Joep Grootjans

Physiology and pathophysiology of splanchnic hypoperfusion and intestinal injury during exercise: strategies for evaluation and prevention

Physical exercise places high demands on the adaptive capacity of the human body. Strenuous physical performance increases the blood supply to active muscles, cardiopulmonary system, and skin to meet the altered demands for oxygen and nutrients. The redistribution of blood flow, necessary for such an increased blood supply to the periphery, significantly reduces blood flow to the gut, leading to hypoperfusion and gastrointestinal (GI) compromise. A compromised GI system can have a negative impact on exercise performance and subsequent postexercise recovery due to abdominal distress and impairments in the uptake of fluid, electrolytes, and nutrients. In addition, strenuous physical exercise leads to loss of epithelial integrity, which may give rise to increased intestinal permeability with bacterial translocation and inflammation. Ultimately, these effects can deteriorate postexercise recovery and disrupt exercise training routine. This review provides an overview on the recent advances in our understanding of GI physiology and pathophysiology in relation to strenuous exercise. Various approaches to determine the impact of exercise on the individual athletes GI tract are discussed. In addition, we elaborate on several promising components that could be exploited for preventive interventions.

Human Intestinal Ischemia-Reperfusion–Induced Inflammation Characterized : Experiences from a New Translational Model

Human intestinal ischemia-reperfusion (IR) is a frequent phenomenon carrying high morbidity and mortality. Although intestinal IR-induced inflammation has been studied extensively in animal models, human intestinal IR induced inflammatory responses remain to be characterized. Using a newly developed human intestinal IR model, we show that human small intestinal ischemia results in massive leakage of intracellular components from ischemically damaged cells, as indicated by increased arteriovenous concentration differences of intestinal fatty acid binding protein and soluble cytokeratin 18. IR-induced intestinal barrier integrity loss resulted in free exposure of the gut basal membrane (collagen IV staining) to intraluminal contents, which was accompanied by increased arteriovenous concentration differences of endotoxin. Western blot for complement activation product C3c and immunohistochemistry for activated C3 revealed complement activation after IR. In addition, intestinal IR resulted in enhanced tissue mRNA expression of IL-6, IL-8, and TNF-alpha, which was accompanied by IL-6 and IL-8 release into the circulation. Expression of intercellular adhesion molecule-1 was markedly increased during reperfusion, facilitating influx of neutrophils into IR-damaged villus tips. In conclusion, this study for the first time shows the sequelae of human intestinal IR-induced inflammation, which is characterized by complement activation, production and release of cytokines into the circulation, endothelial activation, and neutrophil influx into IR-damaged tissue.

Non-invasive assessment of barrier integrity and function of the human gut

Over the past decades evidence has been accumulating that intestinal barrier integrity loss plays a key role in the development and perpetuation of a variety of disease states including inflammatory bowel disease and celiac disease, and is a key player in the onset of sepsis and multiple organ failure in situations of intestinal hypoperfusion, including trauma and major surgery. Insight into gut barrier integrity and function loss is important to improve our knowledge on disease etiology and pathophysiology and contributes to early detection and/or secondary prevention of disease. A variety of tests have been developed to assess intestinal epithelial cell damage, intestinal tight junction status and consequences of intestinal barrier integrity loss, i.e. increased intestinal permeability. This review discusses currently available methods for evaluating loss of human intestinal barrier integrity and function.

Level of Activation of the Unfolded Protein Response Correlates With Paneth Cell Apoptosis in Human Small Intestine Exposed to Ischemia/Reperfusion

BACKGROUND & AIMS In the intestine, Paneth cells participate in the innate immune response. Their highly secretory function makes them susceptible to environmental conditions that cause endoplasmic reticulum (ER) stress. We investigated whether intestinal ischemia/reperfusion (I/R) induces ER stress, thereby activating the unfolded protein response (UPR), and whether excessive UPR activation affects Paneth cells. In addition, we investigated the consequences of Paneth cell compromise during physical barrier damage. METHODS Jejunal I/R was studied using a human experimental model (n = 30 patients). Activation of the UPR was assessed using immunofluorescence for binding protein and quantitative polymerase chain reaction analyses for C/EBP homologous protein (CHOP), growth arrest and DNA-damage inducible protein 34 (GADD34), and X-box binding protein 1 (XBP1) splicing. Paneth cell apoptosis was assessed by double staining for lysozyme and M30. Male Sprague-Dawley rats underwent either intestinal I/R to investigate UPR activation and Paneth cell apoptosis, or hemorrhagic shock with or without intraperitoneal administration of dithizone, to study consequences of Paneth cell compromise during physical intestinal damage. In these animals, bacterial translocation and circulating tumor necrosis factor-α and interleukin-6 levels were assessed. RESULTS In jejunum samples from humans and rats, I/R activated the UPR and resulted in Paneth cell apoptosis. Apoptotic Paneth cells showed signs of ER stress, and Paneth cell apoptosis correlated with the extent of ER stress. Apoptotic Paneth cells were shed into the crypt lumen, significantly lowering their numbers. In rats, Paneth cell compromise increased bacterial translocation and inflammation during physical intestinal damage. CONCLUSIONS ER stress-induced Paneth cell apoptosis contributes to intestinal I/R-induced bacterial translocation and systemic inflammation.

A pilot study on the noninvasive evaluation of intestinal damage in celiac disease using I-FABP and L-FABP.

Background and Goals In the clinical management of celiac disease, new noninvasive tools for evaluation of intestinal damage are needed for diagnosis and for follow-up of diet effects. Fatty acid binding proteins (FABP) are potentially useful for this purpose as these are small cytosolic proteins present in enterocytes and sensitive markers for intestinal mucosal damage. First, the distribution and microscopic localization of FABP in the healthy human intestine was examined. Second, levels of circulating FABP were measured in patients with celiac disease before and after introducing a gluten-free diet (GFD) and in healthy controls. Study The distribution and microscopic localization of FABP in normal human intestinal tissue was assessed using surgical intestinal specimens of 39 patients. Circulating levels of intestinal (I)-FABP and liver (L)-FABP were determined in 26 healthy volunteers and 13 patients with biopsy proven celiac disease. Ten of these patients were reevaluated within 1 year after starting GFD. Results I-FABP and L-FABP are predominantly present in the small intestine, mainly the jejunum. Moreover, FABP are expressed in cells on the upper part of the villi, the initial site of destruction in celiac disease. Circulating levels of FABP are significantly elevated in untreated patients with biopsy proven celiac disease compared with healthy controls (I-FABP: 784.7 pg/mL vs. 172.7 pg/mL, P<0.001; L-FABP: 48.4 ng/mL vs. 10.4 ng/mL, P<0.001). In response to GFD, these concentrations normalize. Conclusions Results of this pilot study strongly suggest that FABP can be used as a noninvasive method for assessment of intestinal damage in celiac disease. Besides an additional role in the diagnosis of celiac disease, FABP potentially enable noninvasive monitoring of the GFD effects.

Ischaemia-induced mucus barrier loss and bacterial penetration are rapidly counteracted by increased goblet cell secretory activity in human and rat colon

Objective Colonic ischaemia is frequently observed in clinical practice. This study provides a novel insight into the pathophysiology of colon ischaemia/reperfusion (IR) using a newly developed human and rat experimental model. Design In 10 patients a small part of colon that had to be removed for surgical reasons was isolated and exposed to 60 min of ischaemia (60I) with/without different periods of reperfusion (30R and 60R). Tissue not exposed to IR served as control. In rats, colon was exposed to 60I, 60I/30R, 60I/120R or 60I/240R (n=7 per group). The tissue was snap-frozen or fixed in glutaraldehyde, formalin or methacarn fixative. Mucins were stained with Periodic Acid Schiff/Alcian Blue (PAS/AB) and MUC2/Dolichos biflorus agglutinin (DBA). Bacteria were studied using electron microscopy (EM) and fluorescent in situ hybridisation (FISH). Neutrophils were studied using myeloperoxidase staining. qPCR was performed for MUC2, interleukin (IL)-6, IL-1β and tumour necrosis factor α. Results In rats, PAS/AB and MUC2/DBA staining revealed mucus layer detachment at ischaemia which was accompanied by bacterial penetration (in EM and FISH). Human and rat studies showed that, simultaneously, goblet cell secretory activity increased. This was associated with expulsion of bacteria from the crypts and restoration of the mucus layer at 240 min of reperfusion. Inflammation was limited to minor influx of neutrophils and increased expression of proinflammatory cytokines during reperfusion. Conclusions Colonic ischaemia leads to disruption of the mucus layer facilitating bacterial penetration. This is rapidly counteracted by increased secretory activity of goblet cells, leading to expulsion of bacteria from the crypts as well as restoration of the mucus barrier.

Urine-based detection of intestinal tight junction loss.

Background Tight junction breakdown, with loss of the important sealing protein claudin-3, is an early event in the development of intestinal damage. Therefore, noninvasive analysis of intestinal tight junction status could be helpful in early detection of intestinal injury. Aim To investigate the usefulness of urinary claudin-3 as marker for intestinal tight junction loss. Methods A rat hemorrhagic shock model and a human setting of known intestinal damage, that is, patients with relapsed inflammatory bowel disease (IBD), were used to investigate intestinal tight junction status by immunohistochemical staining and urinary claudin-3 levels by western blot. Results In rats claudin-3 urine levels increased rapidly after histologically proven intestinal tight junction loss, with significantly elevated levels at 90 minutes after shock compared with sham-operated animals [mean±SEM: 611±101 intensity (INT), n=6 vs. 232±30 INT, n=6; P<0.05]. Moreover, in colonic biopsies of patients with IBD relapse claudin-3 staining was reduced compared with biopsies of patients with IBD without signs of disease. Concomitantly, significantly increased claudin-3 urine levels were found in these patients (502±67 INT, n=10) compared with patients with IBD in remission (219±17 INT, n=10, P<0.001) and healthy volunteers (225±38 INT, n=10, P<0.001). Conclusion Here we show for the first time in both an experimental and clinical setting a strong relation between intestinal tight junction loss and urinary claudin-3 levels. These findings suggest that measurement of urinary claudin-3 can be used as noninvasive marker for intestinal tight junction loss. This offers new opportunities for early diagnosis and follow-up of intestinal injury.

Reduced Paneth cell antimicrobial protein levels correlate with activation of the unfolded protein response in the gut of obese individuals

The intestinal microbiota is increasingly acknowledged to play a crucial role in the development of obesity. A shift in intestinal microbiota composition favouring the presence of Firmicutes over Bacteroidetes has been observed in obese subjects. A similar shift has been reported in mice with deficiency of active Paneth cell α‐defensins. We aimed at investigating changes in Paneth cell antimicrobial levels in the gut of obese subjects. Next, we studied activation of the unfolded protein response (UPR) as a possible mechanism involved in altered Paneth cell function. Paneth cell numbers were counted in jejunal sections of 15 severely obese (BMI > 35) and 15 normal weight subjects. Expression of Paneth cell antimicrobials human α‐defensin 5 (HD5) and lysozyme were investigated using immunohistochemistry, qPCR, and western blot. Activation of the UPR was assessed with western blot. Severely obese subjects showed decreased protein levels of both HD5 and lysozyme, while Paneth cell numbers were unchanged. Lysozyme protein levels correlated inversely with BMI. Increased expression of HD5 (DEFA5) and lysozyme (LYZ) transcripts in the intestine of obese subjects prompted us to investigate a possible translational block caused by UPR activation. Binding protein (BiP) and activating transcription factor 4 (ATF4) levels were increased, confirming activation of the UPR in the gut of obese subjects. Furthermore, levels of both proteins correlated with BMI. Involvement of the UPR in the lowered antimicrobial protein levels in obese subjects was strongly suggested by a negative correlation between BiP levels and lysozyme levels. Additionally, indications of ER stress were apparent in Paneth cells of obese subjects. Our findings provide the first evidence for altered Paneth cell function in obesity, which may have important implications for the obesity‐associated shift in microbiota composition. In addition, we show activation of the UPR in the intestine of obese subjects, which may underlie the observed Paneth cell compromise. Copyright

Starvation Compromises Paneth Cells

Lack of enteral feeding, with or without parenteral nutritional support, is associated with increased intestinal permeability and translocation of bacteria. Such translocation is thought to be important in the high morbidity and mortality rates of patients who receive nothing by mouth. Recently, Paneth cells, important constituents of innate intestinal immunity, were found to be crucial in host protection against invasion of both commensal and pathogenic bacteria. This study investigates the influence of food deprivation on Paneth cell function in a mouse starvation model. Quantitative PCR showed significant decreases in mRNA expression of typical Paneth cell antimicrobials, lysozyme, cryptdin, and RegIIIγ, in ileal tissue after 48 hours of food deprivation. Protein expression levels of lysozyme and RegIIIγ precursor were also significantly diminished, as shown by Western blot analysis and IHC. Late degenerative autophagolysosomes and aberrant Paneth cell granules in starved mice were evident by electron microscopy, Western blot analysis, and quantitative PCR. Furthermore, increased bacterial translocation to mesenteric lymph nodes coincided with Paneth cell abnormalities. The current study demonstrates the occurrence of Paneth cell abnormalities during enteral starvation. Such changes may contribute to loss of epithelial barrier function, causing the apparent bacterial translocation in enteral starvation.

Plasma Intestinal Fatty Acid-Binding Protein Levels Correlate With Morphologic Epithelial Intestinal Damage in a Human Translational Ischemia-reperfusion Model.

Background and Aim: Intestinal fatty acid–binding protein (I-FABP) is a useful marker in the detection of intestinal ischemia. However, more insight into the test characteristics of I-FABP release is needed. This study aimed to investigate the relationship between plasma I-FABP levels and the severity of ischemic mucosal injury, and define the clinical usefulness of systemic I-FABP following ischemia. Methods: In a human experimental model, 6 cm of the jejunum, to be removed for surgical reasons, was selectively exposed to either 15, 30, or 60 minutes of ischemia (I) followed by 30 and 120 minutes of reperfusion (R). Blood and tissue was sampled at all time points. Arteriovenous (V−A) concentration differences of I-FABP were measured. Tissue sections were stained with hematoxylin/eosin, and villus height was measured to score epithelial damage. Results: Histologic analysis showed only minor reversible intestinal damage following 15I and 30I; however, severe irreversible epithelial damage was observed in the jejunum exposed to 60I. I-FABP V−A differences paralleled the degree of tissue damage over time [7.79 (±1.8) ng/mL, 128.6 (±44.2) ng/mL, 463.3 (±139.8) ng/mL for 15I, 30I and 60I, respectively]. A good correlation was found between histologic epithelial damage and V−A I-FABP (r=−0.82, P<0.001). Interestingly, systemic I-FABP levels were significantly increased after 60I of this short small intestinal segment. Conclusions: This study demonstrates the relationship between the duration of ischemia and the extent of tissue damage, which is reflected by I-FABP V−A plasma levels. In addition, systemic I-FABP levels appear valuable in detecting irreversible intestinal ischemia-reperfusion damage.