Extracellular adenosine produced by ecto-5'-nucleotidase (CD73) regulates macrophage pro-inflammatory responses, nitric oxide production, and favors Salmonella persistence.

Surface enzymes CD39 (nucleoside triphosphate dephosphorylase) and CD73 (ecto-5'-nucleotidase) mediate the synthesis of extracellular adenosine that can regulate immune responses. Adenosine produced by CD39/CD73 acts via adenosine receptors (ARs). CD73 is expressed by a variety of cell types and mediates anti-inflammatory responses. Because efficient innate immune responses are required for clearance of Salmonella infection, we investigated the role of CD73 in macrophage function, including phagocytosis, intracellular killing of Salmonella, and anti-bacterial pro-inflammatory responses to Salmonella-whole cell lysate (ST-WCL) or Salmonella infection. Additionally, RAW 264.7 macrophage mRNA expression of CD39, CD73, and all ARs were measured by qPCR after ST-WCL treatment. Pro-inflammatory cytokine mRNA and nitric oxide (NO) production were quantitated in the ST-WCL treated macrophage with and without CD73-inhibitor (APCP) treatment. Phagocytosis and intracellular killing by peritoneal macrophages from CD73-deficent mice were also evaluated using E. coli BioParticles® and GFP-Salmonella infection, respectively. CD73, CD39, and A2BAR mRNA were predominantly expressed in RAW cells. ST-WCL treatment significantly reduced CD73 expression, suggesting endogenous down-regulation of CD73, and an enhanced pro-inflammatory response. ST-WCL treated and CD73-inhibited macrophages produced more NO and a higher level of pro-inflammatory cytokines than CD73-competent macrophages (e.g. IL-1ß, TNF-α). Phagocytosis of E. coli BioParticles® was significantly higher in the macrophages treated with APCP and in the peritoneal macrophages from CD73-deficent mice as compared to APCP-untreated, and CD73-competent macrophages. Internalized bacteria were more efficiently cleared from macrophages in the absence of CD73, as observed by fluorescence-microscopy and Salmonella-DNA measurement by qPCR from the infected cells. CD73 down-regulation or CD73-inhibition of macrophages during Salmonella infection can enhance the production of pro-inflammatory cytokines and NO production, improving intracellular killing and host survivability. Extracellular adenosine synthesized by CD73 suppresses antibacterial responses of macrophages, which may weaken macrophage function and impair innate immune responses to Salmonella infection.

Involvement of Salmonella enterica serovar Typhi RpoS in resistance to NO-mediated host defense against serovar Typhi infection.

The involvement of nitric oxide (NO) in host defense and cytoprotective functions in murine salmonellosis has been reported. Salmonella mutants with the altered sigma factor RpoS (sigmaS) are less virulent and are susceptible to various stresses. This study investigated the role of the rpoS gene of Salmonella enterica serovar Typhi in NO-dependent host defense in vitro and in vivo. Wild-type mice and mice deficient in inducible NO synthase (iNOS) were infected intraperitoneally or orally with serovar Typhi strains. iNOS-deficient mice were more susceptible to infection by both wild-type and rpoS mutant strains of serovar Typhi and showed extensive apoptotic liver damage compared with wild-type mice. Intracellular killing of Salmonella was analyzed with RAW 264 macrophage-like cells and primary peritoneal macrophages from wild-type and iNOS-deficient mice after cells were infected with the serovar Typhi parent or rpoS mutant strain. The rpoS mutant was more susceptible to killing by macrophages than was the wild-type strain. Also, the wild-type strain produced more extensive apoptotic changes in macrophages than did rpoS mutant. These effects were nullified in RAW 264 cells treated with an NOS inhibitor and in iNOS-deficient primary macrophages. Peroxynitrite susceptibility assays of these strains were also performed. The rpoS mutant Typhi strain was more sensitive to in vitro peroxynitrite treatment than was the parent strain. Together these data show that NO has a significant host defense function during serovar Typhi infection, and that Salmonella RpoS, because it reacts to the presence of NO or its reactive derivatives, is thought to have a role in the pathogenicity of serovar Typhi.

Role of nitric oxide in host defense in murine salmonellosis as a function of its antibacterial and antiapoptotic activities.

Host defense functions of nitric oxide (NO) are known for many bacterial infections. In this study, we investigated the antimicrobial effect of NO in murine salmonellosis by using inducible NO synthase (iNOS)-deficient mice infected with an avirulent or virulent Salmonella enterica serovar Typhimurium strain. All iNOS-deficient mice died of severe septicemia within 6 days after intraperitoneal injection with an avirulent strain (LT2) to which wild-type mice were highly resistant; 50% lethal doses (LD(50)s) of the LT2 strain for iNOS-deficient and wild-type mice were 30 CFU and 7 x 10(4) CFU, respectively. Lack of NO production in iNOS-deficient mice was verified directly by electron spin resonance spectroscopy. Bacterial yields in liver and blood were much higher in iNOS-deficient mice than in wild-type mice throughout the course of infection. Very small amounts of a virulent strain of serovar Typhimurium (a clinical isolate, strain Gifu 12142; LD(50), 50 CFU) given orally caused severe septicemia in iNOS-deficient animals; wild-type mice tolerated higher doses (LD(50), 6 x 10(2) CFU). Histopathology of livers from infected iNOS-deficient mice revealed extensive damage, such as diffuse hepatocellular apoptosis and increased neutrophil infiltration, but livers from infected wild-type mice showed a limited number of microabscesses, consisting of polymorphonuclear cells and macrophages and low levels of apoptotic change. The LT2 strain was much more susceptible to the bactericidal effect of peroxynitrite than the Gifu strain, suggesting that peroxynitrite resistance may contribute to Salmonella pathogenicity. These results indicate that NO has significant host defense functions in Salmonella infections not only because of its direct antimicrobial effect but also via cytoprotective actions for infected host cells, possibly through its antiapoptotic effect.