1. Introduction
Epigenetics is within the stable of heritable phenotypic changes in organisms induced through modification of gene expression and intervention of gene translation other than alteration of the structure of DNA, the genetic code itself [
1,
2]. Over the last two decades, clear and definite evidence from numerous studies showed that epigenetics is deeply involved in modulating host immunity and tumorigenesis processes upon infections [
3,
4,
5,
6]. The epigenetic factor microRNA (miRNA) is reportedly involved in various bioprocesses, including oncogene functions and tumorigenesis suppression [
7]. Host cellular miRNAs, like gga-miR-26a, gga-miR-181a, and gga-miR-130a, for instance, play a role in lymphoma cell proliferation suppression [
8,
9].
Marek’s disease (MD) is a contagious disease of domestic chickens caused by an avian α-herpesvirus [
10,
11], commonly known as MD virus (MDV). MD has been controlled primarily by the use of MD vaccines since 1970 [
10], yet sporadic MD outbreaks take place worldwide [
12,
13]. Therefore, the commercial companies are clearly aware that MD remains a threat to the poultry industry, as it continues to impose an annual cost of over USD 2 billion to the industry [
14]. Commonly used commercial MD vaccines include Herpesvirus of turkeys (HVT), SB-1, and CVI988/Rispens, in addition to others that have been under active tests and development [
15]. MD vaccine efficacy is dependent upon multiple factors including host genetics [
16]. Our previous studies showed the protective efficacy of a MD vaccine can differ drastically from one genetic line of birds to the next [
17]. Advancement in understanding the underlying genetic and epigenetic factors that modulate vaccine protective efficacy against tumor incidence would greatly improve the strategy for design and development of new and more potent vaccines, and therefore, would empower better control of infectious diseases, like Marek’s disease, in the chicken.
Genetic resistance to MD is reportedly attributable to major histocompatibility complex haplotypes [
18,
19,
20,
21,
22,
23], a set of quantitative trait loci (QTLs) [
24,
25,
26], copy number variation [
27,
28,
29], single nucleotide polymorphisms (SNPs) [
30,
31,
32,
33], and transcriptomic variation of coding genes [
34,
35,
36]. In addition, like what has been reported in human cancer studies [
37,
38,
39,
40,
41], epigenetics is shown to play an important role in augmenting resistance and susceptibility to MD [
42,
43,
44,
45,
46,
47,
48].
Epigenetic factors include histone modification, DNA methylation, and non-coding RNAs [
49]. Examination of histone modifications and differential chromatin marks in chickens in response to MDV challenge revealed significant differences between inbred lines of chickens in their resistance to MD at both cytolytic and latency phases [
44,
50,
51]. In a whole genome histone modification study, Luo et al. found that trimethylations of histone H3K4me3 and H3K27me3 marks were positively and negatively correlated with expression of protein coding genes, respectively, both in MDV-challenged and MDV-unchallenged chickens [
50]. These reports strongly suggest that histone modifications are highly likely to be involved in MD and potentially associated with tumorigenesis [
52] in chicken. DNA methylation affects DNA transcription, consequently passing on impacts on health and disease status in addition to other phenotypic characteristics [
38,
53]. In a separate study, Luo et al. found that the promoter DNA methylation level of the CD4 gene was downregulated in response to MDV infection, which was negatively correlated with CD4 gene expression in an MD-susceptible line of chickens [
42]. The promoter region of CD30, a key gene likely associated with tumorigenesis in MD, was hypomethylated in response to MDV infection and in MD lymphoma [
46,
54]. DNA methylation level alteration in response to MDV infection was also detected between a genetic line of chickens highly susceptible to MD and another line relatively resistant to MD [
43].
MicroRNA (miRNA) is an important class of short non-coding RNAs. Mature miRNAs are approximately 21–25 nucleotides in length. miRNA negatively regulates gene expression by binding to the 3′-UTR or 5′-UTR of mRNAs to inhibit translation or by initiating mRNA degradation to prevent translation of target genes [
55]. Dysregulated expression of microRNAs has been observed in numerous disease states, particularly in human cancers, neurologic disorders, autoimmune diseases, metabolic diseases, cardiovascular diseases, and stress-induced emotional and suicidal behavior disorders [
56,
57]. It is reported that gga-miR-21 was observed to have significantly upregulated expression in response to MDV infection, and miR-26a expression was consistently downregulated in MD tumors. gga-miR-21 and miR-26a are reportedly known to facilitate an MDV oncogene,
Meq, in lymphomagenesis and to suppress MD tumor formation, respectively [
9,
58].
Two highly inbred lines of chickens, known as line 6
3 and line 7
2, were developed at the USDA, Agricultural Research Service facility, Avian Disease and Oncology Laboratory (ADOL, East Lansing, MI, USA). These lines have subsequently been maintained at both the ADOL and the US National Poultry Research Center (Athens, Georgia) facilities. The 6
3 and 7
2 lines share a common major histocompatibility complex haplotype (
B*2) but differ in susceptibility to avian leukosis virus infection and in resistance to MD [
19,
59]. Our recent studies revealed that MDV infection induced differential expression of nineteen and nine miRNAs in the two genetic lines of birds, respectively [
60], and the MD vaccine HVT and CVI988/Rispens induced differential expression of four and thirteen exclusive microRNAs in the 6
3 line birds, and 0 and one miRNA in the 7
2 line birds, respectively [
61]. To advance the fundamental understanding of microRNAs and microRNA expression in association with MDV infection post-vaccination, this study was designed to profile miRNAs and to identify miRNAs with dysregulated expression in the primary lymphoid organ, bursa of Fabricius, in the two genetically divergent inbred lines of chickens in response to a very virulent plus MDV (vv+MDV) challenge post-vaccination. Predicted target genes of the differentially expressed miRNAs were subjected to Gene Ontology (GO) terms analysis to elucidate the likely biological significance of the differentially expressed miRNAs, potentially in association with MD vaccine protective efficacy.
4. Discussion
As one of the avian tumor virus-induced diseases, Marek’s disease has been well under control since the 1970s in most parts of the world, which is primarily attributable to the wide use of MD vaccines in poultry flocks wherever applicable [
11]. The commonly used commercial MD vaccines include the gold-standard MD vaccine, CVI988/Rispens; the very first anti-tumor vaccine, HVT; and the MDV-2 vaccine, SB-1 [
66,
67]. While most, if not all, researchers and industry professionals fully recognize the great good that MD vaccines have done for the poultry industry, few, if any, claim to thoroughly understand the mechanism of how MD vaccines protect against MDV-induced tumorigenesis and tumor formation in chickens [
68]. The reality that this paradox persistently remains bars the advancement of knowledge-based new vaccine design and development, as well as better control of MD in poultry.
Protective efficacy of different vaccines varies [
69,
70,
71] and protective efficacy of a single MD vaccine also differs between genetic lines of chickens due to host genetics [
16,
17]. The highly inbred line 6
3 and line 7
2 chickens used in this study, sharing a common major histocompatibility complex haplotype (
B*2), strikingly differ in their ability to convey protective efficacy by up to 40% in response to CVI988/Rispens and up to 82% in response to HVT vaccination against very virulent plus MDV challenge [
17,
72]. The observed phenotypic differences in the ability to convey protective efficacy in response to vaccination against MDV-induced tumor formation may result from a rather complex mechanistic system potentially involving host genomic and epigenomic variation and vaccine by chicken line interactions [
61,
73]. This point is well indicated, in turn, by the fact that the MD vaccine’s immunologic mechanism of protection remains poorly understood [
74]. The understanding of genomic and epigenomic mechanisms underlying vaccine protective efficacy is even more limited.
Mounting evidence from research of various biological fields has demonstrated that epigenetic factors like DNA methylations, histone modifications, and non-coding short RNAs, particularly microRNAs, play a pivotal role in differentiation of immune cell subsets and immune functions of those immune cells, which result in alteration of immune responses in reaction to foreign antigens. It is also reported that microRNAs regulate a wide array of cellular processes including, but not limited to, cell proliferation, differentiation, and apoptosis. Dysregulated expression of miRNAs is reportedly associated with malignant transformation and tumor formation [
75,
76]. To investigate the potential roles that miRNAs play in modulating immune response and, subsequently, tumor incidence post-MD vaccination and MDV challenge, two highly inbred lines of White Leghorns, the lines 6
3 and 7
2, were profiled for miRNAs and compared to identify differentially expressed miRNAs in response to HVT or CVI988/Rispens vaccination followed by a vv+MDV challenge. Over six hundred miRNAs were identified among a set of four chicken line by vaccine treatment groups (line 6
3 HVT+MDV, line 6
3 CVI988/Rispens+MDV, line 7
2 HVT+MDV, and line 7
2 CVI988/Rispens+MDV). Although the total numbers of miRNAs identified within each of the line 6
3 and line 7
2 groups subjected to either HVT+MDV or CVI988/Risepens+MDV treatment were similar (
Table 3), the numbers of differentially expressed miRNAs in response to CVI988/Rispens+MDV (in contrast to each line’s non-vaccinated non-challenged control group) differed (14 and 9) between the line 6
3 and line 7
2 groups (
Table 4). It is also noticeable that both HVT+MDV and CVI988/Rispens induced four miRNAs (gga-mir-19b*, gga-mir-425-5p, novelMiR_530, and novelMiR_547) in common with significantly upregulated expression and one miRNA (novelMiR_91) with significantly downregulated expression in common within the line 6
3 birds (
Table 4). Since HVT induced equally good or even better protection of the line 6
3 birds than the CVI988/Rispens vaccination [
72,
73], all or some of these five miRNAs dysregulated in expression between the line 6
3 and line 7
2 birds in response to the same HVT and MDV treatment would be highly probably associated with the previously observed highly protective efficacy against MD in the line 6
3 birds [
17,
72]. Furthermore, of the five differentially expressed miRNAs, the two miRNAs novelMiR_530 and novelMiR_91 were identified in the HVT+MDV treatment group of the line 6
3 birds but not in the same treatment group of the line 7
2 birds. Therefore, these two miRNAs might play an even more critical role in modulating the protective efficacy of HVT vaccination in birds like those of line 6
3 [
17,
72,
73].
Emerging evidence also shows miRNAs play key roles in fine-tuning critical biological processes that affect host immune homeostasis, and consequently infectious diseases and cancer development [
77,
78,
79,
80]. It is also shown that tumor viruses are capable of, on one hand, modulating the expression of cellular miRNAs to facilitate their own infection processes by invading the host immune system, and, on the other, to dysregulate oncogenes and tumor suppressor genes [
81]. Dysregulated expression of miRNAs in response to virus infection is reportedly associated with cancerous disease development [
82,
83,
84] and host antiviral immunity [
85]. It has been shown that the line 6
3 birds are capable of conveying over 80% protection against vv+MDV-induced MD in response to HVT vaccination, while the line 7
2 birds are virtually incapable of conveying any protection, or convey a poorly minimal protection, against the same vv+MDV-induced MD in response to HVT [
72]. Seven and six differentially expressed miRNAs were identified within the line 6
3 and 7
2 birds, respectively, in response to HVT vaccination and vv+MDV challenge in this study (
Table 4), which were primarily or totally different from the sets of differentially expressed miRNAs identified in the two lines of birds in response only to HVT vaccination or only to MDV challenge alone (
Table S14). These results may have shown that the induced dysregulation of miRNA expression in response to HVT and MDV treatment is not a simple sum of the HVT treatment plus the MDV treatment.
Direct comparison between the HVT+MDV treatment groups of the line 6
3 and line 7
2 birds identified five differentially expressed miRNAs. The same comparison between the CVI988/Rispens+MDV treatment groups of line 6
3 and line 7
2 birds identified 22 differentially expressed miRNAs (
Table 6). Only two of the differentially expressed miRNAs (gga-miR-1684a and novelMiR-1062) were in common between the two comparison sets of HVT+MDV and CVI988/Rispens+MDV treatment groups.
Gene Ontology analysis of the target genes of the five differentially expressed miRNAs between the line 6
3 HVT+MDV treatment group and the line 7
2 HVT+MDV group indicated that those miRNAs, through their target genes, are likely involved with multiple signaling pathways, including the MAPK signaling pathway, which elicits many responses in cells evoked by factors including environmental changes and plays a major role in oncogenesis [
86]; the TGF-β signaling pathway, which regulates development, homeostasis, and tissue repairments, and reportedly plays a major role in cancer suppression [
87]; the ErbB signaling pathway, which promotes autophosphorylation and subsequent downstream signaling cascades through binding with numerous signal transducers and was confirmed as having an important role involving cancers [
88,
89,
90]; and the EGFR1 signaling pathway, which reportedly plays a role in immune defense against pathogen infection, diverse cellular processes, including cell apoptosis, proliferation, differentiation, migration, and the cell cycle, and immune regulation in both vertebrates and invertebrates [
91]. Further research is warranted to clarify the details by providing additional experimental evidence as proof of the mechanism used by the vaccine to achieve the various levels of protection. The findings of this study suggest that these five microRNAs are likely to be instrumental for the host epigenetics to interpolate between vaccine protection and virus-induced disease incidence collectively through varied GO terms and pathways including signaling pathways, with which the microRNAs’ target genes are involved.