mediator release, eosinophils also fulfill regulatory and tissue repair roles. Advances in single-cell and spatial technologies have begun to reveal how microenvironmental cues (including cytokines, chemokines, and cell–cell interactions) shape eosinophil behavior in health and disease. These insights are critical for understanding why certain patients respond variably to therapies targeting eosinophils and related type 2 pathways. By dissecting eosinophil heterogeneity in real human tissues, researchers may identify new biomarkers, refine endotyping approaches, and develop more precise therapeutic strategies. This review summarizes emerging concepts of eosinophil biology in inflammatory conditions, highlights the impact of spatial context on eosinophil functions, and discusses the future of advanced phenotyping in guiding personalized treatments.

Biologics targeting interleukin-4 receptor subunit α (IL-4Rα) and interleukin-33 (IL-33) have demonstrated clinical efficacy in asthma, highlighting the importance of IL-4, IL-13, and IL-33 in respiratory diseases. Despite this, few studies have linked preclinical models to human diseases or evaluated disease biology in clinical trials. To address these gaps, we evaluated transcriptional, cellular, and pathophysiological processes driven by IL-4/IL-13 and IL-33 using human innate cells in vitro, a mouse model of airway inflammation, and a bronchial allergen challenge (BAC) in house dust mite (HDM)-sensitized individuals with mild asthma. Our findings in mice revealed that the prophylactic blockade of IL-4/IL-13, but not IL-33, prevented the initiation of HDM-induced type 2 inflammation, whereas blocking IL-4Rα or IL-33 during peak inflammation ameliorated airway inflammation and remodeling. Each pathway had unique and overlapping effects on airway inflammation and remodeling, with combination blockade showing no additional benefit. Initiating either monotherapy during severe, mixed inflammation resulted in partial efficacy, whereas a combination of these two treatments led to a substantial reduction in airway inflammation and remodeling in sensitized mice. Some of these mechanistic observations translated to a human BAC model, where blocking IL-4Rα or IL-33 alone suppressed gene expression in sputum and circulating biomarkers. As observed in mice, combination treatment in individuals with allergic asthma did not provide additional benefit compared to monotherapy. Overall, these results provide insight into the differences in targeting IL-4Rα or IL-33 pathways in asthma independently or in combination.

IntroductionFlexible bronchoscopy is regarded as a safe examination and is commonly used in the diagnostic work-up for lung diseases, but is also important in pulmonary research. We aimed to investigate participants’ experiences when undergoing bronchoscopy in a research setting.MethodsParticipants were recruited from the Swedish CArdioPulmonary bioImage Study (SCAPIS). A subset from this cohort (n = 45, mean age 60.5 years, 20 with normal lung function and 25 with chronic obstructive pulmonary disease, COPD) was selected for bronchoscopy. The procedure was explained both orally and in writing during a pre-procedure visit. The information included premedication, monitoring, local anesthesia, airway sampling [bronchoalveolar lavage (BAL), bronchial wash, and mucosal biopsies], and urine and blood samples. Questionnaires pre- and/or post-procedure were used to assess experiences and health impacts.ResultsIn general, participants found the bronchoscopy procedure acceptable and only a few (18%) found it unpleasant. A majority (80%) reported their experience to be much better or as expected. Almost all participants (93%) were very satisfied with the information provided. Topical anesthesia was seen as more unpleasant (20%) than airway sampling (11%). Notably, more women and participants with normal lung function reported BAL as unpleasant. After the procedure, chills, fever, and hemoptysis were reported, but no serious adverse events occurred. Increased cough and phlegm were noted.ConclusionThe present study, conducted by experienced bronchoscopists and healthcare teams, demonstrates that a bronchoscopy in a research setting in well-informed participants with normal lung function or COPD was well-tolerated.

Background: Biologics targeting IL-33 or IL-4Rα pathways have shown clinical efficacy in asthma and chronic obstructive pulmonary disease (COPD), underscoring the significance of IL-4, IL-13, and IL-33 in respiratory diseases. However, there are limited studies that connect preclinical models to human disease and assess disease biology within clinical trials. Methods: To bridge these gaps, we investigated the transcriptional, cellular, and pathophysiological processes driven by IL-4/IL-13 and IL-33 in a mouse model of airway inflammation, and a Bronchial Allergen Challenge (BAC) in house dust mite (HDM)-sensitized individuals with mild asthma. Results: In the HDM-driven mouse model of asthma, we demonstrate that IL-4/IL-13, but not IL-33 signaling, is required for initiation of allergen-induced Type 2 (T2) inflammation. On the other hand, blockade of either IL-4Rα with dupilumab or IL-33 with itepekimab at the peak of T2 inflammation resulted in improvement of airway inflammation and remodeling in the murine model. Interestingly, each pathway had distinct and similar effects on pathways that contribute to inflammation and remodeling, while combination blockade did not show additional benefit on T2 inflammation and mirrored monotherapy arms. In contrast, initiating either monotherapy treatment during established severe, mixed inflammation resulted in partial efficacy, while combination of these two treatments led to a significant reduction of airway inflammation and remodeling, suggesting a treatment opportunity beyond classical T2 inflammation. Some of these mechanistic observations translated to a human BAC model wherein blockade of either IL-4Rα or IL-33 resulted in suppression of gene expression in induced sputum and circulating biomarkers. Of note, combination treatment in the context of allergic airway inflammation did not provide additional benefit compared to monotherapy. Conclusions: These findings highlight IL-33 and IL-4/IL-13 as key drivers of airway inflammation and remodeling, offering insights into the differential effects of targeting IL-4Rα or IL-33 pathways in asthma, either independently or in combination.

Remission is emerging as a feasible treatment goal in moderate-to-severe asthma, driven by the success of biologic therapies in controlling inflammation and reducing exacerbations. Yet current definitions of remission-focused on symptom control, lung function, and corticosteroid reduction-lack precision, can only be ascertained retrospectively, and do not reflect the underlying mechanisms and pathology that drive disease progression. This gap limits the clinical applicability of these definitions and might obscure opportunities for early, disease-modifying intervention. In this Series paper, we propose a refined framework for understanding and reaching remission, centred on distinguishing modifiable disease activity from irreversible remodelling and comorbidity-related factors that contribute to disease burden. We introduce the concept of at-risk asthma as a crucial phase characterised by high disease activity and immune dysregulation, in which timely intervention might prevent irreversible airway and extrapulmonary damage and support long-term disease modification. We examine how symptoms, lung function impairment, and exacerbations can arise from distinct and overlapping mechanisms, underscoring the need for careful attribution in clinical assessment. We also outline four key pathophysiological domains-airway hyper-responsiveness, immune hyper-responsiveness, immune remodelling, and structural remodelling-and describe their temporal evolution and implications for treatment responsiveness. Finally, we present a domain-based strategy for assessment and intervention, linking targeted therapies to underlying mechanisms. This approach supports more personalised treatment decisions and redefines remission, not simply as the absence of symptoms, but as stabilisation of disease biology. As the field advances towards earlier intervention and more tailored application of biologics in at-risk asthma, such a framework could be essential to improve long-term outcomes and prevent overtreatment of irreversible disease.

Background: During chronic inflammation, lymphoid aggregates (LAs) composed of a large aggregate of B cells are formed at mucosal sites. The presence of LAs has also been reported in the lungs of patients with asthma and severe COPD. However, the functional relevance of LAs in airway inflammation remains unclear. Methods: We exposed mice to HDM extract or saline intranasally three times per week for up to 15 weeks. Localization of LAs and B/PCs within lung tissue was assessed by immunofluorescence microscopy. Transcriptional profiling of lung B/PCs was performed using single-cell RNA-seq. IgG1, IgA, and IgE production was measured in lung tissue by ELISA and ELISPOT. Gene expression of Ighg1, Igha, and Ighe was assessed by TaqMan. Results: B cells accumulated in the lung tissue after HDM exposure, with a subset expressing Fas, Gl7, Aicda, Ki67, and Bcl6 (GC-like markers) and the immunoglobulin transcripts Ighg1, Igha, and Ighe, suggesting the occurrence of local class-switching at mucosal sites. A second subset expressed genes associated with PC differentiation, including Prdm1, Xbp1, Sdc1, and Tnfrsf17. Histological analysis revealed that upon short-term (4 weeks) allergen exposure, B cells formed aggregates together with T cells mainly around the airway epithelial wall. Surprisingly, we found that PCs localized outside of the LAs and produced Der p1-specific antibodies, suggesting that allergen-specific antibodies are produced in the lung outside of LAs. In contrast, long-term allergen exposure (15 weeks) led to the dissemination of these LAs to distal parts of the lung with a different cellular composition compared to acute exposure. Finally, systemic depletion of B cells abolished lung LAs and resulted in changes in airway inflammation and remodeling. Conclusions: These data demonstrate that the lung can serve as a source of B cell class-switching, PC differentiation, and local antibody production during repeated intranasal allergen exposure.

Background Local and systemic side effects of glucose remain major limitations of peritoneal dialysis (PD). Glucose transport during PD is thought to occur via inter-endothelial pathways, but recent results show that phloretin, a general blocker of facilitative glucose channels (glucose transporters [GLUTs]), markedly reduced glucose diffusion capacity indicating that some glucose may be transferred via facilitative glucose channels (GLUTs). Whether such transport mainly occurs into (absorption), or across (trans-cellular) peritoneal cells is as yet unresolved. Methods Here we sought to elucidate whether diffusion of radiolabeled 18F-deoxyglucose ([18F]-DG) in the opposite direction (plasma → dialysate) is also affected by GLUT inhibition. During GLUT inhibition, such transport may either be increased or unaltered (favors absorption hypothesis) or decreased (favors transcellular hypothesis). Effects on the transport of solutes other than [18F]-DG (or glucose) during GLUT inhibition indicate effects on paracellular transport (between cells) rather than via GLUTs. Results GLUT inhibition using phloretin markedly reduced [18F]-DG diffusion capacity, improved ultrafiltration (UF) rates and enhanced the sodium dip. No other solutes were significantly affected with the exception of urea and bicarbonate. Conclusion The present results indicate that part of glucose is transported via the transcellular route across cells in the peritoneal membrane. Regardless of the channel(s) involved, inhibitors of facilitative GLUTs may be promising agents to improve UF efficacy in patients treated with PD.