Comparative transcriptome analysis of PBMCs in cats diagnosed with and recovered from FIPV

Abstract

Background: Feline infectious peritonitis is a viral disease caused by feline coronavirus an enveloped virus with a single-stranded RNA genome that is approximately 30 kb long. Although FCoV generally causes mild symptoms, approximately 5% of cases progress to death in cats worldwide. FCoV shares certain virological features with severe acute respiratory syndrome coronavirus 2 that causes COVID-19, indicating that common therapeutic strategies may be applicable. GS-441524 the parent drug of remdesivir and a competitive inhibitor of nucleoside triphosphates in viral RNA synthesis is a well-known treatment for FIP. However, comparative transcriptome and gene ontology analyses of normal (Normal), FIP-diseased (FIPD), and FIP-recovered (FIPR) cats have not yet been conducted.

Results: In this study, we compared the mRNA expression profiles of peripheral blood mononuclear cells from Normal, FIPD, and FIPR cats to identify immunological alterations. We identified 677 (FIPD/Normal) and 431 (FIPR/FIPD) differentially expressed genes with statistical significance. These data were input into the bioinformatics program. As a result, the analysis revealed statistically significant and contrasting patterns of canonical pathways of neutrophil degranulation and interleukin-8 (IL-8) signaling pathways. Additionally, we observed that kruppel-like factor 6 (KLF6) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were upstream molecules of IL-8, promoting neutrophil activation and function.

Conclusions: This study identified immunological alterations in PBMCs of Normal, FIPD, and FIPR cats. KLF-6 and NF-κB were found to regulate IL-8-mediated neutrophil activation.

Keywords: Feline infectious peritonitis; GS-441524; mRNA sequencing.

Fig. 1

Summarized Transcriptome profiling of PBMCs between FIPD/Normal and FIPD/FIPR comparisons (A) Venn diagram shows the common up- (Italic), down- (Dashed), and contrast-regulated (Red) genes between FIPR/FIPD and FIP/Normal comparisons. Volcano plots representing DEGs pattern between (B) FIPD/Normal and (C) FIPR/FIPD. X-axis indicates log₂ FC, and Y-axis indicates -log (p-value). Up (red) and down (green) regulated genes were filtered based on statistical significance (p < 0.05,|log₂ fold| ≥ 1). (D) Clustering heatmap showing the gene expression profiles of individuals in FIPR, Normal, and FIPD groups

Fig. 2

GO analysis from IPA software in PBMCs between FIPR/FIPD and FIPD/Normal comparisons (A) Top 10 canonical pathways ranked by -log (p-value). A negative z-score indicates an inhibitory signal in each pathway based on the DEGs. The significance threshold indicates -log (p-value) ≥ 1.3. Summarized molecular and pathway network of upregulated immune-related genes in PBMCs between (B) FIPD/Normal and (C) FIPR/FIPD. The color of the arrow and symbol indicates predicted activation (Orange), and inhibition (Blue)

Fig. 3

Top 20 of KEGG pathway enrichment analysis of DEGs ‘Fold enrichment’ and ‘Count of gene’ analysis in FIPD/Normal (A, C) and FIPR/FIPD (B, D) respectively. Functional annotation in the Y-axis is descending in order of -log (p-value) and the X-axis indicates fold enrichment and the count of up- and down-regulated genes associated with each pathway

Fig. 4

Activation of upstream molecules of IL-8 in FIPD/Normal comparison (A, B) Interaction of KLF-6 and NF-κB with downstream molecules in the cellular environment. In both analysis results, IL-8 was commonly detected as an activated molecule. (C) Comprehensive overview of IL-8’s role in FIP condition. The schematic representation demonstrates downstream events mediated by IL-8. The color of the symbol indicates predicted activation (Oragne), predicted inhibition (Blue), activation (Red), and inhibition (Green)

Fig. 5

Validation of mRNA-sequencing data. Differential expression analysis of IL-8, TLR4, TLR8, MyD88, and KLF6 across Normal, FIPD, and FIPR groups. Gene expression levels were quantified as log₂ FC, comparing mRNA-seq (blue bars) and RT- qPCR (red bars) results. Panels display expression patterns for (A) IL-8, (B) TLR4, (C) TLR8, (D) MyD88, and (E) KLF6