g. reactive oxygen species) and non-oxidative (e.g. various proteases) mechanisms. The importance of neutrophil function is evident in individuals who have defects in neutrophil chemotaxis,
phagocytic functions or who have neutropenia.[18, 19] These individuals are more prone to bacterial infections. On the other hand, microbicidal molecules released from activated and dying neutrophils can cause bystander damage see more to healthy tissue. The consequent cell injury and death can itself cause or aggravate disease. Accordingly, it is important to elucidate the factors controlling neutrophilic inflammation. In this study we describe the surprising finding that the gut flora influences the ability of animals to mount a systemic acute neutrophilic inflammatory response
in the peritoneum and characterize the underlying basis for this observation. Specific pathogen free (SPF) C57BL/6 mice and IL-1R−/− mice were purchased from The Jackson Laboratories (Bar Harbor, ME). Germ-free C57BL/6 Navitoclax cell line mice were obtained from The National Gnotobiotic Rodent Resource Center, North Carolina State University Gnotobiotics Unit and Gnotobiotic Research Resource, Medical University of South Carolina. MyD88−/− mice were provided by Dr Shizuo Akira, Osaka University, Osaka, Japan or purchased from The Jackson Laboratories. RIP2−/− mice were provided by Dr Michelle Kelliher and RIG-I−/− and MDA5−/− mice were provided by Dr Kate Fitzgerald (University of Massachusetts Medical School, Worcester, MA). NOD1−/− mice were aminophylline provided by Dr Grace Chen, University of Michigan, Ann Arbor, MI. For generating the tamoxifen-inducible deletion mutant mice of MyD88, we used a strategy similar to the one described
previously. MyD88−/− mice were crossed to the whole tissue, tamoxifen-inducible Cre transgenic mice (Rosa26-Cre/ESR+/+) (provided by Dr Roger Davis, University of Massachusetts Medical School, Worcester, MA). The resultant offspring, MyD88+/− Rosa26-Cre/ESR+/− mice were crossed to the MyD88flox/flox mice (provided by Dr Robert Finberg, University of Massachusetts Medical School, Worcester, MA) to generate the MyD88−/flox Rosa26-Cre/ESR+/− (conditional knockout; cKO). Animals were housed and handled according to protocols approved by the University of Massachusetts animal care and use committee. Mice were injected intraperitoneally with 0·2 mg zymosan (Sigma-Aldrich, St Louis, MO), 0·5 mg silica crystals (Sigma-Aldrich), 0·5 mg monosodium urate crystals or 5 ng recombinant murine MIP-2 (R&D Systems, Minneapolis, MN) in 0·2 ml PBS. For the thioglycollate injections, 1 ml of 3% thioglycollate (Thermoscientific, Lenexa, KS) was used. The monosodium urate crystals were prepared as described before. Mice were killed by exposure to isoflourane 4–16 hr after the injection. The peritoneum was lavaged with 2 ml Dulbecco’s modified Eagle’s medium with 2% fetal calf serum, 3 mm EDTA and 10 U/ml heparin.