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. 2017 Aug 28;12(8):e0179391.
doi: 10.1371/journal.pone.0179391. eCollection 2017.

RNA sequencing demonstrates large-scale temporal dysregulation of gene expression in stimulated macrophages derived from MHC-defined chicken haplotypes

Kristopher J L Irizarry  1   2 , Eileen Downs  3 , Randall Bryden  1 , Jory Clark  1 , Lisa Griggs  1 , Renee Kopulos  4 , Cynthia M Boettger  5 , Thomas J Carr Jr  5 , Calvin L Keeler  5 , Ellen Collisson  1 , Yvonne Drechsler  1
Affiliations

RNA sequencing demonstrates large-scale temporal dysregulation of gene expression in stimulated macrophages derived from MHC-defined chicken haplotypes

Kristopher J L Irizarry et al. PLoS One. .

Abstract

Discovering genetic biomarkers associated with disease resistance and enhanced immunity is critical to developing advanced strategies for controlling viral and bacterial infections in different species. Macrophages, important cells of innate immunity, are directly involved in cellular interactions with pathogens, the release of cytokines activating other immune cells and antigen presentation to cells of the adaptive immune response. IFNγ is a potent activator of macrophages and increased production has been associated with disease resistance in several species. This study characterizes the molecular basis for dramatically different nitric oxide production and immune function between the B2 and the B19 haplotype chicken macrophages.A large-scale RNA sequencing approach was employed to sequence the RNA of purified macrophages from each haplotype group (B2 vs. B19) during differentiation and after stimulation. Our results demonstrate that a large number of genes exhibit divergent expression between B2 and B19 haplotype cells both prior and after stimulation. These differences in gene expression appear to be regulated by complex epigenetic mechanisms that need further investigation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Pattern of 13,618 genes expressed across haplotypes and timepoints.
Visual representation of genes within B2 and B19 haplotypes at each of the time points. Figure includes genes expressed in common, genes expressed only in B2, genes expressed only in B19, all genes expressed in B2, all genes expressed in B19, and total non-redundant genes expressed in either B2 or B19 haplotypes.
Fig 2
Fig 2. Distinct temporal gene expression patterns in B2 versus B19 monocytes/macrophages.
B-locus haplotypes in chickens provide a mechanism for genetically perturbing the cluster of immunologically important genes on chromosome 16 and producing phenotypic variation affecting infectious disease susceptibility and resistance. The heat map allows visualization of gene expression between the two genetically distinct haplotypes. Each row represents a gene within the B-locus (listed on the right) and each column corresponds to a particular time point when cells were collected for RNA sequencing. Black pixels indicate zero gene expression for a particular gene at a specific point in time, and dark blue corresponds to very low expression, while brighter blue indicates the next higher levels. Dark purple represents higher expression levels than blue colors, and pink represents the highest levels of gene expression. Monocytes were obtained from each haplotype of chicken and allowed to differentiate into macrophages in vitro for seven, days beginning on day minus 6 (t-6). RNA was sampled on day t-6, day t-3, and again three days later which is denoted as 0 hours (t0), when IFNγ was initially added to the cultures. On t0, RNA was sampled immediately before stimulation with IFNγ. Subsequent time points correspond to the time following interferon stimulation, in hours (1 hour, 2 hours, 4 hours, 8 hours, 16 hours and 24 hours). As visible on the heat map, there are distinct differences in gene expression between the B2 and B19 cells. The most dramatic difference occurs on day t-6. B2 cells exhibit a rapid burst of gene expression, indicated as a single column of pink on the left most edge of the heat map. In contrast, the B19 cells appear to undergo a much slower and prolonged gene expression program that was not as rapidly down regulated as in genes in the B2 cells. Additional gene expression data for a number of proteins involved in cell growth and apoptosis, is shown in the bottom half of the figure to highlight a similar pattern in gene expression and kinetics. The green border indicates the B2 haplotype expression pattern and the red border corresponds to the B19 expression pattern.
Fig 3
Fig 3. Examples of divergent gene expression patterns observed in B2 and B19 haplotype macrophages.
Four distinct patterns were identified as representative of the types of divergent gene expression that re-occur across many genes involved in macrophage differentiation, activation and function in B2 versus B19 macrophages. 1. Day t-6: B2 high vs B19 low. This divergent pattern exhibits strong expression of genes on day -6 in the B2 birds while relatively low levels of expression are observed in the B19 birds at the same time point. Genes of interest include an adenosine receptor (P2RY12) 2. Day t-6: B2 = 1 day vs. B19 = 3 days. This example of divergent patterns is the single peak of day t-6 gene expression in the B2 haplotype cells compared to the prolonged multiple day expression until day t-3 in the B19 haplotype cells. Genes of interest include macrophage differentiation gene GATA, adenosine receptor A2A and macrophage podosome markers VCL and GSN. 3. Maximum IFNγ Stimulation of B2 at 2–4 h versus 4–8 h in B19 macrophages. Another interesting divergent gene expression pattern observed between the two haplotypes occurs after stimulation by IFNγ. There is a four-hour difference in peak expression timing for a large number of induced genes. In the B2 haplotype macrophages, the peak expression occurs between 2 and 4 hours, while in the B19 macrophages, the peak expression occurs between 4 and 8 hours. 4. Maximum IFNγ Stimulation: B2 = Coherent vs. B19 = Non-Coherent Another discernable difference in post-stimulatory induction of genes between the B2 macrophages compared to the B19 macrophages is one of coherence. Specifically, there are a number of genes for which the B2 macrophages are able to rapidly turn on and reach relatively high levels of expression within 2 to 4 hours of IFNγ stimulation. In contrast, these same genes fail to exhibit a coherent peak of expression, even after 4 to 8 hours, in the B19 cells. Instead, they exhibit a dispersed “smear” of gene expression extending from approximately 1 hour after stimulation to 16 hours post-stimulation.
Fig 4
Fig 4. RT-PCR validation of transcripts identified as significantly expressed in RNA sequencing data.
Gene expression for ATP6V0C, LITAF, IL18R, TLN-1, TLR2, TLR3, TLR4, TLR5, TLR6, and TLR7 was assessed in B2 and B19 monocytes/macrophages following stimulation with IFNγ. Expression was measured at 0 hours, 2 hours and 4 hours. Expression for transcripts in B2 cells are shown in green and expression for transcripts are shown in red. Standard error is shown for each value. Values were considered statistically significant with p<0.05.
Fig 5
Fig 5. IFNγ stimulated vs. cytomegalovirus stimulated macrophage gene expression.
54 genes, for which gene expression changes were previously described following cytomegalovirus stimulation were used as comparisons for the corresponding genes in the B2 and B19 haplotype birds. A total of 25 published genes exhibited decreases in expression following cytomegalovirus stimulation while 29 genes exhibited increased expression following stimulation. All but one gene (FEZ1) in the B2 cells exhibited increased expression following IFNγ stimulation. In contrast, ten genes displayed decreased expression in the B19 cells. Of the ten exhibiting fold-change < 0 in the B19 cells, seven exhibited decreased expression in the cytomegalovirus stimulated cells. Twenty-eight genes (52%) expressed in the B2 cells matched the direction of the fold change reported in the published data while 33 genes (61%) corresponded between the B19 cells and the published data. Of the ten published genes reported as having greater than at least 5-fold increased expression, 90% of the B2 genes exhibited fold-change in the same direction.
Fig 6
Fig 6. Identification of divergent gene expression patterns between B2 and B19 macrophages.
Visualization of divergent gene expression patterns between the B2 and B19 haplotypes. A subset of genes exhibiting divergent gene expression were identified and visualized in heat following hierarchical clustering of the genes (rows), but not the time points (columns). The genes cluster into four major clades (clade1, clade2, clade3, and clade4) with a singleton gene (labelled clade 5). Among these genes, represented in clade1 and clade2, are a number of miRNAs exhibiting strong expression in B2 cells (mir-147, mir-146b, mir-1618, mir-200a, mir-1649, and mir-1648a) compared to the B19 samples. Likewise, miRNAs contained in clade3 and clade4 exhibit greater expression in B19 cells (mir-1627, mir-222b, mir-1633, and mir-19a). Additionally, a number of small nucleolar RNAs (snoRNAs) exhibit similarly dichotomous gene expression patterns (clade4) such that SNORd24, snoZ40, SNORD74, SNORA17, and SNORD12 exhibit substantially higher levels of expression in B19 cells on day -6 compared to B2 cells while B2 cells express such as snoU2_19 (clade2).
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