GALT LEUCOCYTES AS A HEALTH SCREEN FOR FUNCTIONAL FEEDS IN RAINBOW TROUT Oncorhynchus mykiss

Introduction

The advances of functional feeds for farmed fish are now regarded as a key factor in improving fish health and performance. Although there has been significant research on the impact of functional feeds on the immune system, the mechanisms by which these occur is still poorly understood , as such there is a need for further research into the nutrition health interface for aquaculture species. As part of our research to examine direct immune responses to functional feed ingredients, cell lines and primary cell cultures are being used as an alternative method which also addresses 3Rs in reducing whole animal experiments. Cell cultures offer a quick and non-invasive method of identifying immune responses, however, the number of cell lines available is limited and the phenotype of these cells  is likely to have changed from their original in-vivo state, especially in the intestine with the  complex nature and multiple cell types present. Gut associated lymphoid tissues ( GALT) leucocytes have been extracted and have shown to be a good screen to PAMPs in previous studies by Attaya et al, (2018) but their role as a viable alternative to traditional feeding trial methods is yet to be seen.

 The present study was undertaken to identify the suitability of both cell culture and primary GALT leucocyte models as potential alternative methods to test  functional feed ingredients on. In addition to the primary culture cells, a rainbow trout macrophage cell line (RTS11) and a rainbow trout gut epithelial cell line (RTgutGC), were used as the cell line models (Ganassin and Bols , 1998) (Kawano et al., 2011).  The cells were challenged with a variety of PAMPs and multiple types of the prebiotic β glucan to test their suitability as a health screen for functional feeds.

Materials and Methods

RTgutGC cells were cultured in flasks at 20o C in growth media (Leibovitz L-15 media + 10% FBS + 1% Penicillin/Streptomycin). RTS11 were cultured in flasks at 20oC in growth media (Leibovitz L-15 media + 30% FBS + 1% Penicillin/Streptomycin). Fish for GALT leucocytes extraction were maintained in 1 m-diameter fiberglass tanks with recirculating freshwater at 14 °C and fed twice a day with a commercial diet at 1.5% bodyweight. Fish were sampled at the same time of day on each occasion used. GALT leucocytes were extracted from the distal intestine using the method previously described by Attaya et al, 2018 with some alterations: cells were washed in PBS containing 1% Penicillin/Streptomycin, cells were digested for a singular two-hour digestion. GALT leucocytes, RTgutGC and RTS11 cells were added to a 24 well plate at a concentration of 2.5x105 cells/well and resuspended in 1ml total of stimulation media (Leibovitz L-15 media + 1% FBS + 1% Penicilin /Streptomycin). Cells were stimulated with 100µg/ml Poly IC, 10µg/ml PHA, 20µg/ml rIL-1β  (Provided by Dr. Tiehui Wang (Scottish Fish Immunology Research Centre)), and β-glucans 1 and 3 100µg/ml (provided by Skretting Ltd) for 4 hours before RNA extraction. Following cell stimulation cells were lysed directly in TRIzol  and stored at 80o C.  RNA was then extracted following the manufacturer’s instructions Reverse Transcription was completed using the Quantitect® Reverse Transcription Kit (Qiagen) following the manufacturer’s instructions.  Primers for each gene were designed using NCBI primer blast or were identified in published papers and then checked using Primer-Blast. Primers were designed with at least one primer across an exon-exon junction to ensure only the cDNA was being amplified.  qPCR on the cDNA was carried out using the Lightcycler 480 machine (Roche).  Relative gene expression was calculated using GenEx which included the use of three housekeeping genes for normalization.

Results

Stimulations with Poly IC (Figure 1) show a significant upregulation in both  MX and IFN1α  for RTS11 at all time points, RTgut and GALT leucocytes. The results also show that RTgut are more sensitive to Poly IC stimulation at the 4hr time point compared to the GALT leucocytes and RTS11 cell lines. The RTS11 and GALT Leucocytes show similar responses to Poly IC.

Discussion

The results of this study suggest that both cell lines and GALT leucocytes provide a novel insight into the immune response to functional feeds. The results show that whilst both the RTS11 and RTgutGC cell lines do respond to both PAMPs and β-glucans their immune responses are similar but not the same as primary cell cultures, primarily since there is only one cell type rather than multiple cell types that would be seen in-vivo . This suggests that the cell lines are a beneficial precursor to feeding trials if they are used to screen novel feed additives prior to the feeding trial. The GALT leucocytes are a good alternative to identify the immune response of the gut directly rather than looking at the systemic effects. The results from the GALT leucocyte stimulations show that stimulation with the PAMPs; Poly IC, PHA, rIL-1β show expected responses to target genes. Poly IC showed significant upregulation in  MX  and IFN1α  compared to the control in RTS11, RTgut and GALT leucocytes. MX encodes for a guanosine triphosphate (GTP)-metabolizing protein that participates in the cellular antiviral response which is induced by Type I and II interferons such as IFN1α ; an antiviral cytokine that is released as a key part of the innate immune response to viral infections. Other stimulants including the T cell mitogen PHA and proinflammatory cytokine Il-1b were also examined in the model systems which also showed expected immune modulation to PAMPs . Β-glucan stimulated results show similar responses to published data (Douxfils  et  al, 2017, Ji et al, 2019) with β-glucans causing an upregulat ion in  il-1β, il-8, il-4 and tnfα. These results suggest further research into the direct mechanisms of actions of both β-glucans will help define the direct immunological effects of these functional feed components, enabling focussed future designs of diets.

Funding Acknowledgements:

DP is funded by Skretting and University of Aberdeen for a PhD studentship.

References

 Attaya, A., Wang, T., Zou, J., Herath, T., Adams, A., Secombes, C. and Yoon, S., 2018. Gene expression analysis of isolated salmonid GALT leucocytes in response to PAMPs and recombinant cytokines. Fish & Shellfish Immunology, 80, pp.426-436.

Douxfils , J., Fierro-Castro, C., Mandiki , S., Emile, W., Tort, L. and Kestemont , P., 2017. Dietary β-glucans differentially modulate immune and stress-related gene expression in lymphoid organs from healthy and Aeromonas hydrophila -infected rainbow trout ( Oncorhynchus mykiss ). Fish & Shellfish Immunology, 63, pp.285-296.

 Ji, L., Fu, S., Sun, G., Li, X. and Liu, Y., 2019. Dietary β‐glucan modulate haematological parameters, cytokines and gene expression in TLR and ERK pathways of rainbow trout ( Oncorhynchus mykiss ) during infection by Aeromonas salmonicida. Aquaculture Research, 51(3), pp.906-917.

 Kawano, A., Haiduk , C., Schirmer, K., Hanner, R., Lee, L., Dixon, B. and Bols, N., 2011. Development of a rainbow trout intestinal epithelial cell line and its response to lipopolysaccharide. Aquaculture Nutrition , 17(2), pp.e241-e252.

Ganassin , R. and Bols, N., 1998. Development of a monocyte/macrophage-like cell line, RTS11, from rainbow trout spleen. Fish & Shellfish Immunology, 8(6), pp.457-476.