Genomic and functional analysis of the host response to acute simian varicella infection in the lung

Abstract

Varicella Zoster Virus (VZV) is the causative agent of varicella and herpes zoster. Although it is well established that VZV is transmitted via the respiratory route, the host-pathogen interactions during acute VZV infection in the lungs remain poorly understood due to limited access to clinical samples. To address these gaps in our knowledge, we leveraged a nonhuman primate model of VZV infection where rhesus macaques are intrabronchially challenged with the closely related Simian Varicella Virus (SVV). Acute infection is characterized by immune infiltration of the lung airways, a significant up-regulation of genes involved in antiviral-immunity, and a down-regulation of genes involved in lung development. This is followed by a decrease in viral loads and increased expression of genes associated with cell cycle and tissue repair. These data provide the first characterization of the host response required to control varicella virus replication in the lung and provide insight into mechanisms by which VZV infection can cause lung injury in an immune competent host.

Figure 1. SVV infection results in inflammation.

(a) SVV viral loads in lung biopsies, bronchial alveolar lavage (BAL) and blood (WB) were measured by quantitative PCR using primers and probes specific for SVV ORF21 (BAL: n = 14 (0 days post infection, DPI), n = 11 (3 DPI), n = 8 (7 DPI), n = 5 (10 DPI), n = 3 (14 DPI); Lung: n = 3 (0 DPI), n = 3 (3 DPI), n = 3 (7 DPI), n = 2 (10 DPI), n = 3 (14 DPI); WB: n = 14 (0 DPI), n = 11 (3 DPI), n = 8 (7 DPI), n = 5 (10 DPI), n = 3 (14 DPI)) (^#p < 0.05 for left lung; ^$p < 0.05 for BAL; ^&p < 0.05 for WB; *p < 0.05 for right lung relative to day 0). (b) SVV infection results in focal hemorrhage during peak viral replication that largely resolved 14 DPI. (c) H&E staining shows immune infiltrates and lung consolidation during peak viral replication. (d) VZVgB staining showing high levels of viral antigen 7 DPI that were decreased 14 DPI.

Figure 2. SVV infection induces secretion of cytokines, chemokines and growth factors.

Levels of (a) chemokine, (b) cytokines and (c) growth factors in the BAL were measured using Luminex technology. BAL: n = 14 (0 days post infection, DPI), n = 11 (3 DPI), n = 8 (7 DPI), n = 5 (10 DPI), n = 3 (14 DPI) Mean pg/ml ± SEM (*p < 0.05 compared to day 0).

Figure 3. SVV infection induces immune infiltration.

(a,b) The frequencies (means ± SEM) of CD4, CD8 and CD20 positive cells in (a) BAL and (b) the lungs (*p < 0.05 compared to day 0). (c,d) The percentage of naïve, central memory (CM) and effector memory (EM) CD4 and CD8 T cells in the (c) BAL (^&p < 0.05 for CM; *p < 0.05 for EM compared to day 0) and (d) lungs (^&p < 0.05 for CM; *p < 0.05 for EM; ^#p < 0.05 for naïve compared to day 0). (e,f) The percentage of plasmacytoid DCs (pDCs), myeloid DCs (mDCs), and macrophages (MACs) in (e) BAL and (f) lungs (^&p < 0.05 for MACs; *p < 0.05 for pDCs; ^#p < 0.05 for mDCs). BAL: n = 14 (0 days post infection, DPI), n = 11 (3 DPI), n = 8 (7 DPI), n = 5 (10 DPI), n = 3 (14 DPI); Lung: n = 3 (0 DPI), n = 3 (3 DPI), n = 3 (7 DPI), n = 2 (10 DPI), n = 3 (14 DPI). Tissues used were from the infected right lobe.

Figure 4. Immune cells proliferate and exhibit cytotoxicity in SVV infected lungs.

CD3, CD20, CD68, granzyme B and Ki67 staining in lung sections from (a) naïve, (b) 7 DPI and (c) 14 DPI at 20X and 40X magnification.

Figure 5. SVV infection results in robust changes in lung gene expression.

(a) The number of DEGs correlates with viral loads. (b) Venn diagram of DEGs detected 3, 7 and 10 DPI. (c–e) Gene clusters of the 52 common DEGs with human homologs.

Figure 6. DEGs detected 3 DPI play a role in innate immunity and lung development.

(a) Bar graph shows the number of genes mapping to each of the 10 most statistically significant GO terms to which the 106 up-regulated genes enriched. Line represents the −log(p-value) associated with each GO term. (b) Network of DEGs mapping to the GO process “immune system” that directly interact. (c) Bar graph shows the number of genes mapping to each of the 10 most statistically significant GO processes to which the 54 down-regulated genes enriched. Line represents the −log(p-value) associated with each GO term. (d) Heat map of the 30 down-regulated DEGs that enriched to the GO terms described in (c) grouped by the GO term to which they mapped: ED = Embryo development, PSP = pattern specification process, BP = biosynthetic process and LD = Lung development.

Figure 7. DEGs detected 7 DPI are involved in host defense.

(a) Bar graph shows the number of genes mapping to each of the 10 most statistically significant GO terms to which the 380 up-regulated genes enriched. Line represents the −log(p-value) associated with each GO term (b) Heat map of the genes that mapped to the GO process “response to virus” grouped by function (PRR = pathogen recognition receptor; TF = transcription factor; ISG = immune stimulated gene). (c) Bar graph shows the number of genes mapping to each of the 10 most statistically significant disease pathways to which 400 down-regulated genes enriched. Line represents the −log(p-value) associated with each GO term. (d) Heat map of the DEGs with a FC >8 that enriched to “respiratory tract diseases” and “lung diseases”.

Figure 8. DEGs detected 10 DPI are involved in cell cycle and organ development.

(a) Bar graph shows the number of genes mapping to each of the 10 most statistically significant GO terms to which the 300 up-regulated genes enriched. Line represents the −log(p-value) associated with each GO term. (b) Network image of the 50 most up-regulated genes in the GO term “cell cycle”. (c) Bar graph shows the number of genes mapping to each of the 10 most statistically significant GO terms to which the 223 down-regulated genes enriched. Line represents the −log(p-value) associated with each GO term. (d) Heat map of the 20 most down-regulated genes found in the GO terms.