Crosstalk Between TGF-β And Th-related Cytokines During Echinococcus Multilocularis Infection

Objective:To explore the crosstalk between TGF-β and Th-related cytokines during Echinococcuss multilocularis (E. multilocularis) infection. Methods:(1) One hundred and twenty pathogen-free female BALB/c mice (8-10-week old) were housed in cages with a12-h light/dark cycle and provided with rodent chow and water. BALB/c mice were infected by E. multilocularis and tissue samples were collected. For each autopsy time-point, ten experimentally infected mice were used in E. multilocularis group (n=10) and compared with five control mice (n=5), which received an intra-hepatic injection of0.1mL of saline in the anterior liver lobe using the same surgical procedure. Mice were killed at2,8,30,60,90,180,270and360days, respectively. Tissue samples from E. multilocularis lesions were taken and processed for histopathological examination and immunostaining. In addition, liver tissue samples were taken1) close to the parasitic lesions, i.e.1-2mm from the macroscopic changes due to the metacestode/granuloma lesion, thus avoiding liver contamination with infiltrating immune cells and parasitic tissue, and2) distant from the parasitic lesion, in another lobe of the liver, in E. multilocularis infected mice; in control mice, samples were taken in the injected lobe and in a non-injected lobe of the liver.(2) Based on RNA quality control results and histopathological evaluation, RNA extracts from3infected and3control mice were selected for array hybridization, corresponding to30days,60days,90days and180days after infection, according to a previously described procedure.(3) Using Western Blot, qRT-PCR and immunohistochemistry, we measured the levels of TGF-β1, down-stream Smads, MAPKs activation and cell proliferation and cell cycle markers, as well as fibrosis marker expression in both a murine AE model from day2to360post-infection (p.i.) and in AE patients. Using TUNEL, we detected apoptosis of hepatocytes.(4) After co-culturing primary rat hepatocytes with E. multilocularis fluid, we mesured TGF-β1, down-stream Smads, MAPKs activation by using Western Blot. Results:(1) TGF-β1highly expressed around the parasitic lesions. The positive correlation we found between their expression and expression of TGF-β1, both in the experimental model and in human livers, is an indirect argument for a significant role of this cytokine in AE fibrosis.(2) Profiles of mRNA expression in the hepatic parasitic lesions showed that a mix Thl/Th2immune response, characterized by the concomitant presence of IL-12, IFN-gamma and IL-4, was established from a very early stage, at the site of E. multilocularis development; and was then completed by IL-5, IL-10and TGF-P at the middle stage of infection.(3) We also showed for the first time that chemokine expression was part of the observed profiles, and that mRNA levels of both IL-17A and IL-17F were also increased at an early stage then significantly decreased. IL-17expression was highly correlated with the presence of CD4Tcells.(4) TGF-β1, its receptors, and down-stream Smads were markedly expressed in the periparasitic infiltrate and also in the hepatocytes, close to and distant from AE lesions. Fibrosis was significant at180days p.i. in the periparasitic infiltrate and was also present in the liver parenchyma, even distant from the lesions. Over the time course after infection TGF-β1expression was correlated with CD4/CD8T-cell ratio long described as a hallmark of AE severity. The time course of the various actors of the TGF-β/Smad system in the in vivo mouse model as well as down-regulation of Smad7in liver areas close to the lesions in human cases highly suggest that TGF-β plays an important role in AE both in immune tolerance against the parasite and in liver fibrosis.(5) Within the early (day2～60) and middle (day60-180) stages, CyclinBl and CyclinD拾gene expression increased up to day30and then returned to control level after day60; Gadd45β, CyclinA and PCNA increased all over the period; ERK1/2was permanently activated. Meanwhile, p53, p21and Gadd45c gene expression, and caspase3activation, gradually increased in a time-dependent manner. In the late stage (da180～360), p53, p21and Gadd45c gene expression were significantly higher in infected mice; JNK and caspase3were activated. TUNEL analysis showed apoptosis of hepatocytes. No significant change in CyclinE, p53mRNA and p-p38expression were observed at any time. Conclusion:(1) TGF-β plays an important role in AE both in immune tolerance against the parasite and in liver fibrosis.(2) A mix Th1/Th2immune response, characterized by the concomitant presence of IL-12, IFN-gamma and IL-4, was established from a very early stage, at the site of E. multilocularis development.(3) Our results suggest that the surrounding liver contributes significantly to cytokine/chemokine secretion and functional activities within the host-parasite interactions. A TGF-β-related fine tuning of the various isotypes of IL-17could determine the overall balance between tolerance towards the parasite and protection of the host.(4) Fibrosis is a hallmark of AE, leading to a complete disappearance of the liver parenchyma in the periparasitic area, and to fibrosis in portal spaces. Our results highly suggest that TGF-β and its signaling pathway are in the position to play this major role regarding fibrosis in AE.(5) Our data support the concept of a sequential activation of metabolic pathways which1) would first favor parasitic, liver and immune cell proliferation and survival, and thus promote metacestode fertility and tolerance by the host, and2) would then favor liver damage/apoptosis, impairment in protein synthesis and xenobiotic metabolism, as well as promote immune deficiency, and thus contribute to the dissemination of the protoscoleces after metacestode fertility has been acquired. These findings give a rational explanation to the clinical observations of hepatomegaly and of unexpected survival of AE patients after major hepatic resections, and of chronic liver injury, necrosis and of hepatic failure at an advanced stage and in both human patients and experimental animals.