Carian E. Boorsma

Macrophage Heterogeneity in Respiratory Diseases

Macrophages are among the most abundant cells in the respiratory tract, and they can have strikingly different phenotypes within this environment. Our knowledge of the different phenotypes and their functions in the lung is sketchy at best, but they appear to be linked to the protection of gas exchange against microbial threats and excessive tissue responses. Phenotypical changes of macrophages within the lung are found in many respiratory diseases including asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis. This paper will give an overview of what macrophage phenotypes have been described, what their known functions are, what is known about their presence in the different obstructive and restrictive respiratory diseases (asthma, COPD, pulmonary fibrosis), and how they are thought to contribute to the etiology and resolution of these diseases.

Characterization of Macrophage Phenotypes in Three Murine Models of House-Dust-Mite-Induced Asthma

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.

PGE2-treated macrophages inhibit development of allergic lung inflammation in mice

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.

Human asthma is characterized by more IRF5+ M1 and CD206+ M2 macrophages and less IL-10+ M2-like macrophages around airways compared with healthy airways

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.

A Potent Tartrate Resistant Acid Phosphatase Inhibitor to Study the Function of TRAP in Alveolar Macrophages

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.

Dual role of YM1+ M2 macrophages in allergic lung inflammation

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.

Fibrocytes are increased in lung and peripheral blood of patients with idiopathic pulmonary fibrosis

BackgroundFibrocytes 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.MethodsFirst 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.ResultsUsing 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.ConclusionsThese 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.