TRPM2 deficiency contributes to M2b macrophage polarization via the PI3K/AKT/CREB pathway in murine sepsis

Mice

TRPM2 knockout(trpm2^−/−) mice were bred at the Animal Care Centre (Zhejiang University, Hangzhou, China), and age-matched male wild-type (C57BL/6J) mice were purchased from SLAC Biotechnology (Shanghai, China). All the animals were housed in specific pathogens free-facilities, with unrestricted access to food and water, and maintained on a 12-h light/dark cycle. The male mice were aged 7 to 9 weeks, weighted between 22 and 30 grams. This study adhered to the ARRIVE guidelines (https://arriveguidelines.org), and all the animal experiments were approved by the Ethics Committee of Zhejiang University, in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (No. ZJU20210125).

Sepsis model

The cecal ligation and puncture (CLP) model was generated following a previously established protocol. ¹⁷ In brief, mice were anesthetized using intraperitoneal injection of 80 mg/kg pentobarbital, and a 1-cm midline laparotomy incision was made to expose the cecum. The cecum was then ligated and punctured with a 21-gauge needle at a point equidistant between the distal pole and the ileocecal junction, followed by gentle squeezing to expel a small quantity of fecal material. After repositioning the cecum, the abdominal cavity was closed. Mice in the sham CLP group underwent the same procedure, excluding the ligation and puncture steps. In the CLP + IPI-549 group, 1 mg/kg IPI-549 (Selleck, Houston, USA) was administered 2 h before the CLP operation. After the surgical procedure, each mouse received 1 ml of normal saline subcutaneously. The survival rate was monitored over a period of 7 days, and other in vivo experiments were performed 48 h after the procedure. At the end of experiment, euthanasia was performed by inhalation of carbon dioxide, followed by cervical dislocation and execution. To minimize bias, all further experiments were conducted with blinding to murine genotype and treatment.

Cell culture

Bone marrow-derived macrophages (BMDMs) were isolated from both the femurs and tibias of WT and trpm2^−/− mice and then cultured in Dulbecco's modified Eagle medium (DMEM) (Cellgro, Corning, USA) supplemented with 20% heat-inactivated fetal bovine serum (FBS) (Cellgro, Corning, USA), 5% penicillin and streptomycin, and 20% granulocyte-macrophage colony-stimulating factor (GM-CSF) (acquired from the L929 culture medium). The cells were counted with a hemocytometer and seeded into a 10-cm dish at a density of 6.0 × 10⁶ cells per dish. 6–7 ml old medium was discarded and replaced with fresh complete DMEM medium (20% FBS, 5% penicillin and streptomycin) with 20% GM-CSF on days 3 and 5. On day 7, adherent cells were harvested using Trypsin-ethylenediaminetetraacetic acid (EDTA) (0.25% trypsin and 0.02% EDTA, Keyi Co., Ltd, China) for a short duration (≤5 min at 37°C), then collected into 15 ml tubes and centrifuged at 1200×g for 5 min. The supernatant was removed, and the cells were resuspended and seeded into both 6-well and 24-well plates, then incubated overnight at 37°C in a 5% CO₂ incubator. Then, the cells were treated with lipopolysaccharide (LPS) or Escherichia coli (E.coli ）for the subsequent experiment.

Bacterial killing test

BMDMs were seeded into a 24-well plate at a density of 2 × 10⁵ cells/well, and then incubated at 37°C for 2 h after adding E. coli (DH5ɑ) with multiplicity of infection(MOI) of 20. The cells were washed twice with phosphate buffered saline(PBS), and then gentamicin (100 μg/ml) was added to the medium to eliminate extracellular bacteria. After 6 h, the cells were re-washed with PBS and lysed with 500 μl of 0.1% Triton X-100 for 5 min. The cell lysates were serially diluted with 1× PBS, cultured on Luria broth agar plates, which were incubated for another 12 h at 37°C to determine the bacterial killing capacity by counting the colony-forming units (CFUs). For some experiments, the cells were pretreated with IPI-549 for 15 min. After the incubation periods, the supernatant was removed, and the cells were washed for subsequent experiments.

Peritoneal macrophage isolation and peritoneal lavage fluid (PLF) preparation

After 48 h of CLP, euthanasia was performed on the mice. A 25-gauge needle, attached with a vascular clamp, was inserted into the peritoneal cavity, and 5 ml cold 1× PBS was infused. The entire body was gently shaken for 10 s, and then the peritoneal lavage fluid (PLF) was collected. After centrifugation, the supernatant was discarded. The pellet was resuspended and plated in complete DMEM supplemented with 10% FBS (Cellgro, Corning, USA) and 5% penicillin/streptomycin. After 2 h of incubation in a 37°C, 5% CO₂ incubator, the supernatant was discarded, and the adherent cells (considered to be macrophages) were washed twice with 1 × PBS. The macrophages were then harvested for Western blot experiments.

Determination of bacterial burden

The protocol for determining the bacterial burden was described in our previous study. ¹⁷ PLF was collected according to the above method, and 100 μl of PLF was plated on tryptic soy agar plates. The plates were incubated at 37°C for 12 h, after which CFUs were counted. The bacterial burden is presented as CFU per milliliter of PLF.

Hematoxylin-eosin (HE) staining and tissue histological analysis

Lung and liver tissues were fixed in 4% paraformaldehyde for 48 h and then embedded in paraffin after dehydration and clarification. Four μm thick slices were prepared and stained with H&E staining solution (H&E solution; Nanjingjiancheng, China). The slides were examined under a VS 200 digital slide scanner (Olympus, Tokyo, Japan). A scoring system was used to evaluate lung injury based on indicators such as capillary congestion, alveolar congestion, leukocyte infiltration, and alveolar wall thickness: 0 = normal, 1 = mild injury (<25%), 2 = moderate injury (25–50%), 3 = severe injury (50–75%), 4 = very severe injury (>75%). ²¹ Liver damage was assessed using a similar scale based on necrosis, characterized by loss of architecture, vacuolization, and karyolysis: 0 = normal, 1 = mild injury, 2 = moderate injury, 3 = severe injury, 4 = complete necrosis. ²²

Western blot

BMDMs and peritoneal macrophages were lysed with radioimmunoprecipitation assay buffer (Boster, China) containing PhosSTOP (Roche), protease inhibitor cocktail (Roche) and phenylmethylsulfonyl fluoride (Sigma). Protein concentrations were determined, and 40 μg of protein was loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were separated and transferred to nitrocellulose membranes (BioTrace) for 90 min at 300 mA using a Mini-protein II system (Bio-Rad). Later, the membranes were blocked with tris-buffered saline with 0.1% Tween20 detergent (TBST) containing 5% bovine serum albumin for 1 h at room temperature, followed by overnight incubation at 4°C with specific primary antibodies. Details of the primary antibodies are as follows:

Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) (1:5000, db106, Digbio, China),

Afterwards, the membranes were washed six times with 1× TBST and incubated with the appropriate secondary antibodies, IRDye 800CW goat anti-rabbit or IRDye 680CW goat anti-mouse (1:5000, LI-COR Biosciences, USA), for 2 h. The protein bands were visualized using a LI-COR Odyssey infrared imaging system (LI-COR, USA).

Quantitative real-time polymerase chain reaction (qRT–PCR)

Total RNA was isolated from BMDMs and peritoneal macrophages using isoPlus (9109, Takara, Japan). The extracted RNA was reverse transcribed to first-strand cDNA. SYBR Green Master Mix reagent (Takara, Japan) was used for qRT–PCR analysis. GAPDH was used as a normalization control. The PCR mixture consisted of 4.5 μl of cDNA, 5 μL of 2× SYBR Green mix, 0.25 μ of forward primer and reverse primer. The qRT–PCR process was performed under the following thermal cycler program: 95°C for 10 min; 45 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s. All the primers used in this study were synthesized by Sangon Biotech (Shanghai, China). The sequence of the primers listed in Table 1.

Enzyme-linked immunosorbent assay (ELISA)

To determine the levels of cytokines and chemokines, the supernatants from the BMDMs and peritoneal macrophage culture medium were collected. The level of secreted cytokines were detected using ELISA kits. Specifically, tumor necrosis factor alpha (TNF-α), interleukin10 (IL-10), interleukin 6 (IL-6), interleukin12 (IL-12), and C-C motif chemokine ligand 1 (CCL1) were measured using kits from various manufacturers as follows: TNF-α (DY410), IL-10 (DY417) and IL-6 (DY406) from R&D Systems, Minneapolis, MN, USA; IL-12 (431604) from BioLegend, USA; and CCL1 (L210919515) from Cloud-Clone Corp., China. The measurements were done in accordance with the manufacturers instructions, and the optical density was measured at 450/570 nm. The optical density values were normalized to the blank well, and the results are expressed as pg/ml.

Statistical analysis

All the data are presented as the mean ± SD and represent more than three independent experiments. All the statistical analyses were performed using GraphPad Prism 9.0. Two-way ANOVA followed by Bonferroni's post hoc test was used for multiple comparisons. Student's t test was used for unpaired observations. Survival was analyzed by the Kaplan–Meier method and log-rank test. A value of P < 0.05 was considered to indicate statistical significance.

TRPM2 deficiency promotes M2b polarization in BMDMs stimulated with LPS

Previous studies have shown that the biomarkers, such as CCL1 and IL-10, are more prominently in M2b macrophages compared to other macrophage subtypes. ²³ Therefore, CCL1 plays a key role in maintaining the unique characteristics of M2b macrophages.^9,24 In the current study, qRT-PCR analysis revealed that the expression of M1 markers, such as inducible nitric oxide synthase (iNOS), IL-12, was significantly lower in trpm2−/− BMDMs than in WT-BMDMs after LPS stimulation (Figure 1A–C). Notably, these markers showed low expression levels at the 12 h and 24 h time points. In contrast, the expression of M2b markers, including CCL1, IL-10, was markedly higher in trpm2^−/− BMDMs than in WT-BMDMs after LPS stimulation. The levels of these markers at the same time points (12 h and 24 h) in the supernatants exhibited similar trends (Figure 1D–G). These results suggest that TRPM2 deficiency promotes M2b polarization while suppresses M1 polarization in LPS-stimulated BMDMs at 12 h and 24 h.

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Figure 1.

Trpm2^−/− BMDMs were prone to M2b-type macrophage polarization after LPS stimulation. BMDMs were isolated from wild-type (WT) and trpm2^−/− mice and stimulated with 100 ng/ml LPS at different time points. (A) The mRNA expression levels of iNOS, IL-12, CCL1, IL-10, IL-6, and TNF-α in BMDMs were assessed via qRT–PCR and normalized to that of GAPDH. (B) The concentrations of CCL1, IL-10, IL-12, and TNF-α in the culture medium of BMDMs were measured using ELISA. The data are presented as the means ± SD (n = 3). Two-way ANOVA followed by Bonferroni’s post hoc test was used for multiple group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT mice.

TRPM2 deficiency activates the PI3K/AKT/CREB signaling pathway and M2b polarization in BMDMs

M2b macrophage polarization is regulated by several signaling pathways, including the PI3K/AKT pathway and the transcription factor CREB.^11,12 To understand how TRPM2 impacts macrophage polarization, we examined the expression of AKT and CREB in BMDMs from both trpm2^−/− and WT mice, which were stimulated with LPS. Notably, the ratios of p-CREB/CREB and p-AKT/AKT were higher in the trpm2-/- BMDMs compared to WT BMDMs following LPS stimulation. To investigate the relationship between PI3K pathway and TRPM2, we used the PI3K inhibitor IPI-549 to block PI3K/AKT signaling. Our results revealed that pretreatment with IPI-549 significantly reversed the p-AKT/AKT and p-CREB/CREB ratios (Figure 2). These findings indicate a potential correlation between TRPM2 and the PI3K/AKT/CREB pathway.

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Figure 2.

Inhibition of PI3K-γ reversed the change of the AKT/CREB pathway in trpm2^−/− BMDMs. BMDMs from WT and trpm2^−/− mice were pretreated with IPI-549 15 min before stimulation with 100 ng/ml LPS, and determined 30 min after LPS stimulation via western blotting. (A–B) The protein expression and densitometric quantification of AKT, p-AKT. (C–D) The protein expression and densitometric quantification of CREB, and p-CREB. The data are presented as the means ± SD (n = 3–4). Two-way ANOVA followed by Bonferroni’s post hoc test was used for multiple group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001.

To further investigate the effects of TRPM2 and the PI3K/AKT/CREB pathway on M2b polarization, we assessed the mRNA expression of iNOS, IL-12, CCL1 and IL-10 (Figure 3A–D) and the secretory proteins IL-12, CCL1 and IL-10 (Figure 3E–G) by ELISA and qRT–PCR. We found that the expression of typical M2b markers, including CCL1 and IL-10, was dramatically downregulated, while the expression of the M1 markers iNOS and IL-12 was mildly upregulated in the trpm2^−/− BMDMs compared with LPS-treated trpm2^−/− BMDMs. These results suggest that the M2b markers CCL1 and IL-10 are partly attenuated through the inhibition of the AKT/CREB pathway in trpm2^−/− BMDMs.

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Figure 3.

The PI3K inhibitor IPI-549 affect macrophage polarization and bacterial burdens in BMDMs from WT and trpm2^−/− mice. (A–G) Cells were treated with DMSO or IPI-549 for 30 min, then stimulated with 100 ng/ml LPS, cells and supernatants were collected after 12 h. (A–C) mRNA expression of M1/M2b markers in the BMDMs. (D–G) Cytokine, CCL1, IL-10, and IL-12 levels in the supernatants. (H) Bacterial burdens of BMDMs from WT and trpm2^−/− mice. Cells were treated with treated with DMSO or IPI-549 (10 μmol/ml) for 30 min, and subsequently challenged with Escherichia coli (DH5ɑ) for 1 h at 37°C (MOI = 20). Then, gentamicin (100 μg/ml) was added to the cells for 6 h, after which the cell lysates were inoculated on agar plates for 12 h, after which the colony-forming units (CFU) were counted. The data are presented as the means ± SD (n = 3–4). Two-way ANOVA followed by Bonferroni’s post hoc test was used for multiple group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001.

PI3K inhibition enhances bacterial clearance in trpm2^−/− BMDMs

Our previous study confirmed that there is no significant difference in the phagocytic capacity of peritoneal macrophages between trpm2+/+ and trpm2^−/− mice, ²⁴ and TRPM2 participates in intracellular bacterial elimination of peritoneal macrophages. To determine whether the TRPM2-regulated PI3K/AKT/CREB pathway influences the bactericidal activity of macrophages, we performed the intracellular bacterial killing assay. Consistent with our earlier findings, trpm2^−/− BMDMs exhibited a higher bacterial load than WT BMDMs. Furthermore, treatment with the PI3K inhibitor IPI-549 reduced the bacterial burden in trpm2^−/− BMDMs compared to untreated trpm2^−/− BMDMs (Figure 3H). These results indicate that inhibiting the PI3K pathway in trpm2^−/− BMDMs markedly enhanced the bacterial killing ability.

PI3K inhibition reduces bacterial burden and mortality in trpm2^−/− mice after polymicrobial sepsis

Our previous studies demonstrated that TRPM2 plays an essential role in controlling bacterial clearance and host defense during polymicrobial sepsis. ¹⁵ To further validate the involvement of the TRPM2-mediated bacterial clearance pathway via the PI3K/AKT/CREB pathway in the CLP model, we utilized the PI3K inhibitor IPI-549 to assess its impact on bacterial clearance in septic trpm2^−/− mice. Remarkably, trpm2^−/− mice treated with IPI-549 exhibited a significant decrease in bacterial load in the PLF at 48 h after CLP surgery (Figure 4A). Moreover, the survival rate was higher in trpm2^−/− mice treated with IPI-549 compared to those treated with a vehicle control (Figure 4B).

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Figure 4.

The PI3K inhibitor IPI-549 improved survival by promoting bacterial clearance in trpm2^−/− mice after CLP. (A) At 48 h after CLP, bacterial burdens were detected by plating serial dilutions of peritoneal lavage fluid (PLF) on agar plates. CFUs were counted 12 h after inoculation on agar plates. Each dot represents the CFU of one mouse. The horizontal bars represent the means (n = 6). *p < 0.05, Two-way ANOVA followed by Bonferroni’s post hoc test was used for multiple group comparisons. (B) Survival was monitored for 7 days (n = 12). *p < 0.05, Kaplan–Meier log-rank test.

PI3K inhibition partly mitigates tissue damage in trpm2^−/− mice after polymicrobial sepsis

To investigate the pathological role of TRPM2 in the PI3K/AKT/CREB pathway during CLP-induced sepsis, we performed hematoxylin and eosin (H&E) staining to assess lung and liver injuries (Figure 5A). Notably, several indicators of organ injury, including lung injury score, total protein concentration in bronchoalveolar lavage fluid, wet-to-dry weight ratio of lung tissue, liver injury score, and serum alanine aminotransferase concentration (ALT) levels, were significantly decreased in trpm2^−/− mice following treatment with the PI3K inhibitor. (Figure 5B and C). Taken together, these results suggest that the PI3K inhibitor improved pathological parameters and reduced mortality in septic trpm2^−/− mice.

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Figure 5.

The PI3K inhibitor IPI-549 attenuates organ injury in trpm2^−/− mice after CLP wt and trpm2^−/− mice were intraperitoneally injected with 1 mg/kg IPI-549 or vehicle 30 min before CLP. Mice were sacrificed at 48 h after CLP. (A) Lungs and livers were collected for hematoxylin and eosin staining (original magnification, 20×; scale bar, 20 µm). Green arrow points out the neutrophil infitration in the lung . (B) The lung injury score (n = 5), total protein concentration in bronchoalveolar lavage fluid (n = 5), and lung wet-to-dry weight ratio (n = 4) represent the severity of lung injury. (C) The liver injury score (n = 5) and serum alanine aminotransferase concentration (ALT) (n = 4) represent the severity of liver injury. The data represent the means ± SD. Two-way ANOVA followed by Bonferroni’s post hoc test was used for multiple group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001.

PI3K mediated TRPM2 deficiency induced M2b polarization in peritoneal macrophages from septic mice

To confirm these findings in vivo, we developed a CLP model to explore whether the PI3K/AKT/CREB pathway mediates the contribution of TRPM2 to macrophage polarization. At 48 h after CLP, the mRNA expression of M2b markers, including CCL1 and IL-10, was significantly higher in trpm2^−/− mice compared to the WT group. Pretreatment with IPI-549 in trpm2^−/− mice decreased the mRNA levels of M2b markers (Figure 6A–C). Similarly, the levels of CCL1, IL-10, and IL-12 in the supernatants followed the same trend (Figure 6D–F). Furthermore, protein expression of p-AKT and p-CREB in peritoneal macrophages 48 h after CLP was markedly upregulated in the trpm2^−/− macrophages (Figure 7A and B). These findings suggest that TRPM2 deficiency may promote M2b-type macrophage polarization by enhanced phosphorylation of AKT and CREB.

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Figure 6.

M2b polarization was exhibited in peritoneal macrophages from trpm2^−/− mice in the CLP model (A–C) mRNA expression of IL-12 (n = 3), Il-12, CCL1, and IL-10 (n = 5) in peritoneal macrophages 48 h after CLP and quantitative analysis via qRT–PCR. (D–F) Cytokine, CCL1, IL-10, and IL-12 levels in the supernatants were assessed via ELISA. The data represent the means ± SD. Two-way ANOVA followed by Bonferroni’s post hoc test was used for multiple group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001.

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Figure 7.

PI3K/AKT/CREB pathway mediated M2b polarization in peritoneal macrophages from trpm2^−/− mice in the CLP model, and the phenotypic shift from M2b to M1 was facilitated by the PI3Kγ inhibitor. (A) The pres The levels of AKT, phosphorylated AKT (n = 3) (B), CREB, and phosphorylated CREB (n = 4) (C)were detected by immunoblotting, and analysis. The data represent the means ± SD. Two-way ANOVA followed by a Bonferroni comparison test was used. *p < 0.05, **p < 0.01, ***p < 0.001.