RESUMO
BACKGROUND: Schistosomiasis is still one of the most serious parasitic diseases. Evidence showed that the metabolite profile in serum can potentially act as a marker for parasitic disease diagnosis and evaluate disease progression and prognosis. However, the serum metabolome in patients with Schistosoma japonicum infection is not well defined. In this study, we investigated the metabolite profiles of patients with chronic and with advanced S. japonicum infection. METHODS: The sera of 33 chronic S. japonicum patients, 15 patients with advanced schistosomiasis and 17 healthy volunteers were collected. Samples were extracted for metabolites and analyzed with ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). RESULTS: We observed significant differences in metabolite profiles in positive and negative ion modes between patients with advanced and chronic S. japonicum infection. In patients with chronic S. japonicum infection, 199 metabolites were significantly upregulated while 207 metabolites were downregulated in advanced infection. These differential metabolites were mainly concentrated in steroid hormone biosynthesis, cholesterol metabolism and bile secretion pathways. We also found that certain bile acid levels were significantly upregulated in the progression from chronic to advanced S. japonicum infection. In receiver operator characteristic (ROC) analysis, we identified three metabolites with area under the curve (AUC) > 0.8, including glycocholic (GCA), glycochenodeoxycholate (GCDCA) and taurochenodeoxycholic acid (TCDCA) concentrated in cholesterol metabolism, biliary secretion and primary bile acid biosynthesis. CONCLUSIONS: This study provides evidence that GCA, GCDCA and TCDCA can potentially act as novel metabolite biomarkers to distinguish patients in different stages of S. japonicum infection. This study will contribute to the understanding of the metabolite mechanisms of the transition from chronic to advanced S. japonicum infection, although more studies are needed to validate this potential role and explore the underlying mechanisms.
Assuntos
Biomarcadores , Espectrometria de Massas , Metabolômica , Schistosoma japonicum , Esquistossomose Japônica , Humanos , Esquistossomose Japônica/parasitologia , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/sangue , Metabolômica/métodos , Schistosoma japonicum/metabolismo , Masculino , Feminino , Animais , Adulto , Pessoa de Meia-Idade , Espectrometria de Massas/métodos , Biomarcadores/sangue , Metaboloma , Cromatografia Líquida de Alta Pressão/métodos , Idoso , Adulto Jovem , Cromatografia Líquida/métodos , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Schistosomiasis is a parasitic disease characterized by liver fibrosis, a process driven by the activation of hepatic stellate cells (HSCs) and subsequent collagen production. Previous studies from our laboratory have demonstrated the ability of Schistosoma japonicum protein P40 (SjP40) to inhibit HSCs activation and exert an antifibrotic effect. In this study, we aimed to elucidate the molecular mechanism underlying the inhibitory effect of recombinant SjP40 (rSjP40) on HSCs activation. Using a cell model in which rSjP40 inhibited LX-2 cell activation, we performed RNA-seq analyses and identified ATF3 as the most significantly altered gene. Further investigation revealed that rSjP40 inhibited HSCs activation partly by suppressing ATF3 activation. Knockdown of ATF3 in mouse liver significantly alleviated S. japonicum-induced liver fibrosis. Moreover, our results indicate that ATF3 is a direct target of microRNA-494-3p, a microRNA associated with anti-liver fibrosis effects. rSjP40 was found to downregulate ATF3 expression by upregulating microRNA-494-3p in LX-2 cells. This downregulation led to the inhibition of the expression of liver fibrosis proteins α-SMA and COL1A1, ultimately alleviating liver fibrosis caused by S. japonicum.
Assuntos
Fator 3 Ativador da Transcrição , Proteínas de Helminto , Células Estreladas do Fígado , Cirrose Hepática , MicroRNAs , Schistosoma japonicum , Esquistossomose Japônica , Animais , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/parasitologia , Esquistossomose Japônica/parasitologia , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/genética , Cirrose Hepática/parasitologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Camundongos , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Actinas/metabolismo , Actinas/genética , Linhagem Celular , Regulação da Expressão Gênica , Fígado/metabolismo , Fígado/parasitologia , Fígado/patologia , Modelos Animais de Doenças , Antígenos de HelmintosRESUMO
Schistosoma infection is one of the major causes of liver fibrosis. Emerging roles of hepatic progenitor cells (HPCs) in the pathogenesis of liver fibrosis have been identified. Nevertheless, the precise mechanism underlying the role of HPCs in liver fibrosis in schistosomiasis remains unclear. This study examined how autophagy in HPCs affects schistosomiasis-induced liver fibrosis by modulating exosomal miRNAs. The activation of HPCs was verified by immunohistochemistry (IHC) and immunofluorescence (IF) staining in fibrotic liver from patients and mice with Schistosoma japonicum infection. By coculturing HPCs with hepatic stellate cells (HSCs) and assessing the autophagy level in HPCs by proteomic analysis and in vitro phenotypic assays, we found that impaired autophagy degradation in these activated HPCs was mediated by lysosomal dysfunction. Blocking autophagy by the autophagy inhibitor chloroquine (CQ) significantly diminished liver fibrosis and granuloma formation in S. japonicum-infected mice. HPC-secreted extracellular vehicles (EVs) were further isolated and studied by miRNA sequencing. miR-1306-3p, miR-493-3p, and miR-34a-5p were identified, and their distribution into EVs was inhibited due to impaired autophagy in HPCs, which contributed to suppressing HSC activation. In conclusion, we showed that the altered autophagy process upon HPC activation may prevent liver fibrosis by modulating exosomal miRNA release and inhibiting HSC activation in schistosomiasis. Targeting the autophagy degradation process may be a therapeutic strategy for liver fibrosis during Schistosoma infection.
Assuntos
Autofagia , Exossomos , Cirrose Hepática , MicroRNAs , Células-Tronco , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/parasitologia , Camundongos , Humanos , Exossomos/metabolismo , Células-Tronco/metabolismo , Células Estreladas do Fígado/metabolismo , Esquistossomose Japônica/metabolismo , Masculino , Schistosoma japonicum/genética , Feminino , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Esquistossomose/complicações , Fígado/patologia , Fígado/metabolismo , Fígado/parasitologiaRESUMO
Schistosomiasis is a fatal zoonotic parasitic disease that also threatens human health. The main pathological features of schistosomiasis are granulomatous inflammation and subsequent liver fibrosis, which is a complex, chronic, and progressive disease. Extracellular vesicles (EVs) derived from schistosome eggs are broadly involved in host-parasite communication and act as important contributors to schistosome-induced liver fibrosis. However, it remains unclear whether substances secreted by the EVs of Schistosoma japonicum, a long-term parasitic "partner" in the hepatic portal vein of the host, also participate in liver fibrosis. Here, we report that EVs derived from S. japonicum worms attenuated liver fibrosis by delivering sja-let-7 into hepatic stellate cells (HSCs). Mechanistically, activation of HSCs was reduced by targeting collagen type I alpha 2 chain (Col1α2) and downregulation of the TGF-ß/Smad signaling pathway both in vivo and in vitro. Overall, these results contribute to further understanding of the molecular mechanisms underlying host-parasite interactions and identified the sja-let-7/Col1α2/TGF-ß/Smad axis as a potential target for treatment of schistosomiasis-related liver fibrosis.
Assuntos
Vesículas Extracelulares , Cirrose Hepática , Schistosoma japonicum , Esquistossomose Japônica , Animais , Vesículas Extracelulares/metabolismo , Cirrose Hepática/parasitologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/parasitologia , Esquistossomose Japônica/patologia , Camundongos , Interações Hospedeiro-Parasita/fisiologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/parasitologia , Células Estreladas do Fígado/patologia , MicroRNAs/metabolismo , MicroRNAs/genética , Transdução de Sinais , Humanos , Proteínas de Helminto/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Schistosomiasis remains an important public health concern. The eggs deposited in livers invoke a Th2-dominant response, which mediates the fibrotic granulomatous response. However, the mechanisms involved in this immunopathological process are still not perfectly clear. Here, we report a single-cell transcriptional landscape of longitudinally collected BALB/c mouse splenocytes at different time points after Schistosoma japonicum infection. We found that exhausted CD4+ T cells were enriched after infection, changing from coproducing multiple cytokines to predominantly producing the Th2 cytokine IL-4. Regulatory B cells had high expression of Fcrl5, Ptpn22, and Lgals1, potentially regulating exhausted CD4+ T cells via direct PD-1-PD-L2 and PD-1-PD-L1 interactions. Within the myeloid compartment, the number of precursor and immature neutrophils sharply increased after infection. Moreover, dendritic cells, macrophages, and basophils showed inhibitory interactions with exhausted CD4+ T cells. Besides, in mouse livers, we found that exhausted CD4+ T cells were distributed around egg granuloma, promoting collagen expression in primary mouse hepatic stellate cells via IL-4 secretion, resulting in liver fibrosis. Our study provides comprehensive characterization of the composition and cellular states of immune cells with disease progression, which will facilitate better understanding of the mechanism underlying liver fibrotic granulomatous response in schistosomiasis.
Assuntos
Schistosoma japonicum , Esquistossomose Japônica , Esquistossomose , Camundongos , Animais , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/patologia , Linfócitos T CD4-Positivos , Interleucina-4 , Receptor de Morte Celular Programada 1 , Exaustão das Células T , Cirrose Hepática/patologia , Fígado , Fibrose , CitocinasRESUMO
Objectives To investigate the effect of the imbalance of Th17/Treg on egg granuloma formation of liver with Schistosomiasis japonicum. Methods The BALB/c mice were infected with Schistosoma japonicum cercariae to establish a model of Schistosomiasis japonica. The blood samples, liver tissues and spleen tissue were harvested at the 2nd, 4th, 6th, 8th week, respectively. HE staining and Masson staining were performed to assess the pathological characteristics of the liver. Flow cytometry (FCM) was conducted to evaluate the proportion of CD4+ T cell subsets including Th17 cells and Tregs in liver and spleen tissue. The quantitative real-time PCR (qRT-PCR) was carried out to investigate the mRNA level of cytokines including RORγt, FOXP3, IL-6, IL-17, IL-23 and IL-10 in liver tissues. Finally, ELISA was performed to assess the serum level of cytokines including IL-6, IL-17, IL-23 and TGF-ß. Schistosoma japonicium soluble egg antigen (SjSEA) were prepared to stimulate mouse spleen cells in vitro. qRT-PCR was carried out to investigate the mRNA level of cytokine including RORγt and FOXP3 and ELISA was performed to assess the expression level of cytokines including IL-6, IL-17, IL-23 and TGF-ß at different time points. Results HE and Masson staining demonstrated that inflammatory cell infiltration, schistosome egg granuloma formation and the collagen deposition increased in the liver tissue after the 4th week. The longer the infection, the more severe the liver pathology. In the liver and spleen tissues, the percentage of Th17 cells of infection group (2nd, 4th and 6th weeks) were significantly higher than the healthy group. The percentage of Tregs in the liver tissues of infection group (4th, 6th and 8th weeks) were significantly higher than the healthy group, and the percentage of Tregs in the spleen of infection group (2nd and 4th weeks) were significantly higher than the healthy group. Th17/Treg ratios in the liver of infection group were lower than the healthy group. Th17/Treg ratios in the spleen of infection group (2nd and 4th weeks) were lower than the healthy group, while it increased in the 6th week. At the same time, the levels of Th17 cells and Tregs related nuclear transcription factors and cytokines showed similar dynamic changes as the percentages of T cell subsets. SjSEA can induce the differentiation of Th17 and Tregs and the expression of related cytokines and transcription factors. Conclusion Th17 cells may play a major role in liver pathology, and the imbalance of Th17 cells/Tregs was closely related to the schistosome egg granuloma formation.
Assuntos
Schistosoma japonicum , Esquistossomose Japônica , Animais , Citocinas/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Granuloma/metabolismo , Granuloma/patologia , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Interleucina-6/metabolismo , Fígado , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , RNA Mensageiro/metabolismo , Schistosoma japonicum/metabolismo , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/patologia , Linfócitos T Reguladores , Células Th17 , Fator de Crescimento Transformador beta/metabolismoRESUMO
The source of macrophages that contribute to human liver disease remains poorly understood. The purpose of this study is to investigate the functional mechanism of peritoneal macrophages in the development of hepatic immunopathology. By performing the natural infection with the blood fluke Schistosoma japonicum (S. japonicum) and the chemically carbon tetrachloride (CCl4 )-induced liver injured mouse model, we identified the peritoneal cavity as an essential source of hepatic macrophages. Here, we show that a large number of F4/80+ macrophages was accumulated in the peritoneal cavity during liver injury. An unknown source population of macrophages, which highly expressed GATA6 that is specific to peritoneal macrophages, was found to exist in the injured livers. Peritoneal macrophage deletion by injection with clodronate-containing liposomes led to an attenuated hepatic pathology and the inflammatory microenvironment, while adoptive transfer of macrophages into the abdominal cavity, by contrast, results in restoring liver pathology. Importantly, there are set genes of monocyte chemoattractant protein (MCP)-1, -2, and -3 that are highly related to recruit GATA6+ macrophages during S. japonicum infection, while administration of bindarit, a selective inhibitor of MCPs synthesis, dramatically decreased the hepatic expression of GATA6+ macrophages and thus attenuated hepatic pathology. Furthermore, in vivo study showed that peritoneal macrophages promote hepatic immunopathology is dependent on the accumulation of regulatory T cells (Tregs) in the liver. Altogether, these data provide the first clear evidence that GATA6+ peritoneal macrophages play critical roles in both the formation of hepatic immunopathology and the accumulation of Tregs cells.
Assuntos
Schistosoma japonicum , Esquistossomose Japônica , Animais , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Humanos , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Esquistossomose Japônica/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
Schistosoma japonicum infections, which lead to local inflammatory responses to schistosome eggs trapped in host tissues, can result in long-term, severe complications. The development of schistosomiasis may result from a complex interaction between the pathogenic, environmental, and host genetic components. Notably, the genetic factors that influence the development of schistosomiasis complications are poorly understood. Here we performed a genome-wide association study on multiple schistosomiasis-related phenotypes of 637 unrelated schistosomiasis patients in the Chinese population. Among three indicators of liver damage, we identified two novel, genome-wide significant single-nucleotide polymorphisms (SNPs) rs34486793 (P = 1.415 × 10-8) and rs2008259 (P = 6.78 × 10-8) at locus 14q32.2 as well as a gene, PMEPA1, at 20q13.31 (index rs62205791, P = 6.52 × 10-7). These were significantly associated with serum levels of hyaluronic acid (HA). In addition, RASIP1 and MAMSTR at 19q13.33 (index rs62132778, P = 1.72 × 10-7) were significantly associated with serum levels of aspartate aminotransferase (AST), and TPM1 at 15q22.2 (index rs12442303, P = 4.39 × 10-7) was significantly associated with serum levels of albumin. In schistosomiasis clinical signs, ITIH4 at 3p21.1 (index rs2239548) was associated with portal vein diameter (PVD) class, an indicator of portal hypertension, and OGDHL at 10q11.23 (index rs1258172) was related to ascites grade. We also detected an increased expression of these six genes in livers of mice with severe schistosomiasis. Summary data-based Mendelian randomization analyses indicated that ITIH4, PMEPA1 and MAMSTR were pleiotropically associated with PVD class, HA and AST, respectively.
Assuntos
Esquistossomose Japônica , Esquistossomose , Animais , China/epidemiologia , Estudo de Associação Genômica Ampla , Humanos , Fígado/patologia , Proteínas de Membrana/metabolismo , Camundongos , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/patologiaRESUMO
Schistosoma is a genus of parasitic trematodes that undergoes complex migration in final hosts, finally developing into adult worms, which are responsible for egg production and disease dissemination. Recent studies documented the importance of extracellular vesicles (EVs) in the regulation of host-parasite interactions. Herein, we investigated the microRNA (miRNA) profiles of EVs isolated from host plasma at different stages of Schistosoma japonicum infection (lung stage: 3 days post-infection (dpi), and liver stages: 14 and 21 dpi) to identify miRNA cargo potentially involved in the pathogenesis and immune regulation of schistosomiasis. Characterization of the isolated plasma EVs revealed their diameter to be approximately 100 nm, containing typical EV markers such as Hsp70 and Tsg101. Deep sequencing analysis indicated the presence of 811 known and 15 novel miRNAs with an increasing number of differential miRNAs from the lung stage (27 miRNAs) to the liver stages (58 and 96 miRNAs at 14 and 21 dpi, respectively) in the plasma EVs of infected mice compared to EVs isolated from the uninfected control. In total, 324 plasma EV miRNAs were shown to be co-detected among different stages of infection and the validation of selected miRNAs showed trends of abundance similar to deep sequencing analysis. For example, miR-1a-3p and miR-122-5p showed higher abundance, whereas miR-150-3p and miR-126a showed lower abundance in the plasma EVs of infected mice at 3, 14, and 21 dpi as compared to those of uninfected mice. In addition, bioinformatic analysis combined with PCR validation of the miRNA targets, particularly those associated with the immune system and parasitic infectious disease, indicated a significant increase in the expression of Gbp7and Ccr5 in contrast to the decreased expression of Fermt3, Akt1, and IL-12a. Our results suggested that the abundance of miRNA cargo of the host plasma EVs was related to the stages of Schistosoma japonicum infection. Further studies on the roles of these miRNAs may reveal the regulatory mechanism of the host-parasite interaction. Moreover, the differentially abundant miRNA cargo in host EVs associated with S. japonicum infection may also provide valuable clues for identifying novel biomarkers for schistosomiasis diagnosis.
Assuntos
Vesículas Extracelulares , MicroRNAs , Schistosoma japonicum , Esquistossomose Japônica , Animais , Vesículas Extracelulares/metabolismo , Interações Hospedeiro-Parasita , Camundongos , MicroRNAs/metabolismo , Schistosoma japonicum/genética , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/parasitologiaRESUMO
Background: Th cells (helper T cells) have multiple functions in Schistosoma japonicum (S. japonicum) infection. Inducible co-stimulator (ICOS) is induced and expressed in activated T lymphocytes, which enhances the development of B cells and antibody production through the ICOS/ICOSL pathway. It remains unclear about the role and possible regulating mechanism of ICOS+ Th cells in the spleen of S. japonicum-infected C57BL/6 mice. Methods: C57BL/6 mice were infected with cercariae of S. japonicum through the abdomen. The expression of ICOS, activation markers, and the cytokine production on CD4+ ICOS+ Th cells were detected by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR). Moreover, the differentially expressed gene data of ICOS+ and ICOS- Th cells from the spleen of infected mice were obtained by mRNA sequencing. Besides, Western blot and chromatin immunoprecipitation (ChIP) were used to explore the role of Ikzf2 on ICOS expression. Results: After S. japonicum infection, the expression of ICOS molecules gradually increased in splenic lymphocytes, especially in Th cells (P < 0.01). Compared with ICOS- Th cells, more ICOS+ Th cells expressed CD69, CD25, CXCR5, and CD40L (P < 0.05), while less of them expressed CD62L (P < 0.05). Also, ICOS+ Th cells expressed more cytokines, such as IFN-γ, IL-4, IL-10, IL-2, and IL-21 (P < 0.05). RNA sequencing results showed that many transcription factors were increased significantly in ICOS+ Th cells, especially Ikzf2 (P < 0.05). And then, the expression of Ikzf2 was verified to be significantly increased and mainly located in the nuclear of ICOS+ Th cells. Finally, ChIP experiments and dual-luciferase reporter assay confirmed that Ikzf2 could directly bind to the ICOS promoter in Th cells. Conclusion: In this study, ICOS+ Th cells were found to play an important role in S. japonicum infection to induce immune response in the spleen of C57BL/6 mice. Additionally, Ikzf2 was found to be one important transcription factor that could regulate the expression of ICOS in the spleen of S. japonicum-infected C57BL/6 mice.
Assuntos
Fator de Transcrição Ikaros/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Ativação Linfocitária , Schistosoma japonicum/patogenicidade , Esquistossomose Japônica/parasitologia , Baço/parasitologia , Linfócitos T Auxiliares-Indutores/parasitologia , Animais , Sítios de Ligação , Proliferação de Células , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita , Fator de Transcrição Ikaros/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Schistosoma japonicum/imunologia , Esquistossomose Japônica/genética , Esquistossomose Japônica/imunologia , Esquistossomose Japônica/metabolismo , Transdução de Sinais , Baço/imunologia , Baço/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Emerging evidences have highlighted the crucial role of microRNAs (miRNAs) in the liver cirrhosis, but the relationship between miR-130a-3p and liver cirrhosis is not entirely clear. As we all know, schistosomiasis, as one of the zoonoses, can lead to liver cirrhosis when it advances. In this study, we investigated the biological functions of miR-130a-3p on the liver fibrosis of schistosomiasis in vivo and in vitro. The mice infected with Schistosoma japonicum (S. japonicum) were treated with lentivirus vector (LV)-miR-130a-3p by hydrodynamic injection through the tail vein. Our findings showed significantly decreased expression of miR-130a-3p both in the serum of patients with cirrhosis and in the liver of mice infected with S. japonicum. The results showed that LV-miR-130a-3p could effectively enter into the liver and alleviate liver granulomatous inflammation and collagen deposition. Simultaneously, LV-miR-130a-3p-promoted macrophages presented the Ly6Clo phenotype, concomitant with the decreased expression of the tissue inhibitor of metalloproteinases (TIMP) 1, and increased the expression of matrix metalloproteinase (MMP) 2, which contributed to the dissolution of collagen. Furthermore, overexpression of miR-130a-3p not only inhibited the activation and proliferation of hepatic stellate cells (HSCs) but also induced the apoptosis of HSCs. In addition, we also confirmed that miR-130a-3p enables to bind with mitogen-activated protein kinase (MAPK) 1 and transforming growth factor-beta receptors (TGFBR) 1 and TGFBR2 genes and inhibit the expressions of these genes. Our findings suggested that miR-130a-3p might represent as the potential candidate biomarker and therapeutic target for the prognosis identification and treatment of schistosomiasis liver fibrosis.
Assuntos
Antígenos Ly/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/prevenção & controle , Fígado/parasitologia , Macrófagos/metabolismo , MicroRNAs/administração & dosagem , Schistosoma japonicum/patogenicidade , Esquistossomose Japônica/prevenção & controle , Animais , Apoptose , Estudos de Casos e Controles , Linhagem Celular , Proliferação de Células , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Feminino , Células Estreladas do Fígado/imunologia , Células Estreladas do Fígado/parasitologia , Interações Hospedeiro-Parasita , Humanos , Fígado/imunologia , Fígado/metabolismo , Cirrose Hepática/imunologia , Cirrose Hepática/metabolismo , Cirrose Hepática/parasitologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Fenótipo , Schistosoma japonicum/imunologia , Esquistossomose Japônica/imunologia , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/parasitologia , Transdução de SinaisRESUMO
Schistosomiasis is a neglected tropical disease of public health concern. The most devastating pathology in schistosomiasis japonica and mansoni is mainly attributed to the egg-induced granulomatous response and secondary fibrosis in host liver, which may lead to portal hypertension or even death of the host. Schistosome eggs induce M2 macrophages-rich granulomas and these M2 macrophages play critical roles in the maintenance of granuloma and subsequent fibrosis. Reactive oxygen species (ROS), which are highly produced by stimulated macrophages during infection and necessary for the differentiation of M2 macrophages, are massively distributed around deposited eggs in the liver. However, whether ROS are induced by schistosome eggs to subsequently promote M2 macrophage differentiation, and the possible underlying mechanisms as well, remain to be clarified during S. japonicum infection. Herein, we observed that extensive expression of ROS in the liver of S. japonicum-infected mice. Injection of ROS inhibitor in infected mice resulted in reduced hepatic granulomatous responses and fibrosis. Further investigations revealed that inhibition of ROS production in S. japonicum-infected mice reduces the differentiation of M2, accompanied by increased M1 macrophage differentiation. Finally, we proved that S. japonicum egg antigens (SEA) induce a high level of ROS production via both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and mitochondria in macrophages. Our study may help to better understand the mechanism of schistosomiasis japonica-induced hepatic pathology and contribute to the development of potential therapeutic strategies by interfering with ROS production.
Assuntos
Fígado/patologia , Macrófagos/citologia , Óvulo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Schistosoma japonicum/fisiologia , Esquistossomose Japônica/fisiopatologia , Animais , Diferenciação Celular , Humanos , Fígado/metabolismo , Fígado/parasitologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Schistosoma japonicum/genética , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/parasitologiaRESUMO
BACKGROUND: Hepatic stellate cells (HSCs) are one of the main cell types involved in liver fibrosis induced by many factors, including schistosomes. Previous studies in our lab have shown that recombinant P40 protein from Schistosoma japonicum (rSjP40) can inhibit HSC activation in vitro. Let-7b is a member of the let-7 microRNA family and plays an inhibitory role in a variety of diseases and inflammatory conditions. In this study, we investigated the role of let-7b in the inhibition of HSC activation by rSjP40. METHODS: Expression of let-7b was detected by quantitative real-time PCR. A dual luciferase assay was used to confirm direct interaction between let-7b and collagen I. We also used western blot to assess protein levels of TGFßRI and collagen type I α1 (COL1A1). RESULTS: We found that rSjP40 up-regulates expression of let-7b in HSCs. Let-7b inhibits collagen I expression by directly targeting the 3'UTR region of the collagen I gene. Furthermore, we discovered that let-7b inhibitor partially restores the loss of collagen I expression caused by rSjP40. CONCLUSION: Our research clarifies the role of let-7b in the inhibition of HSC activation by rSjP40 and will provide new insights and ideas for the inhibition of HSC activation and treatment of liver fibrosis.
Assuntos
Antígenos de Helmintos/metabolismo , Proteínas de Helminto/metabolismo , Células Estreladas do Fígado/metabolismo , MicroRNAs/metabolismo , Schistosoma japonicum/metabolismo , Esquistossomose Japônica/metabolismo , Regiões 3' não Traduzidas , Animais , Antígenos de Helmintos/genética , Linhagem Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Regulação da Expressão Gênica , Proteínas de Helminto/genética , Células Estreladas do Fígado/parasitologia , Interações Hospedeiro-Parasita , Humanos , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/parasitologia , MicroRNAs/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schistosoma japonicum/genética , Esquistossomose Japônica/genética , Esquistossomose Japônica/parasitologiaRESUMO
BACKGROUND: Hepatic stellate cell (HSC) activation plays a pivotal role in hepatic inflammation and liver fibrosis. TLR4 pathway activation has been reported to be involved in mice liver fibrosis induced by hepatitis virus infection, alcohol abuse, biliary ligation, carbon tetrachloride 4 treatment, and Schistosoma japonicum (Sj) infection. The effect and mechanisms of the cyclooxygenase 2 (COX2)/prostanoid E2 (PGE2) axis on liver fibrosis induced by Sj are still unclear. METHODS: Mice liver fibrosis were induced by cutaneous infection of Sj cercariae. COX-2 inhibitor, NS398 were injected from week 5 to week 7, while TLR4 inhibitor TAK242 were injected from week 4 to week 8 post Sj infection. Human HSCs line, LX-2 cells were cultured and exposed to LPS or synthetic PGE2, or pretreated by TAK242, TLR4-siRNA or NS398. Liver tissue and serum or in vitro cultured cell lysaste were collected at indicated time courses for exploring the relationship between TLR4 and COX2-PGE2 axis through qPCR, western blot, immunohistochemical assay, ect. One-way analysis of variance among multiple groups followed by Uncorrected Fisher's LSD-t test or paired comparisons through t test were performed to tell the statistical differences. RESULTS: This study investigated the link between the COX2/PGE2 axis and TLR4 signaling in the induction of liver fibrogenesis in mice during Sj infection and in vitro culture of HSC strain-LX-2. The COX2/PGE2 axis was positively associated with Sj-induced liver fibrosis. TLR4 pathway activation stimulated the COX2/PGE2 axis in Sj-infected mice and in lipopolysaccharide (LPS)-exposed cultured HSCs. Synthetic PGE2 activated cultured HSCs through upregulation of alpha smooth muscle actin (α-SMA) expression. In LPS-triggered HSCs, NS398, a COX2 inhibitor, led to suppression of PGE2 synthesis and reduced expression of α-SMA and type I collagen (COL I). CONCLUSIONS: These results indicate firstly the positive association of the COX2/PGE2 axis with liver fibrosis induced by Sj infection. TLR4 signaling may at least partially control the COX2/PGE2 axis in Sj-infected mice liver and in vitro cultured HSCs. The COX2/PGE2-EP2/EP4 axis might be a good drug target against liver fibrosis induced by Sj infection.
Assuntos
Ciclo-Oxigenase 2/genética , Dinoprostona/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/parasitologia , Esquistossomose Japônica/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Animais , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Feminino , Células Estreladas do Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Esquistossomose Japônica/imunologia , Receptor 4 Toll-Like/genéticaRESUMO
Background: The host-parasite relationship is based on subtle interplay between parasite survival strategies and host defense mechanisms. It is well known that helminth infection, which afflicts more than one billion people globally, correlates with a decreased prevalence of obesity. Dissecting the underlying mechanisms can provide new targets for treating obesity from the host-parasite interaction perspective. Methods: C57BL/6 mice received a normal or high-fat diet (HFD) with or without Sjp40 (one main component of schistosome-derived soluble egg antigens) treatment. Both the loss and gain-of-function experiments by the inhibitor suppression and lentivirus treatment of miR-802 were utilized to elucidate the role of miR-802/AMPK axis in host lipid metabolism. Hepatocyte lipogenesis assay and metabolic parameters were assessed both in vivo and in vitro. The potential interactions among Sjp40, CD36, miR-802, Prkab1, and AMPK were clarified by pull-down, miRNA expression microarray, quantitative RT-PCR, dual-luciferase reporter assay, and western blotting analysis. Results: We showed a link between decreased miR-802 and impaired lipid metabolism in Schistosoma japonicum infected mice. The decreased miR-802 promotes murine Prkab1 or human Prkaa1 expression, respectively, which increases levels of phosphorylated AMPK, resulting in a decrease in hepatic lipogenesis. Also, injection with schistosome-derived soluble egg antigens (SEA) attenuated metabolism. We demonstrated that Sjp40 as a main component of SEA interacted with CD36 on hepatocytes to inhibit miR-802, resulting in the activation of AMPK pathway and subsequent attenuation of lipogenesis. Collectively: Our study reveals the significant role of miR-802/AMPK axis in hepatic lipid metabolism and identifies the therapeutic potential of Sjp40 in treating obesity-related fatty liver.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , MicroRNAs/metabolismo , Obesidade/metabolismo , Animais , Antígenos CD36/metabolismo , Dieta Hiperlipídica/métodos , Interações Hospedeiro-Parasita/fisiologia , Lipogênese/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Schistosoma japonicum , Esquistossomose Japônica/metabolismoRESUMO
Schistosomiasis is a major public health problem that is included in the neglected tropical diseases. The early diagnosis and detection of the pathogen are of critical importance in the control of the disease. The diagnostic techniques in use include the detection of worm's eggs in fecal examination or detection of circulating antigens in immunological based assays. These traditional strategies lack sensitivity in earlier detection of the schistosomiasis. Cell-free DNA (cfDNA) that includes the fragments of parasitic DNA circulating in the body fluids of host offers an alternative mean for the rapid pathogen detection and thus is a useful diagnostic tool. In this study, we explored the usefulness of the mitochondrial cfDNA markers for the diagnosis of schistosomiasis from the experimentally infected hosts (rabbits and mice). In this study we found mitochondrial DNA fragment cytochrome B gene as persistent and useful cfDNA marker for the early detection of schistosomiasis. We evaluated the sensitivity of cfDNA marker with varying numbers of cercaria. Overall, our results suggest that cfDNA markers can be useful for developing a diagnostic tool for the detection of S. japonicum infection.
Assuntos
Citocromos b/genética , Schistosoma japonicum/genética , Schistosoma japonicum/metabolismo , Esquistossomose Japônica/diagnóstico , Esquistossomose Japônica/metabolismo , Animais , Biomarcadores/sangue , Ácidos Nucleicos Livres/sangue , DNA Mitocondrial/sangue , DNA de Protozoário/sangue , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Técnicas de Diagnóstico Molecular/métodos , Doenças Negligenciadas/diagnóstico , Doenças Negligenciadas/metabolismo , Doenças Negligenciadas/parasitologia , Coelhos , Esquistossomose Japônica/parasitologia , Sensibilidade e EspecificidadeRESUMO
Schistosomiasis is a neglected tropical disease with over 250 million people infected worldwide. The main clinically important species Schistosoma mansoni (S. mansoni) and Schistosoma japonicum (S. japonicum) cause inflammatory responses against tissue-trapped eggs, resulting in formation of granulomas mainly in host liver. Persistent granulomatous response results in severe fibrosis in the liver, leading to irreversible impairment of the liver and even death of the host. CD1d, a highly conserved MHC class I-like molecule, is expressed by both haematopoietic and non-haematopoietic cells. CD1d on antigen-presenting cells (APCs) of haematopoietic origin presents pathogen-derived lipid antigens to natural killer T (NKT) cells, which enables them to rapidly produce large amounts of various cytokines and facilitate CD4+ T helper (Th) cell differentiation upon invading pathogens. Noteworthy, hepatocytes of non-haematopoietic origin have recently been shown to be involved in maintaining liver NKT cell homeostasis through a CD1d-dependent manner. However, whether hepatocyte CD1d-dependent regulation of NKT cell homeostasis also modulates CD4+ Th cell responses and liver immunopathology in murine schistosomiasis remains to be addressed. Here, we show in mice that CD1d expression on hepatocytes was decreased dramatically upon S. japonicum infection, accompanied by increased NKT cells, as well as upregulated Th1 and Th2 responses. Overexpression of CD1d in hepatocytes significantly decreased local NKT numbers and cytokines (IFN-γ, IL-4, IL-13), concomitantly with downregulation of both Th1 and Th2 responses and alleviation in pathological damage in livers of S. japonicum-infected mice. These findings highlight the potential of hepatocyte CD1d-targeted therapies for liver immunopathology control in schistosomiasis.
Assuntos
Antígenos CD1d/metabolismo , Hepatócitos/imunologia , Fígado/imunologia , Schistosoma japonicum/imunologia , Esquistossomose Japônica/imunologia , Animais , Antígenos CD1d/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Hepatócitos/metabolismo , Hepatócitos/patologia , Interações Hospedeiro-Parasita , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Células T Matadoras Naturais/parasitologia , Schistosoma japonicum/patogenicidade , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/parasitologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/parasitologia , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/parasitologiaRESUMO
Bnip3, which is regulated by Hif-1 in cells under oxygen deprivation, is a death related protein associated with autophagy and apoptosis. Hif-1 was reported to regulate autophagy to activate hepatic stellate cells (HSCs), while the specific molecular mechanism is vague. The possible mechanism of Hif-1 regulating autophagy of HSCs via Bnip3 was explored in this study. Bnip3 was detected in fibrotic liver tissues from humans and mice. Hif-1 was inhibited by chemical inhibitor and Bnip3 was detected in activated HSCs. The co-localization of Bnip3 and LC3B was captured by confocal microscopy and autophagic flow was assessed in Bnip3 siRNA transfected cells. Bnip3 interacted proteins were screened with mass spectrometry. The interaction of Bnip3 and vimentin was detected with co-immunoprecipitation and confocal microscopy. The results showed that Bnip3 was increased in fibrotic liver tissues and activated HSCs. Hif-1 inhibition suppressed Bnip3 expression in activated HSCs. Bnip3 was partially co-localized with autophagosomes and Bnip3 inhibition suppessed autophagy in activated HSCs. Bnip3 interacted with vimentin and Bnip3 expression was inhibited as vimentin was inhibited in activated HSCs. Conclusively, this study indicated that Bnip3 promoted autophagy and activation of HSCs, via interacting with vimentin, an intermediate filament protein with highly abundant expression in HSCs.
Assuntos
Autofagia , Células Estreladas do Fígado/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Cirrose Hepática/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Esquistossomose Japônica/metabolismo , Vimentina/metabolismo , Animais , Hipóxia Celular , Linhagem Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Lipopolissacarídeos , Camundongos , Proteínas Associadas aos Microtúbulos , Cultura Primária de CélulasRESUMO
Schistosoma japonicum (S. japonicum) is one of the etiological agents of schistosomiasis, a widespread zoonotic parasitic disease. However, the mechanism of the balanced co-existence between the host immune system and S. japonicum as well as their complex interaction remains unclear. In this study, 16S rRNA gene sequencing, combined with metagenomic sequencing approach as well as ultraperformance liquid chromatography-mass spectrometry metabolic profiling, was applied to demonstrate changes in the gut microbiome community structure during schistosomiasis progression, the functional interactions between the gut bacteria and S. japonicum infection in BALB/c mice, and the dynamic metabolite changes of the host. The results showed that both gut microbiome and the metabolites were significantly altered at different time points after the infection. Decrease in richness and diversity as well as differed composition of the gut microbiota was observed in the infected status when compared with the uninfected status. At the phylum level, the gut microbial communities in all samples were dominated by Firmicutes, Bacteroidetes, Proteobacteria, and Deferribacteres, while at the genus level, Lactobacillus, Lachnospiraceae NK4A136 group, Bacteroides, Staphylococcus, and Alloprevotella were the most abundant. After exposure, Roseburia, and Ruminococcaceae UCG-014 decreased, while Staphylococcus, Alistipes, and Parabacteroides increased, which could raise the risk of infections. Furthermore, LEfSe demonstrated several bacterial taxa that could discriminate between each time point of S. japonicum infection. Besides that, metagenomic analysis illuminated that the AMP-activated protein kinase (AMPK) signaling pathway and the chemokine signaling pathway were significantly perturbed after the infection. Phosphatidylcholine and colfosceril palmitate in serum as well as xanthurenic acid, naphthalenesulfonic acid, and pimelylcarnitine in urine might be metabolic biomarkers due to their promising diagnostic potential at the early stage of the infection. Alterations of glycerophospholipid and purine metabolism were also discovered in the infection. The present study might provide further understanding of the mechanisms during schistosome infection in aspects of gut microbiome and metabolites, and facilitate the discovery of new targets for early diagnosis and prognostic purposes. Further validations of potential biomarkers in human populations are necessary, and the exploration of interactions among S. japonicum, gut microbiome, and metabolites is to be deepened in the future.
Assuntos
Microbioma Gastrointestinal , Interações Hospedeiro-Parasita/imunologia , Metabolômica , Schistosoma japonicum , Esquistossomose Japônica/imunologia , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/parasitologia , Animais , Biomarcadores , Biologia Computacional/métodos , Feminino , Metabolômica/métodos , Metagenoma , Metagenômica/métodos , Camundongos , RNA Ribossômico 16S/genéticaRESUMO
CD4+ T cells differentiate into distinct functional effector and inhibitory subsets are facilitated by distinct cytokine cues present at the time of antigen recognition. Maintaining a balance between T helper 17 (Th17) and regulatory T (Treg) cells are critical for the control of the immunopathogenesis of liver diseases. Here, by using the mouse model of helminth Schistosoma japonicum (S japonicum) infection, we show that the hepatic mRNA levels of P21-activated kinase 1 (PAK1), a key regulator of the actin cytoskeleton, adhesion and cell motility, are significantly increased and associated with the development of liver pathology during S japonicum infection. In addition, PAK1-deficient mice are prone to suppression of Th17 cell responses but increased Treg cells. Furthermore, PAK1 enhances macrophage activation through promoting IRF1 nuclear translocation in an NF-κB-dependent pathway, resulting in promoting Th17 cell differentiation through inducing IL-6 production. These findings highlight the importance of PAK1 in macrophages fate determination and suggest that PAK1/IRF1 axis-dependent immunomodulation can ameliorate certain T cell-based immune pathologies.