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Comparison of anti-inflammatory mechanisms between doxofylline and theophylline in human monocytes
Pulmonary Pharmacology & Therapeutics 59 (2019) 101851
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Comparison of anti-inflammatory mechanisms between doxofylline and theophylline in human monocytes
T
Maria Talmon, Erika Massara, Chiara Brunini, Luigia Grazia Fresu∗ Department of Health Sciences, University of Piemonte Orientale, Via Solaroli 17, 28100, Novara, Italy
A R T I C LE I N FO
A B S T R A C T
Keywords: Monocytes Methylxanthines Theophylline Doxofylline Cytokines Superoxide anion
Background: Methylxanthines are important pharmacological agents in the treatment of asthma and of chronic obstructive pulmonary diseases. The present study was designed to compare the ability of doxofylline and theophylline to modulate inflammatory pathways in human monocytes. Methods: Monocytes isolated from healthy anonymous human buffy coats were treated with doxofylline or theophylline in the presence of phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS), and their phenotype, the oxidative burst, cytokine expression and release, cAMP production, and protein kinase C (PKC) activity were evaluated. Results: Doxofylline and theophylline did not have overlapping effects on human monocytes. While sharing some common characteristics, they differed significantly in their selectivity. Theophylline affected LPS- above PMA-induced cellular responsivity, while doxofylline behaved in the opposite manner. Furthermore, when testing PKC activity, we found an inhibitory effect of doxofylline but not of theophylline, at equimolar doses. Conclusions: In conclusion, our data support the growing hypothesis that doxofylline does not have a superimposable mechanism of action compared to theophylline, and this may both explain some differences in the risk/benefit ratio and may direct studies to tailor therapy for patients.
1. Introduction Methylxanthines are indicated both in GOLD [1] and GINA [2] guidelines as add-on agents in the treatment of asthma and of chronic obstructive pulmonary disease (COPD). Theophylline is the most widely used methylxanthine, although other drugs have also reached the market, including doxofylline, bamifylline and acebrofylline [3]. Recently, there has been a resurgence in the interest in doxofylline and various meta-analyses have shown that doxofylline might not have a superimposable risk/benefit compared to theophylline [4–6]. Chemically, a dioxolane group differentiates doxofylline from theophylline, but the two molecules have been postulated to have a number of distinct pharmacodynamics characteristics. Theophylline has been reported to be a non-selective inhibitor of phosphodiesterases (PDE) and of phosphoinositide 3-kinase-δ (PI3K-δ), an antagonist of adenosine receptors (A1, A2A/B and A3) and a modulator of histone deacetylases (HDACs) [7]. Doxofylline, instead, has been suggested not
to have affinity for most adenosine receptors (with the exception of A2A), to lack significant activity on PDEs, except for PDE2A1, and to have a more prominent anti-inflammatory effect [8,9]. Given the elusive mechanism of methylxanthines, though, it is difficult to ascertain whether these differences may be reconducted to the differences observed in patients. While several studies have evaluated the anti-inflammatory activity of theophylline on monocytes/macrophages [10–12] there are no reports on doxofylline, and it is therefore difficult to ascertain whether the pharmacodynamics differences reported are translated to this cell type, which is increasingly being involved in pulmonary disorders. Studies in murine models have demonstrated that monocytes accumulate in the lung and play a role in allergic reactions both by secreting cytokines and chemokines [13] and by maintaining T cell-mediated immunity [14]. This sustains asthma exacerbations in paediatric patients in which activated circulating monocytes are increased [15]. Monocytes constitute 5–10% of peripheral blood leukocytes and
Abbreviations: COPD, chronic obstructive pulmonary disease; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDTA, ethylene diamine tetraacetic acid; EGTA, ethylene glycol tetraacetic acid; FBS, fetal bovine serum; HDACs, histone deacetylases; LPS, lipopolysaccharide; MOPS, 3-(N-morpholino) propanesulfonic acid; MTT, methylthiazolyldiphenyl-tetrazolium bromide; PDE, phosphodiesterases; PI3K-δ, phosphoinositide 3-kinase-δ; PKC, phosphor kinase C; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; SOD, superoxide dismutase ∗ Corresponding author. Department of Health Sciences, Via Solaroli, 17, 28100, Novara, Italy. E-mail address: [email protected] (L.G. Fresu). https://doi.org/10.1016/j.pupt.2019.101851 Received 1 May 2019; Received in revised form 26 September 2019; Accepted 26 September 2019 Available online 26 September 2019 1094-5539/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Effect of methylxanthines on cell viability. Monocytes were treated for 6 and 24 h with either doxofylline or theophylline at 0.1, 1 or 10 μM. Results are expressed as percentage of living cells. Value are expressed as means ± S.E.M. of 5 independent experiments from distinct donors, analysed by one-way ANOVA with Kruskal Wallis test for multiple comparison.
CD14++CD16− cells (representing about 90% of circulating monocytes) and “non-classical” CD14+CD16+ cells [16]. A third subset, called “intermediate” CD14++CD16+ cells, has been more recently identified [17], and this population has been found increased in severe asthma [18]. The present study was designed to compare the ability of doxofylline and theophylline to modulate a range of pro-inflammatory pathways in human monocytes, thus improving our knowledge on their mechanism of action in immune cells. We report that doxofylline and theophylline share some common features, although they appear to significantly differ in their selectivity. While theophylline appears to inhibit mainly LPS-induced cellular responsivity, doxofylline appears more directed to the activity of PMA. Furthermore, when testing PKC activity, we found an inhibitory effect of doxofylline but not of theophylline, at equimolar doses. In conclusion, our data support the growing hypothesis that doxofylline does not have a superimposable mechanism of action compared to theophylline, and this may both explain some differences in the risk/benefit ratio and may direct studies to tailor therapy for patients. 2. Methods 2.1. Chemicals Doxofylline, theophylline, histopaque, PMA, SOD, cytochrome C, RIPA buffer, LPS, DTT, sodium ortovanadate, EGTA, EDTA, PMSF, MOPS, sodium fluoride, protease inhibitor cocktail cocktail, DMSO, RPMI 1640 medium, MTT were obtained from Sigma-Aldrich (Milan, Italy); glutamine, hepes, streptomycin-penicillin, FBS were purchased from Life Technologies (Monza, Italy); trizol reagent from Fisher Molecular Biology (Rome, Italy). 2.2. Monocyte isolation Fig. 2. Effect of methylxanthines on superoxide anion production. (A) Monocytes were incubated for 1 h with doxofylline or theophylline at 0.1, 1 and 10 μM and analysed for their superoxide anion production in comparison to control (Ctrl, untreated cells). (B) Monocytes were pre-incubated for 1 h with either doxofylline or theophylline, and then stimulated with PMA or LPS for 30 min. Results are expressed as nmoles of reduced cytochrome C/106 cells. Data are means ± SEM of 5 independent experiments from distinct donors, analysed by one-way ANOVA with Kruskal–Wallis test for multiple comparison. Significance levels: ****p < 0.001 vs Ctrl; °°°p < 0.001 vs PMA; #### p < 0.001 vs LPS; ##p < 0.01 vs LPS; #p < 0.05 vs LPS. Ctrl represents basal superoxide anion production in untreated cells.
Thirty-six healthy anonymous human buffy coats were provided by the Transfusion Service of the Ospedale Maggiore della Carità (Novara, Italy) after authorization from the local Ethics Committee (Comitato Etico Interaziendale Maggiore della Carità, Novara; authorization document 88/17). From these, monocytes were isolated. Briefly, after sedimentation by dextran and histopaque (density = 1.077 g cm−3) gradient centrifugation (400×g, 30 min, room temperature), monocytes were recovered by thin suction at the interface and left to adhere (90 min, 37 °C, 5% CO2) in serum free RPMI 1640 medium as described previously [19]. Then, cells were maintained in culture in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS), 2 mM glutamine and 1% antibiotics. Cell viability was assessed by trypan blue dye exclusion. Doxofylline and theophylline concentrations (0.1–10 μM) were taken from previous publications [9,20–23] as were incubation times [19,24,25].
circulate for several days before migrating into tissues and differentiating into mature macrophages. According to the expression of CD14 (the LPS co-receptor) and CD16 (the low-affinity receptor for the Fc region of IgG), monocytes were originally divided into “classical” 2
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Fig. 3. Effects of doxofylline and theophylline on monocyte polarization. Monocytes were incubated in the absence (ctrl) or presence of methylxanthines (1 μM), and then stained for 1 h with anti-CD14 and anti-CD16 antibodies. (A) Gating strategy for FACS analysis. The cell population set by physical parameters (FSC-SSC) was analysed for CD14 and CD16 co-expression (dot plot CD14/CD16). (B) Graphical representation of the percentage of the different monocyte populations after treatment by doxofylline and theophylline. Results are expressed as percentage of CD14+ cells. Data are means ± SEM of 5 independent experiments from distinct donors, analysed by one-way ANOVA with Kruskal–Wallis test for multiple comparison.
2.3. Cell viability
2.5. Superoxide anion (O2−) production
To assess potential drugs toxicity in monocytes, cell viability was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Cells (2.5×105 cells) were challenged for 6 and 24 h with doxofylline and theophylline at 0.1, 1 and 10 μM. Then, the medium was replaced with the MTT assay solution (1 mg ml−1; 2 h, 37 °C 5% CO2). The supernatant was removed and DMSO was added in order to dissolve the purple formazan; the absorbance was read at 570 and 650 nm.
Cells (1×106 cells/plate) were treated for 1 h with doxofylline and theophylline at 0.1, 1 and 10 μM alone or followed by 30 min stimulation with phorbol 12-myristate 13-acetate (PMA) 1 μM or LPS (lipopolysaccharide) (1 μg/ml). Superoxide anion production was then evaluated by the superoxide dismutase (SOD)-sensitive cytochrome C reduction assay and expressed as nmoles cytochrome C reduced/ 106 cells/30 min, using an extinction coefficient of 21.1 mM. 2.6. Quantitative real-time RT-PCR
2.4. Flow cytometric analysis of monocytes
Total RNA was isolated by Trizol from monocytes treated for 6 h with doxofylline and theophylline at 10 μM alone or followed by 30 min challenge with PMA 1 μM or LPS 1 μg/ml. The amount and purity of total RNA were quantified at the spectrophotometer (Nanodrop, Thermo Fisher) by measuring the optical density at 260 and 280 nm cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer's instructions. A two-step cycling real-time PCR was carried out in a volume of 10 μl per well in a 96-well optical reaction plate (Biorad) containing SensiFast No-ROX kit (Bioline) 1x, forward and reverse primer 400 nM, and 1 μl of cDNA template. The primers used were: IL-6 for 5′-GGAGACTTGCCTGGTGAAAA-3′ and rev 5′-GTCAGGGGTGGTTA TTGCAT-3’; IL-1β for 5′-ACAGATGAAGTGCTCCTTCCA-3′ and rev 5′GTCGGAGATTCGTAGCTGGAT-3’; IL-10 for 5′-CATCGATTTCTTCCCT GTGAA-3′ and rev 5′-TCTTGGAGCTTATTAAAGGCATTC-3’; IL-4 for
Analysis of cell phenotype were performed by multi-parametric analysis by flow cytometry (Attune NxT Flow Cytometer – Thermo Fisher Scientific). On the day of collection, treated monocytes were incubated for 1 h with allophycocianin (APC)-conjugated human antiCD14 monoclonal antibody (Invitrogen) and fluorescein isothiocyanate (FITC)-conjugated anti-CD16 monoclonal antibody (Invitrogen) and then washed twice in phosphate-buffered saline (PBS). The cell population was first defined using forward scatter (FSC) and side scatter (SSC) to find viable cells and exclude debris. On this population the coexpression of CD14 and CD16 markers on a dot plot was analysed. Gating strategy was defined first using unstained controls.
3
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Fig. 4. Real time analysis of LPS- or PMA-induced cytokine expression after treatment with doxofylline or theophylline. Monocytes were treated for 6 h with the drugs at 10 μM and then challenged for 30 min with PMA 1 μM or LPS 1 μg/ml. A: IL-6, B: IL-1β, C: TNFα, D: IL-10, E: IL-4. Data are expressed as 2-ΔCt and are means ± SEM of 12 independent experiments from distinct donors, analysed by one-way ANOVA with Kruskal–Wallis test for multiple comparison. Significance levels: °°p < 0.01, °°°p < 0.001 vs Ctrl; *p < 0.05 vs PMA; #p < 0.05 vs LPS.
2.8. Phosphodiesterase (PDE) activity
5′-ACTTTGAACAGCCTCACAGAG-3′ and rev 5′-TTGGAGGCAGCAAAG ATGTC-3’; IL-13 for 5′-TGAGGAGCTGGTCAACATCA-3′ and rev 5′-CAGGTTGATGCTCCATACCAT-3’; TNFα for 5′-CATGATCCGGGACG TGGAGC-3′ and rev 5′-CTGATTAGAGAGAGGTCCCTG-3’; GAPDH 5′-AACGTGTCAGTGGTGGACCTG-3′ and rev 5′- AGTGGGTGTCGCTGT TGAAGT-3’ (this last one was included for each sample and used for normalization). The relative quantification was determined by the 2ΔCT method [26].
Monocytes were treated for 6 h with doxofylline and theophylline at 10 μM, then lysed using RIPA buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (100 mM sodium orthovanadate and 1 mM sodium fluoride). The PDE activity was evaluated on whole cells extract using the PDE activity assay kit (Enzo Life Sciences) following the manufacturer's protocol. 2.9. Cyclic AMP assay
2.7. Cytokine release 106 monocytes were treated with doxofylline and theophylline at 10 μM for 6 h and then lysed with 0.1 M HCl and 1% Triton X-100 for 10 min. The samples were centrifuged at 600 g for 10 min and the supernatants were used for the cyclic AMP assay using a competitive ELISA kit (Invitrogen) following the manufacturer's protocol.
Commercially available kits were used to test the presence of proand anti-inflammatory cytokines. Cell supernatants were collected after monocyte treatment with doxofylline and theophylline (10 μM) for 6 h alone or followed by 30 min challenge with PMA (1 μM) or LPS (1 μg/ ml). Samples were tested for the presence of IL-6 (Human High sensitive Interleukin 6 ELISA Kit), IL-1β (IL-1Beta (human) ELISA Kit), and TNFα (General Tumor Necrosis Factor Alpha ELISA kit), IL-4 (Human High sensitive Interleukin 4 ELISA Kit), and IL-10 (Human Interleukin 10 ELISA Kit) (all LiStarFish), following the manufacturer's instructions.
2.10. Protein kinase C (PKC) assay Monocytes were treated with doxofylline and theophylline at 10 μM for 6 h alone or followed by challenge for 30 min with PMA 1 μM or LPS 4
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Fig. 5. Effect of doxofylline or theophylline on cytokine release. ELISAs were performed on supernatants of monocytes treated for 6 h with doxofylline or theophylline and then stimulated with PMA 1 μM or LPS 1 μg/ml for 30 min. (A) IL-6, (B) IL-1β, (C) TNFα, (D) IL-10, (E) IL-4. Data are means ± SEM of 8 independent experiments from distinct donors analysed by one-way ANOVA with Kruskal–Wallis test for multiple comparison. Significance levels: °p < 0.05 vs Ctrl; *p < 0.05 vs PMA; ***p < 0.001 vs PMA; #p < 0.05 vs LPS.
1 μg/ml. Cells were lysed in lysis buffer containing 20 mM MOPS, 50 mM β-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% NP40, 1 mM dithiothreitol (DTT), protease inhibitor cocktail 1X, 1 mM phenyl methane sulphonyl fluoride (PMSF). PKC activity was measured using the PKC Kinase Activity Assay Kit (Abcam) following the manufacturer's instructions and results were read at a spectrophotometer (Nanodrop, Thermo Fisher) at 450 nm.
3. Results 3.1. Effect of doxofylline and theophylline on cell viability In order to avoid confounding effects attributable to cell toxicity, we first evaluated the effects of the two methylxanthines on cell viability via the MTT assay on monocytes. No significant differences compared to untreated cells were found in the viability of cells after treatment for 6 or 24 h with doxofylline and theophylline at concentrations up to 10 μM (Fig. 1).
2.11. Statistical analysis
3.2. Effect of doxofylline and teophylline on PMA- and LPS-induced oxidative bursts
Statistical analysis was performed using GraphPad Prism 6. Data are expressed as mean ± S.E.M. of ‘n’ independent experiments performed in triplicate. Statistical significance among different cell treatments was assessed by one-way repeated measures ANOVA followed by KruskalWallis multiple comparisons test if more than two treatment groups were compared. Statistical significance was defined as P < 0.05.
PMA and bacterial LPS are known to be strong stimuli that induce a respiratory burst in monocytes via PKC activation [27] or via binding to CD14 (which shuttles the molecule to the Toll-like receptor 4; TLR4), 5
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modest effect at a concentration of 1 μM which was not maintained at higher concentrations. LPS at 1 μg/ml induced a stronger oxidative burst that was significantly reverted by both methylxanthines (Fig. 2B). 3.3. Effects of doxofylline and theophylline on monocyte phenotype Expression of the surface markers CD14 and CD16 was evaluated by FACS to estimate the effect of methylxanthines in driving polarization of monocytes. Fig. 3A reports a representative dot plot for the gating strategy utilised. As shown in Fig. 3B, neither doxofylline nor theophylline induced monocyte polarization, and the proportion of classical (CD14++/CD16-), non-classical (CD14+/CD16+) and intermediate (CD14++/CD16+) cell populations were superimposable in treated and untreated cells. 3.4. Effect of doxofylline and theophylline on cytokine gene expression and release Fig. 6. Effect of doxofylline or theophylline on PDE activity. Monocytes were treated for 6 h with doxofylline and theophylline. (A) Whole protein extracts were prepared to detect the activity of total PDEs. Results are represented as nmoles of 5′-AMP. Data are mean ± SEM of 11 independent experiments from distinct donors. (B) Intracellular cAMP was measured on lysates of treated cells. Data are expressed as pmol/ml and are mean ± SEM of 4 independent experiments from distinct donors.
We next evaluated the expression of cytokines after stimulation in the presence or absence of the two methylxanthines. Doxofylline or theophylline alone did not modify gene expression of the selected cytokines, with the single exception of IL-1β, which was significantly reduced by doxophylline (Fig. 4B). As expected, when monocytes were stimulated with PMA or LPS, a significant increase in IL-6, IL-1β and TNFα was observed (Fig. 4A, B and C respectively). Unexpectedly, doxofylline did not modify the LPS-induced increase while significantly reducing the PMA-induced increase of all the three pro-inflammatory cytokines. Theophylline behaved differently, reducing significantly both PMA- and LPS-induced increases of IL-6 (Fig. 4A), and reducing exclusively IL-1β and TNFα expression, while not affecting the PMAinduced actions (Fig. 4Β, C). It has been hypothesized that IL-10 contributes to the anti-inflammatory action of methylxanthines [29]. In our cell model, neither doxofylline nor theophylline induced changes of IL-10, either alone nor in combination to PMA and LPS (Fig. 4D), in accord to what reported for low-dose theophylline in patients [12]; likewise, the mRNA level of IL-4 was not significantly modified (Fig. 4E) by either methylxanthine. Last, we analysed if doxofylline or theophylline were able to modulate cytokine release from activated monocytes. As depicted in Fig. 5, IL-6 (Fig. 5A), IL-4 (Fig. 5D) and IL-10 (Fig. 5E) release was not affected by treatments, although doxofylline, unlike theophylline, significantly reduced PMA-induced release of IL-1β (Fig. 5B) and TNFα (Fig. 5C). While gene expression is obviously permissive towards cytokine release, the mechanisms governing the two mechanisms are different [30] and therefore it is not surprising that the effects on these two parameters are not superimposable upon methylxanthine exposure.
Fig. 7. Effect of doxofylline or theophylline on PKC activity. Monocytes were treated for 6 h with the drugs at 10 μM and then challenged for 30 min with PMA 1 μM or LPS 1 μg/ml, lysed and PKC activity was measured. Data are expressed as O.D. at 450 nm. Significance levels: °p < 0.05 and °°p < 0.01 vs Ctrl; *p < 0.05 vs PMA.
respectively [28]. Given that human monocytes, as phagocytes, release basal amounts of O2− that increase after stimulation, we investigated the anti-inflammatory actions of doxofylline or theophylline against PMA- and LPS-induced superoxide anion production in human monocytes. Basal O2− production from unstimulated monocytes (ctrl) was unaffected by both methylxanthines at the highest concentration tested (10 μM; Fig. 2A), demonstrating that neither methylxanthine on their own is able to activate monocytes. PMA 1 μM treatment for 30 min (Fig. 2B) led to a significant increase of O2− which was inhibited by doxofylline at the maximal concentration tested (10 μM). Theophylline, instead, showed just a
3.5. Effect of doxofylline and theophylline on PDE activity and cAMP level Among the diverse targets suggested for methylxanthines, inhibition of phosphodiesterases has been postulated as a possible mechanism for the bronchodilator and anti-inflammatory effects of theophylline [31]. We therefore evaluated PDE activity and the level of 5′-AMP in monocytes treated for 6 h with methylxanthines at 10 μM. As shown in Fig. 6, neither doxofylline nor theophylline induced a decrease of 5′AMP (Fig. 6A) or exerted any significant effect against PDE activity
Table 1 Comparison between the effects of doxofylline and theophylline on human monocytes. O2−PMA
DOXOFYLLINE THEOPHYLLINE
++ +/−
LPS
++ ++
IL-6
IL-1β
TNFα
PKC
PMA LPS
PMA LPS
PMA LPS
PMA LPS
++ ++
0 ++
++ 0
6
0 ++
++ 0
0 +/−
+ 0
0 0
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have demonstrated that the anti-inflammatory effect of theophylline may be due to restoration of HDAC activity, thus silencing gene transcription of inflammatory genes and at the same time restoring steroid responses in macrophages [10]. Similarly, To Y et al. [45] have demonstrated that theophylline reduces the oxidative stress in peripheral blood mononuclear cells from patients with COPD via PI3K inhibition, also unlocking HDAC activity and reverting corticosteroid insensitivity. On the contrary, while a steroid sparing effect of doxofylline has also been shown, it has been demonstrated to be PI3K/HDAC and PDE independent [46]. From our data we can therefore suggest that doxophylline in human monocytes is able to inhibit LPS-superoxide anion production in short treatments but its mechanism of action in longer treatments is mainly mediated by modulation of PKC activity. This hypothesis of a switch of mechanism between acute and chronic exposure could also explain the discrepancy of some of our results with those of Riffo-Vasquez et al. [47], that demonstrated that doxofylline inhibited leukocyte count and migration in lungs of mice after 24 h treatments with LPS. It should also be noticed that in vitro short-term experiments are designed to elucidate mechanisms, but in longer treatments in vivo compensatory mechanisms may take place that render mechanism dissection harder. All these data, taken together, suggest, once again, that methylxanthines have a complex mechanism of action.
(Fig. 6B). 3.6. Effect of doxofylline and theophylline on PKC activity The ability of doxofylline to selectively modulate the PMA-pathway prompted us to evaluate at which level of the pathway doxofylline and theophylline diverged. We therefore evaluated PKC activity in monocytes activated by PMA and LPS in the presence or absence of methylxanthines. As shown in Fig. 7, PMA and LPS both significantly increased PKC activity, while doxofylline and theophylline alone did not. When cells were co-stimulated, theophylline did not modulate PKC activity induced by either PMA or LPS, while doxofylline again was able to significantly reduce the effect of PMA but not that of LPS. 4. Discussion The effectiveness of methylxanthines has been initially ascribed to bronchodilatation and, later, also to the ability of these molecules to act as bland, atypical anti-inflammatory drugs [32], but their molecular and cellular mechanism of action remains elusive. In the present manuscript we demonstrate that methylxanthines act on monocytes and that theophylline and doxofylline do not have superimposable effects. The important role of monocytes in asthma is strongly supported by in vivo murine models [13,33,34] and by clinical evidence in patients. In fact, recruitment of monocytes has been demonstrated in the lung of young patients with fatal asthma [15] and an increase in monocyte number has been shown in the circulation of children with asthma exacerbations [14]. The ability to generate and release oxygen free radicals (ROS) is a process known as “respiratory burst” that generally begins with the production of superoxide anion [35], which in turn promotes the phagocytic function of monocytes [36]. Over twenty years ago it was been demonstrated that monocytes seemed to be primed for ROS production in asthmatic patients [37] and therefore an anti-inflammatory effect was pursued in the new anti-asthmatic drugs. A direct inhibitory effect of theophylline on superoxide anion production was demonstrated by Chorostowska-Wynimko et al. [23] and ascribed to PDE inhibition. Our results demonstrate that theophylline or doxofylline alone do not activate monocytes or modify monocyte polarization. This is paralleled by the inability of these two drugs alone to induce superoxide anion production or to increase cytokine expression and release. On the contrary, both drugs are able to reduce the oxidative burst induced by PMA and LPS, and when evaluating their effects on PDE activity neither molecule affected levels of cAMP or 5′AMP. These results are in accordance with previous evidences that reported that the anti-inflammatory effects of doxofylline are PDE-independent [7,21,23,38]. Alongside the above similitudes between doxofylline and theophylline, to our surprise we found that the effect of these two methylxanthines on other circuits was distinct, depending of the stimulus used, PMA or LPS, as summarized in Table 1. Interestingly, doxofylline reverted the effect of PMA on cytokines but had no effect on the actions of LPS while theophylline modulated mainly the effects of LPS. To elucidate the possible molecular mechanism of action of these differences we investigated PKC activation and we found an inhibitory effect of doxofylline that was not reproduced by theophylline. To our knowledge this is the first report on an effect on this pathway by methylxanthines, and we do not know whether it occurs also in other cell types. Moreover, doxofylline alters PKC activation by PMA but not by LPS. This can be explained looking at the different way through which the two stimuli activate PKC. It is well documented that responsiveness of monocytes to LPS occurs after PKC activation through PI3-Kinase [39–41], while PMA stimulates PKC directly [42]. In 2002, Ito K et al. showed that LPS inhibits HDAC activity allowing for the transcription of several markers of inflammation, and that low doses of theophylline were able to revert this effect [43]. In COPD, there is a decreased activity of HDAC [44], and several studies
5. Conclusions Few studies on theophylline in monocytes have been performed and comparative studies with doxofylline in monocytes have never been done previously. In the present study on human monocytes we show that both methylxanthines have anti-inflammatory properties, but that their specificity is not superimposable. From a mechanistic viewpoint, we have identified PKC as a differentiating target as it is solely affected by doxofylline. Declaration of competing interest None. The work was partly supported by a research grant from ABC Farmaceutici. References [1] Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease. Global Initiative for Chronic Obstructive Lung Disease, (Jan 2009) Available from: http://www.goldcopd.com. [2] Global Initiative for Asthma, Pocket Guide for Health Professionals, (2018) available from: https://ginasthma.org/. [3] R.G. Barr, B.H. Rowe, C.A. Camargo Jr., Methylxanthines for exacerbations of chronic obstructive pulmonary disease: meta-analysis of randomised trials, Br. Med. J. 327 (2003) 643. [4] M.D. Faiz Akram, M. Nasirudin, Z. Ahmad, R.A. Khan, Doxofylline and theophylline: a comparative clinical study, J. Clin. Diagn. Res. 6 (2012) 1681–1684. [5] M. Cazzola M, L. Calzetta, P. Rogliani, C. Page, M.G. Matera, Impact of doxofylline in COPD: a pairwise meta-analysis, Pulm. Pharmacol. Ther. 51 (2018) 1–9. [6] P. Rosigliani, L. Calzetta, J. Ora, M. Cazzola, M.G. Matera, Efficacy and safety profile of doxofylline compared to theophylline in asthma: a meta-analysis, Multidiscip Respir Med 3 (2019) 14:25. [7] D. Spina, C. Page, Xanthines and phosphodiesterase inhibitors, Handb. Exp. Pharmacol. 237 (2017) 63–91. [8] D. Shukla, S. Chakraborty, S. Singh, B. Mishra, Doxofylline: a promising methylxanthine derivative for the treatment of asthma and chronic obstructive pulmonary disease, Expert Opin. Pharmacother. 10 (2009) 2343–2356. [9] M.G. Matera, C. Page, M. Cazzola, Doxofylline is not just another theophylline!, Int. J. Chronic Obstr. Pulm. Dis. 12 (2017) 3487–3493. [10] B.G. Cosio, L. Tsaprouni, K. Ito, E. Jazrawi, I.M. Adcock, P.J. Barnes, Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages, J. Exp. Med. 200 (2004) 689–695. [11] B.G. Cosio, B. Mann, K. Ito, E. Jazrawi, P.J. Barnes, K.F. Chung, et al., Histone acetylase and deacetylase activity in alveolar macrophages and blood monocytes in asthma, Am. J. Respir. Crit. Care Med. 170 (2004) 141–147. [12] B. Oliver, K. Tomita, A. Keller, G. Caramori, I. Adcock, K.F. Chung, et al., Low-dose theophylline does not exert its anti-inflammatory effects in mild asthma through upregulation of interleukin-10 in alveolar macrophages, Allergy 56 (2001)
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1087–1090. [13] M. Plantinga, M. Guilliams, M. Vanheerswynghels, K. Deswarte, F. Branco-Madeira, W. Toussaint, et al., Conventional and monocyte-derived CD11b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen, Immunity 38 (2013) 322–335. [14] L.S. Subrata, J. Bizzintino, E. Mamessier, A. Bosco, K.L. McKenna, M.E. Wikström, et al., Interactions between innate antiviral and atopic immunoinflammatory pathways precipitate and sustain asthma exacerbations in children, J. Immunol. 183 (2009) 2793–2800. [15] I. Eguiluz-Gracia, K. Malmstrom, S.A. Dheyauldeen, J. Lohi, A. Sajantila, R. Aalokken, et al., Monocytes accumulate in the airways of children with fatal asthma, Clin Exp Allergy 48 (2018) 1631–1639. [16] L. Ziegler-Heitbrock, Blood monocytes and their subsets: established features and open questions, Front. Immunol. 6 (2015) 423. [17] L. Ziegler-Heitbrock, P. Ancuta, S. Crowe, M. Dalod, V. Grau, D.N. Hart, et al., Nomenclature of monocytes and dendritic cells in blood, Blood 116 (2010) e74–e80. [18] M. Moniuszko, A. Bodzenta-Lukaszyk, K. Kowal, D. Lenczewka, M. Dabrowska, Enhanced frequencies of CD14++CD16+, but not CD14+CD16+, peripheral blood monocytes in severe asthmatic patients, Clin. Immunol. 130 (2009) 338–346. [19] M. Talmon, S. Rossi, A. Pastore, C.I. Cattaneo, S. Brunelleschi, L.G. Fresu, Vortioxetine exerts anti-inflammatory and immunomodulatory effects on human monocytes/macrophages, Br. J. Pharmacol. 175 (2018) 113–124. [20] B.S. Patel, M.J. Kugel, G. Baehring, A.J. Ammit, Doxofylline does not increase formoterol-induced cAMP nor MKP-1 expression in ASM cells resulting in lack of anti-inflammatory effect, Pulm. Pharmacol. Ther. 45 (2017) 34e39. [21] J. van Mastbergen, T. Jolas, L. Allegra, C.P. Page, The mechanism of action of doxofylline is unrelated to HDAC inhibition, PDE inhibition or adenosine receptor antagonism, Pulm. Pharmacol. Ther. 25 (2012) 55e61. [22] M. Umeda, T. Ichiyama, S. Hasegawa, M. Kaneko, T. Matsubara, S. Furukawa, Theophylline inhibits NF-kappaB activation in human peripheral blood mononuclear cells, Int. Arch. Allergy Immunol. 128 (2) (2002) 130–135. [23] J. Chorostowska-Wynimko, J. Kus, E.E. Skopinska-Roewska, Teophylline inhibits free oxygen radical production by human monocytes via phosphodiesterase inhibition, J. Physiol. Pharmacol. 58 (Suppl 5) (2007) 95–103. [24] S. Velnati, E. Ruffo, A. Massarotti, M. Talmon, K.S.S. Varma, A. Gesu, L.G. Fresu, A.L. Snow, A. Bertoni, D. Capello, G.C. Tron, A. Graziani, G. Baldanzi, Identification of a novel DGKα inhibitor for XLP-1 therapy by virtual screening, Eur. J. Med. Chem. 15 (164) (2019) 378–390. [25] J.A. Obeng, A. Amoruso, G.L. Camaschella, D. Sola, S. Brunelleschi, L.G. Fresu, Modulation of human monocyte/macrophage activity by tocilizumab, abatacept and etanercept: an in vitro study, Eur. J. Pharmacol. 5 (780) (2016) 33–37. [26] J.S. Yuan, A. Reed, F. Chen, C.N. Chen Jr., Statistical analysis of real-time PCR data, Bioinformatics 22 (7) (2006) 85. [27] M.A. Myers, L.C. Mcphail, R. Snyderman, Redistribution of protein kinase C activity in human 13 monocytes: correlation with activation of the respiratory burst, J. Immunol. 135 (1985) 3411–3416. [28] M. Caroff, D. Karibian, J.M. Cavaillon, N. Haeffner-Cavaillon, Structural and functional analyses of bacterial lipopolysaccharides, Microb. Infect. 4 (2002) 915–926. [29] J.J. Mascali, P. Cvietusa, J. Negri, L. Borish, Anti-inflammatory effects of theophylline: modulation of cytokine production, Ann. Allergy Asthma Immunol. 77
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Comparison of anti-inflammatory mechanisms between doxofylline and theophylline in human monocytes
T
Maria Talmon, Erika Massara, Chiara Brunini, Luigia Grazia Fresu∗ Department of Health Sciences, University of Piemonte Orientale, Via Solaroli 17, 28100, Novara, Italy
A R T I C LE I N FO
A B S T R A C T
Keywords: Monocytes Methylxanthines Theophylline Doxofylline Cytokines Superoxide anion
Background: Methylxanthines are important pharmacological agents in the treatment of asthma and of chronic obstructive pulmonary diseases. The present study was designed to compare the ability of doxofylline and theophylline to modulate inflammatory pathways in human monocytes. Methods: Monocytes isolated from healthy anonymous human buffy coats were treated with doxofylline or theophylline in the presence of phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS), and their phenotype, the oxidative burst, cytokine expression and release, cAMP production, and protein kinase C (PKC) activity were evaluated. Results: Doxofylline and theophylline did not have overlapping effects on human monocytes. While sharing some common characteristics, they differed significantly in their selectivity. Theophylline affected LPS- above PMA-induced cellular responsivity, while doxofylline behaved in the opposite manner. Furthermore, when testing PKC activity, we found an inhibitory effect of doxofylline but not of theophylline, at equimolar doses. Conclusions: In conclusion, our data support the growing hypothesis that doxofylline does not have a superimposable mechanism of action compared to theophylline, and this may both explain some differences in the risk/benefit ratio and may direct studies to tailor therapy for patients.
1. Introduction Methylxanthines are indicated both in GOLD [1] and GINA [2] guidelines as add-on agents in the treatment of asthma and of chronic obstructive pulmonary disease (COPD). Theophylline is the most widely used methylxanthine, although other drugs have also reached the market, including doxofylline, bamifylline and acebrofylline [3]. Recently, there has been a resurgence in the interest in doxofylline and various meta-analyses have shown that doxofylline might not have a superimposable risk/benefit compared to theophylline [4–6]. Chemically, a dioxolane group differentiates doxofylline from theophylline, but the two molecules have been postulated to have a number of distinct pharmacodynamics characteristics. Theophylline has been reported to be a non-selective inhibitor of phosphodiesterases (PDE) and of phosphoinositide 3-kinase-δ (PI3K-δ), an antagonist of adenosine receptors (A1, A2A/B and A3) and a modulator of histone deacetylases (HDACs) [7]. Doxofylline, instead, has been suggested not
to have affinity for most adenosine receptors (with the exception of A2A), to lack significant activity on PDEs, except for PDE2A1, and to have a more prominent anti-inflammatory effect [8,9]. Given the elusive mechanism of methylxanthines, though, it is difficult to ascertain whether these differences may be reconducted to the differences observed in patients. While several studies have evaluated the anti-inflammatory activity of theophylline on monocytes/macrophages [10–12] there are no reports on doxofylline, and it is therefore difficult to ascertain whether the pharmacodynamics differences reported are translated to this cell type, which is increasingly being involved in pulmonary disorders. Studies in murine models have demonstrated that monocytes accumulate in the lung and play a role in allergic reactions both by secreting cytokines and chemokines [13] and by maintaining T cell-mediated immunity [14]. This sustains asthma exacerbations in paediatric patients in which activated circulating monocytes are increased [15]. Monocytes constitute 5–10% of peripheral blood leukocytes and
Abbreviations: COPD, chronic obstructive pulmonary disease; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDTA, ethylene diamine tetraacetic acid; EGTA, ethylene glycol tetraacetic acid; FBS, fetal bovine serum; HDACs, histone deacetylases; LPS, lipopolysaccharide; MOPS, 3-(N-morpholino) propanesulfonic acid; MTT, methylthiazolyldiphenyl-tetrazolium bromide; PDE, phosphodiesterases; PI3K-δ, phosphoinositide 3-kinase-δ; PKC, phosphor kinase C; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; SOD, superoxide dismutase ∗ Corresponding author. Department of Health Sciences, Via Solaroli, 17, 28100, Novara, Italy. E-mail address: [email protected] (L.G. Fresu). https://doi.org/10.1016/j.pupt.2019.101851 Received 1 May 2019; Received in revised form 26 September 2019; Accepted 26 September 2019 Available online 26 September 2019 1094-5539/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Effect of methylxanthines on cell viability. Monocytes were treated for 6 and 24 h with either doxofylline or theophylline at 0.1, 1 or 10 μM. Results are expressed as percentage of living cells. Value are expressed as means ± S.E.M. of 5 independent experiments from distinct donors, analysed by one-way ANOVA with Kruskal Wallis test for multiple comparison.
CD14++CD16− cells (representing about 90% of circulating monocytes) and “non-classical” CD14+CD16+ cells [16]. A third subset, called “intermediate” CD14++CD16+ cells, has been more recently identified [17], and this population has been found increased in severe asthma [18]. The present study was designed to compare the ability of doxofylline and theophylline to modulate a range of pro-inflammatory pathways in human monocytes, thus improving our knowledge on their mechanism of action in immune cells. We report that doxofylline and theophylline share some common features, although they appear to significantly differ in their selectivity. While theophylline appears to inhibit mainly LPS-induced cellular responsivity, doxofylline appears more directed to the activity of PMA. Furthermore, when testing PKC activity, we found an inhibitory effect of doxofylline but not of theophylline, at equimolar doses. In conclusion, our data support the growing hypothesis that doxofylline does not have a superimposable mechanism of action compared to theophylline, and this may both explain some differences in the risk/benefit ratio and may direct studies to tailor therapy for patients. 2. Methods 2.1. Chemicals Doxofylline, theophylline, histopaque, PMA, SOD, cytochrome C, RIPA buffer, LPS, DTT, sodium ortovanadate, EGTA, EDTA, PMSF, MOPS, sodium fluoride, protease inhibitor cocktail cocktail, DMSO, RPMI 1640 medium, MTT were obtained from Sigma-Aldrich (Milan, Italy); glutamine, hepes, streptomycin-penicillin, FBS were purchased from Life Technologies (Monza, Italy); trizol reagent from Fisher Molecular Biology (Rome, Italy). 2.2. Monocyte isolation Fig. 2. Effect of methylxanthines on superoxide anion production. (A) Monocytes were incubated for 1 h with doxofylline or theophylline at 0.1, 1 and 10 μM and analysed for their superoxide anion production in comparison to control (Ctrl, untreated cells). (B) Monocytes were pre-incubated for 1 h with either doxofylline or theophylline, and then stimulated with PMA or LPS for 30 min. Results are expressed as nmoles of reduced cytochrome C/106 cells. Data are means ± SEM of 5 independent experiments from distinct donors, analysed by one-way ANOVA with Kruskal–Wallis test for multiple comparison. Significance levels: ****p < 0.001 vs Ctrl; °°°p < 0.001 vs PMA; #### p < 0.001 vs LPS; ##p < 0.01 vs LPS; #p < 0.05 vs LPS. Ctrl represents basal superoxide anion production in untreated cells.
Thirty-six healthy anonymous human buffy coats were provided by the Transfusion Service of the Ospedale Maggiore della Carità (Novara, Italy) after authorization from the local Ethics Committee (Comitato Etico Interaziendale Maggiore della Carità, Novara; authorization document 88/17). From these, monocytes were isolated. Briefly, after sedimentation by dextran and histopaque (density = 1.077 g cm−3) gradient centrifugation (400×g, 30 min, room temperature), monocytes were recovered by thin suction at the interface and left to adhere (90 min, 37 °C, 5% CO2) in serum free RPMI 1640 medium as described previously [19]. Then, cells were maintained in culture in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS), 2 mM glutamine and 1% antibiotics. Cell viability was assessed by trypan blue dye exclusion. Doxofylline and theophylline concentrations (0.1–10 μM) were taken from previous publications [9,20–23] as were incubation times [19,24,25].
circulate for several days before migrating into tissues and differentiating into mature macrophages. According to the expression of CD14 (the LPS co-receptor) and CD16 (the low-affinity receptor for the Fc region of IgG), monocytes were originally divided into “classical” 2
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Fig. 3. Effects of doxofylline and theophylline on monocyte polarization. Monocytes were incubated in the absence (ctrl) or presence of methylxanthines (1 μM), and then stained for 1 h with anti-CD14 and anti-CD16 antibodies. (A) Gating strategy for FACS analysis. The cell population set by physical parameters (FSC-SSC) was analysed for CD14 and CD16 co-expression (dot plot CD14/CD16). (B) Graphical representation of the percentage of the different monocyte populations after treatment by doxofylline and theophylline. Results are expressed as percentage of CD14+ cells. Data are means ± SEM of 5 independent experiments from distinct donors, analysed by one-way ANOVA with Kruskal–Wallis test for multiple comparison.
2.3. Cell viability
2.5. Superoxide anion (O2−) production
To assess potential drugs toxicity in monocytes, cell viability was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Cells (2.5×105 cells) were challenged for 6 and 24 h with doxofylline and theophylline at 0.1, 1 and 10 μM. Then, the medium was replaced with the MTT assay solution (1 mg ml−1; 2 h, 37 °C 5% CO2). The supernatant was removed and DMSO was added in order to dissolve the purple formazan; the absorbance was read at 570 and 650 nm.
Cells (1×106 cells/plate) were treated for 1 h with doxofylline and theophylline at 0.1, 1 and 10 μM alone or followed by 30 min stimulation with phorbol 12-myristate 13-acetate (PMA) 1 μM or LPS (lipopolysaccharide) (1 μg/ml). Superoxide anion production was then evaluated by the superoxide dismutase (SOD)-sensitive cytochrome C reduction assay and expressed as nmoles cytochrome C reduced/ 106 cells/30 min, using an extinction coefficient of 21.1 mM. 2.6. Quantitative real-time RT-PCR
2.4. Flow cytometric analysis of monocytes
Total RNA was isolated by Trizol from monocytes treated for 6 h with doxofylline and theophylline at 10 μM alone or followed by 30 min challenge with PMA 1 μM or LPS 1 μg/ml. The amount and purity of total RNA were quantified at the spectrophotometer (Nanodrop, Thermo Fisher) by measuring the optical density at 260 and 280 nm cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer's instructions. A two-step cycling real-time PCR was carried out in a volume of 10 μl per well in a 96-well optical reaction plate (Biorad) containing SensiFast No-ROX kit (Bioline) 1x, forward and reverse primer 400 nM, and 1 μl of cDNA template. The primers used were: IL-6 for 5′-GGAGACTTGCCTGGTGAAAA-3′ and rev 5′-GTCAGGGGTGGTTA TTGCAT-3’; IL-1β for 5′-ACAGATGAAGTGCTCCTTCCA-3′ and rev 5′GTCGGAGATTCGTAGCTGGAT-3’; IL-10 for 5′-CATCGATTTCTTCCCT GTGAA-3′ and rev 5′-TCTTGGAGCTTATTAAAGGCATTC-3’; IL-4 for
Analysis of cell phenotype were performed by multi-parametric analysis by flow cytometry (Attune NxT Flow Cytometer – Thermo Fisher Scientific). On the day of collection, treated monocytes were incubated for 1 h with allophycocianin (APC)-conjugated human antiCD14 monoclonal antibody (Invitrogen) and fluorescein isothiocyanate (FITC)-conjugated anti-CD16 monoclonal antibody (Invitrogen) and then washed twice in phosphate-buffered saline (PBS). The cell population was first defined using forward scatter (FSC) and side scatter (SSC) to find viable cells and exclude debris. On this population the coexpression of CD14 and CD16 markers on a dot plot was analysed. Gating strategy was defined first using unstained controls.
3
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Fig. 4. Real time analysis of LPS- or PMA-induced cytokine expression after treatment with doxofylline or theophylline. Monocytes were treated for 6 h with the drugs at 10 μM and then challenged for 30 min with PMA 1 μM or LPS 1 μg/ml. A: IL-6, B: IL-1β, C: TNFα, D: IL-10, E: IL-4. Data are expressed as 2-ΔCt and are means ± SEM of 12 independent experiments from distinct donors, analysed by one-way ANOVA with Kruskal–Wallis test for multiple comparison. Significance levels: °°p < 0.01, °°°p < 0.001 vs Ctrl; *p < 0.05 vs PMA; #p < 0.05 vs LPS.
2.8. Phosphodiesterase (PDE) activity
5′-ACTTTGAACAGCCTCACAGAG-3′ and rev 5′-TTGGAGGCAGCAAAG ATGTC-3’; IL-13 for 5′-TGAGGAGCTGGTCAACATCA-3′ and rev 5′-CAGGTTGATGCTCCATACCAT-3’; TNFα for 5′-CATGATCCGGGACG TGGAGC-3′ and rev 5′-CTGATTAGAGAGAGGTCCCTG-3’; GAPDH 5′-AACGTGTCAGTGGTGGACCTG-3′ and rev 5′- AGTGGGTGTCGCTGT TGAAGT-3’ (this last one was included for each sample and used for normalization). The relative quantification was determined by the 2ΔCT method [26].
Monocytes were treated for 6 h with doxofylline and theophylline at 10 μM, then lysed using RIPA buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (100 mM sodium orthovanadate and 1 mM sodium fluoride). The PDE activity was evaluated on whole cells extract using the PDE activity assay kit (Enzo Life Sciences) following the manufacturer's protocol. 2.9. Cyclic AMP assay
2.7. Cytokine release 106 monocytes were treated with doxofylline and theophylline at 10 μM for 6 h and then lysed with 0.1 M HCl and 1% Triton X-100 for 10 min. The samples were centrifuged at 600 g for 10 min and the supernatants were used for the cyclic AMP assay using a competitive ELISA kit (Invitrogen) following the manufacturer's protocol.
Commercially available kits were used to test the presence of proand anti-inflammatory cytokines. Cell supernatants were collected after monocyte treatment with doxofylline and theophylline (10 μM) for 6 h alone or followed by 30 min challenge with PMA (1 μM) or LPS (1 μg/ ml). Samples were tested for the presence of IL-6 (Human High sensitive Interleukin 6 ELISA Kit), IL-1β (IL-1Beta (human) ELISA Kit), and TNFα (General Tumor Necrosis Factor Alpha ELISA kit), IL-4 (Human High sensitive Interleukin 4 ELISA Kit), and IL-10 (Human Interleukin 10 ELISA Kit) (all LiStarFish), following the manufacturer's instructions.
2.10. Protein kinase C (PKC) assay Monocytes were treated with doxofylline and theophylline at 10 μM for 6 h alone or followed by challenge for 30 min with PMA 1 μM or LPS 4
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Fig. 5. Effect of doxofylline or theophylline on cytokine release. ELISAs were performed on supernatants of monocytes treated for 6 h with doxofylline or theophylline and then stimulated with PMA 1 μM or LPS 1 μg/ml for 30 min. (A) IL-6, (B) IL-1β, (C) TNFα, (D) IL-10, (E) IL-4. Data are means ± SEM of 8 independent experiments from distinct donors analysed by one-way ANOVA with Kruskal–Wallis test for multiple comparison. Significance levels: °p < 0.05 vs Ctrl; *p < 0.05 vs PMA; ***p < 0.001 vs PMA; #p < 0.05 vs LPS.
1 μg/ml. Cells were lysed in lysis buffer containing 20 mM MOPS, 50 mM β-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% NP40, 1 mM dithiothreitol (DTT), protease inhibitor cocktail 1X, 1 mM phenyl methane sulphonyl fluoride (PMSF). PKC activity was measured using the PKC Kinase Activity Assay Kit (Abcam) following the manufacturer's instructions and results were read at a spectrophotometer (Nanodrop, Thermo Fisher) at 450 nm.
3. Results 3.1. Effect of doxofylline and theophylline on cell viability In order to avoid confounding effects attributable to cell toxicity, we first evaluated the effects of the two methylxanthines on cell viability via the MTT assay on monocytes. No significant differences compared to untreated cells were found in the viability of cells after treatment for 6 or 24 h with doxofylline and theophylline at concentrations up to 10 μM (Fig. 1).
2.11. Statistical analysis
3.2. Effect of doxofylline and teophylline on PMA- and LPS-induced oxidative bursts
Statistical analysis was performed using GraphPad Prism 6. Data are expressed as mean ± S.E.M. of ‘n’ independent experiments performed in triplicate. Statistical significance among different cell treatments was assessed by one-way repeated measures ANOVA followed by KruskalWallis multiple comparisons test if more than two treatment groups were compared. Statistical significance was defined as P < 0.05.
PMA and bacterial LPS are known to be strong stimuli that induce a respiratory burst in monocytes via PKC activation [27] or via binding to CD14 (which shuttles the molecule to the Toll-like receptor 4; TLR4), 5
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modest effect at a concentration of 1 μM which was not maintained at higher concentrations. LPS at 1 μg/ml induced a stronger oxidative burst that was significantly reverted by both methylxanthines (Fig. 2B). 3.3. Effects of doxofylline and theophylline on monocyte phenotype Expression of the surface markers CD14 and CD16 was evaluated by FACS to estimate the effect of methylxanthines in driving polarization of monocytes. Fig. 3A reports a representative dot plot for the gating strategy utilised. As shown in Fig. 3B, neither doxofylline nor theophylline induced monocyte polarization, and the proportion of classical (CD14++/CD16-), non-classical (CD14+/CD16+) and intermediate (CD14++/CD16+) cell populations were superimposable in treated and untreated cells. 3.4. Effect of doxofylline and theophylline on cytokine gene expression and release Fig. 6. Effect of doxofylline or theophylline on PDE activity. Monocytes were treated for 6 h with doxofylline and theophylline. (A) Whole protein extracts were prepared to detect the activity of total PDEs. Results are represented as nmoles of 5′-AMP. Data are mean ± SEM of 11 independent experiments from distinct donors. (B) Intracellular cAMP was measured on lysates of treated cells. Data are expressed as pmol/ml and are mean ± SEM of 4 independent experiments from distinct donors.
We next evaluated the expression of cytokines after stimulation in the presence or absence of the two methylxanthines. Doxofylline or theophylline alone did not modify gene expression of the selected cytokines, with the single exception of IL-1β, which was significantly reduced by doxophylline (Fig. 4B). As expected, when monocytes were stimulated with PMA or LPS, a significant increase in IL-6, IL-1β and TNFα was observed (Fig. 4A, B and C respectively). Unexpectedly, doxofylline did not modify the LPS-induced increase while significantly reducing the PMA-induced increase of all the three pro-inflammatory cytokines. Theophylline behaved differently, reducing significantly both PMA- and LPS-induced increases of IL-6 (Fig. 4A), and reducing exclusively IL-1β and TNFα expression, while not affecting the PMAinduced actions (Fig. 4Β, C). It has been hypothesized that IL-10 contributes to the anti-inflammatory action of methylxanthines [29]. In our cell model, neither doxofylline nor theophylline induced changes of IL-10, either alone nor in combination to PMA and LPS (Fig. 4D), in accord to what reported for low-dose theophylline in patients [12]; likewise, the mRNA level of IL-4 was not significantly modified (Fig. 4E) by either methylxanthine. Last, we analysed if doxofylline or theophylline were able to modulate cytokine release from activated monocytes. As depicted in Fig. 5, IL-6 (Fig. 5A), IL-4 (Fig. 5D) and IL-10 (Fig. 5E) release was not affected by treatments, although doxofylline, unlike theophylline, significantly reduced PMA-induced release of IL-1β (Fig. 5B) and TNFα (Fig. 5C). While gene expression is obviously permissive towards cytokine release, the mechanisms governing the two mechanisms are different [30] and therefore it is not surprising that the effects on these two parameters are not superimposable upon methylxanthine exposure.
Fig. 7. Effect of doxofylline or theophylline on PKC activity. Monocytes were treated for 6 h with the drugs at 10 μM and then challenged for 30 min with PMA 1 μM or LPS 1 μg/ml, lysed and PKC activity was measured. Data are expressed as O.D. at 450 nm. Significance levels: °p < 0.05 and °°p < 0.01 vs Ctrl; *p < 0.05 vs PMA.
respectively [28]. Given that human monocytes, as phagocytes, release basal amounts of O2− that increase after stimulation, we investigated the anti-inflammatory actions of doxofylline or theophylline against PMA- and LPS-induced superoxide anion production in human monocytes. Basal O2− production from unstimulated monocytes (ctrl) was unaffected by both methylxanthines at the highest concentration tested (10 μM; Fig. 2A), demonstrating that neither methylxanthine on their own is able to activate monocytes. PMA 1 μM treatment for 30 min (Fig. 2B) led to a significant increase of O2− which was inhibited by doxofylline at the maximal concentration tested (10 μM). Theophylline, instead, showed just a
3.5. Effect of doxofylline and theophylline on PDE activity and cAMP level Among the diverse targets suggested for methylxanthines, inhibition of phosphodiesterases has been postulated as a possible mechanism for the bronchodilator and anti-inflammatory effects of theophylline [31]. We therefore evaluated PDE activity and the level of 5′-AMP in monocytes treated for 6 h with methylxanthines at 10 μM. As shown in Fig. 6, neither doxofylline nor theophylline induced a decrease of 5′AMP (Fig. 6A) or exerted any significant effect against PDE activity
Table 1 Comparison between the effects of doxofylline and theophylline on human monocytes. O2−PMA
DOXOFYLLINE THEOPHYLLINE
++ +/−
LPS
++ ++
IL-6
IL-1β
TNFα
PKC
PMA LPS
PMA LPS
PMA LPS
PMA LPS
++ ++
0 ++
++ 0
6
0 ++
++ 0
0 +/−
+ 0
0 0
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have demonstrated that the anti-inflammatory effect of theophylline may be due to restoration of HDAC activity, thus silencing gene transcription of inflammatory genes and at the same time restoring steroid responses in macrophages [10]. Similarly, To Y et al. [45] have demonstrated that theophylline reduces the oxidative stress in peripheral blood mononuclear cells from patients with COPD via PI3K inhibition, also unlocking HDAC activity and reverting corticosteroid insensitivity. On the contrary, while a steroid sparing effect of doxofylline has also been shown, it has been demonstrated to be PI3K/HDAC and PDE independent [46]. From our data we can therefore suggest that doxophylline in human monocytes is able to inhibit LPS-superoxide anion production in short treatments but its mechanism of action in longer treatments is mainly mediated by modulation of PKC activity. This hypothesis of a switch of mechanism between acute and chronic exposure could also explain the discrepancy of some of our results with those of Riffo-Vasquez et al. [47], that demonstrated that doxofylline inhibited leukocyte count and migration in lungs of mice after 24 h treatments with LPS. It should also be noticed that in vitro short-term experiments are designed to elucidate mechanisms, but in longer treatments in vivo compensatory mechanisms may take place that render mechanism dissection harder. All these data, taken together, suggest, once again, that methylxanthines have a complex mechanism of action.
(Fig. 6B). 3.6. Effect of doxofylline and theophylline on PKC activity The ability of doxofylline to selectively modulate the PMA-pathway prompted us to evaluate at which level of the pathway doxofylline and theophylline diverged. We therefore evaluated PKC activity in monocytes activated by PMA and LPS in the presence or absence of methylxanthines. As shown in Fig. 7, PMA and LPS both significantly increased PKC activity, while doxofylline and theophylline alone did not. When cells were co-stimulated, theophylline did not modulate PKC activity induced by either PMA or LPS, while doxofylline again was able to significantly reduce the effect of PMA but not that of LPS. 4. Discussion The effectiveness of methylxanthines has been initially ascribed to bronchodilatation and, later, also to the ability of these molecules to act as bland, atypical anti-inflammatory drugs [32], but their molecular and cellular mechanism of action remains elusive. In the present manuscript we demonstrate that methylxanthines act on monocytes and that theophylline and doxofylline do not have superimposable effects. The important role of monocytes in asthma is strongly supported by in vivo murine models [13,33,34] and by clinical evidence in patients. In fact, recruitment of monocytes has been demonstrated in the lung of young patients with fatal asthma [15] and an increase in monocyte number has been shown in the circulation of children with asthma exacerbations [14]. The ability to generate and release oxygen free radicals (ROS) is a process known as “respiratory burst” that generally begins with the production of superoxide anion [35], which in turn promotes the phagocytic function of monocytes [36]. Over twenty years ago it was been demonstrated that monocytes seemed to be primed for ROS production in asthmatic patients [37] and therefore an anti-inflammatory effect was pursued in the new anti-asthmatic drugs. A direct inhibitory effect of theophylline on superoxide anion production was demonstrated by Chorostowska-Wynimko et al. [23] and ascribed to PDE inhibition. Our results demonstrate that theophylline or doxofylline alone do not activate monocytes or modify monocyte polarization. This is paralleled by the inability of these two drugs alone to induce superoxide anion production or to increase cytokine expression and release. On the contrary, both drugs are able to reduce the oxidative burst induced by PMA and LPS, and when evaluating their effects on PDE activity neither molecule affected levels of cAMP or 5′AMP. These results are in accordance with previous evidences that reported that the anti-inflammatory effects of doxofylline are PDE-independent [7,21,23,38]. Alongside the above similitudes between doxofylline and theophylline, to our surprise we found that the effect of these two methylxanthines on other circuits was distinct, depending of the stimulus used, PMA or LPS, as summarized in Table 1. Interestingly, doxofylline reverted the effect of PMA on cytokines but had no effect on the actions of LPS while theophylline modulated mainly the effects of LPS. To elucidate the possible molecular mechanism of action of these differences we investigated PKC activation and we found an inhibitory effect of doxofylline that was not reproduced by theophylline. To our knowledge this is the first report on an effect on this pathway by methylxanthines, and we do not know whether it occurs also in other cell types. Moreover, doxofylline alters PKC activation by PMA but not by LPS. This can be explained looking at the different way through which the two stimuli activate PKC. It is well documented that responsiveness of monocytes to LPS occurs after PKC activation through PI3-Kinase [39–41], while PMA stimulates PKC directly [42]. In 2002, Ito K et al. showed that LPS inhibits HDAC activity allowing for the transcription of several markers of inflammation, and that low doses of theophylline were able to revert this effect [43]. In COPD, there is a decreased activity of HDAC [44], and several studies
5. Conclusions Few studies on theophylline in monocytes have been performed and comparative studies with doxofylline in monocytes have never been done previously. In the present study on human monocytes we show that both methylxanthines have anti-inflammatory properties, but that their specificity is not superimposable. From a mechanistic viewpoint, we have identified PKC as a differentiating target as it is solely affected by doxofylline. Declaration of competing interest None. The work was partly supported by a research grant from ABC Farmaceutici. References [1] Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease. Global Initiative for Chronic Obstructive Lung Disease, (Jan 2009) Available from: http://www.goldcopd.com. [2] Global Initiative for Asthma, Pocket Guide for Health Professionals, (2018) available from: https://ginasthma.org/. [3] R.G. Barr, B.H. Rowe, C.A. Camargo Jr., Methylxanthines for exacerbations of chronic obstructive pulmonary disease: meta-analysis of randomised trials, Br. Med. J. 327 (2003) 643. [4] M.D. Faiz Akram, M. Nasirudin, Z. Ahmad, R.A. Khan, Doxofylline and theophylline: a comparative clinical study, J. Clin. Diagn. Res. 6 (2012) 1681–1684. [5] M. Cazzola M, L. Calzetta, P. Rogliani, C. Page, M.G. Matera, Impact of doxofylline in COPD: a pairwise meta-analysis, Pulm. Pharmacol. Ther. 51 (2018) 1–9. [6] P. Rosigliani, L. Calzetta, J. Ora, M. Cazzola, M.G. Matera, Efficacy and safety profile of doxofylline compared to theophylline in asthma: a meta-analysis, Multidiscip Respir Med 3 (2019) 14:25. [7] D. Spina, C. Page, Xanthines and phosphodiesterase inhibitors, Handb. Exp. Pharmacol. 237 (2017) 63–91. [8] D. Shukla, S. Chakraborty, S. Singh, B. Mishra, Doxofylline: a promising methylxanthine derivative for the treatment of asthma and chronic obstructive pulmonary disease, Expert Opin. Pharmacother. 10 (2009) 2343–2356. [9] M.G. Matera, C. Page, M. Cazzola, Doxofylline is not just another theophylline!, Int. J. Chronic Obstr. Pulm. Dis. 12 (2017) 3487–3493. [10] B.G. Cosio, L. Tsaprouni, K. Ito, E. Jazrawi, I.M. Adcock, P.J. Barnes, Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages, J. Exp. Med. 200 (2004) 689–695. [11] B.G. Cosio, B. Mann, K. Ito, E. Jazrawi, P.J. Barnes, K.F. Chung, et al., Histone acetylase and deacetylase activity in alveolar macrophages and blood monocytes in asthma, Am. J. Respir. Crit. Care Med. 170 (2004) 141–147. [12] B. Oliver, K. Tomita, A. Keller, G. Caramori, I. Adcock, K.F. Chung, et al., Low-dose theophylline does not exert its anti-inflammatory effects in mild asthma through upregulation of interleukin-10 in alveolar macrophages, Allergy 56 (2001)
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