RNA-Seq reveals an integrated immune response in nucleated erythrocytes

Abstract

Background: Throughout the primary literature and within textbooks, the erythrocyte has been tacitly accepted to have maintained a unique physiological role; namely gas transport and exchange. In non-mammalian vertebrates, nucleated erythrocytes are present in circulation throughout the life cycle and a fragmented series of observations in mammals support a potential role in non-respiratory biological processes. We hypothesised that nucleated erythrocytes could actively participate via ligand-induced transcriptional re-programming in the immune response.

Methodology/principal findings: Nucleated erythrocytes from both fish and birds express and regulate specific pattern recognition receptor (PRR) mRNAs and, thus, are capable of specific pathogen associated molecular pattern (PAMP) detection that is central to the innate immune response. In vitro challenge with diverse PAMPs led to de novo specific mRNA synthesis of both receptors and response factors including interferon-alpha (IFNα) that exhibit a stimulus-specific polysomal shift supporting active translation. RNA-Seq analysis of the PAMP (Poly (I:C), polyinosinic:polycytidylic acid)-erythrocyte response uncovered diverse cohorts of differentially expressed mRNA transcripts related to multiple physiological systems including the endocrine, reproductive and immune. Moreover, erythrocyte-derived conditioned mediums induced a type-1 interferon response in macrophages thus supporting an integrative role for the erythrocytes in the immune response.

Conclusions/significance: We demonstrate that nucleated erythrocytes in non-mammalian vertebrates spanning significant phylogenetic distance participate in the immune response. RNA-Seq studies highlight a mRNA repertoire that suggests a previously unrecognized integrative role for the erythrocytes in other physiological systems.

Figure 1. Model description of rainbow trout erythrocytes.

(a) General micrograph of cultured rainbow trout erythrocytes (×50). (b) TEM of trout erythrocyte cultures. Left panel, nucleus show condensed (arrow) and decondensed (arrowhead) chromatin; middle panel, trout erythrocyte nucleus with nuclear pores (arrows); right panel, erythrocyte with ribosomes (arrowheads), smooth endoplasmic reticulum (black arrow) and Golgi apparatus (white arrow). (c) Specific mRNA transcript expression (RT-PCR) in trout and chicken erythrocytes (ENO, enolase; TLR 3, 9 and 21, Toll-like receptors; Mx, myxovirus resistance 1). Left panel, 1) density gradient isolated cultured trout erythrocytes under control conditions; 2) cell sorted trout erythrocyte population from density gradients. Right panel, c) specific mRNA transcript expression in density gradient isolated cultured chicken erythrocytes under control conditions. One representative of four individuals is shown.

Figure 2. Analysis of specific mRNA transcript expression in cultured trout and chicken erythrocytes after PAMP or cytokine stimulation.

(a) Response of trout and (b) chicken erythrocytes after 12 hours exposure to: 50 µg/ml of LPS, 50 µg/ml of poly (I∶C), 5 µg/ml of PGN and 50 ng/ml of rTNF. CCL4, Mx, IFNα, TLR3 and TLR9 (TLR21 in chicken) mRNA abundance was analyzed by RT-PCR and 1.2% agarose gel electrophoresis. One representative of 3 and 4 individuals is shown for trout and chicken respectively. (c) RT-PCR analysis of the tEC response over time (6–24 h) to 50 µg/ml of poly (I∶C), densitometry data shown as fold changes (mean ± std.dev., n = 4 individuals) with respect to 18S rRNA. (d) Absolute Q-PCR analysis of the tEC response over time control (6–24 h) to 50 µg/ml of LPS, fold changes (mean ± std.dev., n = 4 individuals) in respect to specific transcript copy number.

Figure 3. Analysis of polysome-bound mRNA regulation in cultured trout erythrocytes after poly (I∶C) stimulation.

(a) RT-PCR analysis of polysome-bound mRNAs (AIF-1, TNFR-like, IRF1.1 and IFNα) under control and after poly (I∶C) stimulation. M, ribosome free mRNA; D, mono and disome bound mRNA; and P, polysome bound mRNA obtained after polysome gradient centrifugation. NSC119889 (200 µM) was added to inhibit polysome formation. (b) Densitometry analysis (n = 3) of % bound or non-polysome specific mRNA in respect to the total specific mRNA abundance.

Figure 4. Gene Ontology representation of tEC stimulated with or without poly (I∶C).

Pie charts of the percent of transcripts within functional categories for genes regulated >2 fold in the control versus poly (I∶C) libraries. Genes regulated >2 fold were divided into functional categories using CateGOrizer (http://www.animalgenome.org/bioinfo/tools/countgo/).

Figure 5. Effects of conditioned medium from poly (I∶C) stimulated tECs upon the anti-viral response in adherent trout monocyte/macrophages.

(a) Rainbow trout macrophage Mx mRNA abundance analyzed by RT-PCR after 12 h incubation with tEC supernatants. eCM were incubated with benzonase to remove poly (I∶C) (50 µg/ml) and/or incubated at 95°C for 10 min. (b) Mx, IFNα and STAT1α/β mRNAs abundance after 12 h incubation with tEC supernatants. Data shown as fold change (mean ± std.dev, n = 3). A, poly (I∶C) stimulated erythrocyte supernatants benzonase-treated vs control supernatant benzonase-treated; B, poly (I∶C) stimulated erythrocyte supernatants benzonase- and temperature-treated vs control supernatant benzonase- and temperature-treated.