Background
Asthma is a chronic airway inflammation characterized by intense eosinophil, mast cell, and lymphocyte infiltration, mucus hyper-production, and airway hyper-responsiveness [
1]. Asthma symptoms develop when allergens activate antigen-specific helper T-lymphocytes (Th) to produce Th-2 cytokines, such as interleukin (IL)-4, IL-5, and IL13 [
1]. Activated phagocytic cells (neutrophils, eosinophils, monocytes and macrophages) also play a role in the pathophysiology of airway inflammation due their release of large amounts of reactive oxygen species (ROS), lipid mediators, and cytokines [
2,
3]. Airway cells and tissues are also exposed to oxidative stress elicited by environmental pollutants (ozone, cigarette smoke, and dust), infections, inflammatory reactions or decreased levels of antioxidants that lead to enhanced levels of ROS [
3,
4]. It has been shown that ROS can damage DNA, lipids, proteins, and carbohydrates leading to impaired cellular functions and enhanced inflammatory reactions [
3]. Consequently, it has been suggested that ROS play a role in airway disorders such as adult respiratory, distress syndrome (ARDS), cystic fibrosis, idiopathic fibrosis, chronic obstructive pulmonary diseases (COPD), and asthma [
3,
5].
The mammalian super family of transient receptor potential (TRP) cation channels can be subdivided into six subfamilies based on sequence homology: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin) and TRPML (mucolipin) [
6,
7]. TRP channels express in a broad range of cell types including sensory nerves, lung fibroblast, epithelial cells and immune cells [
6,
7]. Relevant to the context of asthma, TRPA1 channels have been implicated in pain and inflammatory responses in the airways in mice [
8]. In addition, the TRPM2 channel has been implicated in stress-related inflammatory and neurodegenerative conditions [
9‐
11]. However, the relevance of TRPM2 in severe asthma pathophysiology has not yet been explored.
TRPM2 is a non-selective calcium (Ca
2+) influx and lysosomal Ca
2+ release channel expressed in neutrophils [
9,
12], monocytes [
9], Jurkat T cells [
13], INS-1 cells [
14] and mouse bone marrow derived-dendritic cells (BMDC) [
15]. TRMP2 is co-activated by intracellular adenosine diphosphate ribose (ADPR) and Ca
2+, downstream of ROS and chemokine signaling pathways [
11,
15‐
17]. TRPM2 activation by ADPR is further facilitated by the presence of nicotinic acid adenine dinucleotide phosphate (NAADP), cyclic ADPR (cADPR), and hydrogen peroxide (H
2O
2) [
18‐
21], whereas adenosine monophosphate (AMP) and permeating protons (pH) negatively regulate TRPM2 activation [
21‐
24].
TRPM2-deficient mice are more resistant to chronic experimental colitis due to defective chemokine (C-X-C motif) ligand 2 (CXCL2) production by monocytes and reduced neutrophil infiltration [
9]. Yet, a recent publication has suggested no role for TRPM2 channel in chronic obstructive pulmonary disease [
25]. Intriguingly, cADPR induces Ca
2+ release in airway smooth muscle (ASM) [
26,
27] and acetylcholine (ACh) and endothelin-1 (ET-1) are considered to regulate airway caliber through cADPR-mediated Ca
2+ release in these cells [
28]. Moreover, mice that lack CD38, an ectoenzyme that generates free ADPR through the hydrolysis of nicotinamide adenine dinucleotide (NAD
+) and its cADPR glycohydrolase activity, exhibit altered airway responsiveness to methacholine [
28,
29].
In the present study, we assessed the role of TRPM2 channels in airway inflammation by using an experimental OVA-induced severe asthma model. We found that airway responsiveness, airway inflammation, production of allergen-specific antibodies, and cytokine response were unaffected in TRPM2-/- mice when compared to OVA-sensitized and challenged WT mice. Our findings suggest that in this experimental model the TRPM2 channel is not required for airway inflammation to occur.
Methods
Animals
C57BL/6
trpm2
+/+
(wild type; WT) and C57BL/6
trpm2
-/-
(knock out; TRPM2
-/-
) mice were bred and housed under pathogen free conditions. TRPM2
-/-
mice were generated as previously described [
9]. All mice were genotyped by PCR before the experiments to confirm disruption of
trpm2
-/-
gene. Mice were 8-12 weeks old at the time of the experiments. All protocols involving rodents were reviewed and approved by The Institutional Laboratory Animal Care and Use Committee (IACUC) at The University of Hawaii and The University of California, San Francisco.
Allergen sensitization and challenge of mice
Sensitization and challenge of mice were performed as previously described [
30]. Briefly, TRPM2
-/- mice and WT littermate were sensitized intraperitoneally with 50 μg ovalbumin (OVA; grade V; Sigma-Aldrich) plus 1 mg Alum (Sigma-Aldrich) in 200 μl 0.9% sodium chloride (saline; Hospira) on Days 0, 7, and 14. On Days 21, 22 and 23, mice were anesthetized with isoflurane (Hospira) and challenged with 100 μg OVA in 50 μl saline by nasal administration. Control groups were treated identically except OVA was missing in the solutions. Mice were euthanized and studied on Day 24.
Measurement of airway hyper-responsiveness
Airway resistance in response to intravenously administered acetylcholine was measured using a flexiVent system (SCIREQ, Montreal, Canada) as previously described [
30]. Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and acepromazine (2-3 mg/kg); paralyzed with pancuronium (0.1 mg/kg intraperitoneally), intubated with a 20G cannula and mechanically ventilated at a frequency of 150 breaths per minute and 2 cmH
2O positive end-expiratory. Lung resistance was measured at baseline and in response to increasing intravenous doses of acetylcholine (0, 0.1, 0.3, 1, 3 and 9.6 μg/g body weight) using the linear single compartment model.
Bronchoalveolar lavage fluid (BAL) leukocytes count
Lungs from sacrificed mice were flushed once with 1 ml PBS/1% fetal calf serum (FCS) to obtain bronchoalveolar lavage (BAL) fluid. The total number of cells was determined by a hemocytometer. A maximum of 2 × 105 cells were centrifuged on a microscope slide and stained with Diff-Quick (Polyscience). Differential cell counts were made at 3400 magnification, and at least 100 cells were counted per slide.
Histology and immunohistochemistry
For histologic analysis of goblet cell hyperplasia, tissue samples were fixed in 4% phosphate-buffered formalin, embedded in paraffin, cut into 5-7 μm sections and stained with periodic acid-Schiff (PAS) reagent (Sigma-Aldrich) following manufacturer instructions. To evaluate inflammatory infiltration, tissue sections were stained with hematoxylin and eosin. Scoring was performed at 200x magnification by examining 40 consecutive fields of the peribronchiolar, perivascular, and alveolar areas. Mast cells were counted at 20x magnification in lung sections stained with toluidine blue.
Detection of serum IgE antibodies
Blood samples were collected using the heart puncture method and serum was separated by centrifugation for 15 minutes at 6000 g. OVA-specific IgE antibodies were measured in a serum dilution series by endpoint titration enzyme-linked immunosorbent assay (ELISA). Briefly, plates were coated with 1 mg/ml OVA and alkaline phosphatase-conjugated anti-mouse isotype specific antibodies (Southern Biotechnology) and 4-nitrophenyl phosphate (Sigma-Aldrich) were used for detection. Absorbance was measured at 405 nm with 492 nm as a reference wavelength.
Cytokine levels
The concentration of interleukin IL-5, IL-6, IL-10, IL-13 and transforming growth factor beta 1 (TGFB1) in the BAL fluid of five independent OVA and saline treated WT and TRPM2-/- mice were measured using the specific Single Analyte ELISArray™ Kit (Qiagen) following manufacturer instructions. Samples were analyzed at 450 nm using a Benchmark plus microplate reader spectrophotometer (BioRad).
Statistical analysis
Data are reported as mean ± SEM. Significance testing was performed by Student’s paired t-test for significant differences between two groups and ANOVA to test significant differences among the groups of mice. A value of p < 0.05 was considered to be statistically significant.
Discussion
Allergen-induced airway inflammation is an eosinophilic inflammation and type 2 T and B lymphocytes-driven response [
1]. In this study, we investigated the role of TRPM2 channels in the pathophysiology of severe asthma by using TRPM2-deficient mice in a model of OVA-induced inflammatory airway disease. Our data indicate that TRPM2 channels are not required for acute airway inflammation to occur. Deletion of TRPM2 did not affect airway resistance (Figure
1B), mucus production (Figure
1C), inflammatory cell infiltration (Figure
2), allergen-induced production of IgE (Figure
3), and cytokine production (Figure
4) following allergen treatment. This is somewhat surprising in light of previous studies that used the dextran sulphate sodium (DSS)-induced chronic experimental colitis mouse model [
9] and the lipopolysaccharide (LPS)-induced lung inflammation mouse model [
32]. In the colitis model, the TRPM2 channel controls CXCL2 production in monocytes and consequently affects neutrophil infiltration, and in the lung inflammation model, TRPM2 plays a protective role by preventing ROS production in neutrophils. DSS induces inflammatory bowel disease-like colitis in mice by causing toxicity in colonic epithelial cells of the basal crypts [
33]. Moreover, the inflammatory response is mediated by monocyte-dependent chemokine production in response to locally generated ROS [
9]. Interestingly, LPS may regulate lung inflammation via Toll-like receptor 4 (TLR4) signaling in alveolar macrophages [
34], whereas allergen-induced airway inflammation is driven by type 2 T lymphocytes and cytokines [
1]. In contrast to the above studies, a recent study of chronic obstructive pulmonary disease (COPD) showed no role for TRPM2 in airway inflammation in mice exposed to ozone, LPS or tobacco smoke [
25]. The CD8+ cell is the accepted crucial lymphocyte subtype in COPD. Therefore, TRPM2 appears to be preferentially involved in chronic inflammatory responses with a strong phagocytic cell component and might not have a prominent role in acute inflammatory processes. It also suggests that redundant mechanisms, including other ion channels, might compensate for the absence of TRPM2 channels during certain inflammatory processes. Other chronic inflammatory models should be used to address this hypothesis, including models of chronic allergen exposure or using alternative allergens such as dust mite and cockroach extracts.
The TRPM2 channel is expressed in the plasma membrane of neutrophils and monocytes/macrophages [
9] and in lysosomes of BMDC [
15] and controls their chemotaxis or cytokine production by regulating intracellular calcium concentration upon cell activation. TRPM2-deficient neutrophils exhibit defective
in vitro chemotactic responses and calcium signals toward N-formyl-methionine-leucine-phenylalanine (fMLP), a peptide chain produced by some bacteria [
9], although CXCL2-mediated responses remained unaffected. TRPM2
-/- neutrophils are also defective in ROS production [
32]. In our model of allergen induced-chronic inflammation, deletion of TRPM2 expression did not affect IL-6, IL-10, IL-13 and TGFβ1 production (Figure
4) or inflammatory cell infiltration into the airway (Figure
2).
A central mediator of asthma is the IgE antibody, which is produced by sensitized allergen-specific B cells [
1]. Allergens increase IgE levels in the serum of susceptible subjects subsequent to stimulation [
1]. IgE antibodies then bind to the high-affinity IgE receptor, Fc epsilon receptor I (FcϵRI), present in mast cells, eosinophils, and basophils, thereby sensitizing these cells to allergen exposure [
1]. IgE-FcϵRI complexes trigger degranulation of cytoplasmic vesicles containing histamine and
de novo formation of eicosanoids and ROS in mast cells, eosinophils, and basophils, resulting in smooth muscle contraction [
1]. Our data indicate that TRPM2 channels have no direct effect on allergen-induced production of IgE (Figure
3).
Airway caliber regulation by ACh and ET-1 occurs through a yet to be elucidated mechanism involving cADPR-mediated Ca
2+ release in ASM cells [
28]. It is known that cADPR activates ryanodine receptors [
35] and can facilitate TRPM2 activation [
36]. In addition, airway responsiveness to methacholine is altered in the absence of CD38, an ectoenzyme that generates free ADPR and cADPR from NAD
+[
28,
29]. In our study, we observed no difference in airway resistance between TRPM2-deficient mice and WT mice challenged with OVA, suggesting that ACh does not require TRPM2 activity to regulate airway caliber. Interestingly, another TRP channel, TRPA-1, appears relevant to allergen-induced asthma models, since TRPA-1 knockout mice were shown to be more resistant to airway inflammation and hyperactivity than WT mice [
8].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
All authors conceived and designed the study. A.S-T, acquired, analyzed and interpreted data and wrote manuscript. R.P., A.F., interpreted data and wrote the paper. All authors read and approved the final manuscript.