Transcriptional profiling and functional characterization of Schistosoma japonicum-stimulated Alternatively Activated Macrophages
ABSTRACT: Alternatively activated macrophages (AAMφs) play important roles in a number of Th2 driven pathologies including asthma and allergy, and a number of parasitic infections. Our studies, and those of others, investigating the pathologies associated with infection with the helminth Schistosoma japonicum implicate a role for AAMφs in fibrosis and immunomodulation.. In the present study we show that S. japonicum-secreted egg antigens are able to induce the alternative activation of macrophages as characterised by the induction of Chi3l3 and Arg1 expression. Retnla, another common marker of AAMφs, was not consistently induced in these macrophages suggesting that the specific function of these cells may differ to those induced by S. mansoni and other parasites. Closer examination of the gene expression profile and functionality of these cells identified pathways independent of Retnla expression that could be important for their immunomodulatory activity such as modulating expression of T-cell co-stimulatory molecules and chemokines. In vivo generated S. japonicum soluble egg antigen stimulated AAMφs also exhibited a reduction in their phagocytic ability likely related to the induction of IL4 and decreased expression of cell surface receptors. Additionally these macrophages exhibited reduced expression of Toll-like receptors (TLRs) and an associated reduction in responsiveness to stimulation with TLR ligands. We did not observe pathways that would suggest that AAMφs have a direct profibrotic activity. Taken together, these data describe a mechanism by which alternative activation of macrophages may be induced during S. japonicum infection and highlight the importance of the context of activation in directing AAMφ phenotype and function. The gene expression profile of Schistosoma japonicum soluble egg antigen (SEA)-stimulated macrophages was compared with that of PBS stimulated controls. Macrophages were isolated from the peritoneal cavity of BALB/c mice (n=6 per group) stimulated by intraperitoneal injection with SEA or PBS. The macrophages were pooled and RNA was extracted from these cells. Microarray analysis was performed on cRNA synthesised from total RNA derived from these macrophages. The experiment was performed twice creating two biological replicates. Fold-change (relative to the respective PBS control) reported in supplementary file linked below.
Project description:Migrating schistosomula are an important stage of the schistosome lifecycle and represent a key target for elimination of infection by natural and vaccine induced host immune responses. To gain a better understanding of how these parasites initiate a primary host immune response we have characterised the host lung response to migrating Schistosoma japonicum schistosomula using a combination of histochemistry, microarrays and quantitative cytokine analysis. Our data suggest that, during a S. japonicum infection, actively migrating schistosomula induce a Type-2 cytokine response in the lung that may support the subsequent development of a CD4+ T helper 2 (Th2) response against egg antigens. This hypothesis is supported by the fact that schistosomula and schistosome eggs are known to express important Th2-inducing antigens such as omega-1, peroxiredoxin, kappa-5 and IPSE/alpha1. The host lung response to migrating schistosomula was associated with increased numbers of macrophages and expression of markers for alternatively activated macrophages (AAMφ) in the lung. Activation of AAMφ in the lung and at the systemic level could lead to the modulation of the host immune response to favour parasite survival. Induction of these cells could also contribute to diminished inflammatory responses to, for example, allergy and asthma that are known to be associated with helminth infections. These data enhance our understanding of the mechanisms whereby schistosomes may evade the immune response and the mechanisms by which schistosome infection can help influence the host response following exposure to allergenic stimuli. The gene expression profile of the murine lung was examined at 3 days weeks post infection with 500 Schistosoma japonicum cercariae in comparison to that of uninfected controls. Microarray analysis was performed on cRNA synthesised from total RNA derived from the lungs of 3 individual mice per group.
Project description:An ex vivo approach to studying hepatic schistosomiasis used thick naive mouse liver sections and sterile culturing. This tissue is unfixed, unfrozen, and alive for subsequent in vitro studies. We have cultured and monitored these sections for up to 48 hrs noting unchanged gross hepato-cellular morphology, and no significant increases in hepatotoxicity as demonstrated by media liver enzyme concentrations. Liver slices were treated with soluble egg antigen from Schistosoma japonicum eggs and cultured for 48hrs. Transcriptional changes were then analysed by microarray. The gene expression profile of the naive mouse liver slices cultered with and without the additional of soluble egg antigen (SEA) from Schistosoma japonicum eggs. Microarray analysis was performed on cRNA synthesised from total RNA. The liver of female 6 week old C57BL/6 mice were removed and then sectioned at 250um thickness using a vibrotome. Sections were cultered at 37oC and 5%CO2 in in William’s Medium E containing 25 mM glucose, 10 mg/ml gentamycin and 10% FBS. SEA was added at 10ug/ml and sections sampled at timepoints 0, 4hr, 24hr and 48hrs. Illumina mouse ref8_V2 arrays were used and fold changes compared to whole intact, precut tissue.
Project description:We report on the small RNA profiles of Schistosoma japonicum (S. japonicum) miRNAs using small RNA deep sequencing in the key stages of male-female pairing, gametogenesis, and egg production. Examination of different miRNAs between males and females in Schistosoma japonicum
Project description:Macrophages initiate, modulate, and also serve as final effector cells in immune responses during course of schistosomal infections. Presently, we discussed the roles of the gene expression profile and functional changes of macrophages in immune responses against the Schistosoma japonicum by microarray experiments. Hierarchical clustering analysis demonstrated that a significant switch in gene transformation associated with a type-1 response and linked with a type-2 cytokine phenotype occurs between 4.5 and 8 weeks post-infection. Moreover, the gene profiles at 3 later time-points following egg challenge were similar in complexity and magnitude. These data also showed that there are mostly inhibition in gene expression related TLR, IFN, MHC and TNFrsf at the switch between 4.5 and 8 weeks post-infection, It is suggested that these immunomodulatory genes may be down-regulated in resistance against S. japonicum eggs and granuloma pathology. The induction of alternatively activated macrophage was important for dampening the inflammation in hepatic granulomas and contributing to a decrease in cytotoxicity. The genes expressions involved in repair/remodeling during liver fibrosis were also observed after eggs production. Understanding these immune mechanisms related to parasite resistance, pathology, and growth with regard to the disease will be helpful in further studies on S. japonicum. Two-condition experiment, Control mice vs. Schistosoma japonicum-infected mice. replicates: 2 infected replicates. Different post-infection weeks
Project description:Chinese and Philippine strains of the blood fluke Schistosoma japonicum present clear and distinctive phenotypes in areas of fecundity, pathology, drug sensitivity and immunology. Despite these differences large scale sequencing efforts have focused solely on Chinese mainland strain of the parasite. We have undertaken a comparative genomic hybridisation (CGH) approach to highlight some of the structural differences in the genome of two of the major geographical isolates of S. japonicum. We identified seven distinct regions of the S. japonicum genome that present differential CGH between Chinese and Philippine strains of the blood fluke Schistosoma japonicum, representing either deletion or duplication regions in the Philippine strain. Within these regions, genes that may be related to phenotypical differences are identified and discussed. Genomic DNA was isolated from adult (7 week post cercarial challenge) Schistosoma japonicum Chinese and Philippine isolates and separate maleand femalesamples comparatively hybridised on an Agilent customn designed oligo microarray.
Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. The gene expression profile of macrophages from C57BL/6 and MD-2-deficient mice following either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 stimulation for 2 hours were compared to PBS-stimulated control cells . In vitro differentiated macrophages from two individual WT and MD-2-deficient mice were cultured and stimulated with agonists separately, comparing the gene expression to PBS-stimulated control cells from the same mouse. Comparisons of PBS-stimulated WT cells to PBS-stimulated MD-2-deficient cells were performed to directly compare basal gene expression in the two genotypes.
Project description:We have used a Schistosoma japonicum infected murine model with in vivo sub-lethal dosages of praziquantel against adult parasites. Differential gene expression of parasites was followed between 30 minutes and 24 hours post- drug administration, using a whole transcriptome microarray platform. Differential gene expression was considered separately between parasite gender. Total RNA was isolated from adult (7 week post cercarial challenge) Schistosoma japonicum male and females. Gene expression was determined through hybridisation on an Agilent custom designed oligo microarray.
Project description:Schistosomiasis japonica remains a significant public health problem in China and Southeast Asian countries. The most typical and serious outcome of the chronic oriental schistosomiasis is the progressive granuloma and fibrosis in the host liver, which has been a major medical challenge. However, the molecular mechanisms that underlie the hepatic pathogenesis induced by schistosomal egg deposition have not yet been well-defined. Using microarrays, we quantified the temporal gene expression profiles in the liver of Schistosoma japonicum-infected BALB/c mice at day 15, 30, and 45 post infection (pi) with that from uninfected mice as controls. Meanwhile, microRNA expression profiles from the same samples were decoded by parallel solexa sequencing. Gene expression alternation associated with liver damage was observed even at early stage of infection (e.g., pi 15), which became more magnificent onset of egg deposition within the liver tissue. Up-regulated genes were dominantly associated with inflammatory infiltration of liver during S. japonicum infection, whereas down-regulated genes primarily led to the hepatic functional disorders. More than 130 miRNAs were differentially expressed during S. japonicum infection, and dynamic miRNA-gene co-expression network has been constructed during the development of hepatic pathology. A four chip study using total RNA recovered from liver tissues of BALB/c mice which were percutaneously infected with 30 ± 2 cercariae of S. japonicum at day 0, 15, 30, and 45 post infection, respectively.
Project description:To confirm the changed gene expression profiles in GPR15 knock out (KO) macrophages, we applied a SurePrint G3 Mouse Gene Expression service from Takara Bio Inc (Kusatsu, Shiga, Japan) to analyze gene expression profiles in lipopolysaccharide (LPS)-stimulated GPR15KO macrophages, comparing with wild type (WT) macrophages. Most significantly up-regulated genes in GPR15KO macropahges included Il6, Il17a, Il23, Tnfsf8, Il1b, Ifna2 and Ccnd2. On the contrary, several inflammation-related genes, including Ccl17, Itgax, Nrp1 and Rasgrf2, were down-regulated in WT macrophages, compared to GPR15 KO macrophages. Abdominal macrophages from WT and GPR15 KO mice were stimulated with PBS or LPS (100 ng/ml) for 4 hrs. Total RNA were extracted using a TRIzol-chloroform based method.
Project description:In schistosomiasis japonica, the egg-induced granulomatous response and the development of extensive hepatic fibrosis is the main pathology. Information regarding the specific mechanisms associated with granuloma regression and the subsequent recovery events in the host liver are still limited. In this study, a murine model of schistosomiasis japonica was used to characterise the multicellular pathways occurring during liver regeneration. Schistosoma japonicum-infected C57BL/6 mice were administered with the drug praziquantel (PZQ), on a daily basis for five consecutive days to eliminate all adult parasites. The pathological changes of PZQ-treated groups after 3, 6 and 7 weeks post PZQ treatment were examined, along with the assessment of cellular infiltration to the liver. PZQ treatment significantly reduced the degree of splenomegaly, granuloma density and the collagen deposition of liver fibrosis. The infiltration of inflammatory cells, including neutrophils, eosinophils and macrophages to the liver were as well significantly decreased. Transcriptomic analysis revealed the significant up-regulation of fatty acid metabolism genes and the identification of peroxisome proliferator-activated receptor alpha (PPAR-α) as the upstream regulator during the process of liver recovery. Aryl hydrocarbon receptor (AhR) signalling pathway that is involved primarily in the regulation of hepatic enzymes responsible for xenobiotic metabolism was as well differentially up-regulated. These findings indicate that schistosome egg-induced fibrogenesis process is reversible, and provide a better understanding of the regression mechanisms associated with hepatic schistosomiasis. These results hold important implications for the future alleviation of this and other fibrotic diseases of clinical significance. C57BL/6 murine model infected with S. japonicum were treated orally with Praziquantel (PZQ) after 7 weeks post infection to examine the hepatic regression process following drug treatment. These PZQ-treated, non PZQ-treated and uninfected mice were then euthanised at 10, 13, and 14 weeks p.i. Livers were collected from each mice, and subjected to total RNA isolation and gene expression analysis. Microarray analysis of this study was performed using samples derived from 3 individual mice per group/time-point.