3. The role of nucleated RBCs in adaptive immune response activation

The adaptive immune system consists of a specialized group of cells responsible of a specific immune response which eliminates and prevents reoccurrence of pathogens by immunological memory [61]. The cells that carry out adaptive immune response are B and T lymphocytes [62]. All nucleated cells are capable of presenting an antigen, through major histocompatibility complex (MHC) molecules [62]. MHCI plays a key role in antigen presentation of intracellular pathogens. Nucleated RBCs can express MHCI, and this molecule has been found on the surface of RBCs from rainbow trout [63], African clawed frogs [64], and chickens [65]. In addition, it has been reported that PRV infection induces genes involved in antigen presentation via MHCI in salmon RBCs [23], and incubation with polyI:C upregulates gene ontology (GO) categories related to antigen processing, antigen presentation, and MHCI receptor activity in rainbow trout RBCs [6]. 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].

Taking the above-said observations into account, these facts indicate that nucleated RBCs might participate in antigen presentation through MHCI and MHCII and suggest that RBCs may be participants in the immunological synapse with T and NK cells. Besides, it has been published that human RBCs could play a biological role in the modulation of T-cell differentiation and survival in the active cell division [68]. 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]. In fact, the identification of clusters of cells composed by RBCs and immune cells, commonly termed rosettes, leads to a crosstalk between RBCs and immune cells [70]. These evidences broaden the horizon of nucleated RBC immune functions as they open a novel topic of investigation where nucleated RBCs may act as professional APCs.

Separately, other molecules related to adaptive immune response have been identified in nucleated RBCs. An example of these molecules is the immunoreceptor tyrosine-based activation motif (ITAM) which is contained in certain transmembrane proteins of the immune system and is important for the signal transduction in immune cells [71]. ITAM-bearing molecules are expressed on rainbow trout RBCs [72]. Another molecule, Epstein–Barr virus G-protein-coupled receptor 2 (EBI2), which plays a critical role in the regulation of T-cell-dependent antibody responses and provides a mechanism to balance short- versus long-term antibody responses [73], has also been reported to be highly expressed in rainbow trout young RBCs

been reported to be expressed in other organs such as cardiac stomach, esophagus [46], heart, gill, spleen, kidney, and peripheral blood leucocytes [47] dependent upon the species. They have been shown to respond to bacterial and viral infections [48]. Regarding RBCs, Nombela and colleagues found that rainbow trout RBCs exposed to VHSV did not vary hepcidin protein levels [26]. Therefore, the possible role of hepcidin in nucleated RBCs against infectious pathogens is not known yet. Histone proteins share all of the essential traits of cationic AMPs (CAMPs); they are hydrophobic and cationic and can form amphipathic alpha-helical structures [49]. Recently, it has been demonstrated that a histone mixture (H1, H2A, H2B, H3, H4, and H5) extracted and purified from chicken RBCs had antimicrobial activity against a variety of Gram-negative and Gram-positive planktonic bacteria [50], as well as eradication activity against Gram-positive bacterial biofilms [51]. It has also been reported that histone H5 from chicken RBCs has a broad-spectrum antimicro-

In addition to AMPs, another protein with antimicrobial activity found in RBCs

In brief, nucleated RBCs can produce antimicrobial molecules in response to pathogens. It therefore supports the important contribution of RBCs in the regula-

The complement system is a component of the innate immune system which is involved in the clearance of pathogens, dying cells and immune complexes through opsonization, induction of an inflammatory response, and formation of a lytic pore. This system is composed by a group of 30 different plasma and membrane proteins, which are involved in three distinct pathways of complement activation: the classical, lectin, and alternative pathway. The classical pathway is activated by immune complexes, by pattern recognition molecules such as C-reactive protein (CRP), or directly by apoptotic cells and microbial surfaces. The lectin pathway is triggered by carbohydrate structures from pathogen, and the alternative pathway is activated by

Autologous cells are protected from complement activation and posterior lysis by regulatory proteins [56]. RBCs are continuously in contact with complement proteins in the blood plasma; therefore, they have complement regulatory proteins on their cell membrane to prevent this activation [55]. It has been reported that human and rainbow trout RBCs highly express the regulatory protein complement receptor 1 (CR1) or CD35 [56, 57]. An important function of RBC CR1 is to eliminate complement-opsonized immune complexes from the circulation. A failure in this receptor can end up in inflammation and damage to healthy tissues [58]. In addition, it has been described that human RBCs can sequester typ. 5 adenovirus (Ad5) through CR1 and Coxsackie virus-adenovirus receptor (CAR), in the presence and absence of anti-Ad5 antibodies and complement, respectively. In this context, human RBCs may act as circulating viral traps or clarifiers and prevent systemic virus infection [59]. The studies of immune complex clearance in rainbow trout showed a similar complement-dependent way to eliminate immune complex as found in humans, suggesting that rainbow trout CR1 has a similar function to

is hemoglobin [53], which is the most abundant protein of RBCs. It has been described that hemoglobin can elicit antimicrobial activity through reactive oxygen species production under pathogen attack [53]. The pathogen clearance from the

bloodstream is also carried out by the hemoglobin oxygen [54].

the spontaneous hydrolysis of the protein C3 (reviewed in [55]).

tion of host defense against pathogens.

Immune Response Activation and Immunomodulation

2.4 Complement regulation on RBCs

bial activity [52].

human CR1 [60].

44

Acknowledgements

Conflict of interest

Author details

Spain

47

María del Mar Ortega-Villaizan\*

provided the original work is properly cited.

Lucas for their technical assistance.

DOI: http://dx.doi.org/10.5772/intechopen.80545

The authors declare no conflict of interest.

Verónica Chico, Ivan Nombela, Sara Puente-Marín and

\*Address all correspondence to: mortega-villaizan@umh.es

Instituto de Biología Molecular y Celular, Universidad Miguel Hernandez, Elche,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

This work was supported by the European Research Council (ERC Starting Grant GA639249). The authors would like to thank Remedios Torres and Efren

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

#### Figure 4.

An overview of protein–protein interaction network of a set of proteins, identified in rainbow trout proteome profiling, related to antigen processing and presentation of exogenous peptide antigen via MHCII. Protein–protein interaction network was constructed using NetworkAnalyst software [75]. Highlighted red nodes represent the input protein-related antigen processing and presentation of exogenous peptide antigen via MHCII pathway (Reactome database). Other nodes represent other protein interactions within the same pathway (red nodes) or related to other pathways (other colors).

[74]. Based on these facts, a role for RBCs in the adaptive immune response may be established. However, the function of these molecules and their effect on the antiviral adaptive immune response of nucleated RBCs remain to be studied.
