Received: 23 June 2022 DOI: 10.1002/ueg2.12329 ORIGINAL ARTICLE - Accepted: 11 October 2022 Acute psychological stress increases paracellular permeability and modulates immune activity in rectal mucosa of healthy volunteers Linda Gerdin1,2,3 | Ana M. González‐Castro4 | Ann‐Charlott Ericson1,2 | Mats Persborn3 | Javier Santos5,6 | Susanna A. Walter1,2,7 | Åsa V. Keita1,2 | Maria Vicario4,6,8 | Johan D. Söderholm1,2 1 Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden 2 Department of Surgery, Linköping University, Linköping, Sweden 3 Surgical Clinic of Jönköping County, Jönköping, Sweden 4 Laboratory of Translational Mucosal Immunology, Digestive Diseases Research Unit, Vall d'Hebron Institut de Recerca, Hospital Universitari Vall d'Hebron & Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain 5 Laboratory of Neuro‐immuno‐gastroenterology, Digestive Diseases Research Unit, Vall d'Hebron Institut de Recerca, Hospital Universitari Vall d'Hebron & Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain 6 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain 7 Department of Gastroenterology, Linköping University, Linköping, Sweden 8 Department of Gastrointestinal Health, Nestlé Institute of Health Sciences, Nestlé Research, Société des Produits Nestlé S.A., Lausanne, Switzerland Correspondence Johan D. Söderholm, Department of Abstract Biomedical and Clinical Sciences, Linköping Background: Psychological stress and increased permeability are implicated as University, Linköping, Sweden. Email: johan.d.soderholm@liu.se contributing factors in the initiation and worsening of gastrointestinal diseases. A link between stress and intestinal permeability has been shown in animal models as Funding information well as in human small intestine, but stress effects on the human colorectal mucosal Fondo de Investigación Sanitaria and CIBERehd, Instituto de Salud Carlos III, barrier has not been reported. Subdirección General de Investigación Objective: To investigate the potential effects of acute psychological stress on Sanitaria, Ministerio de Economía y Competitividad, Grant/Award Numbers: PI17/ colorectal mucosal barrier function and to explore stress‐induced molecular events 0190, PI19/01643; Futurum, Region in the rectal mucosa under healthy conditions. Jönköping County, Grant/Award Numbers: 12406, 1460; Centro de Investigación Methods: Endoscopic biopsies were taken from the rectosigmoid region of healthy Biomédica en Red de Enfermedades Hepáticas volunteers, who had been subjected to dichotomous listening stress and after a y Digestivas, Grant/Award Number: CB06/04/ 0021; Vetenskapsrådet, Grant/Award control session, respectively. Paracellular and transcellular permeability were Numbers: 2014‐02537, 2017‐02475; assessed in modified Ussing chambers. RNA expression (microarray technology Forskningsrådet i Sydöstra Sverige, Grant/ Award Number: 37841 confirmed by quantitative real‐time polymerase chain reaction) and biological pathway analysis were used to investigate the local mucosal response to acute stress. Results: Dichotomous listening stress induced a subjective and objective stress response, and significantly increased paracellular but not transcellular permeability. - This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2022 The Authors. United European Gastroenterology Journal published by Wiley Periodicals LLC. on behalf of United European Gastroenterology. United European Gastroenterol J. 2023;11:31–41. wileyonlinelibrary.com/journal/ueg2 31 32 - UNITED EUROPEAN GASTROENTEROLOGY JOURNAL We also identified a stress‐induced reduction in RNA expression of genes related to immune cell activation and maturation (CR2, CD20, TCLA1, BANK1, CD22, FDCSP), signaling molecules of homing of immune cells to the gut (chemokines: CCL21, CXCL13, and CCL19, and receptors: CCR7, CXCR5), and innate immunity (DUOX2). Eight of the 10 top down‐regulated genes are directly involved in B cell activation, signaling and migration. The systemic stress response correlated positively with paracellular permeability and negatively with DUOX2 expression. Conclusion: Dichotomous listening stress increases paracellular permeability and modulates immune cell activity in the rectal mucosa. Further studies are warranted to identify the primary mechanisms of stress‐mediated reduction of mucosal defensive activity and barrier dysfunction, and their potential implications for gastrointestinal disorders. KEYWORDS intestinal permeability, microarray, mucosal immunity, psychological stress INTRODUCTION Key summary Psychological stress is recognized as a contributing factor in Current knowledge several gastrointestinal (GI) conditions, including irritable bowel � Stress and barrier dysfunction are contributing factors in syndrome (IBS) and inflammatory bowel disease (IBD). IBS patients gastrointestinal diseases. have more stressful life events1,2 and have altered visceral sensi- � There is a link between psychological stress and tivity in response to acute stress.3 Additionally, a recent large increased intestinal permeability, but this has not been Genome‐Wide Association Study confirmed that neuronal path- studied in the human colorectal mucosa. ways, potentially involving enteric nerves, play a dominant role in What are the new findings? the genetics of IBS.4 In ulcerative colitis, persistent life stress in- � Dichotomous listening stress increases paracellular creases the number of exacerbations, and stress resilience is pro- permeability in the colorectal mucosa of healthy tective in IBD.5,6 volunteers. Intestinal barrier dysfunction is also an established factor in the � Modifications of immune activity, mainly implicating B‐ pathophysiology of both IBD7–9 and IBS.10,11 In animal models, it cells, is involved in the initial mucosal response to mild has been established that stress and barrier dysfunction are linked: psychological stress. both acute and chronic psychological stress increase GI perme- ability in rats and predispose to inflammation by inducing IBS‐ and UC‐like colonic mucosal phenotypes in a mast cell‐dependent manner.12,13 In humans, acute stress increases small intestinal secretion14 and permeability via mast cells,15 but stress effects on The aim of this study was to investigate the effects of acute human colorectal mucosal function have not been reported. With psychological stress on human colorectal mucosal barrier function the differences in microanatomy, mucus composition, microbiome and gain insights into stress‐induced modulation of mucosal immu- and microbial load, enteric nerve innervation, etc., the potential for nity. Healthy volunteers were submitted to acute dichotomous dissimilarities between the physiological and pathophysiological ef- listening stress19 and a control session, and colorectal mucosal bi- fects of stress in small bowel and colorectal mucosa is obvious. It is opsies were obtained to assess the impact of stress on barrier also unknown how mucosal responses to acute and chronic function20 and gene expression. stressors are linked in humans, but it was shown that healthy women with high background stress had distorted small bowel epithelial responses to acute stress, a potential initial step in the MATERIALS AND METHODS 16 development of prolonged GI dysfunction and disease. Psycho- logical stress also perturbs intestinal immunity,17 affecting circu- Participants and clinical assessment lating lymphocytes18 and activating mucosal mast cells.15 However, early stress‐induced immune responses in human colorectal mucosa Healthy volunteers were recruited by public advertisement. Exclu- have not been fully elucidated. sion criteria were chronic GI disorders or symptoms, other organic GERDIN ET AL. - 33 diseases, first‐degree relatives with IBD, medication known to affect the task was challenging, that their results would be compared with GI permeability, and pregnancy. Use of NSAIDs was not allowed and other participants, and that if they scored more than 75% correct alcohol was discouraged, 2 weeks and 3 days, respectively, before the answers, they would get a reward. During the control session sub- procedures. jects listened to calm music from a selection as they pleased. The Regional Committee of Human Ethics in Linköping approved the study (Dnr M183‐08) and all subjects gave their written informed consent. Assessment of the stress response Psychological response: Perceived stress was assessed by each Study design participant every 15 min on a visual analog scale (VAS) from 0, no stress, to 100, maximum stress. See overview of study in Figure 1. Before each session, all partici- Biological response: Heart rate (HF) and blood pressure (BP) were pants completed validated questionnaires related to life event measured with OMRON M10‐IT (Omron) at baseline and every 5 min stress,21 current perceived stress the week preceding the experi- during the stress procedure. 22 23 ments and the individual's tendency for stress/anxiety. During In previous studies using laboratory psychological stress17,19 each of the control and the stress sessions, objective and subjective there have been non‐responders in whom the stressor did not elicit a stress variables were monitored to assess the response to acute stress response. We defined responders as those who had both stress. After each control and stress session, rectal biopsies were subjective and objective responses to the stress session. obtained to evaluate intestinal mucosal response to acute stress by assessing epithelial barrier function (transepithelial electrical resis- tance [TER], paracellular and transcellular permeability) and by Biological samples quantifying changes in gene expression profile in colonic biopsies. All samples were codified and blindly analyzed. Immediately following the stress/control session, rectal biopsies were collected via flexible sigmoidoscopy. From every session 6–8 biopsies were directly put in ice‐cold oxygenated Krebs buffer Dichotomous listening stress (115 mM NaCl, 1.25 mM CaCl2, 1.2 mM MgCl2, 2 mM KH2PO4, and 25 mM NaHCO3, pH 7.35) for barrier measurements, 2–3 biopsies Each participant performed a stress and a control session (serving as were placed in RNAlater (Invitrogen, Fisher Scientific, Gothenburg, its own control) with an interval of at least 2 weeks between sessions. Sweden) and maintained at −80°C for subsequent mRNA analysis, Procedures were done between 7:30–09:00 AM in all participants and 2–3 were fixed in 4% paraformaldehyde in Phosphate‐buffered and lasted 1 h. Acute stress was induced by dichotomous listening19: saline for histological evaluation. subjects were exposed to two different audiobooks, one in each ear for 60 min. Which story they should focus on alternated during the session. Volunteers were required to, in alternating periods, either Ussing chamber experiments (see also answer listening comprehension questions or mark every tenth word Supplementary S1) using a custom‐made software that gives immediate feedback. The test leader supervised them and gave continuous feedback on their Rectal biopsies were mounted in modified Ussing chambers as pre- performance. Prior to the stress session, subjects were informed that viously described.20 Tissue viability was monitored by potential F I G U R E 1 Overview of experimental design. Background questionnaires: Current background stress, Life event stress and Trait anxiety (see Table S2 in Supporting information S1) were filled in by all volunteers before each session. Stress and control sessions were conducted using a designed software. Subjective stress was measured using a visual analog scale (VAS; see Table 1). Biopsies were collected by endoscopy immediately following the sessions (see Supporting Information S1) 34 - UNITED EUROPEAN GASTROENTEROLOGY JOURNAL difference (PD) during experiments and forskolin response at the end gene was normalized to the endogenous control and the fold change of experiments. The transcellular marker horseradish peroxidase after stress compared to control was calculated individually. (HRP) (type VI; Sigma Chemical Co) and the paracellular probe 51 Chromium(Cr)‐EDTA (Perkin Elmer) were added to the mucosal sides and serosal samples were collected during 120 min. 51Cr‐EDTA Statistical analysis permeability was measured by gamma‐counting (1282 Compu- gamma) and HRP passage was analyzed with QuantaBlu™ Flourigenic Data are expressed as mean (�SD) or median (Q1–Q3). Normality peroxidase substrate kit (Pierce).8 of data distribution was tested by the Kolmogorov‐Smirnov test. Normally distributed parametric data were compared by paired Student's t‐test; otherwise, the Wilcoxon signed ranks test was RNA isolation, microarray and biological pathway used. p < 0.05 was considered statistically significant. Statistical analysis analysis was performed using GraphPad Prism 8 software (GraphPad Software Inc.). Correlations between variables was In randomly selected biopsies, microarray technology (Applied Bio- assessed by the Spearman test. The Benjamini and Hochberg systems), was applied to identify consistent differential gene method was used to adjust p‐values in the gene expression anal- expression due to experimental stress.24 Total mRNA was isolated ysis and adjusted to obtain strong control over the “false discovery with the RNAeasy Mini Kit (Qiagen). Quality and quantity of mRNA rate” (FDR). were assessed with the Agilent 2100 Bioanalyzer. Sampling for high quality mRNA expression was achieved in 10 included individuals and paired analysis was feasible in samples from seven individuals. To RESULTS identify the gene expression profile, we applied hierarchical clus- tering on the complete set of differentially expressed genes using Study population average linkage and correlation as measures of similarity. Genes with similar expression profiles were grouped together in a hierarchical Twenty‐one (8 male, 13 female) healthy volunteers were recruited, way and differentially expressed genes (p < 0.05) were submitted to and 16 were included in the full analysis (Figure 2). All 16 participants pathway and network analysis using the Ingenuity Pathway Analysis were medication free, including NSAIDs and antibiotics. Also, all (IPA) methodology (IPA 7.0, Ingenuity Systems).24 Genes with a mean included volunteers scored inside the normal range for the scales fold‐change of ≤0.7 and ≥1.4, obtained comparing stress versus measuring background stress levels (Table S2), and there were no sex control were analyzed by IPA software. The list of differentially differences. expressed genes in stress versus control, linked to their approved nomenclature (http://www.genenames.org), and fold‐change was uploaded into the IPA application. For network analysis, IPA provided Dichotomous listening induced a systemic stress a score according to the fit of supplied genes and the list of biological response functions involved. Dichotomous listening increased both objective and subjective stress as compared to control sessions. There were significant differences Quantitative real‐time PCR between control and stress in systolic blood pressure, diastolic blood pressure, and heart rate and subjective stress (VAS) (Table 1). Cor- To further validate microarray results, cDNA synthesis and amplifi- relations between trait anxiety and current perceived stress (r = 0.63, cation was performed using 1 μg of total RNA with the High‐Capacity p = 0.03; n = 16), and HF and SysBP (r = 0.49, p = 0.05; n = 16), Reverse Transcription Reagents Kit (Applied Biosystems). Q‐RT‐PCR supported the validity of the stress model. was performed on an ABI PRISM® 7500 FAST Sequence Detection System (Applied Biosystems) using validated TaqMan Gene Expres- sion Assays, and the human Peptidylprolyl isomerase A (PPIA) Dichotomous listening increased paracellular (cyclophilin A) gene as endogenous control (Applied Biosystems). permeability Sampling for validation Q‐RT‐PCR was achieved in 10 included subjects, and full paired analysis was feasible in seven individuals. The In paired comparisons between stress and control sessions in the 51 selection of genes to quantify (based on microarray, plus tryptase and same individuals, paracellular permeability as measured by Cr‐ four barrier‐related proteins) is shown in supplementary material EDTA was significantly increased by stress, p < 0.05 (Figure 3a); 51 (Tables S1a and S1b). Each sample, including distilled water as Cr‐EDTA permeation was increased in 10/11 participants after negative control, was run in triplicate and data were analyzed by the stress (Figure 3b), but there was no significant difference between 2‐ΔΔCt method, as previously described.25 The expression of each stress and control sessions in TER (Figure 3c,d). GERDIN ET AL. - 35 F I G U R E 2 Flow chart of participants in the study. Sixteen of the 21 recruited healthy volunteers were included in the permeability studies M = male; F = female. IBS, irritable bowel syndrome TABLE 1 Subjective stress and cardiovascular variables during control sessions and stress sessions, respectively Control (median; range) Stress (median; range) p‐value Subjective stress (VAS 0–100) 8.1 (0.5–50) 54.9 (19.5–74.3) <0.0001 Heart frequency (bpm) 72.3 (51.9–85.1) 81.6 (57.5–102) <0.0001 Systolic blood pressure (mmHg) 109.8 (97–129) 122.6 (103–159) <0.0001 Diastolic blood pressure (mmHg) 72.4 (65.2–93.8) 79.4 (61.2–83.3) <0.0001 Note: Subjective stress was measured using a visual analog scale where 0 = no stress and 100 = maximum stress (arbitrary units). Abbreviations: mmHg, millimeter mercury; ppm, beats per minute. Transepithelial passage of HRP (Figure 3e,f) was not significantly affected (down‐regulated) transcription factor (p < 0.001). The list of changed by stress neither in absolute (n = 14) nor in paired (n = 10) differentially expressed genes was overlaid onto the molecular analysis. Despite this, the delta value between stress and control network contained in the IPA, which identified seven networks, based 51 sessions in HRP flux correlated to Δ Cr‐EDTA‐permeability on focus genes interrelationships (Table S3). The most significant (r = 0.63, p = 0.044; n = 11). There were no differences between network (score 32) was associated with functions related to canonical men and women in the permeability response to stress. pathways of the network “Inflammatory Response, Cell Signaling, Cellular Function and Maintenance” (Figure 4). Additionally, the bio- function analysis of the immune response, based on stress‐induced Dichotomous listening modulated mucosal gene changes in gene expression, revealed a gene profile associated with expression profile migration and chemotaxis of immune cells, mainly B and T lympho- cytes, dendritic cells and phagocytes (Table S4). Microarray analysis in biopsies from stress and control sessions Validation by q‐RT‐PCR of the top differentially expressed genes identified 185 genes that responded to experimental stress. Among (Table 2) confirmed that acute stress increased the expression of the these, 171 were eligible for gene interaction and biological function PCK1 gene. Reduced expression was confirmed of genes relating to analysis, and 83 were differentially expressed, that is, p‐value ≤ 0.05 immune cell (mainly B‐cell) activation and maturation (CR2, MS4A1 after FDR adjustment. (CD20), TCLA1, BANK1, CD22, FDCSP), and signaling molecules of The stress‐induced expression profile was mainly characterized homing of immune cells to the gut (chemokines: CCL21, CXCL13, by a reduction of immune‐related genes (Table 2). The only gene found CCL19, and receptors: CCR7, CXCR5) and innate immunity (DUOX2), to be up‐regulated was phosphoenolpyruvate carboxykinase 1 (PCK1), as illustrated in Figure 5. However, after stress, the gene expression involved in the gluconeogenesis process (Table 2). The nuclear factor of tryptase in the rectal mucosa was not significantly modified kappa B subunit 2 (NFkB2) was identified as the only significantly (Control: 1 [0.4–1.7]; Stress: 1 [0.4–2.2], fold change; p = 0.41) 36 - UNITED EUROPEAN GASTROENTEROLOGY JOURNAL F I G U R E 3 Assessment of barrier function in rectal mucosa in control and stress sessions. Results are presented as box plot and paired line chart (control and stress in same individual), respectively, for all variables. (a and b) Paracellular permeability studied using 51Cr‐EDTA (n = 16 subjects, 14 paired analyses, p = 0.0245); (c and d) Transcellular antigen passage studied using horseradish peroxidase (HRP) (n = 14 subjects, 8 paired analyses); (e and f) Transepithelial resistance (TER) (n = 12 subjects, 10 paired analyses). *p < 0.05 (Figure 5). Also, no significant changes were found in mRNA normal background stress levels, using a dichotomous listening stress expression of the apical junctional proteins, JAM‐A, claudin 2, ZO1 protocol. Dichotomous listening is known to induce mild psycholog- and ZO3 (Table S5). ical stress, with previously documented effects on several GI func- tions in humans, for example, absorption and secretion,14 sensitivity and motility,26 and mucosal blood flow,17 but the effects of dichot- Association between systemic and mucosal response omous listening on intestinal permeability have not previously been to acute stress reported. During the stress session, significant but moderate in- creases in subjective (VAS) and objective (heart rate, BP) systemic Changes in mucosal function and/or immunity following stress were stress parameters were found, which validated that the present in some cases statistically correlated to systemic stress response. The experimental protocol induced psychological stress and suggests 51 increase in Cr‐EDTA permeability correlated to increased heart activation of the sympathetic‐adrenomedullary system. Additionally, rate during stress (r = 0.52, p = 0.036; n = 13). Moreover, subjective mucosal expression of PCK‐1, a key enzyme in gluconeogenesis, was stress during dichotomous listening (VAS) correlated with decrease increased after stress, suggesting higher local energy requirements. in FDCSP gene expression (r = 0.93, p = 0.017; n = 6), and perceived Thus, the used methodology was relevant for studies of the effects of stress during the 2 weeks preceding the experiment correlated with low‐grade acute stress on mucosal function. acute stress‐induced reduction in DUOX2 (r = 0.85, p = 0.023; n = 7). Following dichotomous listening, all individuals had permeability values within the range found in healthy volunteers in line with our previous publications.8,11,20 In other words, at a group level, there DISCUSSION were limited differences between control and stress sessions regarding paracellular permeability and no changes in transcellular In this study we show, for the first time, that mild acute laboratory permeability. On the other hand, we did find clearly measurable stress increases paracellular permeability in rectal mucosa and im- changes in paracellular permeability using paired comparisons (10/11 pacts mucosal immunity. We studied healthy volunteers reporting subjects had increased permeability after stress). On the other hand, GERDIN ET AL. - 37 TABLE 2 Top regulated genes by acute experimental stress in the colorectal mucosa of healthy volunteers Gene Fold symbol Gene/protein name change FDR Main function of gene product PCK1 Phosphoenolpyruvate carboxykinase 1.83 0.050 Acts as the rate‐limiting enzyme in gluconeogenesis and controls levels of metabolic intermediates in the citric acid cycle. Regulates formation and maintenance of memory CD8(+) T‐cells. CR2 Complement C3d receptor 2 −2.18 0.021 Receptor for complement C3, on human B‐cells a and T‐cells. Participates in B‐cell activation. CXC13 C‐X‐C motif chemokine 13 −1.97 0.050 Chemotactic for B‐lymphocytes but not for T‐lymphocytes, monocytes and neutrophils. Binds to chemokine receptor CXCR5. CCL21 C‐C motif chemokine 21 −1.82 0.029 Chemotactic for activated T‐cells, B‐cells, and dendritic cells. Binds to atypical chemokine receptor CCR7. MS4A1 Membrane spanning 4‐domains A1 −1.78 0.049 B‐lymphocyte‐specific membrane protein that plays a role in the development, differentiation, and activation of B‐lymphocytes. BANK1 B cell scaffold protein with ankyrin repeat −1.70 0.029 Involved in B‐cell receptor (BCR)‐induced Ca(2+) mobilization from intracellular stores. CCL19 C‐C motif chemokine ligand 19 −1.62 0.022 Potent chemotactic activity for T‐cells and B‐cells. Binds to chemokine receptor CCR7 and ACKR4. DOUX2 Dual oxidase 2 −1.59 0.050 Generates hydrogen peroxide; playing role in Lactoperox‐idase‐mediated antimicrobial defense at mucosal surface. TCLA1 T cell leukemia/lymphoma 1 family AKT −1.51 0.050 Phosphorylation and activation of AKT1, AKT2 and AKT3. Stabilizes coactivator A mitochondrial membrane potential and promotes cell survival. CD22 CD22 molecule −1.51 0.044 Mediates B‐cell–B‐cell interactions. Involved in localization of B‐cells in lymphoid tissues and regulation of B‐cell antigen receptor signaling. Note: Gene symbol and name for the up‐ and down‐regulated genes and their fold‐change expression respect to basal state are indicated. Data are based on analysis of mRNA expression in seven individuals in control and stress sessions, respectively. p values were adjusted to obtain strong control over the “false discovery rate” (FDR) using the Benjamini and Hochberg method. Main protein functions from The Human Protein Atlas (https://www. proteinatlas.org). PCK1 was the only up‐regulated gene, all others were significantly down‐regulated. no significant changes were seen in TER during stress. This could look further into the TJ‐related alterations by stress. Potentially, relate to there being different regulation of permeability pathways, more sustained and/or more pronounced stress would be needed to 27 that is, the leak and the pore pathways, with molecules being able show changes in mRNA expression of the TJ proteins. Also, the stress to pass through the paracelllular pathway without the complete tight responsiveness of the TJ in the small intestinal and colorectal mucosa junction (TJ) network being open at the same time and thereby not would need comparative studies to elucidate differences related to affecting TER. The findings could also be a result of differences in anatomical regions. mucosal TER between stress and control situation being relatively In the present study, using a broad gene expression analysis small in most subjects, and conventional TER measurements not following mild psychological stress, alterations of rectal mucosal B‐ being sensitive enough to fully detect differences between tissues.28 cell immunity were demonstrated. During disease, the intestinal Notably, systemic stress affected physiological variables related to mucosa of both IBS and IBD show higher number of B cells and barrier function: increased heart rate was correlated to the increase plasma cells and increased production of antibodies.30,31 The in paracellular permeability and perceived stress the weeks preced- importance of neuro‐immune interactions in the control of humoral ing the experiment correlated to reduction in DUOX2, an enzyme immunity was recently highlighted,32 and several studies have shown that plays a fundamental role in mucosal host defense against path- a link between stress and B cell responses. Previous studies of ogens by the production of reactive oxygen species. Interestingly, circulating lymphocytes in various types of acute stress models in mutations in DUOX2 have been identified in patients at very early humans,33,34 including dichotomous listening,17 have shown 29 onset of IBD. Based on our findings, our model may be a useful increased numbers and/or activity of natural killer (NK) cells and approach to further identify mechanisms underlying stress‐mediated CD8+ T cells, together with a decrease in CD19+ B cells. At a reduction in relevant innate defensive responses predisposing to GI mucosal level, stress in rodents induce decreased colonic secretion of disease. With changes in paracellular permeability, we had also ex- IgA35 and studies on college students showed decreased secretory pected changes in mRNA expression of proteins of the apical junc- IgA concentrations in saliva at examinations.36 Together, these pre- tional complex. The lack of significant changes in JAM‐A, CLDN2, vious studies suggest that stress diminishes B‐cell activity. Our pre- ZO1 and ZO3 does, however, not rule out changes in phosphoryla- sent data, from analyses of biopsies suggest rapid downregulation of tion, in subunits, in localization etc. More studies will be needed to rectal mucosal NFkB2 and B cell activity in response to mild acute 38 - UNITED EUROPEAN GASTROENTEROLOGY JOURNAL F I G U R E 4 Functional analysis of mucosal transcriptional signature in the colonic mucosa of healthy volunteers induced by dichotomous listening stress. Relationships between differentially expressed genes in the first highest scored network as analyzed by the Ingenuity Pathway Analysis (IPA) application and associated canonical pathways (CP) based on gene expression during control and stress sessions, respectively, in seven individuals. Node (gene) and edge (gene relationship) symbols are described. The color indicates downregulation (green). Genes in uncolored nodes were not identified as differentially expressed and were integrated into the computationally generated networks based on the evidence stored in the IPA knowledge memory indicating relevance for this network stress, since 7 of 10 top down‐regulated genes are directly involved humans, as previously implied.38 Another potential explanation is in B cell activation, signaling and migration (Table 2). Despite changes that, in the colorectal region, mild stress using dichotomous listening in mucosal immunity and barrier function, mRNA expression of mast acts through different mechanisms than stronger stressors and with cell tryptase was unchanged by stress in the present study. Several less pronounced effects on barrier function. previous studies, in animal models12,13 as well as in humans,15,37 Even though our results are obtained from healthy individuals highlighted the importance of neuroimmune interaction involving and most likely are part of a homeostatic response, the stress‐related corticotrpin‐releasing hormone (CRH), mast cells and eosinophils in decrease in expression of follicular dendritic cell secreted protein, the stress response of the mucosal barrier. Animal models of acute that binds to activated B cells, regulating antibody production,39 the stress (e.g., water avoidance restraint stress, crowding stress, etc.) receptors CCR7 and CXCR5, and respective ligands CCL19, CCL21 are different and stronger stressors than the mild experimental and CXCL13, may predispose the intestinal mucosa to deficient stress used in the present study. In addition, there are species dif- defensive activity, as those molecules play a key role in the homing of ferences in the neuro‐immune signaling in the colorectal mucosa lymphocytes and dendritic cells. These immune alterations might, related to CRH and mast cells.8 In humans, small intestinal perme- together with increased permeability, facilitate antigen penetration ability was increased following psychological stress by public speech following acute stress and, thereby, favor mucosal inflammation in at examination via mechanisms involving CRH and mast cells,15 vulnerable individuals exposed to persistent stress. whereas a milder stressor (anticipation of electroshock) had no ef- Dichotomous listening is experimental and may result in other fects on permeability.15 We previously showed that CRH increases responses than real life stressors do, which is a limitation of studies HRP‐uptake in colonic mucosa via mast cells,8,37 while in the present using experimental stress protocols. It is, for example, well estab- study we found no significant changes in HRP permeability. No lished that physical and psychological experimental stressors may detailed analysis of CRH‐mast cell interaction was performed in our affect the intestinal tract in different ways.15,26 Vanuytsel et al.15 also study, but the unaltered mRNA expression of tryptase following showed that the mucosal stress response was related to the systemic dichotomous listening, may imply mast cells not being a major player. cortisol response, with increased small intestinal permeability only in Together, these findings may suggest differences in nerve‐mast cell individuals with elevated salivary cortisol. The authors did not report 15 interactions between the small intestine and the colorectum in effects on the sympathetic‐adrenomedullary system which was GERDIN ET AL. - 39 F I G U R E 5 Validation of microarray data by quantitative real‐time polymerase chain reaction (Q‐RT–PCR) of the top regulated genes by experimental stress, grouped by biological function. Quantitative gene expression in the colonic mucosa of seven individuals with paired analysis, before and after stress, of (a) Gluconeogenesis. (b) Innate immunity. (c) Immune cell activation and maturation. (d) Lymphocyte homing to the gut. BANK1, B‐cell scaffold protein with ankyrin repeats 1; CCL19, C‐C motif chemokine ligand 19; CCL21, C‐C motif chemokine ligand 21; CCR7, C‐C motif chemokine receptor 7; CD22, CD22 molecule; CR2, Complement component 3d receptor 2; CXCL13, C‐X‐C motif chemokine ligand 13; CXCR5, C‐X‐C motif chemokine receptor 5; DUOX2, Dual oxidase 2; FDCSP, Follicular dendritic cell secreted protein; MS4A1, Membrane spanning 4‐domains A1; PCK1, Phosphenolpuruvate carboxy kinase 1; PPIA, Peptidylprolyl isomerase A (Cyclophyllin A); TCLA1, T‐cell leukemia/lymphoma 1A. The PPIA was used as a house‐keeping gene. p‐values are indicated for each analysis 40 - UNITED EUROPEAN GASTROENTEROLOGY JOURNAL activated in the present study as demonstrated by increased blood O R CI D pressure and heart rate and recently shown to play an important role Linda Gerdin https://orcid.org/0000-0002-5758-7303 40 in modulating intestinal barrier function in mice. Future studies to Åsa V. Keita https://orcid.org/0000-0002-6820-0215 investigate mechanisms should, therefore, include assessment of the sympathetic‐adrenomedullary system as well as of the HPA‐axis R EF E R E N CE S during dichotomous listening. 1. Bradford K, Shih W, Videlock EJ, Presson AP, Naliboff BD, Mayer EA, The consequences of the humoral immune inhibition were not et al. Association between early adverse life events and irritable elucidated in our study, and we do not know if dichotomous listening bowel syndrome. Clin Gastroenterol Hepatol. 2012;10(4):385–90e1‐ 3. https://doi.org/10.1016/j.cgh.2011.12.018 reduced antibody production or the release of other defensive me- 2. Parker CH, Naliboff BD, Shih W, Presson AP, Videlock EJ, Mayer EA, diators in the intestinal mucosa. Further research is needed to better et al. Negative events during adulthood are associated with symp- phenotype the mucosal immune response triggered by stress and to tom severity and altered stress response in patients with irritable identify potential therapeutic targets at early stages of colorectal bowel syndrome. Clin Gastroenterol Hepatol. 2019;17(11):2245–52. https://doi.org/10.1016/j.cgh.2018.12.029 dysfunction. However, the fact that mild acute psychological stress 3. Murray CD, Flynn J, Ratcliffe L, Jacyna MR, Kamm MA, Emmanuel AV. leads to rapid changes in paracellular permeability and humoral im- Effect of acute physical and psychological stress on gut autonomic munity in healthy rectal mucosa lends further support to the innervation in irritable bowel syndrome. Gastroenterology. 2004; importance of stress as a contributing factor in GI diseases in pre- 127(6):1695–703. https://doi.org/10.1053/j.gastro.2004.08.057 4. Eijsbouts C, Zheng T, Kennedy NA, et al. Genome‐wide analysis of disposed individuals. 53,400 people with irritable bowel syndrome highlights shared ge- In conclusion, we report, for the first time, that paracellular netic pathways with mood and anxiety disorders. Nat Genet. 2021; permeability was altered in rectal mucosa of healthy volunteers 53:1543–52. immediately following dichotomous listening stress. Additionally, in- 5. Levenstein S, Prantera C, Varvo V, Scribano ML, Andreoli A, Luzi C, et al. Stress and exacerbation in ulcerative colitis: a prospective dications of stress‐induced reduction in mucosal B cell‐activity were study of patients enrolled in remission. Am J Gastroenterol. 2000; demonstrated. Further studies of the importance of humoral immu- 95(5):1213–20. https://doi.org/10.1111/j.1572‐0241.2000.02012.x nity in the early stress response of colorectal mucosa are therefore 6. Melinder C, Hiyoshi A, Fall K, Halfvarson J, Montgomery S. Stress warranted to delineate mechanisms of stress‐mediated mucosal resilience and the risk of inflammatory bowel disease: a cohort study barrier dysfunction. Also, our model may be used in studies of pa- of men living in Sweden. BMJ Open. 2017;7(1):e014315. https://doi. org/10.1136/bmjopen‐2016‐014315 tients with IBS and IBD to further define the role of stress in the 7. Gitter AH, Wullstein F, Fromm M, Schulzke JD. Epithelial barrier initiation and/or perpetuation of these diseases. defects in ulcerative colitis: characterization and quantification by electrophysiological imaging. Gastroenterology. 2001;121(6): A CKN O WL ED G M E N T S 1320–8. https://doi.org/10.1053/gast.2001.29694 8. Wallon C, Persborn M, Jonsson M, Wang A, Phan V, Lampinen M, The authors acknowledge PM Olsson and Johan Hedström for et al. Eosinophils express muscarinic receptors and corticotropin‐ designing the software used to control the dichotomous listening releasing factor to disrupt the mucosal barrier in ulcerative colitis. and control sessions. This work was supported by grants from the Gastroenterology. 2011;140(5):1597–607. https://doi.org/10.1053/ Swedish Research Council (VR‐Medicine and Health, 2014‐02537, j.gastro.2011.01.042 9. Zeissig S, Burgel N, Gunzel D, Richter J, Mankertz J, Wahnschaffe U, 2017‐02475) (Johan D. Söderholm, Åsa V. Keita). Supported in part et al. Changes in expression and distribution of claudin 2, 5 and 8 by Fondo de Investigación Sanitaria and CIBERehd, Instituto de lead to discontinuous tight junctions and barrier dysfunction in Salud Carlos III, Subdirección General de Investigación Sanitaria, active Crohn's disease. Gut. 2007;56(1):61–72. https://doi.org/10. Ministerio de Economía y Competitividad: PI19/01643 (Maria 1136/gut.2006.094375 10. Piche T, Barbara G, Aubert P, Bruley des Varannes S, Dainese R, Vicario); PI17/0190 (Javier Santos); Centro de Investigación Bio- Nano JL, et al. Impaired intestinal barrier integrity in the colon of médica en Red de Enfermedades Hepáticas y Digestivas: CB06/04/ patients with irritable bowel syndrome: involvement of soluble 0021 (Javier Santos & Maria Vicario). Futurum, Region Jönköping mediators. Gut. 2009;58(2):196–201. https://doi.org/10.1136/gut. County 12406 and 11460, (Linda Gerdin, Mats Persborn) Medical 2007.140806 11. Bednarska O, Walter SA, Casado‐Bedmar M, Strom M, Salvo‐Romero Research Council of Southeast Sweden, 37841 (Mats Persborn, E, Vicario M, et al. Vasoactive intestinal polypeptide and mast cells Linda Gerdin). regulate increased passage of colonic bacteria in patients with irrita- ble bowel syndrome. Gastroenterology. 2017;153(4):948–960e3. C ON FL I C T O F IN T E R ES T https://doi.org/10.1053/j.gastro.2017.06.051 The authors disclose no conflicts of interest to study participants and 12. Soderholm JD, Yang PC, Ceponis P, Vohra A, Riddell R, Sherman PM, et al. Chronic stress induces mast cell‐dependent bacterial adher- no financial arrangements with any company whose product figures ence and initiates mucosal inflammation in rat intestine. Gastroen- in the submitted manuscript. Maria Vicario is an employee of Nestlé terology. 2002;123(4):1099–108. https://doi.org/10.1053/gast.200 Research, Switzerland. 2.36019 13. Vicario M, Guilarte M, Alonso C, Yang P, Martinez C, Ramos L, et al. Chronological assessment of mast cell‐mediated gut dysfunction and D AT A A VA I L A B ILI T Y S T A T E M E NT mucosal inflammation in a rat model of chronic psychosocial stress. The data that support the findings of this study are available from the Brain Behav Immun. 2010;24(7):1166–75. https://doi.org/10.1016/j. corresponding author upon reasonable request. bbi.2010.06.002 GERDIN ET AL. - 41 14. Barclay GR, Turnberg LA. Effect of psychological stress on salt and 30. Brandtzaeg P, Carlsen HS, Halstensen TS. The B‐cell system in in- water transport in the human jejunum. Gastroenterology. 1987; flammatory bowel disease. Adv Exp Med Biol. 2006;579:149–67. 93(1):91–7. https://doi.org/10.1016/0016‐5085(87)90319‐2 31. Vicario M, Gonzalez‐Castro AM, Martinez C, Lobo B, Pigrau M, 15. Vanuytsel T, van Wanrooy S, Vanheel H, Vanormelingen C, Ver- Guilarte M, et al. Increased humoral immunity in the jejunum of schueren S, Houben E, et al. Psychological stress and corticotropin‐ diarrhoea‐predominant irritable bowel syndrome associated with releasing hormone increase intestinal permeability in humans by a clinical manifestations. Gut. 2015;64(9):1379–88. https://doi.org/10. mast cell‐dependent mechanism. Gut. 2014;63(8):1293–9. https:// 1136/gutjnl‐2013‐306236 doi.org/10.1136/gutjnl‐2013‐305690 32. Zhang X, Lei B, Yuan Y, Zhang L, Hu L, Jin S, et al. Brain control of 16. Alonso C, Guilarte M, Vicario M, Ramos L, Ramadan Z, Antolin M, humoral immune responses amenable to behavioural modulation. et al. Maladaptive intestinal epithelial responses to life stress may Nature. 2020;581(7807):204–8. https://doi.org/10.1038/s41586‐ predispose healthy women to gut mucosal inflammation. Gastroen- 020‐2235‐7 terology. 2008;135(1):163–172e1. https://doi.org/10.1053/j.gastro. 33. McGregor BA, Murphy KM, Albano DL, Ceballos RM. Stress, cortisol, 2008.03.036 and B lymphocytes: a novel approach to understanding academic 17. Mawdsley JE, Macey MG, Feakins RM, Langmead L, Rampton DS. The stress and immune function. Stress. 2016;19(2):185–91. https://doi. effect of acute psychologic stress on systemic and rectal mucosal org/10.3109/10253890.2015.1127913 measures of inflammation in ulcerative colitis. Gastroenterology. 34. Huang CJ, Webb HE, Garten RS, Kamimori GH, Acevedo EO. Psy- 2006;131(2):410–9. https://doi.org/10.1053/j.gastro.2006.05.017 chological stress during exercise: lymphocyte subset redistribution 18. Marsland AL, Herbert TB, Muldoon MF, Bachen EA, Patterson S, in firefighters. Physiol Behav. 2010;101(3):320–6. https://doi.org/10. Cohen S, et al. Lymphocyte subset redistribution during acute lab- 1016/j.physbeh.2010.05.018 oratory stress in young adults: mediating effects of hemoconcen- 35. Zoppi S, Madrigal JL, Perez‐Nievas BG, Marin‐Jimenez I, Caso JR, tration. Health Psychol. 1997;16(4):341–8. https://doi.org/10.1037/ Alou L, et al. Endogenous cannabinoid system regulates intestinal 0278‐6133.16.4.341 barrier function in vivo through cannabinoid type 1 receptor acti- 19. McRae S, Younger K, Thompson DG, Wingate DL. Sustained mental vation. Am J Physiol Gastrointest Liver Physiol. 2012;302(5): stress alters human jejunal motor activity. Gut. 1982;23(5):404–9. G565–71. https://doi.org/10.1152/ajpgi.00158.2011 https://doi.org/10.1136/gut.23.5.404 36. Jemmott JB, 3rd, Magloire K. Academic stress, social support, and 20. Wallon C, Braaf Y, Wolving M, Olaison G, Soderholm JD. Endo- secretory immunoglobulin A. J Pers Soc Psychol. 1988;55(5): scopic biopsies in Ussing chambers evaluated for studies of 803–10. https://doi.org/10.1037/0022‐3514.55.5.803 macromolecular permeability in the human colon. Scand J Gastro- 37. Wallon C, Yang PC, Keita AV, Ericson AC, McKay DM, Sherman PM, enterol. 2005;40(5):586–95. https://doi.org/10.1080/00365520510 et al. Corticotropin‐releasing hormone (CRH) regulates macromo- 012235 lecular permeability via mast cells in normal human colonic biopsies 21. Holmes THR, Rahe RH. The social readjustment rating scale. J Psy- in vitro. Gut. 2008;57(1):50–8. https://doi.org/10.1136/gut.2006. chosom Res. 1967;11(2):213–18. https://doi.org/10.1016/0022‐399 117549 9(67)90010‐4 38. Keita AV, Carlsson AH, Cigehn M, Ericson AC, McKay DM, Soder- 22. Cohen S, Kamarck T, Mermelstein R. A global measure of perceived holm JD. Vasoactive intestinal polypeptide regulates barrier func- stress. J Health Soc Behav. 1983;24(4):385–96. https://doi.org/10. tion via mast cells in human intestinal follicle‐associated epithelium 2307/2136404 and during stress in rats. Neurogastroenterol Motil. 2013;25(6): 23. Gaudry E, Vagg P, Spielberger CD. Validation of the state‐trait e406–17. https://doi.org/10.1111/nmo.12127 distinction in anxiety research. Multivariate Behav Res. 1975; 39. Al‐Alwan M, Du Q, Hou S, Nashed B, Fan Y, Yang X, et al. Follicular 10(3):331–41. https://doi.org/10.1207/s15327906mbr1003_6 dendritic cell secreted protein (FDC‐SP) regulates germinal center 24. Martinez C, Vicario M, Ramos L, Lobo B, Mosquera JL, Alonso C, and antibody responses. J Immunol. 2007;178(12):7859–67. https:// et al. The jejunum of diarrhea‐predominant irritable bowel syn- doi.org/10.4049/jimmunol.178.12.7859 drome shows molecular alterations in the tight junction signaling 40. Mallesh S, Ten Hove AS, Schneider R, Schneiker B, Efferz P, Kalff JC, pathway that are associated with mucosal pathobiology and clinical et al. Sympathetic innervation modulates mucosal immune homeo- manifestations. Am J Gastroenterol. 2012;107(5):736–46. https:// stasis and epithelial host defense. Cells. 2022;11(16):2606. https:// doi.org/10.1038/ajg.2011.472 doi.org/10.3390/cells11162606 25. Schmittgen TD, Livak KJ. Analyzing real‐time PCR data by the comparative C(T) method. Nat Protoc. 2008;3(6):1101–8. https:// doi.org/10.1038/nprot.2008.73 26. Rao SS, Hatfield RA, Suls JM, Chamberlain MJ. Psychological and S U P P O R T I N G I N F O RM A T I O N physical stress induce differential effects on human colonic motility. Additional supporting information can be found online in the Sup- Am J Gastroenterol. 1998;93(6):985–90. https://doi.org/10.1111/j. porting Information section at the end of this article. 1572‐0241.1998.00293.x 27. Shen L, Weber CR, Raleigh DR, Yu D, Turner JR. Tight junction pore and leak pathways: a dynamic duo. Annu Rev Physiol. 2011;73(1): 283–309. https://doi.org/10.1146/annurev‐physiol‐012110‐142150 How to cite this article: Gerdin L, González‐Castro AM, 28. Keita AV, Gullberg E, Ericson AC, Salim SY, Wallon C, Kald A, et al. Characterization of antigen and bacterial transport in the follicle‐ Ericson A‐C, Persborn M, Santos J, Walter SA, et al. Acute associated epithelium of human ileum. Lab Invest. 2006;86(5): psychological stress increases paracellular permeability and 504–16. https://doi.org/10.1038/labinvest.3700397 modulates immune activity in rectal mucosa of healthy 29. Hayes P, Dhillon S, O'Neill K, Thoeni C, Hui KY, Elkadri A, et al. volunteers. United European Gastroenterol J. 2023;11(1): Defects in NADPH oxidase genes NOX1 and DUOX2 in very early onset inflammatory bowel disease. Cell Mol Gastroenterol Hepatol. 31–41. https://doi.org/10.1002/ueg2.12329 2015;1:489–502.