Gastrointestinal peptides in children before and after hematopoietic stem cell transplantation

Impaired intestinal function is a common complication of hematopoietic stem cell transplantation (HSCT). Damage to the gastrointestinal (GI) mucosa in patients undergoing HSCT is a serious but still poorly understood complication. Toxicity of HSCT conditioning regimens and graft-versus-host disease (GvHD) result in a 5-fold increase of the risk of significant GI complications compared with other cancer survivors [1, 2]. Chemotherapy and total body irradiation (TBI) can damage GI mucosa and cause diffuse inflammation of GI tract. This leads to disruption of integrity of GI mucosa with subsequent transfer of bacterial lipopolysaccharides and other danger/pathogen-associated molecular patterns (DAMPs/PAMPs) into the circulation [3]. The intestine is also known as the largest endocrine organ in the body. It strongly influences other organs, including the brain via the gut-brain axis [4]. The majority of GI regulatory peptides are secreted by strictly defined sections of the intestine [5]. Ghrelin is produced mainly in the stomach by P/D1 cells, cholecystokinin (CCK) is secreted mainly by the I cells of the upper small intestine, while glucagone like peptide-1 (GLP-1) is produced by the endocrine L cells in the lower intestine [6‐11]. Fibroblast growth factor-21 (FGF21) is secreted by several organs, including the liver, pancreas, and adipose tissue [12]. The intensity of GI dysfunction can be assessed using mucositis grading and parenteral nutrition requirements, but these tools cannot identify the most severely affected parts of the GI tract [13]. Endoscopy is rarely performed in the early post-HSCT phase due to the high risk of severe complications. In addition, the test load with nutrients is unreliable in this phase. Due to the differences in the anatomic distribution of intestinal endocrine cells, studies of alterations in GI peptide concentrations might help to localize the affected sections of the gut and assess the severity of inflammation. Thus, there is a need to identify simple and noninvasive tests that can assess the location and severity of gut damage. Additional comparison of marker concentrations before and several months after HSCT can explain the mechanisms of destruction and restoration of the GI tract [14‐16]. The aim of this study was to determine and analyze the selected GI peptides secreted on different levels of the gut in patients before and after HSCT.

When comparing the pre-HSCT and post-HSCT groups and the control group (Table 5), we noted a significantly lower BF mass and BF% measured using bioimpedance (6.46/12.0; 6.65/12.0, p < 0.05). The comparative analysis of the pre-HSCT group and the post-HSCT group showed no significant differences in anthropometric parameters.

Conditioning regimens are highly toxic to GI mucosal cells. The damage to the GI tract as well as other organs causes adverse effects, like nausea, vomiting, or diarrhea [21]. The effect of the treatment (chemotherapy and irradiation) of primary disease and effect of HSCT procedure cannot be easily distinguished. After the treatment of primary disease adverse effects are also observed. In our study 9 of 27 examined patients were without any previous treatment (Table 1). Before HSCT procedure significant difference was noted in CCK concentration in non- neoplastic disease group compare to neoplastic (median of 2.02 vs 1.07 ng/ml, p = 0.003). Same comparison 6 months after HSCT has shown significant difference in FGF-21 concentration in non-neoplastic disease group compare to neoplastic (median of 48.1 vs 114 pg/ml, p = 0.044, Additional File 2). Clinical symptoms of GI tract damage are well described, but there are no precise markers of advanced intestinal involvement and/or recovery. Endoscopic evaluation and intestinal biopsy are not recommended in patients with acute disease due the high risk of bleeding from seriously damaged mucosa and perforation. Therefore, there is a need to define blood biomarker that would correlate with location and severity of mucositis. Recently, serum citrulline (a non-essential amino acid) was proposed as a biomarker of small intestinal enterocyte mass and function [22, 23]. Citrulline indicates damage to the small intestine but is not specific to the intestinal enterocytes, because it is also produced in hepatocytes [24]. Therefore, we looked for other possible markers of GI mucositis dedicated to various levels of the gut. We studied concentrations of ghrelin produced in the stomach, CCK produced in the jejunum, GLP-1 produced in the ileum, and FGF-21 produced in the liver, pancreas, and white and brown adipose tissue [25, 26]. Cells of immune system are directly involved in acute graft versus host disease and they are infiltrating GI tract abundantly, therefore we hypothesized that this could be a cellular source, taking also into the account that gastrointestinal tract is one of the largest organs rich in lymphatic and vascular tissue itself. The expression of GI peptides has been investigated previously, not only in the gastrointestinal tract, but also in other tissue. For example ghrelin mRNA is naturally expressed in intestinal tissue but also in lymphocytes, neutrophils and lymphoid tissue [27]. Cholecytokinin was considered in some studies as a potential marker for Ewing Sarcoma in children [28]. GLPR-1 receptor is expressed on various immune cells and shows anti-inflammatory effects - decreasing proliferation of T- cells, increasing number of T regulatory cells [29]. To our knowledge only FGF-21 was underinvestigated in this matter. From the fact that expression of this peptides was significant in cells circulating in peripheral blood we can draw a conclusion that there is physiological relevance. There is little information in the literature on examined peptides in disease state, especially in metabolically unstable patients.

The amount of body fat did not influence peptide secretion, as the HSCT subgroups did not differ in terms of anthropometric parameters. Our study showed that 6 months after conditioning there was a significant increase in the secretion of ghrelin, CCK, and GLP-1. Plasma concentrations of these peptides were lower in the pre-HSCT group than the post-HSCT (convalescent) group and the control group. Kuruca et al. showed that irradiation during the treatment of intestinal cancers was associated with a decrease in ghrelin concentrations [30]. The low concentrations of ghrelin persisted 3 months after irradiation. Statistical analysis of our data revealed a considerable trend towards significance (p = 0.08) for the decreased ghrelin concentrations in patients with mucositis. Moreover, we found that 6 months after irradiation patients had higher levels of ghrelin compared to the values before conditioning. This suggests recovery of ghrelin secretion. This is favorable because ghrelin reduces intestinal injury and mortality after irradiation in animal models [31]. Interestingly, ghrelin levels were increased in patients with liver aGvHD compared with those with cutaneous or intestinal aGvHD (p = 0.02). This suggests dysregulation of gastric peptide secretion caused by liver damage.

CCK has anti-inflammatory properties and reduces cell apoptosis [32, 33]. We found higher concentrations of CCK after HSCT suggesting regeneration of the upper small intestine. The median concentration of GLP-1 was significantly decreased in patients with aGvHD symptoms. Moreover, GLP-1 levels negatively correlated with severity of aGvHD. In addition, GLP-1 concentrations returned to baseline (the values seen in healthy subjects) 6 months after conditioning. This suggests full recovery of the ileum. Logistic regression model indicates that GLP-1 concentration could be a potential biomarker for progression of aGvHD.

Increased concentrations of FGF-21 before conditioning suggest that hepatic injury may result from prolonged chemotherapy administered before HSCT. Animal models show that liver damage induces FGF-21 expression [34]. Conditioning adds to an additional liver injury. FGF-21is recognized as a stress response hepatokine that reduces hepatic damage through increased glucose uptake by adipose tissue. Normalization of FGF-21 concentrations 6 months after HSCT suggests complete recovery of hepatic function after transplantation. The FGF-21 gene expression data confirm the findings from biochemical analysis. Although we found statistically significant differences in peptide concentrations and gene expression model, the limitation of the current study is small sample size. On the other hand, examined group is unique. The presented results seem promising for establishing new diagnostic tools and provide the background for further investigation.

Conditioning before HSCT and GvHD result in a widespread damage to the GI tract. Our data reveal that the stomach, jejunum, ileum, and liver are affected by chemo- and radiotherapy. Ghrelin may be a biomarker for liver aGvHD, and GLP-1 seems to be a potential biomarker for the progression of aGvHD. The increases in the concentrations of the regulatory peptides secreted in all parts of the GI tract suggest intensive regeneration of the mucosa. These alterations seem to be beneficial. The peptide measurements allow us to monitor intestinal damage and regeneration. Our study also showed that dysregulation of peptide secretion in some segments of the intestine are long-lasting, as 6 months after HSCT increased ghrelin secretion in the stomach, as well as CCK secretion in the jejunum, did not return to the values seen in the control group. The gene expression data are consistent with the biochemical data.