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Comparative Study
. 2014 Dec 1;9(12):e112997.
doi: 10.1371/journal.pone.0112997. eCollection 2014.

Comparison of Temporal Transcriptomic Profiles From Immature Lungs of Two Rat Strains Reveals a Viral Response Signature Associated With Chronic Lung Dysfunction

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Comparative Study

Comparison of Temporal Transcriptomic Profiles From Immature Lungs of Two Rat Strains Reveals a Viral Response Signature Associated With Chronic Lung Dysfunction

Elizabeth A Hines et al. PLoS One. .
Free PMC article

Abstract

Early life respiratory viral infections and atopic characteristics are significant risk factors for the development of childhood asthma. It is hypothesized that repeated respiratory viral infections might induce structural remodeling by interfering with the normal process of lung maturation; however, the specific molecular processes that underlie these pathological changes are not understood. To investigate the molecular basis for these changes, we used an established Sendai virus infection model in weanling rats to compare the post-infection transcriptomes of an atopic asthma susceptible strain, Brown Norway, and a non-atopic asthma resistant strain, Fischer 344. Specific to this weanling infection model and not described in adult infection models, Sendai virus in the susceptible, but not the resistant strain, results in morphological abnormalities in distal airways that persist into adulthood. Gene expression data from infected and control lungs across five time points indicated that specific features of the immune response following viral infection were heightened and prolonged in lungs from Brown Norway rats compared with Fischer 344 rats. These features included an increase in macrophage cell number and related gene expression, which then transitioned to an increase in mast cell number and related gene expression. In contrast, infected Fischer F344 lungs exhibited more efficient restoration of the airway epithelial morphology, with transient appearance of basal cell pods near distal airways. Together, these findings indicate that the pronounced macrophage and mast cell responses and abnormal re-epithelialization precede the structural defects that developed and persisted in Brown Norway, but not Fischer 344 lungs.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Lung sections comparing airway morphology changes in F344-virus and BN-virus lungs at representative time points.
(A-J) H&E stained sections of F344-virus (FV) and BN-virus (BV) lungs at D7 (A-B), D10 (C-D), D14 (E-F), D21 (G-H), and D35 (I-J). Shown scale bars in micrometers (µm).
Figure 2
Figure 2. Gene ontology (GO) analysis of genes differentially expressed between BN-virus and F344-virus.
(A-F) Z-score time course plots are shown for key GO terms related to immune response (A-C) or lung structure (D-F). Within each GO term plot, a higher Z-score indicates that more genes in that term group are differentially expressed between BN-virus and F344-virus.
Figure 3
Figure 3. Analysis of inter-strain and intra-strain gene expression differences for D7 differentially expressed genes.
(A) The 200 genes differentially expressed between BN-virus and F344-virus at D7 were sorted into the sixteen possible categories. Red columns indicate inter-strain comparisons: BN-virus (BV) vs. F344-virus (FV), BN-sal (BS) vs. F344-sal (FS). Blue columns indicate intra-stain comparisons: BN-virus (BV) vs. BN-sal (BS) and F344-virus (FV) vs. F344-sal (FS). The number of genes that fit the criteria in each row is listed on the right of each row. (B) As examples, strain differences in gene expression levels are diagramed for one selected gene for each of the two groups, Category 1 and Category 2, which contained the most genes. The x-axis represents no value other than to allow separate presentation of the two strains. The y-axis represents transcript level.
Figure 4
Figure 4. Representative time course plots for expression pattern groups.
(A-F) Plots of individual genes that represent each pattern groups. Gene names are on top of each plot. The Y-axis indicates transcript level and the X-axis indicates the five time points. Inset in (A) indicates the color key for each line representing the expression level in F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red). (A′-F′) qPCR verification of D7 gene expression levels. (A″-F″) qPCR verification of D21 gene expression levels. Dots in A′-F″ indicate values from four independent lung samples (biological replicates) and the line indicates the median value.
Figure 5
Figure 5. BN-virus lungs exhibit increased macrophage cell number and gene expression.
(A-H) Lung sections stained using anti-CD68 antibody to label macrophages (red), and DAPI to label all nuclei (blue). CD68+ cells were rarely observed in either F344-sal (FS) (A) or BN-sal (BS) (B). A greater number of CD68+ cells were observed in BN-virus (BV) compared with F344-virus (FV) at D7 (C,D), D10 (E,F) and at D21 (G,H). Within each strain, there is a reduction in CD68+ cell number as time progresses after inoculation. (G-O) Gene expression plots of transcript level across five time points: the macrophage marker Cd68 (I); an inductive signal for classically activated macrophages (CAMΦs) Il12a (J); CAMΦ effector genes Il6 (K) and Mmp9 (L); alternatively activated macrophage (AAMΦ) inductive signal receptor Il13ra1 (M); AAMΦ marker Retnla (N); and AAMΦ effector genes Spp1 (O), Mmp12 (P), and Tgfb1 (Q).
Figure 6
Figure 6. BN-virus lungs exhibit increased mast cell gene expression and cell number.
(A,B) Expression plots of mast cell marker genes, protease Mcpt10 (A) and tryptase Tpsab1 (B). (C-J) Lung sections stained with anti-TPSAB1 antibody to label mast cells, and DAPI to label all nuclei (blue). For TPSAB1, no staining was observed in the F344-sal (FS) lung (C), infrequent staining was observed in BN-sal (BS) (D), D7, D10 or D21 F344-virus (FV) (E,G,I), D7 BN-virus (BV) lungs (F), and increased staining was observed in D10 and D21 BN-virus (BV) lungs (H,J).
Figure 7
Figure 7. F344-virus exhibit efficient epithelial repair.
(A-C) Gene plots of differentiated airway cell marker genes Scgb1a1 for club cells (A); progenitor basal cells Krt5 (B), and progenitor variant club cells Upk3a (C). (D-I) Longitudinal (D-G) or transverse (H-I) sections of the airway stained with basal cell markers anti-KRT5 (green) and P63 (red). Blue DAPI staining labels nuclei. Red staining in the mesenchyme in H and I is also observed in sham inoculated controls, and is not nuclear as expected for P63 expression. Thus, we speculate that it is non-specific background staining.

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