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:Cytotoxic type 2 T cells (Tc2) are increasingly recognized as contributors to type 2 inflammation, including asthma, yet the metabolic programs that support their function remain poorly defined. We aimed to define the metabolic requirements of Tc2 cells, identify pathways that regulate their effector function, and assess whether serotonin-modifying therapies are associated with altered Tc2 responses and allergic sensitization in humans. METHODS:Tc2 cells from human peripheral blood and lung tissue were analyzed using Seahorse metabolic assays, Mitotracker staining, flow cytometry, and RNA sequencing. Effector functions were evaluated following inhibition of glycolysis, fatty acid metabolism, and monoamine oxidase A (MAOA) inhibition. In parallel, anti-depressant prescription data were analyzed in the population-based BAMSE cohort to assess associations with allergic sensitization. Peripheral blood mononuclear cells from individuals prescribed selective serotonin reuptake inhibitors (SSRIs) and matched controls were stimulated ex vivo to assess cytokine production. RESULTS:Tc2 cells exhibited a distinct metabolic profile characterized by increased mitochondrial respiration, glycolytic activity, and elevated expression of GLUT1 and CD36. Type 2 cytokine production (IL-4, IL-5, IL-13) was enhanced by alarmins and significantly reduced following inhibition of glycolysis, fatty acid metabolism, or PPARγ activity. RNA sequencing identified high MAOA expression in Tc2 cells, and pharmacological inhibition of MAOA selectively reduced type 2 cytokine production without affecting IFN-γ. In the BAMSE cohort, dispensing of prescribed anti-depressive drugs was associated with reduced IgE sensitization. Consistent with these findings, individuals prescribed SSRIs exhibited reduced IL-5+ and IL-13+ expressing Tc2 cells and increased IFN-γ-producing Tc2 cells following ex vivo stimulation. CONCLUSION:Tc2 cells rely on coordinated lipid metabolism and serotonin catabolism to sustain type 2 cytokine production. Serotonin-modifying therapies were associated with reduced allergic sensitization and altered Tc2 function in humans, consistent with a link between serotonin signaling and type 2 immunity.

BACKGROUND:Concomitant exposure to IL-33, thymic stromal lymphopoietin (TSLP), and IL-25 augments antigen-induced contractions of isolated human small bronchi through enhanced mast cell reactivity. OBJECTIVE:We sought to test the individual contribution of alarmins in antigen-induced airway hyperresponsiveness and hyperosmolarity-induced responses evoked by mannitol, a surrogate model of exercise-induced bronchoconstriction. METHODS:Intact segments of small airways, isolated from fresh human lung tissue, were incubated with IL-33, TSLP, IL-25, or buffer control for 48 hours. Contractile responses to anti-IgE or mannitol were then assessed using myograph systems. Mast cell degranulation and mediator release were analyzed both from the bronchial segments and from isolated primary human lung mast cells as well as from the human mast cell line LAD2 (Laboratory of Allergic Diseases 2). RESULTS:IL-33 increased contractile force (Emax) to anti-IgE and hyperosmolar mannitol by 62% and 78%, respectively. IL-33 also doubled antigen-IgE-induced prostaglandin D2 release from bronchial segments as well as enhanced degranulation, cysteinyl leukotriene, and prostaglandin D2 release from isolated human lung mast cells stimulated by anti-IgE or mannitol. In contrast, TSLP and IL-25 had no effect on contraction, degranulation, or mediator release in response to either stimulus. CONCLUSIONS:IL-33, but not TSLP or IL-25, enhances bronchoconstriction in human small bronchi by amplifying mast cell activation and mediator release in response to both antigen and hyperosmolar challenge.

BACKGROUND:The capacity of human lung mast cells (HLMC) to biosynthesize lipid mediators other than prostaglandin D2 (PGD2) and the cysteinyl leukotrienes is not established. We therefore profiled lipid mediator metabolites in IgE-activated HLMC. METHODS:A liquid chromatography-tandem mass spectrometry platform including 107 metabolites of main endogenous polyunsaturated fatty acids was applied to supernatants of HLMC and for comparison on material from in vitro-developed cord blood mast cells. Involved pathways were defined by the use of specific pharmacologic inhibitors and transcriptomic analysis of the expression of relevant enzymes. RESULTS:IgE activation of HLMC profoundly increased the release of leukotriene C4 (>500 times), PGD2 (>200 times), and thromboxane B2 (>100 times). In contrast, only minimal production of prostaglandin E2, leukotriene B4, or lipoxin A4 was detected. The majority of the quantified compounds (n = 50) were metabolites of arachidonic acid, whereas levels of metabolites from other polyunsaturated fatty acids were low. Despite the COX-2 transcript being the most abundant species, all prostanoids, as well as lipoxin A4 and 15-HETE, were solely biosynthesized in reactions catalyzed by COX-1. There was no apparent shunting between the main enzymatic pathways when COX-1 or 5-LOX were inhibited. Similar results were obtained in the cord blood mast cell model. CONCLUSIONS:There is a high degree of specialization in HLMC with a strong and specific increase in cysteinyl leukotrienes and PGD2 but also the bronchoconstrictor thromboxane A2 after IgE-dependent activation.

BackgroundMast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells.MethodsHuman lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method.ResultsIgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells.ConclusionsFor an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.

OBJECTIVES To assess the feasibility and safety of uniportal video-assisted thoracoscopic pulmonary segmentectomy compared with lobectomy by studying early postoperative outcomes. METHODS We included all patients who underwent uniportal segmentectomy and lobectomy between 2017 and 2022 at Karolinska University Hospital. Early clinical outcomes were compared between the uniportal segmentectomy and lobectomy groups. Differences in baseline characteristics were addressed using inverse probability of treatment weighting. RESULTS A total of 833 patients (232 segmentectomy, 601 lobectomy) were included. The number of uniportal operations increased during the study period. Patients in the segmentectomy and lobectomy groups, respectively, had stage I lung cancer in 65% and 43% of the cases; 97% and 94% had no postoperative complications, the median number of lymph node stations sampled was 4 vs 5, and non-radical microscopic resection occurred in 1.7% vs 1.8%. The drains were removed on postoperative day 1 in 75% vs 72% of the patients following segmentectomy and lobectomy, respectively, and 90% vs 89% were discharged directly home. CONCLUSIONS Uniportal video-assisted segmentectomy was performed with similar early postoperative clinical results compared with uniportal lobectomy in patients with benign, metastatic or early-stage lung cancer.

Lung cancer is a leading cause of cancer-related death worldwide. Despite recent advances in tissue immunology, little is known about the spatial distribution of tissue-resident lymphocyte subsets in lung tumors. Using high-parameter flow cytometry, we identified an accumulation of tissue-resident lymphocytes including tissue-resident NK (trNK) cells and CD8+ tissue-resident memory T (TRM) cells toward the center of human non-small cell lung carcinomas (NSCLC). Chemokine receptor expression patterns indicated different modes of tumor-infiltration and/or residency between trNK cells and CD8+ TRM cells. In contrast to CD8+ TRM cells, trNK cells and ILCs generally expressed low levels of immune checkpoint receptors independent of location in the tumor. Additionally, granzyme expression in trNK cells and CD8+ TRM cells was highest in the tumor center, and intratumoral CD49a+CD16- NK cells were functional and responded stronger to target cell stimulation than their CD49a- counterparts, indicating functional relevance of trNK cells in lung tumors. In summary, the present spatial mapping of lymphocyte subsets in human NSCLC provides novel insights into the composition and functionality of tissue-resident immune cells, suggesting a role for trNK cells and CD8+ TRM cells in lung tumors and their potential relevance for future therapeutic approaches.

Mast cells are tissue-resident cells playing major roles in homeostasis and disease conditions. Lung mast cells are particularly important in airway inflammatory diseases such as asthma. Human mast cells are classically divided into the subsets MC T and MC TC , where MC T express the mast cell protease tryptase and MC TC in addition express chymase, carboxypeptidase A3 (CPA3) and cathepsin G. Apart from the disctintion of the MC T and MC TC subsets, little is known about the heterogeniety of human lung mast cells and a deep analysis of their heterogeniety has previously not been performed. We therefore performed single cell RNA sequencing on sorted human lung mast cells using SmartSeq2. The mast cells showed high expression of classical mast cell markers. The expression of several individual genes varied considerably among the cells, however, no subpopulations were detected by unbiased clustering. Variable genes included the protease-encoding transcripts CMA1 (chymase) and CTSG (cathepsin G). Human lung mast cells are predominantly of the MC T subset and consistent with this, the expression of CMA1 was only detectable in a small proportion of the cells, and correlated moderately to CTSG . However, in contrast to established data for the protein, CPA3 mRNA was high in all cells and the correlation of CPA3 to CMA1 was weak.

Exercise-induced bronchoconstriction (EIB) is thought to be triggered by increased osmolarity at the airway epithelium. The aim of this study was to define the contractile prostanoid component of EIB, using an ex vivo model where intact segments of bronchi (inner diameter 0.5-2 mm) isolated from human lung tissue and subjected to mannitol. Exposure of bronchial segments to hyperosmolar mannitol evoked a contraction (64.3 & PLUSMN; 3.5 %) which could be prevented either by elimination of mast cells (15.8 & PLUSMN; 4.3 %) or a combination of cysteinyl leukotriene (cysLT1), histamine (H1) and thromboxane (TP) receptor antagonists (11.2 & PLUSMN; 2.3 %). Likewise, when antagonism of TP receptor was exchanged for inhibition of either cyclooxygenase-1 (8 & PLUSMN; 2.5 %), hematopoietic prostaglandin (PG)D synthase (20.7 & PLUSMN; 5.6 %), TXA synthase (14.8 & PLUSMN; 4.9 %), or the combination of the latter two (12.2 & PLUSMN; 4.6 %), the mannitol-induced contraction was prevented, suggesting that the TP-mediated component is induced by PGD2 and TXA2 generated by COX-1 and their respective synthases.

Asthma is a chronic inflammatory disease characterised by recurrent airflow obstruction where contraction of the smooth muscle is one main cause of the airway narrowing. Recent observations implicate thymic stromal lymphopoietin (TSLP), interleukin (IL)-33 and IL-25, collectively called epithelial alarmins, as key signalling molecules in asthmatic airway inflammation.1 When airway epithelium is exposed to external stimuli such as allergens, bacteria, viruses and air pollutants, alarmins are released as part of the host defence reaction. The actions of the epithelial alarmins initiate and maintain disease-driving inflammatory processes. Furthermore, tezepelumab, a biologic blocker the TSLP pathway, has recently been introduced as a new therapy in asthma treatment.1 Despite this, the molecular consequences of blocking TSLP or the signalling of other epithelial alarmins in human airways remain incompletely understood. So far, most mechanistic studies of actions of epithelial alarmins in humans have assessed their role in cell models in the context of type 2 (T2) inflammatory processes1; however, their possible effects on physiological processes in intact human airways have not been studied before. The current study was stimulated by the observation that the anti-TSLP antibody tezepelumab can attenuate allergen-induced bronchoconstriction in subjects with asthma.2 We therefore hypothesised that epithelial alarmins may enhance bronchoconstriction by previously unrecognised actions on human bronchi. Consequently, we used isolated human small bronchi to characterise the influence of the exposure to a combination of the three alarmins TSLP, IL-33 and IL-25 on controlled contractile responses. With permission from the regional ethical review board in Stockholm (ref. no. 2018/1819-31/1), tumour-free lung specimens were obtained with patient consent from 17 patients (12 women and 5 men, median age 70 [range 42-76 years]) undergoing lung surgery and consenting to donate removed lung tissue not required for diagnostic purpose. Small airway segments (inner diameter 0.5–2 mm) were isolated and exposed to TSLP, IL-33 and IL-25 (100 ng/mL each; hereafter referred to as the alarmin mix), for 24 or 48 h.3 After the preincubation with the alarmin mix or vehicle, the segments were mounted in separate myographs to study contractile responses. First, cumulative challenge with histamine (10–100 μM) was performed. Following a washout period, the same segments were challenged with two consecutive concentrations of anti-human IgE antibody (anti-IgE), (0.5 and 5 μg/mL, 15 min apart).4 The sensitivity of the bronchial segments to histamine was expressed as the negative logarithm of the concentration of histamine required to reach its half-maximal response (pEC50), and the amplitude of the response to the highest concentration of histamine was presented as the maximal response (Emax) in per cent of the segments' maximal contraction. We have previously shown using the same methodology that challenge of human small bronchi with anti-IgE triggers mast cell-dependent contractions.4 The contractile response to cumulative challenge with two concentrations of anti-IgE was enhanced after the preincubation with the alarmin mix for 48 h (Figure 1A), but not after 24 h (data not shown). In the alarmin mix pretreated group, the total area under the curve (AUC) was increased by 67% compared with the untreated group (1906 ± 274 vs. 1139 ± 185; p = .027; Figure 1B) and the maximal contractile response to the first dose of anti-IgE (0.5 μg/mL) was more than doubled compared with the untreated group (36.9 ± 6.8% vs. 14.5 ± 4.9%; p = .016; Figure 1C). The time to reach the peak contraction was shortened in the alarmin mix pretreated group compared with untreated (24.4 ± 1.0 min vs. 28.2 ± 1.2 min, p = .028; Figure 1D). Hence, the administration of the same concentration of anti-IgE to both treatment groups resulted in an enhanced contractile response in the group pretreated with the alarmin mix. We next investigated whether the enhanced contractile response to the anti-IgE challenge was due to increased release of mast cell mediators or increased responsiveness at the level of bronchial smooth muscle (Figure 2). To monitor mediator release, samples from the organ bath fluid were collected at baseline, 20 and 60 min after the start of the change with anti-IgE. The samples were then analysed for prostaglandin (PG) D2 using ELISA (PGD2-MOX kit: Cayman) and histamine using fluorometric analysis (RefLab ApS). The detected concentrations were normalised to the weight of the respective segment. Increased concentrations of PGD2 were detected in the organ bath fluid of the alarmin mix pretreated group 20 min after the initial challenge with anti-IgE (51.3 ± 14.0 ng/mg tissue; p = .034), but not in the bath fluid of the untreated group (Figure 2A). After 60 min, the concentrations of PGD2 were increased in the bath fluid from both treatment groups (87.4 ± 9.0 ng/mg tissue for vehicle control, p < .001; 146.8 ± 24.3 ng/mg tissue for alarmin mix; p < .001). Notably, at this time, the release of PGD2 was significantly greater in the alarmin mix pretreated group than in the untreated group (p = .031; Figure 2A). These findings support that the alarmin mix enhanced the IgE-triggered release of PGD2, presumably by increasing mast cell reactivity. In the control group, release of histamine into organ bath fluid was not significantly increased from baseline, either at 20 (fold change 1.2 ± 0.3) or at 60 min (fold change 0.8 ± 0.2). In contrast, in the alarmin mix pretreated group, there was a significant increase of histamine release at 60 min after the start of the anti-IgE challenge (fold change 2.9 ± 0.7; p = .045) with a trend of almost reaching significance already at 20 min (fold change 3.3 ± 0.9; p = .059; Figure 2B). The release of histamine in response to IgE-mediated stimulation increased only in the alarmin mix pretreated group, indicating that, as shown for PGD2, the alarmin mix enhanced the IgE-triggered release of histamine, presumably by increasing mast cell reactivity. In contrast to the influence of the alarmins on the indirect contractile response triggered by exposure to anti-IgE, there was no effect of preincubation with the alarmin mix on the responsiveness to histamine. Preincubation with the alarmin mix for neither 24 (data not shown) nor 48 h altered neither the potency (pEC50 6.2 ± 0.1 vs. 6.1 ± 0.1 for vehicle control) nor the efficacy (Emax 99.9 ± 0.1% vs. 98.6 ± 1.1% for vehicle control) of histamine (Figure 2C). In summary, these findings confirmed our hypothesis that the epithelial alarmins have the potential to enhance bronchoconstriction. Specifically, 2 days of combined exposure of isolated small bronchi to TSLP, IL-33 and IL-25 enhanced the contractile response to anti-IgE and the associated release of the mast cell mediators histamine and PGD2, while having no effect on contractions triggered directly at the level of the smooth muscle by exposure to exogenous histamine. The lack of effect of the alarmin mix on responsiveness to histamine in the isolated bronchi is in line with the results of studies of human airway smooth muscle cells, where neither IL-335 nor TSLP has been shown to affect contraction.6 The effect of IL-25 on smooth muscle contractions has not been reported. Using isolated human small bronchi, we have recently shown that preincubation with the T2 cytokines IL-4 and IL-13 increases airway responsiveness to contractile agonists histamine, carbachol and leukotriene D4 that act directly on the smooth muscle.3 While 48-h exposure to IL-4 and IL-13 caused a marked increase in the potency of histamine, the alarmin mix had no direct effect on histamine responsiveness. Instead, the epithelial alarmins selectively enhanced the IgE-mediated indirect contractile response. Our experiments document that pretreatment with the alarmin mix resulted in a more pronounced release of PGD2 and histamine from isolated human bronchi in response to an anti-IgE challenge. This is in line with previous observations in other models, for instance, exposure to the combination of IL-33 and TSLP for 6 h increased release of PGD2 from human mast cells.7 Research on the influence of alarmins on human mast cells is at an early stage and has mostly been performed in combined stimulations. For example, Buchheit et al. showed that only the combined stimulation with IL-33 and TSLP, and not IL-33 alone increased the release of PGD2.7 Likewise, exposure to a combination of IL-33 and TSLP for 1 week before IgE crosslinking caused increase in cysteinyl leukotriene formation in mast cells from patients with exercise-induced bronchoconstriction, whereas exposure to either IL-33 or TSLP alone had no effect.8 The use of the combination of epithelial alarmins has also been found to trigger enhanced release of PGD2 and cytokines in type 2 innate lymphoid cells.9 Thus, for this first proof-of-concept study, it was considered appropriate to use an alarmin mix, such as in previously mentioned studies. This limitation will be addressed in follow-up studies where the actions of the individual epithelial alarmins and their combinations will be defined. There is also a need to establish the influence of longer periods of preincubation as well as the effects of different doses of the alarmins. In conclusion, to the best of our knowledge, this is the first study that explores the effects of epithelial alarmins in intact isolated human small bronchi. The findings implicate that the epithelial alarmins specifically increase the reactivity of airway mast cells. This mode of action may explain why the clinical effects of the first registered anti-alarmin tezepelumab include rapid attenuation of the allergen-induced bronchoconstriction and eosinophil-independent improvement of severe asthma.1, 2 MB performed experiments and data analysis and wrote the first version of the manuscript. MA-A and A-CO provided surgical specimens and contributed to study design and interpretation. MA and SED obtained ethics approval. MA, JS and SED had the general project responsibility, performed experimental design and finalised the manuscript together with MB. All authors read, revised and approved the manuscript. We thank Susanne Hylander for her dedicated management of the logistics for the collection of human lung specimens and all co-workers at the Heart and Vascular Theme as well as at Perioperative Medicine & Intensive Care for their contributions. We also gratefully acknowledge the patients at the Heart and Vascular Theme. This work was supported by Magnus Bergvall Foundation, Konsul Th C Berg Foundation, Swedish Heart-Lung Foundation, Swedish Research Council—Medicine and Health, Stockholm County Council Research Funds (ALF), Swedish Society of Medicine, Centre for Allergy Research Highlights Asthma Markers of Phenotype consortium which is funded by the Swedish Foundation for Strategic Research, Karolinska Institutet, AstraZeneca & Science for Life Laboratory Joint Research Collaboration and Vårdal Foundation. The funding sources were not involved in either of the following: preparation of the article, study design, collection, analysis and interpretation of data, writing of the report and decision to submit the article for publication. The authors have no conflicts of interest to declare. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Background: Recent observations indicate that epithelial alarmins thymic stromal lymphopoetin (TSLP), interleukin (IL)-33, and IL-25 are key signalling molecules in asthmatic airway inflammation. Aim: The aim was to test if combined pre-treatment with TSLP, IL-33 and IL-25 (alarmin mix) affected bronchoconstriction in isolated human small airways. Methods: Human lung tissue was obtained with consent from 17 patients undergoing lobectomies. Intact segments of isolated small bronchi (inner ⌀ 0.5-2 mm) were exposed to the alarmin mix (100 ng/mL each) for 48 hours. The segments were placed in organ baths where contractile responses to first histamine and then anti-human IgE antibody (anti-IgE, 0.5 and 5 μg/mL) were studied in myographs, and mediator release was analysed. Results: Pre-treatment with the alarmin mix enhanced the maximal contractile response (36.9±6.8 vs. 14.5±4.9%) to anti-IgE (0.5 μg/mL) and the time to reach peak contraction in response to anti-IgE (5 μg/mL) was shortened (24.4±1.0 vs. 28.2±1.2 min) compared to untreated. One hour after anti-IgE exposure, levels of prostaglandin (PG)D2 (146.8±24.3 vs. 87.4±9.0 ng/mg tissue) and histamine (2.9±0.7 vs. 0.8±0.2-fold change from baseline) were increased in the bath fluid from the alarmin mix pre-treated group compared to untreated. In contrast, the pre-treatment with the alarmin mix did not affect the histamine-mediated contractile response. Conclusions: Epithelial alarmins enhanced the IgE-dependent bronchoconstriction by increasing the release of mast cell mediators PGD2 and histamine without changing smooth muscle responsiveness to histamine. These findings highlight the potential of alarmins as regulators of airway mast cell responsiveness.

AbstractBackgroundActivation of sphingomyelinase (SMase) as a result of a general inflammatory response has been implicated as a mechanism underlying disease‐related loss of skeletal muscle mass and function in several clinical conditions including heart failure. Here, for the first time, we characterize the effects of SMase activity on human muscle fibre contractile function and assess skeletal muscle SMase activity in heart failure patients.MethodsThe effects of SMase on force production and intracellular Ca2+ handling were investigated in single intact human muscle fibres. Additional mechanistic studies were performed in single mouse toe muscle fibres. RNA sequencing was performed in human muscle bundles exposed to SMase. Intramuscular SMase activity was measured from heart failure patients (n = 61, age 69 ± 0.8 years, NYHA III‐IV, ejection fraction 25 ± 1.0%, peak VO2 14.4 ± 0.6 mL × kg × min) and healthy age‐matched control subjects (n = 10, age 71 ± 2.2 years, ejection fraction 60 ± 1.2%, peak VO2 25.8 ± 1.1 mL × kg × min). SMase activity was related to circulatory factors known to be associated with progression and disease severity in heart failure.ResultsSphingomyelinase reduced muscle fibre force production (−30%, P < 0.05) by impairing sarcoplasmic reticulum (SR) Ca2+ release (P < 0.05) and reducing myofibrillar Ca2+ sensitivity. In human muscle bundles exposed to SMase, RNA sequencing analysis revealed 180 and 291 genes as up‐regulated and down‐regulated, respectively, at a FDR of 1%. Gene‐set enrichment analysis identified ‘proteasome degradation’ as an up‐regulated pathway (average fold‐change 1.1, P = 0.008), while the pathway ‘cytoplasmic ribosomal proteins’ (average fold‐change 0.8, P < 0.0001) and factors involving proliferation of muscle cells (average fold‐change 0.8, P = 0.0002) where identified as down‐regulated. Intramuscular SMase activity was ~20% higher (P < 0.05) in human heart failure patients than in age‐matched healthy controls and was positively correlated with markers of disease severity and progression, and with several circulating inflammatory proteins, including TNF‐receptor 1 and 2. In a longitudinal cohort of heart failure patients (n = 6, mean follow‐up time 2.5 ± 0.2 years), SMase activity was demonstrated to increase by 30% (P < 0.05) with duration of disease.ConclusionsThe present findings implicate activation of skeletal muscle SMase as a mechanism underlying human heart failure‐related loss of muscle mass and function. Moreover, our findings strengthen the idea that SMase activation may underpin disease‐related loss of muscle mass and function in other clinical conditions, acting as a common patophysiological mechanism for the myopathy often reported in diseases associated with a systemic inflammatory response.

Background Immunohistochemical analysis of granule-associated proteases has revealed that human lung mast cells constitute a heterogeneous population of cells, with distinct subpopulations identified. However, a systematic and comprehensive analysis of cell-surface markers to study human lung mast cell heterogeneity has yet to be performed. Methods Human lung mast cells were obtained from lung lobectomies, and the expression of 332 cell-surface markers was analyzed using flow cytometry and the LEGENDScreen™ kit. Markers that exhibited high variance were selected for additional analyses to reveal whether they were correlated and whether discrete mast cell subpopulations were discernable. Results We identified the expression of 102 surface markers on human lung mast cells, 23 previously not described on mast cells, of which several showed high continuous variation in their expression. Six of these markers were correlated: SUSD2, CD49a, CD326, CD34, CD66 and HLA-DR. The expression of these markers was also correlated with the size and granularity of mast cells. However, no marker produced an expression profile consistent with a bi- or multimodal distribution. Conclusions LEGENDScreen analysis identified more than 100 cell-surface markers on mast cells, including 23 that, to the best of our knowledge, have not been previously described on human mast cells. The comprehensive expression profiling of the 332 surface markers did not identify distinct mast cell subpopulations. Instead, we demonstrate the continuous nature of human lung mast cell heterogeneity.

The impact of the microenvironment on innate lymphoid cell (ILC)-mediated immunity in humans remains largely unknown. Here we used full-length Smart-seq2 single-cell RNA-sequencing to unravel tissue-specific transcriptional profiles and heterogeneity of CD127 + ILCs across four human tissues. Correlation analysis identified gene modules characterizing the migratory properties of tonsil and blood ILCs, and signatures of tissue-residency, activation and modified metabolism in colon and lung ILCs. Trajectory analysis revealed potential differentiation pathways from circulating and tissue-resident naïve ILCs to a spectrum of mature ILC subsets. In the lung we identified both CRTH2 + and CRTH2 − ILC2 with lung-specific signatures, which could be recapitulated by alarmin-exposure of circulating ILC2. Finally, we describe unique TCR-V(D)J-rearrangement patterns of blood ILC1-like cells, revealing a subset of potentially immature ILCs with TCR-δ rearrangement. Our study provides a useful resource for in-depth understanding of ILC-mediated immunity in humans, with implications for disease.

Human adaptive-like "memory" CD56dimCD16+ natural killer (NK) cells in peripheral blood from cytomegalovirus-seropositive individuals have been extensively investigated in recent years and are currently explored as a treatment strategy for hematological cancers. However, treatment of solid tumors remains limited due to insufficient NK cell tumor infiltration, and it is unknown whether large expansions of adaptive-like NK cells that are equipped for tissue residency and tumor homing exist in peripheral tissues. Here, we show that human lung and blood contains adaptive-like CD56brightCD16- NK cells with hallmarks of tissue residency, including expression of CD49a. Expansions of adaptive-like lung tissue-resident NK (trNK) cells were found to be present independently of adaptive-like CD56dimCD16+ NK cells and to be hyperresponsive toward target cells. Together, our data demonstrate that phenotypically, functionally, and developmentally distinct subsets of adaptive-like NK cells exist in human lung and blood. Given their tissue-related character and hyperresponsiveness, human lung adaptive-like trNK cells might represent a suitable alternative for therapies targeting solid tumors.

Background: Specific inflammatory pathways are indicated to contribute to severe asthma, but their individual involvement in the development of airway hyperresponsiveness remains unexplored. Objective: This experimental study in human small bronchi aimed to provide insight into which of the type 2 and type 17 cytokines cause hyperresponsiveness of airway smooth muscle. Methods: Explanted small bronchi isolated from human lung tissue and human airway smooth muscle cells were treated for 2 and 1 day(s), respectively, with 100 ng/mL of IL-4, IL-5, IL-13, or IL-17A, and contractile responses, Ca2+ mobilization, and receptor expression were Results: Treatment with IL-13 increased the potency of histamine, carbachol, and leukotriene D-4 as contractile agonists. IL-4, but not IL-5 or IL-17A, also increased the potency of histamine. In human airway smooth muscle cells, IL-13 and IL-4, but not IL-5 and IL-17A, enhanced the histamine-induced Ca2+ mobilization that was accompanied with increased mRNA expression of histamine H-1 and cysteinyl leukotriene CysLT(1) receptors. RNA sequencing of isolated bronchi confirmed the IL-13-mediated upregulation of H-1 and CysLT(1) receptors, without showing an alteration of muscarinic M3 receptors. Dexamethasone had no effects on IL-13-induced hyperresponsiveness in human bronchi, the increased Ca2+ mobilization, or the enhanced receptor expression. In contrast, antagonism of the common receptor for IL-13 and IL-4 by the biologic dupilumab prevented the effects of both IL-13 and IL-4 in human bronchi and human airway smooth muscle cells. Conclusions: The glucocorticoid-insensitive hyperrresponsiveness in isolated human airways induced by IL13 and IL-4 provides further evidence that the IL-4Ra pathway should be targeted as a new strategy for the treatment of airway hyperresponsiveness in asthma.

[This corrects the article DOI: 10.21037/jtd.2018.05.177.].

Background Epithelial cytokines, including IL-33 and TSLP, have attracted interest because of their roles in chronic allergic inflammation-related conditions such as asthma. Mast cells are one of the major targets of IL-33, to which they respond by secreting cytokines. Most studies performed thus far have investigated the acute effects of IL-33 on mast cells. Objective The objective of this study is to investigate how acute versus prolonged exposure of human mast cells to IL-33 and TSLP affects mediator synthesis and IgE-mediated activation. Methods Human lung mast cells (HLMCs), cord blood-derived mast cells (CBMCs), and the ROSA mast cell line were used for this study. Surface receptor expression and the levels of mediators were measured after treatment with IL-33 and/or TSLP. Results IL-33 induced the acute release of cytokines. Prolonged exposure to IL-33 increased while TSLP reduced intracellular levels of tryptase. Acute IL-33 treatment strongly potentiated IgE-mediated activation. In contrast, four days of exposure to IL-33 decreased IgE-mediated activation, an effect that was accompanied by a reduction in FcεRI expression. Conclusion & Clinical Relevance We show that IL-33 plays dual roles for mast cell functions. The acute effect includes cytokine release and the potentiation of IgE-mediated degranulation, whereas prolonged exposure to IL-33 reduces IgE-mediated activation. We conclude that mast cells act quickly in response to the alarmin IL-33 to initiate an acute inflammatory response, whereas extended exposure to IL-33 during prolonged inflammation reduces IgE-mediated responses. This negative feedback effect suggests the presence of a novel IL-33 mediated regulatory pathway that modulates IgE-induced human mast cell responses.

Background: Video-assisted thoracic surgery (VATS) lobectomy is the recommended surgical approach for patients with stage I lung cancer. Whether a multiportal or a uniportal approach is preferable remains unclear. The aim of this study was to evaluate the safety of implementing uniportal VATS lobectomy into the treatment program of lung cancer patients. Methods: We used the national quality register for general thoracic surgery in Sweden and included all patients who underwent VATS lobectomy for lung cancer at the Karolinska University Hospital between 2016-2018. Early postoperative complications were compared in patients undergoing uniportal (n=122) and multiportal (n=211) VATS lobectomy for lung cancer. Inverse probability of treatment weighting and standardized mean differences were used to limit differences in baseline characteristics and to assess balance after weighting. Results: The proportion of uniportal VATS lobectomies increased during the study period and the conversion rates declined significantly. Baseline characteristics were similar in the two groups with the exception of a higher percentage of patients without any comorbidity in the uniportal group (59.8% vs. 44.5%, P=0.010). After inverse probability of treatment weighting the groups were well balanced. Postoperative complications were rare regardless of surgical approach, 94% in both groups had no complications. The 30-day mortality and overall survival at 1 year was 0% and 97% in the uniportal group, and 0.5% and 98% in the multiportal group (P=0.71). Patients undergoing uniportal VATS lobectomy were discharged directly to home to a higher extent than multiportal VATS patients (76.2% vs. 62.1%, P=0.008). Conclusions: We found that uniportal VATS lobectomy was feasible and safe, and might entail advantages in terms of a faster recovery after surgery as compared to multiportal VATS lobectomy in patients with lung cancer.