Characterization of phospholipid-modified lung surfactant in vitro and in a neonatal ARDS model reveals anti-inflammatory potential and surfactant lipidome signatures.

A strong inflammatory immune response drives the lung pathology in neonatal acute respiratory distress syndrome (nARDS). Anti-inflammatory therapy is therefore a promising strategy for improved treatment of nARDS. We demonstrate a new function of the anionic phospholipids POPG, DOPG, and PIP2 as inhibitors of IL-1ß release by LPS and ATP-induced inflammasome activation in human monocyte-derived and lung macrophages. Curosurf® surfactant was enriched with POPG, DOPG, PIP2 and the head-group derivative IP3, biophysically characterized and applicability was evaluated in a piglet model of nARDS. The composition of pulmonary surfactant from piglets was determined by shotgun lipidomics screens. After 72 h of nARDS, levels of POPG, DOPG, and PIP2 were enhanced in the respective treatment groups. Otherwise, we did not observe changes of individual lipid species in any of the groups. Surfactant proteins were not affected, with the exception of the IP3 treated group. Our data show that POPG, DOPG, and PIP2 are potent inhibitors of inflammasome activation; their enrichment in a surfactant preparation did not induce any negative effects on lipid profile and reduced biophysical function in vitro was mainly observed for PIP2. These results encourage to rethink the current strategies of improving surfactant preparations by inclusion of anionic lipids as potent anti-inflammatory immune regulators.

IL-37 regulates allergic inflammation by counterbalancing pro-inflammatory IL-1 and IL-33.

BACKGROUND: Children with asthma have impaired production of interleukin (IL) 37; in mice, IL-37 reduces hallmarks of experimental allergic asthma (EAA). However, it remains unclear how IL-37 exerts its inhibitory properties in asthma. This study aimed to identify the mechanism(s) by which IL-37 controls allergic inflammation. METHODS: IL-37 target cells were identified by single-cell RNA-seq of IL-1R5 and IL-1R8. Airway tissues were isolated by laser-capture microdissection and examined by microarray-based gene expression analysis. Mononuclear cells (MNC) and airway epithelial cells (AECs) were isolated and stimulated with allergen, IL-1ß, or IL-33 together with recombinant human (rh) IL-37. Wild-type, IL-1R1- and IL-33-deficient mice with EAA were treated with rhIL-37. IL-1ß, IL-33, and IL-37 levels were determined in sputum and nasal secretions from adult asthma patients without glucocorticoid therapy. RESULTS: IL-37 target cells included AECs, T cells, and dendritic cells. In mice with EAA, rhIL-37 led to differential expression of >90 genes induced by IL-1ß and IL-33. rhIL-37 reduced production of Th2 cytokines in allergen-activated MNCs from wild-type but not from IL-1R1-deficient mice and inhibited IL-33-induced Th2 cytokine release. Furthermore, rhIL-37 attenuated IL-1ß- and IL-33-induced pro-inflammatory mediator expression in murine AEC cultures. In contrast to wild-type mice, hIL-37 had no effect on EAA in IL-1R1- or IL-33-deficient mice. We also observed that expression/production ratios of both IL-1ß and IL-33 to IL-37 were dramatically increased in asthma patients compared to healthy controls. CONCLUSION: IL-37 downregulates allergic airway inflammation by counterbalancing the disease-amplifying effects of IL-1ß and IL-33.

Mission impossible?: A cultural change to support scientific integrity.

The Research Center Borstel developed a bottom-up approach based on communication and team scouts to create a culture that fosters research integrity.

Constitutive immune activity promotes JNK- and FoxO-dependent remodeling of Drosophila airways.

Extensive remodeling of the airways is a major characteristic of chronic inflammatory lung diseases such as asthma or chronic obstructive pulmonary disease (COPD). To elucidate the importance of a deregulated immune response in the airways for remodeling processes, we established a matching Drosophila model. Here, triggering the Imd (immune deficiency) pathway in tracheal cells induced organ-wide remodeling. This structural remodeling comprises disorganization of epithelial structures and comprehensive epithelial thickening. We show that these structural changes do not depend on the Imd pathway's canonical branch terminating on nuclear factor κB (NF-κB) activation. Instead, activation of a different segment of the Imd pathway that branches off downstream of Tak1 and comprises activation of c-Jun N-terminal kinase (JNK) and forkhead transcription factor of the O subgroup (FoxO) signaling is necessary and sufficient to mediate the observed structural changes of the airways. Our findings imply that targeting JNK and FoxO signaling in the airways could be a promising strategy to interfere with disease-associated airway remodeling processes.

CARM1 regulates senescence during airway epithelial cell injury in COPD pathogenesis.

Chronic obstructive pulmonary disease (COPD) is a life-threatening lung disease. Although cigarette smoke was considered the main cause of development, the heterogeneous nature of the disease leaves it unclear whether other factors contribute to the predisposition or impaired regeneration response observed. Recently, epigenetic modification has emerged to be a key player in the pathogenesis of COPD. The addition of methyl groups to arginine residues in both histone and nonhistone proteins by protein arginine methyltransferases (PRMTs) is an important posttranslational epigenetic modification event regulating cellular proliferation, differentiation, apoptosis, and senescence. Here, we hypothesize that coactivator-associated arginine methyltransferase-1 (CARM1) regulates airway epithelial cell injury in COPD pathogenesis by controlling cellular senescence. Using the naphthalene (NA)-induced mouse model of airway epithelial damage, we demonstrate that loss of CC10-positive club cells is accompanied by a reduction in CARM1-expressing cells of the airway epithelium. Furthermore, Carm1 haploinsuffficent mice showed perturbed club cell regeneration following NA treatment. In addition, CARM1 reduction led to decreased numbers of antisenescent sirtuin 1-expressing cells accompanied by higher p21, p16, and ß-galactosidase-positive senescent cells in the mouse airway following NA treatment. Importantly, CARM1-silenced human bronchial epithelial cells showed impaired wound healing and higher ß-galactosidase activity. These results demonstrate that CARM1 contributes to airway repair and regeneration by regulating airway epithelial cell senescence.

The alpha-melanocyte-stimulating hormone acts as a local immune homeostasis factor in experimental allergic asthma.

BACKGROUND: Originally, the neuropeptide α-melanocyte-stimulating hormone (α-MSH) has been described as a mediator of skin pigmentation. However, recent studies have shown that α-MSH is able to modulate inflammation in various tissues including the lung. So far, it is still not clear whether α-MSH also plays a role in allergic bronchial asthma. OBJECTIVE: This study aimed at investigating the role and regulatory mechanisms of α-MSH in asthma pathogenesis. METHODS: α-MSH levels were measured in bronchoalveolar lavage (BAL) fluid of asthmatic and non-asthmatic individuals as well as of healthy mice and mice with experimental asthma. Wild-type mice were sensitized to ovalbumin (OVA) and exposed to an OVA aerosol in order to induce experimental allergic asthma. α-MSH was administrated intratracheally, the α-MSH antibody intraperitoneally prior each OVA challenge. Airway inflammation, cytokine production, mucus production, airway hyperresponsiveness and receptor expression were assessed. RESULTS: α-MSH levels in BAL of asthmatic individuals and mice were significantly higher compared to healthy controls. In a mouse model of experimental asthma, α-MSH neutralization increased airway inflammation and mucus production, whereas local administration of α-MSH significantly reduced inflammation of the airways. The beneficial effects were further associated with decreased levels of eosinophilic chemoattractant factors that are released by MC5R-positive T helper 2 and airway epithelial cells. CONCLUSION AND CLINICAL RELEVANCE: α-MSH acts as a regulatory factor to maintain local immune homeostasis in allergic bronchial asthma.

Optimising experimental research in respiratory diseases: an ERS statement.

Experimental models are critical for the understanding of lung health and disease and are indispensable for drug development. However, the pathogenetic and clinical relevance of the models is often unclear. Further, the use of animals in biomedical research is controversial from an ethical perspective.The objective of this task force was to issue a statement with research recommendations about lung disease models by facilitating in-depth discussions between respiratory scientists, and to provide an overview of the literature on the available models. Focus was put on their specific benefits and limitations. This will result in more efficient use of resources and greater reduction in the numbers of animals employed, thereby enhancing the ethical standards and translational capacity of experimental research.The task force statement addresses general issues of experimental research (ethics, species, sex, age, ex vivo and in vitro models, gene editing). The statement also includes research recommendations on modelling asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, lung infections, acute lung injury and pulmonary hypertension.The task force stressed the importance of using multiple models to strengthen validity of results, the need to increase the availability of human tissues and the importance of standard operating procedures and data quality.

Low Dose Carbon Black Nanoparticle Exposure Does Not Aggravate Allergic Airway Inflammation in Mice Irrespective of the Presence of Surface Polycyclic Aromatic Hydrocarbons.

Exposure to exogenous noxae, such as particulate matter, can trigger acute aggravations of allergic asthma-a chronic inflammatory airway disease. We tested whether Carbon Black nanoparticles (CBNP) with or without surface polycyclic aromatic hydrocarbons (PAH) aggravate an established allergic airway inflammation in mice. In an ovalbumin mouse model, Printex®90 (P90), P90 coated with benzo[a]pyrene (P90-BaP) or 9-nitroanthracene (P90-9NA), or acetylene soot exhibiting a mixture of surface PAH (AS-PAH) was administered twice (70 µL, 100 µg/mL) during an established allergic airway inflammation. We analyzed the immune cell numbers and chemokine/cytokine profiles in bronchoalveolar lavages, the mRNA expressions of markers for PAH metabolism (Cyp1a1, 1b1), oxidative stress (HO-1, Gr, Gpx-3), inflammation (KC, Mcp-1, IL-6, IL-13, IL-17a), mucin synthesis (Muc5ac, Muc5b), the histology of mucus-producing goblet cells, ciliary beat frequency (CBF), and the particle transport speed. CBNP had a comparable primary particle size, hydrodynamic diameter, and ζ-potential, but differed in the specific surface area (P90 > P90-BaP = P90-9NA = AS-PAH) and surface chemistry. None of the CBNP tested increased any parameter related to inflammation. The unmodified P90, however, decreased the tracheal CBF, decreased the Muc5b in intrapulmonary airways, but increased the tracheal Muc5ac. Our results demonstrated that irrespective of the surface PAH, a low dose of CBNP does not acutely aggravate an established allergic airway inflammation in mice.

Novel therapeutic roles for surfactant-inositols and -phosphatidylglycerols in a neonatal piglet ARDS model: a translational study.

The biological and immune-protective properties of surfactant-derived phospholipids and phospholipid subfractions in the context of neonatal inflammatory lung disease are widely unknown. Using a porcine neonatal triple-hit acute respiratory distress syndrome (ARDS) model (repeated airway lavage, overventilation, and LPS instillation into airways), we assessed whether the supplementation of surfactant (S; poractant alfa) with inositol derivatives [inositol 1,2,6-trisphosphate (IP3) or phosphatidylinositol 3,5-bisphosphate (PIP2)] or phosphatidylglycerol subfractions [16:0/18:1-palmitoyloleoyl-phosphatidylglycerol (POPG) or 18:1/18:1-dioleoyl-phosphatidylglycerol (DOPG)] would result in improved clinical parameters and sought to characterize changes in key inflammatory pathways behind these improvements. Within 72 h of mechanical ventilation, the oxygenation index (S+IP3, S+PIP2, and S+POPG), the ventilation efficiency index (S+IP3 and S+POPG), the compliance (S+IP3 and S+POPG) and resistance (S+POPG) of the respiratory system, and the extravascular lung water index (S+IP3 and S+POPG) significantly improved compared with S treatment alone. The inositol derivatives (mainly S+IP3) exerted their actions by suppressing acid sphingomyelinase activity and dependent ceramide production, linked with the suppression of the inflammasome nucleotide-binding domain, leucine-rich repeat-containing protein-3 (NLRP3)-apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-caspase-1 complex, and the profibrotic response represented by the cytokines transforming growth factor-ß1 and IFN-γ, matrix metalloproteinase (MMP)-1/8, and elastin. In addition, IκB kinase activity was significantly reduced. S+POPG and S+DOPG treatment inhibited polymorphonuclear leukocyte activity (MMP-8 and myeloperoxidase) and the production of interleukin-6, maintained alveolar-capillary barrier functions, and reduced alveolar epithelial cell apoptosis, all of which resulted in reduced pulmonary edema. S+DOPG also limited the profibrotic response. We conclude that highly concentrated inositol derivatives and phosphatidylglycerol subfractions in surfactant preparations mitigate key inflammatory pathways in inflammatory lung disease and that their clinical application may be of interest for future treatment of the acute exudative phase of neonatal ARDS.

Transcriptomic analysis comparing mouse strains with extreme total lung capacities identifies novel candidate genes for pulmonary function.

BACKGROUND: Failure to attain peak lung function by early adulthood is a risk factor for chronic lung diseases. Previously, we reported that C3H/HeJ mice have about twice total lung capacity (TLC) compared to JF1/MsJ mice. We identified seven lung function quantitative trait loci (QTL: Lfnq1-Lfnq7) in backcross/intercross mice derived from these inbred strains. We further demonstrated, superoxide dismutase 3, extracellular (Sod3), Kit oncogene (Kit) and secreted phosphoprotein 1 (Spp1) located on these Lfnqs as lung function determinants. Emanating from the concept of early origin of lung disease, we sought to identify novel candidate genes for pulmonary function by investigating lung transcriptome in C3H/HeJ and JF1/MsJ mice at the completion of embryonic development, bulk alveolar formation and maturity. METHODS: Design-based stereological analysis was performed to study lung structure in C3H/HeJ and JF1/MsJ mice. Microarray was used for lung transcriptomic analysis [embryonic day 18, postnatal days 28, 70]. Quantitative real time polymerase chain reaction (qRT-PCR), western blot and immunohistochemical analysis were used to confirm selected differences. RESULTS: Stereological analysis revealed decreased alveolar number density, elastin to collagen ratio and increased mean alveolar volume in C3H/HeJ mice compared to JF1/MsJ. Gene ontology term "extracellular region" was enriched among the decreased JF1/MsJ transcripts. Candidate genes identified using the expression-QTL strategy include: ATP-binding cassette, sub-family G (WHITE), member 1 (Abcg1), formyl peptide receptor 1 (Fpr1), gamma-aminobutyric acid (GABA) B receptor, 1 (Gabbr1); histocompatibility 2 genes: class II antigen E beta (H2-Eb1), D region locus 1 (H2-D1), and Q region locus 4 (H2-Q4); leucine rich repeat containing 6 (testis) (Lrrc6), radial spoke head 1 homolog (Rsph1), and surfactant associated 2 (Sfta2). Noteworthy genes selected as candidates for their consistent expression include: Wnt inhibitor factor 1 (Wif1), follistatin (Fst), chitinase-like 1 (Chil1), and Chil3. CONCLUSIONS: Comparison of late embryonic, adolescent and adult lung transcript profiles between mouse strains with extreme TLCs lead to the identification of candidate genes for pulmonary function that has not been reported earlier. Further mechanistic investigations are warranted to elucidate their mode of action in determining lung function.

Biological effects of carbon black nanoparticles are changed by surface coating with polycyclic aromatic hydrocarbons.

BACKGROUND: Carbon black nanoparticles (CBNP) are mainly composed of carbon, with a small amount of other elements (including hydrogen and oxygen). The toxicity of CBNP has been attributed to their large surface area, and through adsorbing intrinsically toxic substances, such as polycyclic aromatic hydrocarbons (PAH). It is not clear whether a PAH surface coating changes the toxicological properties of CBNP by influencing their physicochemical properties, through the specific toxicity of the surface-bound PAH, or by a combination of both. METHODS: Printex®90 (P90) was used as CBNP; the comparators were P90 coated with either benzo[a]pyrene (BaP) or 9-nitroanthracene (9NA), and soot from acetylene combustion that bears various PAHs on the surface (AS-PAH). Oxidative stress and IL-8/KC mRNA expression were determined in A549 and bronchial epithelial cells (16HBE14o-, Calu-3), mouse intrapulmonary airways and tracheal epithelial cells. Overall toxicity was tested in a rat inhalation study according to Organization for Economic Co-operation and Development (OECD) criteria. Effects on cytochrome monooxygenase (Cyp) mRNA expression, cell viability and mucociliary clearance were determined in acute exposure models using explanted murine trachea. RESULTS: All particles had similar primary particle size, shape, hydrodynamic diameter and ζ-potential. All PAH-containing particles had a comparable specific surface area that was approximately one third that of P90. AS-PAH contained a mixture of PAH with expected higher toxicity than BaP or 9NA. PAH-coating reduced some effects of P90 such as IL-8 mRNA expression and oxidative stress in A549 cells, granulocyte influx in the in vivo OECD experiment, and agglomeration of P90 and mucus release in the murine trachea ex vivo. Furthermore, P90-BaP decreased particle transport speed compared to P90 at 10 µg/ml. In contrast, PAH-coating induced IL-8 mRNA expression in bronchial epithelial cell lines, and Cyp mRNA expression and apoptosis in tracheal epithelial cells. In line with the higher toxicity compared to P90-BaP and P90-9NA, AS-PAH had the strongest biological effects both ex vivo and in vivo. CONCLUSIONS: Our results demonstrate that the biological effect of CBNP is determined by a combination of specific surface area and surface-bound PAH, and varies in different target cells.

Airway remodeling in asthma: what really matters.

Airway remodeling is generally quite broadly defined as any change in composition, distribution, thickness, mass or volume and/or number of structural components observed in the airway wall of patients relative to healthy individuals. However, two types of airway remodeling should be distinguished more clearly: (1) physiological airway remodeling, which encompasses structural changes that occur regularly during normal lung development and growth leading to a normal mature airway wall or as an acute and transient response to injury and/or inflammation, which ultimately results in restoration of a normal airway structures; and (2) pathological airway remodeling, which comprises those structural alterations that occur as a result of either disturbed lung development or as a response to chronic injury and/or inflammation leading to persistently altered airway wall structures and function. This review will address a few major aspects: (1) what are reliable quantitative approaches to assess airway remodeling? (2) Are there any indications supporting the notion that airway remodeling can occur as a primary event, i.e., before any inflammatory process was initiated? (3) What is known about airway remodeling being a secondary event to inflammation? And (4), what can we learn from the different animal models ranging from invertebrate to primate models in the study of airway remodeling? Future studies are required addressing particularly pheno-/endotype-specific aspects of airway remodeling using both endotype-specific animal models and "endotyped" human asthmatics. Hopefully, novel in vivo imaging techniques will be further advanced to allow monitoring development, growth and inflammation of the airways already at a very early stage in life.

A distinct microbiota composition is associated with protection from food allergy in an oral mouse immunization model.

In our mouse model, gastric acid-suppression is associated with antigen-specific IgE and anaphylaxis development. We repeatedly observed non-responder animals protected from food allergy. Here, we aimed to analyse reasons for this protection. Ten out of 64 mice, subjected to oral ovalbumin (OVA) immunizations under gastric acid-suppression, were non-responders without OVA-specific IgE or IgG1 elevation, indicating protection from allergy. In these non-responders, allergen challenges confirmed reduced antigen uptake and lack of anaphylactic symptoms, while in allergic mice high levels of mouse mast-cell protease-1 and a body temperature reduction, indicative for anaphylaxis, were determined. Upon OVA stimulation, significantly lower IL-4, IL-5, IL-10 and IL-13 levels were detected in non-responders, while IL-22 was significantly higher. Comparison of fecal microbiota revealed differences of bacterial communities on single bacterial Operational-Taxonomic-Unit level between the groups, indicating protection from food allergy being associated with a distinct microbiota composition in a non-responding phenotype in this mouse model.

Effect of IL-37 on Allergic Airway Inflammation.

RATIONALE: The new cytokine IL-37 has been described as a negative regulator of innate immunity. It reduces activation of dendritic cells and the production of proinflammatory mediators in murine and human immune cells. Although recent results from the CLARA childhood asthma cohort suggested an impact of IL-37 on human asthma pathogenesis, the receptor for IL-37 and its implication in adaptive immune responses have not been determined. OBJECTIVES: This study aimed to clarify whether IL-37 also provides antiinflammatory effects on adaptive immune responses and through which receptor it exerts its effects. METHODS: IL-37 levels in supernatants of restimulated peripheral blood mononuclear cells isolated from children with asthma and healthy children were determined. Mice (wild-type, IL-18Rα(-/-), and SIGIRR/IL-1R8(-/-)) were sensitized to ovalbumin (OVA) and challenged with OVA aerosol to induce acute allergic experimental asthma, and IL-37 was applied intranasally during OVA challenge. Airway hyperresponsiveness was determined. A cytometric bead array was used to assess cytokine levels in bronchoalveolar lavage fluid. Epithelial mucus was quantified on the basis of lung sections stained with periodic acid-Schiff reagent, using the newCAST microscope system. MEASUREMENTS AND MAIN RESULTS: Human peripheral blood mononuclear cells of subjects with allergic asthma produce less IL-37 compared with healthy control subjects. In mice, intranasal administration of IL-37 dampened allergic airway inflammation as well as proinflammatory cytokine production, mucus hyperproduction, and airway hyperresponsiveness. However, the antiinflammatory effects of IL-37 were completely abolished in mice deficient for IL-18Rα or SIGIRR/IL-1R8. CONCLUSIONS: This study demonstrates that IL-37 reduces allergic airway inflammation directed by type 2 helper T cells and the hallmarks of experimental asthma in mice, suggesting that IL-37 may be critical for asthma pathogenesis in particular and may have an impact on adaptive immunity in general. Furthermore, these data suggest a mode of action of IL-37 that involves binding to IL-18Rα and subsequent heterodimerization with or activation of SIGIRR/IL-1R8. Therefore, IL-37 or its receptors could be potential targets for asthma intervention.

The H2S-generating enzymes cystathionine ß-synthase and cystathionine γ-lyase play a role in vascular development during normal lung alveolarization.

The gasotransmitter hydrogen sulfide (H2S) is emerging as a mediator of lung physiology and disease. Recent studies revealed that H2S administration limited perturbations to lung structure in experimental animal models of bronchopulmonary dysplasia (BPD), partially restoring alveolarization, limiting pulmonary hypertension, limiting inflammation, and promoting epithelial repair. No studies have addressed roles for endogenous H2S in lung development. H2S is endogenously generated by cystathionine ß-synthase (Cbs) and cystathionine γ-lyase (Cth). We demonstrate here that the expression of Cbs and Cth in mouse lungs is dynamically regulated during lung alveolarization and that alveolarization is blunted in Cbs(-/-) and Cth(-/-) mouse pups, where a 50% reduction in the total number of alveoli was observed, without any impact on septal thickness. Laser-capture microdissection and immunofluorescence staining indicated that Cbs and Cth were expressed in the airway epithelium and lung vessels. Loss of Cbs and Cth led to a 100-500% increase in the muscularization of small- and medium-sized lung vessels, which was accompanied by increased vessel wall thickness, and an apparent decrease in lung vascular supply. Ablation of Cbs expression using small interfering RNA or pharmacological inhibition of Cth using propargylglycine in lung endothelial cells limited angiogenic capacity, causing a 30-40% decrease in tube length and a 50% decrease in number of tubes formed. In contrast, exogenous administration of H2S with GYY4137 promoted endothelial tube formation. These data confirm a key role for the H2S-generating enzymes Cbs and Cth in pulmonary vascular development and homeostasis and in lung alveolarization.

Poly(inosinic-cytidylic) acid-triggered exacerbation of experimental asthma depends on IL-17A produced by NK cells.

Viral infection of the respiratory tract represents the major cause of acute asthma exacerbations. dsRNA is produced as an intermediate during replication of respiratory viruses and triggers immune responses via TLR3. This study aimed at clarifying the mechanisms underlying TLR3 triggered exacerbation of experimental allergic asthma. The TLR3 ligand poly(inosinic-cytidylic) acid was applied intranasally to mice with already established experimental allergic asthma. Airway inflammation, cytokine expression, mucus production, and airway reactivity was assessed in wild-type, IL-17A, or IL-23p19-deficient, and in NK cell-depleted mice. Local application of poly(inosinic-cytidylic) acid exacerbated experimental allergic asthma in mice as characterized by enhanced release of proinflammatory cytokines, aggravated airway inflammation, and increased mucus production together with pronounced airway hyperresponsiveness. This was further associated with augmented production of IL-17 by Th17 cells and NK cells. Whereas experimental exacerbation could be induced in IL-23p19-deficient mice lacking mature, proinflammatory Th17 cells, this was not possible in mice lacking IL-17A or in NK cell-depleted animals. These experiments indicate a central role for IL-17 derived from NK cells but not from Th17 cells in the pathogenesis of virus-triggered exacerbation of experimental asthma.

Cell counting in human endobronchial biopsies--disagreement of 2D versus 3D morphometry.

QUESTION: Inflammatory cell numbers are important endpoints in clinical studies relying on endobronchial biopsies. Assumption-based bidimensional (2D) counting methods are widely used, although theoretically design-based stereologic three-dimensional (3D) methods alone offer an unbiased quantitative tool. We assessed the method agreement between 2D and 3D counting designs in practice when applied to identical samples in parallel. MATERIALS AND METHODS: Biopsies from segmental bronchi were collected from healthy non-smokers (n = 7) and smokers (n = 7), embedded and sectioned exhaustively. Systematic uniform random samples were immunohistochemically stained for macrophages (CD68) and T-lymphocytes (CD3), respectively. In identical fields of view, cell numbers per volume unit (NV) were assessed using the physical disector (3D), and profiles per area unit (NA) were counted (2D). For CD68+ cells, profiles with and without nucleus were separately recorded. In order to enable a direct comparison of the two methods, the zero-dimensional CD68+/CD3+-ratio was calculated for each approach. Method agreement was tested by Bland-Altmann analysis. RESULTS: In both groups, mean CD68+/CD3+ ratios for NV and NA were significantly different (non-smokers: 0.39 and 0.68, p<0.05; smokers: 0.49 and 1.68, p<0.05). When counting only nucleated CD68+ profiles, mean ratios obtained by 2D and 3D counting were similar, but the regression-based Bland-Altmann analysis indicated a bias of the 2D ratios proportional to their magnitude. This magnitude dependent deviation differed between the two groups. CONCLUSIONS: 2D counts of cell and nuclear profiles introduce a variable size-dependent bias throughout the measurement range. Because the deviation between the 3D and 2D data was different in the two groups, it precludes establishing a 'universal conversion formula'.

Systemic hydrogen sulfide administration partially restores normal alveolarization in an experimental animal model of bronchopulmonary dysplasia.

Arrested alveolarization is the pathological hallmark of bronchopulmonary dysplasia (BPD), a complication of premature birth. Here, the impact of systemic application of hydrogen sulfide (H2S) on postnatal alveolarization was assessed in a mouse BPD model. Exposure of newborn mice to 85% O2 for 10 days reduced the total lung alveoli number by 56% and increased alveolar septal wall thickness by 29%, as assessed by state-of-the-art stereological analysis. Systemic application of H2S via the slow-release H2S donor GYY4137 for 10 days resulted in pronounced improvement in lung alveolarization in pups breathing 85% O2, compared with vehicle-treated littermates. Although without impact on lung oxidative status, systemic H2S blunted leukocyte infiltration into alveolar air spaces provoked by hyperoxia, and restored normal lung interleukin 10 levels that were otherwise depressed by 85% O2. Treatment of primary mouse alveolar type II (ATII) cells with the rapid-release H2S donor NaHS had no impact on cell viability; however, NaHS promoted ATII cell migration. Although exposure of ATII cells to 85% O2 caused dramatic changes in mRNA expression, exposure to either GYY4137 or NaHS had no impact on ATII cell mRNA expression, as assessed by microarray, suggesting that the effects observed were independent of changes in gene expression. The impact of NaHS on ATII cell migration was attenuated by glibenclamide, implicating ion channels, and was accompanied by activation of Akt, hinting at two possible mechanisms of H2S action. These data support further investigation of H2S as a candidate interventional strategy to limit the arrested alveolarization associated with BPD.

Peripheral erythrocytes decrease upon specific respiratory challenge with grass pollen allergen in sensitized mice and in human subjects.

BACKGROUND AND AIMS: Specific hyper-responsiveness towards an allergen and non-specific airway hyperreactivity both impair quality of life in patients with respiratory allergic diseases. We aimed to investigate cellular responses following specific and non-specific airway challenges locally and systemically in i) sensitized BALB/c mice challenged with grass pollen allergen Phl p 5, and in ii) grass pollen sensitized allergic rhinitis subjects undergoing specific airway challenge in the Vienna Challenge Chamber (VCC). METHODS AND RESULTS: BALB/c mice (n = 20) were intraperitoneally immunized with grass pollen allergen Phl p 5 and afterwards aerosol challenged with either the specific allergen Phl p 5 (n = 10) or the non-specific antigen ovalbumin (OVA) (n = 10). A protocol for inducing allergic asthma as well as allergic rhinitis, according to the united airway concept, was used. Both groups of exposed mice showed significantly reduced physical activity after airway challenge. Specific airway challenge further resulted in goblet cell hyperplasia, enhanced mucous secretion, intrapulmonary leukocyte infiltration and lymphoid follicle formation, associated with significant expression of IL-4, IL-5 and IL-13 in splenocytes and also partially in lung tissue. Concerning circulating blood cell dynamics, we observed a significant drop of erythrocyte counts, hemoglobin and hematocrit levels in both mouse groups, challenged with allergen or OVA. A significant decrease in circulating erythrocytes and hematocrit levels after airway challenges with grass pollen allergen was also found in grass pollen sensitized human rhinitis subjects (n = 42) at the VCC. The effects on peripheral leukocyte counts in mice and humans however were opposed, possibly due to the different primary inflammation sites. CONCLUSION: Our data revealed that, besides significant leukocyte dynamics, particularly erythrocytes are involved in acute hypersensitivity reactions to respiratory allergens. A rapid recruitment of erythrocytes to the lungs to compensate for hypoxia is a possible explanation for these findings.

Effects of recombinant human keratinocyte growth factor on surfactant, plasma, and liver phospholipid homeostasis in hyperoxic neonatal rats.

Respiratory distress and bronchopulmonary dysplasia (BPD) are major problems in preterm infants that are often addressed by glucocorticoid treatment and increased oxygen supply, causing catabolic and injurious side effects. Recombinant human keratinocyte growth factor (rhKGF) is noncatabolic and antiapoptotic and increases surfactant pools in immature lungs. Despite its usefulness in injured neonatal lungs, the mechanisms of improved surfactant homeostasis in vivo and systemic effects on lipid homeostasis are unknown. We therefore exposed newborn rats to 85% vs. 21% oxygen and treated them systemically with rhKGF for 48 h before death at 7 days. We determined type II pneumocyte (PN-II) proliferation, surfactant protein (SP) mRNA expression, and the pulmonary metabolism of individual phosphatidylcholine (PC) species using [D(9)-methyl]choline and tandem mass spectrometry. In addition, we assessed liver and plasma lipid metabolism, addressing PC synthesis de novo, the liver-specific phosphatidylethanolamine methyl transferase (PEMT) pathway, and triglyceride concentrations. rhKGF was found to maintain PN-II proliferation and increased SP-B/C expression and surfactant PC in both normoxic and hyperoxic lungs. We found increased total PC together with decreased [D(9)-methyl]choline enrichment, suggesting decreased turnover rather than increased secretion and synthesis as the underlying mechanism. In the liver, rhKGF increased PC synthesis, both de novo and via PEMT, underlining the organotypic differences of rhKGF actions on lipid metabolism. rhKGF increased the hepatic secretion of newly synthesized polyunsaturated PC, indicating improved systemic supply with choline and essential fatty acids. We suggest that rhKGF has potential as a therapeutic agent in neonates by improving pulmonary and systemic PC homeostasis.