Histological, Immunological, and Genetic Analysis of Feline Chronic Gingivostomatitis

Abstract

Feline chronic gingivostomatitis (FCGS) is an immune-mediated inflammatory condition affecting the oral mucosa that results in substantial pain and suffering. The goal of this study was to complete an in-depth immunohistochemistry analysis of affected FCGS mucosa, to perform and compare immune cell phenotypes in the blood of FCGS and healthy controls cats, and to determine a transcriptomic profile of the affected and normal oral mucosa of FCGS cats. We hypothesized that cats with FCGS would have circulating activated CD8+ T cells and that tissues would be infiltrated with activated B and T cells with a highly proinflammatory transcriptome. We found that oral mucosal tissues from cats with FCGS have high tissue infiltration of B cells and that T cells include both CD4+ and CD8+ lymphocytes. Cells positive for CD25 (IL2 receptor, indicative of lymphocyte activation) and FOXP3 (indicative of regulatory T cells) were scattered throughout the mucosa. Compared to healthy individuals, cats with FCGS had high circulating CD8+ effector memory cells with a concurrent decrease in central memory cells and evidence of circulating activated CD8+ T cells (CD25+, CD62L-). Gene expression in the affected tissues was enriched for genes associated with T-cell signaling, cell adhesion molecules, leukocyte migration, inflammatory signaling pathways, extracellular matrix-receptor interactions, cytokine-cytokine receptor interactions, and natural killer cell-mediated cytotoxicity, among others. These data are essential to understand disease pathogenesis, to inform mechanism of action studies for future and current therapies, and to help select prognostic biomarkers and potency assays for stem cell treatment of FCGS.

Keywords: chronic feline stomatitis; feline oral mucosal disease; immune-mediated oral mucosal inflammation; immunohistochemistry of feline stomatitis; immunophenotyping of FCGS; transcriptome of chronic gingivostomatitis.

Figure 1

Flow cytometry gating strategy for immunophenotyping of immune cells in the systemic circulation of FCGS (n = 12) patients along with healthy (n = 6) controls. General lymphocyte population was identified first using forward and side scatter. Next, CD8 immunofluorescent cells were selected out of the lymphocyte gate. Within CD8 gate, percentages of effector memory (CD8+CD45-CD62L−) and central memory (CD8+CD45-CD62L+) cells were interrogated. Similarly, within the CD8 positive population, percentages of activated CD8 cells were quantified (CD8+CD25+CD62L−). CD45R antibody utilized here (also known as CD45RABC) recognizes three (A, B, and C) exons of the CD45 protein. These exons are alternatively spliced to generate up to eight different protein products featuring combinations of zero, one, two, or all three exons. CD45 isoforms show cell-type and differentiation-stage specific expression, a pattern which is quite well-conserved in mammals. These isoforms are often used as markers that identify and distinguish between different types of immune cells. Naive T lymphocytes are typically positive for CD45RA, which includes only the A protein region. Activated and memory T lymphocytes express CD45RO, the shortest CD45 isoform, which lacks all three of the A, B, and C regions. This shortest isoform facilitates T cell activation (15).

Figure 2

Low (100x) and high (400x) magnification images of hematoxylin and eosin and immunohistochemical sections from two different FCGS patients. The rectangle on low magnification image indicates the area captured on high magnification. Note the abundance of plasma cells (thick arrow) and occasional mott cells (ellipse) in the sections of feline patients relative to a fewer lymphocytes (thin arrow) and neutrophils (arrow head). Mott cell is a plasma cell which cytoplasm is expanded by accumulation of immunoglobulins. Scale bar = 100 μm on low (100x) magnification captures and 20 μm on high (400x) magnification captures.

Figure 3

Quantitative assessment of selected immunophenotypes in the circulation of FCGS (n = 12) patients compared to healthy controls (n = 6), respectively. Significant differences are highlighted by a line with and asterisk. The Y axes reflect the percentage of immunoreactive cells relative to the total lymphocyte population in the sample. (A) Percentages of central and effector CD8+ cells in the systemic circulation in healthy and FCGS patients. Percentage of the effector memory (CD8+CD45-CD62L−) lymphocytes is significantly higher in FCGS patients than in healthy controls (p < 0.05). Percentages of central memory (CD8+CD45-CD62L+) lymphocytes are significantly lower in FCGS patients than in healthy controls (p < 0.05). (B) Percentages of activated CD8+ cells are significantly higher in the circulation of FCGS patients than in healthy controls (p < 0.05). (C) Percentages of activated CD4+ cells are trending higher than in healthy controls. (D,E) Percentages of CD25+ FOXP3+ CD8+ and CD25+ FOXP3+ CD4+ lymphocytes, respectively, are trending higher in the FCGS patients.

Figure 4

(A) Cluster analysis of differentially regulated genes in healthy and diseased tissue from FCGS cats. Note that diseased tissues are clustering differently from healthy. (B) Principal component analysis of genes in healthy and diseased cat tissues.