Exploring the utility of circulating miRNAs as diagnostic biomarkers of fasciolosis.

Effective management and control of parasitic infections on farms depends on their early detection. Traditional serological diagnostic methods for Fasciola hepatica infection in livestock are specific and sensitive, but currently the earliest detection of the parasite only occurs at approximately three weeks post-infection. At this timepoint, parasites have already entered the liver and caused the tissue damage and immunopathology that results in reduced body weight and loss in productivity. Here, we investigated whether the differential abundance of micro(mi)miRNAs in sera of F. hepatica-infected sheep has potential as a tool for the early diagnosis of infection. Using miRNA sequencing analysis, we discovered specific profiles of sheep miRNAs at both the pre-hepatic and hepatic infection phases in comparison to non-infected sheep. In addition, six F. hepatica-derived miRNAs were specifically identified in sera from infected sheep. Thus, a panel of differentially expressed miRNAs comprising four sheep (miR-3231-3p; miR133-5p; 3957-5p; 1197-3p) and two parasite miRNAs (miR-124-3p; miR-Novel-11-5p) were selected as potential biomarkers. The expression of these candidates in sera samples from longitudinal sheep infection studies collected between 7 days and 23 weeks was quantified using RT-qPCR and compared to samples from age-matched non-infected sheep. We identified oar-miR-133-5p and oar-miR-3957-5p as promising biomarkers of fasciolosis, detecting infection as early as 7 days. The differential expression of the other selected miRNAs was not sufficient to diagnose infection; however, our analysis found that the most abundant forms of fhe-miR-124-3p in sera were sequence variants (IsomiRs) of the canonical miRNA, highlighting the critical importance of primer design for accurate diagnostic RT-qPCR. Accordingly, this investigative study suggests that certain miRNAs are biomarkers of F. hepatica infection and validates miRNA-based diagnostics for the detection of fasciolosis in sheep.

Stage-specific miRNAs regulate gene expression associated with growth, development and parasite-host interaction during the intra-mammalian migration of the zoonotic helminth parasite Fasciola hepatica.

BACKGROUND: MiRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression in organisms ranging from viruses to mammals. There is great relevance in understanding how miRNAs regulate genes involved in the growth, development, and maturation of the many parasitic worms (helminths) that together afflict more than 2 billion people. RESULTS: Here, we describe the miRNAs expressed by each of the predominant intra-mammalian development stages of Fasciola hepatica, a foodborne flatworm that infects a wide range of mammals worldwide, most importantly humans and their livestock. A total of 124 miRNAs were profiled, 72 of which had been previously reported and three of which were conserved miRNA sequences described here for the first time. The remaining 49 miRNAs were novel sequences of which, 31 were conserved with F. gigantica and the remaining 18 were specific to F. hepatica. The newly excysted juveniles express 22 unique miRNAs while the immature liver and mature bile duct stages each express 16 unique miRNAs. We discovered several sequence variant miRNAs (IsomiRs) as well as miRNA clusters that exhibit strict temporal expression paralleling parasite development. Target analysis revealed the close association between miRNA expression and stage-specific changes in the transcriptome; for example, we identified specific miRNAs that target parasite proteases known to be essential for intestinal wall penetration (cathepsin L3). Moreover, we demonstrate that miRNAs fine-tune the expression of genes involved in the metabolic pathways that allow the parasites to move from an aerobic external environment to the anerobic environment of the host. CONCLUSIONS: These results provide novel insight into the regulation of helminth parasite development and identifies new genes and miRNAs for therapeutic development to limit the virulence and pathogenesis caused by F. hepatica.

Fasciola hepatica hijacks host macrophage miRNA machinery to modulate early innate immune responses.

Fasciola hepatica, a global worm parasite of humans and their livestock, regulates host innate immune responses within hours of infection. Host macrophages, essential to the first-line defence mechanisms, are quickly restricted in their ability to initiate a classic protective pro-inflammatory immune response. We found that macrophages from infected animals are enriched with parasite-derived micro(mi)RNAs. The most abundant of these miRNAs, fhe-miR-125b, is released by the parasite via exosomes and is homologous to a mammalian miRNA, hsa-miR-125b, that is known to regulate the activation of pro-inflammatory M1 macrophages. We show that the parasite fhe-miR-125b loads onto the mammalian Argonaut protein (Ago-2) within macrophages during infection and, therefore, propose that it mimics host miR-125b to negatively regulate the production of inflammatory cytokines. The hijacking of the miRNA machinery controlling innate cell function could be a fundamental mechanism by which worm parasites disarm the early immune responses of their host to ensure successful infection.

Neonatal Rats Exhibit a Predominantly Anti-Inflammatory Response following Spinal Cord Injury.

It has been reported that children may respond better than adults to a spinal cord injury (SCI) of similar severity. There are known biomechanical differences in the developing spinal cord that may contribute to this "infant lesion effect," but the underlying mechanisms are unknown. Using immunohistochemistry, we have previously demonstrated a different injury progression and immune cell response after a mild thoracic contusion SCI in infant rats, as compared to adult rats. Here, we investigated the acute inflammatory responses using flow cytometry and ELISA at 1 h, 24 h, and 1 week after SCI in neonatal (P7) and adult (9 weeks) rats, and locomotor recovery was examined for 6 weeks after injury. Adult rats exhibited a pronounced pro-inflammatory response characterized by neutrophils and M1-like macrophage infiltration and Th1 cytokine secretion. Neonatal rats exhibited a decreased pro-inflammatory response characterized by a higher proportion of M2-like macrophages and reduced Th1 cytokine responses, as compared to adults. These results suggest that the initial inflammatory response to SCI is predominantly anti-inflammatory in very young animals.

An Evaluation of the Fasciola hepatica miRnome Predicts a Targeted Regulation of Mammalian Innate Immune Responses.

Understanding mechanisms by which parasitic worms (helminths) control their hosts' immune responses is critical to the development of effective new disease interventions. Fasciola hepatica, a global scourge of humans and their livestock, suppresses host innate immune responses within hours of infection, ensuring that host protective responses are quickly incapacitated. This allows the parasite to freely migrate from the intestine, through the liver to ultimately reside in the bile duct, where the parasite establishes a chronic infection that is largely tolerated by the host. The recent identification of micro(mi)RNA, small RNAs that regulate gene expression, within the extracellular vesicles secreted by helminths suggest that these non-coding RNAs may have a role in the parasite-host interplay. To date, 77 miRNAs have been identified in F. hepatica comprising primarily of ancient conserved species of miRNAs. We hypothesized that many of these miRNAs are utilized by the parasite to regulate host immune signaling pathways. To test this theory, we first compiled all of the known published F. hepatica miRNAs and critically curated their sequences and annotations. Then with a focus on the miRNAs expressed by the juvenile worms, we predicted gene targets within human innate immune cells. This approach revealed the existence of targets within every immune cell, providing evidence for the universal management of host immunology by this parasite. Notably, there was a high degree of redundancy in the potential for the parasite to regulate the activation of dendritic cells, eosinophils and neutrophils, with multiple miRNAs predicted to act on singular gene targets within these cells. This original exploration of the Fasciola miRnome offers the first molecular insight into mechanisms by which F. hepatica can regulate the host protective immune response.