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The immunometabolite itaconate stimulates OXGR1 to promote mucociliary clearance during the pulmonary innate immune response - PubMed

  • ️Sun Jan 01 2023

The immunometabolite itaconate stimulates OXGR1 to promote mucociliary clearance during the pulmonary innate immune response

Yi-Rong Zeng et al. J Clin Invest. 2023.

Abstract

Pathogens and inflammatory conditions rapidly induce the expression of immune-responsive gene 1 (IRG1) in cells of myeloid lineage. IRG1 encodes an aconitate decarboxylase (ACOD1) that produces the immunomodulatory metabolite itaconate (ITA). In addition to rapid intracellular accumulation, ITA is also secreted from the cell, but whether secreted ITA functions as a signaling molecule is unclear. Here, we identified ITA as an orthosteric agonist of the GPCR OXGR1, with an EC50 of approximately 0.3 mM, which was in the same range as the physiological concentration of extracellular ITA upon macrophage activation. ITA activated OXGR1 to induce Ca2+ mobilization, ERK phosphorylation, and endocytosis of the receptor. In a mouse model of pulmonary infection with bacterial Pseudomonas aeruginosa, ITA stimulated Oxgr1-dependent mucus secretion and transport in respiratory epithelium, the primary innate defense mechanism of the airway. Our study thus identifies ITA as a bona fide ligand for OXGR1 and the ITA/OXGR1 paracrine signaling pathway during the pulmonary innate immune response.

Keywords: G protein–coupled receptors; Metabolism.

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Figures

Figure 1
Figure 1. A screen of GPCR receptors discovers OXGR1 as a potential ITA receptor.

(A) In mouse BMDMs challenged with LPS, intracellular and extracellular levels of ITA were collected and quantified. (B) Immunoblot of Irg1 in LPS-challenged mouse BMDMs. (C) Intracellular and extracellular levels of ITA were measured by LC-MS in LPS-challenged BMDMs at the indicated time points. Error bar, SEM. (D) Comparison of the percentage of newly synthesized intracellular and extracellular ITA during LPS stimulation. (E) Schematic illustration of the GPCR screen. A total of 301 plasmids encoding for GPCRs were transfected along with Gα16 and aequorin plasmids. ITA-induced aequorin luminescence was determined. (F) The ITA dose response was determined by the aequorin assay in HEK293 cells overexpressing human OXGR1 (hOXGR1) or mouse OXGR1 (mOXGR1). Vec, empty vector.

Figure 2
Figure 2. ITA acts as an agonist of OXGR1 to stimulate cytosolic calcium, ERK activation, and receptor endocytosis.

(A) Verification of Gq/11 knockdown in HEK293 cells by Western blotting. (B) Aequorin assay showing calcium mobilization by different concentrations of ITA in cells expressing human OXGR1 with or without siRNAs targeting Gq/11. (C) ITA and α-KG induced endocytosis of FLAG-tagged OXGR1, which was detected using anti-FLAG antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. (D) Endocytosis of FLAG-tagged OXGR1 induced by ITA with or without siRNAs targeting β-arrestin 1/2 was detected by anti-FLAG antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. (E) Serum-starved cells were stimulated with different concentrations of ITA (from 5 μM to 1 mM) for 15 minutes (left), or 500 μM ITA for different durations (from 5 to 30 minutes) (right). Cells were harvested, and p-ERK was determined by Western blotting. (F) Serum-starved cells expressing human OXGR1 transfected with siRNAs targeting Gq/11 and β-arrestin 1/2 were stimulated with 500 μM ITA for the indicated durations. Cells were harvested, and p-ERK levels were determined by Western blotting.

Figure 3
Figure 3. ITA activates OXGR1 by interacting with its orthosteric site.

(A) Virtual structure of human OXGR1 predicted by AlphaFold. Amino acid residues C106, R110, H114, Y118, H258, R261, I265, and R288 are labeled in red. (B) Verification of ectopically expressed OXGR1 in HEK293 cells as determined by Western blotting. (C) Aequorin assay showing calcium mobilization in cells expressing WT or mutant OXGR1 in response to the indicated ligands. (D) Endocytosis of FLAG-tagged WT or mutant OXGR1 was detected using anti-FLAG antibody. Nuclei were stained with DAPI. Scale bar: 10 μm.

Figure 4
Figure 4. ITA signals through OXGR1 to stimulate MCC in respiratory epithelium.

(A) Calcium mobilization of primary trachea epithelial cells isolated from mice in response to 670 μM ITA or 100 μM ATP, as determined by Fluo-4AM assay. (B) Tracheas were cultured ex vivo, and cilia beating–derived fluid flow was monitored by tracing fluorescent bead movement under the microscope. The trajectory of 20 individual beads in 1 minute with or without 500 μM ITA is shown. Scale bar: 150 μm. (C) Quantification of bead movement speed in trachea culture from individual Oxgr1+/+ (n = 3) and Oxgr1–/– (n = 3) mice in the presence of 500 μM ITA or 100 μM ATP. Each dot represents an independent biological replicate. *P < 0.05 and **P < 0.01, by paired, 2-tailed Student’s t test. (D) Primary human bronchial epithelium cells were infected by a control lentivirus (Vec) or viruses expressing sgRNAs targeting OXGR1 (sgOXGR1). Calcium mobilization in response to 500 μM ITA was determined by Fluo-4AM assay. (E and F) Comparison of bead movement speed between ALI culture of human bronchial epithelium cells in the presence of 500 μM ITA or 100 μM ATP. **P < 0.01 and ****P < 0.0001, by 1-way ANOVA (E) or unpaired, 2-tailed Student’s t test (F). Data indicate the mean ± SEM. (G–I) Anesthetized mice of the indicated genotype (n = 4–6 per group) were intranasally administered PBS or 500 μM ITA, and their nasal cavities were harvested 1 hour after ITA administration. (G) Sections were taken from the most proximal aspect of the nasal cavities. (H) Representative images of Alcian blue/PAS staining of nasal cavities are shown. (I) Quantification of mucin-containing cells in the indicated groups of animals. ****P < 0.0001, by 2-tailed Student’s t test. Data indicate the mean ± SEM.

Figure 5
Figure 5. Depletion of either Oxgr1 or Irg1 disrupts the clearance of respiratory bacteria.

(A and B) Anesthetized mice of the indicated genotype (n = 4–10 per group) were intranasally administrated the P. aeruginosa strain PAO1 (1 × 107 CFU). BAL and lung tissue were collected 12 hours after infection. Representative images of PAO1 colonies on CTAB-selective plates after overnight culturing of BAL and lung from infected mice (dilution: 104-fold) (A) and quantification of CFU (B) are shown. *P < 0.05 and **P < 0.01, by 1-way ANOVA. Data indicate the mean ± SEM. (C and D) Anesthetized mice of the indicated genotype (n = 4–10 per group) were intranasally administrated 20 μL saline containing PAO1 (1 × 107 CFU) with or without 1 mM ITA. The CFU in BAL (C) and lung tissues (D) were quantified. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA. Data indicate the mean ± SEM.

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