LRRC8A is essential for hypotonicity-, but not for DAMP-induced NLRP3 inflammasome activation
- Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, United Kingdom
- Lydia Becker Institute of Immunology and Inflammation, University of Manchester, United Kingdom
- Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, United Kingdom
- Genome Editing Unit Core Facility, Faculty of Biology, Medicine and Health, University of Manchester, United Kingdom
- EMBL-ROME, Epigenetics and Neurobiology Unit, Adriano Buzzati-Traverso Campus, Italy
Figures
Figure 1 with 1 supplement
Modelling of proposed VRAC inhibitors on the Cryo-EM structure of LRRC8 channels.
(A) Two orthogonal views: a side view with two chains removed from the hexameric LRRC8A protein with bound DCPIB and a top view for the hexameric VRAC channel (PDB:6NZW). (B, D–I) Docked VRAC …
Figure 1—figure supplement 1
Further modelling of VRAC inhibitors.
(A) Top view and (B) side view of the cryo-EM structure of DCPIB in the arginine pore of VRAC (C–I). Molecular surfaces coloured by electrostatic potential for the docked poses of VRAC inhibitors, …
Figure 2
VRAC inhibitors block hypotonicity-induced Cl- channel opening and regulatory volume decrease (RVD).
(A) Cl- channel opening measured in HeLa cells transiently expressing the halide-sensitive EYFP (H148Q/I152L). HeLa cells were pre-treated with a vehicle control (DMSO) or DCPIB (10 µM) and …
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Figure 2—source data 1
- https://cdn.elifesciences.org/articles/59704/elife-59704-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
VRAC inhibitors differentially regulate NLRP3.
(A) IL-1β release was determined by ELISA on supernatants from murine bone marrow derived macrophages (BMDMs). LPS-primed (1 µg mL−1, 4 hr) BMDMs were pre-treated with either a vehicle control …
Figure 3—figure supplement 1
Dose-response curve for DCPIB.
IL-1β release detected by ELISA on supernatants from LPS-primed (1 µg mL−1, 4 hr) bone-marrow-derived macrophages (BMDM) pre-treated with the indicated dose of DCPIB (0.03–100 µM, 15 min) before …
Figure 4 with 1 supplement
Lrrc8a KO macrophages are unable to undergo hypotonicity-induced regulatory volume decrease (RVD).
(A) Generation of LRRC8A conditional allele. LRRC8A is found on mouse chromosome two and consists of four exons. Untranslated sequences are represented by black boxes, and coding sequences by grey …
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Figure 4—source data 1
source data for data shown in Figure 4C.
- https://cdn.elifesciences.org/articles/59704/elife-59704-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Effect of VRAC inhibitors on the RVD response of WT and Lrrc8a KO BMDMs.
Relative cell size of wild-type (WT) or Lrrc8a knockout (KO) murine bone marrow derived macrophages (BMDMs) incubated in isotonic (340 mOsm kg−1) or hypotonic (117 mOsm kg−1) solution, pre-treated …
Figure 5 with 1 supplement
LRRC8A is dispensable for activation of the NLRP3 inflammasome.
(A) IL-1β release was determined by ELISA on supernatants from wild-type (WT) or Lrrc8a knockout (KO) bone-marrow-derived macrophages (BMDMs). Naïve or LPS-primed (1 µg mL−1, 4 hr) BMDMs were …
Figure 5—figure supplement 1
Loss of LRRC8a does not affect myeloid populations in the peritoneum.
(A–B) IL-6 detected by ELISA in the peritoneal lavage (A) or plasma (B) from wild-type mice. Mice were pre-treated intraperitoneally (i.p.) with a vehicle control, NS3728 (NS3, 50 mg kg−1) or MCC950 …
Figure 6
LRRC8A is an essential component of hypotonicity-induced NLRP3 activation.
(A) IL-1β release was determined by ELISA on supernatants from wild-type (WT) or Lrrc8a knockout (KO) bone marrow derived macrophages (BMDMs). LPS-primed (1 µg mL−1, 4 hr) wild-type (WT) or Lrrc8a …
Videos
Video 1
Phase contrast time-lapse of the regulatory volume decrease of wild-type (WT) littermate bone marrow-derived macrophages (BMDMs).
WT BMDMs were incubated in an isotonic buffer (340 mOsm kg−1) for 5 min before dilution to a hypotonic solution (117 mOsm kg−1) for the duration of the recording. Images were captured every minute …
Video 2
Phase contrast time-lapse of the regulatory volume decrease of Lrrc8a knockout (KO) bone-marrow-derived macrophages (BMDMs).
Lrrc8a KO BMDMs were incubated in an isotonic buffer (340 mOsm kg−1) for 5 min before dilution to a hypotonic solution (117 mOsm kg−1) for the duration of the recording. Images were captured every …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | C57BL/6J | Charles River | C57BL/6NCrl | |
| Genetic reagent (Mus musculus) | C57BL/6J.LRRC8AEm1Uman (Lrrc8afl/fl) | This paper | Line maintained by David Brough lab, University of Manchester | |
| Genetic reagent (Mus musculus) | B6J.B6N(Cg)-Cx3cr1tm1.1(cre)Jung/J | Jackson lab | Stock No: 025524 RRID:IMSR_JAX:025524 | Obtained from breeding colony managed by John Grainger lab (University of Manchester) |
| Cell line (Mus musculus) | Bone-marrow-derived macrophages (BMDMs) | In house | Generated from bone marrow from above mouse lines | |
| Cell line (Mus musculus) | Peritoneal macrophages | In house | Generated by peritoneal lavage from above mouse lines | |
| Cell line (Homo Sapien) | HeLa | ATCC | HeLa (ATCC CCL-2) RRID:CVCL_0030 | |
| Antibody | Anti-mouse IL-1β (goat polyclonal) | R and D Systems | AF-401-NA RRID:AB_416684 | (1:500) |
| Antibody | Anti-Caspase1 + p10 + p12 (rabbit monoclonal) | Abcam | ab179515 | (1:1000) |
| Antibody | Anti- mouse GSDMD (rabbit monoclonal) | Abcam | ab209845 RRID:AB_2783550 | (1:1000) |
| Antibody | Anti-mouse ASC/TMS1 (D2W8U) (rabbit monoclonal) | Cell Signaling Technology | 67824 RRID:AB_2799736 | (1:1000) |
| Antibody | LRRC8A (8H9) (mouse monoclonal) | Santa Cruz | sc-517113 | (1:200) |
| Antibody | Anti-β-Actin−Peroxidase (mouse monoclonal) | Sigma | A3854 RRID:AB_262011 | (1:20000) |
| Antibody | Anti-Rabbit Immunoglobulins HRP (goat polyclonal) | Agilent | P044801-2 RRID:AB_2617138 | (1:1000) |
| Antibody | Anti-Mouse Immunoglobulins HRP (rabbit polyclonal) | Agilent | P026002-2 RRID:AB_2636929 | (1:1000) |
| Antibody | Anti-Goat Immunoglobulins HRP (rabbit polyclonal) | Agilent | P044901-2 | (1:1000) |
| Recombinant DNA reagent | pcDNA3.1 Hygro EYFP H148Q/I152L | Addgene | 25874 RRID:Addgene_25874 | A gift from Peter Haggie |
| Commercial assay or kit | CytoTox 96 Non-Radioactive Cytotoxicity (LDH) Assay | Promega | G1780 | |
| Commercial assay or kit | Mouse IL-1β/IL-1F2 DuoSet ELISA | R and D systems | DY401 | |
| Chemical compound, drug | Lipopolysaccharides from Escherichia coli O26:B6 | Sigma | L2654 | For in vitro experiments |
| Chemical compound, drug | Lipopolysaccharides from Escherichia coli O127:B8 (in vivo) | Sigma | L3880 | For in vivo experiments |
| Chemical compound, drug | Adenosine Triphosphate (ATP) | Sigma | A2383 | |
| Chemical compound, drug | Nigericin sodium salt | Sigma | N7143 | |
| Chemical compound, drug | Silica | U.S. Silica | MIN-U-SIL 15 | |
| Chemical compound, drug | Imiquimod | InvivoGen | R837 | |
| Chemical compound, drug | Tamoxifen | Sigma | T5648 | |
| Chemical compound, drug | 4-Sulfonic calix[6]arene Hydrate | Thermo Fisher | 10494735 | |
| Chemical compound, drug | 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB) | Tocris | 1540 | |
| Chemical compound, drug | Flufenamic acid (FFA) | Sigma | F9005 | |
| Chemical compound, drug | NS3728 | David Brough Lab, University of Manchester | ||
| Chemical compound, drug | CP-456773 sodium salt (MCC950) | Sigma | PZ0280 | |
| Chemical compound, drug | NBC19 | David Brough Lab, University of Manchester | PMID:28943355 | |
| Chemical compound, drug | Calcein, AM, cell-permeant dye | Thermo Fisher | C1430 | |
| Chemical compound, drug | Disuccinimidyl suberate (DSS) | Thermo Fisher | 21555 |
