Huiqin Wen

Impact of Schistosoma japonicum Infection on Collagen-Induced Arthritis in DBA/1 Mice: A Murine Model of Human Rheumatoid Arthritis

Background The hygiene hypothesis suggests that helminth infections prevent a range of autoimmune diseases. Methodology/Principal Findings To investigate the effects of S. japonicum infection on collagen-induced arthritis (CIA), male DBA/1 mice were challenged with unisexual or bisexual S. japonicum cercariae two weeks prior to bovine type II collagen (CII) immunization or at the onset of CIA. S. japonicum infection prior to CII immunization significantly reduced the severity of CIA. ELISA (enzyme linked immunosorbent assay) showed that the levels of anti-CII IgG and IgG2a were reduced in prior schistosome-infected mice, while anti-CII IgG1 was elevated. Splenocyte proliferation against both polyclonal and antigen-specific stimuli was reduced by prior schistosome infection as measured by tritiated thymidine incorporation (3H-TdR). Cytokine profiles and CD4+ T cells subpopulation analysis by ELISA and flow cytometry (FCM) demonstrated that prior schistosome infection resulted in a significant down-regulation of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β and IL-6) and Th1 cells, together with up-regulation of the anti-inflammatory cytokine IL-10 and Th2 cells. Interestingly, the expansion of Treg cells and the reduction of Th17 cells were only observed in bisexually infected mice. In addition, prior schistosome infection notably reduced the expression of pro-inflammatory cytokines and receptor activator of NF-κB ligand (RANKL) in the inflamed joint. However, the disease was exacerbated at one week after infection when established CIA mice were challenged with bisexual cercariae. Conclusion/Significance Our data provide direct evidence that the Th2 response evoked by prior S. japonicum infection can suppress the Th1 response and pro-inflammatory mediator and that bisexual infection with egg-laying up-regulates the Treg response and down-regulates the Th17 response, resulting in an amelioration of autoimmune arthritis. The beneficial effects might depend on the establishment of a Th2-dominant response rather than the presence of the eggs. Our results suggest that anti-inflammatory molecules from the parasite could treat autoimmune diseases.

Serological proteome-oriented screening and application of antigens for the diagnosis of Schistosomiasis japonica.

Schistosomiasis remains a major parasitic disease, with 200 million people infected and 779 million people at risk worldwide. The lack of reliable diagnostic techniques makes this disease difficult to control. In an attempt to discover useful candidates for the diagnosis of schistosomiasis, proteomics in combination with western blotting were employed in this study. This serological proteome assay yielded more than 30 immunodominant spots. Ten of these spots were precisely matched with a homologous two-dimensional electrophoresis (2-DE) gel and successfully identified by LC/MS-MS as corresponding to four different proteins. Of these proteins, SjLAP and SjFBPA were successfully expressed, and their recombinant protein products were further applied in the diagnosis of human Schistosomiasis japonica using ELISA. The ELISA results revealed sensitivities of 98.1% and 87.8% for acute and chronic schistosomiasis with rSjLAP and 100% and 84.7% with rSjFBPA, whereas the assays showed a specificity of 96.7% with both recombinant proteins. After treatment with praziquantel, the titres of the antibodies against both antigens declined significantly (P<0.001). Our data therefore suggest that these antibody-oriented recombinant proteins had a high efficacy for the diagnosis of S. japonica, and 2-DE based screening followed by LC/MS-MS has promising potential in the screening of candidate antigens for the diagnosis of schistosomiasis.

Phylogeny and virulence divergency analyses of Toxoplasma gondii isolates from China

BackgroundToxoplasma gondii (T. gondii) is a very successful parasite that can infect virtually all warm blooded animals with a worldwide distribution. It causes a large range of clinical manifestations in both humans and domesticated animals. In addition, marked biological differences exist among T. gondii strains in the pathogenicity and geographical distribution. Molecular epidemiology studies primarily based on restriction fragment length polymorphism (RFLP) method revealed that three main types are predominant in North America and Europe, whereas other diverse genotypes are found in other parts of the world. Microsatellite (MS) as a type of genetic marker has been widely used in many organisms. Limited MS genotyping, however, to fingerprint T. gondii isolates has been reported and little is known about the MS data of the strains predominantly prevalent in China.MethodsGenotyping of twenty-eight Chinese T. gondii isolates were performed using 15 MS markers located on 12 different chromosomes. Results were analyzed in terms of population structure by a Bayesian statistical approach. Phylogenetic analysis was obtained from a Neighbor-Net phylogenetic network. The virulence analyses of some representative isolates were determined by inoculation of mice and cell invasion assays. The gene expressions of some virulence-associated factors (VFs) were performed by quantitative real-time PCR (qRT- PCR).ResultsThree haplogroups were clustered among the 28 isolates although minor genetic differences were found within haplogroups. The majority of strains belong to one haplogroup corresponding to the previously described Chinese 1 type (ToxoDB#9). Phylogenetic networks uncovered a limited diversity of T. gondii strains and the virulence differs in the strains sharing the same genotype. No remarkable difference, however, was noted in the tested VFs except for dense granule protein3 (GRA3), which was found to have a higher expression in low virulent TgCtwh6 (Wh6) strain than that in high virulent TgCtwh3 (Wh3) strain.ConclusionThe profile of microsatellite typing data from Chinese T. gondii strains revealed a limited genetic diversity and the selected VFs and phylogenetic network analyses displayed less divergence, although the strain virulence differs in the Chinese 1 type of T. gondii predominantly prevalent in China.

Variation detection based on next-generation sequencing of type Chinese 1 strains of Toxoplasma gondii with different virulence from China.

BackgroundToxoplasma gondii is an intracellular protozoan that affects most species of endothermic animals including humans with a great infection rate. The vertical transmission of T. gondii causes abortion, constituting a serious threat to humans and leading to great losses in livestock production. Distinct from population structure of T. gondii in North America and Europe, Chinese 1 (ToxoDB #9) is a dominant genotype prevalent in China. Among the isolates of Chinese 1, the Wh3 and Wh6 have different virulence and pathogenicity in mice. However, little has been known about their difference at the genomic level. Thus the next-generation sequencing (NGS) approach was used to discover the association of the phenotypical variations with the genome sequencing data and the expression and polymorphisms of the key effectors.ResultsWe successfully sequenced the genome of Chinese 1 strains of Wh3 and Wh6. The average sequencing depths were 63.91 and 63.61 for Wh3 and Wh6, respectively. The variations of both isolates were identified in comparison with reference genome of type I GT1 strain. There were 505,645 and 505,856 SNPs, 30,658 and 30,004 indels, 4661 and 2320 SVs, and 1942 and 3080 CNVs for Wh3 and Wh6, respectively. In target search variations of particular factors of T. gondii, the dense granule protein 3 (GRA3) and rhoptry neck protein 3 (RON3) were found to have 35 SNPs, 2 indels and 89 SNPs, 6 indels, respectively. GRA3 and RON3 were both found to have higher expression levels in less virulent Wh6 than in virulent Wh3. Both strains of type Chinese 1 share polymorphic GRA15II and ROPI/III with type I, II, and III strains.ConclusionsSequencing of the two strains revealed that genome structure of Chinese 1 and type I strains has considerable genomic variations. Sequencing and qRT-PCR analyses of 26 effectors displayed a remarkable variation that may be associated with phenotype and pathogenic differences.

Paeoniflorin ameliorates schistosomiasis liver fibrosis through regulating IL-13 and its signalling molecules in mice.

Treatment of liver fibrosis associated with Schistosoma japonicum ova-induced granulomas remains a challenging proposition. Paeoniflorin (PAE, C23H28O11) has anti-inflammatory, anti-allergic, and immunoregulatory effects and it is commonly used in Chinese Herbal prescriptions to treat hepatic disorders. The present study was carried out to investigate the effects of PAE on hepatic fibrosis of mice infected with S. japonicum and to explore its possible mechanism. Upon pathological examination of PAE-treated mice, the size of egg granuloma, fibrosis scores, the concentration of IL-13 and hydroxyproline in liver were significantly reduced compared with the model mice. In the primary culture of hepatic stellate cells (HSCs), PAE inhibited IL-13-induced collagen synthesis. These results suggested that PAE might alleviate the hepatic granulomas and fibrosis caused by S. japonicum and the inhibitory effect of PAE on hepatic fibrosis might be associated with its ability to decrease the level of IL-13 and to interfere with the IL-13 signalling molecule in HSCs.

Activated microglia contribute to neuronal apoptosis in Toxoplasmic encephalitis

BackgroundA plethora of evidence shows that activated microglia play a critical role in the pathogenesis of the central nervous system (CNS). Toxoplasmic encephalitis (TE) frequently occurs in HIV/AIDS patients. However, knowledge remains limited on the contributions of activated microglia to the pathogenesis of TE.MethodsA murine model of reactivated encephalitis was generated in a latent infection with Toxoplasma gondii induced by cyclophosphamide. The neuronal apoptosis in the CNS and the profile of pro-inflammatory cytokines were assayed in both in vitro and in vivo experiments.ResultsMicroglial cells were found to be activated in the cortex and hippocampus in the brain tissues of mice. The in vivo expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) were up-regulated in TE mice, and accordingly, the neuronal apoptosis was significantly increased. The results were positively correlated with those of the in vitro experiments. Additionally,apoptosis of the mouse neuroblastoma type Neuro2a (N2a) remarkably increased when the N2a was co-cultured in transwell with microglial cells and Toxoplasma tachyzoites. Both in vivo and in vitro experiments showed that minocycline (a microglia inhibitor) treatment notably reduced microglial activation and neuronal apoptosis.ConclusionsActivated microglia contribute to neuronal apoptosis in TE and inhibition of microglia activation might represent a novel therapeutic strategy of TE.

Paeoniflorin: A Monomer from Traditional Chinese Medical Herb Ameliorates Schistosoma japonicum Egg-Induced Hepatic Fibrosis in Mice

Abstract Treatment of liver fibrosis associated with Schistosoma japonicum ova-induced granulomas remains a challenging proposition. There is a close relationship between high levels of interleukin-13 (IL-13) and the development of severe schistosome fibrosis. In contrast, IL-13 receptor (R) α2 has an effective role in attenuation of profibrosis. Several Chinese herbs have significant beneficial effects in liver disease. Accordingly, the purpose of the present study was to investigate the therapeutic effect of Paeoniflorin (PAE) on liver fibrosis. A mouse model for liver fibrosis was established, using infection with S. japonicum cercariae via the skin. Liver tissue was used to examine the effect of PAE on hydroxyproline, collagen I and III, and IL-13 and IL-13Rα2. The results showed that PAE has significant suppressive effect on the increase of both hepatic hydroxyproline and collagen I and III, which are the main components of extracellular matrix (ECM). Meanwhile, PAE not only inhibits IL-13 production, it also elevates IL-13Rα2 in PAE-pretreated groups compared with controls. These results suggested that PAE can improve liver fibrosis due to S. japonicum infection. The effect of PAE appears to depend on a decrease of IL-13 and an increase of IL-13Rα2.

Polarization of macrophages induced by Toxoplasma gondii and its impact on abnormal pregnancy in rats.

Toxoplasma gondii infection is the leading cause of fetal intrauterine growth retardation among the five kinds of pathogens termed as TORCH, including Toxoplasma, Rubella virus, Cytomegalo virus, herpes virus and others during pregnancy. Pathogens infect the fetus through the placenta. T. gondii infection may result in congenital toxoplasmosis, miscarriage, stillbirth, and preemie, and increase pregnancy complications. Adaptive immune response induced by T. gondii infection stimulates T cells and macrophages to produce high levels of cytokines. Physiologically, the microenvironment of pregnancy was Th2-dominant. Here we set up a pregnant Sprague-Dawley rat model, and reported the polarization of macrophages induced by genotype Chinese 1 strain (Wh6) of Toxoplasma, and its adverse impact on pregnancy. The results showed that Wh6 infection pre- or in-gestation both led to abnormal pregnancy outcomes. Peritoneal macrophages in pre-gestation infection were polarized toward classically activated macrophages (M1), while in-gestation infection drove macrophages to polarize toward M2 activation. The Th2-dominant immune response in pregnant rat somewhat inhibits the excessive bias of the macrophages toward M1, and partially, toward M2. Infection of pre- and in-gestation may alter the physiological immune microenvironment in pregnant rats, giving rise to abnormal pregnancy outcomes.

Toxoplasma gondii GRA15II effector-induced M1 cells ameliorate liver fibrosis in mice infected with Schistosomiasis japonica.

Recent studies indicated that type II Toxoplasma gondii (Tg) GRA15II favored the generation of classically activated macrophages (M1), whereas type I/III TgROP16I/III promoted the polarization of alternatively activated macrophages (M2). A number of studies have demonstrated that M2 cells are involved in the pathogenesis of the liver fibrogenesis caused by Schistosoma japonicum. The purpose of the present study was to explore the inhibitory effect of Toxoplasma-derived TgGRA15II on mouse hepatic fibrosis with schistosomiasis. The gra15II and rop16I/III genes were amplified from strains T. gondii PRU and Chinese 1 Wh3, respectively. Lentiviral vectors containing the gra15II or rop16I/III plasmid were constructed and used to infect the RAW264.7 cell line. The polarization of the transfected cells was evaluated, followed by co-culture of the biased macrophages with mouse hepatic stellate JS1 cells. Then, mice were injected with GRA15II-driven macrophages via the tail vein and infected with S. japonicum cercariae. TgGRA15II induced a M1-biased response, whereas TgROP16I/III drove the macrophages to a M2-like phenotype. The in vitro experiments indicated that JS1 cell proliferation and collagen synthesis were decreased following co-culture with TgGRA15II-activated macrophages. Furthermore, mice inoculated with TgGRA15II-biased macrophages displayed a notable alleviation of collagen deposition and granuloma formation in their liver tissues. Our results suggest that TgGRA15II-induced M1 cells may dampen the M2 dominant pathogenesis of hepatic fibrosis and granulomatosis. These results provide insights into the use of parasite-derived immunomodulators as potential anti-fibrosis agents and to re-balance the schistosomiasis-induced immune response.

Schistosoma japonicum cystatin attenuates murine collagen-induced arthritis

Recombinant SjCystatin (rSjCystatin), a recombinant protein of Schistosoma japonicum cystatin, has been reported to have an effect on immunoregulation mediated by IL-10 induction. Rheumatoid arthritis (RA) is a common autoimmune inflammatory arthropathy, and recombinant immune-modulating drugs for RA treatment are under development. We aimed to study the putative immune regulation of rSjCystatin and its prophylactic/therapeutic effects on murine collagen-induced arthritis (CIA). CIA was induced in DBA/1 mice by inoculation with bovine collagen II (CII). rSjCystatin was administered prior or post development of CIA. The severity of CIA was assessed using established clinical and histopathological scoring systems. The incidence was also determined. The CII-specific antibodies in sera and cytokines in splenocyte culture supernatants were measured by ELISA. Th1/Th2/Th17 cells and Tregs development in splenocytes were monitored by flow cytometry. The inflammatory mediators in the diseased joint were semiquantitated by qPCR. Prophylactic injection of rSjCystatin attenuated paw clinical scores, incidence, and histopathology scores of joints in CIA mice. The arthritis-alleviative effects were closely associated with the augmentation of IL-4, IL-10, and collagen-specific IgG1, and with the distinct reduction of IFN-γ, collagen-specific IgG2a, and the marked decrease of proinflammatory cytokines IL-6, IL-17, and TNF-α and RANKL. The data indicate that rSjCystatin may prevent cartilage destruction and inflammation of joints in CIA mice. The effects are related to the inhibitory modulation of Th1 and Th17 and upregulation of Tregs and Th2 via a shift of cytokines profiling from Th1 to Th2 response.