Inducible nitric oxide synthase response and associated cytokine gene expression in the spleen of mice infected with Clonorchis sinensis

Abstract

Clonorchis sinensis is a food-borne parasite that induces a permanent increase of nitrosation in the body upon infection. The spleen is an important secondary lymphoid organ for the regulation of immune responses locally and in the whole body. However, the functions and mechanisms of the spleen in nitric oxide (NO) responses after C. sinensis infection remain unknown. In this study, BALB/c mice were infected with 20, 40, and 80 C. sinensis metacercariae to simulate mild, moderate, and severe infections, respectively. We examined the expression of inducible nitric oxide synthase (iNOS) in the spleen and the relevant cytokine transcription in splenocytes from the mice infected with different amounts of metacercariae. The iNOS of the mice infected with 80 metacercariae was expressed in the spleen as early as 10 days post-infection (dpi) and gradually increased until 90 dpi. The iNOS expression in the mice infected with 40 metacercariae was detected only at 45 and 90 dpi, but not in the mice infected with 20 metacercariae. The level of interferon (IFN)-γ messenger RNA (mRNA) transcription in splenocytes significantly increased at 10 and 20 dpi (P < 0.05) in response to mild/moderate infection but gradually decreased to normal levels after 45 dpi. The level of IL-12p35 mRNA transcription did not change at 10 and 20 dpi but significantly decreased after 45 dpi under moderate/severe infection (P < 0.05/0.01/0.001). The level of IL-18 mRNA transcription significantly increased at 10 dpi (P < 0.05/0.01) but significantly decreased after 20 dpi (P < 0.05/0.01/0.001). These results suggest that spleen is an important organ for iNOS/NO responses, which correspond to the severity of C. sinensis infection, but cannot be attributed to the expression of the Th1 cytokines.

Fig. 1

Specific IgG against C. sinensis levels in the plasma of mice. The mice were infected with 20, 40, or 80 C. sinensis metacercariae by gavage; uninfected mice were used as controls. The IgG antibodies against C. sinensis were detected by ELISA at 10, 20, 45, and 90 dpi. The relative levels of the specific IgG antibodies were expressed with OD₄₅₀ value. *P < 0.05. dpi days post-infection

Fig. 2

INOS mRNA transcription in the splenocytes of mice infected with C. sinensis. The mice were infected with 20, 40, or 80 C. sinensis metacercariae by gavage. The transcript levels were detected at 10, 20, 45, and 90 dpi using qRT-PCR and expressed as fold change over uninfected controls with SEM bars. Significance was determined by comparison with the uninfected control groups: *P < 0.05, **P < 0.01, ***P < 0.001. dpi days post-infection

Fig. 3

INOS expression in the spleen of mice infected with C. sinensis. Slices of the spleens were incubated with iNOS antibody and HRP-conjugated goat anti-rabbit IgG (H + L) by sequence and then processed with AEC color development reagent to show the iNOS expression in the cytoplasm of splenocytes with red color. Finally, the slices were counterstained with hematoxylin to display the splenocyte nucleus with blue. a At 90 dpi, iNOS expressions were detected in the mice infected with 20, 40, or 80 metacercariae and the uninfected controls. The pictures were labeled as 20–90, 40–90, 80–90, and 0–90 accordingly. b Mice were infected with 80 metacercariae, and iNOS expression was detected at 10, 20, and 45 dpi. The uninfected mice were as controls. The pictures were labeled as 80–10, 80–20, 80–45, and 0–0 accordingly

Fig. 3

INOS expression in the spleen of mice infected with C. sinensis. Slices of the spleens were incubated with iNOS antibody and HRP-conjugated goat anti-rabbit IgG (H + L) by sequence and then processed with AEC color development reagent to show the iNOS expression in the cytoplasm of splenocytes with red color. Finally, the slices were counterstained with hematoxylin to display the splenocyte nucleus with blue. a At 90 dpi, iNOS expressions were detected in the mice infected with 20, 40, or 80 metacercariae and the uninfected controls. The pictures were labeled as 20–90, 40–90, 80–90, and 0–90 accordingly. b Mice were infected with 80 metacercariae, and iNOS expression was detected at 10, 20, and 45 dpi. The uninfected mice were as controls. The pictures were labeled as 80–10, 80–20, 80–45, and 0–0 accordingly

Fig. 4

Quantification of IFN-γ, IL-12p35, and IL-18 transcripts in the splenocytes of mice infected with C. sinensis. BALB/c mice were infected with 20, 40, or 80 C. sinensis metacercariae by gavage. The levels of IFN-γ (a), IL-12p35 (b), and IL-18 transcript (c), were detected at 10, 20, 45, and 90 dpi and expressed as fold change over uninfected controls with SEM bars. The dotted lines represent 1-fold change (or no change) over the normal controls. Significance was determined by comparison with uninfected control groups: *P < 0.05, **P < 0.01, ***P < 0.001. dpi days post-infection