Neutrophil Heterogeneity Is Modified during Acute Lung Inflammation in Apoa1-/- Mice.

Neutrophils play important roles in inflammatory airway diseases. In this study, we assessed whether apolipoprotein A-I modifies neutrophil heterogeneity as part of the mechanism by which it attenuates acute airway inflammation. Neutrophilic airway inflammation was induced by daily intranasal administration of LPS plus house dust mite (LPS+HDM) to Apoa1-/- and Apoa1+/+ mice for 3 d. Single-cell RNA sequencing was performed on cells recovered from bronchoalveolar lavage fluid on day 4. Unsupervised profiling identified 10 clusters of neutrophils in bronchoalveolar lavage fluid from Apoa1-/- and Apoa1+/+ mice. LPS+HDM-challenged Apoa1-/- mice had an increased proportion of the Neu4 neutrophil cluster that expressed S100a8, S100a9, and Mmp8 and had high maturation, aggregation, and TLR4 binding scores. There was also an increase in the Neu6 cluster of immature neutrophils, whereas neutrophil clusters expressing IFN-stimulated genes were decreased. An unsupervised trajectory analysis showed that Neu4 represented a distinct lineage in Apoa1-/- mice. LPS+HDM-challenged Apoa1-/- mice also had an increased proportion of recruited airspace macrophages, which was associated with a reciprocal reduction in resident airspace macrophages. Increased expression of a common set of proinflammatory genes, S100a8, S100a9, and Lcn2, was present in all neutrophils and airspace macrophages from LPS+HDM-challenged Apoa1-/- mice. These findings show that Apoa1-/- mice have increases in specific neutrophil and macrophage clusters in the lung during acute inflammation mediated by LPS+HDM, as well as enhanced expression of a common set of proinflammatory genes. This suggests that modifications in neutrophil and macrophage heterogeneity contribute to the mechanism by which apolipoprotein A-I attenuates acute airway inflammation.

Asthmatic patients with high serum amyloid A have proinflammatory HDL: Implications for augmented systemic and airway inflammation.

RATIONALE: Serum amyloid A (SAA) is bound to high-density lipoproteins (HDL) in blood. Although SAA is increased in the blood of patients with asthma, it is not known whether this modifies asthma severity. OBJECTIVE: We sought to define the clinical characteristics of patients with asthma who have high SAA levels and assess whether HDL from SAA-high patients with asthma is proinflammatory. METHODS: SAA levels in serum from subjects with and without asthma were quantified by ELISA. HDLs isolated from subjects with asthma and high SAA levels were used to stimulate human monocytes and were intravenously administered to BALB/c mice. RESULTS: An SAA level greater than or equal to 108.8 µg/mL was defined as the threshold to identify 11% of an asthmatic cohort (n = 146) as being SAA-high. SAA-high patients with asthma were characterized by increased serum C-reactive protein, IL-6, and TNF-α; older age; and an increased prevalence of obesity and severe asthma. HDL isolated from SAA-high patients with asthma (SAA-high HDL) had an increased content of SAA as compared with HDL from SAA-low patients with asthma and induced the secretion of IL-6, IL-1ß, and TNF-α from human monocytes via a formyl peptide receptor 2/ATP/P2X purinoceptor 7 axis. Intravenous administration to mice of SAA-high HDL, but not normal HDL, induced systemic inflammation and amplified allergen-induced neutrophilic airway inflammation and goblet cell metaplasia. CONCLUSIONS: SAA-high patients with asthma are characterized by systemic inflammation, older age, and an increased prevalence of obesity and severe asthma. HDL from SAA-high patients with asthma is proinflammatory and, when intravenously administered to mice, induces systemic inflammation, and amplifies allergen-induced neutrophilic airway inflammation. This suggests that systemic inflammation induced by SAA-high HDL may augment disease severity in asthma.

Serum levels of small HDL particles are negatively correlated with death or lung transplantation in an observational study of idiopathic pulmonary fibrosis.

BACKGROUND: Serum lipoproteins, such as high-density lipoproteins (HDL), may influence disease severity in idiopathic pulmonary fibrosis (IPF). Here, we investigated associations between serum lipids and lipoproteins and clinical end-points in IPF. METHODS: Clinical data and serum lipids were analysed from a discovery cohort (59 IPF subjects, 56 healthy volunteers) and validated using an independent, multicentre cohort (207 IPF subjects) from the Pulmonary Fibrosis Foundation registry. Associations between lipids and clinical end-points (forced vital capacity, 6-min walk distance, gender age physiology (GAP) index, death or lung transplantation) were examined using Pearson's correlation and multivariable analyses. RESULTS: Serum concentrations of small HDL particles measured using nuclear magnetic resonance spectroscopy (S-HDLPNMR) correlated negatively with the GAP index in the discovery cohort of IPF subjects. The negative correlation of S-HDLPNMR with GAP index was confirmed in the validation cohort of IPF subjects. Higher levels of S-HDLPNMR were associated with lower odds of death or its competing outcome, lung transplantation (OR 0.9 for each 1-µmol·L-1 increase in S-HDLPNMR, p<0.05), at 1, 2 and 3 years from study entry in a combined cohort of all IPF subjects. CONCLUSIONS: Higher serum levels of S-HDLPNMR are negatively correlated with the GAP index, as well as with lower observed mortality or lung transplantation in IPF subjects. These findings support the hypothesis that S-HDLPNMR may modify mortality risk in patients with IPF.

Apolipoprotein E Signals via TLR4 to Induce CXCL5 Secretion by Asthmatic Airway Epithelial Cells.

The primary function of APOE (apolipoprotein E) is to mediate the transport of cholesterol- and lipid-containing lipoprotein particles into cells by receptor-mediated endocytosis. APOE also has pro- and antiinflammatory effects, which are both context and concentration dependent. For example, Apoe-/- mice exhibit enhanced airway remodeling and hyperreactivity in experimental asthma, whereas increased APOE levels in lung epithelial lining fluid induce IL-1ß secretion from human asthmatic alveolar macrophages. However, APOE-mediated airway epithelial cell inflammatory responses and signaling pathways have not been defined. Here, RNA sequencing of human asthmatic bronchial brushing cells stimulated with APOE identified increased expression of mRNA transcripts encoding multiple proinflammatory genes, including CXCL5 (C-X-C motif chemokine ligand 5), an epithelial-derived chemokine that promotes neutrophil activation and chemotaxis. We subsequently characterized the APOE signaling pathway that induces CXCL5 secretion by human asthmatic small airway epithelial cells (SAECs). Neutralizing antibodies directed against TLR4 (Toll-like receptor 4), but not TLR2, attenuated APOE-mediated CXCL5 secretion by human asthmatic SAECs. Inhibition of TAK1 (transforming growth factor-ß-activated kinase 1), IκKß (inhibitor of nuclear factor κ B kinase subunit ß), TPL2 (tumor progression locus 2), and JNK (c-Jun N-terminal kinase), but not p38 MAPK (mitogen-activated protein kinase) or MEK1/2 (MAPK kinase 1/2), attenuated APOE-mediated CXCL5 secretion. The roles of TAK1, IκKß, TPL2, and JNK in APOE-mediated CXCL5 secretion were verified by RNA interference. Furthermore, RNA interference showed that after APOE stimulation, both NF-κB p65 and TPL2 were downstream of TAK1 and IκKß, whereas JNK was downstream of TPL2. In summary, elevated levels of APOE in the airway may activate a TLR4/TAK1/IκKß/NF-κB/TPL2/JNK signaling pathway that induces CXCL5 secretion by human asthmatic SAECs. These findings identify new roles for TLR4 and TPL2 in APOE-mediated proinflammatory responses in asthma.

Apolipoprotein E is a concentration-dependent pulmonary danger signal that activates the NLRP3 inflammasome and IL-1ß secretion by bronchoalveolar fluid macrophages from asthmatic subjects.

BACKGROUND: House dust mite (HDM)-challenged Apoe-/- mice display enhanced airway hyperreactivity and mucous cell metaplasia. OBJECTIVE: We sought to characterize the pathways that induce apolipoprotein E (APOE) expression by bronchoalveolar lavage fluid (BALF) macrophages from asthmatic subjects and identify how APOE regulates IL-1ß secretion. METHODS: Macrophages were isolated from asthmatic BALF and derived from THP-1 cells and human monocytes. RESULTS: HDM-derived cysteine and serine proteases induced APOE secretion from BALF macrophages through protease-activated receptor 2. APOE at concentrations of less than 2.5 nmol/L, which are similar to levels found in epithelial lining fluid from healthy adults, did not induce IL-1ß release from BALF macrophages. In contrast, APOE at concentrations of 25 nmol/L or greater induced nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein (NLRP) 3 and pro-IL-1ß expression by BALF macrophages, as well as the caspase-1-mediated generation of mature IL-1ß secreted from cells. HDM acted synergistically with APOE to both prime and activate the NLRP3 inflammasome. In a murine model of neutrophilic airway inflammation induced by HDM and polyinosinic-polycytidylic acid, APOE reached a concentration of 32 nmol/L in epithelial lining fluid, with associated increases in BALF IL-1ß levels. APOE-dependent NLRP3 inflammasome activation in macrophages was primarily mediated through a potassium efflux-dependent mechanism. CONCLUSION: APOE can function as an endogenous, concentration-dependent pulmonary danger signal that primes and activates the NLPR3 inflammasome in BALF macrophages from asthmatic subjects to secrete IL-1ß. This might represent a mechanism through which APOE amplifies pulmonary inflammatory responses when concentrations in the lung are increased to greater than normal levels, which can occur during viral exacerbations of HDM-induced asthma characterized by neutrophilic airway inflammation.

A Pilot Study To Investigate the Immune-Modulatory Effects of Fasting in Steroid-Naive Mild Asthmatics.

A fasting mimetic diet blunts inflammation, and intermittent fasting has shown ameliorative effects in obese asthmatics. To examine whether canonical inflammatory pathways linked with asthma are modulated by fasting, we designed a pilot study in mild asthmatic subjects to assess the effect of fasting on the NLRP3 inflammasome, Th2 cell activation, and airway epithelial cell cytokine production. Subjects with documented reversible airway obstruction and stable mild asthma were recruited into this study in which pulmonary function testing (PFT) and PBMCextraction was performed 24 h after fasting, with repeated PFT testing and blood draw 2.5 h after refeeding. PFTs were not changed by a prolonged fast. However, steroid-naive mild asthmatics showed fasting-dependent blunting of the NLRP3 inflammasome. Furthermore, PBMCs from these fasted asthmatics cocultured with human epithelial cells resulted in blunting of house dust mite-induced epithelial cell cytokine production and reduced CD4+ T cell Th2 activation compared with refed samples. This pilot study shows that prolonged fasting blunts the NLRP3 inflammasome and Th2 cell activation in steroid-naive asthmatics as well as diminishes airway epithelial cell cytokine production. This identifies a potential role for nutrient level-dependent regulation of inflammation in asthma. Our findings support the evaluation of this concept in a larger study as well as the potential development of caloric restriction interventions for the treatment of asthma.

Low-density lipoprotein receptor-related protein 1 attenuates house dust mite-induced eosinophilic airway inflammation by suppressing dendritic cell-mediated adaptive immune responses.

BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP-1) is a scavenger receptor that regulates adaptive immunity and inflammation. LRP-1 is not known to modulate the pathogenesis of allergic asthma. OBJECTIVE: We sought to assess whether LRP-1 expression by dendritic cells (DCs) modulates adaptive immune responses in patients with house dust mite (HDM)-induced airways disease. METHODS: LRP-1 expression on peripheral blood DCs was quantified by using flow cytometry. The role of LRP-1 in modulating HDM-induced airways disease was assessed in mice with deletion of LRP-1 in CD11c+ cells (Lrp1fl/fl; CD11c-Cre) and by adoptive transfer of HDM-pulsed CD11b+ DCs from Lrp1fl/fl; CD11c-Cre mice to wild-type (WT) mice. RESULTS: Human peripheral blood myeloid DC subsets from patients with eosinophilic asthma have lower LRP-1 expression than cells from healthy nonasthmatic subjects. Similarly, LRP-1 expression by CD11b+ lung DCs was significantly reduced in HDM-challenged WT mice. HDM-challenged Lrp1fl/fl; CD11c-Cre mice have a phenotype of increased eosinophilic airway inflammation, allergic sensitization, TH2 cytokine production, and mucous cell metaplasia. The adoptive transfer of HDM-pulsed LRP-1-deficient CD11b+ DCs into WT mice generated a similar phenotype of enhanced eosinophilic inflammation and allergic sensitization. Furthermore, CD11b+ DCs in the lungs of Lrp1fl/fl; CD11c-Cre mice have an increased ability to take up HDM antigen, whereas bone marrow-derived DCs display enhanced antigen presentation capabilities. CONCLUSION: This identifies a novel role for LRP-1 as a negative regulator of DC-mediated adaptive immune responses in the setting of HDM-induced eosinophilic airway inflammation. Furthermore, the reduced LRP-1 expression by circulating myeloid DCs in patients with eosinophilic asthma suggests a possible role for LRP-1 in modulating type 2-high asthma.

High density lipoproteins and type 2 inflammatory biomarkers are negatively correlated in atopic asthmatics.

Blood eosinophil counts and serum periostin levels are biomarkers of type 2 inflammation. Although serum levels of HDL and apoA-I have been associated with less severe airflow obstruction in asthma, it is not known whether serum lipids or lipoprotein particles are correlated with type 2 inflammation in asthmatics. Here, we assessed whether serum lipids and lipoproteins correlated with blood eosinophil counts or serum periostin levels in 165 atopic asthmatics and 163 nonasthmatic subjects with and without atopy. Serum lipids and lipoproteins were quantified using standard laboratory assays and NMR spectroscopy. Absolute blood eosinophils were quantified by complete blood counts. Periostin levels were measured using the Elecsys® periostin assay. In atopic asthmatics, blood eosinophils negatively correlated with serum HDL cholesterol and total HDL particles measured by NMR spectroscopy (HDLNMR). Serum periostin levels negatively correlated with total HDLNMR In contrast, blood eosinophil counts positively correlated with serum triglyceride levels. This study demonstrates for the first time that HDL particles were negatively correlated, whereas serum triglycerides were positively correlated, with blood eosinophils in atopic asthmatics. This supports the concept that serum levels of HDL and triglycerides may be linked to systemic type 2 inflammation in atopic asthma.

High-density Lipoproteins and Apolipoprotein A-I: Potential New Players in the Prevention and Treatment of Lung Disease.

Apolipoprotein A-I (apoA-I) and high-density lipoproteins (HDL) mediate reverse cholesterol transport out of cells. Furthermore, HDL has additional protective functions, which include anti-oxidative, anti-inflammatory, anti-apoptotic, and vasoprotective effects. In contrast, HDL can become dysfunctional with a reduction in both cholesterol efflux and anti-inflammatory properties in the setting of disease or the acute phase response. These paradigms are increasingly being recognized to be active in the pulmonary system, where apoA-I and HDL have protective effects in normal lung health, as well as in a variety of disease states, including acute lung injury (ALI), asthma, chronic obstructive pulmonary disease, lung cancer, pulmonary arterial hypertension, pulmonary fibrosis, and viral pneumonia. Similar to observations in cardiovascular disease, however, HDL may become dysfunctional and contribute to disease pathogenesis in respiratory disorders. Furthermore, synthetic apoA-I mimetic peptides have been shown to have protective effects in animal models of ALI, asthma, pulmonary hypertension, and influenza pneumonia. These findings provide evidence to support the concept that apoA-I mimetic peptides might be developed into a new treatment that can either prevent or attenuate the manifestations of lung diseases, such as asthma. Thus, the lung is positioned to take a page from the cardiovascular disease playbook and utilize the protective properties of HDL and apoA-I as a novel therapeutic approach.

The A's Have It: Developing Apolipoprotein A-I Mimetic Peptides Into a Novel Treatment for Asthma.

New treatments are needed for patients with asthma who are refractory to standard therapies, such as individuals with a phenotype of "type 2-low" inflammation. This important clinical problem could potentially be addressed by the development of apolipoprotein A-I (apoA-I) mimetic peptides. ApoA-I interacts with its cellular receptor, the ATP-binding cassette subfamily A, member 1 (ABCA1), to facilitate cholesterol efflux out of cells to form nascent high-density lipoprotein particles. The ability of the apoA-I/ABCA1 pathway to promote cholesterol efflux from cells that mediate adaptive immunity, such as antigen-presenting cells, can attenuate their function. Data from experimental murine models have shown that the apoA-I/ABCA1 pathway can reduce neutrophilic airway inflammation, primarily by suppressing the production of granulocyte-colony stimulating factor. Furthermore, administration of apoA-I mimetic peptides to experimental murine models of allergic asthma has decreased both neutrophilic and eosinophilic airway inflammation, as well as airway hyperresponsiveness and mucous cell metaplasia. Higher serum levels of apoA-I have also been associated with less severe airflow obstruction in patients with asthma. Collectively, these results suggest that the apoA-I/ABCA1 pathway may have a protective effect in asthma, and support the concept of advancing inhaled apoA-I mimetic peptides to clinical trials that can assess their safety and effectiveness. Thus, we propose that the development of inhaled apoA-I mimetic peptides as a new treatment could represent a clinical advance for patients with severe asthma who are unresponsive to other therapies.

Acoustofluidic Transfer of Inflammatory Cells from Human Sputum Samples.

For sputum analysis, the transfer of inflammatory cells from liquefied sputum samples to a culture medium or buffer solution is a critical step because it removes the inflammatory cells from the presence of residual dithiothreitol (DTT), a reagent that reduces cell viability and interferes with further sputum analyses. In this work, we report an acoustofluidic platform for transferring inflammatory cells using standing surface acoustic waves (SSAW). In particular, we exploit the acoustic radiation force generated from a SSAW field to actively transfer inflammatory cells from a solution containing residual DTT to a buffer solution. The viability and integrity of the inflammatory cells are maintained during the acoustofluidic-based cell transfer process. Our acoustofluidic technique removes residual DTT generated in sputum liquefaction and facilitates immunophenotyping of major inflammatory cells from sputum samples. It enables cell transfer in a continuous flow, which aids the development of an automated, integrated system for on-chip sputum processing and analysis.

Emerging Roles of Apolipoprotein E and Apolipoprotein A-I in the Pathogenesis and Treatment of Lung Disease.

Emerging roles are being recognized increasingly for apolipoproteins in the pathogenesis and treatment of lung diseases on the basis of their ability to suppress inflammation, oxidative stress, and tissue remodeling, and to promote adaptive immunity and host defense. Apolipoproteins, such as apolipoprotein E (apoE) and apolipoprotein A-I (apoA-I), are important components of lipoprotein particles that facilitate the transport of cholesterol, triglycerides, and phospholipids between plasma and cells. ApoE-containing lipoprotein particles are internalized into cells by low-density lipoprotein receptors (LDLRs), whereas apoA-I can interact with the ATP-binding cassette subfamily A member 1 (ABCA1) transporter to efflux cholesterol and phospholipids out of cells. ApoE and apoA-I also mediate receptor-independent effects, such as binding to and neutralizing LPS. Both apoE and apoA-I are expressed by lung cells, which allows apoE/LDLR- and apoA-I/ABCA1-dependent pathways to modulate normal lung health and the pathogenesis of respiratory diseases, including asthma, acute lung injury, cancer, emphysema, pulmonary fibrosis, and pulmonary hypertension. Data from human studies and research using experimental murine model systems have shown that both apoE and apoA-I pathways play primarily protective roles in lung biology and respiratory disease. Furthermore, apolipoprotein mimetic peptides, corresponding to the LDLR-binding domain of apoE or the class A amphipathic α-helical structure of apoA-I, have antiinflammatory and antioxidant effects that attenuate the severity of lung disease in murine models. Thus, the development of inhaled apolipoprotein mimetic peptides as a novel treatment paradigm could represent a significant advance for patients with respiratory disease who do not respond to current therapies.

An acoustofluidic sputum liquefier.

We demonstrate the first microfluidic-based on-chip liquefaction device for human sputum samples. Our device is based on an acoustofluidic micromixer using oscillating sharp edges. This acoustofluidic sputum liquefier can effectively and uniformly liquefy sputum samples at a throughput of 30 µL min(-1). Cell viability and integrity are maintained during the sputum liquefaction process. Our acoustofluidic sputum liquefier can be conveniently integrated with other microfluidic units to enable automated on-chip sputum processing and analysis.

Dendritic cells induce Th2-mediated airway inflammatory responses to house dust mite via DNA-dependent protein kinase.

DNA-dependent protein kinase (DNA-PK) mediates double-stranded DNA break repair, V(D)J recombination and immunoglobulin class switch recombination, as well as innate immune and pro-inflammatory responses. However, there is limited information regarding the role of DNA-PK in adaptive immunity mediated by dendritic cells (DCs), which are the primary antigen-presenting cells in allergic asthma. Here we show that house dust mite induces DNA-PK phosphorylation, which is a marker of DNA-PK activation, in DCs via the generation of intracellular reactive oxygen species. We also demonstrate that pharmacological inhibition of DNA-PK, as well as the specific deletion of DNA-PK in DCs, attenuates the induction of allergic sensitization and Th2 immunity via a mechanism that involves the impaired presentation of mite antigens. Furthermore, pharmacological inhibition of DNA-PK following antigen priming similarly reduces the manifestations of mite-induced airway disease. Collectively, these findings suggest that DNA-PK may be a potential target for treatment of allergic asthma.

ATP-binding cassette transporter 1 attenuates ovalbumin-induced neutrophilic airway inflammation.

Apolipoprotein A-I (apoA-I) is an important component of high-density lipoprotein particles that mediates reverse cholesterol transport out of cells by interacting with the ATP-binding cassette transporter 1 (ABCA1). apoA-I has also been shown to attenuate neutrophilic airway inflammation in experimental ovalbumin (OVA)-induced asthma by reducing the expression of granulocyte colony-stimulating factor (G-CSF). Here, we hypothesized that overexpression of the ABCA1 transporter might similarly attenuate OVA-induced neutrophilic airway inflammation. Tie2-human ABCA1 (hABCA1) mice expressing human ABCA1 under the control of the Tie2 promoter, which is primarily expressed by vascular endothelial cells, but can also be expressed by macrophages, received daily intranasal OVA challenges, 5 d/wk for 5 weeks. OVA-challenged Tie2-hABCA1 mice had significant reductions in total bronchoalveolar lavage fluid (BALF) cells that reflected a decrease in neutrophils, as well as reductions in peribronchial inflammation, OVA-specific IgE levels, and airway epithelial thickness. The reduced airway neutrophilia in OVA-challenged Tie2-hABCA1 mice was associated with significant decreases in G-CSF protein levels in pulmonary vascular endothelial cells, alveolar macrophages, and BALF. Intranasal administration of recombinant murine G-CSF to OVA-challenged Tie2-hABCA1 mice for 5 days increased BALF neutrophils to a level comparable to that of OVA-challenged wild-type mice. We conclude that ABCA1 suppresses OVA-induced airway neutrophilia by reducing G-CSF production by vascular endothelial cells and alveolar macrophages. These findings suggest that ABCA1 expressed by vascular endothelial cells and alveolar macrophages may play important roles in attenuating the severity of neutrophilic airway inflammation in asthma.