Could Connexin 43 dependent ATP release represent a new therapeutic target for sepsis?

Abstract

Abstract During sepsis, ATP is released into the extracellular space where it modulates immune response via specific receptors and ectonucleotidases present on various immune cells. ATP can be released by hemichannel Connexin 43 (Cx43). Here, we examine the importance of Cx43 mediated ATP release in macrophages and the role of this pathway in modulating innate immune responses in the context of sepsis. For murine sepsis model, caecal ligation and puncture (CLP) was used. Mice were treated with Gap27, a Cx43 inhibitor. Liver, lung, spleen, bone marrow, peritoneal fluid and blood cells expressing Cx43 were characterized (IHC, FACS). Macrophages were isolated from peritoneum and liver of wild-type mice and stimulated with PAMPs. ATP was quantified using luciferin-luciferase assay. Cytokine levels (ELISA), gene expression (qPCR) and protein expression (immunoblot) were assessed. Blocking of Cx43 during CLP prolonged survival up to 48 hours in comparison with 24 hours for controls. In addition, it decreased systemic levels of inflammatory cytokines (TNFa, IL1b and IL6) and chemokines (CCL2) and lowered bacterial load (CFU/ml) in the blood and peritoneal fluid. Cx43 was not constitutively expressed in the liver, but was induced following CLP. Cells expressing Cx43 in septic livers were identified as infiltrating M1 macrophages and neutrophils. Ex vivo, Cx43 expression was upregulated in primary macrophages upon stimulation with LPS. Macrophages were actively releasing ATP in response to TLR 4 and 2 agonists, and Cx43 inhibition reduced extracellular ATP levels. We observed improved survival to sepsis by blocking Cx43. By mediating release of ATP, and potentially other DAMP, Cx43 might overactivate macrophages and exacerbate the immune response.