Research ArticleRESPIRATORY DISEASE

Pathophysiological regulation of lung function by the free fatty acid receptor FFA4

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Science Translational Medicine  19 Aug 2020:
Vol. 12, Issue 557, eaaw9009
DOI: 10.1126/scitranslmed.aaw9009
  • Fig. 1 FFA4 receptors are expressed in murine lung and localize primarily to the epithelial layer.

    (A) FFA1, FFA4, and tubulin (control) transcripts were identified in lung from wild-type and FFA4-KO(βgal) mice using RT-PCR. Shown is a representative gel from three independent experiments. Expected sizes of PCR products are as follows: FFA4, 800 base pairs (bp), tubulin, 750 bp, and tubulin, 530 bp. (B) qRT-PCR of lung tissue samples from wild-type and FFA4-KO(βgal) mice using the GAPDH housekeeping gene as a control. Data are means ± SD of n = 4 [FFA4-KO(βgal)] and n = 10 (WT animals). (C) Representative confocal image of lung tissue sections obtained from FFA4-KO(βgal) mice and stained for β-galactosidase activity on 5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside substrate (XGAL) as a surrogate for FFA4 expression. (D) Representative images of lung tissue sections obtained from wild-type (left) and FFA4-KO(βgal) (right) mice costained with an in-house generated mouse FFA4-specific antiserum (green), α-actin antibody to stain ASM (red), and DAPI to identify nuclei (blue). (E) Immunofluorescence costaining of mouse FFA4 and club cell marker (CC10). Images are representative of at least three independent experiments.

  • Fig. 2 Pharmacology of FFA4 agonist ligands in cell-based assays and ex vivo murine lung tissue preparations.

    (A and B) Calcium concentration-response curves in Flp-In T-REx 293 cells expressing mouse FFA1 or FFA4 and stimulated with (A) TUG-891 or (B) TUG-1197. (C) ERK1/2 concentration-response curves measured using Homogeneous Time Resolved Fluorescence FRET (HTRF) of CHO Flp-In cells expressing mouse FFA4 after 5 min stimulation with TUG-891 or TUG-1197. (D) Kinetics of TUG-891– and TUG-1197–mediated ERK1/2 responses at maximal concentration (1 μM) of each ligand. HTRF ratio corresponds to the transfer of energy from the donor-conjugated antibody to acceptor-conjugated antibody and is directly proportional to the amounts of ERK1/2 phosphorylation (E) Arrestin3 recruitment to FFA4 concentration response curves of Flp-In T-REx 293 cells expressing recombinant mouse FFA4 and stimulated with varying concentrations of TUG-891 or TUG-1197. (F) FFA4 phosphorylation in response to TUG-891 and TUG-1197 in Western blots of lysates prepared from Flp-In T-REx 293 expressing mouse FFA4 and probed with an anti-phospho antiserum to pThr347/pSer350 on mouse FFA4. Parallel immunoblotting to detect GAPDH (glyceraldehyde-3-phosphate dehydrogenase) acted as a loading control. (G) Internalization of enhanced yellow fluorescent protein (eYFP)–tagged mouse FFA4 expressed in Flp-In T-REx 293 cells after 30-min application of TUG-891 or TUG-1197. (H to J) Influx of intracellular calcium evoked by TUG-1197 in precision cut lung slices from (H) wild-type mice, (I) FFA4 KO(βgal) mice, and (J) wild-type mice in the presence of the Gq inhibitor FR900359. The white boxes indicate the area of interest where the fluorescence changes are illustrated in the graph to the right of the images. The FURA-2 ratio represents changes in 340 of 380 nm emission from the FURA2-AM calcium indicator. Arrows indicate the time point at which the FFA4 agonist was added. The experiments shown are representative of at least three independent experiments. Data in (A) to (E) are the means ± SEM.

  • Fig. 3 Activation of FFA4 leads to airway relaxation in precontracted murine airways.

    (A and B) Representative images of the concentration-dependent relaxation responses to (A) TUG-891 and (B) TUG-1197 in precision cut lung slices (PCLS) precontracted with carbachol (CCh). (C and D) Quantification of the data in (A) and (B). Data presented are means ± SEM (n = 5). (E) Representative images of the effect of the FFA4 antagonist AH7614 on the TUG-1197–mediated relaxation response. (F) Quantification of the experiment in (E) from n = 6 mice per group. (G) Representative images of the TUG-1197–mediated relaxation responses in PCLS from wild-type and FFA4-KO(βgal) mice. (H) Quantification of the experiment in (G) from n = 6 mice. Data in (F) and (H) are means ± SEM *P < 0.05 and **P < 0.01 as determined by ANOVA with Bonferroni post hoc test. n.s., not significant.

  • Fig. 4 FFA4 agonism reduces airway resistance in healthy mice and produces broncho-relaxant, anti-inflammatory, and prophylactic effects in an ozone model of inflammatory lung disease.

    (A) Broncho-relaxant effect of TUG-1197 (0.364 mg/ml) on acetylcholine-induced (185 mg/ml) airway resistance in healthy wild-type and FFA4-KO(βgal) mice. (B) Broncho-relaxant effect of TUG-1197 (0.364 mg/ml) on acetylcholine-induced (185 mg/ml) airway resistance in wild-type and FFA4-KO(βgal) mice that had been exposed to ozone (3 ppm, 3 hours) 24 hours before the lung resistance experiments. (C to J) Anti-inflammatory effect of repeated administration of TUG-891 in a 3-week ozone exposure model. Mice were exposed to either control air or 3 ppm ozone for 3 hours, twice a week for 3 weeks. Before each exposure, mice were treated with either vehicle or TUG-891 (0.036 mg/ml) by intranasal administration. Cells in BAL fluid were obtained and enumerated for total cell counts (C) and analyzed using flow cytometry to identify (D) alveolar macrophages, (E) monocyte-derived macrophages (MDM), and (F) neutrophil populations. Lung tissue (left lobe) was isolated, and RT-qPCR analysis was performed to identify changes in the expression of (G) KC/CXCL1 and (H) TNFα genes. (I) Prophylactic and (J) broncho-relaxant effect of TUG-891 in 3-week ozone model where TUG-891 (0.036 mg/ml) was administered 1 hour before each ozone exposure (I) and at the end of the ozone exposure (J) during acetylcholine-induced (50 mg/ml) lung resistance measurements. Data are means ± SEM of n = 4 to 7 animals. *P < 0.05, **P < 0.01, ***P < 0.001, Bonferroni multiple comparison test.

  • Fig. 5 FFA4 agonism reduces increased airway resistance and lung inflammation induced by chronic cigarette exposure as well as airway resistance caused by exposure to HDMs.

    Mice were exposed to air or cigarette smoke for 8 weeks. (A) Transpulmonary (Rrs) airway resistance was measured in mice exposed to either control air or cigarette smoke treated for 3 min with nebulized TUG-1197 (0.364 mg/ml), followed by 3 min of nebulized methacholine (30 mg/ml). Mice were administered daily with TUG-891 (0.364 mg/ml) before each smoke exposure, and the effect of chronic TUG891 dosing on (B) macrophages and (C) neutrophil infiltration (in BAL fluid) was measured. Mice were sensitized with subcutaneous injection of either PBS or HDM extracts supplemented with Freunds complete adjuvant (CFA; 1:1 mixture 100 μg HDM) and then challenged with intranasal delivery of PBS or HDM (25 μg). Mice were euthanized and total cell counts (D) in BAL fluid were measured. CD4+ T cells (E) and eosinophils (F) were enumerated by flow cytometry. Mice were administered daily with either vehicle or TUG-891 (0.182 mg/ml) between the sensitization and final HDM challenge, and the prophylactic effect (G) of TUG-891 on lung resistance was measured. Data are means ± SD of three to nine animals. *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA with Bonferroni post hoc test.

  • Fig. 6 FFA4 is expressed in HBECs and human smooth muscle cells and can be detected at both transcript and protein levels.

    (A) Transcript abundances of FFA4 and 18S ribosomal RNA (rRNA) (as control) in bronchial epithelial cells (n = 5 donors) and ASM cells (n = 5 donors) using RT-PCR. A representative gel is shown. The expected sizes of PCR products are FFA4, 251 bp; 18S rRNA, 93 bp. (B) qRT-PCR of bronchial epithelial cells (n = 4 donors) and ASM cells (n = 5 donors) using ACTB housekeeping gene as a control. Data are means ± SEM. (C) Western blot of human FFA4 transiently transfected in human embryonic kidney–293 cells using an antiserum selective for FFA4 developed in-house (hFFA4-AB). (D) Immunofluorescence images of human FFA4 stably transfected in CHO-Flip-In cells (CHO-FFA4) and control nontransfected CHO cells (CHO-NT). (E and F) Example fluorescent histogram of FFA4 expression (black trace) in bronchial epithelial cells and ASM by flow cytometry versus isotype control antibody (gray shading); fold increase in geometric mean fluorescence intensity (GMFI) of anti-FFA4/isotype control antibody (95% confidence interval) 3.019 (2.042 to 3.996) (n = 11 donors, P < 0.01) and ASM cells 2.241 (1.662 to 2.819) (n = 14 donors, P < 0.001) by two-tailed Student’s t test against isotype control. PE, phycoerythrin. (G and H) Representative photomicrograph (×40 magnification) showing FFA4 expression (red; isotype control antibody inserts) in bronchial epithelial cells (n = 5 donors) and ASM cells (n = 4 donors) with nuclei stained blue by 4′,6-diamidino-2-phenylindole (DAPI) immunofluorescence. (I to L) Representative photomicrographs of normal human bronchial biopsy sections stained with (I) isotype control antiserum, (J) human FFA4-selective antiserum (4 μg/ml), and (K) zoom of selected region of the epithelium from (L). EC, epithelial cell layer; ASM, airway smooth muscle; LP, Lamina propria.

  • Fig. 7 Functional responses mediated by FFA4 in isolated human lung tissues and cells.

    (A) Intracellular calcium (iCa2+) elevation in bronchial epithelial cells (n = 6 donors) and (B) ASM cells (n = 6 donors) in response to TUG-891 treatment or ionomycin (1.5 μg/ml) as a positive control. GMFI equates to total stimulated GMFI minus matched baseline minus vehicle control. Data are plotted as means ± SEM. (C) Representative gel photographs taken at 0 hours (basal) and 24 hours either uncontracted or precontracted with carbachol (CCh, 100 μM) followed by treatment of TUG-891 (50 μM). (D) Percentage contraction of collagen gels in the presence of ASM cells (n = 6 donors) either uncontracted or precontracted with carbachol (CCh, 100 μM) followed by treatment of TUG-891 (50 μM). Data are plotted as means ± SEM. (E) Concentration-response data for TUG-891–mediated relaxation of human airway strips precontracted with carbachol (100 μM). Data in (E) are means ± SEM of at least three independent experiments from healthy donors. *P < 0.05, **P < 0.01, ANOVA with Bonferroni post hoc test.

  • Fig. 8 FFA4-mediated airway relaxation in mouse is partly dependent on PGE2 and the prostanoid receptor EP2.

    (A) PGE2 release from PCLS obtained from the lungs of wild-type mice and FFA4 KO(βgal) mice detected by enzyme-linked immunosorbent assay. (B) Effects of PGE2 (500 nM) treatment of PCLS precontracted with carbachol (CCh). (C) TUG-891 response on PCLS pretreated with the selective EP2 receptor antagonist PF-04418948 (10 μM) for 1 hour. (D) PGE2 release into the bronchoalveolar lavage fluid (BALF) of mice exposed to normal air or chronically exposed to ozone and treated with either vehicle (1% dimethyl sulfoxide) or TUG891 (100 μM). Data are means ± SD of at least n = 4 animals. *P < 0.05, **P < 0.01, ANOVA with Bonferroni post hoc test.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/557/eaaw9009/DC1

    Materials and Methods

    Fig. S1. FFA4 activation does not cause airway contraction in murine PCLS.

    Fig. S2. TUG-891-mediated relaxation in precontracted PCLS was reduced by the FFA4 antagonist AH7614.

    Fig. S3. FFA4 agonist compound A mediates ASM relaxation in precontracted PCLS.

    Fig. S4. FFA4 agonism mediates relaxation in airways precontracted with serotonin.

    Fig. S5. FFA4 agonist TUG-891 reduces airway resistance in wild-type C57BL6/N mice.

    Fig. S6. FFA4 agonism did not alter transcript abundance of 5-Lox, BLT1, BLT2, or IL17a but increased expression of LTB4 in a chronic ozone exposure model.

    Fig. S7. FFA4 is expressed in Gr-1+, CD11c+, and Siglec-F+ immune cells.

    Fig. S8. FFA4 is normally expressed and fully functional in ozone-exposed mice.

    Fig. S9. Expression of FFA4 in human lung.

    Fig. S10. PGE2 receptors are expressed in HBECs and human ASM cells.

    Fig. S11. FFA4 agonism up-regulates COX-2 but not other PGE2-related transcripts in a chronic ozone model.

    Fig. S12. FFA4 activation mimics the anti-inflammatory effects of a COX-2 inhibitor but is not altered by the presence of COX-2 inhibition.

    Table S1. FFA4 agonists do not act as off-target agonists at prostanoid receptors.

    Table S2. FFA4 agonists do not act as off-target antagonists at prostanoid receptors.

    Data file S1. Raw data from figures.

    References (6265)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. FFA4 activation does not cause airway contraction in murine PCLS.
    • Fig. S2. TUG-891-mediated relaxation in precontracted PCLS was reduced by the FFA4 antagonist AH7614.
    • Fig. S3. FFA4 agonist compound A mediates ASM relaxation in precontracted PCLS.
    • Fig. S4. FFA4 agonism mediates relaxation in airways precontracted with serotonin.
    • Fig. S5. FFA4 agonist TUG-891 reduces airway resistance in wild-type C57BL6/N mice.
    • Fig. S6. FFA4 agonism did not alter transcript abundance of 5-Lox, BLT1, BLT2, or IL17a but increased expression of LTB4 in a chronic ozone exposure model.
    • Fig. S7. FFA4 is expressed in Gr-1+, CD11c+, and Siglec-F+ immune cells.
    • Fig. S8. FFA4 is normally expressed and fully functional in ozone-exposed mice.
    • Fig. S9. Expression of FFA4 in human lung.
    • Fig. S10. PGE2 receptors are expressed in HBECs and human ASM cells.
    • Fig. S11. FFA4 agonism up-regulates COX-2 but not other PGE2-related transcripts in a chronic ozone model.
    • Fig. S12. FFA4 activation mimics the anti-inflammatory effects of a COX-2 inhibitor but is not altered by the presence of COX-2 inhibition.
    • Table S1. FFA4 agonists do not act as off-target agonists at prostanoid receptors.
    • Table S2. FFA4 agonists do not act as off-target antagonists at prostanoid receptors.
    • References (6265)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Raw data from figures.

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