In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling

Abstract

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation.

Characterization of Sialic Acid-Binding Immunoglobulin-Type Lectins in Fish Reveals Teleost-Specific Structures and Expression Patterns

“…The main function of mammalian erythrocytes is the transportation of oxygen, whereas teleostean erythrocytes also exert immunological functions [40][41][42][43]. This may be due to the fact that teleostean erythrocytes contain nuclei and are capable of regulating their gene expression if necessary [44], in contrast to their mammalian counterparts [45,46].…”

“…This may be due to the fact that teleostean erythrocytes contain nuclei and are capable of regulating their gene expression if necessary [44], in contrast to their mammalian counterparts [45,46]. In particular, fish erythrocytes are considered as antigen-presenting cells, recognize pathogen-associated molecular patterns (PAMPs), phagocytose, and influence the activity of other immune cells [40][41][42][43]. It is likely that Siglecs are also involved in these erythrocyte-pathogen interactions.…”

Nucleated Red Blood Cells Contribute to the Host Immune Response Against Pathogens

“…Unlike MHCI, MHCII molecules are generally restricted to some endothelial cells and a subset of antigen-presenting cells (APCs), such as macrophages, dendritic cells, and B cells [66]. However, MHCII transcripts have been detected in chicken [10] and rainbow trout RBCs [67]. Moreover, in rainbow trout RBCs, a combination of transcriptomeand proteome-sequencing data identified functional pathways related to antigen presentation via major histocompatibility complex class II.…”

“…The set of genes/proteins identified were ARP1 actin-related protein 1 homolog B (ACTR1B), adaptor-related protein complex 1 beta 1 subunit (AP1B1), adaptor-related protein complex 2 alpha 1 subunit (AP2A1), adaptor-related protein complex 2 alpha 2 subunit (AP2A2), ADP ribosylation factor 1 (ARF1), calnexin (CANX), capping actin protein of muscle Z-line alpha subunit 1 (CAPZA1), clathrin light chain A (CLTA), clathrin heavy chain (CLTC), cathepsin D (CTSD), dynamin 2 (DNM2), dynein cytoplasmic 1 heavy chain 1 (DYNC1H1), dynein light chain LC8 typ. 2 (DYNLL2), and member RAS oncogene family (RAB7A) ( Figure 4) [67].…”

“…Also, natural killer enhancing factor (NKEF) protein in human RBC cytosol mediates enhancement of NK cell activity [69]. In addition, in rainbow trout RBCs, functional pathways related to regulation of leukocyte activation were identified by a combination of transcriptome-and proteome-sequencing data [67]. Separately, rainbow trout RBCs have been reported to use phagocytosis to bind and engulf Candida albicans and present it to macrophages [70].…”