AUTHOR=Prochazkova Petra , Roubalova Radka , Skanta Frantisek , Dvorak Jiri , Pacheco Natividad Isabel Navarro , Kolarik Miroslav , Bilej Martin 

TITLE=Developmental and Immune Role of a Novel Multiple Cysteine Cluster TLR From Eisenia andrei Earthworms

JOURNAL=Frontiers in Immunology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2019.01277

DOI=10.3389/fimmu.2019.01277

ISSN=1664-3224

ABSTRACT=<p>Earthworms are not endowed with adaptive immunity and they are rely on the tools of innate immunity. Cells of the innate immune system utilize pattern recognition receptors, such as Toll-like receptors, to detect the pathogen-associated molecular patterns (PAMPs). The first earthworm TLR was isolated from <italic>Eisenia andrei</italic> earthworms (<italic>Ea</italic>TLR), which belongs to the single cysteine cluster TLR (sccTLR). Here, we identified a new multiple cysteine cluster TLR (mccTLR) in <italic>E. andrei</italic> earthworms. Phylogenetic DNA analysis revealed that it has no variability within one earthworm as well as in the population. By screening of the tissue expression profile, the TLR was expressed primarily in earthworm seminal vesicles and receptacles suggesting a connection to sperm cells. Seminal vesicles are often heavily infected by gregarine parasites. As a sign of immune response, a strong melanization reaction is visible around parasites. Stimulation experiments with profilin from related parasite <italic>Toxoplasma gondii</italic>, led to the upregulation of mcc<italic>Ea</italic>TLR in the earthworm seminal vesicles. Also, profilin activated prophenoloxidase cascade, the efficient mechanism of innate immunity. However, its involvement in the NF-κB signaling was not proven. Further, we provide evidence that the antibiotics metronidazole and griseofulvin destroyed the developing spermatocytes. The observed decrease in the mcc<italic>Ea</italic>TLR mRNA levels after the antibiotic treatment of parasites is caused by the decline of sperm cells numbers rather than by diminution of the parasites. Since earthworms with extensively reduced parasite load had a similar amount of mcc<italic>Ea</italic>TLR mRNA, presumably, earthworm sperm cells have a certain level of mcc<italic>Ea</italic>TLR expressed as a standard, which can be augmented by particular antigenic stimulation. Also, mcc<italic>Ea</italic>TLR was expressed mainly in the early stages of earthworm development and presumably is primarily involved in early embryonic development. Expression of mcc<italic>Ea</italic>TLR in seminal vesicles correlates with the expression of endothelial monocyte-activation polypeptide II. High-throughput sequencing of gregarine DNA from seminal vesicles of individual earthworms resulted in great diversity of the observed genotypes. Phylogenetically, all observed OTUs belong to the clade of earthworm gregarines suggesting host specificity. Overall, mcc<italic>Ea</italic>TLR is supposed to play a function role in early embryonic development and potentially it participates in immune response against parasites.</p>