Fecal and Serum Calprotectin Concentrations in Cats With Chronic Enteropathies Before and During Treatment

Abstract

Chronic enteropathy (CE) in cats is a collective term used to describe a diverse group of gastrointestinal (GI) diseases in cats that result in chronic (longer than 3 weeks' duration) clinical signs of intestinal dysfunction, such as decreased appetite or anorexia, diarrhea, vomiting, weight loss, or some combination of these signs. Chronic enteropathies have been categorized into chronic inflammatory enteropathy (CIE) and small cell GI lymphoma (SCGL) [1-6]. Further subclassification of CIE is based on response to treatment and is divided into immunosuppressant-responsive enteropathy (IRE) and food-responsive enteropathy (FRE) [1]. Small cell GI lymphoma is the most common GI neoplasm in cats, and its prevalence has increased during the past two decades [7, 8]. Progression of CIE to SCGL over months to years has long been suspected, but this progression has not been definitively proven as of yet [9, 10]. Currently, establishment of a definitive diagnosis of CIE or SCGL is based on histopathology with immunohistochemistry and sometimes clonality testing [5, 9, 11-13].

The ability to predict response to treatment and monitor disease activity and treatment efficacy with non-invasive tools is highly desirable. In 2001, the Biomarker Definitions Working Group defined a biomarker as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathologic processes, or pharmacologic response to a therapeutic intervention [14]. In inflammatory conditions, proteins, such as calprotectin, are commonly released by inflammatory cells or in response to tissue dysfunction, and some of them may serve as biomarkers [15, 16].

In humans with Crohn's disease, evaluation and monitoring of response to treatment by use of a tight control algorithm that includes both clinical signs and inflammatory biomarkers increases the possibility of disease remission compared with only using clinical signs for treatment monitoring [17]. In cats with CE, noninvasive biomarkers for treatment monitoring have not been systematically evaluated [16].

Calprotectin is a protein that belongs to the damage-associated molecular pattern (DAMP) molecules of the innate immune response. Although the development and validation of assays for the measurement of calprotectin concentrations in cat feces and serum have been reported, their utility as biomarkers in treatment monitoring in cats with CE has only been poorly evaluated to date [18]. In a recent study, fecal calprotectin was evaluated in cats with CIE and SCGL [19], and in a more recent study, fecal calprotectin was evaluated before and after treatment in 17 cats with CE [20]. The results support the utility of fecal calprotectin as a surrogate biomarker to assess disease severity in cats with CE [20]. No studies have evaluated serum calprotectin concentration in cats with CE before and after treatment.

Our hypothesis was that fecal and serum calprotectin concentrations could be used as biomarkers for the diagnosis, prediction of response to treatment, and monitoring of treatment in cats with CE. Therefore, our aims were: (a) to measure serum and fecal calprotectin concentrations in cats with CE and compare them with those of healthy control cats; (b) to compare fecal and serum calprotectin concentrations between cats with CIE and those with SCGL; and (c) to evaluate changes in fecal and serum calprotectin concentrations before and during treatment in cats with CE.

A total of 43 cats with CE were included in the study. Of these, feces were available from 41 cats and serum from 40 cats at baseline. Based on clinical response, histopathology, and immunochemistry results, 25 cats were diagnosed with CIE (58%), (19 IRE [76%] and 6 FRE [24%]), and 18 with SCGL (42%; Table 1).

In our prospective study, we investigated whether serum or fecal calprotectin concentrations could serve as biomarkers to differentiate the different forms of CE and monitor treatment in cats with CE.

In our study, fecal calprotectin concentrations were significantly increased at diagnosis in cats with CE, compared with healthy control cats. These findings are similar to those reported in a recent study in cats, in which cats with IRE, FRE, and SCGL had higher fecal calprotectin concentrations than healthy cats [19]. Our results are also similar to those described in two previous studies in dogs in which fecal calprotectin concentrations of dogs with inflammatory bowel disease (IBD) [25] or dogs with chronic diarrhea [26] were compared to fecal calprotectin concentrations in healthy dogs. In humans with IBD, fecal calprotectin is a promising biomarker and has been widely studied [27, 28]. Fecal calprotectin in humans can distinguish GI signs caused by organic disorders (such as IBD) from those resulting from functional disorders (e.g., irritable bowel syndrome) with high sensitivity and specificity [29]. Our results support the hypothesis that fecal calprotectin is increased in cats with CE compared with healthy controls, but both seemed to decrease with treatment. Whether or not either fecal or serum calprotectin concentration could serve as biomarkers for objectively monitoring disease activity in cats with either form of CE remains to be determined. Serum and fecal calprotectin concentrations failed to differentiate CIE enteropathies from SCGL.

In cats with CE, response to treatment generally is based on evaluation of clinical activity. Because repeated endoscopies and histopathological evaluations are unrealistic in clinical practice, biological markers that are able to indirectly evaluate GI pathology are highly desirable. In our study, fecal calprotectin concentrations significantly decreased during treatment in cats with CE. These findings are similar to those reported in a recent study of 17 cats with CE, 12 with FRE, 4 with IRE, and 1 with unspecified disease, all diagnosed retrospectively based on treatment response [20]. In this study, fecal calprotectin concentrations were decreased in cats that responded to treatment [20]. Our results are similar to those described in dogs with IBD, where fecal calprotectin concentrations significantly decreased after treatment, and dogs showed clinical improvement [25]. In another study in dogs with CE, fecal calprotectin concentrations were correlated with disease clinical activity [30]. The same findings also have been described in human patients, where fecal calprotectin has been used as a non-invasive marker to monitor clinical disease severity and to differentiate active and quiescent Crohn's disease in adults and children [31, 32]. Based on the above, fecal calprotectin seems to be a promising biomarker for monitoring treatment in cats with CE. Further investigation is needed to determine if calprotectin concentration can be used for long-term follow-up of treatment.

In contrast to fecal calprotectin concentrations, serum calprotectin concentrations did not differ between cats with CE and healthy control cats at baseline and did not decrease in response to treatment in cats with CE. Our results are in contrast with a study in dogs with CIE, in which serum calprotectin concentrations were increased compared with controls [33], but similar to a more recent study in dogs, in which dogs with CIE had increased serum calprotectin concentrations that were not different from controls and did not decrease in response to treatment [25].

Our results are in contrast with those in humans with IBD (ulcerative colitis and Crohn's disease). In humans, serum calprotectin has gained more attention as a serum-based biomarker for IBD, because in humans it may be more convenient in routine practice to acquire blood samples than fecal samples. In one study in humans with IBD, serum calprotectin concentration was significantly increased compared with controls [34]. In another study in humans, serum calprotectin concentration strongly correlated with fecal calprotectin and was the strongest predictor of IBD diagnosis in comparison with other biomarkers such as C-reactive protein and albumin in 156 patients [35]. The same study concluded that a diagnostic and prognostic model with a combination of serum calprotectin and other blood-based biomarkers is capable of predicting the inflammatory burden in IBD patients, as well as predicting disease and its outcome [35]. However, in a subset of 50 patients with paired serum and fecal calprotectin concentrations, fecal calprotectin was a better discriminating marker for the differentiation of IBD from controls [36]. The reasons for this apparent difference in the utility of serum calprotectin between species are not known.

Calprotectin is an inflammatory marker that is mainly produced by myeloid cells, predominately neutrophils, monocytes, and macrophages. It is a calcium- and zinc-binding protein consisting of two small anionic proteins (S100A8 and S100A9), the S100A8/A9 protein complex, and it belongs to the damage-associated molecular pattern (DAMP) molecules of the innate immune response. In IBD in humans, where the main histopathological finding is mucosal neutrophilic inflammation, fecal calprotectin has proven to be a promising biomarker. Studies in humans have correlated calprotectin with the severity of histologic lesions [37]. In cats with CE, the main type of inflammation is lymphocytic. Regardless, fecal calprotectin concentration was increased compared with controls in our study and in a previous study [19]. Our results support previous evidence suggesting that lymphocytes may express or be associated with calprotectin expression under certain pathological or inflammatory conditions, thus making calprotectin, and especially fecal calprotectin, a promising biomarker in these cases [38-40].

Although it is found in various body fluids, calprotectin concentration in feces is six times higher than in blood in healthy humans [27, 41], which may be one of the reasons why serum calprotectin concentration does not appear to be a good biomarker for cats with CE, compared with fecal calprotectin. In humans, the increase of calprotectin concentration in fecal samples during inflammatory diseases is proportional to its increase in other biological fluids in the body, but in our study, the results were in contrast with findings in humans. In a study in dogs, serum calprotectin concentration was increased with corticosteroid administration [31]. In this study, calprotectin concentrations were increased in serum after treatment with prednisolone or a combination of prednisolone and metronidazole, despite clinical improvement [33]. No studies are available regarding the effect of corticosteroids on serum calprotectin concentrations in cats, but considering the results in dogs, serum calprotectin concentrations should be interpreted with caution in animals receiving corticosteroids. In our study, 21 of 27 cats with CE (77%) received prednisolone for treatment of CIE or SCGL, which could have affected our results. However, the fact that no difference was found in serum calprotectin concentration in cats with CE compared to healthy control cats at baseline makes this possibility less likely. In addition, because treatment with prednisolone is standard care in cats with CIE or SCGL, a practical marker for treatment monitoring should not be affected by corticosteroid treatment.

Our study failed to find any differences in serum and fecal calprotectin concentrations between cats with CIE and cats with SCGL. This finding might be because CIE and SCGL represent different stages of the same condition and that SCGL is a disease process of CIE, instead of being distinct diseases [1]. Another possible explanation could be that in both conditions (CIE and SCGL) the predominant inflammatory cells that infiltrate the mucosa are lymphocytes, and thus the secretion of calprotectin could be equally influenced in both conditions. In our study, only one cat had eosinophilic CIE, whereas all of the other cats with CIE had lymphoplasmacytic enteritis. Our results are similar to those of a recent study in cats with CE, in which no difference in fecal calprotectin concentrations was found between cats with CIE and cats with SCGL [19].

Our study had some limitations. One limitation is the relatively small number of cats with CIE and SCGL and the even smaller number of cats that completed the 3-month follow-up period. This factor potentially could have led to type 2 statistical error. However, ours was a prospective study, with relatively strict inclusion criteria and a 3-month follow-up period, and therefore it was challenging to enroll larger numbers of animals. Another limitation of our study is that the lower detection limit of the assay used was relatively high. Regardless, fecal calprotectin concentrations were significantly higher in cats with CE compared with controls and lower in cats with CE after treatment compared with baseline.

Another potential limitation of our study was that cats enrolled in the control group were not age-matched with the cats in the CE group. This factor might have affected our results, but it is currently unknown whether age affects calprotectin concentrations in serum or feces in cats. A study in healthy humans reported that fecal calprotectin concentrations increase with age in adults [42]. The age difference between CE cats and healthy cats could have influenced our results regarding fecal calprotectin concentrations. However, even in humans, no age-specific reference intervals are available, and fecal calprotectin is considered a very useful noninvasive biomarker in adult humans at any age [32, 42]. Moreover, in our study, in cats with CE, fecal calprotectin concentrations decreased during treatment, suggesting that calprotectin concentrations were influenced by disease activity and not simply by the age of the cats.

Finally, several of the cats in our study did not consume the hydrolyzed protein diet (Anallergenic, Royal Canin). This problem is commonly encountered in clinical practice when attempting to change the diet of cats that are hyporexic. Although making diet transitions very gradually and trying different diets may help with diet acceptance, many of the cats enrolled in our study were very ill, and taking more time for dietary trials could have been detrimental to those cats.

In conclusion, fecal calprotectin concentration could be useful as a biomarker for the diagnosis and treatment monitoring in cats with CE because it was increased in cats with CE compared with healthy controls and decreased during treatment. Serum calprotectin concentrations failed to differentiate cats with CE from healthy control cats and were found to not be helpful for treatment monitoring in cats with CE. Neither fecal nor serum calprotectin concentrations could differentiate cats with CIE from cats with SCGL. Additional studies are needed to investigate the potential use of fecal calprotectin concentrations in cats with CE as a marker for response to treatment.

Authors declare no off-label use of antimicrobials.

Study protocol approved by the Animal Ethics Committee of the University of Thessaly, Greece (AUP number:115/6.10.2020). Authors declare human ethics approval was not needed.

Jonathan A. Lidbury, Jan S. Suchodolski and Joerg M. Steiner are employes at Gastroenterology (GI) Laboratory Texas A&M University that offers laboratory testing including calprotectin on a fee for service basis. Shelley Newman is a consultant as an anatomic pathologist at GI Laboratory Texas A&M University that offers histopathology and immunohistochemistry examination on a fee for service basis. All authors except Dimitra A. Karra were blinded on each cat's history and clinical signs, and each cats group.