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Dual Role of Hepatic Macrophages in the Establishment of the Echinococcus multilocularis Metacestode in Mice.

ABSTRACT: Echinococcus multilocularis larvae, predominantly located in the liver, cause a tumor-like parasitic disease, alveolar echinococcosis (AE), that is characterized by increased infiltration of various immune cells, including macrophages, around the lesion that produces an "immunosuppressive" microenvironment, favoring its persistent infection. However, the role of hepatic macrophages in the host defense against E. multilocularis infection remains poorly defined. Using human liver tissues from patients with AE and a hepatic experimental mouse model of E. multilocularis, we investigated the phenotype and function of hepatic macrophages during the parasite infection. In the present study, we found that a large number of CD68⁺ macrophages accumulated around the metacestode lesion in the liver of human AE samples and that both S100A9⁺ proinflammatory (M1 phenotype) and CD163⁺ anti-inflammatory (M2 phenotype) macrophages were significantly higher in close liver tissue (CLT) than in distant liver tissue (DLT), whereas M2 macrophages represent the dominant macrophage population. Furthermore, E. multilocularis-infected mice exhibited a massive increase in macrophage (F4/80⁺) infiltration in the liver as early as day 5, and the infiltrated macrophages were mainly monocyte-derived macrophages (CD11b^hi F4/80^int MoMFs) that preferentially differentiated into the M1 phenotype (iNOS⁺) at the early stage of E. multilocularis infection and then polarized to anti-inflammatory macrophages of the M2 phenotype (CD206⁺) at the chronic stage of infection. We further showed that elimination of macrophages by treatment of mice with clodronate-liposomes before E. multilocularis infection impaired worm expulsion and was accompanied by a reduction in liver fibrosis, yielding a high parasite burden. These results suggest that hepatic macrophages may play a dual role in the establishment and development of E. multilocularis metacestodes in which early larvae clearance is promoted by M1 macrophages while persistent metacestode infection is favored by M2 macrophages.

SUBMITTER: Wang H

PROVIDER: S-EPMC7820908 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Dual Role of Hepatic Macrophages in the Establishment of the Echinococcus multilocularis Metacestode in Mice.

Wang Hui H Zhang Chuan-Shan CS Fang Bin-Bin BB Hou Jiao J Li Wen-Ding WD Li Zhi-De ZD Li Liang L Bi Xiao-Juan XJ Li Liang L Abulizi Abuduaini A Shao Ying-Mei YM Lin Ren-Yong RY Wen Hao H

Frontiers in immunology 20210108

Echinococcus multilocularis larvae, predominantly located in the liver, cause a tumor-like parasitic disease, alveolar echinococcosis (AE), that is characterized by increased infiltration of various immune cells, including macrophages, around the lesion that produces an "immunosuppressive" microenvironment, favoring its persistent infection. However, the role of hepatic macrophages in the host defense against E. multilocularis infection remains poorly defined. Using human liver tis ...[more]

PMID: 33488594

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Project description:The neglected zoonotic disease alveolar echinococcosis (AE) is caused by the metacestode stage of the tapeworm parasite Echinococcus multilocularis. MicroRNAs (miRNAs) are small non-coding RNAs with a major role in regulating gene expression in key biological processes. We analyzed the expression profile of E. multilocularis miRNAs throughout metacestode development in vitro, determined the spatial expression of miR-71 in metacestodes cultured in vitro and predicted miRNA targets. Small cDNA libraries from different samples of E. multilocularis were sequenced. We confirmed the expression of 37 miRNAs in E. multilocularis being some of them absent in the host, such as miR-71. We found a few miRNAs highly expressed in all life cycle stages and conditions analyzed, whereas most miRNAs showed very low expression. The most expressed miRNAs were miR-71, miR-9, let-7, miR-10, miR-4989 and miR-1. The high expression of these miRNAs was conserved in other tapeworms, suggesting essential roles in development, survival, or host-parasite interaction. We found highly regulated miRNAs during the different transitions or cultured conditions analyzed, which might suggest a role in the regulation of developmental timing, host-parasite interaction, and/or in maintaining the unique developmental features of each developmental stage or condition. We determined that miR-71 is expressed in germinative cells and in other cell types of the germinal layer in E. multilocularis metacestodes cultured in vitro. MiRNA target prediction of the most highly expressed miRNAs and in silico functional analysis suggested conserved and essential roles for these miRNAs in parasite biology. We found relevant targets potentially involved in development, cell growth and death, lifespan regulation, transcription, signal transduction and cell motility. The evolutionary conservation and expression analyses of E. multilocularis miRNAs throughout metacestode development along with the in silico functional analyses of their predicted targets might help to identify selective therapeutic targets for treatment and control of AE.

Project description:Extracellular RNAs (ex-RNAs) are secreted by cells through different means that may involve association with proteins, lipoproteins or extracellular vesicles (EV). In the context of parasitism, ex-RNAs represent new and exciting communication intermediaries with promising potential as novel biomarkers. In the last years, it was shown that helminth parasites secrete ex-RNAs, however, most work mainly focused on RNA secretion mediated by EV. Ex-RNA study is of special interest in those helminth infections that still lack biomarkers for early and/or follow-up diagnosis, such as echinococcosis, a neglected zoonotic disease caused by cestodes of the genus Echinococcus. In this work, we have characterised the ex-RNA profile secreted by in vitro grown metacestodes of Echinococcus multilocularis, the casuative agent of alveolar echinococcosis. We have used high throughput RNA-sequencing together with RT-qPCR to characterise the ex-RNA profile secreted towards the extra- and intra-parasite milieus in EV-enriched and EV-depleted fractions. We show that a polarized secretion of small RNAs takes place, with microRNAs mainly secreted to the extra-parasite milieu and rRNA- and tRNA-derived sequences mostly secreted to the intra-parasite milieu. In addition, we show by nanoparticle tracking analyses that viable metacestodes secrete EV mainly into the metacestode inner vesicular fluid (MVF); however, the number of nanoparticles in culture medium and MVF increases > 10-fold when metacestodes show signs of tegument impairment. Interestingly, we confirm the presence of host miRNAs in the intra-parasite milieu, implying their internalization and transport through the tegument towards the MVF. Finally, our assessment of the detection of Echinococcus miRNAs in patient samples by RT-qPCR yielded negative results suggesting the tested miRNAs may not be good biomarkers for this disease. A comprehensive study of the secretion mechanisms throughout the life cycle of these parasites will help to understand parasite interaction with the host and also, improve current diagnostic tools.

Project description:BACKGROUND: Alveolar echinococcosis (AE) is a severe chronic hepatic parasitic disease currently emerging in central and eastern Europe. Untreated AE presents a high mortality (>90%) due to a severe hepatic destruction as a result of parasitic metacestode proliferation which behaves like a malignant tumor. Despite this severe course and outcome of disease, the genetic program that regulates the host response leading to organ damage as a consequence of hepatic alveolar echinococcosis is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used a mouse model of AE to assess gene expression profiles in the liver after establishment of a chronic disease status as a result of a primary peroral infection with eggs of the fox tapeworm Echinococcus multilocularis. Among 38 genes differentially regulated (false discovery rate adjusted p</=0.05), 35 genes were assigned to the functional gene ontology group <immune response>, while 3 associated with the functional group <intermediary metabolism>. Upregulated genes associated with <immune response> could be clustered into functional subgroups including <macrophages>, <APCs>, <lymphocytes, chemokines and regulation>, <B-cells> and <eosinophils>. Two downregulated genes related to <lymphocytes, chemokines and regulation> and <intermediary metabolism>, respectively. The <immune response> genes either associated with an <immunosupression> or an <immunostimulation> pathway. From the overexpressed genes, 18 genes were subsequently processed with a Custom Array microfluidic card system in order to assess respective expression status at the mRNA level relative to 5 reference genes (Gapdh, Est1, Rlp3, Mdh-1, Rpl37) selected upon a constitutive and stable expression level. The results generated by the two independent tools used for the assessment of gene expression, i.e., microarray and microfluidic card system, exhibited a high level of congruency (Spearman correlation rho = 0.81, p = 7.87e-5) and thus validated the applied methods. CONCLUSIONS/SIGNIFICANCE: Based on this set of biomarkers, new diagnostic targets have been made available to predict disease status and progression. These biomarkers may also offer new targets for immuno-therapeutic intervention.

Project description:The lethal zoonosis alveolar echinococcosis (AE) is caused by tumor-like, infiltrative growth of the metacestode larval stage of the tapeworm Echinococcus multilocularis. We previously showed that the metacestode is composed of posteriorized tissue and that the production of the subsequent larval stage, the protoscolex, depends on re-establishment of anterior identities within the metacestode germinative layer. It is, however, unclear so far how protoscolex differentiation in Echinococcus is regulated. We herein characterized the full complement of E. multilocularis TGFβ/BMP receptors, which is composed of one type II and three type I receptor serine/threonine kinases. Functional analyzes showed that all Echinococcus TGFβ/BMP receptors are enzymatically active and respond to host derived TGFβ/BMP ligands for activating downstream Smad transcription factors. In situ hybridization experiments demonstrated that the Echinococcus TGFβ/BMP receptors are mainly expressed by nerve and muscle cells within the germinative layer and in developing brood capsules. Interestingly, the production of brood capsules, which later give rise to protoscoleces, was strongly suppressed in the presence of inhibitors directed against TGFβ/BMP receptors, whereas protoscolex differentiation was accelerated in response to host BMP2 and TGFβ. Apart from being responsive to host TGFβ/BMP ligands, protoscolex production also correlated with the expression of a parasite-derived TGFβ-like ligand, EmACT, which is expressed in early brood capsules and which is strongly expressed in anterior domains during protoscolex development. Taken together, these data indicate an important role of TGFβ/BMP signalling in Echinococcus anterior pole formation and protoscolex development. Since TGFβ is accumulating around metacestode lesions at later stages of the infection, the host immune response could thus serve as a signal by which the parasite senses the time point at which protoscoleces must be produced. Overall, our data shed new light on molecular mechanisms of host-parasite interaction during AE and are relevant for the development of novel treatment strategies.

Project description:BackgroundAlveolar echinococcosis (AE), caused by the metacestode of the tapeworm Echinococcus multilocularis, is a lethal zoonosis associated with host immunomodulation. T helper cells are instrumental to control the disease in the host. Whereas Th1 cells can restrict parasite proliferation, Th2 immune responses are associated with parasite proliferation. Although the early phase of host colonization by E. multilocularis is dominated by a potentially parasitocidal Th1 immune response, the molecular basis of this response is unknown.Principal findingsWe describe EmTIP, an E. multilocularis homologue of the human T-cell immunomodulatory protein, TIP. By immunohistochemistry we show EmTIP localization to the intercellular space within parasite larvae. Immunoprecipitation and Western blot experiments revealed the presence of EmTIP in the excretory/secretory (E/S) products of parasite primary cell cultures, representing the early developing metacestode, but not in those of mature metacestode vesicles. Using an in vitro T-cell stimulation assay, we found that primary cell E/S products promoted interferon (IFN)-γ release by murine CD4+ T-cells, whereas metacestode E/S products did not. IFN-γ release by T-cells exposed to parasite products was abrogated by an anti-EmTIP antibody. When recombinantly expressed, EmTIP promoted IFN-γ release by CD4+ T-cells in vitro. After incubation with anti-EmTIP antibody, primary cells showed an impaired ability to proliferate and to form metacestode vesicles in vitro.ConclusionsWe provide for the first time a possible explanation for the early Th1 response observed during E. multilocularis infections. Our data indicate that parasite primary cells release a T-cell immunomodulatory protein, EmTIP, capable of promoting IFN-γ release by CD4+ T-cells, which is probably driving or supporting the onset of the early Th1 response during AE. The impairment of primary cell proliferation and the inhibition of metacestode vesicle formation by anti-EmTIP antibodies suggest that this factor fulfills an important role in early E. multilocularis development within the intermediate host.

Project description:We present a comprehensive analysis of the hepatic miRNA transcriptome at one month post-infection of experimental primary alveolar echinococcosis (AE), a parasitic infection caused upon ingestion of E. multilocularis eggs. Liver tissues were collected from infected and non-infected C57BL/6 mice, then small RNA libraries were prepared for next-generation sequencing (NGS). We conducted a Stem-loop RT-qPCR for validation of most dysregulated miRNAs. In infected mice, the expression levels of 28 miRNAs were significantly altered. Of these, 9 were up-regulated (fold change (FC) ≥ 1.5) and 19 were down-regulated (FC ≤ 0.66) as compared to the non-infected controls. In infected livers, mmu-miR-148a-3p and mmu-miR-101b-3p were 8- and 6-fold down-regulated, respectively, and the expression of mmu-miR-22-3p was reduced by 50%, compared to non-infected liver tissue. Conversely, significantly higher hepatic levels were noted for Mus musculus (mmu)-miR-21a-5p (FC = 2.3) and mmu-miR-122-5p (FC = 1.8). In addition, the relative mRNA expression levels of five genes (vegfa, mtor, hif1-α, fasn and acsl1) that were identified as targets of down-regulated miRNAs were significantly enhanced. All the five genes exhibited a higher expression level in livers of E. multilocularis infected mice compared to non-infected mice. Finally, we studied the issue related to functionally mature arm selection preference (5p and/or 3p) from the miRNA precursor and showed that 9 pre-miRNAs exhibited different arm selection preferences in normal versus infected liver tissues. In conclusion, this study provides first evidence that miRNAs are regulated early in primary murine AE. Our findings raise intriguing questions such as (i) how E. multilocularis affects hepatic miRNA expression;(ii) what are the alterations in miRNA expression patterns in more advanced AE-stages; and (iii) which hepatic cellular, metabolic and/or immunologic processes are modulated through altered miRNAs in AE. Thus, further research on the regulation of miRNAs during AE is needed, since miRNAs constitute an attractive potential option for development of novel therapeutic approaches against AE.

Dataset Information

Dual Role of Hepatic Macrophages in the Establishment of the Echinococcus multilocularis Metacestode in Mice.

Publications

Dual Role of Hepatic Macrophages in the Establishment of the <i>Echinococcus multilocularis</i> Metacestode in Mice.

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