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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.

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.

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.

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.