High-Dimensional Mass Cytometry Reveals Emphysema-associated Changes in the Pulmonary Immune System.

RATIONALE: Chronic inflammation plays an important role in alveolar tissue damage in emphysema, but the underlying immune alterations and cellular interactions are incompletely understood. OBJECTIVE: To explore disease-specific pulmonary immune cell alterations and cellular interactions in emphysema. METHODS: We used single-cell mass cytometry to compare the immune compartment in alveolar tissue from 15 patients with severe emphysema and 5 controls. Imaging mass cytometry (IMC) was applied to identify altered cell-cell interactions in alveolar tissue from emphysema patients (n=12) compared to controls (n=8). MEASUREMENTS AND MAIN RESULTS: We observed higher percentages of central memory CD4 T cells in combination with lower proportions of effector memory CD4 T cells in emphysema. In addition, proportions of cytotoxic central memory CD8 T cells and CD127+CD27+CD69- T cells were higher in emphysema, the latter potentially reflecting an influx of circulating lymphocytes into the lungs. Central memory CD8 T cells, isolated from alveolar tissue from emphysema patients exhibited an IFN-γ-response upon anti-CD3/anti-CD28 activation. Proportions of CD1c+ dendritic cells (DC), expressing migratory and costimulatory markers, were higher in emphysema. Importantly, IMC enabled us to visualize increased spatial colocalization of CD1c+ DC and CD8 T cells in emphysema in situ. CONCLUSION: Using single-cell CyTOF, we characterized the alterations of the immune cell signature in alveolar tissue from patients with COPD stage III/IV emphysema versus control lung tissue. These data contribute to a better understanding of the pathogenesis of emphysema and highlight the feasibility of interrogating the immune cell signature using single-cell and IMC in human lung tissue.

Organoid-based expansion of patient-derived primary alveolar type 2 cells for establishment of alveolus epithelial Lung-Chip cultures.

Development of effective treatment strategies for lung tissue destruction as seen in emphysema would greatly benefit from representative human in vitro models of the alveolar compartment. Studying how cellular cross talk and/or (altered) biomechanical cues affect alveolar epithelial function could provide new insight for tissue repair strategies. Preclinical models of the alveolus ideally combine human primary patient-derived lung cells with advanced cell culture applications such as breathing-related stretch, to reliably represent the alveolar microenvironment. To test the feasibility of such a model, we isolated primary alveolar type 2 cells (AEC2s) from patient-derived lung tissues including those from patients with severe emphysema, using magnetic bead-based selection of cells expressing the AEC2 marker HTII-280. We obtained pure alveolar feeder-free organoid cultures using a minimally modified commercial medium. This was confirmed by known AEC2 markers as well as by detection of lamellar bodies using electron microscopy. Following (organoid-based) expansion, cells were seeded on both cell culture inserts and the Chip-S1 Organ-Chip that has a flexible polydimethylsiloxane (PDMS) membrane enabling the application of dynamic stretch. AEC2s cultured for 7 days on inserts or the chip maintained expression of HTII-280, prosurfactant protein C (SP-C), SP-A and SP-B, and zonula occludens-1 (ZO-1) also in the presence of stretch. AEC2s cultured on the chip showed lower expression levels of epithelial-mesenchymal transition-related vimentin expression compared with static cultures on inserts. The combination of a straightforward culture method of patient-derived AEC2s and their application in microfluidic chip cultures supports successful development of more representative human preclinical models of the (diseased) alveolar compartment.

Highlights of the ERS Lung Science Conference 2022.

This article presents the highlights of the ERS Lung Science Conference 2022, including a session organised by the Early Career Member Committee (ECMC) dedicated to career development https://bit.ly/3tarCXc.

Disease modeling following organoid-based expansion of airway epithelial cells.

Air-liquid interface (ALI) cultures are frequently used in lung research but require substantial cell numbers that cannot readily be obtained from patients. We explored whether organoid expansion [three-dimensional (3D)] can be used to establish ALI cultures from clinical samples with low epithelial cell numbers. Airway epithelial cells were obtained from tracheal aspirates (TA) from preterm newborns and from bronchoalveolar lavage (BAL) or bronchial tissue (BT) from adults. TA and BAL cells were 3D-expanded, whereas cells from BT were expanded in 3D and 2D. Following expansion, cells were cultured at ALI to induce differentiation. The impact of cell origin and 2D or 3D expansion was assessed with respect to 1) cellular composition, 2) response to cigarette smoke exposure, and 3) effect of Notch inhibition or IL-13 stimulation on cellular differentiation. We established well-differentiated ALI cultures from all samples. Cellular compositions (basal, ciliated, and goblet cells) were comparable. All 3D-expanded cultures showed a similar stress response following cigarette smoke exposure but differed from the 2D-expanded cultures. Higher peak levels of antioxidant genes HMOX1 and NQO1 and a more rapid return to baseline, and a lower unfolded protein response was observed after cigarette smoke exposure in 3D-derived cultures compared to 2D-derived cultures. In addition, TA- and BAL-derived cultures were less sensitive to modulation by DAPT or IL-13 than BT-derived cultures. Organoid-based expansion of clinical samples with low cell numbers, such as TA from preterm newborns is a valid method and tool to establish ALI cultures.

Acute and chronic effects of treatment with mesenchymal stromal cells on LPS-induced pulmonary inflammation, emphysema and atherosclerosis development.

BACKGROUND: COPD is a pulmonary disorder often accompanied by cardiovascular disease (CVD), and current treatment of this comorbidity is suboptimal. Systemic inflammation in COPD triggered by smoke and microbial exposure is suggested to link COPD and CVD. Mesenchymal stromal cells (MSC) possess anti-inflammatory capacities and MSC treatment is considered an attractive treatment option for various chronic inflammatory diseases. Therefore, we investigated the immunomodulatory properties of MSC in an acute and chronic model of lipopolysaccharide (LPS)-induced inflammation, emphysema and atherosclerosis development in APOE*3-Leiden (E3L) mice. METHODS: Hyperlipidemic E3L mice were intranasally instilled with 10 µg LPS or vehicle twice in an acute 4-day study, or twice weekly during 20 weeks Western-type diet feeding in a chronic study. Mice received 0.5x106 MSC or vehicle intravenously twice after the first LPS instillation (acute study) or in week 14, 16, 18 and 20 (chronic study). Inflammatory parameters were measured in bronchoalveolar lavage (BAL) and lung tissue. Emphysema, pulmonary inflammation and atherosclerosis were assessed in the chronic study. RESULTS: In the acute study, intranasal LPS administration induced a marked systemic IL-6 response on day 3, which was inhibited after MSC treatment. Furthermore, MSC treatment reduced LPS-induced total cell count in BAL due to reduced neutrophil numbers. In the chronic study, LPS increased emphysema but did not aggravate atherosclerosis. Emphysema and atherosclerosis development were unaffected after MSC treatment. CONCLUSION: These data show that MSC inhibit LPS-induced pulmonary and systemic inflammation in the acute study, whereas MSC treatment had no effect on inflammation, emphysema and atherosclerosis development in the chronic study.

The Effects of Selective Hematopoietic Expression of Human IL-37 on Systemic Inflammation and Atherosclerosis in LDLr-Deficient Mice.

The human cytokine interleukin (IL)-37 has potent anti-inflammatory capacities, and hematopoietic cell-specific transgenic overexpression of IL-37 in mice protects against septic shock and colitis. In the present study we investigated the effect of hematopoietic expression of IL-37 on atherosclerosis development under low-grade inflammatory conditions. Low-density lipoprotein receptor (LDLr)-deficient mice were lethally irradiated and transplanted with bone marrow from IL-37-transgenic or control wild-type mice and fed a Western-type diet (WTD; 1% cholesterol) for eight weeks. Metabolic and inflammatory parameters were monitored and atherosclerosis was assessed in the aortic valve area. Hematopoietic IL-37 expression did not influence body weight, food intake and plasma cholesterol levels during the study. Plasma soluble E-selectin levels were increased with WTD-feeding as compared to chow-feeding, but were not influenced by IL-37 expression. IL-37 expression reduced the inflammatory state as indicated by reduced white blood cell counts and by reduced basal and lipopolysaccharide-induced cytokine response by peritoneal macrophages ex vivo. IL-37 expression did not influence the atherosclerotic lesion area. Lesion composition was marginally affected. Smooth muscle cell content was decreased, but macrophage and collagen content were not different. We conclude that under low-grade inflammatory conditions, hematopoietic IL-37 expression reduces the inflammatory state, but does not influence atherosclerosis development in hyperlipidemic LDLr-deficient mice.

Murine models of cardiovascular comorbidity in chronic obstructive pulmonary disease.

Patients with chronic obstructive pulmonary disease (COPD) have an increased risk for cardiovascular disease (CVD). Currently, COPD patients with atherosclerosis (i.e., the most important underlying cause of CVD) receive COPD therapy complemented with standard CVD therapy. This may, however, not be the most optimal treatment. To investigate the link between COPD and atherosclerosis and to develop specific therapeutic strategies for COPD patients with atherosclerosis, a substantial number of preclinical studies using murine models have been performed. In this review, we summarize the currently used murine models of COPD and atherosclerosis, both individually and combined, and discuss the relevance of these models for studying the pathogenesis and development of new treatments for COPD patients with atherosclerosis. Murine and clinical studies have provided complementary information showing a prominent role for systemic inflammation and oxidative stress in the link between COPD and atherosclerosis. These and other studies showed that murine models for COPD and atherosclerosis are useful tools and can provide important insights relevant to understanding the link between COPD and CVD. More importantly, murine studies provide good platforms for studying the potential of promising (new) therapeutic strategies for COPD patients with CVD.

Splenic autonomic denervation increases inflammatory status but does not aggravate atherosclerotic lesion development.

UNLABELLED: The brain plays a prominent role in the regulation of inflammation. Immune cells are under control of the so-called cholinergic anti-inflammatory reflex, mainly acting via autonomic innervation of the spleen. Activation of this reflex inhibits the secretion of proinflammatory cytokines and may reduce the development of atherosclerosis. Therefore, the aim of this study was to evaluate the effects of selective parasympathetic (Px) and sympathetic (Sx) denervation of the spleen on inflammatory status and atherosclerotic lesion development. Female APOE*3-Leiden.CETP mice, a well-established model for human-like lipid metabolism and atherosclerosis, were fed a cholesterol-containing Western-type diet for 4 wk after which they were subdivided into three groups receiving either splenic Px, splenic Sx, or sham surgery. The mice were subsequently challenged with the same diet for an additional 15 wk. Selective Px increased leukocyte counts (i.e., dendritic cells, B cells, and T cells) in the spleen and increased gene expression of proinflammatory cytokines in the liver and peritoneal leukocytes compared with Sx and sham surgery. Both Px and Sx increased circulating proinflammatory cytokines IL-1ß and IL-6. However, the increased proinflammatory status in denervated mice did not affect atherosclerotic lesion size or lesion composition. CONCLUSION: Predominantly selective Px of the spleen enhances the inflammatory status, which, however, does not aggravate diet-induced atherosclerotic lesion development.

Brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development.

Brown adipose tissue (BAT) combusts high amounts of fatty acids, thereby lowering plasma triglyceride levels and reducing obesity. However, the precise role of BAT in plasma cholesterol metabolism and atherosclerosis development remains unclear. Here we show that BAT activation by ß3-adrenergic receptor stimulation protects from atherosclerosis in hyperlipidemic APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism that unlike hyperlipidemic Apoe(-/-) and Ldlr(-/-) mice expresses functional apoE and LDLR. BAT activation increases energy expenditure and decreases plasma triglyceride and cholesterol levels. Mechanistically, we demonstrate that BAT activation enhances the selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT, subsequently accelerating the hepatic clearance of the cholesterol-enriched remnants. These effects depend on a functional hepatic apoE-LDLR clearance pathway as BAT activation in Apoe(-/-) and Ldlr(-/-) mice does not attenuate hypercholesterolaemia and atherosclerosis. We conclude that activation of BAT is a powerful therapeutic avenue to ameliorate hyperlipidaemia and protect from atherosclerosis.

Brown adipose tissue takes up plasma triglycerides mostly after lipolysis.

Brown adipose tissue (BAT) produces heat by burning TGs that are stored within intracellular lipid droplets and need to be replenished by the uptake of TG-derived FA from plasma. It is currently unclear whether BAT takes up FA via uptake of TG-rich lipoproteins (TRLs), after lipolysis-mediated liberation of FA, or via a combination of both. Therefore, we generated glycerol tri[(3)H]oleate and [(14)C]cholesteryl oleate double-labeled TRL-mimicking particles with an average diameter of 45, 80, and 150 nm (representing small VLDL to chylomicrons) and injected these intravenously into male C57Bl/6J mice. At room temperature (21°C), the uptake of (3)H-activity by BAT, expressed per gram of tissue, was much higher than the uptake of (14)C-activity, irrespective of particle size, indicating lipolysis-mediated uptake of TG-derived FA rather than whole particle uptake. Cold exposure (7°C) increased the uptake of FA derived from the differently sized particles by BAT, while retaining the selectivity for uptake of FA over cholesteryl ester (CE). At thermoneutrality (28°C), total FA uptake by BAT was attenuated, but the specificity of uptake of FA over CE was again largely retained. Altogether, we conclude that, in our model, BAT takes up plasma TG preferentially by means of lipolysis-mediated uptake of FA.

The effect of PPE-induced emphysema and chronic LPS-induced pulmonary inflammation on atherosclerosis development in APOE*3-LEIDEN mice.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by pulmonary inflammation, airways obstruction and emphysema, and is a risk factor for cardiovascular disease (CVD). However, the contribution of these individual COPD components to this increased risk is unknown. Therefore, the aim of this study was to determine the contribution of emphysema in the presence or absence of pulmonary inflammation to the increased risk of CVD, using a mouse model for atherosclerosis. Because smoke is a known risk factor for both COPD and CVD, emphysema was induced by intratracheal instillation of porcine pancreatic elastase (PPE). METHODS: Hyperlipidemic APOE*3-Leiden mice were intratracheally instilled with vehicle, 15 or 30 µg PPE and after 4 weeks, mice received a Western-type diet (WTD). To study the effect of emphysema combined with pulmonary inflammation on atherosclerosis, mice received 30 µg PPE and during WTD feeding, mice were intranasally instilled with vehicle or low-dose lipopolysaccharide (LPS; 1 µg/mouse, twice weekly). After 20 weeks WTD, mice were sacrificed and emphysema, pulmonary inflammation and atherosclerosis were analysed. RESULTS: Intratracheal PPE administration resulted in a dose-dependent increase in emphysema, whereas atherosclerotic lesion area was not affected by PPE treatment. Additional low-dose intranasal LPS administration induced a low-grade systemic IL-6 response, as compared to vehicle. Combining intratracheal PPE with intranasal LPS instillation significantly increased the number of pulmonary macrophages and neutrophils. Plasma lipids during the study were not different. LPS instillation caused a limited, but significant increase in the atherosclerotic lesion area. This increase was not further enhanced by PPE. CONCLUSION: This study shows for the first time that PPE-induced emphysema both in the presence and absence of pulmonary inflammation does not affect atherosclerotic lesion development.