Inducible nitric oxide synthase deficiency in mice increases resistance to chronic infection with Echinococcus multilocularis - PubMed (original) (raw)

Inducible nitric oxide synthase deficiency in mice increases resistance to chronic infection with Echinococcus multilocularis

Wen J Dai et al. Immunology. 2003 Feb.

Abstract

The production of nitric oxide (NO) by intraperitoneal macrophages of mice during secondary infection with Echinococcus multilocularis mediates immunosuppression at early and late stages of infection. We addressed the role of NO in host resistance against this extracellular metazoan parasite by infecting inducible nitric oxide synthase knockout ((iNOS-KO) mice (of the C57BL/6 background) with 100 metacestode vesicles. The parasite weight was significantly lower in iNOS-KO mice when compared with wild-type (WT) mice at 4 months postinfection (late stage), thus demonstrating that iNOS deficiency confers a certain degree of resistance against persistent chronic infection. However, histological analysis of periparasitic tissue showed no differences between WT and iNOS-KO mice, as both exhibited granuloma formation and the presence of giant cells. Together with histology, the production of a high level of interferon-gamma (IFN-gamma) in infected iNOS-KO mice upon stimulation with concanavalin A (Con A) and VF-antigen indicated normal T-cell signalling in these animals. As expected, peritoneal exudate cells (PEC) from infected iNOS-KO mice produced no detectable NO, while the PEC from infected WT mice produced high levels of NO after stimulation with lipopolysaccharide (LPS) and parasite protein or carbohydrate antigen, or even without in vitro stimulation. Consequently, the high level of NO production observed during chronic infection in WT mice appears to contribute more to immunosuppression than to limitation of parasite growth. This is also reflected by the fact that splenocyte proliferation was significantly higher and parasite masses lower in iNOS-KO mice (at 1 and 4 months postinfection) than in WT mice.

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Figures

Figure 1

Figure 1

Mean metacestode tissue masses (in mg) at 1 and 4 months postinfection (p.i.) (1

M

and 4

M

, respectively). White bars represent the values obtained from wild-type (WT) mice, black bars the values from inducible nitric oxide synthase-knockout (iNOS-KO) mice. Indicated are the mean values + standard errors per group of 20 mice (data represent four independent experiments with five animals per each group). *Statistically significant differences (P < 0.05).

Figure 2

Figure 2

Comparative histopathological investigation of _Echinococcus multilocularis_-infected wild-type (WT) mice (panels a, c and e) and inducible nitric oxide synthase (iNOS)-deficient mice (panels b, d and f) showed no difference between lesions of WT and iNOS-knockout (KO) mice at 1 and 4 months postinfection (p.i.). Panels (a), (b), (c) and (d) show lesions of mice at 1 month p.i. at low and high magnification. There was a massive fibrosis (panels a and b) with numerous multinucleated giant cells and epithelioid cells (panels c and d) in both wild-type (2a, 2c) and iNOS-KO mice (2b, 2d). There was still no significant difference between lesions of WT (Fig. 2e) and KO (Fig. 2f) mice at 4 months p.i. The magnification was ×160 for (a) and (b), and ×320 for (c), (d) and (f).

Figure 3

Figure 3

Interferon-γ (IFN-γ) production in infected wild-type (WT) and inducible nitric oxide synthase-knockout (iNOS-KO) mice upon stimulation with concanavalin A (Con A) and VF-antigen in vitro. Splenocytes were collected at 4 months postinfection (p.i.). Identical results were obtained from animals at 1 month p.i. (data not shown). The figure shows results from one representative out of two independent experiments. *, † and ‡ indicate statistically significant differences, assessed upon five different measurement points per group (P < 0.05).

Figure 4

Figure 4

Determination of nitric oxide (NO) production in culture supernatants. Peritoneal exudate cells (PEC) from 1-month-infected mice were left unstimulated (–) or were stimulated in vitro with lipopolysaccharide (LPS) or parasite VF- and Em2(G11)-antigen. White bars refer to non-infected control mice, black bars to wild-type (WT) mice and striped bars to inducible nitric oxide synthase-knockout (iNOS-KO) mice. Each point was measured in quadruplicate. Supernatants were harvested after 24, 48 and 72 hr (LPS) or 96 hr (VF-antigen and Em2(G11)-antigen), respectively; nitrite (NO2−) accumulation was assessed using the Griess method. This is one representative out of four independent experiments with similar patterns of NO production.

Figure 5

Figure 5

The proliferative responses to concanavalin A (Con A)- and parasite VF- and Em2-antigen stimulation of spleen cells from non-infected (white bars), infected wild type (WT) (black bars) and infected inducible nitric oxide synthase-knockout (iNOS-KO) mice (striped bars) were determined at different time-points [1 and 4 months postinfection (p.i.)]. The figure shows results from one representative out of four independent experiments exhibiting similar results. The suppression of concanavalin A (Con A)-derived proliferation at an early stage was repeated in four independent experiments, with comparable results obtained. *, †, ‡ and § indicate statistically significant differences. The results were calculated as the mean counts per minute (c.p.m.) of quadruplicate wells and expressed as geometric mean minus background (Δc.p.m.), where background = c.p.m. of wells containing pulsed but unstimulated cells.

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