Locust Hemolymph Conveys Erythropoietin-Like Cytoprotection via Activation of the Cytokine Receptor CRLF3

Abstract

The cytokine receptor-like factor 3 (CRLF3) is an evolutionary conserved class 1 cytokine receptor present in all major eumetazoan groups. Endogenous CRLF3 ligands have not been identified and the physiological responses mediated by mammalian CRLF3 are poorly characterized. Insect CRLF3 is activated by erythropoietin (Epo) and several related molecules that protect mammalian neurons from stress-induced apoptosis. However, insects neither express Epo nor “classical” Epo receptor. Cell-protective effects of insect hemolymph have been described for several species. In this study, we explored the possibility that the endogenous CRLF3 ligand is contained in locust hemolymph. PCR analyses confirmed expression of crfl3-transcripts in neurons and hemocytes of Locusta migratoria and Tribolium castaneum. Survival of locust hemocytes in primary cultures was significantly increased by supplementation of culture medium with locust hemolymph serum. Locust primary neuron cultures were also protected by locust hemolymph, though preceding exposure to fetal bovine serum changed the hemolymph dose-dependency of neuroprotection. Direct comparison of 10% hemolymph serum with recombinant human Epo in its optimal neuroprotective concentration revealed equivalent anti-apoptotic effects on hypoxia-exposed locust neurons. The same concentration of locust hemolymph serum also protected hypoxia-exposed T. castaneum neurons. This indicates that the neuroprotective factor in locust hemolymph is sufficiently conserved in insects to allow activation of neuroprotective receptors in different species. Locust hemolymph-induced neuroprotection in both L. migratoria and T. castaneum was abolished after RNAi-mediated suppression of crlf3-expression. In summary, we report the presence of a conserved endogenous cytokine in locust hemolymph that activates CRLF3 and connected anti-apoptotic processes in hemocytes and neurons. Identification and characterization of the CRLF3 ligand will promote knowledge about cytokine evolution and may unravel cell-protective agents with potential clinical application.

Protection of insect neurons by erythropoietin/CRLF3-mediated regulation of pro-apoptotic acetylcholinesterase

“…Insect primary brain cell culture. Primary neuron cultures were established as previously described 19,32,33,60,73 . In brief, 20 tribolium or 2 locust brains per culture were dissected and collected in Leibowitz 15 medium (Gibco; Life Technologies, Darmstadt, Germany) supplemented with 1% penicillin/streptomycin and 1% amphotericin B (both Sigma-Aldrich, Munich, Germany) (from now referred to as L15 medium).…”

“…To reduce expression of respective protein, 10 ng/ml dsRNA targeting either one of the genes was added from the beginning of the experiment and renewed with each medium change. Successful interference with protein expression by "soaking RNAi" was previously demonstrated in primary cultured T. castaneum neurons 19,73 . Cells were exposed to hypoxia on day 5 for 36 h (O 2 < 0.3%) before being fixed and analyzed for cell survival.…”

“…From each cell culture, two rows of non-overlapping photographs were taken (for locust cultures ~ 80 images using × 40 magnification; for tribolium cultures ~ 120 images using × 63 magnification). Cell survival was assessed by the DAPI stained chromatin structure as described previously 19,32,33,60,73 . In brief, alive and dead/dying cells can be identified by chromatin condensation, which is represented by increasingly uniform and intense DAPI staining of nuclei (see Fig.…”

“…RNA isolation and cDNA synthesis. RNA of cell cultures and brains was isolated using Trizole (Thermo Fisher Scientific, Schwerte, Germany) as described previously 60,73 . For tissue specimen 15 brains of late T. cas- www.nature.com/scientificreports/ taneum pupae were extracted and collected in RNALater (Sigma-Aldrich, Munich, Germany).…”

Cytokine Receptor-Like Factor 3 (CRLF3) Contributes to Early Zebrafish Hematopoiesis

“…This is particular true in the CHD that incorporates the archetypal WSXWS domain, but also extends to residues in the C-terminus, suggesting both are important for function. Indeed, evidence from insect systems suggest that CRLF3 is activated by cytokines, with both exogenous mammalian erythropoietin and an as yet unidentified endogenous cytokine in the hemolymph able to elicit CRLF3-dependent responses ( 57 ), suggesting the CHD is functional in cytokine binding. The insect CRLF3 has also been shown to lie upstream of the JAK-STAT pathway ( 8 ), while mammalian studies have also identified STAT3 as a downstream mediator ( 58 ), actions presumably mediated by the C-terminus of the protein.…”