Chanzyme TRPM7 Mediates the Ca2+ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation

doi:10.1016/j.immuni.2017.11.026

. 2018 Jan 16;48(1):59-74.e5.

doi: 10.1016/j.immuni.2017.11.026.

Chanzyme TRPM7 Mediates the Ca²⁺ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation

Affiliations

¹ Pharmacology Department, University of Virginia, Jordan Hall, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA.
² Pharmacology Department, University of Virginia, Jordan Hall, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA; Carter Immunology Center, University of Virginia, 345 Crispell Dr. MR-6, Charlottesville, VA 22908, USA; Robert M. Berne Cardiovascular Research Center, University of Virginia, 415 Lane Rd, Charlottesville, VA 22908, USA.
³ Pharmacology Department, University of Virginia, Jordan Hall, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA; Carter Immunology Center, University of Virginia, 345 Crispell Dr. MR-6, Charlottesville, VA 22908, USA; Robert M. Berne Cardiovascular Research Center, University of Virginia, 415 Lane Rd, Charlottesville, VA 22908, USA. Electronic address: bdesai@virginia.edu.

PMID: 29343440
PMCID: PMC5783319
DOI: 10.1016/j.immuni.2017.11.026

Chanzyme TRPM7 Mediates the Ca²⁺ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation

Michael S Schappe et al. Immunity. 2018.

. 2018 Jan 16;48(1):59-74.e5.

doi: 10.1016/j.immuni.2017.11.026.

Affiliations

¹ Pharmacology Department, University of Virginia, Jordan Hall, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA.
² Pharmacology Department, University of Virginia, Jordan Hall, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA; Carter Immunology Center, University of Virginia, 345 Crispell Dr. MR-6, Charlottesville, VA 22908, USA; Robert M. Berne Cardiovascular Research Center, University of Virginia, 415 Lane Rd, Charlottesville, VA 22908, USA.
³ Pharmacology Department, University of Virginia, Jordan Hall, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA; Carter Immunology Center, University of Virginia, 345 Crispell Dr. MR-6, Charlottesville, VA 22908, USA; Robert M. Berne Cardiovascular Research Center, University of Virginia, 415 Lane Rd, Charlottesville, VA 22908, USA. Electronic address: bdesai@virginia.edu.

PMID: 29343440
PMCID: PMC5783319
DOI: 10.1016/j.immuni.2017.11.026

Abstract

Toll-like receptors (TLRs) sense pathogen-associated molecular patterns to activate the production of inflammatory mediators. TLR4 recognizes lipopolysaccharide (LPS) and drives the secretion of inflammatory cytokines, often contributing to sepsis. We report that transient receptor potential melastatin-like 7 (TRPM7), a non-selective but Ca²⁺-conducting ion channel, mediates the cytosolic Ca²⁺ elevations essential for LPS-induced macrophage activation. LPS triggered TRPM7-dependent Ca²⁺ elevations essential for TLR4 endocytosis and the subsequent activation of the transcription factor IRF3. In a parallel pathway, the Ca²⁺ signaling initiated by TRPM7 was also essential for the nuclear translocation of NFκB. Consequently, TRPM7-deficient macrophages exhibited major deficits in the LPS-induced transcriptional programs in that they failed to produce IL-1β and other key pro-inflammatory cytokines. In accord with these defects, mice with myeloid-specific deletion of Trpm7 are protected from LPS-induced peritonitis. Our study highlights the importance of Ca²⁺ signaling in macrophage activation and identifies the ion channel TRPM7 as a central component of TLR4 signaling.

Keywords: LPS; TLR4; TRP channel; TRPM7; endotoxin; inflammation; ion channel; sepsis; toll-like receptor.

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Conflict of interest statement

The authors declare that they have no conflicts of interest to disclose.

Figures

**Figure 1. Characterization of *Trpm7^fl/fl (LysM Cre)* mice and *Trpm7*-deficient macrophages**
(A) Schematic showing generation of *Trpm7^fl/fl (LysM Cre)* mice and bone-marrow-derived macrophage (BMDM) culture. (B) A representative I-V relationship of whole-cell Mg²⁺-inhibitable I_TRPM7 in freshly isolated *Trpm7^fl/fl* [*Trpm7^+/+*] and *Trpm7^fl/fl (LysM Cre)* [*Trpm7^−/−*] peritoneal macrophages (left panel). The statistics of TRPM7 current densities (n=5) are shown as box charts (right panel). (C) Schematic of inflammasome activation assay used for results shown in *panel D*. The inflammasome activating ligands were added after 3h of LPS priming (100 ng/mL). The secreted IL-1β in the culture supernatants (collected at 24h) was quantified by ELISA. (D) Quantification of secreted IL-1β in the supernatants of *Trpm7^+/+* and *Trpm7^−/−* BMDMs, stimulated with indicated inflammasome activating ligands, as depicted in *panel C*. Error bars represent SEM, n=4. (E) Gene expression analysis (qRT-PCR) of *Trpm7^+/+* and *Trpm7^−/−* BMDMs after stimulation with 100 ng/mL LPS (3h). Changes in mRNA levels, relative to untreated BMDMs are shown. Error bars represent SD (Means representative of n=3 independent experiments). (F) Gene expression analysis (qRT-PCR) of *Trpm7^+/+* and *Trpm7^−/−* BMDMs after stimulation with indicated ligand (3h). BMDMs treated with LPS (100 ng/ml), Pam3CSK4 (100 ng/mL), PolyI:C (25 μg/mL), or ODN 1826 (1 μM). Changes in mRNA levels, relative to untreated BMDMs are shown. Error bars represent SD (Means representative of n=3 independent experiments).

**Figure 2. TRPM7 regulates TLR4 endocytosis and downstream NFκB and IRF3 phosphorylation**
(A) Flow cytometry-based quantification of TLR4/MD2 dimers on the BMDM cell surface at indicated times after LPS (1 μg/mL) treatment. The change in percentage of cell surface TLR4/MD2 dimers was derived by staining the cells with an antibody specific for TLR4/MD2 dimers and calculations were based on the Mean fluorescence intensities (MFI). Error bars represent SEM (n=3). (B) Flow cytometry-based quantification of TLR4 on the BMDM cell surface at indicated times after LPS (1 μg/mL) treatment. An anti-TLR4 antibody was used for staining. The error bars represent SEM (n=4). (C) Flow cytometry-based quantification of CD14 on the BMDM cell surface at indicated times after LPS (1 μg/mL) treatment using an anti-CD14 antibody. Analysis was similar to *panel B*. Error bars represent SEM (n=5). (D) Immunoblot analysis of NFκB p65 phosphorylation at S276 and S534 (in humans S536), P-IRF3 at S388, and IκBα protein levels from whole cells lysates of indicated BMDMs. Cells were stimulated with LPS (100 ng/mL) as indicated. Blots are representative of at least three independent experiments (n>3). Densitometry values indicate phospho-protein levels relative to total protein (ratios) which were then normalized to *Trpm7^+/+* at 0 min. Grey-dotted line denotes that the bottom gels were obtained from independent samples obtained using identical conditions. (E) Quantification of immunoblots shown in *Panel D*. The densitometric values were calculated by taking the ratios of phospho-protein levels relative to total protein and then normalizing the ratios to *Trpm7^+/+* at 0 min. Bar charts represent means from at least three independent experiments (n= 4 for P-IRF3, n = 3 for others). Error bars are SEM. See also Fig. S3D.

**Figure 3. LPS-induced nuclear translocation of NFκB p65 and IRF3 is defective in *Trpm7*-deficient BMDMs**
(A) Schematic of ImageStream analysis of LPS-induced NFκB translocation. Quantification of nuclear translocation is reflected by and directly proportional to the similarity scores of NFκB and nuclear staining. Similarity scores of individual cells are displayed together as a histogram. (B) Representative images (n> 2000 cells) taken from ImageStream flow cytometry analysis of indicated BMDMs, stimulated with LPS (100 ng/mL, 30m) and stained with anti-NFκB p65 antibody and DRAQ5 (nuclear stain) *(left panels)*. An overlaid image of NFκB p65 staining (green) and DRAQ5 (red) is shown and the single channel images of that cell are shown on the right in smaller sizes. The overlay of histograms depicts the representative similarity scores derived for each condition (*right panel*), quantified in *Panel C* (n=4). (C) Quantification of ImageStream flow cytometry results depicted in *Panel B*. Error bars are SEM (n=4). (D) Confocal immunofluorescence microscopy images of fixed BMDMs, treated or untreated with LPS (100 ng/ml, 30m) as indicated, and stained with anti-NFκB p65 and Hoechst nuclear stain. Images shown are a single optical section (0.25 μm). Scale bar = 15 μm. Typical results of n=3 experiments. (E) Confocal immunofluorescence microscopy images of fixed BMDMs, treated or untreated with LPS (100 ng/ml, 60m) and stained with anti-IRF3 and Hoechst nuclear stain. Images shown are a single optical section (0.25 μm). Scale bar = 25 μm. (F) Immunoblot analysis of NFκB p65 present in cytosolic and nuclear fractions obtained from indicated BMDMs at various time points after LPS (100 ng/ml) treatment. Immunoblots of β-actin in each fraction is also shown. These are representative results of independent experiments (n=3). (G) Linear intensity analysis of IRF3 nuclear localization. Fluorescent intensity of IRF3 signal (cyan) was measured with a single x-y plane line trace; location of nuclei is overlayed (red). Line traces depict typical cells from *panel E* and are illustrated in Fig. S4H.

**Figure 4. LPS signaling and NFκB translocation are abrogated by clamping intracellular Ca²⁺ but there is no further decrease in *Trpm7*-deficient macrophages**
(A) Flow cytometry-based quantification of phospho-NFκB p65, as indicated by MFI, in DMSO (vehicle control) and BAPTA-AM loaded BMDMs in response to LPS (100 ng/mL, indicated time points). The error bars represent SEM (n=3). (B) Representative images (n>5000 cells) from ImageStream analysis of DMSO and BAPTA-AM loaded cells are shown (*left panel*). Cells were stimulated with LPS (100 ng/ml, 30m) and then stained with anti-NFκB p65 antibody and DRAQ5 (a nuclear stain), prior to ImageStream analysis. To the right of the merged image of NFκB p65 (green) and DRAQ5 (red), single channel images of that cell are shown in smaller sizes. Overlaid histograms of similarity scores derived from each condition are shown (*right panels*); unstimulated (grey-filled), BAPTA-AM pre-treated (orange), and LPS treated (blue-wt; red-KO) are shown. The data are representative and typical of independent experiments (n=2). (C) Quantification of similarity scores from *panel B*, reflecting the degree of NFκB p65 translocation in indicated conditions. Error bars represent SEM (n=3). (D) qRT-PCR analysis of indicated inflammatory genes in *Trpm7^+/+* and *Trpm7^−/−* BMDMs treated as depicted. Prior to LPS treatment (100 ng/mL, 3h), BMDMs were pre-treated with DMSO or BAPTA-AM for 30 min in serum-free media. Error bars represent SD (n=3). (E) qRT-PCR analysis of indicated inflammatory genes in *Trpm7^+/+* and *Trpm7^−/−* BMDMs treated as indicated in the schematic of the experimental design *(left panel)*. Mean expression relative to untreated condition (*right panel*). Ionomycin is added with LPS to induce Ca²⁺ influx. Error bars represent SD (n=3). (F) Flow cytometry-based quantification of cell surface TLR4 in RAW 264.7 cells at indicated times after LPS (1 μg/mL) treatment. Cells were treated with BAPTA-AM or DMSO as indicated prior to LPS treatment. Extracellular Ca²⁺-free conditions supplemented 10 mM EGTA. The error bars reflect SEM (n=3). * is BAPTA-AM treatment and ^# is low Ca²⁺ outside relative to wt. * indicates p<0.05, *** or ^### indicates p<0.001. (G) Flow cytometry-based quantification of cell surface TLR4 in RAW 264.7 cells at indicated times after LPS (1 μg/mL) treatment. Cells were treated with BAPTA-AM, EGTA-AM, or DMSO as indicated prior to LPS treatment. The error bars reflect SEM (n=3). * is BAPTA-AM treatment and # is EGTA-AM relative to wt. * or ^# indicates p<0.05, ** or ^## indicates p<0.01.

**Figure 5. LPS-induced Ca²⁺ elevations are highly compromised in *Trpm7*-deficient macrophages**
(A) Relative changes in [Ca²⁺]_i over time in WT BMDMs treated with indicated TLR ligands for 5 min. Cells were treated with either LPS (100 ng/ml), Pam3CSK4 (100 ng/ml), ODN 1826 (1 μM), or bath solution (untreated). *Left panel:* trace represents mean ∆F340/F380 ratio from all samples and error bars are SEM; ionomycin (1 μM) was perfused as a positive control. The arrow indicates the time point of peak [Ca²⁺]_i, these values were used for statistical analysis. *Right panel:* Quantification of peak [Ca²⁺]_i after LPS stimulation; box-whisker plot is overlaid on individual measurements. One-way ANOVA statistical analysis and n values (number of cells) are indicated in the figure. Results are a compilation of three independent experiments. (B) Relative changes in [Ca²⁺]_i over time in *Trpm7^+/+* and *Trpm7^−/−* BMDMs treated with LPS (100 ng/ml) for 5 min. *Left panel:* trace represents mean ∆F340/F380 ratio from all samples (n=79 and n=138 for *Trpm7^+/+* and *Trpm7^−/−*, respectively) and error bars are SEM. Ionomycin (1 μM) was perfused as a positive control. *Right panel:* Quantification of peak [Ca²⁺]_i after LPS stimulation; box-whisker plot is overlaid on individual measurements; n values (number of cells analyzed) indicated in figure. Results are a compilation of three independent experiments. (C) Relative changes in the fluorescence of Fluo-4AM-loaded BMDMs, reflecting changes in intracellular Ca²⁺ concentration [Ca²⁺]_i BMDMs after LPS (1 μg/mL) treatment for 15 min. Error bars reflect SEM (n=146 and n=179 for *Trpm7^+/+* and *Trpm7^−/−*, respectively). These represent typical results from four independent experiments. (D) Representative fluorescence images from indicated time points in the experiments shown in *panel C*. (E) Statistical representation of mean peak intensities of Fluo-4 fluorescence after LPS treatment from *panel C*. (F) Relative changes in [Ca²⁺]_i over time in *Trpm7^+/+* and *Trpm7^−/−* BMDMs treated with LPS (1 μg/mL) for 60s. Error bars are SEM (n=217 and n=190 for *Trpm7^+/+* and *Trpm7^−/−*, respectively). These represent typical results from four independent experiments. (G) Representative images from indicated time points in *panel F*. (H) Statistical representation of mean peak intensities after LPS treatment from *panel F*. (I) Relative changes in [Ca²⁺]_i over time in *Trpm7^+/+* and *Trpm7^−/−* BMDMs treated with Pam3CSK4 (100 ng/ml) for 5 min. *Left* and *Right panels* depicted as in Fig. 5B. (J) Relative changes in [Ca²⁺]_i over time in *Trpm7^+/+* and *Trpm7^−/−* BMDMs treated with ODN 1826 (1 μM) for 5 min. *Left* and *Right panels* depicted as in Fig. 5B.

**Figure 6. Blocking TRPM7 channel activity abrogates LPS-induced Ca²⁺ entry and TLR4 endocytosis**
(A) Relative changes in [Ca²⁺]_i over time in C57BL/6 BMDMs treated with LPS (100 ng/ml) or LPS+FTY720 (5μM) for 5 min. Trace represents mean ∆F340/F380 ratio from all samples and error bars are SEM. Ionomycin (1 μM) was perfused as a positive control. Results are from n=3 independent experiments; n value indicated in figure. (B) Quantification of peak [Ca²⁺]_i after LPS stimulation from Fig 6A. Box-whisker plot is overlaid on individual measurements. (C) Flow cytometry based measurement of cell surface TLR4 levels in BMDMs, pre-treated with either vehicle (DMSO), FTY720, or BAPTA-AM for 15 min, and then stimulated with LPS as indicated. The relative change in the percentage of cell surface TLR4 levels was inferred based on mean MFI values. Error bars represent SEM (n=3; * indicates p < 0.05, ** p < 0.01). * indicates significance from WT relative to BAPTA-AM; #, FTY720 group. These results represent typical results obtained in n=2 independent experiments. (D) Gene expression analysis (qRT-PCR) of human THP-1 monocytes stimulated with LPS (100 ng/ml; 3h) with FTY720 pre-treatment as indicated. Bar charts represent mean of n=3 independent experiments. Error bars are SEM (n=3). (E) Relative changes in [Ca²⁺]_i over time in BMDMs treated with LPS (100 ng/ml) for 5 min. Ionomycin (1 μM) was perfused as a positive control. Traces represent mean ∆F340/F380 ratio from all samples measured and error bars are SEM. Results are representative from n=5 independent experiments. (F) Quantification of peak [Ca²⁺]_i after LPS stimulation from Fig 6E. Violin plot is shown to illustrate range and distribution of data points; diamond indicates mean value for group. One-way ANOVA (F[2,851] = 266.23) indicates p = 1.6e–90. T-test used to compare groups with C57BL/6J BMDMs.

**Figure 7. *Trpm7^fl/fl (LysM Cre)* mice are resistant to LPS-induced peritonitis**
(A) Schematic of LPS-induced peritonitis mouse model and analysis. (B) After a sub-lethal dose of LPS (0.2 mg/kg, i.p), the mice were observed for 24 hrs by a double-blinded experimenter to record clinical scores in accord with an index described in Table S1. Error bars represent SEM (n>5). (C) Statistical box charts showing composite clinical scores from individual mice, at 4h of observation with indicated treatments (see panel A for details). Data were compiled from three independent cohorts of mice. Box chart parameters are described in methods. (D) ELISA measurements of indicated cytokines in the serum collected after LPS (0.2 mg/kg) injections at indicated time points. Error bars represent SEM (n>6 compiled from three independent cohorts). (E) Gene expression analysis (qRT-PCR) of cells collected via a peritoneal lavage, after LPS (0.2 mg/kg, 4h) injections. Quantification is relative to PBS-injected controls (not shown). Error bars represent SEM (n=4 mice). (F) Flow cytometry-based immunophenotypic analysis of peritoneal macrophages for the indicated cell surface markers, after gating on CD45+ cells. The legacy of gates is shown in Figure S5. Cells were isolated from peritoneal lavage after LPS (0.2 mg/kg, 24h). The data represent typical results from independent experiments (n=3 mice). (G) The mean percentage of peritoneal hematopoietic (CD45+) and macrophages (CD45+ CD11b+ F4/80+) determined by flow cytometry analysis shown in Figure S5. Error bars represent SEM (n=3 mice). (H) Mean numbers of total cells, hematopoietic (CD45+) and macrophages (CD45+ CD11b+ F4/80+) in peritoneal lavages, as determined by flow cytometry analysis shown in *Panel F*. Cell counts were calculated by accounting for the number of cells analyzed, percentages of cells in legacy gates, and recovery volume of peritoneal lavage. Error bars represent SEM (n=3 mice). See also Figure S7 and Table S1.

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The Family of LPS Signal Transducers Increases: the Arrival of Chanzymes.
Granucci F. Granucci F. Immunity. 2018 Jan 16;48(1):4-6. doi: 10.1016/j.immuni.2017.12.016. Immunity. 2018. PMID: 29343439

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References

1. Afonina IS, Muller C, Martin SJ, Beyaert R. Proteolytic Processing of Interleukin-1 Family Cytokines: Variations on a Common Theme. Immunity. 2015;42:991–1004. - PubMed
1. Akashi S, Saitoh S, Wakabayashi Y, Kikuchi T, Takamura N, Nagai Y, Kusumoto Y, Fukase K, Kusumoto S, Adachi Y, et al. Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2: Higher Affinity than That with MD-2 or CD14. The Journal of experimental medicine. 2003;198:1035–1042. - PMC - PubMed
1. Akashi S, Shimazu R, Ogata H, Nagai Y, Takeda K, Kimoto M, Miyake K. Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages. Journal of immunology. 2000;164:3471–3475. - PubMed
1. Barton GM, Kagan JC. A cell biological view of Toll-like receptor function: regulation through compartmentalization. Nature reviews Immunology. 2009;9:535–542. - PMC - PubMed
1. Brinkmann V, Davis MD, Heise CE, Albert R, Cottens S, Hof R, Bruns C, Prieschl E, Baumruker T, Hiestand P, et al. The immune modulator FTY720 targets sphingosine 1-phosphate receptors. The Journal of biological chemistry. 2002;277:21453–21457. - PubMed

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[1] Afonina IS, Muller C, Martin SJ, Beyaert R. Proteolytic Processing of Interleukin-1 Family Cytokines: Variations on a Common Theme. Immunity. 2015;42:991–1004. - PubMed

[2] Afonina IS, Muller C, Martin SJ, Beyaert R. Proteolytic Processing of Interleukin-1 Family Cytokines: Variations on a Common Theme. Immunity. 2015;42:991–1004. - PubMed

[3] Akashi S, Saitoh S, Wakabayashi Y, Kikuchi T, Takamura N, Nagai Y, Kusumoto Y, Fukase K, Kusumoto S, Adachi Y, et al. Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2: Higher Affinity than That with MD-2 or CD14. The Journal of experimental medicine. 2003;198:1035–1042. - PMC - PubMed

[4] Akashi S, Saitoh S, Wakabayashi Y, Kikuchi T, Takamura N, Nagai Y, Kusumoto Y, Fukase K, Kusumoto S, Adachi Y, et al. Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2: Higher Affinity than That with MD-2 or CD14. The Journal of experimental medicine. 2003;198:1035–1042. - PMC - PubMed

[5] Akashi S, Shimazu R, Ogata H, Nagai Y, Takeda K, Kimoto M, Miyake K. Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages. Journal of immunology. 2000;164:3471–3475. - PubMed

[6] Akashi S, Shimazu R, Ogata H, Nagai Y, Takeda K, Kimoto M, Miyake K. Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages. Journal of immunology. 2000;164:3471–3475. - PubMed

[7] Barton GM, Kagan JC. A cell biological view of Toll-like receptor function: regulation through compartmentalization. Nature reviews Immunology. 2009;9:535–542. - PMC - PubMed

[8] Barton GM, Kagan JC. A cell biological view of Toll-like receptor function: regulation through compartmentalization. Nature reviews Immunology. 2009;9:535–542. - PMC - PubMed

[9] Brinkmann V, Davis MD, Heise CE, Albert R, Cottens S, Hof R, Bruns C, Prieschl E, Baumruker T, Hiestand P, et al. The immune modulator FTY720 targets sphingosine 1-phosphate receptors. The Journal of biological chemistry. 2002;277:21453–21457. - PubMed

[10] Brinkmann V, Davis MD, Heise CE, Albert R, Cottens S, Hof R, Bruns C, Prieschl E, Baumruker T, Hiestand P, et al. The immune modulator FTY720 targets sphingosine 1-phosphate receptors. The Journal of biological chemistry. 2002;277:21453–21457. - PubMed

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Chanzyme TRPM7 Mediates the Ca²⁺ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation

Affiliations

Chanzyme TRPM7 Mediates the Ca²⁺ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation

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