Transient receptor potential melastatin 2 is a Ca2+-permeable cation channel abundantly expressed in macrophages. Trpm2−/− mice showed exacerbated infection and mortality during polymicrobial sepsis, which is associated with inefficient bacterial killing in macrophages. However, the mechanism of transient receptor potential melastatin 2 regulating bacterial killing remains unknown.
Trpm2−/− mice were intraperitoneally injected with Escherichia coli. The survival rate (n = 21) and bacterial burden (n = 5) were assessed. The processes of phagosome maturation and phagosome–lysosome fusion in peritoneal macrophages were extensively studied. The impact of increasing intracellular Ca2+ concentration on bacterial clearance in macrophages (n = 3) and on survival rate of Trpm2−/− mice infected with E. coli (n = 21) was investigated.
Trpm2−/− mice exhibited increased mortality (85% vs. 54%; P < 0.01) and aggravated bacterial burden during E. coli sepsis. Trpm2−/− peritoneal macrophages infected with E. coli showed dampened recruitment of lysosomal-associated membrane protein 1 and impaired phagosome maturation evidenced by a decrease in the accumulation of early endosome antigen 1, whereas a normal acquisition of Ras-related protein in brain 5. Increasing the cytosolic Ca2+ concentration in Trpm2−/− peritoneal macrophages via ionomycin treatment facilitated early endosome antigen 1 recruitment to Ras-related protein in brain 5 and phagosomal localization of lysosomal-associated membrane protein 1 and consequently enhanced bactericidal activity. Adoptive transfer of ionomycin-treated Trpm2−/− peritoneal macrophages improved bacterial clearance and survival (67% vs. 29%; P < 0.01) in Trpm2−/− mice challenged with E. coli.
Transient receptor potential melastatin 2 plays a critical role in host defense against invading bacteria via promoting phagosome maturation through facilitation of early endosome antigen 1 recruitment.
Transient receptor potential melastatin 2 is a Ca2+-permeable channel expressed in macrophages, which is important in control of sepsis; however, its mechanism of action is unknown
In a study of Escherichia coli sepsis using transient receptor potential melastatin 2 (TRPM2) knockout mice, the TRPM2 channel facilitated increased cytosolic calcium and phagosome maturation, which in turn augmented bactericidal action and improved survival
SEPSIS is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.1 It remains the leading cause of death in critically ill patients worldwide, despite early and aggressive antibiotic treatments to control bacterial infection.2–5 Recent studies have revealed that septic patients have impaired immune responses to eradicate invading pathogens and suggest that improving host immunity might increase the survival of sepsis.6–9
The innate immune system acts as the first line of host defense against bacterial infection. One of the most important initial responses in innate immunity is the phagocytosis of pathogens by residing macrophages.10 During phagocytosis, the engagement of cell membrane receptors by cognate ligands on the surface of pathogens leads to their internalization into a membrane-bound vacuole named the phagosome.11 Nascent phagosomes lack the ability to kill engulfed pathogens. They then acquire new components through sequential fusions with endosomes, in a process described as “phagosome maturation.”12,13 Maturing phagosomes ultimately fuse with lysosomes to kill internalized microorganisms.13 Because phagocytosis depends on a network of endocytic vesicles to deliver cargo from nascent phagosomes to lysosomes for degradation, maintaining a normal phagolysosome fusion process is especially critical for bacterial killing in septic patients and may provide targets for developing new therapeutic strategies for sepsis.
The transient receptor potential melastatin-2 (TRPM2) channel is a nonselective, Ca2+-permeable cation channel with unique gating properties conferred by a functional adenosine diphosphoribose hydrolase domain in its C terminus.14,15 Previous studies reported a ubiquitous expression of functional TRPM2 channels in immunocytes, particularly in cells of the monocytic lineage.15,16 The expression level of TRPM2 in human monocytes can be further increased upon exposure to bacterial components and their synthetic analog such as lipopolysaccharide and Pam3-CysSK4.17 The TRPM2 channel contributes to multiple cellular functions including cytokine production, insulin release, cell motility, and cell death.17–21 It is required for the lipopolysaccharide-induced production of interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor-α in THP-1 monocytes (a human monocytic leukemia cell line).17 TRPM2-deficient macrophages showed impaired function in terms of cytokine release and chemokine production upon H2O2 stimulation.18 A study has also shown that loss of TRPM2 attenuated phagocytic ability in resident macrophages.20 Moreover, we recently reported that nonsurvivors of septic patients had much lower TRPM2 levels in monocytes than those survived and nonseptic controls.22 Trpm2−/− mice experienced aggravated bacterial burden and worsened survival during polymicrobial sepsis, which may result from impaired intracellular bactericidal activity rather than from impaired uptake in macrophages, as demonstrated using bone marrow–derived macrophages.22 Thus, we deduced that disruption of TRPM2 may compromise the normal process of phagolysosome fusion in macrophages.
In the current study, we further tested whether disruption of TRPM2 impacted the outcome of Escherichia coli–induced peritoneal sepsis. We then investigated in detail how TRPM2 disruption affected the process of phagolysosome fusion in peritoneal macrophages. Finally, we explored whether increasing intracellular Ca2+ in Trpm2−/− macrophage could restore its bactericidal activity and rescue mice from lethal sepsis induced by E. coli.
Materials and Methods
C57BL/6 mice were obtained from Experimental Animal Center of Zhejiang Province (Hangzhou, China). Trpm2−/− mice were kindly provided by Dr. Yasuo Mori (Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan) and were backcrossed to the C57BL/6 stain for more than 12 generations. Trpm2−/− mice and wild-type littermates (Trpm2+/+) were housed in pathogen-free cages with free access to water and food. All animal experiments were approved by the Institutional Animal Care and Use Committees of Zhejiang University (Hangzhou, China).
To elicit primary peritoneal macrophages, mice were treated with 2.0 ml thioglycollate medium (EMD Chemicals, Germany), 3%, injected intraperitoneally (ip). After 72 h, cells were isolated by peritoneal lavage and cultured in Dulbecco Modified Eagle Medium (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco). In some experiments, the primary macrophages were treated with 2 μM ionomycin or vehicle for 5 min in the presence of extracellular Ca2+ (2 mM) before they were applied in further studies.
Infection with E. coli
Trpm2−/− and Trpm2+/+ mice were challenged with ip injection of 2 × 107 colony-forming units (CFU) of E. coli (ATCC 25922). After 2, 6, or 12 h, peritoneal cavity was lavaged with sterile phosphate-buffered saline (PBS) and the remained E. coli in the peritoneal lavage fluid were detected. In some experiments, mice were treated in the peritoneal cavity with primary macrophages (5 × 105 cells) that had been preincubated with ionomycin or vehicle (dimethyl sulfoxide), 2 h before E. coli challenge. Survival rates were monitored for 72 h. The mice were randomly assigned to experimental groups throughout the study, and all further experiments were blinded to murine genotype and treatment.
Bacterial Killing Assay
Primary peritoneal macrophages were counted and cultured in a 24-well plate (Corning, USA) at an approximate density of 5 × 105 cells per well. Two h later, nonadherent cells were removed by washing twice with PBS. The remaining cells were cultured for another 12 h for next experiments. To determine bacterial killing ability, 5 × 106 CFUs of E. coli were added in the well. After centrifuging at 300g for 1 min, the cells were incubated at 37°C for 1 h. Then the medium was changed to Dulbecco Modified Eagle Medium (Gibco) containing 100 μg/ml gentamycin for additional culture. After washing with ice-cold PBS, the cells were lysed with 0.3% (vol/vol) Triton X-100 for 5 min. Cell lysates were serially diluted with PBS and inoculated on Luria broth agar plates. Bacterial CFUs were counted after incubation at 37°C for 16 h.
Fluorescent polystyrene microspheres (Life Technologies, USA) were added into primary macrophages at multiplicity of infection of 10:1 and incubated at 37°C for 1 h. After three washes with PBS, fluorescence was observed under a fluorescence microscope.
Transmission Electron Microscopy
Peritoneal macrophages isolated from experimental mice were fixed in 2.5% glutaraldehyde and postfixed in aqueous 1% OsO4 followed by 2% uranyl acetate. After ethanol and propylene oxide dehydration and embedding in polybed 812 resin, ultrathin (80-nm) sections were poststained with 2% uranyl acetate followed by 0.3% lead citrate. Sections were imaged using a Hitachi H-7650 transmission electron microscope at 80 kV. The micrographs were magnified at ×40,000.
Western Blot and Immunoprecipitation
Western blot was performed as described previously.22 Briefly, the proteins from lysed cells were added into a 12% bis-tris polyacrylamide gel (Novex, USA) and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Afterward, the proteins were transferred to a polyvinyl difluoride membrane (Millipore, USA), blocked using Tris-buffered saline with 0.1% Tween-20 (Sigma-Aldrich, USA) containing 5% nonfat dry milk for 1 h, and incubated with corresponding primary and secondary antibodies. The specific bands were detected by enhanced chemiluminescence solution (Thermo Scientific, Inc., USA) and Kodak film (Kodak, USA).
For immunoprecipitation, cells were lysed with 1% NP-40 lysis buffer containing protease inhibitors (Beyotime Biotechnology, China). After centrifugation, the supernatants were incubated with the related primary antibody overnight and then Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, USA) at 4°C for 5 h. After this, the immunocomplexes were washed and analyzed using Western blot.
The phagosome isolation was performed according to the previously published method.23 Plated macrophages were incubated with 3-μm latex beads (Sigma-Aldrich) coated with a crude preparation of E. coli outer membrane extracts. Cells were then scraped with 30% (w/v) sucrose buffer (Sigma-Aldrich) overlaid with a linear 30 to 0% sucrose gradient, followed by centrifugation at 270,000g for 45 min. An equal number of bead containing phagosomes were loaded for each condition for Western blot analysis.
Lysosome isolation from subcellular fractionation of primary macrophages was performed with a lysosome isolation kit (Sigma-Aldrich) according to the manufacturer’s manual. After a discontinuous iodixanol gradient centrifugation using Optima MAX-XP Benchtop Ultracentrifuge (Beckman Coulter, USA) with MLS-50 rotor at 150,000g and 4°C for 4 h, the sample was divided into nine fractions (0.5 ml each) for further Western blot analysis.
Primary peritoneal macrophages were cultured in a 96-well black plate with clear bottom (Corning) and treated with pHrodo-conjugated E. coli (Life Technologies). After three washes in cold PBS, the plate was inserted into a SpectraMax M5 (Molecular Devices, USA) and analyzed immediately. Values were compared with a standard curve obtained by resuspension of the cells for 2 h at a fixed pH in PBS containing 0.1% (vol/vol) Triton X 100.23
After 4% paraformaldehyde fixation and blocking with 5% bovine serum albumin in PBS/0.3% Triton X-100 for 30 min, cultured cells were incubated with primary antibodies for 2 h at room temperature. After washing with PBS, the cells were incubated with goat anti-rabbit Alexa Fluor 488 or goat anti-rat Cy3 (Millipore) for 1 h at room temperature, followed by staining with 0.5 µg/ml 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) in PBS for 5 min. Isolated phagosomes were fixed with 70% cold methanol and 30% cold acetone and then immunostained as described in Immunofluorescent Analysis (“incubated with primary antibodies for 2 h at room temperature…”). All samples were imaged on a Nikon A1 confocal microscope outfitted with a Plan-Apochromat ×60 1.4NA oil immersion objective (Nikon, USA). Data were collected using Nikon software NIS-Elements viewer 4.20 and processed in Image J and Photoshop CS6 (Adobe, USA).
Acridine Orange and Lysotracker Staining
Primary peritoneal macrophages were grown in glass-bottomed dishes (Nest Biotechnology, China) and incubated with 5 µg/ml acridine orange (Life Technologies) or 50 nM LysoTracker Red DND-99 (Life Technologies) labeling at 37°C for 15 min. Cells were stained with 0.5 µg/ml 4′,6-diamidino-2-phenylindole, washed three times with PBS, and then visualized using confocal microscopy.
Calcium Imaging with Fluo-3 AM
Primary peritoneal macrophages were loaded with 5 µM Fluo-3 AM (Dojindo Laboratories, Japan) premixed with Pluronic F-127 (Dojindo Laboratories) in Hank Balanced Salt Solution for 30 min at 37°C. Cells were then washed three times with Hank Balanced Salt Solution and incubated for another 20 min to allow complete deesterification of intracellular AM esters. After treatment with Tyrode solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose, and 10 mM HEPES) with or without 2 µM ionomycin for 5 min, the cells were imaged at 37°C using confocal microscopy.
Data are presented as mean ± SD. There are no data lost for the analyses. Comparisons of differences in continuous variables were conducted using an unpaired Student’s t test or ANOVA with Bonferroni post hoc test where appropriate. Survival curves (Kaplan–Meier plots) were analyzed using the log-rank test. The sample sizes used in the current study were based on the previous experience. Statistical analysis was performed using SPSS 20.0 (SPSS Inc., USA), Prism 6.0 (GraphPad Software Inc., USA) and Stata 11.0 (Stata Corporation, USA). Two-tailed value of P < 0.05 was considered statistically significant.
Disruption of TRPM2 Aggravated the Outcome of E. coli Sepsis and Decreased Bacterial Elimination in Peritoneal Macrophages
To investigate the role of TRPM2 in E. coli sepsis, the survival rate was first investigated. As shown in fig. 1A, most of the Trpm2−/− mice (85%) succumbed to sepsis within 36 h after E. coli infection, while 46% of the wild-type (Trpm2+/+) mice survived more than 72 h. Consistent with this and with our previous findings in the cecal ligation and puncture-induced sepsis model,22 the bacterial loads in peritoneal lavage fluid from Trpm2−/− mice at 2, 6, and 12 h after E. coli challenge were significantly higher than those from Trpm2+/+ mice (fig. 1B). These results indicate that the antibacterial immune response is dampened in Trpm2−/− mice.
Our previous study found that TRPM2 was a key factor involved in antimicrobial immune defense in bone marrow–derived macrophages. Thus, we evaluated the role of TRPM2 in bactericidal activity in the primary peritoneal macrophages. The gentamicin protection assay showed that after infection with live E. coli for 2 and 6 h, there were around 2.5- and 4-fold more bacteria, respectively, left in Trpm2−/− peritoneal macrophages than in Trpm2+/+ peritoneal macrophages (fig. 1C). The defect in bacterial killing in Trpm2−/− peritoneal macrophages could not be attributed to a difference in bacterial uptake since intracellular bacterial burdens were comparable in Trpm2−/− and Trpm2+/+ peritoneal macrophages at 1 h after E. coli infection (fig. 1D). Furthermore, no difference was observed in the ingestion of fluorescent polystyrene microspheres between the two types of peritoneal macrophages (fig. 1E). In all, these findings suggest that TRPM2 participates in intracellular bacterial elimination by peritoneal macrophages.
Disruption of TRPM2 Affected Phagolysosomal Acidification
To determine whether Trpm2−/− peritoneal macrophages are indeed defective in bacterial killing, we first examined the ultrastructure of Trpm2−/− and Trpm2+/+ peritoneal macrophages using transmission electron microscopy. One hour after E. coli infection, phagosomes had formed normally in both Trpm2−/− and Trpm2+/+ peritoneal macrophages. However, the phagosomes in Trpm2+/+ peritoneal macrophages were partially degraded at 6 h after uptake, while they remained unchanged in Trpm2−/− peritoneal macrophages (fig. 2A). Since the degradative capacity of phagosomes is dependent on the optimal functioning of degradative enzymes facilitated by phagosomal acidification, we next monitored phagosomal pH using pHrodo-conjugated E. coli. The pHrodo dye is nonfluorescent at neutral pH, and its fluorescence intensity increases with an increased acidity. The maturation of E. coli–containing phagosome involves increasing acidity in the phagosomal compartment and therefore increased fluorescence intensity is detectable as phagosome maturation progresses. Indeed, there was no difference in phagosomal pH between Trpm2−/− and Trpm2+/+ peritoneal macrophages in the early stages of phagosomes (fig. 2B). In contrast, 60 min later, the phagosomal pH in Trpm2+/+ peritoneal macrophages was lower than that in Trpm2−/− peritoneal macrophages, and significant differences were observed at 80 and 100 min (fig. 2B). These findings suggest that phagosome acidification in the later stages of E. coli uptake was affected in Trpm2−/− peritoneal macrophages, which might result from a defect in either lysosomal acidification or phagosome–lysosome fusion.
It has been reported that TRPM2 not only localizes to the plasma membrane, but is also present in the lysosomal membrane.24,25 After purifying and separating lysosomes by an iodixanol gradient, we also confirmed that TRPM2 was enriched in the lysosome fractions (fig. 2C). Thus, we further investigated whether disruption of TRPM2 impacts lysosome function. We first checked the acidification of lysosomes in Trpm2−/− and Trpm2+/+ peritoneal macrophages using acridine orange. When acridine orange enters acidic lysosomes, the green fluorescence is converted to bright red fluorescence. No differences were observed in the number of red dots between Trpm2−/− and Trpm2+/+ peritoneal macrophages in steady-state conditions (fig. 2D). Since lysosomal enzymes require an acidic environment for their maturation and activities, maintenance of acidity is a hallmark required for functional lysosomes. We next investigated the maturation of the lysosomal hydrolase cathepsin D (CTSD). CTSD is synthesized in the endoplasmic reticulum as an inactive glycosylated propeptide, transported into endosomes, and then cleaved into an intermediate form which is further cleaved to form the mature enzyme in the acidic environment of mature lysosomes.26 Thus, any change in the mature form of CTSD reflects altered late endocytic trafficking or perturbed lysosomal acidification. As predicted, the expression levels of mature CTSD showed no difference between Trpm2−/− and Trpm2+/+ peritoneal macrophages with or without E. coli stimulation (fig. 2E). Moreover, lysosomal acidification was further assessed by staining the cells with another fluorescent dye, Lyso-Tracker Red DND-99. As shown in fig.2F, no differences in Lysotracker-positive dots were detected between Trpm2−/− and Trpm2+/+ peritoneal macrophages under steady-state conditions. When the macrophages were incubated with Alexa Fluor–conjugated E. coli, the majority of engulfed E. coli were contained inside phagosomes that efficiently fused with lysosomes in Trpm2+/+ peritoneal macrophages. However, in Trpm2−/− macrophages, only a minority of the E. coli–containing vacuoles acquired the Lysotracker dye (fig. 2G). Taken together, these data indicate that disruption of TRPM2 does not directly affect lysosome acidification, but might impact the fusion of lysosomes with E. coli–containing phagosomes.
Disruption of TRPM2 Impaired Phagosome–Lysosome Fusion
Proper fusion of E. coli–containing phagosomes with lysosomes is a prerequisite for efficient bacterial killing. Thus, we assessed whether phagolysosomal maturation was dysregulated in the absence of TRPM2. First, the primary peritoneal macrophages were treated with Alexa Fluor–conjugated E. coli and then colocalization of E. coli–containing phagosomes with lysosome marker lysosomal-associated membrane protein 1 (LAMP 1) was evaluated using confocal microscopy. In Trpm2−/− peritoneal macrophages, only 25% of the E. coli–containing phagosomes colocalized with LAMP 1, significantly lower than that in Trpm2+/+ peritoneal macrophages (66%; P < 0.0001; fig. 3A). Next, we evaluated LAMP-1 staining in isolated bead-containing phagosomes by fluorescence confocal microscopy. A significant reduction in the recruitment of LAMP 1 to phagosomes was also observed in Trpm2−/− peritoneal macrophages (fig. 3B). Consistent with this result, bead-containing phagosomes isolated from Trpm2−/− peritoneal macrophages had lower LAMP 1 content than those isolated from Trpm2+/+ peritoneal macrophages (fig. 3C). Furthermore, the expression level of CTSD was significantly decreased in bead-containing phagosomes isolated from Trpm2−/− peritoneal macrophages (fig. 3D). Thus, these findings indicate that the fusion of phagosomes with lysosomes is impaired in Trpm2−/− primary macrophages.
Disruption of TRPM2 Impeded Phagosome Maturation
It is well known that nascent phagocytic vacuoles should undergo maturation to acquire microbicidal and degradative activity. Maturation entails the orderly sequential fusion of the phagosomal vacuole with early and late endosomes to remodel its membrane and contents, a process that culminates in the formation of phagolysosomes. Thus, we next asked whether the impaired phagosome–lysosome fusion in Trpm2−/− peritoneal macrophages resulted from a damaged maturation process. As a member of the Ras-related protein in brain (Rab) family of small guanosine triphosphate hydrolases, Rab5 is important for mediating fusion conversion between the maturation stages.27 Rab5 is a characteristic of the early endosome and facilitates its fusion with phagosomes through the recruitment of endosomal early antigen 1 (EEA1),28,29 which is important for maturation and is required for the transition to the late endosomal stage identified by the presence of Rab7.13 As expected, the absence of TRPM2 led to sharp decreases in the distribution and expression of Rab7 on phagocytotic vesicles prepared from Trpm2−/− peritoneal macrophages (fig. 4, A and B). In the very early stage of phagosome maturation, phagosomes isolated from both Trpm2+/+ and Trpm2−/− peritoneal macrophages possessed similar abilities to activate Rab5. During phagosome maturation, phagosomes isolated from Trpm2+/+ peritoneal macrophages displayed a time-course–dependent decrease in the level of Rab5, which paralleled a similar gradual reduction in the level of EEA1. On the contrary, in Trpm2−/− peritoneal macrophages, a persistently high level of active Rab5 was detected in isolated phagosomes, which was accompanied by a steadily lowered level of EEA1 (fig. 4, C and D). Interestingly, in Trpm2−/− peritoneal macrophages, immunoprecipitation of Rab5 resulted in an obvious decrease in coimmunoprecipitation of endogenous EEA1 upon E. coli stimulation for 30 min (fig. 4E). These findings demonstrated that the interaction between Rab5 and EEA1 was impaired in Trpm2−/− peritoneal macrophages, due to the steadily lowered level of EEA1 in these cells.
Increasing Intracellular Ca2+ Facilitated Phagosome Maturation in Trpm2−/− Peritoneal Macrophages
Because monocytes isolated from Trpm2−/− mice are defective in intracellular Ca2+ levels upon receiving related stimuli,30 we asked whether increasing intracellular Ca2+ could restore bactericidal activity in Trpm2−/− peritoneal macrophages. Ionomycin is a calcium ionophore facilitating Ca2+ diffusion across cell membranes. Peritoneal macrophages were first cultured with medium containing Ca2+ in the presence of 2 μM ionomycin. Five minutes later, elevated intracellular Ca2+ influx was recorded using the calcium indicator Fluo-3 AM (Dojindo Laboratories; fig. 5A). Moreover, ionomycin treatment induced a strongly rising level of LAMP-1 in isolated phagosomes (fig. 5, B and C). Strikingly, increased intracellular Ca2+ facilitated EEA1 recruitment to Rab5 after E. coli infection, as manifested by coimmunoprecipitation analysis (fig. 5D). Consequently, the inefficient bactericidal ability of Trpm2−/− peritoneal macrophages was rescued after ionomycin treatment (fig. 5E). These observations indicate that TRPM2 promotes phagosome maturation by regulating intracellular Ca2+ current.
Adoptive Transfer of Ionomycin-Treated Trpm2−/− Peritoneal Macrophages Ameliorated E. coli Sepsis in Trpm2−/− Mice
To further ascertain the role of TRPM2 and intracellular Ca2+ in bacterial killing by macrophages, ionomycin-treated Trpm2−/− peritoneal macrophages were administered in vivo 2 h before E. coli challenge. Notably, ionomycin-treated macrophages significantly protected 67% of the mice against E. coli–induced sepsis in Trpm2−/− mice (fig. 6A). Furthermore, as expected, the bacterial burden in the peritoneal cavity was significantly lower than that in control mice at 12 h after sepsis onset (fig. 6B).
The current study confirmed that Trpm2−/− mice exhibited obviously increased mortality after ip infection with E. coli and failed to eradicate E. coli from the peritoneal cavity. Disruption of TRPM2 inhibited the recruitment of EEA1 to early endosomes and tethering with Rab5 on the membrane, which impaired phagosome maturation and subsequent phagolysosomal fusion and finally resulted in inefficient bacterial killing in peritoneal macrophages. Increasing cytosolic Ca2+ concentration could restore the bactericidal activity of Trpm2−/− peritoneal macrophages and thus improve the outcome of E. coli–induced abdominal sepsis in Trpm2−/− mice. These findings indicate that TRPM2 plays a vital role in the elimination of invading pathogens via regulating phagosome maturation in macrophages.
TRPM2 is a calcium-permeable nonselective cation channel widely expressed in immune cells and in tissues such as brain and lung.21 TRPM2 channels have been implicated in the development of various inflammatory diseases such as ulcerative colitis,18 myocardial ischemia/reperfusion injury,31 neuropathic pain,32 and autoimmune encephalomyelitis.33 Our previous study discovered the protective role of TRPM2 in controlling bacterial clearance during polymicrobial sepsis, possibly by regulating heme oxygenase-1 (HO-1) expression.22 However, HO-1 is an inducible stress-responsive enzyme whose inducibility is time dependent, which led us to speculation regarding the existence of other mechanisms mediating bacterial clearance before the stable expression of HO-1 during pathogen infection. Using tissue-resident peritoneal macrophages, the current study confirmed that TRPM2 did not participate in the uptake of E. coli, consistent with the previous finding observed in bone marrow–derived macrophages.22 Because the TRPM2 channel mediates Ca2+ influx into cell,32 the current finding is also in line with reports that particle ingestion is largely Ca2+ independent.
Although phagocytic ingestion appears to be largely Ca2+ independent, studies revealed that the process of phagosome maturation is regulated more stringently by cytosolic Ca2+ level.34,35 In particular, release of Ca2+ from lumens such as lysosomes is required for several steps in intracellular trafficking, including fusion and fission events.36,37 The TRPM2 channel is also found in mammalian lysosomes and functions as an important lysosomal Ca2+-release channel.24,25 Indeed, the expression of TRPM2 was observed in lysosomes of primary peritoneal macrophages. However, the absence of TRPM2 did not directly impact lysosomal function, as demonstrated by the fact that neither acidification nor mature CTSD enzyme was impacted in lysosomes from Trpm2−/− peritoneal macrophages. Therefore, inefficient bacterial killing in Trpm2−/− peritoneal macrophages cannot be attributed to disabled lysosomal digestive ability.
Phagosome degradation of engulfed bacteria requires the precise fusion of lysosomes with phagosomes. The level of the lysosomal membrane protein LAMP-1 was markedly decreased in phagosomes of Trpm2−/− peritoneal macrophages, indicating a direct role for TRPM2 in phagolysosomal fusion. One reason for this trafficking problem is due to a defect in phagosome maturation in Trpm2−/− peritoneal macrophages. Notably, the phagosome maturation process that endows the phagosome with lytic activity depends critically on the interaction of the nascent vacuole with endocytic compartments. By analogy with the endocytic pathway, phagosome maturation is segregated into early, late, and lysosome-interacting stages.13,38 EEA1 is a membrane-tethering factor required for the fusion and maturation of early endosomes and a core component of endosome docking through interaction with the early endosome-specific small guanosine triphosphate hydrolase Rab5.28,39 In the current study, phagosomes isolated from Trpm2−/− peritoneal macrophages showed decreased levels of EEA1 but persistent levels of Rab5 after E. coli infection. The reduced recruitment of EEA1 to Rab5 in the early endosome-interacting stage subsequently led to an impaired interaction with the late endosome, as evidenced by a lower level of Rab7, and finally hindered the normal fusion of the phagosome with the lysosome. Thus, the disabled bactericidal activity in Trpm2−/− peritoneal macrophages was attributed to a loss of EEA1 recruited for the fusion of phagosomes with early endosomes.
Ca2+ is a ubiquitous second messenger that controls multiple processes in immune cells, including chemotaxis and adhesion.40 Ca2+ is also an essential requirement in membrane fusion, acting through binding proteins such as calmodulin.41 Lawe et al.42 have shown that the inhibition of Ca2+/calmodulin could profoundly inhibit EEA1 binding to endosomal membranes. Mills et al.43 have reported a Ca2+-dependent manner for calmodulin binding to the IQ domain of EEA1 to promote endosome fusion. Because disruption of TRPM2 led to a diminished level of cytosolic Ca2+, our results also indicate that increasing the intracellular Ca2+ concentration could restore the recruitment of EEA1 for the fusion of phagosomes with the endocytic pathway in Trpm2−/− peritoneal macrophages. These processes ultimately enhance intracellular bacterial killing of the macrophages, leading to improved outcomes in Trpm2−/− mice suffering from E. coli sepsis. These findings demonstrate that by regulating the intracellular Ca2+ concentration, TRPM2 controls the process of phagosome maturation and ultimately impacts the bactericidal activity of professional phagocytes such as macrophages. Moreover, in septic patients, nonsurvivors showed much lower TRPM2 levels in monocytes/macrophages, which may compromise immune defense against invading pathogens in these patients.22 Thus, the current findings provide a new therapeutic strategy for modulating intracellular Ca2+ levels in order to improve the innate immune response in critically ill septic patients, which is valuable for further investigation.
In summary, the current study found that TRPM2 regulates phagosome maturation via modulating the cytosolic Ca2+ concentration and is required for bacterial clearance in E. coli sepsis. These findings not only provide new insight into the role of TRPM2 in the pathogenesis of sepsis, but also offer new therapeutic direction for sepsis and other infectious diseases.
Supported by programs (Nos. 81471838 and 81130036) from the National Natural Science Foundation of China, Beijing, China; Research Fund for the Doctoral Program of Higher Education of China (No. 20130101110147), Beijing, China; and the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (No. 2012BAI11B05), Beijing, China.
The authors declare no competing interests.