Determination and Evaluation of Mucosal Matrix Metalloproteinase -2 and -9, S100A12 and Myeloperoxidase in the Intestine of Dogs with Chronic Enteropathies and Healthy Beagles

Abstract

Chronic enteropathy (CE) in dogs refers to a group of inflammatory conditions of the intestinal tract with unknown etiology. However, the occurrence of an aberrant immune response to antigens derived from endogenous microbiota is likely to play an important role in the pathogenesis of canine CE. Thus, finding inflammatory markers that reflect disease severity would be clinically useful. Matrix metalloproteinase (MMP) -2 and -9 degrade extracellular matrix under both physiological and pathological conditions. Mucosal MMP-2 and -9 activities have been reported to be upregulated in the intestine of humans with inflammatory bowel disease (IBD) and also in animal models of human IBD. However, their identification in the intestinal mucosa of healthy Beagles and their involvement in the pathogenesis of canine CE are unknown. Elevated intestinal mucosal levels of S100A12 and myeloperoxidase, as markers of gut inflammation, have been reported in human patients with IBD. Also, increased concentrations of S100A12 in feces and serum have been reported in dogs with CE. However, intestinal mucosal S100A12 concentrations and MPO activities have not previously been investigated in dogs with CE and in healthy Beagles. The aims of this project were to validate laboratory methods for the determination of MMP-2 and -9, S100A12, and MPO in the intestinal mucosa samples of healthy Beagles, to measure their mucosal levels in dogs with CE, and to compare these results to healthy Beagles. The project also sought to determine the relationship between the levels of the four markers and the canine clinical IBD activity index (CIBDAI), histopathologic findings, clinical outcome, and serum albumin concentrations in dogs with CE. Intestinal mucosal biopsies were collected from 40 dogs with CE (duodenum [n = 35], ileum [n = 12], colon [n = 15], and cecum [n = 6]). Stored intestinal tissue samples from 18 healthy Beagle dogs served as controls (duodenum, ileum, colon [n = 18, each], and cecum [n = 6]). MMP-2 and -9 activities, S100A12 concentrations, and MPO activities were measured using gelatin zymography, ELISA, and spectrophotometric methods, respectively. The methods for determination of MMP-2 and -9, S100A12, and MPO were successfully validated in the intestinal mucosa samples of healthy Beagles. Compared to healthy Beagles, mucosal pro- and active MMP-2 positive samples were significantly higher in duodenum, ileum, and colon of dogs with CE, while mucosal pro-MMP-9 positive samples were significantly higher in the duodenum and colon. None of the intestinal mucosal samples in healthy Beagles showed gelatinolytic activity corresponding to the control bands of active MMP-2 and -9. In dogs with CE, however, mucosal active MMP-9 activities showed a significant positive association with the severity of neutrophils infiltration in duodenum, eosinophils in the cecum. Ileum activities were positively associated with the CIBDAI score. Compared with healthy controls, both mucosal S100A12 concentrations and MPO activities were increased in the duodenum and colon of dogs with CE, while the mucosal MPO activity was also increased in the ileum and cecum. In dogs with CE, mucosal S100A12 concentrations had an association with the severity of epithelial injury and total histopathological injury in the colon; and with the presence of neutrophils and macrophages in the duodenal mucosa or with hypoalbuminemia. Moreover, mucosal MPO activity had a relationship with the severity of epithelial injury and total histopathological injury in the duodenum of dogs with CE. Overall, the results of this project demonstrate an upregulation of mucosal pro- and active MMP-2 and pro-MMP-9, S100A12, and MPO in the intestine of dogs with CE compared to healthy Beagles and it seems that they are involved in the pathogenesis of canine chronic enteropathies. These results provide supporting evidence to more deeply assess the clinical utility of MMP-2 and -9, S100A12, and MPO as possible diagnostic biomarkers in dogs with CE