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

Abstract

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.

Developmental regulation and functional prediction of microRNAs in an expanded Fasciola hepatica miRNome

“…Note that the total of 89 sequences considers 5p and 3p sequences from the same hairpin as separate mature sequences. Note that after we completed this part of the study in 2020, a similar analysis was published by other authors (Ricafrente et al, 2020).…”

“…Ovchinnikov et al focused on adult EV miRNAs, uding PITA and TargetSCAN to link 24 miRNAs to 321 cow and human mRNAs, including targets within the Wnt signalling pathway and the immune system (Ovchinnikov et al, 2020). Similarly, (Ricafrente et al, 2020) used miRTarget to link 38 NEJ expressed miRNAs (Fontenla et al, 2015a) with 26 target genes expressed in innate immune cells. Our work builds on previous studies to catalogue new miRNAs within an expanded F. hepatica miRNome, alongside a developmental expression profile of these in muiltiple intramammalian life stages.…”

“…This fundamental process occurs throughout eukaryotes, but our ability to probe and understand it in liver fluke specifically has been hindered by the lack of appropriate functional genomic protocols and an incomplete understanding of the miRNome. To date, 89 F. hepatica miRNAs have been reported in adult and newly excysted juvenile (NEJ) parasites (Xu et al, 2012b;Fontenla et al, 2015a;Fromm et al, 2015b;Ovchinnikov et al, 2020;Ricafrente et al, 2020). Given that miRBase (release 22.1) reports more than 100 miRNAs in other flatworm species, this is probably not the full extent of the F. hepatica miRNome.…”

“…Following the first report of extracellular vesicle (EV) release from F. hepatica (Marcilla et al, 2012), miRNAs were soon observed amongst the cargo molecules of these membrane bound carriers (Fromm et al, 2015b). These miRNAs have been analysed in terms of their potential to target immune transcripts of host organisms (Fromm et al, 2015b;Fromm et al, 2017;Ovchinnikov et al, 2020;Ricafrente et al, 2020), with data now showing that fhe-mir-125b can enter host immune cells and attach to host argonaut protein, forming a potentially functional silencing unit (Tran et al, 2021). Additional functional insight has come from using miRNA target prediction algorithms to identify the host mRNAs that are targeted for silencing by fluke secreted miRNAs.…”

Pathogenicity and virulence of the liver flukesFasciola hepaticaandFasciolaGiganticathat cause the zoonosis Fasciolosis

“…This is comparable to the in vitro apoptotic effects of the ES proteins on peritoneal eosinophils and macrophages [ 222–224 ]. Furthermore, transcriptional analysis revealed that pro-apoptotic signals are increased in peripheral blood mononuclear cells recovered from F. hepatica infected sheep and cattle [ 225 , 226 ]. The specific proteins that bring about this cellular apoptosis have yet to be characterized, and further studies are required to determine whether similar parasite proteins are involved within the different immune compartments of the infected animal.…”