Dynamics Of Cytokine Secretion In Mice Infected With Schistosoma Japonicum By In Vivo Cytokine Capture Assay (IVCCA) And Their Relation To Hepatic Fibrosis

Schistosomiasis is a chronic helminthic disease that affects about two hundred millions of people in the world. The pathogenesis of Schistosome infection is primarily due to hepatic and intestinal granuloma formation around deposited eggs and subsequent fibrosis. It is known that CD4+ T cell subsets play critical roles in the host immunity and immunopathogenesis to Schistosome infections, in which T helper 1(Th1) and Th2 cells are major effector T cell subsets, whereas T regulatory(Treg) cells exert immunosuppressive roles in general. The recently discovered Th17 cells are also actively involved in the immune responses to the infection. During the infection, these T cell subsets cross-talk and exhibit different kinetics and roles in the control and regulation of infection progress and fibrosis.The host’s immune responses to schistosome infections are predominantly mediated by Th1, Th2, Treg and Th17 cells and their effector cytokines. In the acute phase of infection, that is, the first three weeks after infection, the host’s immune response is mainly targeted on the migrating immature and mature parasites. The dominant response is Th1-like. When the parasites begin to produce eggs beginning 3~4 weeks(wk) post infection(PI), the Th1-like response is altered, with the emergence of a stronger Th2 response which is primarily induced by egg antigens. At the same time, the granulomas form around the eggs in the liver. CD4+ T cells can be induced to differentiate into CD4+CD25+ T regulatory(Treg) cells, which have immunosuppressive activities and down-regulate immune responses to inhibit immunopathology. Th17 cells have recently emerged as a third independent effector Th subset, which differentiate from CD4+ T cells upon antigen stimulation. Emerging data suggest that Th17 cells and heir effector cytokine IL-17 play an important role in host defenses to infections, such as schistosomiasis. The granuloma responses around deposited eggs in the liver are reported to be positively regulated by Th17 cells and their secreted IL-17 cytokine. The use of either Th17 knockout mice or neutralizing antibodies to IL-17 can significantly reduce the inflammatory response and granuloma size. Therefore, it is very important for the future development of novel therapy for hepatic fibrosis in Schistosome infection if we can accurately evaluate the dynamics of cytokines secreted during Schistosoma japonicum infection in vivo. Currently, most cytokine assays are based on in vitro stimulation of spleen or lymph node cells, which have been applied to infer in vivo cytokine secretion levels in Schistosome infections. These methods include ELISA, intracellular cytokine staining, real-time PCR, etc. Results from these assays can provide important and useful information, but they can not accurately reflect in vivo cytokine secretion. In addition, all of these assays examine cytokine production from single organ rather than the whole-body level. To overcome the limitations of the above methods, this study intends to establish in vivo cytokine capture assays(IVCCA) to measure the dynamic changes of cytokine production in mice infected with Schistosoma japonicum. The sensitivity of IVCCA is about 30 to 1,000-fold higher than regular ELISA. We can explore, more accurately, the effects of various cytokines in the pathogenesis of schistosomiasis by the use of IVCCA. This assay is particularly valuable for the detection of low levels of some cytokines in vivo. In summary, the dynamic changes of CD4+ subsets and their effector cytokines in relation to hepatic fibrosis needs further investigation in schistosome, especially Schistosoma japonicum, infections. This research is divided into the following two parts:Part I Establishment of IVCCA and comparison to in vitro assaysObjective: To establish IVCCA for the evaluation of dynamic changes of IFN-g, IL-4, IL-10 and IL-17 in mice infected with S. japonicum, and to compare with in vitro assays.Methods: Mice were infected with 15 cercariae of S. japonicum through the abdominal skin. At 0, 3, 5, 7, 10 and 12 wk post-infection(PI), five infected plus three age- and sex-matched normal mice were randomly selected for IVCCA assay to measure the levels of IFN-g, IL-4, IL-10 and IL-17 in the circulating blood. The mice were sacrificed to prepare single cell suspensions of spleen. Cells were cultured for 48 hours in 24 well plates in the presence of antiCD3/anti-CD28 or PMA /ionomycin. The culture supernatants were collected and kept at-80 oC for ELISA assay; Cells were cultured for 6 hours in 24 well plates in the presence of PMA /ionomycin and Golgistop. The culture cells were collected for intracellular cytokine staining.Results: For IVCCA, mouse serum IFN-g level continued to increase post-infection and reached a peak at 5 wk PI(P<0.05), then gradually decreased and remained at a high level. Mouse serum IL-4 and IL-17 levels continued to increase post-infection, and reached a peak at7 wk PI(P<0.05), then gradually decreased and remained at a high level. Serum IL-10 level continued to increase after infection. The above cytokine levels in the culture supernatants continued to increase after infection as analyzed by ELISA. Intracellular cytokine staining datas show that the proportion of the Treg,Th1,Th2 and Th17 cells in the total splenic CD4+T cell population showed a continuous increase after infection.Conclusion: We have successfully established IVCCA for dynamic detections of in vivo cytokines after S. japonicum infection and compared with in vitro assay of culture supernatants by regular ELISA and intracellular cytokine staining. IVCCA has the advantages of high sensitivity and dynamic detection of in vivo cytokine levels without the need of sacrificing mice. This assay may provide a valuable tool for the study of T cells and cytokines involved in the immunopathogenesis in infections by schistosomes and perhaps other parasites or microbes.Part II Dynamics of cytokine secretion in mice infected with Schistosoma japonicum and their relation to hepatic fibrosisObjective: To analyze the dynamic changes of cytokine secretion in C57BL/6 mice infected with S. japonicum and their relation to hepatic fibrosisMethods: C57BL/6 mice were infected with 15 cercariae of S. japonicum through the abdominal skin. At 0, 3, 5, 7, 10 and 12 weeks post infection, five infected plus three age- and sex-matched normal mice were randomly selected and sacrificed. Part of each liver was processed for paraffin sections for hematoxylin and eosin(H&E) and Masson Trichrome staining. Another part of 2~3 grams of liver was digested with 4% KOH at 37 oC overnight and the egg numbers were counted by microscopic examination. Other two parts of liver were used for hydroxyproline detection and q PCR analysis for TGF-β, Col I and Col III. The mice were sacrificed to prepare single cell suspensions of spleen. Cells were cultured for 72 hours in 24 well plates in the presence of SEA. Culture supernatants were collected for ELISA.Results: The granuloma formation around the deposited eggs in the liver was observed at 5 wk PI. The size of the granulomas reached a peak at 7 wk PI, then the gradually reduced. By Masson Trichrome staining, a small amount of blue fiber cord was observed around the eggs 5 wk PI. This fibrosis deposition continued to increase upon infection progress. The m RNA levels of several genes related to hepatic fibrosis, such as TGF-b and Col III, were increased continually and reached a peak at 7 wk PI(P<0.05), then gradually decreased and remained at high levels; whereas Col I mRNA expression peaked at 10 wk PI(P<0.05). The results of in vitro assays showed that low levels of IFN-g were induced when the spleen cells were stimulated with SEA(P>0.05). The secretion of IL-4 and IL-17 upon SEA stimulation were increased markedly after infection and reached a peak at 7 wk PI(P<0.05), then gradually reduced but remained at higher levels as compared to uninfected group. However, secretion of IL-10 levels continued to increase after infection.Conclusion: After C57BL/6 mice were infected with S. japonicum, granuloma formation around the deposited eggs in the liver was observed at 5 wk PI. The size of the granulomas reached a peak at 7 wk PI, then gradually reduced. The inflammatory cells around the egg were significantly decreased and replaced by a large number of collagen cells. Furthermore, the dynamic changes of fibrosis is correlated with the results of IVCCA. Our findings also indicated that the IL-4, IL-17 and IL-10 might be involved in the pathogenesis of granuloma formation and fibrosis when the spleen cells were stimulated with SEA. SEA may directly activate Th2, Th17 and Treg cells, etc. On the other hand, SEA did not significantly induce the production of Th1 representative cytokine IFN-?. Our findings suggested that the hepatic inflammatory granuloma responses might reflect, to some extent, the dynamics of interaction between antiinflammatory(such as IL-10 and TGF-?1) and proinflammatory(such as IL-17) cytokines. From 7 wk post-infection, proinflammatory factors reduced; whereas, immunosuppressive factors increased gradually. Therefore, the granuloma responses down-regulated and their size reduced, reaching to an immunomodulation status.