Macrophages were infected with LVS (MOI = 5; confirmed by colony count) and incubated at 37 C

Macrophages were infected with LVS (MOI = 5; confirmed by colony count) and incubated at 37 C. humans, yet causes a disease resembling human tularemia in mice when administered by some routes of contamination (4). Contamination of mice with LVS results in a pronounced inflammatory response, yet the organism is able to persist within host cells (6,7). Macrophages differentiate into functionally unique immunological populations depending on the cytokine environment. Macrophages exposed to IFN- and LPS become classically activated (CA-M) and function predominantly in LDN-192960 hydrochloride inflammation, tissue damage, killing of intracellular microbes, and increased tumoricidal activity (rev. in 8). Conversely, certain cytokines, specifically IL-4 and IL-13, induce an alternatively activated phenotype in macrophages. AA-M are principally associated with allergic and parasitic immune responses, tissue remodeling, angiogenesis, tumor promotion, and humoral immunity (rev. in 8), and more recently, have been associated with Alzheimers Disease (9,10). Some organisms, such as may avoid macrophage-mediated killing by altering the macrophage differentiation state from one that is classically activated to one that is alternatively activated. We observed that after an initial, strong proinflammatory response, LVS contamination induces expression of markers associated with AA-M both in vitro and in vivo. This reprogramming of macrophage differentiation promotes the survival and replication of the bacterium while mitigating the pro-inflammatory response. The failure of LVS contamination to induce AA-M in IL-4R?/? or STAT6?/? LDN-192960 hydrochloride macrophages results in a sustained CA-M phenotype and clearance of the bacterium. In vivo, IL-4R?/? mice exhibited increased survival compared to WT mice. Antibody-mediated neutralization of IL-4 and IL-13 also reversed the AA-M phenotype in wild-type (WT) macrophages, blocked production of IL-4 and IL-13 by macrophages, increased IL-12 p70 secretion, and curtailed intracellular replication. Our data support the notion that macrophage differentiation is usually malleable, allowing for rapid responses to environmental conditions. This study provides new insights into the innate immune response to LVS contamination and the mechanism by which LVS evades the host innate immune response. Materials and Methods Reagents Murine rIL-4 and rIFN- were purchased from R&D Systems, Inc. K235 LPS was prepared as previously explained (15). Rabbit anti-mouse FIZZ1 ELF3 antibody was isolated from serum (kindly provided by Dr. Steven Kunkel, Univ. of Michigan) using an ImmunoPure (A) IgG Purification kit (Pierce Endogen) per the manufacturers protocol. The following antibodies were purchased: anti-murine arginase-1 MAb (IgG1) (BD Biosciences); polyclonal goat anti-mouse IL-12 p70, anti-IL-4 MAb, anti-IL-13 MAb (R&D Systems, Inc.); anti-mouse CD206 (mannose receptor):FITC (IgG2a) (AbD Serotec); anti-murine F4/80 MAb (IgG2b) (Abcam); mouse IgG2a and IgG1, rat IgG2b, goat IgG, and rabbit IgG isotype control Abs (Sigma); Cy2-conjugated donkey anti-rabbit IgG, Cy2-conjugated donkey anti-mouse IgG, and Cy3-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Labs). Frozen aliquots of LVS (ATCC 29684; American Type Culture Collection were kindly provided by Dr. Karen Elkins (FDA, Rockville, MD) and stored as previously explained (16). Macrophage cell culture Six to 8-week aged C57BL/6J, TLR2?/? (B6.129-Tlr2 tm1Kir /J), and BALB/cByJ mice were purchased from your Jackson Laboratory. Breeding pairs of IL-4R?/? and STAT6?/? mice on a BALB/c background were obtained from Dr. LDN-192960 hydrochloride Nancy Noben-Trauth (George Washington University or college Medical Center, Washington, DC) and Dr. William E. Paul (Laboratory of Immunology, NIAID, NIH, Bethesda, MD), respectively, and bred within UMBs accredited facility. Peritoneal macrophages isolated from mice 4 days after i.p. injection of sterile 3% thioglycollate were cultured as explained previously (17). Macrophages were plated in 6-well (4 106 cells/well) or 12-well (2 106 cells/well) tissue.