Redox signal-mediated sensitization of transientreceptor potential melastatin 2 (TRPM2) totemperature affects macrophage functionsMakiko 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; cBiomedicalLaboratory, Division of Biomedical Research, Kitasato Institute Medical Center Hospital, Kitasato University, Saitama 108-8641, Japan; and dDepartment ofSynthetic 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 survivaland efficient metabolism. Body temperatures profoundly affectmany physiological functions, including immunity. Transient re-ceptor potential melastatin 2 (TRPM2) is a thermosensitive, Ca2+-permeable cation channel expressed in a wide range of immuno-cytes. TRPM2 is activated by adenosine diphosphate ribose and hy-drogen peroxide (H2O2), although the activation mechanism byH2O2 is not well understood. Here we report a unique activationmechanism in which H2O2 lowers the temperature threshold forTRPM2 activation, termed “sensitization,” through Met oxidationand adenosine diphosphate ribose production. This sensitization iscompletely abolished by a single mutation at Met-214, indicatingthat the temperature threshold of TRPM2 activation is regulated byredox signals that enable channel activity at physiological bodytemperatures. Loss of TRPM2 attenuates zymosan-evoked macro-phage functions, including cytokine release and fever-enhancedphagocytic activity. These findings suggest that redox signals sen-sitize TRPM2 downstream of NADPH oxidase activity and makeTRPM2 active at physiological body temperature, leading to in-creased cytosolic Ca2+ concentrations. Our results suggest thatTRPM2 sensitization plays important roles inmacrophage functions.

calcium | immune cells

The capacity to sense temperature is essential for organismsurvival and efficient metabolism, and body temperature has

profound effects on many physiological functions, including im-munity. Paradoxically, lowering body temperature with cyclo-oxygenase inhibitors worsens survival rates for bacterial infection(1), whereas fever elevates immune reactivity (2). Together, theseeffects suggest that elevated body temperature has beneficialeffects for the immune system, although the molecular mecha-nisms underlying these effects remain largely unknown.Transient receptor potential melastatin 2 (TRPM2) is a ther-

mosensitive, Ca2+-permeable cation channel expressed by a widerange of immunocytes, including macrophages, whose function isgradually being clarified (3–9). We previously reported that heatstimulation activates TRPM2 in the presence of low concen-trations of agonists, such as adenosine diphosphate ribose(ADPR) and related molecules (10). These agonists are believedto act on a unique C-terminal pyrophosphatase domain in TRPM2(Nudix-like domain) (11–13). Temperature-dependent activationof TRPM2 plays significant roles in cellular functions, includinginsulin release from pancreatic β cells (10, 14). TRPM2 channelscan be activated by hydrogen peroxide (H2O2) and are reported tobe involved in cell death caused by oxidative stress via mechanismsthat remain to be clarified (15, 16). ADPR released from in-tracellular organelles, such as the nucleus and mitochondria, mayplay a primary role in TRPM2 activation by H2O2 (17–19), al-though one report suggests involvement of anADPR-independentactivation mechanism (20).

H2O2, a reactive oxygen species (ROS) produced by NADPHoxidase (Nox), is crucial for microorganism removal, given thatdefects in H2O2 production lead to persistent infections (21). Asthe first line of defense against infections, the Toll-like receptors(TLRs) of phagocytes, including macrophages, recognize com-mon microbial components, such as pathogen-associated mo-lecular patterns. Then infective organisms are phagocytosed andcleared by systems in which Nox activity is engaged. Along withH2O2’s important role in microbicidal function inside the phag-osomes, membrane-diffusible H2O2 also could play roles in cellsignaling outside the phagosomes by acting on various proteins(22). ROS such as H2O2 are now considered to be signalingmolecules, in parallel with reactive nitrogen species. These cel-lular “redox” signals play important roles in a wide range ofphysiological functions, including ion channel activity (23). Wehypothesized that redox signals generated by microbicidal ac-tivity in macrophages could regulate the function of TRPM2,which is expressed in macrophages (8). To test the hypothesis, weinvestigated the regulation mechanisms of TRPM2.Here we describe a unique mechanism for TRPM2 activation in

which its temperature threshold is regulated dynamically by H2O2,termed “sensitization.” Sensitization of TRPM2 is caused by a re-duction in its temperature threshold through oxidation of a sin-gle methionine at Met-214, and is partially attenuated by a poly(ADP ribose) polymerase (PARP) inhibitor. The loss of TRPM2attenuates macrophage functions such as cytokine release at 37 °Cand enhancement of phagocytic activity at febrile temperatures.We suggest that TRPM2 is sensitized by redox signals downstreamof Nox activity, and contributes to macrophage functions.

ResultsH2O2 Sensitizes TRPM2 to Heat. We first examined the effects ofH2O2 on heat-evoked TRPM2 activities using a Ca2+-imagingmethod. Heat stimulation of up to ∼41 °C was applied before andafter H2O2 treatment of mouse TRPM2-expressing HEK293 cells.Heat-evoked [Ca2+]i increases were dramatically enhanced byH2O2 treatment in a dose-dependent manner, whereas heatstimulation without H2O2 treatment caused only slight activation(Fig. 1 A and B). In addition to the concentration dependence, theduration of H2O2 treatment also affected the responses; increasingH2O2 (30 μM) treatment from 1 min to 5 min proportionally en-hanced heat (∼41 °C)-evoked responses (Fig. 1C–E).We observed

Author contributions: M.K., T.S., and M.T. designed research; M.K., K.S., and N.F. per-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.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Freely available online through the PNAS open access option.1To whom correspondence should be sent. E-mail: tominaga@nips.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1114193109/-/DCSupplemental.

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no [Ca2+]i increases in DsRed-negative TRPM2-nonexpressingcells, and H2O2 treatment for 1 min at room temperature failed toincrease [Ca2+]i, even in TRPM2-expressing cells (Fig. 1A). Fur-thermore, the heat-evoked [Ca2+]i increases were not observed ineither vector-transfected cells or TRPM2-expressing cells in theabsence of extracellular Ca2+ (Fig. 1B), suggesting that Ca2+ influxthrough TRPM2 caused the increase in heat-evoked [Ca2+]i.H2O2-dependent enhancement of heat-evoked TRPM2 responseswas also observed in whole-cell patch-clamp recordings, confirm-ing an event across the plasma membrane (Fig. S1). The heat-evoked TRPM2 currents gradually returned to basal levels afterthe temperature reduction, and the sustained currents were com-pletely inhibited by the TRPM2 inhibitor 2-aminoethoxydiphenylborate (2-APB; Fig. S1) (24), suggestingmediation of the sustainedcurrents by TRPM2.The observation that TRPM2 was significantly activated by

heat stimulation after H2O2 treatment, whereas heat stimulation(∼41 °C) alone evoked only slight TRPM2 activation (Fig. 1A),might be explained if H2O2 reduces the temperature thresholdfor TRPM2 activation. Indeed, heat stimulation with higher

temperatures induced potent [Ca2+]i increases even withoutH2O2treatment (Fig. 2A, upper trace). When temperature thresholdswere determined from temperatures causing [Ca2+]i increases inexcess of those observed for DsRed-negative cells, the averagethresholdwas 47.2± 0.2 °C (n=5) (Fig. 2B). TreatmentwithH2O2for 1min significantly lowered this threshold [100 μM:41.7± 0.1 °C(n=5); 3mM: 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 timedependence of heat-evoked responses (Fig. 1 C–E), temperaturethreshold reductions also depended on the duration of H2O2treatment (Fig. 2B). Tomore precisely determine the temperaturethresholds, we used the heat-evoked currents observed in whole-cell patch-clamp recordings to generate Arrhenius plots, whichdisplayed an explicit flex point during heating (Fig. 2C). Thereductions in temperature thresholds were recapitulated in whole-cell patch-clamp recordings in which cells were exposed toH2O2 inthe pipette solution [100 μM: 40.2± 1.3 °C (n= 11); 3 mM: 36.3±0.6 °C (n=10);P< 0.01] (Fig. 2C andD).Ofnote, the sensitizationof heat-evoked currents was more easily reproduced by lowerconcentrations when H2O2 was applied in the pipette solutionrather than extracellularly (Fig. 2C and Fig. S1). In the whole-cellrecordings, higher concentrations of H2O2 are needed whenH2O2is applied extracellularly, because H2O2 entering the cell can bediluted by the pipette solution. This suggests an intracellular sitefor H2O2 action. H2O2-mediated reduction in the temperaturethreshold for TRPM2 activation could explain the increasedTRPM2activity underphysiological temperatures, as shown inFig.S2A. Therefore, the effect of H2O2 on TRPM2 can be viewed asa “sensitization” to physiological body temperature.

Molecular Mechanism of TRPM2 Sensitization to Heat.Most previousstudies have suggested that TRPM2 activation by H2O2 is causedby ADPR release from intracellular organelles (17–19). To testthis possibility, we evaluated the effects of H2O2 in inside-outsingle-channel recordings in which intracellular components areabsent. Consistent with the data from whole-cell recordings,heat-evoked currents in an inside-out configuration were dra-matically enhanced by H2O2 treatment, whereas heat stimulationalone caused only slight activation (Fig. 3A). Single-channelopenings of the heat-evoked current after H2O2 treatment wereseen at temperatures as low as 37 °C, and the calculated con-ductance was 118.4 ± 10.1 pS (n = 7), higher than the reported58 pS of the ADPR-evoked current of human TRPM2 at roomtemperature (12). In addition, the single-channel conductanceincreased concurrently with temperature (Fig. S3). Data from thesingle-channel recordings provide significant evidence that sen-sitization of TRPM2 could be caused independently of cytosolicADPR, although ADPR production also could be involved inTRPM2 sensitization with intracellular components. In addition,TRPM2 sensitization in single-channel recordings was detectedas long as 5 min after H2O2 removal (Fig. S4A), suggesting thatH2O2 acts by oxidative modification of target amino acids.The major candidate targets of ROS-mediated protein oxida-

tion are cysteine (Cys) and Met residues (25). Thus, we evaluatedthe effects of various oxidants to identify the residues possiblyinvolved in TRPM2 sensitization. Chloramine-T, a membrane-permeant oxidant that preferentially oxidizes Met residues, sen-sitized TRPM2 in both single-channel and whole-cell recordings(Fig. 3 B and C). In contrast, 5,5′-dithiobis-2-nitrobenzoic acid,a membrane-impermeant Cys-specific oxidant, did not inducesensitization of TRPM2 in either type of recording (Fig. 3 D andE). These data suggest that sensitization of TRPM2 can be me-diated by direct oxidation of Met rather than by Cys. UnlikeH2O2, S-nitroprusside, an NO donor, did not induce TRPM2sensitization (Fig. S4B). In addition, even though the amino acidsequence of TRPM2 is very close to that of TRPM8, cold-evokedTRPM8 responses were not affected by H2O2 (Fig. S4C). Thesedata suggest that H2O2-induced sensitization is unique to andcharacteristic of TRPM2.Met-Alamutagenesis was performed to identify theMet residue(s)

in TRPM2 involved inH2O2-induced sensitization.Mouse TRPM2

Fig. 1. Heat-evoked responses of TRPM2 were elevated by H2O2 in a concen-tration- and time-dependentmanner. (A) H2O2 (100μM)enhancedheat-evokedincreases in intracellular Ca2+ concentrations ([Ca2+]i) in DsRed(+) TRPM2-expressing cells (Left and Right Upper). Representative pseudocolor images offluorescence intensity during heat stimulation before (a) and after (b) H2O2

treatment. (B) Each H2O2 concentration (10 μM, 30 μM, 60 μM, 100 μM, 1 mM,and 3 mM) was applied for 1 min as in A, and the heat-evoked [Ca2+]i increasesafter H2O2 treatment were normalized to the values in response to ionomycinfor each experiment. Enhancement of the heat-evoked response was not ob-served in vector-transfected control cells (vector) or in TRPM2-expressing cells inthe absence of extracellular Ca2+ (0 Ca2+), even at the highest H2O2 concen-tration (3mM). Data aremean± SEM (n = 5–13). (C andD) Representative tracesof [Ca2+]i changes in TRPM2-expressing cells in response toheatbefore andafterH2O2 (30 μM) treatment for 1 min (C) or 5 min (D) and later exposure to ion-omycin (5 μM). (E) Heat-evoked responses of TRPM2 were elevated by pro-longing H2O2 treatment. Data are mean ± SEM (n = 6 or 7). R2 = 0.97.

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has 35 Met residues, 22 of which are conserved between humansandmice. Given thatH2O2-induced sensitization was also observedin HEK293 cells expressing human TRPM2 in both whole-cell andsingle-channel recordings (Fig. S5 A and B), Ala substitutions wereintroduced at 21 of the conserved Met residues (the first Met wasexcluded; Fig. 4A), and the sensitization of each mutant was eval-uated with a Ca2+-imaging method. In WT TRPM2, H2O2 (100μM) treatment significantly reduced the temperature threshold for[Ca2+]i increases in a time-dependent manner (Fig. 4 B and D).Among the 21mutants, onlyM214A completely lost H2O2-inducedsensitization even after long exposure (3 min) (Fig. 4C andD), andthe corresponding human mutant M215A also showed similarproperties (Fig. S5C). In addition, M214A did not exhibit H2O2-evoked [Ca2+]i increases at physiological temperature (Fig. S2B).We could not evaluate sensitization in two mutations (M815A andM1044A) because of their limited channel activity. ADPR releasefrom organelles is thought to be involved in sensitization because itcan be induced by H2O2 in [Ca2+]i imaging, and the threshold shiftwas partly attenuated by the PARP inhibitor PJ-34, which inhibitsADPR release from the nucleus (Fig. 4E, Left). Nevertheless, thesignificant threshold reductions seen in the presence of PJ-34 (Fig.4E, Right) indicate that Met oxidation could be crucial for sensiti-zation, consistent with the finding that oxidants, including H2O2and chloramine-T, sensitized TRPM2 in a membrane-delimitedmanner. Unexpectedly, the density of ADPR (100 μM)-evokedcurrents was significantly reduced (5.5 ± 1.1 pA/pF) for M214A

compared with WT (521.2 ± 153.1 pA/pF), although M214Ademonstrated ADPR sensitivity to higher concentrations (500 μM)of ADPR (128.0 ± 89.7 pA/pF) (Fig. S6). This result suggestsa possible interaction between M214 and the Nudix motif. Tofurther confirm the importance ofM214, we examined the TRPM2splice variant with a C-terminal deletion (Δ1288–1321; ΔC) (18,20). TRPM2ΔC still showed sensitization to H2O2 treatment whilelosing ADPR sensitivity, as reported previously (Fig. S7). Thisfinding, along with rapid increases in H2O2-evoked [Ca2+]i atphysiological temperature (Fig. S2A), support the possibility thatthe TRPM2 temperature threshold could be regulated in anADPR-independent way.

TRPM2 Sensitization in Peritoneal Macrophages. We performed ad-ditional studies to determine whether H2O2-induced sensitizationcould be recapitulated in native cells using peritoneal macrophagesthat endogenously produce ROS on phagocytosis. TRPM2 ex-pression was detected by RT-PCR in freshly prepared WT mac-rophages but not in TRPM2-deficient cells, even though the twocell types had similar morphology (Fig. S8 A–C). Heat-evokedresponses in WT macrophages were enhanced by H2O2 in Ca2+imaging (Fig. 5A) andwhole-cell patch-clampmethods (densities ofheat-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 tothe 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 wereinhibited by 2-APB. Although 2-APB is not specific to TRPM2 andalso affects store-operated Ca2+ entry (26), TRPM2 could mediatethe heat-evoked responses (Fig. 5 A and C), given that TRPM2-deficientmacrophages did not show such sensitization (Fig. 5B andD). In addition, sensitization of heat-evoked responses in WTmacrophages was not induced in the absence of extracellular Ca2+(Fig. S8D). Together, these data indicate that the sensitization wasinducible in murine macrophages by H2O2, and that these heat-evoked responses are attributable to endogenous TRPM2.

TRPM2-Dependent Regulation of Macrophage Functions. On in-fection, macrophage activation induces the release of cytokines forthe recruitment and activation of immune cells. To examine theinvolvement of TRPM2, we compared the release of cytokines inWT and TRPM2-deficient macrophages using ELISA of culturemedia. In these assays, macrophages were stimulated for 24 h at37 °C with the TLR2 agonist zymosan (50 μg/mL), which inducesNox activation and ROS production (27). ROS generation down-streamof TLR2 activation could causeCa2+ influx throughTRPM2sensitization, which in turn would enhance macrophage functions.As such, we focused on the cytokines regulated by NF-ĸB, whoseactivity is regulated by cytosolic Ca2+ levels (28). Macrophagestimulation with zymosan elicited the release of granulocyte col-ony stimulating factor (G-CSF), TNFα, IL-1α, IL-1β, macrophageinflammatory protein-2 (CXCL2), and monocyte chemotacticprotein-1 (CCL2) (Fig. 6 A–F). Among them, release of G-CSF,CXCL2, and IL-1αwere significantly reduced in TRPM2-deficientmacrophages compared withWT cells. IL-1β release tended to belower in TRPM2-deficient macrophages without statistical sig-nificance (P=0.10; values were 163± 43 and 65± 20 pg/mL) (Fig.6E). These data suggest that TRPM2-mediated pathways, in-cluding ROS-induced sensitization, contribute to the increasedrelease of G-CSF, CXCL2, IL-1α, and possibly IL-1β. ConsideringTRPM2 sensitization, temperature elevation should affect H2O2-evoked [Ca2+]i increases in macrophages. Indeed, temperatureelevations as small as 1.3 °C enhancedH2O2 (30 μM)-evoked [Ca2+]iincreases (Fig. 6G). These data indicate that sensitized-TRPM2channel function can be further enhanced by temperature eleva-tion, suggesting that redox signals and fever can act cooperativelyon macrophage functions via TRPM2. To examine the effectsof small temperature increases on other macrophage functions,we examined phagocytic activity at normal (37 °C) and febrile(38.5 °C) temperatures (2). Phagocytosis was significantly in-creased at 38.5 °C compared with 37 °C in WT macrophages,

Fig. 2. H2O2 reduced the temperature threshold for TRPM2 activation. (A)Representative traces of temperature-response profiles in heat stimulationwithout H2O2 (Top) or after H2O2 treatment at 100 μM (Middle) or 3 mM(Bottom) for 1 min. (B) Averaged data for heat stimulation without and afterH2O2 treatment at 100 μM or 3 mM for 1 min and at 60 μM for 1, 3, and 5min. Mean ± SEM (n = 5–8). ###P < 0.001 vs. H2O2-untreated; ***P < 0.001between indicated pairs (ANOVA). (C) A heat-evoked current after 1 minexposure to pipette solution containing 100 μM H2O2 (Top) obtained bywhole-cell recording. Representative Arrhenius plot traces are shown fortemperature vs. density of heat-evoked currents with 100 μM H2O2 (Middle,using upper trace) or 3 mM (Bottom) after 1 min exposure. 2-APB, a TRPM2inhibitor. (D) Averaged data for whole-cell recordings of heat-evoked cur-rents after H2O2 treatment at 100 μM or 3 mM for 1 min. Data are mean ±SEM (n = 10 or 11). *P < 0.05 (t test).

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whereas TRPM2-deficient macrophages showed no such tem-perature-dependent effect (Fig. 6H). These data suggest thatTRPM2 could mediate the enhanced phagocytic activity atelevated temperatures.

DiscussionIn the present study, we have identified a unique mechanism for“sensitization” of TRPM2. In the absence of H2O2 treatment, thetemperature threshold for TRPM2 activation remained at supra-physiological temperature levels, whereas H2O2 treatment low-ered the threshold to physiological temperatures. Our presentresults differ somewhat from those of our previous study, whichshowed that TRPM2 was activated by heat alone at around bodytemperature and maximal single-channel openings occurred at∼36 °C (10). However, in that study, to obtain sufficient currentsize, HEK293 cells were cultured for longer periods (more than 36h) after transfection, compare with only 20–36 h in the presentstudy. These different culture conditions might have affected thesensitivity to heat stimulation.Although TRPM2 is activated by ROS and is involved in cell

death after oxidative stress (15, 16), the activation mechanismsinvolved are unclear (17–20). The primary activator of TRPM2 isthought to be ADPR, with most previous studies suggesting thatthe release of ADPR from the nucleus and mitochondria playsa primary role in TRPM2 activation by H2O2 (17–19), althoughone study has reported that H2O2 acts on TRPM2 directly (20).We show here that H2O2 can sensitize TRPM2 in the absence ofthese organelles and activate it at physiological temperatures in

a membrane-delimited manner by reducing the temperaturethreshold for activation. We also found that PARP inhibitionattenuates H2O2-evoked reductions in the temperature threshold.Nevertheless, we believe thatADPRparticipation ismodest, giventhat single-channel activation is induced at ∼37 °C after H2O2treatment (Fig. 3A), and that the PARP inhibitor used might havean additional effect in this system. These results suggest that H2O2causes TRPM2 sensitization through two different mechanismsin parallel, which might explain the different proposals for theaction of H2O2 on TRPM2 (17–20). Along with PARP-dependentADPR release from the nucleus, ADPR release from mitochon-dria is also reportedly involved in TRPM2 activation by H2O2.Although our results using intact cells cannot rule out mitochon-drial involvement, the fact that H2O2-evoked activation ofTRPM2 at physiological temperature is more rapid (Fig. S2) thanTRPM2 currents mediated by ADPR released from mitochon-dria (18) make its participation less likely. Interestingly, TRPM2

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-outconfiguration. Magnified traces (a and b) correspond to the currents shownby the arrows in the left trace. (B and C) Heat-evoked responses of TRPM2were sensitized by chloramine-T, a membrane-permeant oxidant that pref-erentially oxidizes Met residues, in both whole-cell (B) and inside-out single-channel (C) recordings, where the magnified traces (a and b) correspond tothe currents shown by the arrows in the above trace. (D and E) 5,5′-Dithiobis-2-nitrobenzoic acid, a membrane-impermeant Cys-specific oxidant, did notsensitize the heat response of TRPM2 in either whole-cell (D) or inside-outsingle-channel (E) recordings.

Fig. 4. Structural basis for TRPM2 sensitization by H2O2. (A) Putativemembrane topology of TRPM2, with the Met residues conserved betweenmouse and human TRPM2 indicated. Mutations to Ala were introducedat the Met residues indicated by red circles. (B–D) Reduction in the tem-perature threshold for TRPM2 activation by H2O2 treatment (100 μM for1 or 3 min) was completely abolished in the M214A mutant. Shown aretemperature-response profiles of heat-evoked [Ca2+]i increases observed inDsRed(+), WT (B), or M214A-expressing cells (C) and nonexpressing DsRed(−) cells (D). Data are mean ± SEM (n = 4 or 5). P < 0.05, Student t testor ANOVA, unless noted otherwise, between without and after treatmentwith H2O2. NS, not significant (ANOVA). (E ) Sensitization of WT TRPM2 inthe presence (+) or absence (−) of PJ-34. In the PJ(+) groups, the potentPARP inhibitor PJ-34 (1 μM) was present during the entire experiment.Data are mean ± SEM (n = 4–8). *P < 0.05; **P < 0.01; ***P < 0.001 vs.0 min (ANOVA).

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sensitization was completely abolished by a single mutation atMet-214 (M214A) (Fig. 4) and a corresponding mutation in hu-man TRPM2 (M215A) (Fig. S5C), strongly supporting the im-portance of this Met residue in TRPM2 sensitization. Previousstudies on a TRPM2 splice variant with diminishedADPR-evokedactivation also support our idea that ADPR-independent sensiti-zation of TRPM2 occurs through Met oxidation. Unexpectedly,the M214 mutant affected TRPM2 activation by ADPR, eventhough 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 oxida-tion is involved in regulation of the temperature threshold for ac-tivation of thermosensitive TRP channels (thermo-TRPs). Amongthe known thermo-TRPs, TRPV1 was found to exhibit a reductionin temperature threshold through serine phosphorylation by pro-tein kinasesA andConproinflammatorymediator production (30,31). Thus, the temperature thresholds for activating thermo-TRPsmight be regulated by various mechanisms depending on thechannel and its cellular environment.Sensitization of TRPM2 was found to be involved in macro-

phage functions. Pathogen-associated molecular patterns of in-vading microorganisms can activate the TLR pathway, leading toproduction of ROS for microbicidal activity. Among the producedROS, H2O2 has weaker reactivity and higher membrane perme-ability, allowing it to diffuse over long distances (32), thus makingH2O2 a suitable signaling molecule (33). H2O2 concentrations arereported to reach mM levels within phagosomes (34) and 10–100μM in inflammatory environments (35), which would be sufficientto sensitize TRPM2. We first hypothesized that TRPM2 activitycould enhance cytokine release, given that intracellular Ca2+

activates NF-kB (28), which in turn regulates the expression ofvarious cytokines (36). However, we found that the loss of TRPM2affected the release of cytokine subsets. One possible explanationfor this finding is that direct regulation of transcription factors byredox signals (37) leads to complex changes in cytokine release.Other mechanisms lying downstream of TRPM2 activity could beinvolved as well, given that cytokine release is not caused simplyby effects on transcription regulation. For example, the calcium-

Fig. 5. Sensitization is observed in WT macrophages, but not in TRPM2-deficient cells. (A and B) H2O2-induced sensitization of heat-evoked [Ca2+]iincreases was observed in WT (A), but not in TRPM2-deficient (B) macro-phages. (C and D) H2O2-induced sensitization of heat-evoked current wasobserved in WT (C), but not in TRPM2-deficient (D) macrophages. (C, Inset) Amagnified trace corresponding to the red box shown in the upper trace.

Fig. 6. Zymosan-induced cytokine release and phagocytic activity in WTand TRPM2-deficient macrophages. (A–F) Amount of released cytokinesfrom unstimulated and zymosan (50 μg/mL)-stimulated macrophages fromWT and TRPM2-deficient mice. Data are mean ± SEM (n = 4–5). *P < 0.05;**P < 0.01 (ANOVA). (G) A small temperature elevation caused furtherenhancement of H2O2 (30 μM)-induced [Ca2+]i increases in WT macrophages.Mean values for the normal and elevated temperatures are reported asmean ± SD. The lower panels show pseudocolor images of fluorescenceintensity corresponding to the time points in the upper trace (a–c) anda phase-contrast image. Colored wedges in the phase-contrast image in-dicate the cells corresponding to each colored ratio trace. (H) Enhancementof phagocytic activity by elevated temperature (38.5 °C) was abolished inTRPM2-deficient macrophages. The ratio of cells that phagocytized zymo-san particle(s) was normalized to the average values at 37 °C in each ge-notype. Data are mean ± SEM (n = 3). *P < 0.05; NSP > 0.05 for 37 °C vs.38.5 °C (Student t test).

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dependent proteases calpains are involved in the maturation andrelease of IL-1α (38) aswell as in phagocytosis (39).A recent in vivostudy reported significantly enhanced susceptibility to Listeriamonocytogenes in TRPM2-deficient mice (40), which can be partlyexplained by the impaired macrophage functions observed in thepresent study.Of note, TRPM2 is expressed by lymphocytes, neutrophils, and

monocytes/macrophages (3–8), whose activities have a strongrelationship with body temperature (2, 41). This suggests thatTRPM2 might have a broader role in the temperature sensitivityof the immune system. Fever or hyperthermia is a widely con-served phenomenon involved in host defenses against infections inboth endotherms and ectotherms (42, 43) and is considered toenhance immune reactivity (2). Thus, fever is considered a bene-ficial response in host defenses, but the underlying mechanismremains unclear. Given that TRPM2 is conserved among a widerange of species (44) and is thought to be widely expressed inimmunocytes, ROS-sensitized TRPM2 can act as a thermosensorto regulate immune reactivities at body temperatures ranging fromnonfebrile to febrile. Redox signals are also known to affect Ca2+release from Ca2+ stores (37), suggesting that ROS can regulateCa2+ signals in various ways. TRPM2 could play a part in thisregulatory system, as suggested by a recent report demonstratingnegative regulation of ROS by TRPM2 (9). Our findings suggest

that the study of TRPM2 sensitization might identify uniqueapproaches for determining the physiological function of TRPM2that focus on body temperature and redox signals.

Materials and MethodsHEK293T cells transfected with cDNAs or peritoneal macrophages preparedfrom female C57BL/6NCr and TRPM2-deficient mice (7) were used for Ca2+

imaging with fura-2 and patch-clamp recordings to study TRPM2-mediatedchannel properties. Zymosan-evoked cytokine release and phagocytic ac-tivity were compared in WT and TRPM2-deficient macrophages. Data arepresented as mean ± SEM or mean ± SD. Statistical analysis was performedusing the Student t test or ANOVA, followed by the Bonferroni-type mul-tiple t test. P values < 0.05 were considered significant. Synthetic oligonu-cleotide primers constructing specific mutations and splice variant are shownin Table S1. A detailed description of the experimental procedures is pro-vided in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Drs. Shin-ichiro Saitoh (Tokyo University),Masatsugu Ohora (Tokyo Medical and Dental University), Yoshihiro Kubo,and Masaki Fukata (National Institute for Physiological Sciences) for theirhelpful advice. This work was supported by grants from the JapaneseMinistry of Education, Culture, Sports, Science and Technology (to M.T.) andthe Mitsubishi Foundation (to M.T.); and by a postdoctoral fellowship fromthe Japan Society for Promotion of Science postdoctoral fellowship (to M.K.).

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