Bisphosphonate drugs have actions in the lung and inhibit the mevalonate pathway in alveolar macrophages
- Garvan Institute of Medical Research and St Vincent’s Clinical School, UNSW Sydney, Australia
- Victor Chang Cardiac Research Institute Innovation Centre, Australia
- School of Pharmacy, University of Queensland, Australia
- BioVinc, United States
- University of Southern California, United States
- Centre for Inflammation, Centenary Institute and University of Technology Sydney, Australia
Figures
Figure 1 with 1 supplement
Bisphosphonate is internalised by alveolar and peritoneal macrophages in vivo.
(a) An in vitro prenylation assay detects unprenylated Rab GTPases (uRabs) in bone marrow-derived macrophages after culture for 24 hr with 1, 5, or 10 μM zoledronic acid (ZOL) compared to 1, 5, or 10 μM AF647-ZOL. (b) FACS plots showing the percentage of labelled single cells in bronchoalveolar lavage (BAL) samples after one intravenous (i.v.) dose of AF647-ZOL compared to saline-treated mice. (c) AF647-ZOL-positive cells were predominantly alveolar macrophages (AMΦ), that is, B220-TCRb-, CD11c+F4/80+ singlets. (d) Histograms show AF647-ZOL mean fluorescence intensity (MFI) of AMΦ in BAL samples from n = 3 control mice (white) and n = 3 AF647-ZOL-treated mice (grey). (e) FACS plot illustrating the percentages of small peritoneal macrophages/SPMΦ (B220-TCRb- singlets, CD11bintF4/80int) and large peritoneal macrophages/LPMΦ (B220-TCRb- singlets, CD11bhiF4/80hi) in peritoneal lavage. (f) Percentage of labelled peritoneal cells 4 hr after one i.v. injection of saline (left) or AF647-ZOL (right). (g) The labelled cell population (AF647-ZOL+, 82.4%) in (f) consists predominantly of CD11bhiF4/80hi LPMΦ. (h) Histograms show the MFI of LPMΦ from saline- (white) and AF647-ZOL-treated (grey) mice. (i) MFI (AF647 MFI) values from SPMΦ and LPMΦ isolated from saline- or AF647-ZOL-treated animals. Bars represent mean ± SD (n = 3 mice per group in (h, i); each symbol represents the measurement from an individual mouse). FACS plots in (b, c, e–g) are representative of three mice per group.
-
Figure 1—source data 1
In vitro prenylation assay of BMDM following treatment with ZOL or AF647-ZOL.
- https://cdn.elifesciences.org/articles/72430/elife-72430-fig1-data1-v1.zip
-
Figure 1—source data 2
In vitro prenylation assay of BMDM following treatment with ZOL or AF647-ZOL, showing cropped regions of the blot (uRabs and loading control).
- https://cdn.elifesciences.org/articles/72430/elife-72430-fig1-data2-v1.zip
Figure 1—figure supplement 1
Percentage of macrophages in bronchoalveolar lavage (BAL) and peritoneal lavage (PL) samples from mice.
BAL and PL samples from untreated mice (n = 4 mice per group) were analysed by flow cytometry. The graph shows the percentage of AMΦ (B220-TCRb-CD11chiF4/80+Siglec-Fhi singlets) and PMΦ (total peritoneal monocyte/macrophages: B220-TCRb-Siglec-F-Ly6G- singlets), large PMΦ (LPMΦ: B220-TCRb-Siglec-F-Ly6G-CD11bhiF4/80hi singlets), and small PMΦ (SPMΦ: B220-TCRb-Siglec-F-Ly6G-CD11bloF4/80lo singlets). Bars represent mean ± standard deviation; each symbol represents the measurement from an individual mouse.
Figure 2
Systemically administered bisphosphonate has pharmacological activity on the mevalonate pathway in alveolar and peritoneal macrophages in vivo.
(a) Flux though the mevalonate pathway (black arrows) enables prenylation of Rab GTPases by utilising geranylgeranyl diphosphate (GGPP) (step 1); inhibition of farnesyl diphosphate (FPP) synthase by nitrogen-containing bisphosphonate (N-BP) (step 2) causes upstream build-up of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (step 3) and prevents downstream Rab prenylation by reducing GGPP synthesis (red arrows, step 4). (b) Detection of IPP/DMAPP (arrowhead) by liquid chromatography tandem mass spectrometry (LC-MS/MS) in cells from bronchoalveolar lavage (BAL) (left) and peritoneal lavage (PL) (right) samples 48 hr after intravenous (i.v.) zoledronic acid (ZOL) or saline treatment. Coloured peaks in the chromatogram depict the relative abundance of IPP/DMAPP. (c) Detection of unprenylated Rab GTPases (uRabs) in cells from BAL (left) and PL (right) samples 48 hr after i.v. ZOL or saline treatment. Data are representative of three separate experiments.
-
Figure 2—source data 1
Original blots of the in vitro prenylation assay of BAL cells and PL cells, following treatment of mice with a single i.v. dose of ZOL.
- https://cdn.elifesciences.org/articles/72430/elife-72430-fig2-data1-v1.zip
-
Figure 2—source data 2
Original blots of the in vitro prenylation assay of BAL cells and PL cells following treatment of mice with a single i.v. dose of ZOL, showing cropped regions of the blots (uRabs and loading control) and molecular mass markers.
- https://cdn.elifesciences.org/articles/72430/elife-72430-fig2-data2-v1.zip
Figure 3 with 1 supplement
Zoledronic acid (ZOL) treatment enhances the production of inflammatory cytokines in response to endotoxin challenge in vivo.
(a) Schedule of intravenous (i.v.) ZOL administration 48 hr prior to intranasal (i.n.) or intraperitoneal (i.p.) lipopolysaccharide (LPS) treatment and subsequent collection of bronchoalveolar lavage (BAL) or peritoneal lavage (PL) fluid, respectively. (b) Multiplex analysis of cytokines and chemokines in BAL fluid from saline- or ZOL-treated mice after i.n. LPS challenge, and (c) in peritoneal fluid, and serum after i.p. LPS challenge. In (b), bars represent mean ± SD, n = 8–9 mice per group with LPS, or n = 3–5 mice per group with ZOL/saline alone; ***p<0.001, ****p<0.0001, ANOVA with Tukey’s post hoc test. In (c), bars represent mean ± SD, n = 5 mice per group; *p<0.05, unpaired t-test with Welch’s correction; each symbol represents the measurement from an individual mouse. (d) Western blot detection of mature, extracellular IL-β in conditioned medium from PL cells, isolated from a ZOL- or saline-treated mouse, then stimulated ex vivo with LPS or LPS+ nigericin. Relative levels of IL-β were calculated by densitometry and are shown below each lane. The blot shown is representative of three independent experiments.
-
Figure 3—source data 1
Western blot of conditioned media using anti-IL-1β shows a single 17 kDa band of cleaved IL-1β.
- https://cdn.elifesciences.org/articles/72430/elife-72430-fig3-data1-v1.zip
Figure 3—figure supplement 1
Zoledronic acid (ZOL) treatment does not affect the percentage or number of alveolar macrophages or cell viability in vivo.
Analysis of AMΦ in bronchoalveolar lavage (BAL) samples from mice injected intravenously (i.v.) with saline (white bars) or ZOL (grey bars) 48 hr prior to intranasal (i.n.) administration of saline (square symbols) or lipopolysaccharide (LPS) (round symbols) (n = 5 mice per group). (a) Percentage (left) and total number (right) of AMΦ (B220-TCRb-CD11chiF4/80+Siglec-Fhi singlets) measured by flow cytometry. (b) Viability (left) and total number (right) of cells in BAL fluid (BALF) measured by trypan blue exclusion. Bars represent mean ± standard deviation; each symbol represents the measurement from an individual mouse.
Figure 4
Potential routes of antimicrobial activity of nitrogen-containing bisphosphonate (N-BP) via effects on the mevalonate pathway in alveolar macrophages.
Inhibition of farnesyl diphosphate (FPP) synthase by N-BP prevents the biosynthesis of isoprenoid lipids required for normal protein prenylation (step 1). Lack of prenylation leads to enhanced inflammasome activation and increased IL-1β release in response to bacterial endotoxin, boosting the initial inflammatory response and pathogen clearance. Lack of isoprenoid biosynthesis may also hinder the propagation of intracellular pathogens that depend on the host cells' mevalonate pathway. Inhibition of FPP synthase by N-BP causes the accumulation of isopentenyl diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) phosphoantigens (step 2) capable of triggering activation and proliferation of human Vγ9VδT cells with antimicrobial activity. Inhibition of FPP synthase by N-BP may also mimic the endogenous antiviral effect of 25-hydroxycholesterol (25HC), one of the routes by which IFN signalling suppresses the flux through the mevalonate pathway.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | BUV395 anti-CD11b (rat monoclonal) | BD Biosciences | Clone M1/70 | Flow cytometry (1:200) |
| Antibody | Biotin anti-F4/80(rat monoclonal) | BioLegend | Clone BM8 | Flow cytometry(1:200) |
| Antibody | FITC anti- I-A/I-E (MHC-II) (rat monoclonal) | BD Biosciences | Clone 2G9 | Flow cytometry (1:200) |
| Antibody | BB515 anti-Siglec-F (rabbit monoclonal) | BD Biosciences | CloneE50-2440 | Flow cytometry(1:200) |
| Antibody | PE anti-CD11c(hamster monoclonal) | BD Biosciences | Clone N418 | Flow cytometry(1:200) |
| Antibody | PerCP-Cy5.5 anti-Ly6G(rat monoclonal) | BioLegend | Clone IA8 | Flow cytometry(1:200) |
| Antibody | BUV737 anti-B220(rat monoclonal) | BD Biosciences | CloneRA3-6B2 | Flow cytometry (1:300) |
| Antibody | PE-Cy7 anti-Ly6C(rat monoclonal) | BD Biosciences | Clone AL-21 | Flow cytometry(1:200) |
| Antibody | APC-Cy7 anti- TCRb(hamster monoclonal) | BD Biosciences | CloneH57-597 | Flow cytometry(1:300) |
| Antibody | Anti-CD16/CD32 (rat monoclonal Fc block) | BD Biosciences | Clone 2.4G2 | Flow cytometry(1:200) |
| Peptide, recombinant protein | Streptavidin | BD Biosciences | BV421-streptavidin | Flow cytometry (1:400) |
| Chemical compound, drug | Zombie Aqua | BioLegend | Cat#: 423101 | Flow cytometry(1:700) |
| Chemical compound, drug | AF647-ZOL | BioVinc, CA | Cat#: SKU BV501001 | Flow cytometry(47.5 μg/40 nmoles per mouse i.v. dose) |
| Chemical compound, drug | Zoledronic acid | Sigma-Aldrich | Cat#: SML0223 | 500 μg/kgi.v. dose |
| Chemical compound, drug | LPS (Escherichia coli) | Sigma-Aldrich | O111:B4 | 10 μg i.n. or 100 μg i.p. per mouse |
| Chemical compound, drug | Biotin-GPP | doi.org.10.1080/21541248.2015.1085485 | Prof Kirill Alexandrov | |
| Peptide, recombinant protein | REP-1(zebrafish) | doi.org.10.1080/21541248.2015.1085485 | Prof Kirill Alexandrov | |
| Peptide, recombinant protein | GGTase-II(rat) | doi.org.10.1080/21541248.2015.1085485 | Prof Kirill Alexandrov | |
| Peptide, recombinant protein | Streptavidin-680RD | LiCOR | P/N: 925-68079 | Western blotting (1:20,000) |
| Antibody | Anti-IL-1β(goat polyclonal) | R&D Systems | Cat#:AF-401-NA | Western blotting (1:1000) |
| Antibody | HRP anti-goat IgG(donkey polyclonal) | Thermo Fisher | Cat#:A15999 | Western blotting (1:5000) |
| Commercial assay or kit | SuperSignal West Pico substrate | Thermo Fisher | Cat#: 34580 | Western blotting |
| Commercial assay or kit | Bio-Plex immunoassay kit | Bio-Rad | Cat#: M60009RDPD | Cytokine and chemokine assay |
| Recombinant protein | M-CSF (human) | Sino Biological | Cat#: 11792-HNAH | Cell culture supplement (50 ng/mL) |
| Chemical compound | Nigericin | Sigma-Aldrich | Cat#: N7143 | |
| Chemical compound | IPP triammonium salt | Sigma-Aldrich | Cat#: I0503-1VL | LC-MS/MS standard |
| Chemical compound | DMAPP triammonium salt | Toronto Research Chemicals | Cat#: 63180-1MG | LC-MS/MS standard |
| Software, algorithm | FlowJo software | Becton Dickinson | RRID:SCR_008520 | Version 10.6.2 |
| Software, algorithm | MassHunter Quantitative Analysis software | Agilent | Version B08.00.00 |
Additional files
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Bisphosphonate drugs have actions in the lung and inhibit the mevalonate pathway in alveolar macrophages
eLife 10:e72430.
https://doi.org/10.7554/eLife.72430
