T-Cell responses during Trypanosoma brucei infections in mice deficient in inducible nitric oxide synthase

Abstract

We have investigated the possibility that nitric oxide (NO) synthesis may affect the course of a trypanosome infection via T-cell responses using mice deficient in inducible NO synthase (iNOS). Parasitemia levels increased at the same rate in both iNOS-deficient homozygous and control heterozygous mice, and peak parasitemia values were the same in both groups. However, the heterozygous mice maintained higher parasitemia levels after the peak of an infection than the homozygous mice due to a decrease in the rate of clearance of parasites. In iNOS-deficient mice there was an increase in the numbers of total CD4(+) cells and activated (interleukin-2 receptor-expressing) CD4(+) cells in infected mice compared with the numbers in uninfected mice. Spleen cells from infected iNOS-deficient mice displayed increased proliferative responses and gamma interferon secretion when stimulated in vitro than those of control mice. These data suggest that NO production depresses T-helper 1-like responses generated during Trypanosoma brucei infections, thus promoting the survival of the parasite.

FIG. 1

Time course of T. brucei parasitemia in iNOS-deficient (○) and heterozygous control (●) mice inoculated with 10⁴ GUTat 7.2 parasites. Results are expressed as geometric means ±2 SE for groups of five mice.

FIG. 2

Numbers of CD4⁺ (light) or CD8⁺ (dark) cells per spleen (A) and numbers of activated CD4⁺ (light) or CD8⁺ (dark) cells per spleen, with CD25 as a marker for cell activation (B). Numbers were determined by flow cytometry of mononuclear splenocytes of iNOS-deficient (−/−) and control (+/−) mice, either uninfected or infected with GUTat 7.2 (results are for day 11 of infection). Mononuclear splenocytes were counted, the lymphocyte population was gated and checked to exclude autofluorescence, and relative numbers were determined. From these data, cell numbers per spleen were calculated and are expressed as geometric means ±2 SE for groups of five mice.

FIG. 3

Proliferative responses of mononuclear splenocytes from trypanosome-infected iNOS-deficient (−/−) (dark) and control (+/−) (light) mice harboring infections of GUTat 7.2. Splenocytes were cultured in the presence of either medium or ConA (8 μg/ml). Proliferative responses of mononuclear splenocytes from uninfected mice were <2,000 cpm in the absence of stimulation. Results are expressed as the geometric means ±2 SE for groups of five mice.

FIG. 4

IFN-γ production by mononuclear splenocytes from iNOS-deficient mice and their heterozygous counterparts harboring infections with GUTat 7.2. IFN-γ concentrations were assayed by enzyme-linked immunosorbent assay. Cells were stimulated with medium, ConA (8 μg/ml), or paraformaldehyde-fixed trypanosomes (2 × 10⁶/ml) expressing the homologous VAT or one of the two heterologous VATs. Supernatants were harvested 72 h after stimulation. Results are expressed as means ±2 SE for groups of five mice.