A role for connexin43 in macrophage phagocytosis and host survival after bacterial peritoneal infection.

The pathways that lead to the internalization of pathogens via phagocytosis remain incompletely understood. We now demonstrate a previously unrecognized role for the gap junction protein connexin43 (Cx43) in the regulation of phagocytosis by macrophages and in the host response to bacterial infection of the peritoneal cavity. Primary and cultured macrophages were found to express Cx43, which localized to the phagosome upon the internalization of IgG-opsonized particles. The inhibition of Cx43 using small interfering RNA or by obtaining macrophages from Cx43 heterozygous or knockout mice resulted in significantly impaired phagocytosis, while transfection of Cx43 into Fc-receptor expressing HeLa cells, which do not express endogenous Cx43, conferred the ability of these cells to undergo phagocytosis. Infection of macrophages with adenoviruses expressing wild-type Cx43 restored phagocytic ability in macrophages from Cx43 heterozygous or deficient mice, while infection with viruses that expressed mutant Cx43 had no effect. In understanding the mechanisms involved, Cx43 was required for RhoA-dependent actin cup formation under adherent particles, and transfection with constitutively active RhoA restored a phagocytic phenotype after Cx43 inactivation. Remarkably, mortality was significantly increased in a mouse model of bacterial peritonitis after Cx43 inhibition and in Cx43 heterozygous mice compared with untreated and wild-type counterparts. These findings reveal a novel role for Cx43 in the regulation of phagocytosis and rearrangement of the F-actin cytoskeleton, and they implicate Cx43 in the regulation of the host response to microbial infection.

Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide.

Enterocytes exist in close association with tissue macrophages, whose activation during inflammatory processes leads to the release of nitric oxide (NO). Repair from mucosal injury requires the migration of enterocytes into the mucosal defect, a process that requires connexin43 (Cx43)-mediated gap junction communication between adjacent enterocytes. Enterocyte migration is inhibited during inflammatory conditions including necrotizing enterocolitis, in part, through impaired gap junction communication. We now hypothesize that activated macrophages inhibit gap junctions of adjacent enterocytes and seek to determine whether NO release from macrophages was involved. Using a coculture system of enterocytes and macrophages, we now demonstrate that "activation" of macrophages with lipopolysaccharide and interferon reduces the phosphorylation of Cx43 in adjacent enterocytes, an event known to inhibit gap junction communication. The effects of macrophages on enterocyte gap junctions could be reversed by treatment of macrophages with the inducible nitric oxide synthase (iNOS) inhibitor l-Lysine omega-acetamidine hydrochloride (l-NIL) and by incubation with macrophages from iNOS(-/-) mice, implicating NO in the process. Activated macrophages also caused a NO-dependent redistribution of connexin43 in adjacent enterocytes from the cell surface to an intracellular location, further suggesting NO release may inhibit gap junction function. Treatment of enterocytes with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) markedly inhibited gap junction communication as determined using single cell microinjection of the gap junction tracer Lucifer yellow. Strikingly, activated macrophages inhibited enterocyte migration into a scraped wound, which was reversed by l-NIL pretreatment. These results implicate enterocyte gap junctions as a target of the NO-mediated effects of macrophages during intestinal inflammation, particularly where enterocyte migration is impaired.

Hypoxia causes an increase in phagocytosis by macrophages in a HIF-1alpha-dependent manner.

Phagocytosis is the process by which microbial pathogens are engulfed by macrophages and neutrophils and represents the first line of defense against bacterial infection. The importance of phagocytosis for bacterial clearance is of particular relevance to systemic inflammatory diseases, which are associated with the development of hypoxia, yet the precise effects of hypoxia on phagocytosis remain largely unexplored. We now hypothesize that hypoxia inhibits phagocytosis in macrophages and sought to determine the mechanisms involved. Despite our initial prediction, hypoxia significantly increased the phagocytosis rate of particles in vitro by RAW264.7 and primary peritoneal macrophages and increased phagocytosis of labeled bacteria in vivo by hypoxic mice compared with normoxic controls. In understanding the mechanisms involved, hypoxia caused no changes in RhoA-GTPase signaling but increased the phosphorylation of p38-MAPK significantly. Inhibition of p38 reversed the effects of hypoxia on phagocytosis, suggesting a role for p38 in the hypoxic regulation of phagocytosis. Hypoxia also significantly increased the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in macrophages, which was reversed after p38 inhibition, suggesting a link between p38 activation and HIF-1alpha expression. It is striking that small interfering RNA knockdown of HIF-1alpha reversed the effects of hypoxia on phagocytosis, and overexpression of HIF-1alpha caused a surprising increase in phagocytosis compared with nontransfected controls, demonstrating a specific role for HIF-1alpha in the regulation of phagocytosis. These data indicate that hypoxia enhances phagocytosis in macrophages in a HIF-1alpha-dependent manner and shed light on an important role for HIF-1alpha in host defense.