The ion channel CALHM6 controls bacterial infection‐induced cellular cross‐talk at the immunological synapse
Abstract
Synopsis
Introduction
Results
CALHM6 expression in myeloid cells is upregulated by pro‐inflammatory and downregulated by anti‐inflammatory signals
Calhm6−/− mice show normal immune development at steady state
CALHM6 is important for the early innate control of L. monocytogenes infection
CALHM6 controls the kinetics of IFN‐γ production by NK cells
Both Calhm6−/− macrophages and NK cells can respond normally to activating signals when stimulated in isolation in vitro, suggesting a defect in their cell–cell communication in vivo
CALHM6 in CD11c+ dendritic cells is dispensable for NK cell activation and IFN‐γ production
CALHM6 is recruited to immunological synapses formed by activated macrophages
CALHM6 forms an ion channel in the plasma membrane of Xenopus oocytes, but is sequestered in intracellular compartments of mammalian cells prior to activation
CALHM6 channel facilitates ATP release from activated macrophages but, unlike other CALHMs, reduction of extracellular Ca2+ is not sufficient to open CALHM6
Discussion
Materials and Methods
Reagents and Tools table
Reagent or resource | Source | Identifier |
---|---|---|
Antibodies and reagent for immunoblotting | ||
4–20% miniprotean tgx precast gels 12 well | BioRad | 4568095 |
4–20% miniprotean tgx precast gels 15 well | BioRad | 4568096 |
4× Laemmli Sample Buffer, 1610747 | BioRad | 1610747 |
Anti‐HA Tag antibody | Merck | 05‐904 RRID:AB_417380 |
cOmplete EDTA‐free protease inhibitor tablets | Sigma‐Aldrich | 11873580001 |
DTT | Sigma‐Aldrich | GE17‐1318‐02 |
Peroxidase AffiniPure Goat Anti‐Rabbit IgG | Jackson Immuno/Stratech Scientific | 111‐035‐144‐JIR RRID:AB_2307391 |
Goat anti‐mouse IgG1‐hrp | SouthernBiotech | 1071‐05 |
Mouse Anti‐FAM26F Antibody (G‐12) | Santa Cruz | sc‐515780 |
Nativepage novex 4–16% bis‐tris protein gels, 1.0 | BioRad | BN1002BOX |
Nativepage running buffer kit | Thermo Fisher Scientific | BN2007 |
Nativepage sample prep kit | Thermo Fisher Scientific | BN2008 |
Rabbit GAPDH antibody | CST | 2118 RRID:AB_561053 |
Flow cytometry antibodies and reagents | ||
16% Formaldehyde (w/v), Methanol‐free‐10× | Thermo Fisher Scientific | 28908 |
APC anti‐Mouse CD215 (Il‐15rα) | Biolegend | 153505 RRID:AB_2734220 |
APC anti‐mouse NK‐1.1 antibody | Biolegend | 108710 RRID:AB_313397 |
BD cytofix/cytoperm | BD | 554722 |
Brefeldin 1000× Protein Secretion Inhibitor | Biolegend | 420601 |
CD11b anti‐mouse BV711 | Biolegend | 101242 RRID:AB_2563310 |
CD11b anti‐mouse BV785 | Biolegend | 101243 RRID:AB_2561373 |
CD11c anti‐mouse PE | Biolegend | 117307 RRID:AB_313776 |
CD19 anti‐mouse APC‐Cy7 | Biolegend | 115530 RRID:AB_830707 |
CD27 anti‐mouse PE/Dazzle | Biolegend | 124227 RRID:AB_2565793 |
CD3 anti‐mouse PE‐Cy7 | Biolegend | 100220 RRID:AB_1732057 |
CD4 anti‐mouse PE/Dazzle | Biolegend | 100566 RRID:AB_2563685 |
CD8a anti‐mouse BV605 | Biolegend | 100743 RRID:AB_2561352 |
DNAse I, grade ii | Sigma‐Aldrich | 10104159001 |
F4/80 anti‐mouse FITC | Biolegend | 123108 RRID:AB_893502 |
Granzyme B anti‐mouse FITC | Biolegend | 515403 RRID:AB_2114575 |
IFN‐g anti‐mouse PE | Biolegend | 505807 RRID:AB_315401 |
Liberase | Sigma‐Aldrich | 5401135001 |
LIVE/DEAD® Fixable Near‐IR Dead Cell Stain Kit, for 633 or 635 nm excitation | Thermo Fisher Scientific | L10119 |
Ly6C anti‐mouse BV605 | Biolegend | 127639 RRID:AB_2565880 |
Ly‐6c anti‐mouse PE/Dazzle | Biolegend | 128044 RRID:AB_2566577 |
P2X7R anti‐mouse PE | BioLegend | 148703 RRID:AB_2650951 |
Perm wash | BD | 554723 |
Purified rat anti‐mouse CD16/CD32 | BD | 553142 RRID:AB_394657 |
Ultracomp ebeads™ compensation beads | eBioscience | 01‐2222‐42 |
Microscopy antibodies and reagents | ||
Actin Red 555 | Thermo Fisher Scientific | R37112 |
Alexa Fluor® 647 Goat anti‐mouse IgG (minimal x‐reactivity) Antibody | Biolegend | 405322 RRID:AB_2563045 |
Diluent c for general membrane labelling | Thomas Scientific | C914J95 |
Latex beads, polystyrene | Sigma‐Aldrich | LB30‐1ML |
Mouse Anti‐FAM26F Antibody (G‐12) Alexa Fluor® 647 | Santa Cruz | sc‐515780 AF647 |
Mouse Anti‐Flag Antibody, (M2) | Sigma‐Aldrich | F3165 RRID:AB_439685 |
PKH26 red fluorescent cell linker mini kit for general cell membrane labelling | Sigma‐Aldrich | MINI26‐1KT |
Alexa Fluor 647 anti‐DYKDDDDK Tag Antibody | Biolegend | 637315 RRID:AB_2716154 |
Prolong® diamond antifade mounting with DAPI | Thermo Fisher Scientific | P36971 |
Sheep blood in alsevers solution liquid oxoid | Thermo Fisher Scientific | 12977755 |
Chemicals, peptides, and recombinant proteins | ||
Recombinant M‐CSF | Immunotools | 11343118 |
Ampicillin | Sigma‐Aldrich | A9518 |
E. coli LPS 055:B5 | Sigma‐Aldrich | L2637 |
ARL67156 trisodium salt ATPase | Bio‐Techne (R&D Systems) | 1283/10 |
Cell Activation Cocktail (Without Brefeldin A) 500× | Biolegend | 423302 |
Poly(I:C) | Sigma‐Aldrich | P1530‐25mg; CAS:42424‐50‐0 |
Gentamicin | Sigma‐Aldrich | G1397‐10ML |
Ionomycin, calcium salt | Thermo Fisher Scientific | I24222 |
Nigericin | Sigma‐Aldrich | N7143‐5MG; CAS:28643‐80‐3 |
Pen/strep glutamine | Thermo Fisher Scientific | 10378016 |
Puromycin dihydrochloride | PeproTech | P8833 |
Recombinant mouse ifn‐γ (carrier‐free) | Biolegend | 575304 |
Recombinant mouse il‐10 (carrier‐free) | Biolegend | 575804 |
Recombinant mouse il‐12 (p70) (carrier‐free) | Biolegend | 577004 |
Recombinant mouse il‐18 (carrier‐free) | Biolegend | 592102 |
HEPES (for transfection mix for retroviral transduction) | Thermo Fisher Scientific | 15630056 |
Polybrene (for transfection mix for retroviral transduction) | Merck | TR‐1003‐G; CAS:28728‐55‐4 |
DSS (Disuccinimidyl suberate) Crosslinking reagent | Thermo Fisher Scientific | 21655 |
ATP | Thermo Fisher Scientific | 18330019 |
Recombinant mouse il‐4 (carrier‐free) | Biolegend | 574304 |
Recombinant mouse TNF (carrier‐free) | Biolegend | 575204 |
Recombinant mouse TGF‐β1 (carrier‐free) | Biolegend | 763104 |
Recombinant murine GM‐CSF | PeproTech | 250‐05 |
Zymosan a from saccharomyces cerevisiae | Sigma‐Aldrich | Z4250 |
Fura‐2 AM | Invitrogen | F1225 |
Enzyme and reagents for transfection and transduction | ||
BamHI‐HF | NEB | R3136S |
Lipofectamine 2000 | Thermo Fisher Scientific | 11668019 |
Lipofectamine 3000 | Thermo Fisher Scientific | L3000008 |
Neb 5‐alpha competent E. coli (high efficiency) | NEB | C2987H |
Phusion polymerase | NEB | M0530S |
T4 dna ligase | NEB | M0202L |
Polybrene | Merck | TR‐1003‐G |
NotI HF | NEB | R3189S |
Smart MMLV reverse transcriptase 20,000 | Takara Bio Europe SAS | 639524 |
Collagenase type 2 | Worthington‐Biochem | CLS‐2 |
Agel‐HF | NEB | R3552S |
HindIII‐HF | NEB | R3104S |
SalI‐HF | NEB | R3138S |
Assays and kits | ||
IL‐1 beta mouse uncoated ELISA Kit | Thermo Fisher Scientific | 88‐7013‐22 |
Cytotox 96® non‐radioactive cytotoxicity assay (ldh assay) | Promega | G1780 |
Enliten rluciferase/luciferin reagent | Promega | FF2021 |
Magnisort™ mouse CD49b positive selection kit | eBioscience | 8802‐6862‐74 |
Magnisort™ mouse F4/80 positive selection kit | eBioscience | 8802‐6863‐74 |
Mouse IFNg elisa | eBioscience | 88‐7314‐77 |
Mouse IL‐18 elisa | eBioscience | BMS618‐3TEN |
pHrodo Red Dextran, 10,000 MW, for Endocytosis | Thermo Fisher Scientific | P10361 |
pHrodo Red Zymosan Bioparticles Conjugate for Phagocytosis | Thermo Fisher Scientific | P35364 |
Qiagen plasmid plus maxi kit | Qiagen | 12963 |
NEBNext rRNA Depletion Kit (Human/Mouse/Rat) | NEB | #E7400 |
NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina | NEB | #E7765 |
Anti‐F4/89 MicroBeads UltraPure, mouse | Miltenyi Biotec | 130‐110‐443 |
MS columns | Miltenyi Biotec | 130‐042‐201 |
LEGENDplex Mouse inflammation panel | BioLegend | 740150 |
ATP Bioluminescent Assay Kit | Sigma | FLAA‐1KT |
Griess Reagent System | Promega | G2930 |
PowerUp SYBR Green Master Mix | Thermo Fisher Scientific | A25777 |
Qiaquick gel extraction kit | Qiagen | 28704 |
Rneasy mini kit | Qiagen | 74106 |
Maxi Prep Kit | Qiagen | 12123 |
RNA kit‐mMessage mMachine SP6 | Invitrogen | AM1340 |
Experimental models: cell lines | ||
HEK293T cells | Sigma Aldrich | 12022001‐1VL |
N2a (Neuro‐2a) cells | ATCC | CCL‐131 |
Platinum‐E (PlatE) retroviral packaging cell line | eBioscience | RV‐101 |
S‐S.1 (TIB‐111) | American Type Culture Collection | TIB‐111 |
Xenopus oocytes from Xenopus Laevis | Xenopus 1Corp | 4121 |
Experimental models: organisms/strains | ||
C57BL/6J Flp mice (ACTB:FLPe B6J) | The Jackson Laboratory | #005703 |
C57BL/6J Itgaxcre/− mice (B6.Cg‐Tg(Itgax‐cre)1‐1Reiz/J) | The Jackson Laboratory | #008068 |
C57BL/6J PGKCre/− mice | The Jackson Laboratory | #020811 |
C57BL/6J Rag−/− mice (B6.129S7‐Rag1tm1Mom/J) | The Jackson Laboratory | #002216 |
C57BL/6N FAM26Ffl/fl mice | From EUCOMM repository | Generated by Jelena Bezbradica in the Medzhitov lab (Yale School of Medicine, New Haven, CT) |
Plasmids | ||
pMIGRMCS retroviral packaging plasmid expressing GFP, CALHM6‐FLAG, CALHM6‐HA, CALHM1‐HA | Plasmid backbone from Prof Kate Schroder lab (University of Queensland, AU) | Sequence available upon request |
pMMLV mammalian gene expression plasmid expressing IRES eGFP and CALHM6‐eGFP | VectorBuilder | Sequence available upon request |
mCALHM6 untagged plasmid | Origene Technologies Inc | MR204443 |
WT‐mCALHM6 and E119R‐mCALHM6 WT‐mCALHM6‐GFP and E11R‐mCALHM6‐GFP in pBF vector | Subclone with Sall and Agel sites into pBF vector | Sequence available upon request |
mCALHM6‐GFP in pCMV6‐A‐BSD vector | Subclone with BamHI and HindIII sites into pCMV6‐A‐BSD vector | Sequence available upon request |
hCALHM1 and mCALHM6 in pIRES2‐AcGFP1 vector | Subclone with SalI and BamHI sites into pIRES2‐AcGFP1 vector | Sequence available upon request |
Software and algorithms | ||
GraphPad Prism | http://www.graphpad.com/ | RRID: SCR_002798 |
BioRender | http://biorender.com | RRID:SCR_018361 |
SRplot | http://www.bioinformatics.com.cn | |
Fiji | https://fiji.sc/ | RRID: SCR_002285 |
FlowJo | https://fiji.sc/ | RRID: SCR_008520 |
Pulse 8.80 | http://heka.com/ | Pulse 8.80 version |
Igor program | https://www.wavemetrics.com/ | Igor 8.0 version |
Primers for qPCR | ||
Gene | Forward | Reverse |
Calhm1 | TGAGATCTATGATGGGAACTG | CAATGTCAATGTAGTGGGAC |
Calhm2 | AGTATGAGTCTCAGCTCTTC | AGTAATGTTTGAGGCACTTG |
Calhm3 | TGATGGTGGAGGAGTGGC | GCACACACAAAACACTTGCC |
Calhm4 | CACAGATACCAGTCACAAATG | CGATGTCTAATAAGAAGTTGGG |
Calhm5 | GATAACACAGTGGGAAACTG | CTTACAAATCGTGGCAGACC |
Calhm6 | CTCTTTCTACCAATGTGCTG | CTATCAGAATCCAACCGAAC |
Hprt | GTTGGATACAGGCCAGACTTTGTTG | CCAGTTTCACTAATGACACAAACG |
Ifna14 | TCTCTCTCAGGTAAACAGTG | TAGACTCCTTCTGCAATGAC |
Ifna4 | CAAAATCCTTCCTGTCCTTC | ATGATAGAGCTACTACTGGTC |
Ifnb1 | AACTCTGTTTTCCTTTGACC | AACTTCCAAAACTGAAGACC |
Il10 | CAGGACTTTAAGGGTTACTTG | ATTTTCACAGGGGAGAAATC |
Il1r2 | GAAGAGACTTCTTTGACTGTG | AAAACTATGTGGAAGTGTCG |
Il12a | GAAGACATCGATCGATCATGAAGAC | CTCTTGTTGTGGAAGAATC |
Il12b | CATCAGGGACATCATCAAAC | CTCTGTCTCCTTCATCTTTTC |
Primers for genotyping | ||
Calhm6 primer 1 | GTGTAGCAAAGCCTCAGGACAGGT | |
Calhm6 primer 2 | CCAACTGACCTTGGGCAAGAACAT | |
Calhm6 primer 3 | CACACCTCCCCCTGAACCTGAAA | |
Calhm6 primer 7 | CTTTCTGCCCAATCCTCCTG | |
Calhm6 primer 8 | GTTTCCCTAACTGGGCTGTC | |
Cd11c‐control | CAAATGTTGCTTGTCTGGTG | GTCAGTCGAGTGCACAGTTT |
Cd11c‐Cre | ACTTGGCAGCTGTCTCCAAG | GCGAACATCTTCAGGTTCTG |
Flp | ATAGCAGCTTTGCTCCTTCG | TGGCTCATCACCTTCCTCTT |
Flp‐Control | CTAGGCCACAGAATTGAAAGATCT | GTAGGTGGAAATCTAGCATCATCC |
LacZ cassette | GGTAAACTGGCTCGGATTAGGG | TTGACTGTAGCGGCTGATGTTG |
Pgk‐Cre | CGTTTTCTGAGCATACCTGGA | ATTCTCCCACCGTCAGTACG |
Rag | TGGATGTGGAATGTGTGCGAG | CATTCCATCGCAAGACTCCT |
Rag‐control | TCTGGACTTGCCTCCTCTGT | CATTCCATCGCAAGACTCCT |
Methods and Protocols
In vitro stimulation of BMDM, BMDC and NK cells
Quantitative real‐time PCR
Immunoblots
Mouse generation
Listeria monocytogenes infection
Griess assay
Co‐culture experiments
HEK293T transfection and BMDM transduction
N2a cell culture and transfection
RNA sequencing
Confocal imaging
Phagocytosis assays
Ca2+ dependent ATP release assay
Macrophage culture and stimulation for steady state ATP release
Single‐cell calcium imaging
Inflammasome activation
Statistical analysis
Data availability
Author contributions
Disclosure and competing interests statement
Acknowledgements
Supporting Information
References
Information & Authors
Information
Published In
This cover highlights the article DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma by Clara Taffoni, Johanna Marines, Nadine Laguette and colleagues. Two matches (DNA-PK and cGAS) light a candle (tumour). The candle both glows and melts away, representing the pro- or anti-tumorigenic effects of the DNA-PK - cGAS cooperation. (Cover concept by the authors; scientific illustration by Sandra Krahl)
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