Xin Hou

IL‐17 neutralization significantly ameliorates hepatic granulomatous inflammation and liver damage in Schistosoma japonicum infected mice

IL‐17 is a signature cytokine of Th17 cells implicated in the induction and progression of chronic inflammatory diseases. Several studies in C57BL/6 mice, immunized with soluble schistosome egg Ags (SEA) in complete Freunds adjuvant (CFA), and subsequently infected with Schistosoma mansoni (S. mansoni) have shown that severe hepatic granulomatous inflammation is correlated with high levels of IL‐17. Here, using a Schistosoma japonicum (S. japonicum) larvae infection model in C57BL/6 mice, we analyzed the dynamic expression of IL‐17 in infected livers by RT‐qPCR and ELISA. Our results showed that IL‐17 expression was elevated during the course of infection. The temporal expression of IL‐17 and cytokines/chemokines involved in the induction and effector function of Th17 cells was paralleled with hepatic granulomatous inflammation. Treatment of S. japonicum infected mice with IL‐17‐neutralizing mAb resulted in significant downmodulation of granulomatous inflammation and hepatocyte necrosis. The protection was associated with lower expression of proinflammatory cytokines/chemokines, such as IL‐6, IL‐1β, CXCL1, and CXCL2 and a reduced number of infiltrating neutrophils. Anti‐IL‐17 mAb significantly ameliorated hepatic granulomatous inflammation, partly through the downregulation of proinflammatory cytokines/chemokines and recruitment of neutrophils. Our data indicate a pathogenic role of Th17/IL‐17 in hepatic immunopathology in S. japonicum infected mice.

Negative Regulation of Schistosoma japonicum Egg-Induced Liver Fibrosis by Natural Killer Cells

The role of natural killer (NK) cells in infection-induced liver fibrosis remains obscure. In this study, we elucidated the effect of NK cells on Schistosoma japonicum (S. japonicum) egg-induced liver fibrosis. Liver fibrosis was induced by infecting C57BL/6 mice with 18–20 cercariae of S. japonicum. Anti-ASGM1 antibody was used to deplete NK cells. Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly I∶C) was used to enhance the activation of NK cells. Results showed that NK cells were accumulated and activated after S. japonicum infection, as evidenced by the elevation of CD69 expression and IFN-γ production. Depletion of NK cells markedly enhanced S. japonicum egg-induced liver fibrosis. Administration of poly I∶C further activated NK cells to produce IFN-γ and attenuated S. japonicum egg-induced liver fibrosis. The observed protective effect of poly I∶C on liver fibrosis was diminished through depletion of NK cells. Disruption of IFN-γ gene enhanced liver fibrosis and partially abolished the suppression of liver fibrosis by poly I∶C. Moreover, expression of retinoic acid early inducible 1 (RAE 1), the NKG2D ligand, was detectable at high levels on activated hepatic stellate cells derived from S. japonicum-infected mice, which made them more susceptible to hepatic NK cell killing. In conclusion, our findings suggest that the activated NK cells in the liver after S. japonicum infection negatively regulate egg-induced liver fibrosis via producing IFN-γ, and killing activated stellate cells.

Lack of IL-17 signaling decreases liver fibrosis in murine schistosomiasis japonica

Accumulating evidence has identified the profibrogenic properties of IL-17A in organ fibrosis. However, the role of IL-17A signal in liver fibrosis induced by Schistosoma japonicum infection remains unclear. In this study, we investigated liver fibrosis in wild-type (WT) and IL-17RA(-/-) mice upon S. japonicum infection. Hepatic IL-17A, IL-17C, IL-17E (IL-25), IL-17F, IL-17RA, IL-17RB and IL-17RC transcript levels were determined by RT-PCR. IL-17A(+) cells were analyzed by flow cytometry and confocal microscopy among granuloma cells. Immunostaining of IL-17R was performed on liver sections. Collagen deposition was assessed by Van Giesons staining. IL-17A, IL-17C, IL-17E, IL-17F, IL-17RA and IL-17RC mRNA levels were dramatically increased in fibrotic livers. Among granuloma cells, CD3(+) and CD3(-) lymphocytes, neutrophils and macrophages were found to express IL-17A. Compared to WT, IL-17RA(-/-) mice displayed attenuated granulomatous inflammation, liver fibrosis, improved liver function and high survival. Meanwhile, α-smooth muscle actin staining and the expression of fibrogenic genes (transforming growth factor β, IL-13 and collagen-I) as well as IL-17A-induced proinflammatory mediators (IL-1β, IL-6, tumor necrosis factor α, CXCL1 and CXCL2) and proteinases (MMP3 and TIMP1) involved in fibrosis were markedly reduced in IL-17RA(-/-) mice. In addition, Th2 cytokines IL-4 and IL-17E (IL-25) were also decreased in IL-17RA(-/-) mice. These results indicated that IL-17A signal contributes to the pathogenesis of liver fibrosis in murine schistosomiasis. This effect was induced possibly by activating hepatic stellate cells and stimulating the release of proinflammatory cytokines and chemokines. Furthermore, the Th2 response was also enhanced by IL-17A signals. Our data demonstrate that IL-17A may serve as a promising target for antifibrotic therapy.

CD4(+)Foxp3(+) Tregs protect against innate immune cell-mediated fulminant hepatitis in mice.

UNLABELLED Foxp3(+) Tregs play important roles in maintaining homeostasis by suppressing excessive immune responses that result in serious tissue damage; yet, it is largely unknown about the impact of Tregs on innate immune cells in hepatitis models in vivo. In this study, we examined the effect of hepatic Tregs on innate immune-mediated liver injury by using the murine model of polyI:C and d-galactosamine (d-GalN)-induced hepatitis. Administration of polyI:C/d-GalN increased the number of CD4(+)Foxp3(+) Tregs in the liver. Depletion of Tregs leaded to higher levels of proinflammatory cytokine expression and severer liver injury, whereas adoptive transfer of Foxp3(+) Tregs attenuated liver injury in polyI:C/d-GalN-treated mice. In addition, depletion of Tregs leaded to a reduction in TGF-β and IL-10 expression in polyI:C/d-GalN-treated mice. Both of these cytokines were important for suppression of polyI:C/d-GalN-induced liver injury. TGF-β was derived from Tregs. IL-10 was derived from active Kupffer cells, and coincubation of Kupffer cells with Tregs increased IL-10 secretion. Furthermore, TGF-β blockade abrogated Treg-mediated suppression of proinflammatory cytokine production by innate immune cell in vitro. CONCLUSION CD4(+)Foxp3(+) Tregs modify innate immune responses in polyI:C/d-GalN-induced fulminant hepatitis via producing TGF-β and enhancing IL-10 secretion by Kupffer cells.

A boswellic acid-containing extract ameliorates schistosomiasis liver granuloma and fibrosis through regulating NF-κB signaling in mice.

Boswellic acid (BA)-containing extracts such as BSE have anti-inflammatory and immunomodulatory activity. In chronic schistosomiasis, the hepatic granuloma and fibrosis induced by egg deposition in the liver is the most serious pathological manifestations. However, little is known regarding the role of BAs in Schistosoma japonicum (S. japonicum) egg-induced liver granuloma and fibrosis. In order to investigate the effect of a water-soluble complex preparation of BSE, BSE-CD, on S. japonicum egg-induced liver pathology, liver granuloma and fibrosis were induced by infecting C57BL/6 mice with 18–22 cercariae of S. japonicum. S. japonicum cercariae infected mice were injected with BSE-CD at the onset of egg granuloma formation (early phase BSE-CD treatment after 4 weeks infection) or after the formation of liver fibrosis (late phase BSE-CD treatment after 7 weeks infection). Our data show that treatment of infected mice with BSE-CD significantly reduced both the extent of hepatic granuloma and fibrosis. Consistent with an inhibition of NF-κB signaling as evidenced by reduced IκB kinase (IKK) activation, the mRNA expression of VEGF (vascular endothelial growth factor, VEGF), TNF-α (tumor necrosis factor-alpha TNF-α) and MCP-1 (monocyte chemotactic protein 1, MCP-1) was decreased. Moreover, immunohistochemical analysis (IHC) revealed that the content of α-SMA in liver tissue of BSE-CD treated mice was dramatically decreased. Our findings suggest that BSE-CD treatment attenuates S. japonicum egg-induced hepatic granulomas and fibrosis, at least partly due to reduced NF-κB signaling and the subsequently decreased expression of VEGF, TNF-α, and MCP-1. Suppression of the activation of hepatic stellate cells (HSC) may also be involved in the therapeutic efficacy of BSE-CD.

A boswellic acid-containing extract attenuates hepatic granuloma in C57BL/6 mice infected with Schistosoma japonicum

Granuloma formation has been shown to be induced and elicited by schistosome egg antigens, and it finally develops into fibrosis in intestine and the liver. Hepatic fibrosis is the main cause of increased morbidity and mortality in humans infected with schistosomes. Boswellic acid (BA)-containing extracts such as extracts of the oleogum resin from Boswellia serrata (BSE) have anti-inflammatory and immunomodulatory activity. However, little is known about the role of such extracts in schistosome egg-induced granulomatous inflammation. In order to investigate the effect of a watersoluble cyclodextrin complex preparation of BSE (BSE-CD) on Schistosoma japonicum (S. japonicum) egg-induced liver granuloma, mice infected with S. japonicum cercariae were injected with BSE-CD during egg granuloma formation. The data showed that BSE-CD significantly reduced the size of liver granuloma and levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST); however, BSE-CD treatment had no effect on worm load and egg burden. The data also showed that BSE-CD significantly decreased the expression of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2), as well as the expression of matrix metallopeptidase 9 (MMP-9) in liver both on the mRNA and protein level. Thus, BSE-CD can significantly attenuate S. japonicum egg-induced hepatic granuloma, which may be partly dependent on the downregulation of some biochemical mediators.

Identification of pro-inflammatory CD205 + macrophages in livers of hepatitis B virus transgenic mice and patients with chronic hepatitis B

Hepatic macrophages play a central role in disease pathogenesis during hepatitis B virus (HBV) infection. Our previous study found that CD205+ macrophages in the liver of hepatitis B surface antigen transgenic (HBs-Tg) mice increased significantly compared with those in wild-type mice, and these increased CD205+ macrophages were involved in CpG-oligodeoxynucleotide-induced liver injury in HBs-Tg mice. Here, we analysed the phenotype and function of CD205+ macrophages derived from the liver of HBs-Tg mice and patients with chronic hepatitis B (CHB). We found that HBs-Tg mice-derived hepatic macrophages produced larger amounts of pro-inflammatory cytokines, including IL-6, IL-12, TNF-α, and of the anti-inflammatory cytokine IL-10 after stimulation with CpG-oligodeoxynucleotides or commensal bacteria DNA than B6 mice-derived hepatic macrophages. Furthermore, hepatic CD205+ macrophages from HBs-Tg mice showed an activated phenotype and expressed higher levels of inflammatory cytokine genes, chemokine genes, and phagocytosis-related genes than hepatic CD205− macrophages. In addition, CD205+ macrophages displayed an inflammatory phenotype and were increased in the liver of patients with CHB compared with those in healthy controls. Our data suggest that hepatic CD205+ macrophages are a unique pro-inflammatory subset observed during HBV infection. Thus, development of intervention targeting these cells is warranted for immunotherapy of HBV-induced liver diseases.

Molecular signature and functional analysis of uterine ILCs in mouse pregnancy

In addition to natural killer cells, other innate lymphoid cells have recently been identified in the mouse and human uterus, but their roles in successful pregnancy remain poorly defined. In this study, we examined the dynamic changes of uterine innate lymphoid cells throughout pregnancy in mice. We found that the total number of uterine innate lymphoid cells markedly increased at early-gestation. Among the three groups of uterine innate lymphoid cells, the number of the group 2 uterine innate lymphoid cells increased the most during pregnancy. We also determined that the depletion of uterine innate lymphoid cells in Rag1-/- mice resulted in impaired uterine spiral artery remodeling. These results suggest that uterine innate lymphoid cells may play an important role in mouse reproduction.

Polyinosinic–polycytidylic acid attenuates hepatic fibrosis in C57BL/6 mice with Schistosoma japonicum infection

The development of hepatic fibrosis is the principal cause of morbidity and mortality in human beings infected with schistosoma. In this study, we investigated the effect of polyinosinic-polycytidylic acid (poly I:C) on Schistosoma japonicum (S. japonicum) egg-induced liver fibrosis. S. japonicum cercariae infected mice were injected with poly I:C at the onset of egg granuloma formation (early phase poly I:C treatment) or after the formation of liver fibrosis (late phase poly I:C treatment). Our results showed that both early and late phase poly I:C treatment significantly reduced collagen deposition and hepatic stellate cell activation in the liver. Poly I:C is one of the most effective adjuvants for Th1 type responses, and its protective effect on liver fibrosis was accompanied by increased IFN-α, IFN-β, IFN-γ, IL-12, TNF-α, and IL-10 mRNA expression, and decreased IL-4 and IL-5 mRNA expression. Moreover, poly I:C injection also enhanced the mRNA expression of natural killer group 2 member D (NKG2D) and tumor necrosis factor related apoptosis-inducing ligand (TRAIL). Therefore, it is indicated that poly I:C can significantly attenuate S. japonicum egg-induced hepatic fibrosis, which may be partly dependent on the increased Th1 response and decreased Th2 response.

Functions of the Vasa gene in Schistosoma japonicum as assessed by RNA interference

Vasa, an enzyme belonging to the helicase family, contributes to the regulation of reproductive system development in many species. Thus, we hypothesized that the Vasa3 gene may function in the reproductive system of the parasite Schistosoma japonicum (S. japonicum), which is a major causative agent of schistosomiasis. It is a severe disease globally affecting humans and animals. To test this hypothesis, we firstly conducted whole mount in situ hybridization analyses and found that the S. japonicum Vasa3 (SjVasa3) gene was expressed mainly in the reproductive organs. We then explored the reproductive functions of Vasa3 in S. japonicum using RNA interference (RNAi) techniques. Coupled schistosomes collected from mice 28days post infection (dpi) were transfected three times with SjVasa3-specific small interfering RNA (siRNA) and cultured in vitro for up to 10days. As measured by quantitative PCR (qPCR) and Western blot analysis, levels of SjVasa3 mRNA and protein in Vasa siRNA treated worms were significantly reduced compared with untreated and scrambled siRNA treated worms. Confocal laser scanning microscopy (CLSM) images showed markedly siRNA induced changes in the morphology of the reproductive organs, especially in the female ovary, vitellarium and the male testes. SjVasa3 gene silencing also significantly reduced egg production. These data demonstrate that SjVasa3 is essential in reproductive organ development and egg production in S. japonicum, and could be a potential target for developing novel compounds to treat schistosomiasis.