6 figures, 1 table and 3 additional files

Figures

Figure 1 with 2 supplements
Small peritoneal macrophages release ATP during sepsis via Connexin-43 hemichannels.

(A) Intraperitoneal cell fractions characterised by flow cytometry in controls (green) and 10 hr after caecal ligation and puncture (CLP, orange) in a tSNE plot of viable (AmCyanlow) CD45high cells. …

https://doi.org/10.7554/eLife.42670.002
Figure 1—figure supplement 1
Flow cytometry gating strategy for peritoneal inflammatory cells and characterization of ATP release from peritoneal macrophages.

(A) Flow cytometry gating strategy for inflammatory cells isolated from the peritoneal cavity. Identified peritoneal macrophages subsets in controls versus 10 hr after caecal ligation and puncture …

https://doi.org/10.7554/eLife.42670.003
Figure 1—figure supplement 2
Generation of conditional MAC-CX43 KO.

(A) Schematic representation of our conditional Connexin-43 KO mouse model (MAC-CX43 KO). (B) Gja1 (Cx43) mRNA expression in LPS (1 µg/ml)-stimulated peritoneal macrophages isolated from MAC-CX43 KO …

https://doi.org/10.7554/eLife.42670.004
Figure 2 with 1 supplement
Connexin-43 expression is induced on macrophages in a MyD88/TRIF dependent manner.

(A) Flow cytometry gating strategy for human inflammatory cells isolated from fluids obtained from peritoneal lavage during abdominal operations. Macrophages were defined as viable (AmCyanlow), CD45h…

https://doi.org/10.7554/eLife.42670.005
Figure 2—figure supplement 1
Connexin-43 expression on peritoneal macrophages.

(A) Increased expression of CX43 in peritoneal macrophages after 5 hr LPS stimulation (1 µg/ml). (B) Single channel staining for PE-Texas Red-labelled CX43, FITC-labelled F4/80 and DAPI.

https://doi.org/10.7554/eLife.42670.006
Figure 3 with 1 supplement
Connexin-43 expressing macrophages are recruited systemically during sepsis.

(A) Elevated CX43 protein expression in the liver 5 and 10 hr after CLP compared to controls (N = 3 animals in each control group from the gels at 5 hr and 10 hr, all used for the calculation of the …

https://doi.org/10.7554/eLife.42670.007
Figure 3—figure supplement 1
Connexin-43 expression in the liver.

(A) Expression of CX43 in the liver 10 hr after CLP. Data are representative of >3 experiments. (B–C) Following CLP (orange), CD8 positive cytotoxic T cells decreased in the liver, while NK cells …

https://doi.org/10.7554/eLife.42670.008
Figure 4 with 3 supplements
Connexin-43-mediated local and systemic ATP release contributes to macrophage over-activation via P2Y1.

(A) Extracellular ATP levels as assessed by luminescence of i.p. injected 5 × 106 HEK293-pmeLUC D-luciferin activated cells (in counts per second (cps)). Luminescence was significantly higher 10 hr …

https://doi.org/10.7554/eLife.42670.009
Figure 4—figure supplement 1
Characterization of peritoneal macrophage activation. 

(A–B) Quantification of functional markers of M1 macrophage differentiation Inos and Il12rb, in peritoneal macrophages with CX43 blocking or deletion by qPCR (unpaired t-test)*. (C–E) Markers of M2 …

https://doi.org/10.7554/eLife.42670.010
Figure 4—figure supplement 2
Regulation of P2-type receptors on peritoneal macrophages.

(A–B) TNF-alpha release from peritoneal macrophages in response to LPS (1 µg/ml) and by blocking specifically purinergic receptors including P2X (A) and P2Y (B) receptors (N = 4)*. (C) Purinergic …

https://doi.org/10.7554/eLife.42670.011
Figure 4—figure supplement 3
No consequences of Connexin-43 deletion on function of CD73 and CD39 in peritoneal macrophages.

(A–B) CD73 and CD39 expression was assessed by qPCR (each dot is representative of a single independent biological samples, unpaired t-test). (C–D) Kinetic of extracellular ATP degradation from WT …

https://doi.org/10.7554/eLife.42670.012
Figure 5 with 3 supplements
Improved survival and decreased local and systemic cytokine secretion in response to Connexin-43 blocking or deletion during abdominal sepsis.

(A–B) Clinical outcome using Murine Sepsis Score and weight loss following CLP (N = 5 animals per group, two-way ANOVA). (C) Survival of mice following caecal ligation and puncture (CLP). Mice …

https://doi.org/10.7554/eLife.42670.013
Figure 5—figure supplement 1
No consequences of Connexin-43 deletion on local and systemic bacterial load.

(A–B) No difference in bacterial load as determined by CFUs after serial dilution of blood and peritoneal fluid 10 hr post CLP (N = 4 animals per group, Mann-Whitney-test).

https://doi.org/10.7554/eLife.42670.014
Figure 5—figure supplement 2
Impact of CX43 on inflammatory cell recruitment to the peritoneal cavity.

(A) Schematic representation of clustering after CyTOF analysis and dot plot representation of tSNE analysis showing different clusters in the peritoneal cavity 10 hr after CLP compared to control …

https://doi.org/10.7554/eLife.42670.015
Figure 5—figure supplement 3
Consequences of peritonitis on distribution of T cells subsets, IL1beta, IL-6 and MPO.

(A–B) Impact of CX43 on T-lymphocyte recruitment in the peritoneal cavity, CD4 positive T helper and CD8 cytotoxic T cells were similar between MAC-CX43 KO and WT mice (N = 5 animals per group, …

https://doi.org/10.7554/eLife.42670.016
Graphical summary.

(1) Following caecal ligation and puncture (CLP), bacteria invade the peritoneal cavity. (2) Bacteria-derived products, for example LPS, interact with TLR-4 to increase CX43 synthesis in a …

https://doi.org/10.7554/eLife.42670.017

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-Connexin-43
(rabbit, polyclonal)
Cell Signalling3512,
RRID:AB_2294590
WB (1:850)
IHC/IF (1:100)
AntibodyAnti-β-actin
HRP-conjugated
(mouse, monoclonal)
Sigma-AldrichA2228,
RRID:AB_476697
(1:5000)
AntibodyAnti-rabbit
HRP-conjugated
(goat, polyclonal)
DakoP0448,
RRID:AB_2617138
(1:5000)
AntibodyAnti-rabbit
biotinylated
(goat, polyclonal)
DakoE0432,
RRID:AB_2313609
(1:200)
AntibodyAnti-rabbit IgG
FITC-conjugated
(goat, polyclonal)
Sigma-AldrichF0382,
RRID:AB_259384
(1:200)
Cell line
(Homo sapiens)
HEK293-pmeLUCdoi:10.1371/journal.pone.0002599
Chemical
compound
ARL 67156Sigma-Aldrich67156
Chemical
compound
TRIzolRoche11667165001
Chemical
compound
Omniscript RT
Kit 200
Quiagen205113
Chemical
compound
Lipopolysaccharides
E.coli O111:B4
Sigma-AldrichL4130
Chemical
compound
Pam3CSK4Invivogentlrlpms
Chemical
compound
18-alpha-
glycyrrhetinic acid
Sigma-AldrichG8503
Chemical
compound
Gap27Tocris-Bioscience1476
Chemical
compound
ProbenecidSigma-AldrichP8761
Chemical
compound
CarbenoxoloneSigma-AldrichC4790
Commercial
assay
ATP Reagent SLBioThema144–041
Commercial
assay
Resazurin assaySigma-Aldrich199303
Commercial
kit
V-PLEX Mouse
Cytokine 19-Plex kit
Meso Scale
Discovery
K15255D-1
Commercial
kit (ELISA)
TNF-alphaInvitrogen88-7324-22
Commercial
kit (ELISA)
IL-6Invitrogen88-7064-22
Commercial
kit (ELISA)
IL-10Invitrogen88-7105-22
Commercial
kit (ELISA)
IL-1 betaInvitrogen88-7013-86
Commercial
assay
Phagocytic assayCayman Chemical500290
OtherBrewer thioglycollateSigma-AldrichB2551
OtherDMEM/F12 GlutaMAXThermoFisher
Scientific, Gibco
10565018
OtherCTAD tubesBD Vacutainer367599
OtherPeroxidase
labelled streptavidin
Seracare71-00-38(1:200)
OtherDAPI stainSigma-AldrichD9542(1:1000)
OtherFicollGE Healthcare17-5442-02
OtherMyeloperoxidase
assay
doi:10.1097/TP.0000000000000101
Strain, strain
background
(Mus musculus)
Wild-type miceHarlan, NetherlandsC57BL/6J
Strain, strain
background
(Mus musculus)
MyD88/TRIF
double-knockout
mice
Mucosal Immunology,
University of Bern
B6.129-MyD88tm1Aki x
B6-Ticam1lps2
Strain, strain
background
(Mus musculus)
Caspase-1/11
double-knockout
mice
Mucosal Immunology,
University of Bern
B6N.129S2-Casp1tm1Flv
Strain, strain
background
(Mus musculus)
MAC-CX43
knockout mice
Visceral Surgery,
University of Bern
B6.129P2-Lyz2tm1(cre)Ifo/J x
B6.129S7-Gja1tm1Dlg/J
Cell-specific conditional
MAC-CX43 KO mouse model
developed in our laboratory
Strain, strain
background
(Mus musculus)
Lyz2-cre miceJackson LaboratoryB6.129P2-Lyz2tm1(cre)Ifo/J
Strain, strain
background
(Mus musculus)
Gja1 floxed miceJackson LaboratoryB6.129S7-Gja1tm1Dlg/J

Additional files

Supplementary file 1

Anti-mouse antibodies used for Time-of-Flight Mass Cytometry.

(A) Diagnosis of human patients included in the study. (B) P2 receptors antagonists. (C) Visceral Surgery Murine Sepsis Score Sheet. (D) Anti-mouse antibodies used for flow cytometry (E) Anti-human antibodies used for flow cytometry (F) Anti-mouse antibodies used for Time-of-Flight Mass Cytometry

https://doi.org/10.7554/eLife.42670.018
Source data 1

Full uncut western blot membranes.

https://doi.org/10.7554/eLife.42670.019
Transparent reporting form
https://doi.org/10.7554/eLife.42670.020

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