4E). These data indicate that the overall number of responding T cells is constant between WT and CGD, but that the inflammatory signal amplitude (i.e. IFN-γ) is increased within individual T cells in proportion to the CGD-associated increase in NO production within APCs. To directly test whether CGD APCs drive increased T-cell-dependent abscess formation in CGD, splenocytes were harvested from WT and CGD animals and depleted of both neutrophils

and T cells. The remaining cells (B cells, macrophages, and DCs; data not shown) were adoptively transferred into WT animals buy AZD1152-HQPA and then each animal was challenged with a seven-fold dilution of the GlyAg and SCC inoculum, which generated an abscess in 0–10% of WT animals (see Fig. 1A). We found that when WT APCs were transferred into the WT animals, 1 out of 8 mice developed an abscess, as before. In contrast, 75% of the WT animals receiving CGD APCs developed an abscess https://www.selleckchem.com/products/nu7441.html (Fig. 4F). These findings demonstrate that CGD APCs are sufficient to transfer the CGD phenotype characterized by increased GlyAg-induced abscess formation. Based on our findings, attenuation of NO production in the first 24 h post challenge should reduce T-cell activation and abscess incidence in CGD. We therefore performed in vitro T-cell activation experiments with CGD cells with and without the specific iNOS inhibitor 1400W.

We found that 1400W reduced the amount of IFN-γ produced by up to 50% as compared with mock-treated cultures (Fig. 5A). Next, L-gulonolactone oxidase WT and CGD animals were challenged with a four-fold dilution of the standard inoculum and compared with another group of CGD animals also treated 0 and 6 h post challenge with 0.5 mg 1400W. Twenty-four hours later, peritoneal lavage fluid was collected and analyzed for NO production. We found a large increase in NO production in CGD animals over

WT (Fig. 5B), reflecting increased iNOS expression (Fig. 3). In addition, 1400W did not eliminate NO production, but reduced NO levels to that seen in WT animals (Fig. 5B). Reducing NO production to WT levels in already immunocompromised CGD mice could result in the inability to clear bacterial challenge, thus we examined bacterial clearance in CGD animals treated with 1400W. Mice were challenged with 106 live B. fragilis, a leading cause of peritonitis associated with intestinal leakage 30, 31, and were treated with 0.5 mg 1400W or PBS vehicle at 0, 6, and 24 h post inoculation. All mice maintained body weight (Fig. 5C and D) and no overt change in activity levels was seen over a 10-day period. On day 8, one mouse in each group was sacrificed and blood agar plates were streaked with tissue samples of blood, liver, spleen, and peritoneal lavage and incubated under anaerobic conditions for 48 h. No bacterial growth was detected from any tissue sample from the PBS or 1400W treated mice (not shown), indicating that 1400W had no deleterious effect on the ability to clear B. fragilis.