Elsevier

Vaccine

Volume 35, Issue 5, 1 February 2017, Pages 729-737
Vaccine

H9N2 avian influenza virus enhances the immune responses of BMDCs by down-regulating miR29c

https://doi.org/10.1016/j.vaccine.2016.12.054Get rights and content

Abstract

Avian influenza virus (AIV) of the subtypes H9 and N2 is well recognised and caused outbreaks-due to its high genetic variability and high rate of recombination with other influenza virus subtypes. The pathogenicity of H9N2 AIV depends on the host immune response. Dendritic cells (DCs) are major antigen presenting cells that can significantly inhibit H9N2 AIV replication. MicroRNAs (miRNAs) influence the ability of DCs to present antigens, as well as the ability of AIVs to infect host cells and replicate. Here, we studied the molecular mechanism underlying the miRNA-mediated regulation of immune function of mouse DCs. We first screened for and verified the induction of miRNAs in DCs after H9N2 AIVstimulation. We also constructed miR29c, miR339 and miR222 over-expression vector and showed that only the induction of miR29c lead to a hugely increased expression of surface marker MHCII and CD40. Whilst the inhibition of miR29c, miR339 and miR222 in mouse DCs would repressed the expression of DCs surface markers. Moreover, we found that miR29c stimulation not only up-regulate MHCII and CD40, but also enhance the ability of DCs to activate lymphocytes and secrete cytokines IL-6 or TNF-a. Furthermore, we found that Tarbp1 and Rfx7 were targeted and repressed by miR29c. Finally, we revealed that the inhibition of miR29c marvelously accelerated virus replication. Together, our data shed new light on the roles and mechanisms of miR29c in regulating DC function and suggest new strategies for combating AIVs.

Introduction

The H9N2 subtype avian influenza virus (AIV), classified as a low pathogenic AIV, is still prevalent since its isolation in 1966 [1], [2]. It has high genetic variability and has shown both increases in virulence and ability to cross the host barrier, including transmission to swine and mammals, such as hamsters and ferrets [3], [4], [5], [6], [7], [8]. The frequent recombination of H9N2 AIV plays a major role in reassorting new AIV strains that enables them to cross the interspecies barrier [4], [6]. Six segments of the H7N9 virus are from H9N2 origin. But again H9N2 is not a pandemic strain. Since the H7N9 and H10N8 AIV outbreaks in 2013 resulted from recombination between H9N2 and other influenza subtypes [4], [9], H9N2 AIV is a subject of intense research.

There is a constant struggle between viruses and the host immune system, and the pathogenicity of a virus is determined not only by its characteristics but also by the host immune response [10]. Dendritic cells (DCs), the professional and effective antigen-presenting cells in vivo, play an essential role in the innate immune response [11]. AIV infection affects the maturation, antigen presenting ability, and cytokine secretion of DCs [12]. Hundreds of host proteins have been characterised and a functional map of host-influenza interactions has been drawn for epithelial cells. The binding of pathogen-associated molecular patterns to receptors expressed by DCs may activate DCs [13], but it remains unclear how AIVs produce changes in DCs and how DCs respond to AIV infection.

MicroRNAs (miRNAs) have emerged as key regulators of diverse biological processes, including innate immune responses [14], [15]. miRNAs affect the development of DCs and their ability to present antigens and secrete cytokines [16]. For example, the disruption of miR155 caused defective DC-mediated antigen presentation and reduced the numbers of germinal center B cells and Th2 T cell responses [17]. In addition, miR148 and miR152 can influence DC functions, including the capture of antigens via Toll-like receptors and the processing and presentation of antigens to T cells. Furthermore, AIV infection leads to the differential expression of cellular miRNAs in chickens and mice, and miR491 and miR654 inhibit the replication of H1N1 virus through binding to PB1 in MDCK cells [18]. The purpose of our study was to investigate how miRNAs regulate the immune response of DCs to H9N2 AIV.

Section snippets

Virus and animals

Influenza A virus (A/duck/Nanjing/01/1999(H9N2)) was provided by the Institute of Animal Husbandry and Veterinary Medicine, Jiangsu Academy of Agricultural Science (Nan Jing, China). Allantoic fluid was concentrated 10-fold (109 egg infectious doses 50 (EID50)/0.1 ml) and purified on a discontinuous sucrose density gradient as described [19]. The viral 50% -tissue culture infectious dose (TCID50) of the purified H9N2 was calculated using the Reed and Muench method [20]. SPF C57BL/6 and BALB/c

The miRNAs expression following H9N2 AIV infection

Previously, we studied the influence of H9N2 AIV infection on global RNA expression in mice. Of total 349 conserved miRNAs, we found that 9 miRNAs were significantly up-regulated by viral infection, whilst 8 were down-regulated (Fig. 1A and Supplementary Table 4). Then, we selected 8 down-regulated miRNAs and 1 up-regulated miR222 (positive control) for further experiment. Quantitative PCR (qPCR) showed that miR181b1, miR339, miR375, miR29c and miR24-1 were significantly decreased, which was

Discussion

The interactions between cellular factors, such as miRNAs, and H9N2 AIV are important for AIV infection. In this report, we focused on 9 miRNAs whose expressions were altered by AIV in BMDCs. DCs represent a central element in the generation and maintenance of immune responses [16]. Our study suggests that the immune function of DCs, including phenotypic alteration and T lymphocyte activation, was greatly stimulated by miR29c. Members of the miR29 family can be activated by IFN signaling,

Ethics approval and consent to participate

This study was approved by the Ethics Committee for Animal Experiments of the College of Life Science, Nanjing Agricultural University. All animal care and use were conducted in strict accordance with the Animal Research Committee Guidelines of the College of Life Science, Nanjing Agricultural University.

Competing interests

The authors of this editorial have no conflicts of interest to declare. The authors have no other relevant affiliations or financial involvement in any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Authors' contributions

Jian Lin design and carried out the molecular genetic studies, bio-information analyses, data analyses and manuscript drafting. Jing Xia and Ya T Chen developed chicken dendritic cells and performed qRT-PCR analyses. Keyun Zhang and Yan Zeng anticipated in the interpretations of the data and participated in its design and coordination experiment in the lab. Qian Yang is the principal investigator of the project, who conceived the study, participated in its design and helped to draft the

Acknowledgments

This work was supported by the National Science Foundation of P.R. China (No. 31372465), the National Science Foundation of P.R. China (No. 31570843), the National Science Foundation of Jiangsu Province (No. BK20150666) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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