March 2017 | Volume 8 | Article 2461
Original researchpublished: 21 March 2017
doi: 10.3389/fimmu.2017.00246
Frontiers in Immunology | www.frontiersin.org
Edited by: Wenzhe Ho,
Temple University School of Medicine, USA
Reviewed by: Dane Parker,
Columbia University, USA Debashree Chatterjee,
Memorial Sloan Kettering Cancer Center, USA
*Correspondence:Frank Pessler
[email protected], [email protected]
Present address: Matthias Preusse,
Department of Molecular Bacteriology, Helmholtz Centre for
Infection Research, Braunschweig, Germany
Specialty section: This article was submitted to
Microbial Immunology, a section of the journal
Frontiers in Immunology
Received: 03November2016Accepted: 20February2017
Published: 21March2017
Citation: PreusseM, SchughartK and
PesslerF (2017) Host Genetic Background Strongly Affects
Pulmonary microRNA Expression before and during
Influenza A Virus Infection. Front. Immunol. 8:246.
doi: 10.3389/fimmu.2017.00246
host genetic Background strongly affects Pulmonary microrna expression before and during influenza a Virus infectionMatthias Preusse1,2, Klaus Schughart3,4,5 and Frank Pessler1,2,6*
1 Institute for Experimental Infection Research, TWINCORE Center for Experimental and Clinical Infection Research, Hannover, Germany, 2 Helmholtz Centre for Infection Research, Braunschweig, Germany, 3 Department of Infection Genetics, Helmholtz Centre for Infection Research, Braunschweig, Germany, 4 University of Veterinary Medicine Hannover, Hannover, Germany, 5Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Centre, Memphis, TN, USA, 6 Centre for Individualised Infection Medicine, Hannover, Germany
Background: Expression of host microRNAs (miRNAs) changes markedly during influ-enza A virus (IAV) infection of natural and adaptive hosts, but their role in genetically determined host susceptibility to IAV infection has not been explored. We, therefore, compared pulmonary miRNA expression during IAV infection in two inbred mouse strains with differential susceptibility to IAV infection.
results: miRNA expression profiles were determined in lungs of the more sus-ceptible strain DBA/2J and the less susceptible strain C57BL/6J within 120h post infection (hpi) with IAV (H1N1) PR8. Even the miRNomes of uninfected lungs differed substantially between the two strains. After a period of relative quiescence, major miRNome reprogramming was detected in both strains by 48 hpi and increased through 120 hpi. Distinct groups of miRNAs regulated by IAV infection could be defined: (1) miRNAs (n=39) whose expression correlated with hemagglutinin (HA) mRNA expression and represented the general response to IAV infection indepen-dent of host genetic background; (2) miRNAs (n=20) whose expression correlated with HA mRNA expression but differed between the two strains; and (3) remarkably, miR-147-3p, miR-208b-3p, miR-3096a-5p, miR-3069b-3p, and the miR-467 fam-ily, whose abundance even in uninfected lungs differentiated nearly perfectly (area under the ROC curve>0.99) between the two strains throughout the time course, suggesting a particularly strong association with the differential susceptibility of the two mouse strains. Expression of subsets of miRNAs correlated significantly with peripheral blood granulocyte and monocyte numbers, particularly in DBA/2J mice; miR-223-3p, miR-142-3p, and miR-20b-5p correlated most positively with these cell types in both mouse strains. Higher abundance of antiapoptotic (e.g., miR-467 family) and lower abundance of proapoptotic miRNAs (e.g., miR-34 family) and those regulating the PI3K-Akt pathway (e.g., miR-31-5p) were associated with the more susceptible DBA/2J strain.
conclusion: Substantial differences in pulmonary miRNA expression between the two differentially susceptible mouse strains were evident even before infection, but evolved
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Preusse et al. Host miRNAs in IAV Infection
Frontiers in Immunology | www.frontiersin.org March 2017 | Volume 8 | Article 246
inTrODUcTiOn
microRNAs (miRNAs) are short regulatory RNA molecules that occur in most eukaryotic cells and may have profound effects on a broad range of biological processes, including apoptosis, develop-ment, differentiation, proliferation, and immune responses (14). It, therefore, comes as no surprise that major changes in miRNA expression and activity have been shown to occur as part of the host cell response to infection by a variety of viruses (57). Increasing attention has been paid to the role(s) of miRNAs in influenza virus infection, with most studies having focused on influenza A virus (IAV). Studies have involved cells or tissues from natural hosts, such as humans (810), birds (11, 12), pigs (13), and macaques (14) and mice as adaptive hosts (15, 16). Major insights have been gained by comparing differences in the miRNome responses to IAV strains of differential pathogenicity (1417) or by compar-ing effects of different host backgrounds (16). The importance of some of the differentially expressed miRNAs has also been validated experimentally [e.g., Buggele etal. (18)], thus underscor-ing that miRNAs may substantially influence the course of IAV infection (19). However, in spite of this ever growing interest in the interactions between IAV and the host miRNome, there have been no studies investigating differences in the miRNA response in hosts of well characterized differential genetic susceptibility to IAV. This is all the more surprising since recent work with inbred mouse strains of differential susceptibility to IAV infection clearly showed that the transcriptomic (mRNA) response to IAV is to a great extent determined genetically (20, 21). It is therefore quite plausible that major genetic differences might also manifest at the level of the miRNomes response to IAV infection and that the description of this miRNome will enable identification of regula-tory miRNAmRNA networks that are critical for successful host defense against IAV infection and, perhaps, related viruses. Based on a well-established mouse model of IAV infection, and using deep sequencing of small RNAs (22), we have therefore used two inbred mouse strains of well characterized different genetic susceptibility to IAV infection to identify host strain-specific differences in the host miRNA response to this virus. We find substantial differences in the miRNA populations of the two strains, both at baseline and during infection, and an overall more rapid reprogramming in the more susceptible DBA/2J strain. The results suggest that host-strain-specific differences in abundance and regulation of miRNAs provide a link to host susceptibility to IAV infection.
MaTerials anD MeThODs
availability of DataSmall RNA sequencing data, a miRNA expression table, and tables with expression differences and correlations with hematological
parameters in peripheral blood, including adjusted p values, are available in the Gene Expression Omnibus repository (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE89064).
Data tables contain the 473 miRNAs that passed pre-analytic filtering. Columns within the table with expression differences (GSE89064_edgeR_miR.xls) represent the effect of infection or mock treatment compared to untreated mice, differences between infection and mock treatment at each time point, and differences between mouse strains after mock treatment, infection, or without treatment. All results were obtained with the edgeR package. Data table (GSE89064_miRNA_vs_blood_spearman_correlations.xlsx) contains Spearman correlation coefficients of all miRNAs with peripheral blood indices.
statement on ethics approvalAll animal work was conducted according to the national guidelines of the animal welfare law of the Federal Republic of Germany and approved by the Niederschsisches Landesamt fr Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany (Permit Numbers: 3392 42502-04-13/1234 and 33.19- 42502-04-13/1234).
VirusOriginal stocks of mouse adapted A/Puerto Rico/8/34 (H1N1, PR8) virus were obtained from Stefan Ludwig (University of Mnster) (23). Virus stocks were propagated in the chorioal-lantoic cavity of 10-day-old pathogen-free embryonated chicken eggs for 48h at 37C as described previously (24).
animal ProceduresInfections were essentially carried out as described previously (20, 25). Briefly, female 12- to 13-week-old C57BL/6J and DBA/2J mice (n=56 per time point and treatment) were anesthetized by intraperitoneal injection of 10 l/g body weight of a stock solution of 0.5 ml ketamine (50 mg/ml, Invesa Arzneimittel GmbH, Freiburg, Germany), 0.5ml 2% xylazine hydrochloride (Bayer Health-Care, Leverkusen, Germany), and 9 ml sterile NaCl 0.9% (Delta-Select GmbH, Dreieich, Germany). For intranasal infec
