Detrimental role of endogenous nitric oxide in host defence against Sporothrix schenckii

Abstract

We earlier demonstrated that nitric oxide (NO) is a fungicidal molecule against Sporothrix schenckii in vitro. In the present study we used mice deficient in inducible nitric oxide synthase (iNOS-/-) and C57BL/6 wild-type (WT) mice treated with Nomega-nitro-arginine (Nitro-Arg-treated mice), an NOS inhibitor, both defective in the production of reactive nitrogen intermediates, to investigate the role of endogenous NO during systemic sporotrichosis. When inoculated with yeast cells of S. schenckii, WT mice presented T-cell suppression and high tissue fungal dissemination, succumbing to infection. Furthermore, susceptibility of mice seems to be related to apoptosis and high interleukin-10 and tumour necrosis factor-alpha production by spleen cells. In addition, fungicidal activity and NO production by interferon-gamma (IFN-gamma) and lipopolysaccharide-activated macrophages from WT mice were abolished after fungal infection. Strikingly, iNOS-/- and Nitro-Arg-treated mice presented fungal resistance, controlling fungal load in tissues and restoring T-cell activity, as well as producing high amounts of IFN-gamma Interestingly, macrophages from these groups of mice presented fungicidal activity after in vitro stimulation with higher doses of IFN-gamma. Herein, these results suggest that although NO was an essential mediator to the in vitro killing of S. schenckii by macrophages, the activation of NO system in vivo contributes to the immunosuppression and cytokine balance during early phases of infection with S. schenckii.

Figure 1

Absence of iNOS leads to increased resistance and T cell responsiveness after infection with yeast cells. (a) Survival of WT mice (n = 30) and iNOS^–/– mice (n = 10) following i.v. infection with 5 × 10⁶ yeast cells. Deficient mice had an increased survival rate compared with wild-type controls (*, P < 0·05, log rank test). These data are representative of two independent experiments. (b) CFU counts of S. schenckii in organs of WT and iNOS^–/– mice at 14th day of infection are expressed as means of CFU counts per g/tissue ± SD for groups of five animals each. P < 0·05 (ANOVA followed by Bonferroni's t-test) when compared to WT mice (*). (c) At 14th day of infection, spleen cells from WT (c) and iNOS^–/– (d) mice were harvested and cultured with medium alone, fungal antigen or Con A for 36 hr as described in Methods. The values represent the mean ± SD of three experiments with groups of three mice for each one. P < 0·05 (ANOVA followed by Bonferroni's t-test) when compared to non-infected mice (*) or to WT infected mice (**).

Figure 2

Absence of the iNOS gene reduces apoptosis induced by yeast cells in murine spleen cells. At 14th day of infection, percent apoptosis was determined by measuring Annexin V labelling of freshly explanted spleen cells from WT and iNOS^–/– mice. P < 0·05 (ANOVA followed by Bonferroni's t-test) when compared to non-infected mice (*).

Figure 3

Different cytokine production by spleen cells of WT and iNOS^–/– infected mice. At 14th day of infection, TNF-α (a), IL-10 (b) and IFN-γ (c) production were determined in culture supernatants of spleen cells from WT and iNOS^–/– mice cultured with fungal antigen (a) or Con A (b and c) by 48 hr. Values represent the mean ± SD of three mice in one experiment representative of three performed separately. P < 0·05 (ANOVA followed by Bonferroni's t-test) when compared to non-infected mice (*) or to WT infected mice (#).

Figure 4

Fungicidal activity of macrophages in the absence of iNOS gene is related to IFN-γ induced IDO activity. Peritoneal cells were harvested from WT and iNOS^–/– mice infected (closed symbols) or not (open symbols) with yeast cells for 14 days. Macrophages were activated or not with different doses of IFN-γ and LPS (20 ng/ml) for 4 hr before addition of yeast cells. The percentage of killing (a) and nitrite concentration (b) in supernatants were determined after 18 hr of culture as described in Material and methods. No detected amounts of nitrite accumulation in supernatant of iNOS^–/– mice were detected. *P < 0·05 when compared to non-infected WT mice (ANOVA followed by Bonferroni's t-test).

Figure 5

Treatment of S. schenckii-infected mice with a NO inhibitor also prevents T-cell unresponsiveness and improves fungal disease. (a) Survival of WT mice (n = 30) following i.v. infection with 5 × 10⁶ yeast cells. One group of mice (n = 30) was treated daily with 50 mg/kg of Nitro-Arg to abrogate NO production in vivo. Curves of mortality rate were followed for 30 days after infection. All groups of non-infected mice were inoculated with PBS and presented no mortality and no CFU counts throughout the time. Similar results were obtained in a second experiment. Treated mice had an increased mortality compared with WT controls (*, P < 0·05, log rank test). (b) CFU counts in organs of untreated and Nitro-Arg-treated mice at 14th day of infection are expressed as means of CFU counts per g/tissue ± SD for groups of five animals each. P < 0·05 (a, log rank test; b and c, ANOVA followed by Bonferroni's t-test), when compared to untreated mice (*). (c) At the 0, 7th, 10th, 14th, and 21st day of infection, spleen cells were harvested from WT mice that were treated with PBS or Nitro-Arg. Cultures were incubated for 36 hr in the presence of fungal antigen or Con A as described in Methods. Lymphocyte proliferation was determined by [³H]-thymidine (³HTdR) incorporation during the last 12 hr. Spleen cells incubated with medium alone presented background ³HTdr uptakes of 600 ± 205 and 800 ± 105. P < 0·05 (a, log rank test; b and c, ANOVA followed by Bonferroni's t-test), when compared to non-infected mice (*).