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Case Study About Acid Phosphatase Assay

TRAP expression is higher in smokers and in patients with COPD

To assess whether TRAP mRNA expression is changed in COPD versus control lung tissue, we did a single gene look-up for TRAP in a genome wide gene expression dataset comparing 311 COPD patients and 270 non-COPD controls31. Among the upregulated genes, TRAP was identified as significantly higher in COPD patients compared to control patients (Fig. 2a). To investigate the effect of current smoking on TRAP expression, we additionally compared control individuals currently smoking with individuals that had stopped smoking for at least 5 years in the same dataset. This comparison showed significantly higher expression of TRAP in the individuals that are currently smoking versus ex-smokers (Fig. 2b). A similar analysis among the COPD patients showed no differences between current and ex-smokers (data not shown).

In addition, we examined whether TRAP mRNA expression correlated with lung function in COPD patients (as defined by FEV1) and found a significant but weak negative correlation, meaning higher TRAP expression was linked with lower FEV1 values (Fig. 2c). This correlation is mainly caused by the high expression of TRAP in lung tissue of patients with severe COPD: patients with the most severe disease, i.e. highest GOLD stage and therefore lowest FEV1 value, had significantly higher expression of TRAP in lung tissue as compared to nonCOPD controls, while the patients with less severe COPD had similar TRAP expression as compared to controls (Fig. 2d).

Patients dying of asthma have more TRAP-active macrophages in lung tissue

To assess whether asthma is also characterized by changes in TRAP, we investigated the number of cells staining positive for TRAP activity in lung sections of patients who had died from an asthma attack or had died of non-pulmonary causes. The sections showed that only alveolar macrophages stained positive for active TRAP enzyme, as judged by their morphology and location in the tissue, though not all alveolar macrophages were positive for TRAP activity (Fig. 3a and b, some are indicated by arrows). In addition, the number of macrophages positive for TRAP activity was higher in lung tissue from patients with fatal asthma as compared to control subjects (Fig. 3c).

The number of TRAP-active cells is higher in mouse models for COPD and asthma

To check if higher expression/activity of TRAP in humans with pulmonary disease was a general phenomenon that could be extrapolated to mouse models, we examined TRAP activity in lungs of mice exposed to either cigarette smoke for 9 months (COPD model) or house dust mite (HDM) for 2 weeks (asthma model). Again, we stained for active TRAP enzyme and found that alveolar macrophages, as judged by their morphology and location in the tissue, stained strongly positive for active TRAP enzyme, though not all of them were positive for TRAP activity (Fig. 4a,b,d,e, some are indicated by arrows). In lung tissue of mice that were exposed to cigarette smoke (Fig. 4c or HDM (Fig. 4f) we found significantly more TRAP-positive macrophages than in lung tissue of the relevant control mice. In lung tissue of HDM-exposed mice faint staining for active TRAP enzyme could also be noticed in inflammatory infiltrates and in epithelial cells of the large airways (Fig. 4e).

TRAP expression is upregulated by RANKL and oxidative stress

In order to study what causes the higher activity and/or expression of TRAP in alveolar macrophages, we exposed murine MPI alveolar-like macrophages (Max Planck Institute, a kind gift from Dr. Gyorgy Fejer32) and murine precision-cut lung slices to various stimuli related to COPD and asthma, namely IL-4, M-CSF and RANKL, the damage-associated molecular pattern ATP, and oxidative stress mimicked by the xanthine/xanthine oxidase (X/XO) system. Notably, TRAP mRNA expression in MPI alveolar-like macrophages was significantly higher after stimulation with RANKL and the X/XO system (Fig. 5A). M-CSF stimulation resulted in a trend towards lower TRAP mRNA expression. No significant effects were observed after stimulation with ATP or IL-4.

To study whether changes in mRNA expression would also lead to changes in active enzyme, we used precision-cut lung slices to study the effects of RANKL, ATP and oxidative stress on TRAP activity (Fig. 5B). Only RANKL treatment resulted in significantly higher TRAP activity in lung slices as compared to control conditions. Conversely, ATP treatment and induction of oxidative stress with X/XO treatment did not lead to significant changes in TRAP activity.

The Au(III) compound AubipyOMe inhibits TRAP activity

Having a potent and specific inhibitor of TRAP can greatly benefit studies into its function, and we therefore investigated whether we could improve on the currently known inhibitors of TRAP15,20,21,22,23,24. The most potent inhibitor previously reported is the inorganic complex NaAuCl420, but this Au(III) reactive compound is prone to reduction in biological environments and features unspecific protein binding and oxidative damage, which may interfere with many different cellular pathways33. Therefore, a series of gold coordination compounds, more stable in biological environments compared to NaAuCl4, were evaluated as possible TRAP activity inhibitors: these included mono- and di-nuclear Au(III) compounds with N-donor ligands and the previously tested anti-rheumatic agent sodium aurothiomalate (Myochrysine®, see Supplementary Fig. S1 for the structures of the compounds tested)20. The initial screening using commercially bought recombinant TRAP revealed that the compound AubipyOMe possessed the best TRAP inhibition activity described to date, similar to NaAuCl4, being able to inhibit the protein activity with IC50 in the nanomolar range (see Supplementary Fig. S2 for inhibition curves of all compounds tested).

Thus, we continued our investigations with AubipyOMe, and NaAuCl4 as reference compound, to further assess its selectivity for the TRAP isoforms 5a and 5b. AubipyOMe inhibited TRAP5a activity with an IC50 value of 1.3 ± 0.5 μM and TRAP5b with an IC50 value of 1.8 ± 0.3 μM (Fig. 6a and b). These IC50 values were comparable to the values found for NaAuCl4 (see Table 1 and Supplementary Fig. S3 for the individually fitted curves used to calculate IC50 values). To assess the inhibition potencies in more relevant biological settings, we continued testing AubipyOMe and NaAuCl4, in cell and tissue lysates. TRAP activity in cell lysates of MPI alveolar macrophages was significantly inhibited in the presence of AubipyOMe and NaAuCl4 with IC50 values of 1.7 ± 0.4 μM and 0.7±0.0 μM, respectively (Fig. 6c, Table 1, and Supplementary Fig. S3 for the individually fitted curves used to calculate IC50 values). Importantly, the inhibitory effects of AubipyOMe and NaAuCl4 were also tested on TRAP activity in pooled lung tissue lysates from COPD patients. AubipyOMe significantly inhibited TRAP activity in these lysates with an IC50 value of 4.8 ± 1.3 μM, while NaAuCl4 inhibited the activity with an IC50 value of 3.6 ± 0.0 μM (Fig. 6d, table, and Supplementary Fig. S3 for the individually fitted curves used to calculate IC50 values). At concentrations around these IC50 values, both AubipyOMe and NaAuCl4 had no cytotoxic effects on RAW264.7 macrophages (Fig. 6e). Only in very high concentrations AubipyOMe showed some cytotoxicity (IC50 around 35 μM) and NaAuCl4 did not display significant toxicity (IC50 > 200 μM).

Macrophage migration depends on TRAP activity and is inhibited by AubipyOMe

Osteoclast migration was previously shown to be TRAP-dependent through the ability of TRAP to dephosphorylate osteopontin2,14. To investigate if this is also the case for macrophages we used RAW264.7 macrophages because we could modulate TRAP expression and activity from low to high by pretreatment with RANKL in these cells34. We subsequently investigated the effects of having TRAP activity and inhibition of this TRAP activity on macrophage migration in a transwell and live cell-imaging setup (Fig. 7).

RAW264.7 macrophages pretreated with RANKL for 72 hours migrated significantly more through an osteopontin-coated membrane as compared to unstimulated cells (Fig. 7a). This effect was osteopontin-specific, as migration over a membrane coated with collagen was not affected by RANKL pretreatment (Fig. 7b). RANKL pretreatment and the presence of our proposed TRAP inhibitor AubipyOMe led to significantly less migration of RAW264.7 macrophages as compared to RANKL pretreatment alone. Accordingly, the presence of the previously published TRAP-inhibitor 5-PNA, that inhibits TRAP-dependent migration of TRAP-overexpressing cancer cells also inhibited macrophage migration induced by RANKL pretreatment15. Treatment of the cells with either inhibitors alone did not affect macrophage migration and neither did AubipyOMe affect migration when cells were grown on collagen-coated membranes, indicating that the inhibition was not unspecific (Fig. 7).

Furthermore, we investigated the involvement of TRAP in macrophage migration using live cell imaging. RAW264.7 macrophages plated on wells coated with osteopontin showed more migratory behavior after RANKL pretreatment than control cells not pretreated with RANKL (Fig. 7c, movie 1 versus 2 in supplementary information). The observed increased migratory behavior was not recorded when RANKL-pretreated cells were in the presence of AubipyOMe (Fig. 7c, movie 3, supplementary information). The presence of AubipyOMe did not affect the migratory behavior of control cells (Fig. 7c, movie 4, supplementary information).

For both the transwell migration experiments as well as the live cell imaging, pretreatment with RANKL for 48–72 hours did not lead to the development of multinucleated osteoclast-like cells. This can also be appreciated from the images in Fig. 7c and the movies in the supplementary information available.

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