(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. Populations were defined based on F4/80 (BUV395), CD11b (APC/Cy7), Ly6G (PE-Cy7) and CD11c (Pacific blue). In response to CLP, elevated levels of neutrophils (1)/(2) (F4/80low, CD11bhigh, Ly6Ghigh) and small (infiltrating) peritoneal macrophages (3) (F4/80int, CD11bint) were observed but not large (resident) peritoneal macrophages (4) (F4/80high, CD11bhigh) or dendritic cells (5) (F4/80low, CD11blow, Ly6Glow, CD11chigh) (N = 5 animals per group). (B) Relative frequency of large (resident) peritoneal macrophages (LPM) and small (infiltrating) peritoneal macrophages (SPM) in the peritoneal cavity of controls (green) and 10 hr after CLP (orange) (N = 5 animals per group, one-way ANOVA). (C) Extracellular ATP levels in the supernatant of peritoneal macrophages after 30 min stimulation with LPS (TLR-4 agonist) or Pam3CSK4 (PAM, TLR-2 agonist). Cells were isolated from C57 Bl/6 WT mice (each dot is representative of an independent biological replicate, representative of more than five independent experiments, unpaired t-test). (D) Dose-dependent ATP release by peritoneal macrophages in response to LPS as quantified by a luciferin-luciferase assay (N = 12 (control) and 6 (LPS))*. (E) LPS-induced ATP release by macrophages can be blocked by carbenoxolone ((Carbeno) combined pannexin/connexin blockade), 18-alpha-GA (18-a-GA) global connexin blockade), Gap27 (specific blockade of Connexin 43 (CX43)) but not by probenecid ((Prob) pannexin channel blocker) and N-Ethylmaleimide ((NEM) blockade of vesicular exocytosis) (N = 10 (negative and positive controls) and 5 (blockers))*. (F–G) Non-specific (18-a-GA) and specific (Gap27) blocking of connexin hemichannels decreases LPS-induced ATP release from WT peritoneal macrophages in a dose-dependent manner (N = 12 (negative control) and 6 (treated groups), negative and positive controls were the same for both experiments)*. (H) LPS-induced ATP release from WT compared to MAC-CX43 KO peritoneal macrophages with and without specific CX43 blocking (N = 8)*. (I) LPS-induced ATP release has no impact on intracellular ATP levels (N = 8)*. *Data represent independent biological replicates, compared by unpaired t-test and are representative of three or more independent experiments.