Search

Rationale Idiopathic Pulmonary Fibrosis (IPF) is thought to be triggered by repeated alveolar epithelial cell injury. Current evidence suggests that aberrant immune activation may contribute. However, the role of B-cell activation remains unclear. We determined the phenotype and activation status of B-cell subsets and evaluated the contribution of activated B-cells to the development of lung fibrosis both in humans and in mice. Methods B-cells in blood, mediastinal lymph node, and lung single-cell suspensions of IPF patients and healthy controls (HC) were characterized using 14-color flow cytometry. Mice were exposed to bleomycin to provoke pulmonary fibrosis. Results More IgA+ memory B-cells and plasmablasts were found in blood (n = 27) and lungs (n = 11) of IPF patients compared to HC (n = 21) and control lungs (n = 9). IPF patients had higher levels of autoreactive IgA in plasma, which correlated with an enhanced decline of forced vital capacity (p = 0.002, r = − 0.50). Bruton’s tyrosine kinase expression was higher in circulating IPF B-cells compared to HC, indicating enhanced B-cell activation. Bleomycin-exposed mice had increased pulmonary IgA+ germinal center and plasma cell proportions compared to control mice. The degree of lung fibrosis correlated with pulmonary germinal center B-cell proportions (p = 0.010, r = 0.88). Conclusion Our study demonstrates that IPF patients have more circulating activated B-cells and autoreactive IgA, which correlate with disease progression. These B-cell alterations were also observed in the widely used mouse model of experimental pulmonary fibrosis. Autoreactive IgA could be useful as a biomarker for disease progression in IPF.

Osteoprotegerin (OPG), a decoy receptor for receptor activator of NF-kB ligand (RANKL), is used as a biomarker for assessing severity of liver fibrosis. However, its expression and role in pulmonary fibrosis are unknown. We hypothesized that OPG also has a role in pulmonary fibrosis.Human and mouse control and fibrotic lung tissue were used to examine OPG expression, and mouse precision-cut lung slices to study OPG regulation in pulmonary fibrosis. Serum from idiopathic pulmonary fibrosis (IPF) patients and controls was analysed to investigate whether OPG levels correlate with disease status as measured by lung function.OPG-protein levels were significantly higher in mouse and human fibrotic lung tissue compared to control. OPG-mRNA and protein production were induced in mouse precision-cut-lung slices upon TGFβ stimulation and could be inhibited with galunisertib, a TGFβ receptor kinase inhibitor. OPG-protein levels in fibrotic mouse lung tissue correlated with degree of fibrosis. Isolated lung fibroblasts from IPF patients had higher OPG-protein levels than control fibroblasts. Serum OPG levels in IPF patients, at first presentation, negatively correlated with diffusing capacity to carbon monoxide. Finally, serum OPG levels higher than 1234 pg/ml at first presentation were associated with progression of disease in IPF patients.In conclusion, OPG is produced in lung tissue, associates with fibrosis, and may be a potential prognostic biomarker for IPF disease progression. Validation in a larger cohort is warranted to further explore the role of OPG in pulmonary fibrosis and its potential for assessing the prognosis of fibrotic lung disease in individual patients.Take home messageOsteoprotegerin is present in fibrotic lung tissue and high serum levels correlate with low lung function and IPF disease progression in this small study, indicating osteoprotegerin may have value as a biomarker to predict IPF progression

Higher levels of receptor activator for NF-κβ ligand (RANKL) are found in serum of emphysema patients. RANKL is a well-known stimulator of bone tissue degradation, possibly explaining the association of COPD with osteoporosis. However, RANKL is also reported to be involved in epithelial cell regeneration in breast and thymus. Given RANKL is produced directly in lung tissue, we hypothesized a role for RANKL in lung epithelial cell regeneration. The aim of this study was to elucidate the role of RANKL in lung epithelial repair. Mouse soluble RANKL (sRANKL) treatment of murine precision-cut lung slices resulted in a higher number of proliferating cells compared to untreated controls. We also found that sRANKL stimulated proliferation of type II alveolar (A549 cells) but not airway (16HBE) epithelial cells. Using an organoid model of epithelial development by co-culturing primary EpCAM+ cells with fibroblasts, we found higher numbers of alveolar organoids in cultures derived from murine epithelial cells upon sRANKL treatment compared to control. Importantly, this effect was similar in RANKL-treated organoids derived from epithelial cells isolated from lung tissue of COPD patients. The effect of sRANKL was abrogated upon addition of osteoprotegerin, the soluble, inhibitory, receptor for RANKL. In conclusion, we found that sRANKL promotes type II alveolar epithelial cell proliferation and may therefore contribute to lung tissue repair. Our data suggest that the higher levels of RANKL found in emphysema, may reflect an attempt at epithelial repair by the lung to counteract the lung tissue destruction that characterizes emphysema.

The enzyme tartrate resistant acid phosphatase (TRAP, two isoforms 5a and 5b) is highly expressed in alveolar macrophages, but its function there is unclear and potent selective inhibitors of TRAP are required to assess functional aspects of the protein. We found higher TRAP activity/expression in lungs of patients with chronic obstructive pulmonary disease (COPD) and asthma compared to controls and more TRAP activity in lungs of mice with experimental COPD or asthma. Stimuli related to asthma and/or COPD were tested for their capacity to induce TRAP. Receptor activator of NF-κb ligand (RANKL) and Xanthine/Xanthine Oxidase induced TRAP mRNA expression in mouse macrophages, but only RANKL also induced TRAP activity in mouse lung slices. Several Au(III) coordination compounds were tested for their ability to inhibit TRAP activity and [Au(4,4′-dimethoxy-2,2′-bipyridine)Cl2][PF6] (AubipyOMe) was found to be the most potent inhibitor of TRAP5a and 5b activity reported to date (IC50 1.3 and 1.8 μM respectively). AubipyOMe also inhibited TRAP activity in murine macrophage and human lung tissue extracts. In a functional assay with physiological TRAP substrate osteopontin, AubipyOMe inhibited mouse macrophage migration over osteopontin-coated membranes. In conclusion, higher TRAP expression/activity are associated with COPD and asthma and TRAP is involved in regulating macrophage migration.

Alternatively activated (M2 or YM1+) macrophages have been associated with the development of asthma but their contribution to disease initiation and progression remains unclear. To assess the therapeutic potential of modulating these M2 macrophages, we have studied inhibition of M2 polarisation during and after development of allergic lung inflammation by treating with cynaropicrin, a galectin-3 pathway inhibitor. Mice that were treated with this inhibitor of M2 polarisation during induction of allergic inflammation developed less severe eosinophilic lung inflammation and less collagen deposition around airways, while the airway α-smooth muscle actin layer was unaffected. When we treated with cynaropicrin after induction of inflammation, eosinophilic lung inflammation and collagen deposition were also inhibited though to a lesser extent. Unexpectedly, both during and after induction of allergic inflammation, inhibition of M2 polarisation resulted in a shift towards neutrophilic inflammation. Moreover, airway hyperresponsiveness was worse in mice treated with cynaropicrin as compared to allergic mice without inhibitor. These results show that M2 macrophages are associated with remodeling and development of eosinophilic lung inflammation, but prevent development of neutrophilic lung inflammation and worsening of airway hyperresponsiveness. This study suggests that macrophages contribute to determining development of eosinophilic or neutrophilic lung inflammation in asthma.

Draijer C, Hylkema MN, Boorsma CE, Klok PA, Robbe P, Timens W, Postma DS, Greene CM, Melgert BN. Sexual maturation protects against development of lung inflammation through estrogen. Am J Physiol Lung Cell Mol Physiol 310: L166-L174, 2016. First published November 25, 2015; doi:10.1152/ajplung. 00119.2015.-Increasing levels of estrogen and progesterone are suggested to play a role in the gender switch in asthma prevalence during puberty. We investigated whether the process of sexual maturation in mice affects the development of lung inflammation in adulthood and the contributing roles of estrogen and progesterone during this process. By inducing ovalbumin-induced lung inflammation in sexually mature and immature (ovariectomized before sexual maturation) adult mice, we showed that sexually immature adult mice developed more eosinophilic lung inflammation. This protective effect of "puberty" appears to be dependent on estrogen, as estrogen supplementation at the time of ovariectomy protected against development of lung inflammation in adulthood whereas progesterone supplementation did not. Investigating the underlying mechanism of estrogenmediated protection, we found that estrogen-treated mice had higher expression of the anti-inflammatory mediator secretory leukoprotease inhibitor (SLPI) and lower expression of the proasthmatic cytokine IL-33 in parenchymal lung tissue and that their expressions colocalized with type II alveolar epithelial cells (AECII). Treating AECII directly with SLPI significantly inhibited IL-33 production upon stimulation with ATP. Our data suggest that estrogen during puberty has a protective effect on asthma development, which is accompanied by induction of anti-inflammatory SLPI production and inhibition of proinflammatory IL-33 production by AECII.

Background Fibrocytes are implicated in Idiopathic Pulmonary Fibrosis (IPF) pathogenesis and increased proportions in the circulation are associated with poor prognosis. Upon tissue injury, fibrocytes migrate to the affected organ. In IPF patients, circulating fibrocytes are increased especially during exacerbations, however fibrocytes in the lungs have not been examined. Therefore, we sought to evaluate if fibrocytes can be detected in IPF lungs and we compare percentages and phenotypic characteristics of lung fibrocytes with circulating fibrocytes in IPF. Methods First we optimized flow cytometric detection circulating fibrocytes using a unique combination of intra- and extra-cellular markers to establish a solid gating strategy. Next we analyzed lung fibrocytes in single cell suspensions of explanted IPF and control lungs and compared characteristics and numbers with circulating fibrocytes of IPF. Results Using a gating strategy for both circulating and lung fibrocytes, which excludes potentially contaminating cell populations (e.g. neutrophils and different leukocyte subsets), we show that patients with IPF have increased proportions of fibrocytes, not only in the circulation, but also in explanted end-stage IPF lungs. These lung fibrocytes have increased surface expression of HLA-DR, increased intracellular collagen-1 expression, and also altered forward and side scatter characteristics compared with their circulating counterparts. Conclusions These findings demonstrate that lung fibrocytes in IPF patients can be quantified and characterized by flow cytometry. Lung fibrocytes have different characteristics than circulating fibrocytes and represent an intermediate cell population between circulating fibrocytes and lung fibroblast. Therefore, more insight in their phenotype might lead to specific therapeutic targeting in fibrotic lung diseases. Electronic supplementary material The online version of this article (10.1186/s12931-018-0798-8) contains supplementary material, which is available to authorized users.

In healthy lungs, many macrophages are characterized by IL-10 production, and few are characterized by expression of IFN regulatory factor 5 (formerly M1) or YM1 and/or CD206 (formerly M2), whereas in asthma, this balance shifts toward few producing IL-10 and many expressing IFN regulatory factor 5 or YM1/CD206. In this study, we tested whether redressing the balance by reinstating IL-10 production could prevent house dust mite-induced allergic lung inflammation. PGE2 was found to be the best inducer of IL-10 in macrophages in vitro. Mice were then sensitized and challenged to house dust mites during a 2 wk protocol while treated with PGE2 in different ways. Lung inflammation was assessed 3 d after the last house dust mite challenge. House dust mite-exposed mice treated with free PGE2 had fewer infiltrating eosinophils in lungs and lower YM1 serum levels than vehicle-treated mice. Macrophage-specific delivery of PGE2 did not affect lung inflammation. Adoptive transfer of PGE2-treated macrophages led to fewer infiltrating eosinophils, macrophages, (activated) CD4+, and regulatory T lymphocytes in lungs. Our study shows that the redirection of macrophage polarization by using PGE2 inhibits development of allergic lung inflammation. This beneficial effect of macrophage repolarization is a novel avenue to explore for therapeutic purposes.

In asthma, macrophage polarization is altered from a state associated with anti-inflammatory responses to a state associated with inflammation as compared to control. Macrophage polarization states is linked to disease severity, sex and ICS treatment.

Macrophages are abundantly present in the airways of asthmatics and they can respond to allergens with different states of activation at the cost of the anti-inflammatory state. These states have not been quantified in lung tissue of asthmatics versus healthy controls yet, and it is unknown how they relate to disease severity and treatment responses. We quantified activated and anti-inflammatory macrophages in asthma patients and healthy controls and studied associations with lung function, sex and corticosteroid use. 138 bronchial biopsies from clinically well-characterized asthma patients and 50 matched healthy controls were stained for activated macrophages defined by IRF5+CD68+ or CD206+CD68+ and anti-inflammatory macrophages defined by IL-10+CD68+. Double positive cells were counted and expressed as positive cells per millimeter basement membrane. Higher numbers of IRF5+ and CD206+ macrophages were found in biopsies of asthma patients compared to healthy individuals, with males having more IRF5+ and females more CD206+ macrophages. Asthma patients had lower numbers of IL-10+ macrophages than healthy individuals, with more IL10+ macrophages in corticosteroid users than in non-users. Higher numbers of IRF5+ macrophages are associated with more severe airflow obstruction as reflected by lower FEV1 and FEV1/FVC values, while the number of IL-10+ macrophages correlated positively with FEV1/FVC ratio (Pearson r=0.18, p This study suggests that macrophages are important in asthma. Their activation state can be linked with disease severity, sex and corticosteroid treatment. Especially increasing the number of anti-inflammatory macrophages by therapeutic intervention may be a novel way of treating asthma.

M2 macrophages have been implicated in the development of asthma but their contribution to disease initiation and progression remains unclear. To assess the therapeutic potential of modulating M2 macrophages, we have studied inhibition of M2 polarization during and after development of murine allergic lung inflammation by treating with cynaropicrin, a galectin-3 pathway inhibitor. Female Balb/c mice were exposed i.n. to house dust mite (HDM) or phosphate-buffered saline (PBS) for 2 weeks. Mice were treated with cynaropicrin (2.5 mg/kg), PBS or vehicle (4% DMSO in PBS) during or after the second week of HDM exposures. Severity of lung inflammation, airway remodeling and airway hyperresponsiveness were assessed 3 days after the last HDM exposure. Activity of M2 macrophages was assessed by YM1 production. Mice treated with cynaropicrin during and after HDM exposures had less eosinophils and Th2 cytokines in lung tissue than vehicle-treated mice. However, both sets of mice did have more neutrophils and higher levels of IL-12 present in lung tissue. Moreover, airway hyperresponsiveness was worse in mice treated with cynaropicrin as compared to vehicle-treated mice. Less airway remodeling was observed in mice treated with cynaropicrin as compared to vehicle-treated mice. These results suggest that M2 macrophages contribute to both induction and progression of eosinophilic lung inflammation and protect against development of neutrophilic inflammation. In addition, our results suggest that M2 macrophages contribute to airway remodeling and thereby protect against development of more severe airway hyperresponsiveness. This study reveals the important dual role of M2 macrophages in asthma.

Alveolar macrophages express Receptor Activator of NF-κB (RANK), which is a known regulator of bone matrix turnover. Upon binding of its ligand RANKL, extracellular matrix (ECM) degradation can be induced. This interaction is inhibited by osteoprotegerin (OPG), the decoy receptor of RANKL and previously found to be elevated in pulmonary fibrosis (PF). We hypothesized that OPG in PF dampens ECM degradation by macrophages leading to fibrosis and proceeded to characterize and influence the system in lung tissue. We compared RANKL and OPG protein levels in lung tissue of PF patients (n=11) and controls (n=16), and control and PF mice (n=6). Exact sources were determined by immunohistochemistry, ELISA, and flow cytometry. Furthermore, mice with established PF were treated with soluble RANKL followed by total lung collagen analysis to assess PF. PF patients and mice had significantly higher OPG levels in lung tissue compared to their respective controls (p ECM modulator OPG is differentially expressed in healthy versus fibrotic conditions and induction of OPG following RANKL treatment suggests that the balance with RANKL is tightly regulated. These results support the importance of a RANKL-OPG balance in PF and may constitute a new target for treatment.

In asthma, an important role for innate immunity is increasingly being recognized. Key innate immune cells in the lungs are macrophages. Depending on the signals they receive, macrophages can at least have an M1, M2, or M2-like phenotype. It is unknown how these macrophage phenotypes behave with regard to (the severity of) asthma. We have quantified the phenotypes in three models of house dust mite (HDM-)induced asthma (14, 21, and 24 days). M1, M2, and M2-like phenotypes were identified by interferon regulatory factor 5 (IRF5), YM1, and IL-10, respectively. We found higher percentages of eosinophils in HDM-exposed mice compared to control but no differences between HDM models. T cell numbers were higher after HDM exposure and were the highest in the 24-day HDM protocol. Higher numbers of M2 macrophages after HDM correlated with higher eosinophil numbers. In mice with less severe asthma, M1 macrophage numbers were higher and correlated negatively with M2 macrophages numbers. Lower numbers of M2-like macrophages were found after HDM exposure and these correlated negatively with M2 macrophages. The balance between macrophage phenotypes changes as the severity of allergic airway inflammation increases. Influencing this imbalanced relationship could be a novel approach to treat asthma.