Feline Chronic Gingivostomatitis Diagnosis and Treatment through Transcriptomic Insights

Abstract

Feline chronic gingivostomatitis (FCGS) is a debilitating inflammatory oral mucosal disease with a multifactorial etiology. The clinical diagnosis of FCGS is made based on inspection of severe inflammatory lesions and histological confirmation rather than a molecular diagnostic outcome. This gap limits the ability to provide an early diagnosis. In this report, we seek to provide additional diagnostic tools using genomics to aid in providing clinically relevant information. The use of in-depth diagnostic tools, like transcriptomics of diseased tissues, to diagnose FCGS and stratify patients into predictive treatment response groups would dramatically improve both clinical decisions and patient outcomes. In this study, we addressed the gap in diagnostic options using transcriptomic analysis of caudal oral mucosal swab specimens coupled to detailed medical record linkage of FCGS-affected cats undergoing tooth extractions and in some cases administration of mesenchymal stromal cells (MSCs). To better identify markers of disease and potential response to treatment, the transcriptomes of FCGS-afflicted cats were compared to those of healthy cats and those with chronic periodontitis to clearly establish diagnostic biomarker signal transduction connections. Phosphatidylinositol 3-kinase/Ak strain transforming (PI3K/AKT) and stress-activated protein kinases/Jun N-terminal kinase (SAP/JNK) signaling pathways were significantly differentially regulated in FCGS-afflicted cats. Activation of these pathways also differed in the treatment response groups. In conjunction, the enzymes Caspase 4 (CASP4), matrix metalloproteinase-8 (MMP8), and prostaglandin-endoperoxide synthase 2 (PTGS2) were identified as potential biomarkers for the prediction of treatment response outcomes. The observations in the case study support the use of transcriptomics of FCGS patients to contribute to improved molecular diagnostics for the diagnosis and treatment of FCGS.

Keywords: MSC; biomarkers; dentistry; feline; gingivitis; stem cells; stomatitis; transcriptomics.

Figure 1

Clinical pictures of feline chronic gingivostomatitis (FCGS)-afflicted oral cavities. (Left) Manifestation of FCGS at time of diagnosis with all teeth present. (Right) FCGS oral lesions in refractory patient despite undergoing full-tooth extractions.

Figure 2

Method schematic detailing the sample numbers and basic collection and sequencing scheme. A total of 42 unique samples were collected, with 9 more samples derived from the same sampling population at contralateral locations or times. Healthy cats were those with no diagnosed dental disease or with mild to moderate periodontitis. Feline chronic gingivostomatitis (FCGS) cats were those with gingivostomatitis and underwent either tooth extraction or mesenchymal stromal cell (MSC) treatment after not responding to extractions. Swabs were taken from the oral mucosa lateral to the palatoglossal folds. mRNA was extracted from the swabs, sequenced, and analyzed.

Figure 3

Overview of differentially expressed genes in cats with feline chronic gingivostomatitis (FCGS) compared to healthy controls. IPA (Ingenuity Pathway Analysis) was used to determine the top significantly (−log10p > 10) expressed pathways in FCGS patients as compared to healthy samples (top). Coloration represents the Z-score of each pathway; orange denoting positive values and gray showing no clear activity pattern in that pathway, with color intensity positively correlated to Z-score. General expression patterns across healthy and FCGS samples were also evaluated and displayed using a heatmap (bottom). R (version 4.2.2) package pheatmap (version 1.0.12), with Euclidean distance clustering of both genes and samples with log2 normalized read counts as input data was used to generate the map.

Figure 4

Overview of differentially expressed genes in patients that responded to extractions (top) and those that underwent mesenchymal stromal cell (MSC) therapy (bottom). Ingenuity Pathway Analysis (IPA) was used to determine the top significantly (−log10p > 10) expressed pathways in extraction responsive vs. non-responsive cats and MSC-treated vs. extraction-responding cats. Coloration represents the Z-score of each pathway; orange denoting positive values, blue denoting negative values, and gray showing no clear activity pattern in that pathway, with color intensity positively correlated to Z-score. General expression patterns across healthy and feline chronic gingivostomatitis (FCGS) samples was also evaluated by heatmap. R (version 4.2.2) package pheatmap (version 1.0.12) with Euclidean distance clustering of both genes and samples with log2 normalized read counts as input data was used to generate the map.

Figure 5

Canonical signaling pathways from gene and pathway enrichment (IPA) comparing feline chronic gingivostomatitis (FCGS) samples to healthy samples. Significance cutoff of -log10p >10. The top axis represents the percentage of the pathway annotated in the gene expression data set that was downregulated (teal), upregulated (red), or not represented in the data (white). The total gene count in each pathway is noted on the right-hand side of the figure. The bottom axis represents the significance of each pathway and corresponds to the orange line on each figure, and pathways are ordered from most significant to least from top to bottom.

Figure 6

Canonical signaling pathways from Ingenuity Pathway Analysis (IPA) comparing extraction-responsive samples to non-responder samples (left) and those that received mesenchymal stromal cells (MSC) treatment after extraction compared to those that received extractions only (right). Significance cutoff of -log10p >10. The top axis represents percentage of the pathway annotated in the gene expression data set that was downregulated (teal), upregulated (red), or not represented in the data (white). The total gene count in each pathway is noted on the right-hand side of the figure. The bottom axis represents the significance of each pathway and corresponds to the orange line on each figure, and pathways are order from most significant to least from top to bottom.

Figure 7

Ingenuity Pathway Analysis (IPA) pathway of stress-activated protein kinases/Jun N-terminal kinase (SAP/JNK) signaling overlayed with gene expression data from extraction and mesenchymal stromal cell (MSC) treatment samples. Significance cutoff of −log10P > 1.3, and data shown as log2 fold change expression. Red represents increased measurement of that gene, while teal indicates decreased measurement. Orange and blue overlays represent predicted activation or inhibition, respectively. (Top) Comparison of differentially expressed genes from patients that responded to extraction treatment versus those who had no successful therapeutic response to extractions. (Bottom) Comparison of differentially expressed genes from non-responder extractions patients that underwent MSC therapy versus patients that underwent only extraction therapy.