Background: MicroRNAs (miRNAs) play critical roles in a wide spectrum of biological processes and have been shown to be important effectors in the intricate host-pathogen interaction networks. Avian influenza virus (AIV) not only causes significant economic losses in poultry production, but also is of great concern to human health. The objective of this study was to identify miRNAs associated with AIV infections in chickens.

BackgroundAvian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited.ResultsTotal RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6 M and 3.3 M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher’s exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44 K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection.ConclusionsA comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122–1, 122–2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.

Material Supplementary 6.DC1http://www.jimmunol.org/content/suppl/2010/10/28/jimmunol.090129References

Naive CD4 T cell responses, especially their ability to help B cell responses, become compromised with aging. We find that using APC pre-treated ex vivo with TLR agonists, polyI:C and CpG, to prime naive CD4 T cells in vivo, restores their ability to expand and become germinal center T follicular helpers and enhances B cell IgG antibody production. Enhanced helper responses are dependent on IL-6 production by the activated APC. Aged naive CD4 T cells respond sub-optimally to IL-6 compared to the young, such that higher doses are required to induce comparable signaling. Pre-activating APC overcomes this deficiency. Responses of young CD4 T cells are also enhanced by pre-activating APC with similar effects but with only partial IL-6 dependency. Strikingly, introducing just the activated APC into aged mice significantly enhances otherwise compromised antibody production to inactivated influenza vaccine. These findings reveal a central role for production of IL-6 by APC during initial cognate interactions in the generation of effective CD4 T cell help, which becomes greater with age. Without APC activation aging CD4 T cell responses shift towards IL-6-independent Th1 and ThCTL responses. Thus, strategies that specifically activate and provide antigen to APC could potentially enhance Ab mediated protection in vaccine responses.

Aging leads to reduced immunity, especially adaptive responses. A key deficiency is the poor ability to mount robust antibody response. Although intrinsic alterations in B cells with age are in part responsible, impaired CD4 T cell help makes a major contribution to the poor antibody response. Other CD4 effector responses and memory generation are also impaired. We find delayed and reduced development of CD4 T follicular help (Tfh) cells in aged mice in response to influenza infection with reduction of long-lived plasma cells. When we examine CD4 subsets we also find shift towards Th1 and cytotoxic CD4 (ThCTL) responses. We summarize strategies to circumvent the CD4 T cell defect in aged, including adjuvants and proinflamatory cytokines. We find that we can strongly enhance responses of aged naïve CD4 T cells by using Toll-like receptor (TLR) activated dendritic cells (DC) as APC in vivo and that this leads to improved germinal center B cells and IgG antibody responses. The enhanced response of aged naïve CD4 T cells is dependent on IL-6, production by the DC.