| ?Backgrounds & Aims? Schistosomiasis is a serious parasitic disease throughout the world's tropical and subtropical regions,affecting more than 200 million people worldwide.Schistosome worms lay their eggs in the mesenteric and portal veins of their human host,and the eggs are trapped in liver sinusoids.Here,larval miracidia within the mature eggs secrete soluble egg antigen(SEA)that elicit host immune responses including granulomatous inflammation and fibrotic reactions.Intestinal and hepatic schistosomiasis are the most common forms of chronic disease.Intestinal schistosomiasis is an acute or chronic,specific enteropathy caused by the deposition of schistosome ovum on the colon and rectal walls.Gastrointestinal symptoms may include abdominal pain,altered bowel habits,and bloody stools.The egg-induced hepatic fibrosis,which can lead to portal hypertension and variceal bleeding,is the primary cause of morbidity and mortality associated with this chronic disease.Several studies by our and other groups have shown that the regulators of schistosomiasis hepatic fibrosis are far more complicated than expected.Splenomegaly is a consequence and an important clinical indicator of portal hypertension.Previous studies have suggested that splenic abnormalities may promote the progression of liver fibrosis to cirrhosis and exacerbate disease prognosis through multiple possible pathways.However,the role of the spleen in severe schistosomiasis has been little explored and probably involves more than just a contribution to portal blood hypertension.In past decades,splenectomy has been utilized to ameliorate the fatal complications of fibrosis-associated portal hypertension.However,many fibrosis patients present with contraindications that preclude splenectomy.Thus,it remains critical for us to identify alternative novel and non-surgical methods for the treatment of liver fibrosis.Although chemotherapy can effectively target and kill schistosomes,the progression of hepatic fibrosis persists.Since anti-fibrotic therapies for schistosomiasis have been neglected,new prospective drugs are urgently required that can reverse the fibrosis.Endothelin receptors(ETRs)are class A G-protein-coupled receptors(GPCRs)activated by the vasoactive peptide hormones endothelins.Two subtypes of ETRs,endothelin A receptor(ETAR)and endothelin B receptor(ETBR),are involved in various functions,such as regulation of blood pressure,sodium excretion,cell proliferation,and neural crest development.Endothelin-1(ET-1)is a peptide that is produced primarily by vascular endothelial cells and is characterized as a powerful smooth muscle vasoconstrictor and mitogen.In addition,autocrine and paracrine signaling of ET-1 exerts its effects by binding to ETRs,thus triggering the downstream signaling in cells.Both receptors are found in vascular smooth muscle where they mediate vasoconstriction,whereas in the endothelium,ETBR mediates vasodilatation in part through nitric oxide release.Studies have found that ET-1 and its receptors are related to liver fibrosis or cirrhosis.Plasma ET-1 levels are increased in cirrhosis and correlate with the severity of liver disease and portal pressure.Several studies by our and other groups have shown that elevated concentrations of ET-1 act on upregulated ETRs on hepatic stellate cells(HSCs)to cause increased contractility and intrahepatic sinusoidal resistance,resulting in portal hypertension.The spleen plays a critical role in development of liver fibrosis and cirrhosis.A previous study has shown that the high concentration of ET-1 in cirrhosis with portal hypertension could be due to increased splenic production of this endothelial factor.The splenic cells involved in the increased endothelin production are endothelial cells of the vein sinuses and B-cells in the germinal centers and in the marginal zone of lymphoid sheaths and follicles.These findings prompted us to ask whether the splenocytes express ETRs and are involved in the progression of hepatic fibrosis or cirrhosis.In addition,the role of ETRs in schistosomiasis has been little explored,and no study thus far has localized ETAR and ETBR in Schistosoma(S.)japonicum-infected spleen tissues.As the largest lymphoid organ in the body,the spleen contains highly elaborate tissue structures and is anatomically linked to the liver via the portal vein system.B cells,representing a major immune cell population,are well known for their ability to differentiate into antibody-secreting plasma cells in response to foreign antigens or pathogens.However,recent studies have suggested that B cells appear to regulate both protective and pathologic immune responses by antibody-independent mechanisms.Helminth infections,particularly infection with schistosomes,are well-known to induce regulatory B cells,a relatively new member of the network of regulatory immune cells.B cells suppress pro-inflammatory immune responses via several mechanisms,of which the best described are the expression of the regulatory cytokine interleukin-10(IL-10)and induction of regulatory T(Treg)cells.In the current study,we used both human schistosomiasis and a mouse model to investigate the role of ETAR and ETBR.We hypothesized that ETRs play an important role in the pathogenesis of schistosomiasis.We attempted to apply endothelin receptor antagonists,including BQ-123,a selective ETA antagonist,and BQ-788,a selective ETB antagonist,to demonstrate our hypothesis and further elucidate the molecular pathogenesis pathways through which ETRs may cause fibrotic changes of humans and mice upon exposure to S.japonicum infection.?Methods?1.The degree of fibrosis in chronic schistosomiasis(CS)patients was observed.The expression of ETAR and ETBR in liver tissues was detected by immunohistochemistry.Furthermore,the pathological staining,collagen deposition and the expression of ET-1 and ETRs in liver tissues caused by schistosomiasis and chronic hepatitis B(CHB)were studied.2.The spleen tissues of patients with CS and CHB were collected to observe the changes of spleen structure and collagen deposition.We further detected the expression of ET-1 and ETRs.The localization and expression of ETAR and ETBR in spleen tissues were detected by immunofluorescence.Finally,the results of abdominal ultrasonography in the three groups of patients with splenectomy were detected,and the correlation between the results of patients with CS and the m RNA levels of ETRs was analyzed.3.We applied the well-established model of S.japonicum-induced for hepatic fibrogenesis in mice.Liver and spleen samples were harvested at 4,6,8 and 12 weeks post-infection from mice infected with S.japonicum.We observed the morphological changes of the liver and spleen tissues,weighed the weight to calculate the liver and spleen indexes,and detected the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the serum of mice to assess liver injury.Pathological changes,granuloma area and collagen deposition were observed by H&E,Masson and Sirius Red staining.The changes of hydroxyproline content in liver tissues,and the expression of collagen ?,collagen ?,liver portal vein diameter and pressure of mice were also detected.We evaluated the expression of ET-1 and ETRs of liver tissues in mice.We further examined the levels of splenic fibrosis in our model.The changes of spleen structure and collagen deposition were observed.The expression levels of ET-1 and ETRs were further detected.4.The anthelmintic effect of praziquantel(PZQ)was observed.At 6 weeks post-infection,the infected mice were treated with PZQ to kill the parasites and then were necropsied at 12 weeks post-infection.We observed the morphological changes of the liver and spleen tissues,weighed the weight to calculate the liver and spleen indexes,and detected the levels of ALT and AST in serum of mice to assess liver injury.Liverportal vein pressure and diameter were also detected.Pathological changes,granuloma area and collagen deposition were observed by H&E,Masson and Sirius Red staining.The expression of hydroxyproline content in liver tissues was also detected.Quantification of the area of liver fibrosis and the expression of collagen were assayed.The expression levels of ET-1,ETAR and ETBR in liver tissues were detected by q PCR,western blot and immunohistochemistry.The serum level of SEA-specific Ig G was assayed.The egg burden in the liver was counted.Spleen tissues of infected mice in different groups were collected.We observed the changes of spleen structure and collagen deposition,and further detected the expression of ET-1 and ETRs.5.The location of ETAR and ETBR in spleen tissues was verified by immunofluorescence.Mice were injected with anti-CD20 to deplete B cells.After 7 days,isolated mouse splenic mononuclear cells were cultured in the presence of SEA.Supernatants were stored for IL-10 analysis by ELISA.Mouse CD19+ B cells were isolated from the spleen at different time points during schistosome infection.Supernatants were stored for IL-10 analysis by ELISA.The expression levels of IL-10 and CD86 in splenic B lymphocytes were detected by flow cytometry.The expression of IL-10,Ig G and Ig M in the supernatant of cell culture were detected by ELISA.SEA-restimulated B cells were co-cultured for 4 days with CD25-depleted CD4 T cells.The frequency of CD25+Foxp3+ Treg cells after co-culture was detected by flow cytometry.The expression of IL-10 in the supernatant of cell culture was detected by ELISA.?Results?1.Representative photomicrographs revealed that pathological lesions and collagen deposition in liver biopsy samples of patients with CS significantly increased with the aggravation of liver fibrosis.ETAR and ETBR-positive cells were localized mainly in areas of inflammation and fibrosis.Fibrotic liver tissues had larger fibrotic areas and more collagen deposition and S.japonicum eggs were observed in liver specimen sections of patients with CS.Our data revealed that the expression levels of ET-1,ETAR and ETBR were significantly higher in the fibrotic groups.In addition,the schistosome-induced liver fibrosis group appeared to have slightly stronger ET-1 and ETBR expression than the HBV-induced group.2.In CS and CHB spleen tissues,splenic red pulp was dilated by congestion,and the size of the splenic white pulp was reduced and collagen deposition was increased compared with control(Cont)tissues.We found the expression levels of Collagen ??Collagen ??ET-1?ETAR and ETBR were upregulated in both patients with CS and CHB,with a greater increase in CS patients.Double staining using immunofluorescence displayed a co-localization of ETRs and CD20 staining,indicating that human splenic B cells express ETAR and ETBR.Abdominal ultrasonography indicated that the portal vein diameter and spleen thickness diameter were remarkably higher in CS and CHB patients than Cont.The m RNA levels of ETAR and ETBR were positively correlated with the portal vein diameter and spleen thickness diameter in patients with CS.3.The results showed that we had successfully established a mouse model of S.japonicum infection in which hepatic injury and spleen enlargement could be observed.These results were confirmed by the elevation level of ALT and AST in mouse serum and further confirmed by the liver and spleen indexes.The results showed that liver portal vein pressure and diameter were significantly increased after schistosome infection.Importantly,hydroxyproline quantification,Masson's trichrome and Sirius Red staining revealed that S.japonicum infection induced progressive hepatic injury and collagen deposition,and the size of the hepatic granulomas was increased.These results were confirmed by enhanced expression of ET-1,ETAR,ETBR,Col1?1 and Col3?1 over the course of S.japonicum infection in mice.Microscopic findings of spleen sections of S.japonicum showed congestion of red blood cells and disruption of lymphoid follicles.Increases in trabeculae and sinus hyperplasia were also noted.However,collagen deposition was not significantly altered in these mice.The expression levels of ET-1,ETAR and ETBR in infected spleen tissues were elevated.4.PZQ significantly reduced the worm burden and egg count.Morphological changes in liver and spleen samples showed a moderate granulomatous response and splenomegaly in the endothelin receptor antagonist-treated mice.Mice receiving endothelin receptor antagonists displayed a significant reduction in liver portal vein pressure and diameter,as well as extracellular matrix(ECM)deposition as shown by hydroxyproline quantification,Masson's trichrome and Sirius Red staining,whereas the size of the hepatic granulomas in all groups was similar as shown by H&E staining and livers showed no significant change in egg burden.In addition,we found that the serum level of SEA-specific Ig G was decreased in the presence of endothelin receptor antagonists.The amounts of ET-1,ETAR,ETBR,Col1?1 and Col3?1 were dramatically reduced in livers of mice treated with endothelin receptor antagonists.Next,we evaluated the levels of splenic fibrosis in these mice.Consistent with the attenuated fibrotic phenotype,a reduction of structural damage was detected in spleens of mice treated with endothelin receptor antagonists as shown by H&E staining,while collagen deposition was not significantly altered in these mice by Masson's trichrome staining.The expression levels of ET-1,ETAR and ETBR were decreased in the spleen tissues of mice treated with endothelin receptor antagonists.5.We found that B cells in mice spleen tissues expressed ETAR and ETBR in vivo based on immunofluorescence staining.SEA-stimulated splenic mononuclear cells from mice depleted of B cells exhibited lower levels of IL-10 in culture supernatants compared to control mice,suggesting that IL-10-producing B cells may play an important role in the process of schistosomiasis infection.Elevated IL-10 characterizes chronic stages of schistosome infection and is produced by B cells starting from week 8 during infection.The frequencies of B cells expressing intracellular IL-10 protein in the endothelin receptor antagonist-treated mice were reduced compared with the vehicle group.Additionally,the surface activation marker CD86,which is often upregulated on activated B cells,was decreased on splenic B cells in response to endothelin receptor antagonist treatment.Splenic B cells from the endothelin receptor antagonist-treated mice showed significantly decreased IL-10 secretion,as measured in culture supernatants.Compared with the vehicle group,endothelin receptor antagonist-treated groups significantly decreased the ability of B cells to secrete Ig G and Ig M antibodies.Splenic B cells from endothelin receptor antagonist treated,but not vehicle group,mice showed decreased development of CD25+ Foxp3+ Treg cells during 4 days of co-culture with CD25-depleted CD4 T cells.In addition,the IL-10 protein concentration in the co-culture supernatants was decreased in the presence of endothelin receptor antagonists.?Conclusions?1.Our results showed that the expression levels of ETAR and ETBR were upregulated in liver and spleen tissues of patients with CS and CHB,with a greater increase in CS patients.The endothelin receptor-producing cells are mainly located in splenic B cells.Their expression also appeared to be linked to the histological degree of schistosomiasis hepatic fibrosis.This outcome provided evidence of their involvement in the progression of schistosomiasis.2.Our study indicated that schistosome-infected mice showed higher ETAR and ETBR expression than the uninfected group both in liver and spleen tissues,and they also showed an association with advanced stages of liver fibrosis.We further demonstrated that elevated levels of ETRs are predominantly expressed on B cells in spleen tissues during infection.3.Endothelin receptor antagonists caused a significant reduction of hepatocellular damage and partially alleviated schistosome-induced hepatic fibrosis.A reduction of structural damage was detected in spleens of mice treated with endothelin receptor antagonists.We further found that the expression levels of ETRs in the treated liver and spleen tissues were down-regulated.4.Schistosome infection induced B cell activation.Endothelin receptor antagonists were capable to suppress the activation of splenic B cells characterized by IL-10 secretion and Treg cell-inducing capacity.5.These data demonstrate that endothelin receptor antagonists can partially alleviate schistosomiasis-induced hepatic fibrosis by suppressing the activation of splenic B cells. |
PDF Full Text Request