Re-examination of the rainbow trout (Oncorhynchus mykiss) immune response to flagellin: Yersinia ruckeri flagellin is a potent activator of acute phase proteins, anti-microbial peptides and pro-inflammatory cytokines in vitro

Abstract

Flagellin is the principal component of bacterial flagellum and a major target of the host immune system. To provide new insights into the role of flagellin in fish immune responses to flagellated microorganisms, a recombinant flagellin from Yersinia ruckeri (rYRF) was produced and its bioactivity investigated in the trout macrophage cell line RTS-11 and head kidney cells. rYRF is a potent activator of pro-inflammatory cytokines, acute phase proteins, antimicrobial peptides and subunits of the IL-12 cytokine family. This and the synergy seen with IFN-γ to enhance further expression of specific IL-12 and TNF-α isoforms may suggest that flagellin could be a useful immune stimulant or adjuvant for use in aquaculture. Gene paralogues were often differentially modulated, highlighting the need to study all of the paralogues of immune genes in fish to gain a full understanding of the effects of PAMPs or other stimulants, and the potential immune responses elicited.

Gene expression analysis of isolated salmonid GALT leucocytes in response to PAMPs and recombinant cytokines

“…CATH-2 is a potent AMP in salmonids, able to kill a range of fish bacterial pathogens as part of the innate immune response [55]. It has been reported to be upregulated by IL-6 and flagellin (YRF) in a trout macrophage (RTS-11) cell line [29,26], in keeping with the present findings. However, the effects of rIL-6 on CATH-2 expression were quite marginal, and indeed both pro-inflammatory cytokines tested (rIL-1β and rIL-6) induced relatively few/small effects.…”

“…Purified Atlantic salmon GALT leucocytes obtained using protocol-3, were suspended in RPMI-1640 containing P/S and 10% FBS, then plated at 2x10 6 cells /well into 12 well plates and cultured at 20 o C. The cells, from individual fish, were then stimulated with 10 μg /ml phytohaemagglutinin from Phaseolus vulgaris (PHA, Sigma), 50 μg /ml LPS, 100 μg /ml polyinosinic acid: polycytidylic acid (poly I:C, Sigma), 100 ng /ml recombinant flagellin from Yersinia ruckeri (rYRF, [26], 25 ng /ml IL-1β1 [27], 100 ng /ml IL-2B [28], 100 ng /ml IL-6 [29], 200 ng /ml IL-21 [30] and 20 ng /ml IFN-α2/IFN2 [31]. All doses used were previously shown to be optimal for trout and Atlantic salmon cells [32,33,34], and the cytokine paralogues chosen have known bioactivity on leucocytes from other tissues.…”

Dissecting the immune pathways stimulated following injection vaccination of rainbow trout (Oncorhynchus mykiss) against enteric redmouth disease (ERM)

“…The expression of a set of genes for acute phase proteins (APPs), antimicrobial peptides (AMPs) and cytokines using real time PCR were performed as described previously [25,[32][33]. Briefly, the PCR amplification was performed using a LightCycler® 480 Instrument II (Roche Applied Science) and 384 multiwell plates in a µl reaction using SYBR® Green I (Invitrogen™, Carlsbad, USA) and IMMOLASE ™ DNA Polymerase (Bioline, UK), and expression levels calculated using the 'LightCycler® 480 software version 1.5.…”

“…The biggest differences were seen when one of the paralogues was responsive and the other not, as with IL-1β1 and 2 vs IL-1β3, IL-4/13A vs IL-4/13B1 and 2, IL-10A vs IL-10B, IL-12 p40B2 vs p40B1 and p40C, IL-17A/F1A vs IL-17A/F1B, IL-17A/F2A vs IL-17A/F2B, and SAP1 vs SAP2. Differential responses of paralogues have been seen in responses to PAMPs [22,33], infection [24,26] and cytokine stimulation [25][26]. These differences likely reflect differences in the promoters, with some of the paralogues becoming more or less responsive to particular signalling pathways, perhaps in particular cell types, or genes that are being pseudogenised.…”

Evolution of Th2 responses: characterization of IL-4/13 in sea bass (Dicentrarchus labrax L.) and studies of expression and biological activity

“…Following transformation of the plasmid into BL21 Star (DE3) competent cells (Invitrogen) and the induction of recombinant protein production, a first purification was performed under denaturing conditions. Successively we undertook a refolding process, a re-purification under native conditions, a SDS-PAGE analysis of the purified proteins and a quantification of the protein concentration as described in detail previously 45–47 . The refolding buffer contained 50 mM Tris-HCl (pH 7.5), 10% glycerol, 0.5 M arginine monohydrochloride, and 5 mM 2-mercaptoethanol.…”