The redox-sensitive cation channel TRPM2 modulates phagocyte ROS production and inflammation

Abstract

The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is critical for host defense, but ROS overproduction can also lead to inflammation and tissue injury. Here we report that TRPM2, a nonselective and redox-sensitive cation channel, inhibited ROS production in phagocytic cells and prevented endotoxin-induced lung inflammation in mice. TRPM2-deficient mice challenged with endotoxin (lipopolysaccharide) had an enhanced inflammatory response and diminished survival relative to that of wild-type mice challenged with endotoxin. TRPM2 functioned by dampening NADPH oxidase-mediated ROS production through depolarization of the plasma membrane in phagocytes. As ROS also activate TRPM2, our findings establish a negative feedback mechanism for the inactivation of ROS production through inhibition of the membrane potential-sensitive NADPH oxidase.

Figure 1. TRPM2 deletion augmented endotoxin-induced lung inflammation and injury

(a-c). Augmented LPS-induced production of MIP-2 (a), TNFα (b), and IL-6 (c) in mouse lung after LPS (10 mg/kg, i.p.) challenge in Trpm2^−/− mice. (a). *p = 0.036 (n = 6), **p = 0.0003 (n = 6), ***p = 0.0008 (n = 6), compared to Trpm2^+/+ group; (b). *p = 0.036 (n = 6), **p = 0.037 (n = 6), compared to Trpm2^+/+ group; (c). *p = 0.018 (n = 6), **p = 0.005 (n = 6), compared to Trpm2^+/+ group. (d) Lung PMN sequestration as measured by tissue MPO activity. Mice were challenged with LPS (10 mg/kg, i.p.) for the times indicated. *p = 0.055 (n = 3), **p = 0.022 (n = 5), compared to Trpm2^+/+ group. (e) H&E (hematoxylin and eosin) staining of lung tissue sections isolated from Trpm2^+/+ and Trpm2^−/− mice challenged with LPS (20 mg/kg, i.p.) for 20 hr. Note the enhanced inflammatory cell infiltration in Trpm2^−/− lung after LPS challenge. Scale bar, 200 μm. (f) Pulmonary edema formation in Trpm2^+/+ and Trpm2^−/− lungs after LPS challenge (20 mg/kg, i.p.). Edema was measured by increase in wet weight of lungs. *p = 0.006 (n = 3). (g). TRPM2 expression protects mice from LPS-induced death. Both Trpm2^+/+ and Trpm2^−/− mouse survival rates were calculated after LPS i.p. injection (30 mg/kg). Differences in mortality were determined by log-rank test (p = 0.0007, n = 40 each).

Figure 2. TRPM2 deletion enhanced ROS generation and oxidative lung inflammatory injury

(a-d). TRPM2 ablation enhances ROS release from Trpm2^−/− PMN stimulated with LPS (1 μg/ml, a-b) and Trpm2^−/− BMDM stimulated with PMA (1 μM, c-d) compared with cells from Trpm2^+/+ mice. cps=counts per second of light emitted. DPI prevented the increased ROS production in both Trpm2^+/+ and Trpm2^−/− BMDM. Note that pretreatment with DPQ (100 μM, 30 min at 37⁰C) augmented ROS release in Trpm2^+/+ cells but not in Trpm2^−/− cells. *p = 0.00007 (n= 3) in b and *p = 0.007 (n = 6) in d. e-f. Immunohistochemical detection of 8-OHdG in lung sections from Trpm2^+/+ and Trpm2^−/− mice challenged with LPS (20 mg/kg) for 20 hr with lower magnification in e (scale bar, 100 μm) and higher magnification in f (scale bar, 20 μm). Nuclei were stained with hematoxylin (blue color, labeled as “N” in the figure). 8-OHdG (brown color) expression was weak in untreated mouse lung (control) and was increased after LPS stimulation in both Trpm2^−/− and Trpm2^+/+ group. (g). Augmented LPS-induced production of TNFα from mouse BMDM was NADPH oxidase sensitive. *p = 0.00003 (n = 7) compared to Trpm2^+/+ + LPS group; **p = 0.046 (n = 3) compared to Trpm2^+/+ + LPS group. ***p = 0.013 (n = 3) compared to Trpm2^−/− + LPS group.

Figure 3. TRPM2 had no effect on phosphorylation of PKCα and p47^phox and high extracellular potassium inhibited ROS production in both Trpm2^−/− and Trpm2^+/+ macrophages

(a) Phosphorylation of PKCα of BMDM from Trpm2^+/+ or Trpm2^−/− mice. BMDM were stimulated with PMA (1 μM) for 2 min. Equal protein loading was verified by membrane probing with anti-PKCα antibodies. TRPM2 expression did not affect phosphorylation of PKCα in BMDM stimulated with PMA. (b) Phosphorylation of p47^phox of BMDM pretreated with PMA (1 μM) for 2 min. Phosphorylation was evaluated by immunoprecipitation of p47^phox and immunoblotting with antibodies against phosphoserine and p47^phox. Note that TRPM2 expression did not regulate the phosphorylation of p47^phox in BMDM stimulated with PMA. (c) Representative curves of ROS release in BMDM from both Trpm2^+/+ BMDM and Trpm2^−/− BMDM under different extracellular potassium concentration ([K⁺]_out). These curves showed that pre-depolarization of BMDM by incubating cells with high K⁺ (120 mM) abrogated the differences in ROS production between Trpm2^+/ and Trpm2^−/− BMDM stimulated with PMA (1 μM). cps, counts per second of light emitted. (d) Summary of data obtained in (C). The figure shows ROS generation decreased with high extracellular concentration of K⁺ which depolarized the cell plasma membrane. *p = 0.015 (n = 5) compared with Trpm2^+/+ group.

Figure 4. TRPM2 inhibited ROS production in macrophages through plasma membrane depolarization

(a-b) The TRPM2 activator ADPR failed to depolarize the plasma membrane in the absence of TRPM2 expression in BMDM. Representative curves showing plasma membrane potential changes are shown in (a) and the summary is shown in (b). *p = 0.0005 (n = 6) compared with Trpm2^−/− + ADPR group. (c-d) PMA (1 μM) failed to fully depolarize the plasma membrane in the absence of TRPM2 expression in BMDM. Representative curves showing changes in plasma membrane potential are shown in (c) and summary is shown in (d). *p = 0.0005 (n = 15) compared with Trpm2^+/+ group. (e) Correlation of membrane potential with ROS production in BMDM. Figure is reconstituted partly from data shown in Figure 3d and Figure 4d. Cells were stimulated with PMA (1 μM) in the presence of 5 mM K⁺ in the extracellular solution. (f-g) ROS release in both Trpm2^+/+ and Trpm2^−/− BMDM stimulated with PMA (1 μM) under physiological and controlled membrane potential. These curves show that pre-depolarization of BMDM by incubating cells with gramicidin (GC, 40μg/ml) not only inhibited ROS release from both Trpm2^+/+ BMDM and Trpm2^−/− BMDM, but also abrogated the differences in ROS production between Trpm2^+/+ and Trpm2^−/− BMDM. Note that replacement of Na⁺ with NMDG augmented ROS release in Trpm2^+/+ BMDM while it had little effect of ROS release from Trpm2^−/− BMDM. *p = 0.0003 (n = 6) and **p = 0.0006 (n = 4) compared to Trpm2^+/+ + PMA group.

Figure 5. LPS -induced Ca²⁺ entry in macrophages was dependent on TRPM2 expression

(a-b). TRPM2 ablation reduced LPS-induced ROS-dependent [Ca²⁺]_i increase in mouse macrophages. Typical recordings are displayed in a and the average results are shown in b. LPS (1μg/ml) induced a rapid [Ca²⁺]_i increase in Trpm2^+/+ BMDM but [Ca²⁺]_i increase induced by LPS was significantly reduced in Trpm2^−/− BMDM. The [Ca²⁺]_i increase in Trpm2^+/+ cells pretreated with DPI (1μM) was reduced to the same level as in Trpm2^−/− cells treated with same amount of DPI. Data are mean of changes in ratiometric values from basal to steady-state [Ca²⁺]_i . *p = 0.001 (n = 5) compared with Trpm2^−/− group. (c-d). DPQ-pretreatment reduced H2O2-induced [Ca²⁺]_i increase in mouse macrophages. Typical recordings are displayed in c and the average results are shown in d. *p = 0.008 (n = 7) compared with Trpm2^−/− group. (e-f). TRPM2-ablation did not altered PAF-induced [Ca²⁺]_i increase in mouse macrophages. Typical recordings are displayed in c and average results are shown in d. PAF (0.1μM) induced a rapid [Ca²⁺]_i increase followed by a slow [Ca²⁺]_i increase in both Trpm2^+/+ BMDM and Trpm2^−/− BMDM. The amplitudes of the two peaks were calculated by measuring changes in ratiometric values from basal to steady-state [Ca²⁺]_i and summarized in d. Data are mean of changes in ratiometric values from basal to steady-state [Ca²⁺]_i (n = 4-5 ).