Figure 4. 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 after CLP compared to sham-operated controls. (B) Direct measurement of extracellular ATP in the plasma collected from vena cava inferior is elevated in response to CLP and higher in WT mice compared to MAC-CX43 KO mice (each dot is representative for a single animal, unpaired t-test). (C–D) Inhibition (Gap27 (1 µM)) or genetic deletion of CX43 decreased TNF-alpha (C) und IL-6 (D) secretion from peritoneal macrophages in response to stimulation with LPS (1 μg/ml) for 3 and 6 hr (removal of 7 outliers after ROUT test in Figure 4D)*. (E) IL-6 release was restored by activation of purinergic receptors by exogenous administration of ATPуS (10 nM) and abrogated in response to apyrase (100 IU/ml) (each dot is representative of an independent biological replicate, two-way ANOVA, representative of three experiments). (F–G) IL-6 release from peritoneal macrophages in response to LPS (1 µg/ml) and by blocking major P2X (G) and P2Y (H) purinergic receptors (N = 4)*. *Data represent independent biological replicates, compared by unpaired t-test and are representative of three or more independent experiments.

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 differentiation Arg1, Tgfb and Il-10 (unpaired t-test)*. (F) Phagocytic activity of peritoneal macrophages assessed using IgG coated latex beads. (G) TNF-alpha release was not increased by exogenous administration of ATPуS (10 nM) but was abrogated in response to apyrase (100 IU/ml) (N = 5, unpaired t-test)*. *Data represent independent biological replicates and are representative of three or more independent experiments.

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 receptors expression in LPS (1 µg/ml)-stimulated peritoneal macrophages isolated from WT mice treated with 1 µM Gap27 (B = CX43 Blocking) or not (W = Wild type) or isolated from MAC-CX43 KO mice (K = Knock out). Expression was assessed using qPCR (unpaired t-test)*. *Data represent independent biological replicates and are representative of three or more independent experiments.

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 and MAC-CX43 KO peritoneal macrophages after 3 and 6 hr of LPS stimulation and the addition 100 µM ATP (each dot is representative of 5 independent biological samples, one-way ANOVA).