Role for inducible nitric oxide synthase in protection from chronic Chlamydia trachomatis urogenital disease in mice and its regulation by oxygen free radicals

Abstract

It has been previously reported that although inducible nitric oxide synthase (iNOS) gene knockout (NOS2(-/-)) mice resolve Chlamydia trachomatis genital infection, the production of reactive nitrogen species (RNS) via iNOS protects a significant proportion of mice from hydrosalpinx formation and infertility. We now report that higher in vivo RNS production correlates with mouse strain-related innate resistance to hydrosalpinx formation. We also show that mice with a deletion of a key component of phagocyte NADPH oxidase (p47(phox-/-)) resolve infection, produce greater amounts of RNS in vivo, and sustain lower rates of hydrosalpinx formation than both wild-type (WT) NOS2(+/+) and NOS2(-/-) controls. When we induced an in vivo chemical block in iNOS activity in p47(phox-/-) mice using N(G)-monomethyl-L-arginine (L-NMMA), a large proportion of these mice eventually succumbed to opportunistic infections, but not before they resolved their chlamydial infections. Interestingly, when compared to WT and untreated p47(phox-/-) controls, L-NMMA-treated p47(phox-/-) mice resolved their infections more rapidly. However, L-NMMA-treated p47(phox-/-) mice lost resistance to chronic chlamydial disease, as evidenced by an increased rate of hydrosalpinx formation that was comparable to that for NOS2(-/-) mice. We conclude that phagocyte oxidase-derived reactive oxygen species (ROS) regulate RNS during chlamydial urogenital infection in the mouse. We further conclude that while neither phagocyte oxidase-derived ROS nor iNOS-derived RNS are essential for resolution of infection, RNS protect from chronic chlamydial disease in this model.

FIG. 1

Urine nitrite excretion in response to C. trachomatis MoPn urogenital infection in disease-susceptible C3H/HeN mice and resistant C57BL/6 mice. Urine was collected daily from 6 days prior to infection until day 42 postinfection. Nitrite levels were assessed by the Greiss reaction and standardized according to urine creatinine concentration. The solid line represents the response for C57BL/6 mice. The dashed line represents that for C3H/HeN mice. Following clearance of dietary nitrates and nitrites (day −7 to day zero), a significantly higher level of excretion of nitrite was observed with C57BL/6 mice when the main effects of strain and time were compared (P = 0.0006; Kruskal-Wallis one-way ANOVA on ranks).

FIG. 2

Course of infection as assessed by quantitation of viable C. trachomatis chlamydia shed from the urogenital tract. C. trachomatis MoPn was isolated from cervical-vaginal swabs collected at 4, 7, 10, and 14 days and every 7 days thereafter through day 56. Each data point represents the mean and standard deviation of IFU enumerated in HeLa 229 cultures of swabs from culture-positive mice. Overall, a significantly lower IFU count was observed when the main effects of treatment group and time were compared (P = <0.0001, two-factor ANOVA). The asterisks designate significant differences in the quantitative recovery of viable MoPn chlamydia at the indicated time points postinfection (two-tailed t test).

FIG. 3

Urine nitrite excretion in response to C. trachomatis MoPn urogenital infection in phagocyte oxidase deficient mice. Urine was collected daily from 6 days prior to infection until day 55 postinfection. Nitrite levels were assessed by the Greiss reaction and standardized according to urine creatinine concentration. The solid line represents the response for p47^phox−/− mice treated with either 50 mM L-NMMA (as labeled) or 50 mM l-arginine (labeled p47^phox−/−) given in their drinking water. The dashed line represents that of C57BL/6 mice similarly treated with 50 mM l-arginine. Significantly elevated excretion of nitrite was observed with the l-arginine treated p47^phox−/− mice compared to similarly treated C57BL/6 mice (P < 0.00001; Kruskal-Wallis one-way ANOVA on ranks). This response was significantly blunted by treatment with L-NMMA compared to that of the C57BL/6 or p47^phox−/− controls (P = 0.00001; Kruskal-Wallis one-way ANOVA on ranks).

FIG. 4

Protection from chronic chlamydial disease correlates with higher in vivo iNOS activity. The mean urine nitrite response (day 7 through day 54 postinfection) was calculated for each experimental group from the data displayed in Fig. 3 and is shown here as the solid line graph. The percent hydrosalpinx formation for each group from Table 2 is displayed as the open bars. A negative correlation exists between the mean nitrite excretion during infection and hydrosalpinx formation (correlation coefficient = −0.99952).