Nucleated Teleost Erythrocytes Play an Nk-Lysin- and Autophagy-Dependent Role in Antiviral Immunity

Abstract

With the exception of mammals, vertebrate erythrocytes are nucleated. Nevertheless, these cells are usually considered as mere carriers of hemoglobin. In this work, however, we describe for the first time an unrecognized role of teleost red blood cells (RBCs). We found that Nk-lysin (Nkl), an antimicrobial peptide produced by NK-cells and cytotoxic T-lymphocytes, was also expressed in flatfish turbot (Scophthalmus maximus) erythrocytes. Although the antiviral role of Nkl remains to be elucidated, we found a positive correlation between the transcription of nkl and the resistance to an infection with Rhabdovirus in a teleost fish. Surprisingly, Nkl was found to be present in the autophagolysosomes of erythrocytes, and therefore this higher resistance provided by Nkl could be related to autophagy. The organelles of RBCs are degraded through autophagy during the maturation process of these cells. In this work, we observed that the blockage of autophagy increased the replication of viral hemorrhagic septicemia virus in nucleated teleost erythrocytes, which suggests that this mechanism may also be a key process in the defense against viruses in these cells. Nkl, which possesses membrane-perturbing ability and was affected by this modulation of RBC autophagy, could also participate in this process. For the first time, autophagy has been described not only as a life cycle event during the maturation of erythrocytes but also as a pivotal antiviral mechanism in nucleated erythrocytes. These results suggest a role of erythrocytes and Nkl in the antiviral immunity of fish and other vertebrates with nucleated RBCs.

Keywords: Nk-lysin; autophagolysosome; autophagy; erythrocytes; granulysin; red blood cells; teleost.

Figure 1

Characterization of turbot Nkl. (A) The complete coding region of turbot nkl (444-bp long) encodes a protein of 147 residues. The signal peptide is underlined and the SapB domain is highlighted. (B) 3D structure of turbot Nkl using the pig protein as a template (TM score = 0.582). The tertiary structure comprises six α-helices. (C) Structure of the turbot nkl gene and alignment between the coding region and the corresponding genomic sequence. This gene contains five exons and four introns. The 5′ and 3′ UTRs are represented as gray boxes, the CDSs of the exons as white boxes, and introns as solid lines. The length (bp) of the CDSs and introns is also reflected in the figure. CDSs, coding DNA sequences; Nkl, Nk-lysin; SapB, saposin B; UTRs, untranslated regions.

Figure 2

Tissue distribution of the nkl expression, induction after a VHSV challenge in lymphoid tissues, and relation between nkl transcription and resistance against VHSV. (A) Constitutive expression of nkl in different tissues from healthy adult turbot. Normalized expression values are represented as the mean of three individuals plus SD. (B,C) Modulation of nkl gene expression in the head kidney (C) and spleen (B) after VHSV infection. Data are expressed as fold-change regarding the values obtained in controls at each sampling point. Graphs represent the mean of five biological replicates plus SD. Significant overexpression (p < 0.05) are represented with asterisks. (D) Normalized expression of nkl gene in head kidney samples from resistant families (1 and 4) and susceptible families (2 and 3), before and after VHSV infection. Graph represents the mean of five biological replicates plus SD. Letters (A,B) indicate significant differences (p < 0.05) between the families in naïve conditions. Asterisks indicate significant differences (p < 0.05) between the naïve and VHSV-infected condition in each family. (E) Correlation between nkl transcription level in blood cells (before infection) and the day of death after VHSV challenge in juvenile turbot. The Spearman’s Rho correlation test showed a significant (p = 0.008) but moderate (R = 0.438) positive correlation between both variables. VHSV, viral hemorrhagic septicemia virus.

Figure 3

Modulation of Nkl during vaccination and/or infection in different tissues by flow cytometry and qPCR. (A–C) head kidney; (D–F), spleen; (G–I), blood. (A,D,G) represent the percentage of Nkl-positive cells in the three tissues. (B,E,H) represent the median value of the intensity of fluorescence in positive cells in the three tissues. (C,F,I) represent the expression of nkl in the tissues by qPCR. The positive region in the FL1-H histogram was adjusted using the signal registered in the same samples stained with the preimmune serum. Gating values for the positive region were 300-9910 for the head kidney, 514-9910 for spleen, and 262-9910 for blood samples. In all figures, the mean value of six samples plus SD is represented. Asterisks indicate significant differences (p < 0.05) between experimental groups. Nkl, Nk-lysin; qPCR, quantitative polymerase chain reaction.

Figure 4

Cell distribution of Nkl by flow cytometry in head kidney and blood samples and confocal images of cells stained with the anti-Nkl antibody. (A) A FL1-H histogram was used to compare the fluorescence profile of head kidney samples stained with the preimmune serum or the polyclonal antibody. FL1-H positive cells were gating at 300-9910. (B) Distribution of head kidney cell populations using SSC-H vs. FL1-H density plots. The FSC-H/SSC-H position of Nkl-positive stained cells is boxed. (C) Fluorescence profile of blood samples stained with preimmune serum and polyclonal antibody. Positive cells for Nkl were gated at 262-9910. (D) Dot plot representing the position of the two Nkl-positive cell populations (red: cell population enriched in erythrocytes or red cells; green: cell population enriched in white cells). (E) Mean fluorescence value registered in blood cell populations. Asterisks indicate significant differences (p < 0.05). (F,G) Confocal microscopy images of a head kidney and a blood sample, respectively, showing white and red cells stained with the anti-Nkl polyclonal antibody. Green: Nkl. Blue; DAPI. Scale bar, 10 µm. FSC, forward-light-scatter; Nkl, Nk-lysin; SSC, side-light-scatter.

Figure 5

Analysis of Nkl in erythrocytes by confocal microscopy, qPCR and flow cytometry, and the characterization of the cytoplasmic structures containing Nkl. (A,B) Immunocytofluorescence of purified erythrocytes stained with the rabbit preimmune serum (A) or the anti-Nkl polyclonal antibody (B), and the secondary antibody Alexa Fluor 488 goat anti-rabbit IgG. Nuclei were stained with DAPI. Scale bar, 10 µm. The nonspecific fluorescence of the preimmune serum was not registered in samples stained with the polyclonal antibody. (C) Nkl was mainly detected in a large, spherical structure. Moreover, small puncta were also present in the cytoplasm. Scale bar, 10 µm. (D) Normalized expression of nkl in total blood cells and in purified erythrocytes. Bars represents the mean plus SD of 3 individual samples. (E) FL1-H fluorescence profile of purified erythrocytes stained with preimmune serum and polyclonal antibody. A specific signal of the antibody was clearly registered. (F) FSC/SSC density plot of FL1-H positive cells gated between 262-9910 log scale. (G) Description of the Nkl-positive vesicles by TEM. Almost all turbot erythrocytes present a large, double-membrane bound structure in the cytoplasm, although a few smaller ones are also observed (H) LysoSensor Blue staining of turbot erythrocytes. The spherical structures containing Nkl are LysoSensor-positive acidic vesicles. Scale bar, 10 µm. (I) Immunocytofluorescence of purified erythrocytes stained with the rabbit anti-LC3A/B polyclonal antibody, and the Alexa Fluor 546 goat anti-rabbit IgG as secondary antibody. Nuclei were stained with DAPI. Scale bar, 10 µm. Nkl, Nk-lysin; qPCR, quantitative polymerase chain reaction; TEM, transmission electron microscopy.

Figure 6

Nkl associated with autophagolysosomes in erythrocytes. (A) Immunofluorescence confocal images of turbot erythrocytes in the absence of treatment (PBS) or after incubation with chloroquine or rapamycin. The cells were immunostained with anti-Nkl and anti-LC3B antibodies (Alexa Fluor 546 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG as secondary antibodies, respectively). Nuclei were stained with DAPI. Merged images showed co-localization of Nkl and LC3B in the erythrocytes incubated with rapamycin. Red: Nkl, Green: LC3B, Blue: DAPI. Scale bar, 10 µm. (B) 3D reconstruction of confocal images of erythrocytes incubated with rapamycin showing the double-positive (Nkl and LC3B) autophagolysosomes (white arrows). Nkl, Nk-lysin; PBS, phosphate-buffered saline.

Figure 7

VHSV infecting and replicating in erythrocytes. (A) Confocal images of double-immunofluorescence staining of VHSV nucleoprotein −N− and Nkl in blood cells. The virus was stained with the mouse anti-N VHSV monoclonal antibody 3E7 and the secondary antibody Alexa Fluor 635 goat anti-mouse IgG, and Nkl with the rabbit anti-Nkl polyclonal antibody and Alexa Fluor 488 goat anti-rabbit IgG. Nuclei were stained with DAPI. Red: N-VHSV, Green: Nkl, Blue: DAPI. Scale bar, 10 µm. (B) The replication of VHSV in erythrocytes and total blood cells after an in vitro infection was measured by qPCR detection of the VHSV glycoprotein −G− gene. Bars represents the mean plus SD of three individual samples. (C,D) Effect of chloroquine and rapamycin in the transcription of nkl (C) and in the VHSV replication (D) in erythrocytes. Bars represents the mean plus SD of three individual samples. Nkl, Nk-lysin; qPCR, quantitative polymerase chain reaction; VHSV, viral hemorrhagic septicemia virus.