Both inducible nitric oxide synthase and NADPH oxidase contribute to the control of virulent phase I Coxiella burnetii infections

Abstract

Host control of Coxiella burnetii infections is believed to be mediated primarily by activated monocytes/macrophages. The activation of macrophages by cytokines leads to the production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that have potent antimicrobial activities. The contributions of ROI and RNI to the inhibition of C. burnetii replication were examined in vitro by the use of murine macrophage-like cell lines and primary mouse macrophages. A gamma interferon (IFN-gamma) treatment of infected cell lines and primary macrophages resulted in an increased production of nitric oxide (NO) and hydrogen peroxide (H2O2) and a significant inhibition of C. burnetii replication. The inhibition of replication was reversed in the murine cell line J774.16 upon the addition of either the inducible nitric oxide synthase (iNOS) inhibitor NG-monomethyl-L-arginine (NGMMLA) or the H2O2 scavenger catalase. IFN-gamma-treated primary macrophages from iNOS-/- and p47phox-/- mice significantly inhibited replication but were less efficient at controlling infection than IFN-gamma-treated wild-type macrophages. To investigate the contributions of ROI and RNI to resistance to infection, we performed in vivo studies, using C57BL/6 wild-type mice and knockout mice lacking iNOS or p47phox. Both iNOS-/- and p47phox-/- mice were attenuated in the ability to control C. burnetii infection compared to wild-type mice. Together, these results strongly support a role for both RNI and ROI in the host control of C. burnetii infection.

FIG. 1.

Estimated generation times for C. burnetii in J774 cells and primary murine macrophages. Generation times were calculated by the formula G = t/[3.3 × log(b/B)], where G is the generation time, t is the time interval, in hours, b is the number of bacteria at the end of the time interval, and B is the number of bacteria at the beginning of the time interval. (A) Replication rates in J774.16 cells. J774.16, 45 h; J774D.9, 20 h. (B) Replication rates in peritoneal macrophages. Wild-type PMφ, 26 h; p47^phox−/− PMφ, 19 h; iNOS^−/− PMφ, 17 h. C. burnetii replication rates in cells lacking either iNOS or NADPH oxidase were significantly (P < 0.05) faster than replication rates in wild-type cells.

FIG. 2.

Nitrite and hydrogen peroxide levels in J774.16 and J774D.9 cells. The cells were infected for 24 h with virulent phase I C. burnetii and then were treated with 100 U of murine rIFN-γ/ml in thepresence or absence of the iNOS inhibitor NMMLA (1 mM) or the hydrogen peroxide scavenger catalase (100 μM). The stimulation of iNOS was indirectly determined by measuring nitrite, which is a stable end product of nitric oxide production. Hydrogen peroxide levels were determined by measuring the horseradish peroxidase-dependent oxidation of phenol red. (A) Nitrite levels in J774.16 cells. (B) Nitrite levels in J774D.9 cells. (C) Hydrogen peroxide levels in J774.16 cells. (D) Hydrogen peroxide levels in J774D.9 cells. (A and B) Stippled bars, no treatment; diagonally striped bars, treatment with 100 U of IFN-γ/ml; vertically striped bars, treatment with 100 U of IFN-γ/ml and 1 mM NMMLA. (C) Stippled bars, no treatment; diagonally striped bars, treatment with 100 U of IFN-γ/ml; vertically striped bars, treatment with 100 U of IFN-γ/ml and 100 μM catalase. (D) Stippled bars, no treatment; diagonally striped bars, treatment with 100 U of IFN-γ/ml. The results are expressed as means and standard errors for three replicates from two independent experiments. Asterisks indicate significant differences between treated and untreated samples within the same time points.

FIG. 3.

Effects of IFN-γ treatment on C. burnetii growth in J774.16 and J774D.9 cells. At 48, 96, and 144 h postinfection, infected cells were harvested, and the numbers of C. burnetii organisms, represented as numbers of Coxiella genomes, were determined by real-time PCR. (A) J774.16 cells were infected for 24 h with virulent phase I C. burnetii and then were treated with 100 U of murine rIFN-γ/ml in the presence or absence of the iNOS inhibitor NMMLA (1 mM) or the hydrogen peroxide scavenger catalase (100 μM). (B) J774D.9 cells were infected for 24 h with virulent phase I C. burnetii and then were treated with 100 U of murine rIFN-γ/ml in the presence or absence of the iNOS inhibitor NMMLA (1 mM) or the ROI donor HX/XO (250 μM/30 μM). The results are expressed as means and standard errors for three replicates from two independent experiments. Asterisks indicate significant differences between treated and untreated samples within the same time points.

FIG. 4.

Growth of virulent phase I C. burnetii in primary murine peritoneal macrophages. Macrophages were infected for 24 h with virulent phase I bacteria and then treated for 5 days with or without 100 U of IFN-γ/ml. Nitrite and hydrogen peroxide levels were determined for primary murine peritoneal and bone marrow-derived macrophages. Levels of nitrite (A) and hydrogen peroxide (B) were measured at 144 h postinfection as described for the J774 cell lines. Stippled bars, no treatment; diagonally striped bars, treatment with 100 U of IFN-γ/ml. (C) At 144 h postinfection, peritoneal macrophages were harvested, and the numbers of C. burnetii were determined by real-time PCR. Stippled bars, wild type; diagonally striped bars, p47^phox−/−; vertically striped bars, iNOS^−/−. The results are expressed as means and standard errors for three replicates. Asterisks indicate significant differences between treated and untreated samples.

FIG. 5.

Splenomegaly in C57BL/6, iNOS^−/−, and p47^phox−/− mice. Mice were challenged intraperitoneally with 10⁴ or 10⁷ C. burnetii cells. At 14 (A) and 28 (B) dpi, spleens were collected and evaluated for splenomegaly. Stippled bars, wild type; diagonally striped bars, iNOS^−/−; vertically striped bars, p47^phox−/−. The results are expressed as means and standard errors for four mice. Asterisks indicate significant differences between wild-type and knockout mice.

FIG. 6.

Immunolocalization of C. burnetii in cardiac valve sections from mice challenged with 10⁷ C. burnetii organisms at 28 days postinfection. C. burnetii was detected by the ABC method (Vector Laboratories). (A) Valve section from wild-type mouse; (B) valve section from iNOS^−/− mouse; (C) valve section from p47^phox−/− mouse. The red to rust-colored deposits represent the presence of C. burnetii (arrows denote examples). At, atrial side; V, ventricular side. Magnification, ×40.