Identification of diverse defense mechanisms in rainbow trout red blood cells in response to halted replication of VHS virus

Abstract

Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Rainbow trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling. Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of type I interferon ( ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs, previously exposed to UV-inactivated VHSV, and TSS (stromal cell line from spleen) revealed IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs. iTRAQ profiling revealed that VHSV exposure can induce a global protein downregulation in rainbow trout RBCs, mainly related to RNA stability and proteasome pathways. Antioxidant/antiviral response is also suggested to be involved in the response of rainbow trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of rainbow trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail.

Rainbow Trout Erythrocytes ex vivo Transfection With a DNA Vaccine Encoding VHSV Glycoprotein G Induces an Antiviral Immune Response

“…Gene expression of antioxidant enzymes such as sod1, nkef , prdx6 , and trx appeared to be upregulated in GVHSV-transfected RBCs. This mechanism has been reported in rainbow trout RBCs exposed to VHSV ( 8 ), where protective antioxidant enzymes were implicated in the response of RBCs to the induction of ROS after viral exposure. However, in this study, it is important to note that the antigen GVHSV encoded by this DNA vaccine is able to induce ROS signaling and homeostasis.…”

“…Also, RNA sequencing data showed the downregulation of genes related to interferon alpha/beta signaling such as irf1, ifnar1 , and jak1 . However, VHSV has been reported to induce ifn1 downregulation in rainbow trout RBCs ( 8 ). These differences between ifn1 and ISG gene expression could be due to the effort of the immune system to maintain homeostasis or to the differential regulation of these genes.…”

“…Peripheral blood was collected from the caudal vein using insulin syringes (NIPRO, Bridgewater, NJ, USA). RBCs were purified by two density gradient centrifugations (1,600 rpm, Ficoll 1.007; Lymphoprep, Reactiva, Sigma-Aldrich) as previously described ( 8 ). Purified RBCs were placed in RPMI-1640 medium (Dutch modification) (Gibco, Thermo Fisher Scientific Inc., Carlsbad, CA) supplemented with 10% gamma irradiated fetal bovine serum (FBS) (Cultek, Madrid, Spain), 1 mM pyruvate (Gibco), 2 mM L-glutamine (Gibco), 50 μg/mL gentamicin (Gibco), 2 μg/mL fungizone (Gibco), 100 U/mL penicillin (Sigma-Aldrich), and 100 μg/mL streptomycin (Sigma-Aldrich).…”

“…RNA extraction, cDNA synthesis and RT-qPCR analyses were performed as previously described ( 8 ). Briefly, E.Z.N.A.® Total RNA Kit (Omega Bio-Tek, Inc., Norcross, GA) was used together with DNAse (TURBO™ DNase, Ambion, Thermo Fisher Scientific, Inc.) for RNA extraction.…”

Aeromonas Hydrophila Infection Activates Death Receptor Apoptosis Pathway in the RBCs of Grass Carp (Ctenopharyngodon idellus)

“…Even though the principle function of red blood cell is the gas exchange, numerous other biological functions for red blood cells which nuclei are associated with immune responses have been recently uncovered [5,6]. Nucleated RBCs can thus modulate leukocyte activity, respond to different pattern recognition receptors (PRRs), including peptidoglycan recognition protein (PGRP) receptor families [7], Toll-like receptors (TLRs) [8], and then release cytokines [9][10][11]. Therefore, sh RBCs are excellent models for exploring the immunity features of nucleated RBCs.…”