The Search for Innate Immune Receptors

on Earthworm Coelomocytes

Joe W. Francis¹, Stephen Wreesman^1,3, Katie Reigstad ^1,3, Rebekah Eggar^1,3, Michael Enos^1,4 Stephan Krutzik² and E.L. Cooper²

The Master’s College, Santa Clarita California¹,

David Geffen School Of Medicine at UCLA University of California, Los Angeles², Undergraduate researchers³,

Advantage Preparatory Schools, Santa Clarita, California⁴

Pattern recognition receptors are known to play major roles in modulating both innate and adaptive immune responses.  Much attention has focused on a family of receptors called the Toll-like receptors (TLR).  TLR expression has been detected in a variety of different vertebrate and invertebrate species as well as in plants. In some invertebrate species, TLR gene expression has been detected but their cell surface expression on immune cells has not been noted.  TLRs as well as several other innate immune receptors have not yet been detected on earthworm coelomocytes.¹  We have  employed a group of commercially available polyclonal and monoclonal antibodies (mAb) directed against  mammalian TLRs It is expected that many mAb directed against mammalian TLRs may not interact with invertebrate TLRs since mAbs typically identify only a single epitope on a given antigen and it is very possible that TLRs from different species only share a small subset of epitopes.  Using fluorescence microscopy, we recently obtained some preliminary data that suggests that a mAb  directed against TLR2  binds to roughly 20% of the coelomocytes of Eisenia foetida.  Meanwhile, we did not detect expression of TLR4, 4, or 9. We are in the process of confirming the expression of TLR2 using flow cytometry. In positive control experiments, CD 90 and a series of recently developed mAb (EFCC) directed against coelomocyte surface markers were detected on the  surface of coelomocytes from both earthworm species.²  (CD-90 has been shown to be a surface marker present on Eisenia coelomocytes but to our knowledge it has not been shown to be a surface marker on Lumbricus terrestris coelomocytes). To further demonstrate that earthworm coelomocytes possess unique TLRs (largely undetectable with currently available mAB) or receptors which bind to TLR ligands we have constructed a fluorescent flagellin probe (TLR 5 ligand) and a fluorescent peptidoglycan probe (TLR 2 ligand).  Our preliminary results show that a subset (10-20%) of both  Lumbricus and Eisenia coelomocytes bind the flagellin probe.  Taken together our results suggest that invertebrate coelomocytes may express pattern recognition receptors capable of detecting pathogens and providing a mechanism of host defense.