TLR2 signal influences the iNOS/NO responses and worm development in C57BL/6J mice infected with Clonorchis sinensis

Although the responses of inducible nitric oxide synthase (iNOS) and associated cytokine after Clonorchis sinensis infection have been studied recently, their mechanisms remain incompletely understood. In this study, we investigated the effects of toll-like receptor 2 (TLR2) signals on iNOS/nitric oxide (NO) responses after C. sinensis infection. We also evaluated the correlations between iNOS responses and worm development, which are possibly regulated by TLR2 signal.

TLR2 wild-type and mutant C57BL/6 J mice were infected with 60 C. sinensis metacercariae, and the samples were collected at 30, 60, 90 and 120 days post-infection (dpi). The total serum NO levels were detected using Griess reagent after nitrate was reduced to nitrite. Hepatic tissue samples from the infected mice were sliced and stained with hematoxylin and eosin (HE) to observe worm development in the intrahepatic bile ducts. The iNOS mRNA transcripts in the splenocytes were examined by real time reverse transcriptase polymerase chain reaction (qRT-PCR), and iNOS expression was detected by immunohistochemistry.

Developing C. sinensis juvenile worms were more abundant in the intrahepatic bile ducts of TLR2 mutant mice than those of TLR2 wild-type mice. However, no eggs were found in the faeces of both mice samples. The serum levels of total NO significantly increased in TLR2 mutant mice infected with C. sinensis at 30 (t ₍₅₎ = 2.595, P = 0.049), 60 (t ₍₅₎ = 7.838, P = 0.001) and 90 dpi (t ₍₅₎ = 3.032, P = 0.029). Meanwhile, no changes occurred in TLR2 wild-type mice compared with uninfected controls during the experiment. The iNOS expression in splenocytes showed unexpected higher background levels in TLR2 mutant mice than those in TLR2 wild-type mice. Furthermore, the iNOS mRNA transcripts in splenocytes were significantly increased in the TLR2 wild-type mice infected with C. sinensis at 30 (t ₍₅₎ = 5.139, P = 0.004), 60 (t ₍₅₎ = 6.138, P = 0.002) and 90 dpi (t ₍₅₎ = 6.332, P = 0.001). However, the rising of iNOS transcripts dropped under the uninfected control level in the TLR2 mutant mice at 120 dpi (t ₍₅₎ = -9.082, P < 0.0001). Both total NO and iNOS transcripts were significantly higher in the TLR2 mutant mice than those in the TLR2 wild-type mice at 30 (t ₍₅₎ = 3.091/2.933, P = 0.027/0.033) and 60 dpi (t ₍₅₎ = 2.667/6.331, P = 0.044/0.001), respectively. In addition, the remarkable increase of iNOS expressions was immunohistochemically detected in the splenic serial sections of TLR2 wild-type mice at 30 and 60 dpi. However, the expressions of iNOS were remarkably decreased in the splenocytes of both TLR2 wild-type and mutant mice at 120 dpi.

These results demonstrate that TLR2 signal plays an important role in the regulation of iNOS expression after C. sinensis infection. TLR2 signal is also beneficial to limiting worm growth and development and contributing to the susceptibility to C. sinensis in which the iNOS/NO reactions possibly participate.