IPA, a web-based bioinformatics tool, was used to further analyze the enrichment of gene sets and functions in our RNA-seq data
[16][5]. We identified activated
HIFα (−log
p-value = 2.6, Z-score = 1.67) and inhibited natural killer cell signaling (−log
p-value = 2.7, Z-score = 1.67) as the most significant canonical pathways affected by DEGs in our dataset. A bar chart was constructed, and STRING analysis was performed to visualize these observations (
Figure 6). Examination of the genes associated with these pathways revealed that most fell within the log2 fold change threshold (1 to −1) and were not significantly deregulated. Genes associated with
HIFα signaling that were downregulated in response to
ARG1 (
AKT1,
HSPA8 and
RPS6KB2) have classical roles in promoting cell proliferation and growth (
Figure 6a). In contrast, genes involved in the HIFα signaling that were upregulated in response to
ARG1 (
EDN1,
IL6,
NCF2,
SERPINE1,
SLC2A3 and
VIM) were generally more pleiotropic and were associated with metabolism, epithelial-to-mesenchymal transition and angiogenesis (
Figure 6b). Genes involved in the natural killer cell signaling pathway that were downregulated in response to
ARG1 (
AKT1,
HSPA8,
ITGB1 and
NFATC3) have well-established roles in promoting cell proliferation and growth, as well as in the regulation of transcriptional activation (
Figure 6c). In comparison, DEGs involved in the natural killer cell signaling pathway that were upregulated in response to
ARG1 (
WIPF1,
LAT,
HLA-B,
HLA-C and
HLA-F) have traditional roles in antigen presentation and mediation of T-cell receptor signaling (
Figure 6c,d). Beyond the interaction with
HIFα and natural killer cell signaling,
ARG1 overexpression was also associated with upregulation of genes involved in regulating transcription, including prospero homeobox protein 2 (
PROX2), histone H2B type 2-E1 (
H2BE1) and GLIS family zinc finger 2 (
GLIS2).
We also analyzed upstream regulators of DEGs by using the upstream regulator analysis tool in IPA
(Supplementary Table S2). This tool associated the upstream regulators with downstream functions to generate regulator effects hypotheses with predicted activation of upstream regulators. Type I interferon family genes,
IFNα2,
IFNλ1 and
IFNλ receptor 1, important regulators of innate antiviral and antibacterial immunity, were predicted to be positive regulators upon
ARG1 overexpression. Similarly,
MAP3K7 was predicted as another positive regulator and is an important mediator of cellular responses evoked by changes in the environment. It is noteworthy that
IFNL1 was identified as the most important activated upstream regulator (
p = 1.32 x 10
−13) and is predicted to be responsible for the gene expression changes observed in the experimental dataset. Beyond this, IPA predicted that
IFNL1 is most likely to be associated with elevated levels of IL-12 in the circulation and decreased viral replication (
Figure 7). This prediction tool also revealed that
IRGM and
MAPK1 may be significant negative upstream regulators.
IRGM is a putative GTPase involved in orchestrating an innate immune response and regulating proinflammatory cytokine production.
MAPK1 is an essential component of the MAP kinase signal transduction pathway and mediates diverse biological functions such as cell growth, adhesion, survival and differentiation through the regulation of transcription, translation and cytoskeletal rearrangements.