The ion channel transient receptor potential melastatin-2 does not play a role in inflammatory mouse models of chronic obstructive pulmonary diseases
© Hardaker et al; licensee BioMed Central Ltd. 2012
Received: 19 December 2011
Accepted: 4 April 2012
Published: 4 April 2012
There is strong evidence that oxidative stress is associated with the pathogenesis of chronic obstructive pulmonary disease (COPD). The transient receptor potential melastatin-2 (TRPM2) is an oxidative stress sensing channel that is expressed in a number of inflammatory cells and therefore it has been suggested that inhibition of TRPM2 could lead to a beneficial effect in COPD patients. In this study, we have investigated the role of TRPM2 in a variety of mouse models of oxidative stress and COPD using TRPM2-deficent mice.
Mice were exposed to ozone (3 ppm for 4 h) or lipopolysaccharide (LPS, 0.3 mg/kg, intranasaly). In another model, mice were exposed to tobacco smoke (750 μg/l total wet particulate matter) for 30 min twice a day on three consecutive days. For the exacerbation model, the smoke exposure on the morning of day 3 animals was replaced with intranasal administration of LPS (0.3 mg/kg). Animals were killed 3 and 24 h after the challenge (ozone and LPS model) or 18 h after the last tobacco smoke exposure. In vitro neutrophil chemotaxis and monocyte activation were also studied using cells isolated from wild type and TRPM2-deficient animals. Statistical significance for the in vivo data (P < 0.05) was determined using analysis of variance with Kruskal-Wallis and Dunns multiple comparison test.
In all models studied, no difference in the bronchoalveolar lavage inflammation could be evidenced when comparing wild type and TRPM2-deficient mice. In addition, no difference could be seen in the lung inflammation as assessed by the measurement of various cytokines/chemokines. Similarly in various in vitro cellular activation assays using isolated neutrophils and monocytes no significant differences could be observed when comparing wild type and TRPM2-deficient mice.
We have shown, in all the models tested, no difference in the development of airway inflammation or cell activation between TRPM2-deficient mice and their wild type counterparts. These results would suggest that inhibiting TRPM2 activity in COPD would have no anti-inflammatory effect.
KeywordsTransient receptor potential melastatin-2 Oxidative stress Lung inflammation
Chronic obstructive pulmonary disease (COPD) is a multifactorial disease involving inflammation, mucus production dysregulation, lung parenchymal destruction and systemic effects, all of which contribute to airflow limitation. In developed countries, cigarette smoking is the main aetiological agent of COPD and is believed to account for approximately 90% of the cases . The burden of COPD on the healthcare system is enormous and it is predicted that COPD will become the third leading cause of death and the fourth commonest cause of disability in the world by 2020 . Despite that, pharmacotherapies available to these patients are mainly symptomatic and none of them have been shown to alter the course of the disease . There is strong evidence that oxidative stress from cigarette smoking is associated with the pathogenesis of COPD. In addition to this exogenous source of oxidative stress, the chronic inflammation that takes place in the lungs of COPD patients also contributes to the oxidative stress and the pathogenesis of COPD . This would suggest that targeting the oxidative stress burden in COPD patients could lead to beneficial effect.
The vertebrate transient receptor potential ion channel family is divided into six subfamilies; ankyrin (TRPAs), canonical (TRPCs), melastatin (TRPM), mucolipin (TRPMLs), polycystickidneydisease (TRPPs) and vanilloid (TRPVs). This classification is based on protein sequence homology and all family members have six putative transmembrane domains and a pore region between the fifth and the sixth transmembrane domains. These proteins assemble into homo- or hetero-tetramers to form active cation channels . A number of TRP subfamilies serve as a sensor for a variety of chemical (menthol or acrolein) and physical stimuli such as oxidative stress, temperature, membrane potential changes and osmotic stress. Three of the subfamilies have members reported to play a role in the oxidative stress sensing. They are TRPA, TRPM and TRPV . Among the oxidative stress sensors, TRPM2 is of interest since it has been shown to be expressed in a number of inflammatory cells (i.e., neutrophil, macrophage, monocyte). In addition, TRPM2 has also been shown to be activated by pro-inflammatory stimuli such as lipopolysaccharide (LPS, a major component of the outer cell wall of Gram-negative bacteria) and TNF-α. Since all the above inflammatory cells and stimuli are thought to be of importance in COPD pathogenesis, it has been suggested that inhibition of TRPM2 could lead to a beneficial effect in COPD patients .
In this study, we have investigated the role of TRPM2 in a variety of mouse models of COPD and in vitro cell-based assays using TRPM2-deficient mice . Our results show, in all the models tested, no difference in the development of airway inflammation or cell activation between TRPM2-deficient mice and their wild type counterpart. These results would suggest that inhibiting TRPM2 activity in COPD would have no anti-inflammatory effect.
Male and female TRPM2-deficient mice  and their wild type counterparts were backcrossed onto BALB/C using the speed congenics service, MAX-BAX® (Charles River). The genotype was confirmed using DNA isolated from blood samples. Animals were kept at an ambient temperature of 22 ± 2°C under a 12 hours normal phase light-dark cycle. Food and drinking water was freely available and animals were acclimatized for a period of at least 7 days upon arrival in the animal house prior to initiation of any experimental work. The ozone exposure experiments were carried out at Novartis Basel (Switzerland) with the approval of the Veterinary Authority of the City of Basel (Kantonales Veterinaeramt, Basel-Stadt). All other experiments were carried out at Novartis Horsham (UK) under a Project License issued by the United Kingdom Home Office under the Animal (Scientific Procedures) Act 1986 and approved by local ethical review processes.
The protocol was modified from Williams et al. . Unrestrained mice were placed in plastic exposure chamber of the following dimensions: 23 cm wide, 48 cm deep and 17 cm height (0.1 m2) (maximum of 5 mice per chamber) and exposed to 3 parts per million (ppm) ozone or room air for 4 hours. Ozone was generated, at a flow of 480 l/h, and monitored using a computer-controlled ozone simulator system (SIM6050-T; Anseros GmbH, Germany). Animals were killed 3 and 24 hours post exposure.
Mice were challenged intranasally, as previously described , with 0.3 mg/kg of lipopolysaccharide (E. coli serotype 0111:B4) or its vehicle (isotonic saline, 25 μl). Animals were killed at 3 and 24 hours post challenge.
Tobacco smoke exposure and exacerbation models
The protocol was modified from our previous studies performed in rats . Unrestrained mice were placed in plastic wholebody exposure chambers (maximum of 10 mice per chamber) and exposed to mainstream tobacco smoke for 30 minutes, twice a day on three consecutive days with at least a 5 hours gap between each exposure. Tobacco smoke was generated, at a flow rate of 0.6 l/min, using 1R3F Kentucky Research cigarettes and monitored using an EMMS (Bordon UK) total particulate matter transducer (mean wet total particulate matter of 406 ± 7 μg/l). Sham animals were exposed to room air only. For the exacerbation model, on the morning of day 3 animals were administered LPS (E. coli serotype 0111:B4; 0.3 mg/ml or saline, intranasally). Five hours after the LPS challenge, animals were exposed to tobacco smoke for 30 minutes. In both models, animals were killed 18 hours after the last tobacco smoke exposure.
Assessment of airway inflammation
At the indicated time point, terminal anesthesia was induced with pentobarbitone sodium (60 mg/kg, intraperitoneally). The trachea was cannulated and bronchoalveolar lavage (BAL) was performed by instilling g 3 times 0.4 ml of phosphate buffered saline into the lung via the trachea. Total cell counts were measured and cytospins prepared. Cells were stained with Diff-Quik and a differential count of 200 cells performed using standard morphological criteria. Immediately after BAL was performed, the top right lung lobe was removed and snap frozen in liquid nitrogen for subsequent analysis. This lung lobe was homogenized in phosphate buffered saline containing a protease inhibitor cocktail (Roche, UK). For the ozone experiment, CXCL1, CXCL2, IL-6 (R&D Systems, Abingdon, UK and plasma albumin (Bethyl laboratories, Montgomery, TX, USA) were determined using ELISAs according to the manufacturer's instructions. For the tobacco smoke experiments cytokine analysis was performed using 7-plex pro-inflammatory cytokine mesoscale discovery (MSD) assay following manufacturer's instructions. Lactate dehydrogenase activity was assessed using a Cytotoxicity detection kit following manufacturer's instructions (Roche, UK).
In vitro functional assays
Peripheral blood was collected from the abdominal vena cava of mouse. Pooled blood from 4 mice was used to assess neutrophil shape change. Agonists (10 μl) were added to 80 μl of blood for 5 minutes at 37°C followed by fixation with 2.5% paraformaldehyde (Cell Fix™, BD). Red blood cells were lysed using ice cold lysis buffer (1.5 mM NH4Cl and 0.1 mM KHC03). Forward scatter/side scatter (FSC/SSC) plots were used to gate granulocytes (FACSCalibur, Becton Dickinson). The gated neutrophils were distinguished in FSC/FL2 plots and the mean FSC value was used as a measure of shape change.
Mouse bone marrow neutrophils were isolated from femurs and tibias from 4 mice. Bone marrow cells were pelleted by centrifugation and suspended in 5 ml of PBS. Thirteen ml of 65% Percoll (GE Healthcare) was layered on to 13 ml of a 75% Percoll solution. The cell suspension was layered on top of the gradient. After centrifugation at 1600 × g for 30 min, the neutrophil fraction between the 75 and 65% layer was collected. HTS-transwell-96 well plates with 3 μm pore size membranes (Costar) were used to determine chemotaxis of calcein-AM (0.5uM) labelled neutrophils. The medium containing chemoattractant was added to the lower chamber. Neutrophils (5 × 105 cells) were allowed to migrate from the upper to lower chamber for 1 hour and the number of migrating cells was quantitated by reading the lower plate on a fluorescence plate reader (BioTek Synergy) at 485 nm excitation and 520 nm emission.
Mouse bone marrow monocytes were isolated from femurs and tibias from 4 mice. Bone marrow cells were pelleted by centrifugation. The collected bone marrow cells were counted and the monocytes were isolated by CD11b positivity using a magnetic activated cell sorting system (Miltenyi Biotech). CD11b+ cells were seeded into 96 well plates at 1.105 per well in RPMI 1640 medium (supplemented with 10% fetal bovine serum, 5 mM L-glutamine, 30 units/ml penicillin, and 30 mg/ml streptomycin). Cells were stimulated with LPS (E. Coli, 055:B5) for a period of 6 or 24 hours. Cell free supernatants were collected and assayed for CXCL2 (R&D Systems Duo set ELISA kit) and IL-10, IL-6 and TNF-α (Mesoscale Discovery).
Data are expressed as mean ± standard error of the mean (SEM). Statistical significance (P < 0.05) was determined by analysis of variance with Kruskal-Wallis and Dunns multiple comparison test (GraphPad Software, San Diego, CA, USA).
Results and discussion
In vivo models
Utilizing different models of either exogenous or endogenous oxidative stress-induced airway inflammation, we could not demonstrate a role for TRPM2 in the inflammatory response. This is somewhat in contrast to a recent publication showing that TRPM2-deficient mice had an attenuated neutrophil infiltration and CXCL2 production in a dextran sulfate sodium-induced ulcerative colitis model . However, it is interesting to note that the beneficial effect observed with the TRPM2-deficient mice was almost exclusively driven by an inhibition of CXCL2 production and other pro-inflammatory cytokines such as IL-6 or several CCL chemokines were not affected . This would suggest that TRPM2 inhibition would not have a general anti-inflammatory effect but rather selectively inhibits some of the pro-inflammatory mediators. The results obtained in the dextran sulfate sodium model are not surprising and in line with the published literature. Indeed, the production and release of reactive oxygen species by immune cells play a crucial role in the development of colitis  and CXCL2 has a detrimental role in this model  whereas other neutrophil chemoattractants such as CXCL1 have a protective role . This is in contrast to several mouse airways inflammatory models where both CXCL1 and CXCL2 have been shown to have a crucial role in the development of the neutrophilic inflammation . Based on these observations, one would expect a selective inhibition of CXCL2 in models of airway inflammatory diseases not to translate into a meaningful effect on the overall inflammation. Nevertheless, in our in vivo models, no difference in CXCL2 production or release of other pro-inflammatory mediators was observed between wild type mice and TRPM2-deficient mice. In order to further investigate these discrepancies, we studied the role of TRPM2 in in vitro assays of neutrophil chemotaxis and CD11 + cells activation.
In vitro neutrophil chemotaxis and CD11+ cells activation
Although, the in vitro experiments described above suggest some difference in the activation of CD11+ cells when comparing wild type mice with TRPM2-deficient mice, these differences were minute and stimuli-dependent. It is therefore unsurprising that they do not translate into a relevant physiological effect. Hence, our in vitro data is supportive of the lack of effect of TRPM2 deficiency observed in all the in vivo models of oxidative stress-induced lung inflammation used in the present study. Since several studies have linked TRPM2 activation and oxidative stress , the lack of effect of TRPM2 deficiency in our in vivo and in vitro models could seems somewhat surprising. However, a closer look at the literature might well support our data. In addition to the CXCL2-selective effect described above , in the human monocytic cell line THP-1, only a partial inhibition of LPS-induced pro-inflammatory cytokine release was observed following downregulation of TRPM2 using shRNA despite a full inhibition of the LPS-induced intracellular calcium increase . In human neutrophils, it was shown that fMLP-induced calcium influx and chemotaxis was TRPM2 dependent [19, 20]. In contrast stimulation of human neutrophil with PAF was independent of TRPM2 . Consistent with the human situation, fMLP- but not CXCL2-induced calcium and migration responses were reduced in neutrophils from TRPM2-deficient mice . All these data, including ours, suggest that the contribution of TRPM2 to neutrophil and/or monocyte responses is highly dependent on the stimuli but also on the read out studied. In addition, more recent data suggest that another ion channel, ICRAC, that is expressed in neutrophils , is more important than TRPM2 in mediating intracellular calcium increase in responses to H2O2 .
We have shown that in a mechanistic model of oxidative stress-induced lung injury (ozone exposure) and in several models thought to reproduce aspects of the inflammation observed in COPD, TRPM2-deficiency did not affect the development of airway inflammation. These data suggest that TRPM2 blockers may not have beneficial effects in patients with COPD.
Chronic obstructive pulmonary disease
Standard error of the mean
Transient receptor potential melastatin-2.
The authors would like to thank Chris Poll and Pamela Tranter for many helpful discussions.
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