Identification of differentially expressed miRNAs in chicken lung and trachea with avian influenza virus infection by a deep sequencing approach

Avian influenza virus (AIV) is a type A virus of the family Orthomyxovirida. Although wild aquatic birds such as water fowls and sea gulls are their natural reservoir [1], land-based birds including chickens may also be infected, which cause significant economic losses to the poultry industry, and raise a great public health threat due to potential host jump from animals to humans [2].

miRNAs are non-coding, single-stranded RNAs of 19~23 nucleotides which represent a novel class of gene regulators and play important roles in a variety of biological processes in both plants and animals [3–5]. miRNAs modulate gene expression largely at the post-transcriptional level by different mechanisms including direct cleavage of targeted mRNAs [4], inhibition of translation [6] or even up-regulation of translation [7]. miRNAs are involved in different biological activities such as development, differentiation, growth and metabolism [8–11]. Recently, in mammals, miRNAs have been reported to participate in the regulation of immunity, including development and differentiation of lymphocytes, monocytes and neutrophils, and modulation of inflammation [11]. MiR-150 expresses in mature B and T cells derived from mouse hematopoietic stem cells, and is able to block early B cell development when expressed prematurely [12]. MiR-181a is an intrinsic modulator of T cell sensitivity and selection in mice [13]. After exposure of THP-1 (human acute monocytic leukemia cell line) cells to lipopolysaccharides (LPS), miR-146 was identified as an inhibitor of signalling proteins of the innate immune responses by NF-kappaB [14]. miRNAs have also been found to be critical effectors in the regulation of viral pathogenesis. Two human encoded miRNAs (miR-136 and miR-507), have been shown to have potential binding sites for the genes that code for the polymerase basic 2 (PB2) and hemmagglutinin (HA) proteins and are reported to be involved in the pathogenesis of H5N1 AIV [15]. All of these evidences suggest that certain miRNAs might be important in the modulation of AIV infections in chickens.

In order to effectively control AIV infection in poultry, it is essential to elucidate the mechanisms of virus pathogenesis in chickens. However, how host cells interact with AIVs during infection in poultry remains poorly understood. Identification of differentially expressed miRNAs in AIV infected chickens will pave a novel avenue to understand host-virus interaction. With the development of next generation sequencing, massively parallel sequencing holds great promise for expression profiling [16] and it can provide a superior sensitivity at high sequencing depth to discover especially those miRNAs with low abundance and novel miRNAs that are not able to be identified using traditional cloning approaches. Deep sequencing has been previously used to profile both chicken miRNAs and Marek's disease virus miRNAs [17, 18]. In the current study, a Solexa Sequencer was used to deep sequence differentially regulated chicken miRNAs in H5N3 infected and non-infected SPF chickens. Our results will expand the list of miRNAs which might be related to the host immune responses in animals.

The impact of miRNAs expression on the understanding of molecular mechanisms in gene regulations has been remarkable. Although thousands of small RNAs have been identified over the last decade, the challenge remains to fully identify all small nuclear RNAs, especially very low abundant ones and to determine their individual functions. The majority of known miRNAs have been identified through traditional cloning method, which is both time consuming and labor intensive. The advantages of next-generation sequencing technologies have provided an innovative tool to look into the genome with unprecedented depth of coverage. Solexa deep sequencing is one of these high throughput technologies, by which miRNAs can be detected in any organism without prior sequence or secondary structure information. This technology has been used in many species including human, mice and birds [17, 18, 26–28]. Expression of miRNAs varies in different developmental stages [29–31]. Chicken miRNAs identified in the present study provided novel information in the profiling of miRNAs not only in AIV infected chickens, but also the two tissues (lung and trachea) that have not been previously examined for miRNA profiling in chickens. To our knowledge, this is the first study to profile chicken miRNAs in AIV infected chickens by deep sequencing approach. There are 474 chicken miRNAs predicted in miRBase 13.0 [19–22]. The deep sequencing results in the current study experimentally confirmed 377 miRNAs in chicken lungs and 149 miRNAs in chicken tracheae, and the approach is more powerful than other conventional technologies previously used in birds [30]. The identification of these chicken miRNAs will be very useful in further investigating the functions and regulatory mechanisms of miRNAs in the chicken.

Growing evidence has suggested a relationship between differential miRNA expression and human diseases [32, 33]. miRNAs can regulate many aspects of the immune response, including the development and differentiation of B and T cells, proliferation of monocytes and neutrophils, antibody switching and the release of inflammatory mediators by regulating basic component of host immune system [34–36]. MiR-155 has been reported by several groups to play important roles in both innate and adaptive immune responses in mammals [11, 37, 38]. MiR-155 deficient mice lacked the capability to generate defensive immune responses and to develop lymphocytes, especially B-cell, and antigen-presenting cell functions [39]. However, miR-155 showed very low abundance in both lungs and tracheae and no significant differential expression was observed in the present study. Over expression of miR-181a in mature mouse T cells can augment the sensitivity to peptide antigens, while suppressing miR-181a expression can reduce sensitivity and impair both positive and negative selection [13]. Selective expressions of miR-181a in the thymus and miR-223 in the bone marrow have been shown to be involved in the differentiation of pluripotent hematopoietic stem cells into the various blood cells lineages including B and T cells [40, 41]. In the present study, miR-223 was not significantly regulated while miR-181a was down-regulated in both infected lungs and tracheae. In addition, miR-181a had a higher expression level in lungs than in tracheae under both infected and non-infected states. The expression levels of miR-181a, 181a* and 181b were investigated in LPS activated and CD40-lignad activated macrophages of chickens, respectively [42]. Only miR-181b was expressed in the macrophage cell line HD11 as well in the spleen adherent cells and that its expression increased after activation by LPS or CD40-ligand [42]. In the current study, miR-181b had same expression pattern with miR-181a in both lung and trachea comparisons. These results suggest that miR-181a and miR-181b may be strong miRNA candidates that regulate host response to AIV infection, and warrant further investigation of their targets and regulation mechanism in chickens.

Although the interaction between miRNA expression and virus infection remains to be elucidated, we speculated that miRNA might target immune related genes or modulate virus replication. Sequencing of chicken miRNAs in Marek's disease virus (MDV) infected and non-infected chicken embryo fibroblast (CEF) indicated that more miRNAs were up-regulated in MDV infected cells [17]. These results differ from the current study in which most differentially expressed miRNAs (55 out of 73 in lungs and 27 out of 36 in tracheae) were down-regulated in AIV infected tissues. These results indicate that the mechanisms of miRNA regulation of the host response to different types of virus in chickens are different. Chicken miR-221 and miR-222, the most abundant miRNAs in the CEF small RNA libraries, had significantly higher reads in MDV infected than non-infected CEF [17]. While both miR-221 and 222 had relatively lower abundance in the present study. These results demonstrate that miRNA expression can be tissue-specific with high abundance of miR-221 and 222 in the CEF libraries and low abundances in lungs and tracheae. It can also be speculated that host miRNAs expression may be suppressed by AIV replication based on the miRNA expression patterns observed in the current study.

Some miRNAs have been shown to be directly involved in virus replication. A liver specific miRNA (miR-122) was shown to be required for Hepatitis C virus (HCV) replication in humans [43]. MiR-122 can positively affect the viral replication and has become a therapeutic target for the treatment of HCV infection [44]. In the current study, miR-122 specifically expressed in chicken lungs compared to tracheae under both infected and non-infected states. These data suggest miR-122 might play a more important role in tissue distribution than the responses to AIV infection in chickens. Another two human miRNAs (miR-507 and miR-136) have potential target binding sites in polymerase basic 2 (PB2) and hemagglutinin (HA) genes of AIV, respectively [15]. Unfortunately, these two miRNAs are absent in the chicken genome, which might indicate different infectivity and lethality of the virus between chickens and humans.

Although in the present study most differentially expressed miRNA were down-regulated during AIV infection, some miRNAs were also up-regulated. MiR-1a, miR-140 and miR-449 were significantly up-regulated in both tissues, while miR-455, miR-34b and miR-34c were only up-regulated with AIV infection in tracheae. This suggests different miRNA regulation mechanisms might exist on host response to virus infection. These up-regulated miRNAs might inhibit gene expression of their target genes; therefore down-regulation of these target genes might help the host to inhibit virus replication.

Different tissues serve different biological functions in animals and the expression patterns of miRNAs can vary in different tissues [23, 30]. miRNAs in bursa and spleen of developing chicken embryo have been recently identified, and diverse expression patterns of these miRNAs between different immune organs were observed, suggesting that miRNAs may function as dynamic regulators of the vertebrate immune system [45]. Some miRNAs show tissue-specific distribution in mouse, suggesting specific functions within these tissues [46]. In the current study, chicken lung and trachea were examined, as they are both part of the respiratory system and important sites for AIV replication. There was a significant difference in miRNA expression between lung and trachea with more miRNAs expressed in lungs (377 miRNAs identified) than tracheae (149 miRNAs identified), although only small percentage of miRNAs (19% in lung and 24% in trachea) were significantly differentially expressed in AIV infected samples.

When tissues in the state of virus infection were compared, 28 and 23 miRNAs were specifically and highly expressed in lungs, respectively, and only 6 miRNAs (miR-1a-1 and 2, miR-1b, miR-34b, 34c and miR-449) were highly expressed in tracheae (Table 5). When tissues were compared under the non-infected state, all differentially expressed miRNAs were expressed at higher levels in lungs than tracheae with the only exception of miR-206, which showed a higher expression level in non-infected trachea than lung (Table 6). More interestingly, miR-206 was up-regulated in virus infected vs. non-infected lungs and was down-regulated in infected vs. non-infected tracheae. We can conclude that miR-206 has an opposite regulatory role in lungs and tracheae or might have different targets in different tissues and therefore play different roles in host-virus interactions. MiR-1458 and miR-1612 were up-regulated in AIV infected chicken lungs, while they were specifically expressed in non-infected tracheae not the infected one (Tables 3 and 4). The different regulation of miR-1458 and miR-1612 between lung and trachea suggests they may also have different mechanisms in response to AIV infection between tissues.

We hypothesize that miR-34b, miR-34c, miR-206, miR-1458 and miR-1612 might be some of the most important miRNAs associated with AIV infection. Significantly different miRNA expression pattern between lung and trachea suggests the regulatory mechanism of miRNAs on host response to the AIV infection between lung and trachea is distinct. However, similar regulatory mechanism might also exist in these two tissues. Within the down-regulated miRNAs in infected vs. non-infected lungs and tracheae, there were 18 miRNAs which overlapped in both tissues. This suggests that these 18 miRNAs might have common modulation mechanisms with the AIV infection in chickens.

GO term enrichment analysis has been widely used in functional analysis and allows the identification of important categories associated with functions of interests. GO terms enriched by the target genes of differentially expressed miRNAs in the current study can provide useful information for the follow-up study to elucidate the regulatory mechanism of miRNAs in host immune response to AIV infection. During AIV infection, the host immune system is stimulated to develop a defensive mechanism, which might be the reason why genes involved in immune system development were enriched in all comparisons.

With virus infection, more immune related GO terms were enriched by the targets of repressed miRNAs in lungs than in tracheae (15 terms in lung comparison and 6 terms in trachea comparison) (Figures 3 and 4). Response to virus was identified as the most enriched term (15 fold enrichment) in lung comparison, confirming that genes related to virus infection were regulated by miRNAs. The hyperinduction of proinflammatory cytokines such as TNF-α and IFN-β in human macrophages and respiratory epithelial cells by the highly pathogenic AIV H5N1 was believed to contribute to its high pathogenecity [47]. Lymphocytes were also reported to be suppressed by AIV [48]. Enrichments of T-cell and leukocytes activation and cytokines activities terms identified in the comparison of infected vs. non-infected lungs might be an indication of host immune system response against virus infection. Meanwhile, GO terms involved in lung development and epithelium morphogenesis were enriched, suggesting the genes associated with lung epithelium development in lungs may be important for the recovery from AIV infection in chickens.

It was interesting that two GO terms, interleuklin-12 production and biosynthetic process, were enriched in the infected tissue comparison, which were not included in the non-infected comparison. These two terms were also enriched in the comparison of infected vs. non-infected trachea instead of the lung comparison. IL-12 plays a pivotal regulatory role in the anti-viral response due to its induction of IFN-γ, an anti-viral cytokine [49]. These may suggest that a different defensive mechanism against virus infection might occur in trachea compared to lungs.

The two terms, response to virus and T-cell activation were also enriched by immune related genes differentially expressed in the early immune responses to H9N2 infection in tracheal organ cultures (TOC) [50]. Host immune response, showed as adaptive immune responses in the current study, was enriched by differentially expressed genes in H5N1 infected chicken embryo fibroblasts (CEF) as well [51]. Influenza virus triggered a cascade of both innate and specific immune responses. Then both immune related genes and miRNAs who might regulate these genes maybe involved in similar biological processes with the same GO terms.

Of special note, NF-KappaB binding was also enriched in the comparison between lung and trachea under non-infected state. A similar GO term, regulation of NF-KappaB, was enriched in the previous TOC model with the infection of AIV H9N2 [50]. Activation of NF-KappaB pathway is an essential immediate early step of immune activation. Many viruses have developed strategies to manipulate NF-KappaB signalling through the use of multifunctional viral proteins that target the host innate immune response pathways [52]. Enrichment of GO term NF-KappaB binding suggests these two tissues might utilize this signal pathway differently.

Post-transcriptional gene activity can be regulated through the interaction of regulatory RNA-binding proteins and small non-coding RNAs such as miRNAs [4, 53]. miRNAs can modulate protein activities by altering mRNA stability, translational efficiency or localization [53, 54]. The 3' untranslated regions (3' UTR) are widely accepted as important post-transcriptional regulatory regions of mRNAs, which are particularly rich in cis-acting regulatory elements [55, 56]. miRNAs can regulate their target genes through the cis-acting regulatory elements [57]. miRNAs within the same cluster might share the same cis-regulatory elements [23], and therefore, might have the same regulatory mechanism for their target genes. Out of the 18 miRNA clusters identified in lungs and 12 miRNA clusters identified in tracheae, there were 7 miRNA clusters differentially expressed in different comparisons. The miRNAs from five of these clusters (mir-16-1-mir-15a, mir-16-2-mir-15b, let-7f-let-7a-1, let-7j-let-7k and mir-23b-mir-27b-mir-24) identified in both lungs and tracheae were significantly down-regulated in infected lungs compared to non-infected lungs and also had higher expression levels in non-infected lungs than non-infected tracheae. The mir-181a-1-mir181b-1 cluster was significantly down-regulated in both infected lungs and tracheae. And the mir-34b-mir-34c cluster was the only significantly up-regulated cluster in the AIV infected trachea. Different miRNA clusters had different regulation direction in AIV infected tissues in the present study. This illustrates that, during AIV infection, different modulation mechanisms among different miRNA clusters might coexist in both lungs and tracheae.

It is interesting to note that when considering the miRNA clusters that were most active in chicken lung and trachea, mir-17-92 cluster (consisting of six miRNAs) and mir-302b-mir-302c-mir-1811-mir-302a-mir-302d-mir-367 cluster are highly associated with cell proliferation and self-renewal of stem cells and cancer cells [58–61]. In addition the miRNAs clusters that were significantly down-regulated miR-15/16 and let-7 are typically down-regulated in stem cells and cancer [62–64]. These results suggest that AIV infection in chickens may instigate cell proliferation and self-renewal like behaviour in chicken lung epithelium and the newly recruited T lymphocytes.

miRNAs have recently been implicated in the intricate cross-talk between host and pathogen in viral infections and are critical in viral pathogenesis. In AIV infected tissues, expression patterns of some host miRNAs were significantly differentially regulated in the current study, supporting the hypothesis that certain miRNAs are essential in the host-pathogen interactions. Once the role of these miRNAs in the regulation of host-AIV interaction has been determined, it will improve the protective strategies in AIV infection in poultry.