Despite its clinical relevance, the etiopathogenesis of FCGS remains unresolved. In this study, we performed bulk RNA-seq molecular profiling of affected tissues derived from a cohort of client-owned cats with FCGS and used tissues from cats with PER and unaffected tissues as comparative sets, to identify candidate genes and pathways that can help guide future exploration of novel clinical solutions for FCGS. We complemented transcriptomic findings with IHC and ISH assays to better understand the biological significance of the data and performed RNA-seq validation of selected differentially expressed genes using qPCR assays to demonstrate technical reproducibility.
Overall, the transcriptional profiles of FCGS tissues were largely dominated by immune- and inflammation-related genes and signaling pathways including NFKB, JAK/STAT and IFN type I and II signaling, indicating that FCGS is an inflammatory disease potentially related to antigenic stimulation.6,30,31 As was expected, cluster analysis showed that all samples segregated according to group assignment, indicating that FCGS and PER have distinct molecular phenotypes despite their inflammatory nature and observed transcriptional commonalities. Additionally, there were general similarities in the expression patterns of FCGS when compared to those previously reported in three cats that were refractory to surgical therapy,7 suggesting that the molecular mechanisms underlying FCGS are maintained during the natural course of the disease as long as local tissue inflammation persists and regardless of historical surgical interventions.
Notably, most inflammation-related pathways found to be enriched in FCGS tissues appeared to be heavily influenced by IL6. This is a relevant finding given that dysregulated expression of IL-6 (encoded by IL6) could underlie some of the local and systemic events known to occur in cats with FCGS. In general, IL-6 is a finely regulated pleiotropic cytokine that signals via the JAK/STAT pathway. IL-6 can be produced by multiple cell types including mesenchymal, endothelial, epithelial, and immune cells.32–34 Under normal circumstances, IL-6 blood levels are hardly detectable but can rapidly increase upon stress, tissue injury or antigenic stimulation. Once in circulation, IL-6 activates hepatocytes to produce acute-phase proteins and modulates innate and adaptive immunity to promote healing and help eliminate infections.35,36
Overexpression of IL-6 has been shown to promote chronic inflammation and increase susceptibility to viral infections.32,34,36,37 It is possible that excessive production of IL-6 in cats with FCGS explains the chronic FCV and PFFV infection and elevated globulin and acute-phase protein levels in blood typically found and may be directly involved in perpetuation of oral mucosal inflammation and ulceration. Interestingly, recombinant feline IFN-omega has been shown to reduce IL6 expression and IL-6 levels,38 which could at least partially explain the clinical response reported in some cats with FCGS. 14,15
Another interesting finding in FCGS tissues in agreement with a previous report7 was overexpression of IL17A and enrichment of its corresponding signaling pathway. Importantly, IL-17 (encoded by IL17A) is considered the signature cytokine of Th17 cells.39 Although Th17 cells play an important protective role against microbial and viral pathogens, they are also implicated in autoinflammatory and autoimmune pathology.39–42 IL-6 promotes polarization of naïve CD4 + T cells towards a Th17 phenotype,32,34,35 thus the increased IL17A activity observed in FCGS tissues may be due to IL-6 signaling. However, the biological impact of IL17A overexpression in FGCS, and whether activation of the IL-17 pathway plays a pathogenic or protective role in affected cats is unknown but warrants further investigation.
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nciding with previous reports,18–22 this study showed an association between FCGS and FCV and PFFV infection. However, the very low numbers of RNA-seq reads mapping to corresponding viral genomes in affected tissues, and the fact that IHC and ISH failed to detect FCV antigen and genome signals, respectively, suggest that at least FCV does not replicate in areas of mucosal ulceration, or that it is present in such low numbers that it is undetectable using the IHC and ISH techniques used in this study. Regardless, the consistent overexpression of IFNG, which encodes IFN-gamma, and enrichment of IFN type I and II pathways suggest an immune response to viral stimulation.43,44 Given that IFNG induces expression of CXCL9, CXCL10, and other chemotactic molecules,45 IFN-gamma signaling is likely to play an important role in immune cell traffic in affected oral mucosal tissues, including attraction of T and B cells. Taken together, these observations implicate viral infection as a possibly required or aggravating element in the pathogenesis of FCGS.
Unexpectedly, the gene expression signatures observed in FCGS tissues revealed a predominantly myeloid lineage identity, which suggests that despite the heavy presence of lymphoid infiltrates, the innate system is transcriptionally more active and is thus likely to be an important driver of the dysregulated immune response. This finding conflicts with the hypothesis that FCGS is primarily a T cell driven disease.7 As macrophages and monocytes are the main producers of IL-6,33,34 these new findings support a hypothesis that IL-6 dysregulation underlies some of the pathological events.
The caudal oral mucosa of cats is a relatively thin, non-masticatory mucosal barrier that is exposed to mechanical trauma during mastication, and to pathogenic toxins (e.g., LPS) derived from the oral microbiota.46 Given this environment, it is possible that the caudal oral mucosa is particularly susceptible to immune stimulation, and that in some cases such stimuli result in chronic inflammation. This could explain why extraction of premolar and molar teeth, which typically come in direct contact with caudal and buccal oral mucosal surfaces, results in remission in some animals.9,10 Alternatively or in addition, based on the transcriptional signatures observed in this study and the epidemiological patterns previously reported,17 it is possible that viruses and environmental stressors are involved in the pathogenesis of FCGS; both are likely to contribute to upregulation of IL-6.46 Such a scenario raises the question of why, when subject to similar conditions (i.e., multi-cat environments), only some individuals develop FCGS. One possibility is that some cats are genetically predisposed.
From a translational medicine perspective, the results of this study provide rational targets for clinical diagnostics and therapy in cats with FCGS. For example, IL-6 could be investigated as a potential diagnostic and prognostic marker that could be used to stage and grade FCGS, determine the best treatment modalities or predict therapeutic response. Similarly, given the precedent of successful targeted inhibition of IL-6 signaling in people diagnosed with certain chronic inflammatory conditions and different forms of cancer,32,33,35,47 similar approaches could be tested in cats with FCGS. It should be noted that the expression signatures reported here capture global trends, but further studies are required to determine their biological impact. Importantly, bulk RNA-seq techniques using clinical samples do not allow single-cell inferences or insights of how different cell types interact with each other. Therefore, the observations made here represent hypotheses that will require testing using targeted, single-cell and/or spatially resolved approaches, and ideally in vitro and in vivo validation experiments.
We conclude that the transcriptomic profile of oral mucosal tissues in cats with FCGS is enriched with immune- and inflammation-related genes and pathways that appear to be largely influenced by IL6, and include NFKB, JAK/STAT, IL-17 and IFN type I and II signaling.