Reactive nitrogen species contribute to innate host defense against Campylobacter jejuni

Abstract

Campylobacter jejuni, a gram-negative, invasive organism, is a common cause of food-borne bacterial diarrheal disease. However, the relationship between C. jejuni and the innate immune system is not well described. To better characterize host defense against C. jejuni, we investigated the ability of nitric oxide/reactive nitrogen species to kill two strains of C. jejuni. C. jejuni viability was measured after exposure to reactive nitrogen species produced biochemically as acidified nitrite and by bone marrow-derived macrophages. We report that acidified nitrite caused a 3-log-increased kill of C. jejuni (P < 0.05) at doses that did not affect the viability of Salmonella enterica serovar Typhimurium. Expression of NOS2, the gene responsible for the production of inducible nitric oxide, was increased >100-fold in murine macrophages after incubation with C. jejuni (P < 0.001). These macrophages effected a 2-log-increased kill of C. jejuni over 24 h compared to that by NOS2-/- macrophages unable to produce nitric oxide (P < 0.05). These findings suggest that the mammalian host upregulates the production of nitric oxide in response to exposure to C. jejuni and that nitric oxide and reactive nitrogen species comprise part of the innate defense mechanisms that contribute to the resolution of C. jejuni infection.

FIG. 1.

C. jejuni is susceptible to killing by acidified nitrite. C. jejuni (CJ) 81-176, C. jejuni 84-25, and S. enterica serovar Typhimurium (ST) 1344 were grown to the late log phase, diluted to 10⁶/ml in brucella broth adjusted to pH 5.0 (A) or 7.0 (B), and incubated with increasing doses of sodium nitrite (NaNO₂) for 150 min at 37°C. Viability is shown as numbers of CFU per ml. The data shown for C. jejuni 81-176 are the means ± SD from six experiments performed in duplicate or triplicate; for C. jejuni 84-25 and S. enterica serovar Typhimurium, the data shown are the means from two experiments performed in triplicate. *, P was <0.05 in a comparison with strain 81-176.

FIG. 2.

C. jejuni is efficiently killed by wild-type macrophages induced to produce NO^·/RNS. C. jejuni 81-176 (A and B) and 84-25 (C and D) were cocultured at an MOI of 1:1 with macrophages pretreated with IFN-γ and LPS or not treated for 3 h prior to the addition of bacteria, and the surviving bacteria were enumerated in the gentamicin protection assay after 2 h (A and C) or 24 h (B and D). The data shown in panels A and B are the means ± standard errors of the means from three experiments performed in duplicate or triplicate; panels C and D show the means ± standard errors of the means from two experiments performed in triplicate. WT, wild-type macrophages; KO, knockout macrophages; *, P was <0.05 in a comparison of results for WT macrophages without IFN-LPS and NOS2^−/− macrophages with and without IFN-LPS; †, P was <0.01 in a comparison of results for the 2-h cocultured WT macrophages with and without IFN-LPS.

FIG. 3.

C. jejuni (CJ) induces NOS2 expression in macrophages. Wild-type BMDM stimulated with 10 ng/ml IFN-γ and 100 ng/ml LPS or not stimulated were incubated at an MOI of 100:1 with C. jejuni 81-176, C. jejuni 84-25, or medium alone for 2 h, followed by the replacement of the medium containing 100 μg/ml gentamicin for 1 h. NOS2 induction was normalized to GAPDH expression and is expressed relative to NOS2 expression in BMDM neither stimulated with IFN-γ-LPS nor infected with C. jejuni by using the Pfaffl equation. For 81-176, the data shown are the geometric means + SD from five experiments performed in duplicate or triplicate, yielding at least 11 independent samples; for 84-25, the data shown are the geometric means + SD from three experiments performed in duplicate or triplicate, yielding at least seven independent samples. *, P was <0.001 in a comparison with unstimulated, uninfected macrophages; †, P was <0.05 in a comparison with C. jejuni 81-176 without IFN-γ-LPS.

FIG. 4.

C. jejuni (CJ) augments RNS production from IFN-LPS-stimulated macrophages. Wild-type macrophages were incubated in medium without IFN-γ-LPS or with IFN-γ-LPS and infected with C. jejuni 81-176 at an MOI of 1:1 or not infected, as described in the legend to Fig. 2B and D. Total nitrite in the supernatant was detected via the Griess reaction after reduction of the nitrite to nitrate. Data shown are the means + SD from two experiments performed in triplicate. *, P was <0.05 in a comparison with unstimulated, uninfected macrophages.