Expression of c-Kit discriminates between two functionally distinct subsets of human type 2 innate lymphoid cells.

Human type 2 innate lymphoid cells (ILC2) are the only ILC subset that shows heterogeneous expression of the SCF receptor c-Kit (CD117). Despite its use as surface marker to distinguish ILC populations, its influence on ILC2 biology has not been investigated. Here, we show that c-Kit expression of peripheral blood ILC distinguishes two functionally distinct ILC2 subsets (c-Kithi and c-Kitlo ). When examined for their potential for functional plasticity we found that c-Kitlo ILC2 displayed greater potential to produce type 2 cytokines, possibly representing fully mature and lineage committed ILC2. On the other hand, c-Kithi ILC2 coexpressed the ILC3-marker and chemokine receptor CCR6 and were able to mount a significant IL-17A response under ILC3-promoting conditions. In addition, c-Kithi ILC2 produced higher levels of IFN-γ than c-Kitlo ILC2 under ILC1-conditions. Although costimulation with SCF did not further influence ILC2 plasticity, it augmented type 2 cytokine production. We conclude that c-Kit marks distinct subpopulations of ILC2, which has therapeutic implications for conditions in which ILC2 are involved, such as allergy and asthma.

Activation of group 2 innate lymphoid cells after allergen challenge in asthmatic patients.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are effective producers of IL-5 and IL-13 during allergic inflammation and bridge the innate and adaptive immune responses. ILC2 numbers are increased in asthmatic patients compared with healthy control subjects. Thus far, human data describing their phenotype during acute allergic inflammation in the lung are incomplete. OBJECTIVES: This study aims to characterize and compare blood- and lung-derived ILC2s before and after segmental allergen challenge in patients with mild-to-moderate asthma with high blood eosinophil counts (≥300 cells/µL). METHODS: ILC2s were isolated from blood and bronchoalveolar lavage (BAL) fluid before and after segmental allergen challenge. Cells were sorted by means of flow cytometry, cultured and analyzed for cytokine release or migration, and sequenced for RNA expression. RESULTS: ILC2s were nearly absent in the alveolar space under baseline conditions, but numbers increased significantly after allergen challenge (P < .05), whereas at the same time, ILC2 numbers in blood were reduced (P < .05). Prostaglandin D2 and CXCL12 levels in BAL fluid correlated with decreased ILC2 numbers in blood (P = .004, respective P = .024). After allergen challenge, several genes promoting type 2 inflammation were expressed at greater levels in BAL fluid compared with blood ILC2s, whereas blood ILC2s remain unactivated. CONCLUSION: ILC2s accumulate at the site of allergic inflammation and are recruited from the blood. Their transcriptional and functional activation pattern promotes type 2 inflammation.

Noninvasive quantification of airway inflammation following segmental allergen challenge with functional MR imaging: a proof of concept study.

PURPOSE: To evaluate oxygen-enhanced T1-mapping magnetic resonance (MR) imaging as a noninvasive method for visualization and quantification of regional inflammation after segmental allergen challenge in asthmatic patients compared with control subjects. MATERIALS AND METHODS: After institutional review board approval, nine asthmatic and four healthy individuals gave written informed consent. MR imaging (1.5 T) was performed by using an inversion-recovery snapshot fast low-angle shot sequence before (0 hours) and 6 hours and 24 hours after segmental allergen challenge by using either normal- or low-dose allergen or saline. The volume of lung tissue with increased relaxation times was determined by using a threshold-based method. As a biomarker for oxygen transfer from the lungs into the blood, the oxygen transfer function ( OTF oxygen transfer function ) was calculated. After the third MR imaging examination, eosinophils in bronchoalveolar lavage fluid were counted. Differences between times and segments were analyzed with nonparametric Wilcoxon matched-pairs test and Spearman correlation. RESULTS: In lung segments treated with the standard dose of allergen, the OTF oxygen transfer function was decreased at 6 hours in asthmatic patients, compared with saline-treated segments (P = .0078). In asthmatic patients at 24 hours, the volume over threshold was significantly increased in normal allergen dose-treated segments compared with saline-treated segments (P = .004). In corresponding lung segments, the volume over threshold at 24 hours in the asthmatic group showed a positive correlation (r = 0.65, P = .0001) and the OTF oxygen transfer function at 6 hours showed an inverse correlation (r = -0.67, P = .0001) with the percentage of eosinophils in the bronchoalveolar lavage fluid. CONCLUSION: OTF oxygen transfer function and volume over threshold are noninvasive MR imaging-derived parameters to visualize and quantify the regional allergic reaction after segmental endobronchial allergen challenge.

Pathogen reduction by ultraviolet C light effectively inactivates human white blood cells in platelet products.

BACKGROUND: Residual white blood cells (WBCs) in cellular blood components induce a variety of adverse immune events, including nonhemolytic febrile transfusion reactions, alloimmunization to HLA antigens, and transfusion-associated graft-versus-host disease (TA-GVHD). Pathogen reduction (PR) methods such as the ultraviolet C (UVC) light-based THERAFLEX UV-Platelets system were developed to reduce the risk of transfusion-transmitted infection. As UVC light targets nucleic acids, it interferes with the replication of both pathogens and WBCs. This preclinical study aimed to evaluate the ability of UVC light to inactivate contaminating WBCs in platelet concentrates (PCs). STUDY DESIGN AND METHODS: The in vitro and in vivo function of WBCs from UVC-treated PCs was compared to that of WBCs from gamma-irradiated and untreated PCs by measuring cell viability, proliferation, cytokine secretion, antigen presentation in vitro, and xenogeneic GVHD responses in a humanized mouse model. RESULTS: UVC light was at least as effective as gamma irradiation in preventing GVHD in the mouse model. It was more effective in suppressing T-cell proliferation (>5-log reduction in the limiting dilution assay), cytokine secretion, and antigen presentation than gamma irradiation. CONCLUSIONS: The THERAFLEX UV-Platelets (MacoPharma) PR system can substitute gamma irradiation for TA-GVHD prophylaxis in platelet (PLT) transfusion. Moreover, UVC treatment achieves suppression of antigen presentation and inhibition of cytokine accumulation during storage of PCs, which has potential benefits for transfusion recipients.

Effects of ultrafine particles on the allergic inflammation in the lung of asthmatics: results of a double-blinded randomized cross-over clinical pilot study.

BACKGROUND: Epidemiological and experimental studies suggest that exposure to ultrafine particles (UFP) might aggravate the allergic inflammation of the lung in asthmatics. METHODS: We exposed 12 allergic asthmatics in two subgroups in a double-blinded randomized cross-over design, first to freshly generated ultrafine carbon particles (64 µg/m³; 6.1 ± 0.4 × 105 particles/cm³ for 2 h) and then to filtered air or vice versa with a 28-day recovery period in-between. Eighteen hours after each exposure, grass pollen was instilled into a lung lobe via bronchoscopy. Another 24 hours later, inflammatory cells were collected by means of bronchoalveolar lavage (BAL). ( TRIAL REGISTRATION: NCT00527462) RESULTS: For the entire study group, inhalation of UFP by itself had no significant effect on the allergen induced inflammatory response measured with total cell count as compared to exposure with filtered air (p = 0.188). However, the subgroup of subjects, which inhaled UFP during the first exposure, exhibited a significant increase in total BAL cells (p = 0.021), eosinophils (p = 0.031) and monocytes (p = 0.013) after filtered air exposure and subsequent allergen challenge 28 days later. Additionally, the potential of BAL cells to generate oxidant radicals was significantly elevated at that time point. The subgroup that was exposed first to filtered air and 28 days later to UFP did not reveal differences between sessions. CONCLUSIONS: Our data demonstrate that pre-allergen exposure to UFP had no acute effect on the allergic inflammation. However, the subgroup analysis lead to the speculation that inhaled UFP particles might have a long-term effect on the inflammatory course in asthmatic patients. This should be reconfirmed in further studies with an appropriate study design and sufficient number of subjects.

Impact of a Met(11)Thr single nucleotide polymorphism of surfactant protein D on allergic airway inflammation in a murine asthma model.

The surfactant-associated proteins SP-A and D are pattern recognition molecules with collectin structure. A single nucleotide polymorphism (SNP) exchanging a methionine (Met) for a threonine (Thr) in the amino-terminal SP-D domain influences the oligomeric structure and function of the protein. In this study, we investigated the susceptibility of mice transgenic for the human SP-D Met(11)Thr SNP to allergic airway inflammation and consequences for microRNA (miRNA, miR) expression. Mice expressing either human Met or Thr SP-D were sensitized and challenged with ovalbumin (OVA) in an acute model of allergic asthma. The influence of the SP-D polymorphism on the allergic airway inflammation was evaluated by lung function measurement, pulmonary inflammation parameters, morphological analysis and miRNA expression. Airway hyperresponsiveness, allergic inflammation, and mucus metaplasia were not significantly different between mice expressing one or the other allelic variant of SP-D. OVA sensitization and challenge led to significant airway hyperresponsiveness in wildtype mice and significantly lower eosinophil numbers and interleukin 5 levels in Thr SP-D mice. OVA challenge induced an upregulation of miR-21 and 155 in Thr SP-D mice and a downregulation of miR-21 in Met SP-D mice. Our results show that murine expression of human polymorphic SP-D variants does not significantly influence the severity of allergic airway inflammation. MiR-21 and 155 are differentially regulated in transgenic mice in response to allergic inflammation. Further studies are required to elucidate the impact of this SNP on inflammatory conditions of the lung.

Impact of endobronchial allergen provocation on macrophage phenotype in asthmatics.

BACKGROUND: The role of M2 polarized macrophages (MΦ) during the allergic airway inflammation has been discussed in various animal models. However, their presence and relevance during the chronic and acute phase of allergic airway inflammation in humans has not been fully elucidated so far. In the present study we phenotypically characterized macrophages with regard to M2 polarization in mice, a human in vitro and a human ex vivo model with primary lung cells after endobronchial provocation. RESULTS: Macrophages remained polarized beyond clearance of the acute allergic airway inflammation in mice. Alveolar macrophages of asthmatics revealed increased mRNA expression of CCL13, CCL17 and CLEC10A in response to allergen challenge as well as increased surface expression of CD86. Further, mRNA expression of CCL13, CCL17, and CLEC10A was increased in asthmatics at baseline compared to healthy subjects. The mRNA expression of CCL17 and CLEC10A correlated significantly with the degree of eosinophilia (each P < .01). Furthermore, macrophages from asthmatics released significant amounts of CCL17 protein in vitro which was also found increased in BAL fluid after allergen provocation. CONCLUSIONS: This study supports previous findings of M2 macrophage polarization in asthmatic subjects during the acute course of the allergic inflammation and provides evidence for their contribution to the Th2 inflammation.

Quantification of pulmonary inflammation after segmental allergen challenge using turbo-inversion recovery-magnitude magnetic resonance imaging.

RATIONALE: There is a need to develop novel noninvasive imaging biomarkers that help to evaluate antiinflammatory asthma treatments. OBJECTIVES: To investigate whether the extent of the segmental lung edema measured noninvasively using turbo-inversion recovery-magnitude magnetic resonance imaging (TIRM MRI) corresponds to the severity of the regional allergic reaction determined by the percentage of eosinophils in bronchoalveolar lavage fluid (BAL) 24 hours after segmental allergen challenge in patients with asthma compared with normal control subjects. METHODS: Eleven volunteers with allergic asthma and five healthy volunteers underwent segmental challenges with different allergen doses by two bronchoscopies 24 hours apart. They had lung MRI at baseline and 6 and 24 hours after segmental challenge. MRI TIRM scores were correlated with the eosinophilic response at 24 hours. MEASUREMENTS AND MAIN RESULTS: In patients with asthma, there were significant differences of eosinophil percentages in BAL at 24 hours from segments given standard-dose, low-dose, or no allergen (saline) (P < 0.001). Correspondingly significant differences between the TIRM score in allergen standard-dose, low-dose, and saline-treated segments were observed at 24 hours post-challenge (P < 0.001). With increasing TIRM score at 24 hours the percent eosinophils per segment 24 hours post-challenge also increased accordingly (P < 0.001). There was interobserver agreement for TIRM score grading (kappa = 0.72 for 24-h time point). CONCLUSIONS: The MRI-based noninvasive TIRM score is a promising biomarker for the noninvasive detection of the inflammatory response after segmental allergen challenge in patients with asthma and may serve to monitor the therapeutic effectiveness of novel antiinflammatory drugs in future human trials.

Surfactant and allergic airway inflammation.

Pulmonary surfactant is a complex mixture of unique proteins and lipids that covers the airway lumen. Surfactant prevents alveolar collapse and maintains airway patency by reducing surface tension at the air-liquid interface. Furthermore, it provides a defence against antigen uptake by binding foreign particles and enhancing cellular immune responses. Allergic asthma is associated with chronic airway inflammation and presents with episodes of airway narrowing. The pulmonary inflammation and bronchoconstriction can be triggered by exposure to allergens or pathogens present in the inhaled air. Pulmonary surfactant has the potential to interact with various immune cells which orchestrate allergen- or pathogen-driven episodes of airway inflammation. The complex nature of surfactant allows multiple sites of interaction, but also makes it susceptible to external alterations, which potentially impair its function. This duality of modulating airway physiology and immunology during inflammatory conditions, while at the same time being prone to alterations accompanied by restricted function, has stimulated numerous studies in recent decades, which are reviewed in this article.

Low-dose endotoxin inhalation in healthy volunteers--a challenge model for early clinical drug development.

BACKGROUND: Inhalation of endotoxin (LPS) induces a predominantly neutrophilic airway inflammation and has been used as model to test the anti-inflammatory activity of novel drugs. In the past, a dose exceeding 15-50 µg was generally needed to induce a sufficient inflammatory response. For human studies, regulatory authorities in some countries now request the use of GMP-grade LPS, which is of limited availability. It was therefore the aim of this study to test the effect and reproducibility of a low-dose LPS challenge (20,000 E.U.; 2 µg) using a flow- and volume-controlled inhalation technique to increase LPS deposition. METHODS: Two to four weeks after a baseline sputum induction, 12 non-smoking healthy volunteers inhaled LPS on three occasions, separated by at least 4 weeks. To modulate the inflammatory effect of LPS, a 5-day PDE4 inhibitor (Roflumilast) treatment preceded the last challenge. Six hours after each LPS inhalation, sputum induction was performed. RESULTS: The low-dose LPS inhalation was well tolerated and increased the mean percentage of sputum neutrophils from 25% to 72%. After the second LPS challenge, 62% neutrophils and an increased percentage of monocytes were observed. The LPS induced influx of neutrophils and the cumulative inflammatory response compared with baseline were reproducible. Treatment with Roflumilast for 5 days did not have a significant effect on sputum composition. CONCLUSION: The controlled inhalation of 2 µg GMP-grade LPS is sufficient to induce a significant neutrophilic airway inflammation in healthy volunteers. Repeated low-dose LPS challenges potentially result in a small shift of the neutrophil/monocyte ratio; however, the cumulative response is reproducible, enabling the use of this model for "proof-of-concept" studies for anti-inflammatory compounds during early drug development.

Comprehensive characterisation of pulmonary and serum surfactant protein D in COPD.

BACKGROUND: Pulmonary surfactant protein D (SP-D) is considered as a candidate biomarker for the functional integrity of the lung and for disease progression, which can be detected in serum. The origin of SP-D in serum and how serum concentrations are related to pulmonary concentrations under inflammatory conditions is still unclear. METHODS: In a cross-sectional study comprising non-smokers (n=10), young--(n=10), elderly smokers (n=20), and smokers with COPD (n=20) we simultaneously analysed pulmonary and serum SP-D levels with regard to pulmonary function, exercise, repeatability and its quaternary structure by native gel electrophoresis. Statistical comparisons were conducted by ANOVA and post-hoc testing for multiple comparisons; repeatability was assessed by Bland-Altman analysis. RESULTS: In COPD, median (IQR) pulmonary SP-D levels were lower (129(68) ng/ml) compared to smokers (young: 299(190), elderly: 296(158) ng/ml; p<0.01) and non-smokers (967(708) ng/ml; p<0.001). The opposite was observed in serum, with higher concentrations in COPD (140(89) ng/ml) as compared to non-smokers (76(47) ng/ml; p<0.01). SP-D levels were reproducible and correlated with the degree of airway obstruction in all smokers. In addition, smoking lead to disruption of the quaternary structure. CONCLUSIONS: Pulmonary and serum SP-D levels are stable markers influenced by smoking and related to airflow obstruction and disease state. Smaller subunits of pulmonary SP-D and the rapid increase of serum SP-D levels in COPD due to exercise support the translocation hypothesis and its use as a COPD biomarker. TRIAL REGISTRATION: no interventional trial.

Segmental allergen challenge alters multimeric structure and function of surfactant protein D in humans.

RATIONALE: Surfactant protein D (SP-D), a 43-kD collectin, is synthesized and secreted by airway epithelia as a dodecamer formed by assembly of four trimeric subunits. We have previously shown that the quaternary structure of SP-D can be altered during inflammatory lung injury through its modification by S-nitrosylation, which in turn alters its functional behavior producing a proinflammatory response in effector cells. OBJECTIVES: We hypothesized that alterations in structure and function of SP-D may occur in humans with acute allergic inflammation. METHODS: Bronchoalveolar lavage (BAL) fluid was collected from 15 nonsmoking patients with mild intermittent allergic asthma before and 24 hours after segmental provocation with saline, allergen, LPS, and mixtures of allergen and LPS. Structural modifications of SP-D were analyzed by native and sodium dodecyl sulfate gel electrophoresis. MEASUREMENTS AND MAIN RESULTS: The multimeric structure of native SP-D was found to be disrupted after provocation with allergen or a mixture of allergen and LPS. Interestingly, under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that 7 of 15 patients with asthma developed an abnormal cross-linked SP-D band after segmental challenge with either allergen or a mixture of allergen with LPS but not LPS alone. Importantly, patients with asthma with cross-linked SP-D demonstrated significantly higher levels of BAL eosinophils, nitrogen oxides, IL-4, IL-5, IL-13, and S-nitrosothiol-SP-D compared with patients without cross-linked SP-D. CONCLUSIONS: We conclude that segmental allergen challenge results in changes of SP-D multimeric structure and that these modifications are associated with an altered local inflammatory response in the distal airways.

Surfactant protein D modulates pulmonary clearance of pollen starch granules.

Pollen starch granules (PSGs) are allergen particles that get into contact with pulmonary surfactant and phagocytes in the terminal airways. In this study, the effects of surfactant protein D (SP-D) on the interaction of PSGs with phagocytes and on the pulmonary clearance of PSGs were determined. Fluorescently labeled PSGs were incubated in vitro with murine lung macrophages or dendritic cells (DCs) ± recombinant rat SP-D (rrSP-D). In addition, the effect of SP-D on uptake of PSGs by lung macrophages and DCs was studied in vivo. Furthermore, PSGs were instilled in Balb/c mice and the effects of SP-D on total lung clearance were assessed by optical imaging. SP-D treatment increased the number of PSG-positive macrophages and DCs in vitro. Furthermore, SP-D accelerated uptake/binding by alveolar macrophages and reduced the number of PSG-positive tissue macrophages and DCs at 24 hours. However, SP-D did not affect total lung clearance of PSGs and it did not enhance the T-cell proliferation induced by PSG-positive DCs. In conclusion, SP-D increased PSG-positive cells in vitro and accelerated PSG binding/uptake in vivo. The observed effects were limited to cellular clearance mechanisms and did not affect the total clearance of PSGs from the lung.

Allergen particle binding by human primary bronchial epithelial cells is modulated by surfactant protein D.

BACKGROUND: Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. Our previous work demonstrated that SP-D increases the uptake of SPP by alveolar macrophages. In the present study, we investigated the uptake of SPP in human primary epithelial cells and the potential modulation by SP-D. The patho-physiological consequence was evaluated by measurement of pro-inflammatory mediators. METHODS: SPP were isolated from timothy grass and subsequently fluorescently labelled. Human primary bronchial epithelial cells were incubated with SPP or polystyrene particles (PP) in the presence and absence of surfactant protein D. In addition, different sizes and surface charges of the PP were studied. Particle uptake was evaluated by flow cytometry and confocal microscopy. Soluble mediators were measured by enzyme linked immunosorbent assay or bead array. RESULTS: SPP were taken up by primary epithelial cells in a dose dependent manner. This uptake was coincided with secretion of Interleukin (IL)-8. SP-D increased the fraction of bronchial epithelial cells that bound SPP but not the fraction of cells that internalized SPP. SPP-induced secretion of IL-8 was further increased by SP-D. PP were bound and internalized by epithelial cells but this was not modulated by SP-D. CONCLUSIONS: Epithelial cells bind and internalize SPP and PP which leads to increased IL-8 secretion. SP-D promotes attachment of SPP to epithelial cells and may thus be involved in the inflammatory response to inhaled allergen.