TRPM2) to Temperature Affects Macrophage Functions

Redox signal-mediated sensitization of transient receptor potential melastatin 2 (TRPM2) to temperature affects macrophage functions

Makiko Kashioa, Takaaki Sokabea, Kenji Shintakua,b, Takayuki Uematsuc, Naomi Fukutaa, Noritada Kobayashic, Yasuo Morid, and Makoto Tominagaa,b,1

aDivision of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), National Institutes of Natural Sciences, Okazaki 444-8787, Japan; bDepartment of Physiological Sciences, Graduate University for Advanced Studies, Okazaki 444-8585, Japan; cBiomedical Laboratory, Division of Biomedical Research, Kitasato Institute Medical Center Hospital, Kitasato University, Saitama 108-8641, Japan; and dDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8530, Japan

Edited* by David Julius, University of California, San Francisco, CA, and approved March 15, 2012 (received for review August 30, 2011)

The ability to sense temperature is essential for organism survival H2O2, a reactive oxygen species (ROS) produced by NADPH and efﬁcient metabolism. Body temperatures profoundly affect oxidase (Nox), is crucial for microorganism removal, given that many physiological functions, including immunity. Transient re- defects in H2O2 production lead to persistent infections (21). As ceptor potential melastatin 2 (TRPM2) is a thermosensitive, Ca2+- the ﬁrst line of defense against infections, the Toll-like receptors permeable cation channel expressed in a wide range of immuno- (TLRs) of phagocytes, including macrophages, recognize com- cytes. TRPM2 is activated by adenosine diphosphate ribose and hy- mon microbial components, such as pathogen-associated mo-

drogen peroxide (H2O2), although the activation mechanism by lecular patterns. Then infective organisms are phagocytosed and cleared by systems in which Nox activity is engaged. Along with H2O2 is not well understood. Here we report a unique activation H O ’s important role in microbicidal function inside the phag- mechanism in which H2O2 lowers the temperature threshold for 2 2 TRPM2 activation, termed “sensitization,” through Met oxidation osomes, membrane-diffusible H2O2 also could play roles in cell and adenosine diphosphate ribose production. This sensitization is signaling outside the phagosomes by acting on various proteins PHYSIOLOGY completely abolished by a single mutation at Met-214, indicating (22). ROS such as H2O2 are now considered to be signaling molecules, in parallel with reactive nitrogen species. These cel- that the temperature threshold of TRPM2 activation is regulated by “ ” redox signals that enable channel activity at physiological body lular redox signals play important roles in a wide range of physiological functions, including ion channel activity (23). We temperatures. Loss of TRPM2 attenuates zymosan-evoked macro- hypothesized that redox signals generated by microbicidal ac- phage functions, including cytokine release and fever-enhanced tivity in macrophages could regulate the function of TRPM2, phagocytic activity. These findings suggest that redox signals sen- which is expressed in macrophages (8). To test the hypothesis, we sitize TRPM2 downstream of NADPH oxidase activity and make investigated the regulation mechanisms of TRPM2. TRPM2 active at physiological body temperature, leading to in- Here we describe a unique mechanism for TRPM2 activation in creased cytosolic Ca2+ concentrations. Our results suggest that which its temperature threshold is regulated dynamically by H2O2, TRPM2 sensitization plays important roles in macrophage functions. termed “sensitization.” Sensitization of TRPM2 is caused by a reduction in its temperature threshold through oxidation of a sin- calcium | immune cells gle methionine at Met-214, and is partially attenuated by a poly (ADP ribose) polymerase (PARP) inhibitor. The loss of TRPM2 he capacity to sense temperature is essential for organism attenuates macrophage functions such as cytokine release at 37 °C Tsurvival and efficient metabolism, and body temperature has and enhancement of phagocytic activity at febrile temperatures. profound effects on many physiological functions, including im- We suggest that TRPM2 is sensitized by redox signals downstream munity. Paradoxically, lowering body temperature with cyclo- of Nox activity, and contributes to macrophage functions. oxygenase inhibitors worsens survival rates for bacterial infection (1), whereas fever elevates immune reactivity (2). Together, these Results fi H2O2 Sensitizes TRPM2 to Heat. We first examined the effects of effects suggest that elevated body temperature has bene cial 2+ H2O2 on heat-evoked TRPM2 activities using a Ca -imaging effects for the immune system, although the molecular mecha- ∼ nisms underlying these effects remain largely unknown. method. Heat stimulation of up to 41 °C was applied before and after H2O2 treatment of mouse TRPM2-expressing HEK293 cells. Transient receptor potential melastatin 2 (TRPM2) is a ther- 2+ mosensitive, Ca2+-permeable cation channel expressed by a wide Heat-evoked [Ca ]i increases were dramatically enhanced by H O treatment in a dose-dependent manner, whereas heat range of immunocytes, including macrophages, whose function is 2 2 stimulation without H O treatment caused only slight activation gradually being clarified (3–9). We previously reported that heat 2 2 (Fig. 1 A and B). In addition to the concentration dependence, the stimulation activates TRPM2 in the presence of low concen- duration of H O treatment also affected the responses; increasing trations of agonists, such as adenosine diphosphate ribose 2 2 H2O2 (30 μM) treatment from 1 min to 5 min proportionally en- (ADPR) and related molecules (10). These agonists are believed hanced heat (∼41 °C)-evoked responses (Fig. 1 C–E). We observed to act on a unique C-terminal pyrophosphatase domain in TRPM2 (Nudix-like domain) (11–13). Temperature-dependent activation fi of TRPM2 plays signi cant roles in cellular functions, including Author contributions: M.K., T.S., and M.T. designed research; M.K., K.S., and N.F. per- insulin release from pancreatic β cells (10, 14). TRPM2 channels formed research; Y.M. contributed new reagents/analytic tools; M.K., K.S., T.U., and N.K. analyzed data; and M.K., T.S., and M.T. wrote the paper. can be activated by hydrogen peroxide (H2O2) and are reported to be involved in cell death caused by oxidative stress via mechanisms The authors declare no conflict of interest. that remain to be clarified (15, 16). ADPR released from in- *This Direct Submission article had a prearranged editor. tracellular organelles, such as the nucleus and mitochondria, may Freely available online through the PNAS open access option. play a primary role in TRPM2 activation by H2O2 (17–19), al- 1To whom correspondence should be sent. E-mail: [email protected]. though one report suggests involvement of an ADPR-independent This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. activation mechanism (20). 1073/pnas.1114193109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1114193109 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 2+ temperatures induced potent [Ca ]i increases even without H2O2 treatment (Fig. 2A, upper trace). When temperature thresholds 2+ were determined from temperatures causing [Ca ]i increases in excess of those observed for DsRed-negative cells, the average threshold was 47.2 ± 0.2 °C (n = 5) (Fig. 2B). Treatment with H2O2 for 1 min signiﬁcantly lowered this threshold [100 μM: 41.7 ± 0.1 °C (n = 5); 3 mM: 36.3 ± 0.4 °C (n = 8); P < 0.001 vs. H2O2 untreated] in a dose-dependent manner (Fig. 2 A and B). Similar to the time dependence of heat-evoked responses (Fig. 1 C–E), temperature threshold reductions also depended on the duration of H2O2 treatment (Fig. 2B). To more precisely determine the temperature thresholds, we used the heat-evoked currents observed in whole- cell patch-clamp recordings to generate Arrhenius plots, which displayed an explicit ﬂex point during heating (Fig. 2C). The reductions in temperature thresholds were recapitulated in whole- cell patch-clamp recordings in which cells were exposed to H2O2 in the pipette solution [100 μM: 40.2 ± 1.3 °C (n = 11); 3 mM: 36.3 ± 0.6 °C (n = 10); P < 0.01] (Fig. 2 C and D). Of note, the sensitization of heat-evoked currents was more easily reproduced by lower concentrations when H2O2 was applied in the pipette solution rather than extracellularly (Fig. 2C and Fig. S1). In the whole-cell recordings, higher concentrations of H2O2 are needed when H2O2 is applied extracellularly, because H2O2 entering the cell can be diluted by the pipette solution. This suggests an intracellular site for H2O2 action. H2O2-mediated reduction in the temperature threshold for TRPM2 activation could explain the increased TRPM2 activity under physiological temperatures, as shown in Fig. S2A. Therefore, the effect of H2O2 on TRPM2 can be viewed as a “sensitization” to physiological body temperature.

Molecular Mechanism of TRPM2 Sensitization to Heat. Most previous studies have suggested that TRPM2 activation by H2O2 is caused by ADPR release from intracellular organelles (17–19). To test this possibility, we evaluated the effects of H2O2 in inside-out

Fig. 1. Heat-evoked responses of TRPM2 were elevated by H2O2 in a concen- single-channel recordings in which intracellular components are tration- and time-dependent manner. (A)H2O2 (100 μM) enhanced heat-evoked absent. Consistent with the data from whole-cell recordings, 2+ 2+ increases in intracellular Ca concentrations ([Ca ]i) in DsRed(+) TRPM2- heat-evoked currents in an inside-out configuration were dra- expressing cells (Left and Right Upper). Representative pseudocolor images of matically enhanced by H2O2 treatment, whereas heat stimulation fl uorescence intensity during heat stimulation before (a) and after (b) H2O2 alone caused only slight activation (Fig. 3A). Single-channel μ μ μ μ treatment. (B)EachH2O2 concentration (10 M, 30 M, 60 M, 100 M, 1 mM, openings of the heat-evoked current after H O treatment were 2+ 2 2 and 3 mM) was applied for 1 min as in A, and the heat-evoked [Ca ]i increases seen at temperatures as low as 37 °C, and the calculated con- after H2O2 treatment were normalized to the values in response to ionomycin ductance was 118.4 ± 10.1 pS (n = 7), higher than the reported for each experiment. Enhancement of the heat-evoked response was not ob- 58 pS of the ADPR-evoked current of human TRPM2 at room served in vector-transfected control cells (vector) or in TRPM2-expressing cells in 2+ 2+ temperature (12). In addition, the single-channel conductance the absence of extracellular Ca (0 Ca ), even at the highest H2O2 concen- ± – increased concurrently with temperature (Fig. S3). Data from the tration (3 mM). Data are mean SEM (n =5 13). (C and D) Representative traces fi of [Ca2+] changes in TRPM2-expressing cells in response to heat before and after single-channel recordings provide signi cant evidence that sen- i sitization of TRPM2 could be caused independently of cytosolic H2O2 (30 μM) treatment for 1 min (C)or5min(D) and later exposure to ionomycin (5 μM). (E) Heat-evoked responses of TRPM2 were elevated by pro- ADPR, although ADPR production also could be involved in 2 longing H2O2 treatment. Data are mean ± SEM (n =6or7).R =0.97. TRPM2 sensitization with intracellular components. In addition, TRPM2 sensitization in single-channel recordings was detected as long as 5 min after H2O2 removal (Fig. S4A), suggesting that 2+ fi no [Ca ]i increases in DsRed-negative TRPM2-nonexpressing H2O2 acts by oxidative modi cation of target amino acids. cells, and H2O2 treatment for 1 min at room temperature failed to The major candidate targets of ROS-mediated protein oxida- 2+ increase [Ca ]i, even in TRPM2-expressing cells (Fig. 1A). Fur- tion are cysteine (Cys) and Met residues (25). Thus, we evaluated 2+ thermore, the heat-evoked [Ca ]i increases were not observed in the effects of various oxidants to identify the residues possibly either vector-transfected cells or TRPM2-expressing cells in the involved in TRPM2 sensitization. Chloramine-T, a membrane- absence of extracellular Ca2+ (Fig. 1B), suggesting that Ca2+ influx permeant oxidant that preferentially oxidizes Met residues, sen- through TRPM2 caused the increase in heat-evoked [Ca2+] . sitized TRPM2 in both single-channel and whole-cell recordings i ′ H2O2-dependent enhancement of heat-evoked TRPM2 responses (Fig. 3 B and C). In contrast, 5,5 -dithiobis-2-nitrobenzoic acid, was also observed in whole-cell patch-clamp recordings, confirm- a membrane-impermeant Cys-specific oxidant, did not induce ing an event across the plasma membrane (Fig. S1). The heat- sensitization of TRPM2 in either type of recording (Fig. 3 D and evoked TRPM2 currents gradually returned to basal levels after E). These data suggest that sensitization of TRPM2 can be me- the temperature reduction, and the sustained currents were com- diated by direct oxidation of Met rather than by Cys. Unlike pletely inhibited by the TRPM2 inhibitor 2-aminoethoxydiphenyl H2O2, S-nitroprusside, an NO donor, did not induce TRPM2 borate (2-APB; Fig. S1) (24), suggesting mediation of the sustained sensitization (Fig. S4B). In addition, even though the amino acid currents by TRPM2. sequence of TRPM2 is very close to that of TRPM8, cold-evoked The observation that TRPM2 was significantly activated by TRPM8 responses were not affected by H2O2 (Fig. S4C). These heat stimulation after H2O2 treatment, whereas heat stimulation data suggest that H2O2-induced sensitization is unique to and (∼41 °C) alone evoked only slight TRPM2 activation (Fig. 1A), characteristic of TRPM2. might be explained if H2O2 reduces the temperature threshold Met-Ala mutagenesis was performed to identify the Met residue(s) for TRPM2 activation. Indeed, heat stimulation with higher in TRPM2 involved in H2O2-induced sensitization. Mouse TRPM2

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1114193109 Kashio et al. Downloaded by guest on September 30, 2021 compared with WT (521.2 ± 153.1 pA/pF), although M214A demonstrated ADPR sensitivity to higher concentrations (500 μM) of ADPR (128.0 ± 89.7 pA/pF) (Fig. S6). This result suggests a possible interaction between M214 and the Nudix motif. To further conﬁrm the importance of M214, we examined the TRPM2 splice variant with a C-terminal deletion (Δ1288–1321; ΔC) (18, 20). TRPM2ΔC still showed sensitization to H2O2 treatment while losing ADPR sensitivity, as reported previously (Fig. S7). This 2+ ﬁnding, along with rapid increases in H2O2-evoked [Ca ]i at physiological temperature (Fig. S2A), support the possibility that the TRPM2 temperature threshold could be regulated in an ADPR-independent way.

TRPM2 Sensitization in Peritoneal Macrophages. We performed additional studies to determine whether H2O2-induced sensitization could be recapitulated in native cells using peritoneal macrophages that endogenously produce ROS on phagocytosis. TRPM2 expression was detected by RT-PCR in freshly prepared WT macrophages but not in TRPM2-deficient cells, even though the two cell types had similar morphology (Fig. S8 A–C). Heat-evoked 2+ responses in WT macrophages were enhanced by H2O2 in Ca imaging (Fig. 5A) and whole-cell patch-clamp methods (densities of heat-evoked current before and after H2O2 application were 4.1 ± 0.4 and 46.9 ± 22.2 pA/pF, respectively; n =3)(Fig.5C), similar to the response of HEK293 cells expressing TRPM2 (Fig. 1A and Fig. S1). Single-channel openings were detected in heat-evoked whole- cell currents. The sustained currents in WT macrophages were inhibited by 2-APB. Although 2-APB is not specifictoTRPM2and 2+ also affects store-operated Ca entry (26), TRPM2 could mediate PHYSIOLOGY the heat-evoked responses (Fig. 5 A and C), given that TRPM2- Fig. 2. H2O2 reduced the temperature threshold for TRPM2 activation. (A) fi Representative traces of temperature-response profiles in heat stimulation de cient macrophages did not show such sensitization (Fig. 5 B and μ D). In addition, sensitization of heat-evoked responses in WT without H2O2 (Top) or after H2O2 treatment at 100 M(Middle)or3mM 2+ (Bottom)for1min.(B) Averaged data for heat stimulation without and after macrophages was not induced in the absence of extracellular Ca

H2O2 treatment at 100 μM or 3 mM for 1 min and at 60 μM for 1, 3, and 5 (Fig. S8D). Together, these data indicate that the sensitization was ### min. Mean ± SEM (n =5–8). P < 0.001 vs. H2O2-untreated; ***P < 0.001 inducible in murine macrophages by H2O2, and that these heat- between indicated pairs (ANOVA). (C) A heat-evoked current after 1 min evoked responses are attributable to endogenous TRPM2.

exposure to pipette solution containing 100 μMH2O2 (Top) obtained by whole-cell recording. Representative Arrhenius plot traces are shown for TRPM2-Dependent Regulation of Macrophage Functions. On in- μ temperature vs. density of heat-evoked currents with 100 MH2O2 (Middle, fection, macrophage activation induces the release of cytokines for using upper trace) or 3 mM (Bottom) after 1 min exposure. 2-APB, a TRPM2 the recruitment and activation of immune cells. To examine the inhibitor. (D) Averaged data for whole-cell recordings of heat-evoked cur- involvement of TRPM2, we compared the release of cytokines in μ ± rents after H2O2 treatment at 100 M or 3 mM for 1 min. Data are mean WT and TRPM2-deficient macrophages using ELISA of culture SEM (n = 10 or 11). *P < 0.05 (t test). media. In these assays, macrophages were stimulated for 24 h at 37 °C with the TLR2 agonist zymosan (50 μg/mL), which induces Nox activation and ROS production (27). ROS generation down- has 35 Met residues, 22 of which are conserved between humans stream of TLR2 activation could cause Ca2+ influx through TRPM2 and mice. Given that H2O2-induced sensitization was also observed sensitization, which in turn would enhance macrophage functions. in HEK293 cells expressing human TRPM2 in both whole-cell and As such, we focused on the cytokines regulated by NF-ĸB, whose single-channel recordings (Fig. S5 A and B), Ala substitutions were 2+ fi activity is regulated by cytosolic Ca levels (28). Macrophage introduced at 21 of the conserved Met residues (the rst Met was stimulation with zymosan elicited the release of granulocyte col- excluded; Fig. 4A), and the sensitization of each mutant was eval- α α β 2+ ony stimulating factor (G-CSF), TNF , IL-1 , IL-1 , macrophage uated with a Ca -imaging method. In WT TRPM2, H2O2 (100 inflammatory protein-2 (CXCL2), and monocyte chemotactic μM) treatment significantly reduced the temperature threshold for – 2+ protein-1 (CCL2) (Fig. 6 A F). Among them, release of G-CSF, [Ca ]i increases in a time-dependent manner (Fig. 4 B and D). CXCL2, and IL-1α were significantly reduced in TRPM2-deficient Among the 21 mutants, only M214A completely lost H2O2-induced macrophages compared with WT cells. IL-1β release tended to be sensitization even after long exposure (3 min) (Fig. 4 C and D), and lower in TRPM2-deficient macrophages without statistical sig- the corresponding human mutant M215A also showed similar nificance (P = 0.10; values were 163 ± 43 and 65 ± 20 pg/mL) (Fig. properties (Fig. S5C). In addition, M214A did not exhibit H2O2- 2+ 6E). These data suggest that TRPM2-mediated pathways, in- evoked [Ca ]i increases at physiological temperature (Fig. S2B). cluding ROS-induced sensitization, contribute to the increased We could not evaluate sensitization in two mutations (M815A and release of G-CSF, CXCL2, IL-1α, and possibly IL-1β. Considering M1044A) because of their limited channel activity. ADPR release TRPM2 sensitization, temperature elevation should affect H2O2- 2+ from organelles is thought to be involved in sensitization because it evoked [Ca ]i increases in macrophages. Indeed, temperature 2+ 2+ can be induced by H2O2 in [Ca ]i imaging, and the threshold shift elevations as small as 1.3 °C enhanced H2O2 (30 μM)-evoked [Ca ]i was partly attenuated by the PARP inhibitor PJ-34, which inhibits increases (Fig. 6G). These data indicate that sensitized-TRPM2 ADPR release from the nucleus (Fig. 4E, Left). Nevertheless, the channel function can be further enhanced by temperature eleva- significant threshold reductions seen in the presence of PJ-34 (Fig. tion, suggesting that redox signals and fever can act cooperatively 4E, Right) indicate that Met oxidation could be crucial for sensiti- on macrophage functions via TRPM2. To examine the effects zation, consistent with the finding that oxidants, including H2O2 of small temperature increases on other macrophage functions, and chloramine-T, sensitized TRPM2 in a membrane-delimited we examined phagocytic activity at normal (37 °C) and febrile manner. Unexpectedly, the density of ADPR (100 μM)-evoked (38.5 °C) temperatures (2). Phagocytosis was significantly in- currents was significantly reduced (5.5 ± 1.1 pA/pF) for M214A creased at 38.5 °C compared with 37 °C in WT macrophages,

Kashio et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 Fig. 3. H2O2 sensitizes TRPM2 to heat in a membrane-delimited manner. (A) A heat-evoked current in a TRPM2-expressing cell at −60 mV in an inside-out configuration. Magnified traces (a and b) correspond to the currents shown by the arrows in the left trace. (B and C) Heat-evoked responses of TRPM2 were sensitized by chloramine-T, a membrane-permeant oxidant that preferentially oxidizes Met residues, in both whole-cell (B) and inside-out single- channel (C) recordings, where the magnified traces (a and b) correspond to

the currents shown by the arrows in the above trace. (D and E)5,5′-Dithiobis- Fig. 4. Structural basis for TRPM2 sensitization by H2O2.(A)Putative 2-nitrobenzoic acid, a membrane-impermeant Cys-speciﬁc oxidant, did not membrane topology of TRPM2, with the Met residues conserved between sensitize the heat response of TRPM2 in either whole-cell (D) or inside-out mouse and human TRPM2 indicated. Mutations to Ala were introduced single-channel (E) recordings. at the Met residues indicated by red circles. (B–D) Reduction in the tem-

perature threshold for TRPM2 activation by H2O2 treatment (100 μMfor 1 or 3 min) was completely abolished in the M214A mutant. Shown are fi 2+ whereas TRPM2-deficient macrophages showed no such tem- temperature-response pro les of heat-evoked [Ca ]i increases observed in perature-dependent effect (Fig. 6H). These data suggest that DsRed(+), WT (B), or M214A-expressing cells (C) and nonexpressing DsRed TRPM2 could mediate the enhanced phagocytic activity at (−) cells (D). Data are mean ± SEM (n =4or5).P < 0.05, Student t test elevated temperatures. or ANOVA, unless noted otherwise, between without and after treatment with H2O2.NS,notsignificant (ANOVA). (E) Sensitization of WT TRPM2 in Discussion the presence (+) or absence (−) of PJ-34. In the PJ(+) groups, the potent PARP inhibitor PJ-34 (1 μM) was present during the entire experiment. In the present study, we have identified a unique mechanism for ± – < < < “ ” Data are mean SEM (n =48). *P 0.05; **P 0.01; ***P 0.001 vs. sensitization of TRPM2. In the absence of H2O2 treatment, the 0 min (ANOVA). temperature threshold for TRPM2 activation remained at supra- physiological temperature levels, whereas H2O2 treatment lowered the threshold to physiological temperatures. Our present a membrane-delimited manner by reducing the temperature results differ somewhat from those of our previous study, which threshold for activation. We also found that PARP inhibition showed that TRPM2 was activated by heat alone at around body attenuates H2O2-evoked reductions in the temperature threshold. temperature and maximal single-channel openings occurred at Nevertheless, we believe that ADPR participation is modest, given ∼36 °C (10). However, in that study, to obtain sufficient current that single-channel activation is induced at ∼37 °C after H2O2 size, HEK293 cells were cultured for longer periods (more than 36 treatment (Fig. 3A), and that the PARP inhibitor used might have h) after transfection, compare with only 20–36 h in the present an additional effect in this system. These results suggest that H O study. These different culture conditions might have affected the 2 2 sensitivity to heat stimulation. causes TRPM2 sensitization through two different mechanisms in parallel, which might explain the different proposals for the Although TRPM2 is activated by ROS and is involved in cell – death after oxidative stress (15, 16), the activation mechanisms action of H2O2 on TRPM2 (17 20). Along with PARP-dependent involved are unclear (17–20). The primary activator of TRPM2 is ADPR release from the nucleus, ADPR release from mitochon- thought to be ADPR, with most previous studies suggesting that dria is also reportedly involved in TRPM2 activation by H2O2. the release of ADPR from the nucleus and mitochondria plays Although our results using intact cells cannot rule out mitochon- a primary role in TRPM2 activation by H2O2 (17–19), although drial involvement, the fact that H2O2-evoked activation of one study has reported that H2O2 acts on TRPM2 directly (20). TRPM2 at physiological temperature is more rapid (Fig. S2) than We show here that H2O2 can sensitize TRPM2 in the absence of TRPM2 currents mediated by ADPR released from mitochon- these organelles and activate it at physiological temperatures in dria (18) make its participation less likely. Interestingly, TRPM2

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1114193109 Kashio et al. Downloaded by guest on September 30, 2021 Fig. 5. Sensitization is observed in WT macrophages, but not in TRPM2- 2+ deﬁcient cells. (A and B)H2O2-induced sensitization of heat-evoked [Ca ]i increases was observed in WT (A), but not in TRPM2-deﬁcient (B) macro-

phages. (C and D)H2O2-induced sensitization of heat-evoked current was observed in WT (C), but not in TRPM2-deﬁcient (D) macrophages. (C, Inset)A magniﬁed trace corresponding to the red box shown in the upper trace. PHYSIOLOGY

sensitization was completely abolished by a single mutation at Met-214 (M214A) (Fig. 4) and a corresponding mutation in human TRPM2 (M215A) (Fig. S5C), strongly supporting the importance of this Met residue in TRPM2 sensitization. Previous studies on a TRPM2 splice variant with diminished ADPR-evoked activation also support our idea that ADPR-independent sensitization of TRPM2 occurs through Met oxidation. Unexpectedly, the M214 mutant affected TRPM2 activation by ADPR, even though the site is apart from the C-terminal Nudix-like domain, suggesting that an interaction between the TRPM2 N- and C- terminal regions regulates TRPM2 activity. Although Met oxidation in the regulation of TRPM6 has been reported previously (29), our study demonstrates that Met oxidation is involved in regulation of the temperature threshold for activation of thermosensitive TRP channels (thermo-TRPs). Among the known thermo-TRPs, TRPV1 was found to exhibit a reduction in temperature threshold through serine phosphorylation by protein kinases A and C on proinflammatory mediator production (30, 31). Thus, the temperature thresholds for activating thermo-TRPs might be regulated by various mechanisms depending on the channel and its cellular environment. Sensitization of TRPM2 was found to be involved in macrophage functions. Pathogen-associated molecular patterns of in- vading microorganisms can activate the TLR pathway, leading to production of ROS for microbicidal activity. Among the produced ROS, H2O2 has weaker reactivity and higher membrane perme- Fig. 6. Zymosan-induced cytokine release and phagocytic activity in WT ability, allowing it to diffuse over long distances (32), thus making and TRPM2-deficient macrophages. (A–F) Amount of released cytokines μ H2O2 a suitable signaling molecule (33). H2O2 concentrations are from unstimulated and zymosan (50 g/mL)-stimulated macrophages from reported to reach mM levels within phagosomes (34) and 10–100 WT and TRPM2-deficient mice. Data are mean ± SEM (n =4–5). *P < 0.05; μMininflammatory environments (35), which would be sufficient **P < 0.01 (ANOVA). (G) A small temperature elevation caused further μ 2+ to sensitize TRPM2. We first hypothesized that TRPM2 activity enhancement of H2O2 (30 M)-induced [Ca ]i increases in WT macrophages. could enhance cytokine release, given that intracellular Ca2+ Mean values for the normal and elevated temperatures are reported as mean ± SD. The lower panels show pseudocolor images of fluorescence activates NF-kB (28), which in turn regulates the expression of – various cytokines (36). However, we found that the loss of TRPM2 intensity corresponding to the time points in the upper trace (a c) and a phase-contrast image. Colored wedges in the phase-contrast image in- affected the release of cytokine subsets. One possible explanation fi dicate the cells corresponding to each colored ratio trace. (H) Enhancement for this nding is that direct regulation of transcription factors by of phagocytic activity by elevated temperature (38.5 °C) was abolished in redox signals (37) leads to complex changes in cytokine release. TRPM2-deficient macrophages. The ratio of cells that phagocytized zymo- Other mechanisms lying downstream of TRPM2 activity could be san particle(s) was normalized to the average values at 37 °C in each ge- involved as well, given that cytokine release is not caused simply notype. Data are mean ± SEM (n = 3). *P < 0.05; NSP > 0.05 for 37 °C vs. by effects on transcription regulation. For example, the calcium- 38.5 °C (Student t test).

Kashio et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 dependent proteases calpains are involved in the maturation and that the study of TRPM2 sensitization might identify unique release of IL-1α (38) as well as in phagocytosis (39). A recent in vivo approaches for determining the physiological function of TRPM2 study reported significantly enhanced susceptibility to Listeria that focus on body temperature and redox signals. monocytogenes in TRPM2-deficient mice (40), which can be partly explained by the impaired macrophage functions observed in the Materials and Methods present study. HEK293T cells transfected with cDNAs or peritoneal macrophages prepared Of note, TRPM2 is expressed by lymphocytes, neutrophils, and from female C57BL/6NCr and TRPM2-deficient mice (7) were used for Ca2+ monocytes/macrophages (3–8), whose activities have a strong imaging with fura-2 and patch-clamp recordings to study TRPM2-mediated relationship with body temperature (2, 41). This suggests that channel properties. Zymosan-evoked cytokine release and phagocytic ac- TRPM2 might have a broader role in the temperature sensitivity fi of the immune system. Fever or hyperthermia is a widely con- tivity were compared in WT and TRPM2-de cient macrophages. Data are ± ± served phenomenon involved in host defenses against infections in presented as mean SEM or mean SD. Statistical analysis was performed both endotherms and ectotherms (42, 43) and is considered to using the Student t test or ANOVA, followed by the Bonferroni-type mul- enhance immune reactivity (2). Thus, fever is considered a bene- tiple t test. P values < 0.05 were considered significant. Synthetic oligonu- ficial response in host defenses, but the underlying mechanism cleotide primers constructing specific mutations and splice variant are shown remains unclear. Given that TRPM2 is conserved among a wide in Table S1. A detailed description of the experimental procedures is pro- range of species (44) and is thought to be widely expressed in vided in SI Materials and Methods. immunocytes, ROS-sensitized TRPM2 can act as a thermosensor to regulate immune reactivities at body temperatures ranging from ACKNOWLEDGMENTS. We thank Drs. Shin-ichiro Saitoh (Tokyo University), nonfebrile to febrile. Redox signals are also known to affect Ca2+ Masatsugu Ohora (Tokyo Medical and Dental University), Yoshihiro Kubo, release from Ca2+ stores (37), suggesting that ROS can regulate and Masaki Fukata (National Institute for Physiological Sciences) for their 2+ helpful advice. This work was supported by grants from the Japanese Ca signals in various ways. TRPM2 could play a part in this Ministry of Education, Culture, Sports, Science and Technology (to M.T.) and regulatory system, as suggested by a recent report demonstrating the Mitsubishi Foundation (to M.T.); and by a postdoctoral fellowship from negative regulation of ROS by TRPM2 (9). Our findings suggest the Japan Society for Promotion of Science postdoctoral fellowship (to M.K.).

1. Kluger MJ, Kozak W, Conn CA, Leon LR, Soszynski D (1998) Role of fever in disease. 24. Togashi K, Inada H, Tominaga M (2008) Inhibition of the transient receptor potential Ann N Y Acad Sci 856:224–233. cation channel TRPM2 by 2-aminoethoxydiphenyl borate (2-APB). Br J Pharmacol 153: 2. Hasday JD, Fairchild KD, Shanholtz C (2000) The role of fever in the infected host. 1324–1330. Microbes Infect 2:1891–1904. 25. Spickett CM, Pitt AR, Morrice N, Kolch W (2006) Proteomic analysis of phosphoryla- 3. Heiner I, Eisfeld J, Lückhoff A (2003) Role and regulation of TRP channels in neutro- tion, oxidation and nitrosylation in signal transduction. Biochim Biophys Acta 1764: – phil granulocytes. Cell Calcium 33:533 540. 1823–1841. 4. Inada H, Iida T, Tominaga M (2006) Different expression patterns of TRP genes in 26. DeHaven WI, Smyth JT, Boyles RR, Bird GS, Putney JW, Jr. (2008) Complex actions of – murine B and T lymphocytes. Biochem Biophys Res Commun 350:762 767. 2-aminoethyldiphenyl borate on store-operated calcium entry. J Biol Chem 283: 5. Carter RN, et al. (2006) Molecular and electrophysiological characterization of tran- 19265–19273. sient receptor potential ion channels in the primary murine megakaryocyte. J Physiol 27. Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM (2003) Collaborative 576:151–162. induction of inflammatory responses by dectin-1 and Toll-like receptor 2. J Exp Med 6. Lange I, Penner R, Fleig A, Beck A (2008) Synergistic regulation of endogenous TRPM2 197:1107–1117. channels by adenine dinucleotides in primary human neutrophils. Cell Calcium 44: – 28. Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI (1997) Differential activation of 604 615. + + transcription factors induced by Ca2 response amplitude and duration. Nature 386: 7. Yamamoto S, et al. (2008) TRPM2-mediated Ca2 influx induces chemokine pro- – duction in monocytes that aggravates inflammatory neutrophil infiltration. Nat Med 855 858. 14:738–747. 29. Cao G, et al. (2010) Methionine sulfoxide reductase B1 (MsrB1) recovers TRPM6 – 8. Sumoza-Toledo A, Penner R (2011) TRPM2: A multifunctional ion channel for calcium channel activity during oxidative stress. J Biol Chem 285:26081 26087. signalling. J Physiol 589:1515–1525. 30. Tominaga M, Wada M, Masu M (2001) Potentiation of capsaicin receptor activity by 9. Di A, et al. (2012) The redox-sensitive cation channel TRPM2 modulates phagocyte metabotropic ATP receptors as a possible mechanism for ATP-evoked pain and hy- ROS production and inflammation. Nat Immunol 13:29–34. peralgesia. Proc Natl Acad Sci USA 98:6951–6956. 10. Togashi K, et al. (2006) TRPM2 activation by cyclic ADP-ribose at body temperature is 31. Sugiura T, Tominaga M, Katsuya H, Mizumura K (2002) Bradykinin lowers the involved in insulin secretion. EMBO J 25:1804–1815. threshold temperature for heat activation of vanilloid receptor 1. J Neurophysiol 88: 11. Perraud AL, et al. (2001) ADP-ribose gating of the calcium-permeable LTRPC2 channel 544–548. revealed by Nudix motif homology. Nature 411:595–599. 32. Winterbourn CC (2008) Reconciling the chemistry and biology of reactive oxygen 12. Sano Y, et al. (2001) Immunocyte Ca2+ influx system mediated by LTRPC2. Science 293: species. Nat Chem Biol 4:278–286. 1327–1330. 33. Forman HJ, Maiorino M, Ursini F (2010) Signaling functions of reactive oxygen species. 13. Fleig A, Penner R (2004) The TRPM ion channel subfamily: Molecular, biophysical and Biochemistry 49:835–842. functional features. Trends Pharmacol Sci 25:633–639. 34. Haas A, Goebel W (1992) Microbial strategies to prevent oxygen-dependent killing by 14. Uchida K, et al. (2011) Lack of TRPM2 impaired insulin secretion and glucose me- phagocytes. Free Radic Res Commun 16:137–157. – tabolisms in mice. Diabetes 60:119 126. 35. Nathan CF, Root RK (1977) Hydrogen peroxide release from mouse peritoneal mac- + 15. Hara Y, et al. (2002) LTRPC2 Ca2 -permeable channel activated by changes in redox rophages: Dependence on sequential activation and triggering. J Exp Med 146: status confers susceptibility to cell death. Mol Cell 9:163–173. 1648–1662. 16. Kolisek M, Beck A, Fleig A, Penner R (2005) Cyclic ADP-ribose and hydrogen peroxide 36. Shoelson SE, Lee J, Goldfine AB (2006) Inflammation and insulin resistance. J Clin synergize with ADP-ribose in the activation of TRPM2 channels. Mol Cell 18:61–69. Invest 116:1793–1801. 17. Fonfria E, et al. (2004) TRPM2 channel opening in response to oxidative stress is de- 37. Kamata H, Hirata H (1999) Redox regulation of cellular signalling. Cell Signal 11:1–14. pendent on activation of poly(ADP-ribose) polymerase. Br J Pharmacol 143:186–192. 38. Carruth LM, Demczuk S, Mizel SB (1991) Involvement of a calpain-like protease in the 18. Perraud AL, et al. (2005) Accumulation of free ADP-ribose from mitochondria medi- processing of the murine interleukin 1α precursor. J Biol Chem 266:12162–12167. ates oxidative stress-induced gating of TRPM2 cation channels. J Biol Chem 280: 39. Dewitt S, Hallett MB (2002) Cytosolic free Ca(2+) changes and calpain activation are 6138–6148. required for β integrin–accelerated phagocytosis by human neutrophils. J Cell Biol 19. Buelow B, Song Y, Scharenberg AM (2008) The poly(ADP-ribose) polymerase PARP-1 is – required for oxidative stress-induced TRPM2 activation in lymphocytes. J Biol Chem 159:181 189. 283:24571–24583. 40. Knowles H, et al. (2011) Transient Receptor Potential Melastatin 2 (TRPM2) ion 20. Wehage E, et al. (2002) Activation of the cation channel long transient receptor channel is required for innate immunity against Listeria monocytogenes. Proc Natl potential channel 2 (LTRPC2) by hydrogen peroxide: A splice variant reveals a mode of Acad Sci USA 108:11578–11583. activation independent of ADP-ribose. J Biol Chem 277:23150–23156. 41. Blatteis CM (1986) Fever: Is it beneficial? Yale J Biol Med 59:107–116. 21. Roos D, et al. (1996) Mutations in the X-linked and autosomal recessive forms of 42. Dinarello CA (2004) Infection, fever, and exogenous and endogenous pyrogens: Some chronic granulomatous disease. Blood 87:1663–1681. concepts have changed. J Endotoxin Res 10:201–222. 22. Dröge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 43. Vaughn LK, Bernheim HA, Kluger MJ (1974) Fever in the lizard Dipsosaurus dorsalis. 82:47–95. Nature 252:473–474. 23. Brown DI, Griendling KK (2009) Nox proteins in signal transduction. Free Radic Biol 44. Saito S, Shingai R (2006) Evolution of thermoTRP ion channel homologs in Med 47:1239–1253. vertebrates. Physiol Genomics 27:219–230.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1114193109 Kashio et al. Downloaded by guest on September 30, 2021