Mice lacking inducible nitric oxide synthase demonstrate impaired killing of Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis is a primary etiological agent of generalized severe periodontitis, and emerging data suggest the importance of reactive oxygen and nitrogen species in periodontal tissue damage, as well as in microbial killing. Since nitric oxide (NO) released from inducible NO synthase (iNOS) has been shown to possess immunomodulatory, cytotoxic, and antibacterial effects in experimental models, we challenged iNOS-deficient (iNOS(-/-)) mice with P. gingivalis by using a subcutaneous chamber model to study the specific contribution of NO to host defense during P. gingivalis infection. iNOS(-/-) mice inoculated with P. gingivalis developed skin lesions and chamber rejection with higher frequency and to a greater degree than similarly challenged C57BL/6 wild-type (WT) mice. Chamber fluid from iNOS(-/-) mice possessed significantly more P. gingivalis than that of WT mice. The immunoglobulin G responses to P. gingivalis in serum was similar in WT and iNOS(-/-) mice, and the inductions of tumor necrosis factor alpha, interleukin-1 beta and interleukin-6, and prostaglandin E(2) were comparable between the two mouse strains. Although no differences in total leukocyte counts in chamber fluids were observed between iNOS(-/-) and WT mice, the percentage of dead polymorphonuclear leukocytes (PMNs) was significantly greater in iNOS(-/-) mouse chamber fluids than that of WT samples. Interestingly, casein-elicited PMNs from iNOS(-/-) mice released more superoxide than did WT PMNs when stimulated with P. gingivalis. These results indicate that modulation of superoxide levels is a mechanism by which NO influences PMN function and that NO is an important element of the host defense against P. gingivalis.

FIG. 1.

Increased numbers of P. gingivalis were detected in chamber fluids of iNOS^−/− mice compared to those of WT mice. Subcutaneous chambers were inoculated on day 0 with 10⁸ CFU of P. gingivalis strain A7436, and aliquots of chamber fluid were collected on days 1, 3, 7, and 11. CFU/milliliter values were determined by bacterial growth on anaerobic blood agar plates (n = 10 mice for each group; ✽, P < 0.01 [ANOVA]).

FIG. 2.

IgG titers in serum against P. gingivalis. There was no detectable anti-P. gingivalis IgG before inoculation (day 0) or in the saline-injected mice of either genotype. After P. gingivalis inoculation, WT and iNOS^−/− mice showed increased levels of anti P. gingivalis IgG in serum, as determined by quantitative ELISA on days 7 and 11 after inoculation. No statistical differences were observed in the levels of P. gingivalis-specific IgG in serum between WT and iNOS^−/− mice.

FIG. 3.

Cytokine and PGE₂ levels in chamber fluids of WT and iNOS^−/− mice after P. gingivalis challenge as measured by ELISA. Chambers were inoculated with 10⁸ CFU of P. gingivalis or vehicle on day 0. (A) TNF-α levels measured 1 and 3 days postinoculation; (B) IL-1β levels measured 1 and 3 days postinoculation; (C) IL-6 expression measured 1 and 3 days postinoculation; (D) PGE₂ expression measured in vehicle or in P. gingivalis-inoculated chambers 3 days after inoculation. No statistically significant differences in cytokine levels were found between WT and iNOS^−/− mice after P. gingivalis challenge.

FIG. 4.

Inflammatory cell infiltrate in the chambers of mice challenged with P. gingivalis. (A) Total white blood cell counts were similar between the two genotypes as determined by Trypan blue staining and cell counting. (B) Differential cell counts demonstrated that the dominant leukocyte type in the infected chambers is the PMN. There were no differences in the proportions of PMNs and mononuclear cells in the chamber fluids of WT and iNOS^−/− mice. (C) The ratio of live and dead PMNs in the chamber fluid samples was decreased in iNOS^−/− samples at day 3, as determined by counting the number of dead PMNs out of 100 PMNs after propidium iodine staining (n = nine mice for each group; ✽, P < 0.05 [Student t test]).

FIG. 5.

Peak superoxide release by PMNs in response to fMLP (1 μM) or P. gingivalis (multiplicity of infection = 100). O₂⁻ was measured by using a cytochrome c reduction assay. Data are expressed as the percent increase over baseline O₂⁻ release. P. gingivalis induced significantly greater peak O₂⁻ responses in iNOS^−/− PMNs compared to WT PMNs (n = five mice for each group; ✽, P < 0.05 [iNOS^−/− mice versus WT mice, ANOVA]).