Elevated levels of colonic interleukin-1beta and interleukin-8 in isolated REM sleep behavior disorder without associated changes in permeability

Elevated levels of colonic interleukin-1beta and interleukin-8 in isolated REM sleep behavior disorder without associated changes in permeability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Elevated levels of colonic interleukin-1beta and interleukin-8 in isolated REM sleep behavior disorder without associated changes in permeability Loïc Sellier Montaigne, Simon Lassozé, Adrien de Guilhem de Lataillade, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7829903/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Apr, 2026 Read the published version in npj Parkinson's Disease → Version 1 posted 9 You are reading this latest preprint version Abstract Some recent studies suggest that gastrointestinal inflammation could play a role in the development of Parkinson’s disease (PD) by increasing intestinal permeability and triggering early neuropathological changes in the gut. Therefore, we set out to analyze gut inflammation as well as intestinal barrier function and integrity in patients with isolated REM sleep behavior disorder (iRBD), a prodromal stage of PD. Sigmoid colon biopsy specimens from patients with iRBD (n = 20), patients with PD (n = 34) and controls (n = 20) were analyzed by qPCR to measure the expression levels of proinflammatory cytokines and enteric glial markers. Fecal calprotectin levels were also measured to further assess gastrointestinal inflammation. Gut permeability was evaluated in biopsies mounted in Ussing chambers, and the integrity of the intestinal epithelial barrier was assessed by analyzing the expression of tight junction proteins by western blot. We found that the mRNA expression levels of interleukin-1β and interleukin-8 were increased in both patients with iRBD and PD relative to controls; the expression of TNF-α was also higher in PD but not in iRBD patients. We did not observe any differences in colonic permeability, tight junction proteins expression and calprotectin levels between iRBD, PD and control participants. Our study is the first to characterize the inflammatory profile in the gut in iRBD. As a whole, our findings provide evidence that enteric inflammation is present at a moderate level in prodromal PD, not higher than in individuals with established PD, and without concurrent changes in intestinal permeability. Health sciences/Diseases Health sciences/Gastroenterology Biological sciences/Immunology Health sciences/Neurology Biological sciences/Neuroscience idiopathic REM sleep behavior disorder Parkinson’s disease GI inflammation intestinal permeability enteric glial cells tight junctions Figures Figure 1 Figure 2 Figure 3 Introduction Over the last decade it has become increasingly clear that the gastrointestinal (GI) tract plays a central role in Parkinson’s disease (PD) (see 1 – 3 for reviews). GI symptoms occur in almost every PD patient during the course of the disease 4 , 5 and autopsy studies have consistently reported the presence of alpha-synuclein deposits, the pathological hallmark of the disease, in the enteric nervous system 6 – 9 . Not only has the functional and pathological involvement of the GI tract been observed in patients with PD but also in subjects with isolated REM sleep behavior disorder (iRBD), which is considered a prodromal stage of the disease 10 , 11 . These and other observations led Braak 12 and more recently Borghammer 13 to hypothesize that PD pathology may originate in the GI tract, at least in a patient subpopulation. In addition to alpha-synuclein pathology, accumulating evidence indicates the presence of a low-grade inflammatory state of the gut in PD 14 . Increased expression levels of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and interferon-gamma (IFN-γ) have been reported in sigmoid biopsies from PD patients 15 , 16 . Additionally, higher levels of interleukin-1 alpha, IL-8 and calprotectin (a marker of gut inflammation used to monitor inflammatory bowel disease) have been reported in the stool of patients with PD compared with controls 17 – 19 . In addition to immune cells, enteric glial cells (EGC) play crucial roles in regulating GI inflammation 20 and this logically prompted the study of the expression of EGC markers, such as glial fibrillary acidic protein (GFAP) and S100-β in the PD gut. Higher mRNA and protein levels of both GFAP 15 , 21 – 23 and S100-β 15,24 have been found in GI biopsies of PD patients. These data suggest that in the PD gut, EGC respond to intestinal inflammation by converting to a ‘reactive phenotype’ 20 . Both the enteric nervous system and gut inflammation play a regulatory role in maintaining the function and integrity of the intestinal barrier (reviewed in 25 ). These findings provided the framework for investigating the intestinal barrier in PD. Intestinal barrier function and integrity are maintained via protein networks called tight junctions (TJs), which primarily consist of transmembrane proteins, including occludin and claudin and scaffold proteins such as zonula-occludens-1 (ZO-1) 26 . Existing studies on intestinal permeability in PD have yielded conflicting results. While some authors have reported evidence of increased colonic permeability in PD 16,27 , others reports have been inconclusive 28 , 29 or have shown no difference between PD and control subjects 30 . From a structural standpoint, reduced levels of claudin-1 24 , occludin 30 or ZO-1 16 have been observed in colonic biopsies from patients with PD when compared with controls. Taken together, these findings support the hypothesis that GI inflammation could be an early event in PD, triggering the disease process in the gut. In this scenario, enteric inflammation is thought to increase intestinal permeability, providing access to the nearby enteric neurons and triggering alpha-synuclein pathology. While all studies published to date on GI inflammation in PD have been carried out in patients with overt disease, no data are available for iRBD patients. Here, we examined the expression levels of a panel of proinflammatory cytokines and glial markers in sigmoid colon biopsy specimens from iRBD patients and compared them with samples from PD patients and controls. Intestinal barrier function and integrity were evaluated by measuring gut permeability in sigmoid biopsies mounted in Ussing chambers and by analyzing the expression levels of TJ proteins, respectively. Additional experiments were performed to measure fecal calprotectin in patients with iRBD, PD and controls. Patients and methods Study design and participants This study was a monocentric, prospective interventional study with minimal risks and burdens whose objectives were exploratory. From December 2020 to October 2023, a total of 74 participants in three groups were recruited: 20 participants with iRBD, 34 patients with PD and 20 healthy volunteers. Patients with PD and iRBD, aged over 18 years were recruited from the movement disorder clinic and sleep disorder unit at Nantes University Hospital, France, respectively. The diagnosis of PD was made according to criteria provided by the United Kingdom Parkinson’s Disease Survey Brain Bank. The diagnosis of REM sleep behavior disorder (RBD) followed the International Classification of Sleep Disorders – third edition (ICSD-3) criteria, and neurological examination ruled out secondary causes. The collected demographic data included sex, age at onset and disease duration, as well as age at colonoscopy. Controls were healthy participants who had a clinical evaluation by a gastroenterologist and a routine colonoscopy performed for colorectal cancer screening. All control participants underwent a detailed neurological examination to rule out symptoms of PD, cognitive impairment, and RBD. Participants were not included if they had suffered from irritable bowel syndrome for more than 5 years prior to PD or iRBD, had a history of colorectal cancer, had a history of antibiotic treatment, acute gastrointestinal illness, or hospitalization for an acute medical condition or surgery in the previous month, had a Mini-Mental State Examination (MMSE) score of less than 24. The study protocol was approved in September 2020 by the National Committee on Ethics and Human Research (Comité de Protection des Personnes Nord Ouest IV) and registered on ClinicalTrials.gov (identifier NCT04652843). Written informed consent was obtained from each patient and from each control volunteer according to the principles of Helsinki. Healthy participants received a compensation for taking part in the study. The clinical trial consisted of an inclusion visit, followed by a rectosigmoidoscopy or a colonoscopy (during which biopsies were taken) within 90 days after inclusion, and ended with a phone call visit for safety. Participants who did not have biopsies taken during either rectosigmoidoscopy or colonoscopy were excluded from the trial. Clinical assessment All participants were screened for specific symptoms of RBD using the RBD screening questionnaire (RBDSQ) 31 . The Unified Parkinson's Disease Rating Scale part III (UPDRS-III) was used to assess motor features of PD; the UPDRS-III axial subscore was calculated as the sum of items 18, 19, 22 and 27–30, which evaluates symptoms such as dysarthria or postural instability 32 . Global cognition was assessed with the Montreal Cognitive Assessment (MoCA) and the MMSE. Olfaction was assessed using the Sniffin’ Sticks Identification 12-Test 33 . The SCOPA-Aut questionnaire was used to evaluate autonomic symptoms 34 . Constipation was diagnosed as defined by the Rome IV Diagnostic Criteria for Functional Constipation 35 . Endoscopic procedure and processing of colonic biopsies Seven sigmoid colon biopsies were collected from each subject during rectosigmoidoscopy, for patients with PD and iRBD, or colonoscopy, for controls. Three of these biopsies were immediately processed for the assessment of para- and transcellular permeability in Ussing chambers (Fig. 1 ). Two other biopsies were pooled, immediately frozen in RA1 buffer (Macherey-Nagel, Hoerdt, France) and stored indefinitely at -80°C (Fig. 1 ). For qPCR and western blot analyses, RA1-preserved biopsies were lysed using a “Precellys 24” tissue homogenizer (Bertin Technologies, St Quentin-en-Yvelines, France) followed by sonication with a “Vibracell 75 186” device (Sonics, Newton, CT, USA). RNA and protein extraction was then performed with the NucleoSpin RNA/Protein Kit (Macherey-Nagel, Hoerdt, France) according to the manufacturer’s instructions. The two remaining biopsies were pooled, snap-frozen on dry-ice, and stored at − 80°C for further western blot analysis (Fig. 1 ). Snap-frozen biopsies were lysed and sonicated in radioimmunoprecipitation assay buffer (RIPA, Merck Millipore, Fontenay sous Bois, France, Cat# 20–188) supplemented with 2 mM orthovanadate (Sigma, Cat# S6505), Phosphatase Inhibitor Cocktail 3 (Sigma, Cat# P5726), and cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Sigma, Cat# 11697498001). Protein concentration was assessed using a BCA Protein Assay Kit (Thermo Scientific™ Pierce™). Samples were diluted with adjusted volumes of Sample Reducing Agent (Life Technologies, Saint-Aubin, France, Cat# NP0009) and NuPAGE Sample Buffer (Life Technologies, Cat# NP0008), incubated at 95°C for 5 min and indefinitely stored at -20°C. Para- and transcellular permeability of colonic biopsies in Ussing chambers Three biopsies per subject were mounted in Ussing chambers (World Precision Instruments; WPI, Hertfordshire, UK) exposing a surface of 0.0314 cm². Both the apical and the basolateral sides of each tissue were incubated in 2 ml of Dulbecco’s Modified Eagle Medium F12 (DMEM F12; ThermoFisher Scientific, Cat# 31330038) supplemented with 0.1% (v/v) fetal bovine serum (Biowest, Nuaillé, France, Cat# S1400), 2 mM Glutamine (ThermoFisher Scientific, Cat# 25030024) and 45 g/L of NaHCO 3 (Sigma S5761). The medium was continuously oxygenated with a gas flow (95% O 2 /5% CO 2 ) and maintained at 37°C. After equilibration for 30 min, 300 µl of apical medium was replaced with 300 µl of medium containing FITC-5,6-sulfonic acid (0.4 kDa, final concentration 0.1 mg/mL, ThermoFischer Scientific, Cat# F1130), TRITC-Dextran (4 kDa, Sigma, Cat# 60842-46-8) and horse radish peroxidase (HRP, Life Technologies Cat# …?). Aliquots of 150 µL conditioned media were collected, analyzed and returned to the basolateral compartment every 30 min for a total of 3 h. Paracellular transit from the apical compartment was assessed by measuring FITC and TRITC fluorescence intensities (excitation/emission wavelengths: 487/528 nm and 544/585 nm, respectively) using a BioTek Synergy H1 microplate reader (Agilent). Transcellular transit from the apical compartment was assessed on 30 µl samples of conditioned basolateral media by performing an HRP enzymatic activity assay using the 3,3’,5,5’-tetramethylbenzidine substrate (TMB; BD Bioscience, Le Pont de Claix, France). TMB absorbance was measured at 450 nm on a BioTek Synergy H1 microplate reader. Paracellular and transcellular permeabilities were determined, respectively, by averaging the temporal gradient of fluorescence and absorbance intensities of three technical replicates using a linear regression fit model (GraphPad Prism 5, La Jolla, USA) qRT-PCR Total RNA obtained from the two pooled biopsies stored in RA1 buffer was quantified using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific). Purified mRNA was denatured and processed for reverse transcription using Superscript III reverse transcriptase (ThermoFisher Scientific). PCR amplifications were performed using the Fast SYBR™ Green kit (ThermoFisher Scientific) and run on a StepOnePlus system (ThermoFisher Scientific). The following primers were used: S6 forward : 5’-AAGCACCCAAGATTCAGCGT-3’ S6 reverse : 5’-TAGCCTCCTTCATTCTCTTGGC-3’ TNF-α forward : 5’-CCCGAGTGACAAGCCTGTAG-3’ TNF-α reverse : 5’-TGAGGTACAGGCCCTCTGAT-3’ IL-6 forward : 5’-CAATGAGGAGACTTGCCTGGTGAA − 3’ IL-6 reverse : 5’-TGTGGTTGGGTCAGGGGTGGTT-3’ IFN-γ forward : 5’-CCAGAGCATCCAAAAGAGTGTGGAG-3’ IFN-γ reverse : 5’-GCTGGCGACAGTTCAGCCATCA-3’ IL-1β forward : 5’-GAGCAACAAGTGGTGTTCTCC-3’ IL-1β reverse : 5’-TTGGGATCTACACTCTCCAGC-3’ IL-8 forward : 5’-CTGGCCGTGGCTCTCTTGG-3’ IL-8 reverse : 5’-ATTTCTGTGTTGGCGCAGTGTG-3’ IL-10 forward : 5’-TGAAAACAAGAGCAAGGCCG-3’ IL-10 reverse : 5’-GCCACCCTGATGTCTCAGTT-3’ GFAPk forward : 5’-GGACTGAGGATCAGGGCAAA-3’ GFAPk reverse : 5’-CACCCAGTTCTGCTGTCGAA-3’ Western Blot For the analysis of ZO-1 and occludin expression levels, protein fractions obtained with the NucleoSpin RNA/Protein Kit™ were precipitated using Protein Precipitator buffer (PP; NucleoSpin RNA/Protein Kit) and resuspended in Protein Solving Buffer supplemented with tris(2-carboxyethyl)phosphine reducing agent (PSB/TCEP; NucleoSpin RNA/Protein Kit™) according to the manufacturer’s instructions. Protein concentration was quantified using a Nanodrop2000 spectrophotometer (ThermoFisher Scientific, Cillebon sur Yvette, France). Equal amounts of purified protein extract were separated using NuPAGE Tris-Acetate 3–8% gels™ (Invitrogen) together with NuPAGE Tris-Acetate SDS running buffer™ (Invitrogen) and then transferred onto nitrocellulose membranes with the iBlot™ Dry Blotting System (ThermoFisher Scientific). For the analysis of claudin-1 and 2 expression, Equal amounts of RIPA protein lysates were separated using the NuPAGE™ Bis-Tris Mini 4–12%™ Protein Gels (Invitrogen), together with NuPAGE MOPS SDS running buffer™ (ThermoFisher Scientific) before electrophoretic transfer to nitrocellulose membranes. Membranes were blocked with tris-buffered saline (TBS) with 0.1% (v/v) Tween-20 and 5% (w/v) non-fat dry milk and incubated overnight at 4°C with mouse monoclonal antibody against ZO-1 (1:200, Cat# 33-9100, Thermo Fisher Scientific), rabbit polyclonal antibody against occludin (1:250, Cat# ab31721, Abcam, Paris, France), rabbit polyclonal anti-claudin-1 (1:250, Cat# 51-9000, ThermoFisher Scientific) or rabbit polyclonal anti-claudin-2 (1:250, Cat# 51-6100, ThermoFisher Scientific). To confirm equal protein loading, membranes were probed with mouse monoclonal anti-GAPDH antibody (1:2000, Cat# ab8245, Abcam). Bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit (1:5000; Life technologies, Cat# 31460) or anti-mouse (1:5000; Sigma, Cat# A9044) antibodies and visualized by enhanced chemiluminescent detection using either Clarity™ Western ECL (Biorad, Marnes-la-Coquette, France, Cat# 1705060) or Supersignal™ West Femto (ThermoFisher Scientific, Cat# 34094) substrates. For quantification, the relevant immunoreactive bands were quantified with laser-scanning densitometry and analyzed with imageJ software (NIH, Bethesda, MD; version 1.51). The values of ZO-1, occludin, claudin-1 and 2 immunoreactivities were normalized to the amount of GAPDH. To allow comparison between different membranes, the density of the bands was expressed as a percentage of the average of controls. Lysates of Caco-2 cells, a human epithelial cell line that expresses the main TJs proteins 36 were used as a positive control to confirm co-migration with ZO-1, occludin and claudins from human colonic biopsies (Supplementary Fig. 1). Calprotectin measures For some iRBD, PD and control participants, a stool sample, ideally the first stool of the morning, was collected and transported to the laboratory at + 4°C within 24 hours of collection. Fecal extracts were obtained using the B-CAL-Ex extraction buffer (CALEX® Cap device, BÜHLMANN Laboratories AG, Schönenbuch, Switzerland). Levels of calprotectin in the patients' fecal extracts were measured using particle-enhanced turbidimetric immunoassay (PETIA; BUHLMANN fCAL® turbo, BÜHLMANN Laboratories AG, Schönenbuch, Switzerland) on the Optilite® automated analyzer (Binding site, Birmingham, UK). Results were reported in micrograms/gram of stool. Statistics The exploratory study was designed to include 20 participants in 4 groups: iRBD, PD within less than 5 years from diagnosis, PD with 5 to more years from diagnosis, and healthy (non-PD, non iRBD) volunteers. The total sample size of 80 participants was deemed sufficient to reach exploratory conclusions. Continuous parameters (expression levels of cytokines, inflammation and gut permeability markers) were compared across groups (iRBD, PD and controls) using Kruskall-Wallis nonparametric test. No imputation for missing values was used. Linear correlations between two continuous parameters were estimated using Pearson’s correlation and tested towards 0 (two-tailed) for normal values (all participants), and Spearman’s correlation in subgroups analysis. Associations between categorical variables (constipation, straining during defecation), inflammation and permeability parameters were tested using Wilcoxon-Mann-Whitney test. All statistical analyses were performed using R Studio software, version 4.4.2 from the R Foundation for Statistical Computing (Vienna, Austria). The level of statistical significance was set at 5% for all analyses. No adjustment for multiplicity was used. Results Inclusions Seventy-five participants were enrolled between December 2020 and October 2023, 20 patients with iRBD, 34 patients with PD and 21 healthy controls. One control participant was subsequently excluded because no biopsies were taken (withdrew consent before colonoscopy). Population Description Demographic and clinical characteristics, including motor and cognitive screening scores are reported in Table 1 . There were no differences in demographics between groups. Among patients with PD, 15 individuals (44%) had probable RBD, as indicated by an RBDSQ score ≥ 5. MoCA scores were lower in patients with iRBD compared to patients with PD. As expected, both patients with iRBD and PD had higher scores on the UPDRS part III (both total and axial subscore), RBDSQ, and the Rome IV constipation questionnaire as well as lower scores on the Sniffin’ Sticks Identification Test when compared to controls. Patients with PD had higher scores on the UPDRS part III (both total and axial subscore) in comparison to patients with iRBD. MMSE (on pairwise comparisons) and SCOPA-Aut did not differ between groups. Table 1 Patient demographics and clinical characteristics. iRBD: isolated rapid eye movement sleep behavior disorder; MMSE: Mini Mental State Examination; MoCA: Montreal Cognitive Assessment; PD: Parkinson’s disease; RBDSQ: RBD screening questionnaire; SCOPA-AUT: Scale for Outcomes in Parkinson's disease for Autonomic symptoms; SSIT: Sniffin’ Sticks Identification 12-Test; UPDRS part III total: MDS-UPDRS III Movement Disorder Society’s Unified Parkinson’s Disease Rating Scale Part III, N/A not available. iRBD PD Controls p-value Number 20 34 20 Male/female (%) 16/4 (80/20) 24/10 (71/29) 14/6 (70/30) 0.89 Age, y 68.3 ± 8.2 63.6 ± 7.9 67.3 ± 8 0.08 Body mass index, kg/m² 26 ± 3.8 24.5 ± 3.4 26.3 ± 4.1 0.21 Symptom duration, y 7.3 ± 4.9 5.9 ± 4.9 N/A UPDRS part III total (0-132) 4 ± 3.3 19.2 ± 11.1 0.7 ± 1.2 < 0.001 iRBD vs C, PD vs C, iRBD vs PD UPDRS part III axial (0–36) 1.4 ± 1.5 4.4 ± 3.6 0.2 ± 0.5 < 0.001 iRBD vs C, PD vs C, iRBD vs PD RBDSQ (0–10) 9.8 ± 2.1 5.3 ± 3.9 2.2 ± 1.4 < 0.001, iRBD vs C, PD vs C MMSE (0–30) 28.3 ± 1.3 29 ± 1.1 29.2 ± 0.7 0.04 MoCA (0–30) 26.4 ± 2.2 28.4 ± 1.6 27.2 ± 2.9 0.002, iRBD vs PD SSIT (0–12) 5.4 ± 2.7 6 ± 2.5 10.1 ± 1.9 < 0.001, iRBD vs C, PD vs C SCOPA-Aut (0–69) 18 ± 10.5 15.3 ± 9.1 10.3 ± 5.1 0.07 Constipation Rome IV (no., %) 11 (55) 13 (38) 0 < 0.001, iRBD vs C, PD vs C IL-1β and IL-8 levels in colonic biopsies are higher in iRBD patients compared to controls First, we used qPCR to analyze the expression levels of TNF-α, IL-1β, IL-8, IL-6, IFN-γ and interleukin-10 (IL-10) in colonic biopsies from patients with iRBD and PD as well as from controls. This panel of cytokines was selected because it has been consistently associated with alteration in gut permeability 37 . mRNA expression levels of IL-1β and IL-8 were elevated in both patients with iRBD (p = 0.02 and p = 0.04, respectively) and patients with PD compared with controls (p < 0.001 for both cytokines, Fig. 2 a). No significant differences in IL-1β or IL-8 expression were observed between the iRBD and PD groups (Fig. 2 a). TNF-α expression was increased in patients with PD compared with controls (p = 0.006), but not in the iRBD group (Fig. 2 a). The mRNA expression levels of IL-6, IFN-γ and IL-10 did not differ across groups (p = 0.93, p = 0.4 and p = 0.23, respectively) (Supplementary Fig. 2a). Except for TNF-α expression, which was higher in men with iRBD (1.25 ± 0.82 vs 0.54 ± 0.20 in women, p = 0.049), no difference in cytokine levels was observed between men and women (Supplementary Table 1). The mRNA expression levels for IL-1β and IL-8 were strikingly variable among iRBD and PD patients; some showed levels comparable to controls while others showed a 3- to 6-fold increase (Fig. 2 a). We therefore performed a correlation analysis of IL-1β and IL-8 mRNA expression levels to determine whether these cytokines are upregulated in the same individuals. IL-1β and IL-8 expression positively correlated in both iRBD and PD patient groups (r = 0.78; p < 0.0001 for iRBD; r = 0.92; p < 0.0001 for PD; Fig. 2 b). In addition, a correlation between IL-1β and TNF-α (r = 0.35, p = 0.02) was observed in the PD group (Supplementary Fig. 3). IL-1β, IL-8, IL-6 and IL-10 mRNA levels were positively associated with UPDRS Part III total and axial scores (see Supplementary Table 2 for details), while IL-10 was negatively correlated with olfaction scores in the entire population. However, these correlations were no longer significant when we performed subgroup analyses. In the PD group, IL-8 mRNA levels were positively associated with SCOPA-Aut (p = 0.04, r = 0.36). No significant correlations were found between cytokine levels and age, symptom duration, UPDRS motor and axial scores, MMSE, MoCA, olfaction score, or SCOPA-Aut in the iRBD group. Constipation, as evaluated by Rome-IV criteria, was only observed in patients with PD or iRBD (Table 1 ) and was not associated with changes in cytokine expression levels in those patients (Supplementary Table 2). Straining during defecation (Rome IV criteria), which occurred in 32 participants (iRBD n = 13, PD n = 16, controls n = 3) was associated with increased expression levels of IL-8 in the entire study population (p = 0.04); this relationship was driven more by controls (p = 0.004) than by patients with PD (p = 0.39) or with iRBD (p = 0.44) (Supplementary Fig. 4a). Enteric glial cells, which are involved in the regulation of intestinal permeability 25 , respond to gut inflammation by adopting a ‘reactive phenotype’ 20,38 . This phenotypic switch is associated with the upregulation of several glial markers including GFAP 39 (which includes GFAPκ, the main isoform expressed by enteric glial cells 21 ), S100β 40 , and Sox10 41 . qPCR analysis of colonic biopsies revealed no differences in the expression levels of reactive glial markers were between groups (p = 0.14 for GFAPκ, p = 0.95 for S100β and p = 0.98 for Sox10) (Supplementary Fig. 2b). No associations were found between glial markers and sex, age, BMI, symptom duration, UPDRS motor and axial scores, MMSE, MoCA, olfaction score, SCOPA-Aut, or constipation (Rome IV criteria, Supplementary Table 2). However, straining during defecation (Rome IV criteria) was associated with higher expression levels of Sox-10 in patients with PD (p = 0.02, Supplementary Fig. 4b). Fecal calprotectin levels, which were measured in a subset of participants (14 iRBD, 25 PD and 19 controls), did not differ between groups (158 ± 32 µg/g, 132 ± 37 µg/g and 101 ± 26 µg/g, respectively, p = 0.14, Fig. 2 c). Colonic barrier function and integrity are comparable between subjects with iRBD and controls To assess intestinal barrier function and integrity, we evaluated colonic permeability and analyzed the expression levels of key TJ proteins (Fig. 3 a). The para- and transcellular permeability of colonic biopsies from iRBD, PD and control subjects were measured in Ussing chambers using sulfonic acid/dextran and HRP, respectively (Fig. 3 a). No differences in sulfonic acid, dextran or HRP flux were observed between groups (p = 0.33, p = 0.16, p = 0.05, respectively, Fig. 3 b). No associations were found between permeability measures and demographic or clinical characteristics, or with mRNA levels of cytokines and glial markers (Supplementary Table 3). No differences in the expression levels of the four main TJ proteins- ZO-1, occludin, claudin-1 claudin-2- were observed between groups (p = 0.20, p = 0.63, p = 0.29 and p = 0.19, respectively, Fig. 3 a, c). Discussion Several studies have reported abnormalities in gut inflammatory markers and intestinal barrier in patients with overt PD (reviewed in 14,42 ). Here, we have used sigmoid colon biopsy specimens from patients with iRBD and compared them to samples from patients with PD and controls to show for the first time that low-grade intestinal inflammation is present in prodromal PD. Among the cytokines and glial markers tested, only IL-8 and IL-1β were augmented in the gut of iRBD patients. In addition to IL-1β and IL-8, TNF-α gene expression was significantly increased in GI biopsies from PD patients. IL-1β is a pro-inflammatory cytokine primarily produced by monocytes, macrophages and dendritic cells 43 , whereas, IL-8 is a powerful chemoattractant for neutrophils and is commonly released in the gut by macrophages and epithelial cells 44 . However, it is unsurprising, that these two cytokines are concomitantly upregulated, as IL-1β tightly regulates IL-8 expression in various cell types, including intestinal epithelial cells 45 . The strong correlation between IL-1β and IL-8 expression observed in both iRBD and PD patients supports the hypothesis that a shared underlying inflammatory pathway is activated early in disease progression in certain individuals. These results are consistent with our previous study, in which we found increased expression levels of both IL-1β and TNF-α expression in colon biopsies from subjects with symptomatic PD versus controls (IL-8 expression was not measured) 15 . Furthermore, our data corroborate other publications showing increased TNF-α and IL-1β levels in stool samples from PD patients compared to controls 19 , 46 . We observed higher TNF-α levels in men with iRBD. Although limited data are available on sex differences in iRBD 47 , the higher level of gut inflammation observed in men with prodromal synucleinopathy is consistent with previously reported sex differences in immune reactivity and PD vulnerability 48 . Fecal calprotectin, an established marker of intestinal inflammation, did not differ between groups. This discordance may indicate that immune activity in iRBD and PD is highly localized or below the detection threshold of this assay. Interestingly, we observed a significant correlation between IL-8 expression and straining during defecation. Unexpectedly, the strongest association was observed in the control group. While straining is generally regarded as a benign functional symptom, our findings suggest that altered defecatory function may be associated with subclinical gut immune activation, even in individuals without neurological symptoms. One possible interpretation is that straining could be an early marker of gut dysfunction linked to neurodegenerative disease, given some epidemiological evidence linking constipation with an increased risk of PD 49 . An alternative hypothesis is that the upregulation of IL-8 in association with straining in controls may reflect a pathological change in the gut of individuals without neurodegenerative pathological features who are subjected to mechanical stress and remodeling, as observed in patients with spinal cord injury 50 . We observed a positive correlation between enteric IL-8 mRNA levels and dysautonomic symptoms in the PD group, indicating a widespread pattern of peripheral alterations—including in the gut—in some PD patients. However, in a previous study, no association was found between dysautonomic symptoms and colonic alpha-synucleinopathy 51 . Unlike other observations based on blood or CSF samples in patients with PD, we did not observe any correlations between inflammatory cytokines and the motor or cognitive phenotype. Previous reports have associated worse motor function with higher blood levels of IL-6, while some studies also demonstrated associations with higher blood levels of IL-1 β or TNFα, or higher CSF levels of IL-6 and IL-8 (reviewed in 52 ). Similarly, altered cognitive function or dementia has been correlated with higher blood levels of IL-6, TNF-α, or higher CSF levels of IL-6 or IL-8 (reviewed in 52 ). Although IL-1β is a known modulator of intestinal barrier function 53 , we could not find evidence of intestinal barrier dysfunction and/or alteration in iRBD and PD patients. Beyond the present work, 5 other studies have evaluated the intestinal barrier function in PD and they have provided conflicting results 16 , 27 – 30 . A recent review has discussed this inconsistency in detail, attributing it primarily to small sample sizes and methodological heterogeneity across studies 42 . In this study, intestinal permeability was assessed using a validated method (Ussing chambers) 54 in a relatively large number of subjects, and no differences were observed between iRBD, PD and controls. In line with this, no changes in the expression levels of TJs proteins were detected. The absence of significant findings underscores the importance of employing more sensitive methods to determine whether subtle intestinal barrier dysfunction occurs in PD and prodromal PD 42 . Moreover, and in sharp contrast to previous reports, we found no evidence of increased expression of glial markers, including as GFAP and S-100β in the PD gut. Likewise, these markers were unchanged in iRBD. Although there is no definitive explanation for these diverging results, two points are worth discussing. First, symptomatic PD are clinically and pathologically heterogeneous 55 , which can cause variability between studies. Second, prior studies analyzed glial markers expression in the GI tract of patients with advanced PD, whose mean disease duration ranged from 8 to 13 years 23 , 24 . By contrast, our study included non-demented patients with a mean disease duration of 5.9 years. Third, autopsy studies have shown that levels of GFAP in the substantia nigra, caudate and putamen of subjects with PD were positively correlated with the extent of dopamine loss 56 . It is therefore plausible that activation of GFAP-positive cells in both the CNS and peripheral autonomic networks represents a late event in PD. Regarding the correlations analyses between glial markers and clinical characteristics, the only significant relationship was observed is the positive association between straining during defecation and Sox10 expression in PD patients. While this finding may reflect local glial activation induced by mechanical stress, it also raises the possibility of reciprocal influences between specific motor disturbances in the colon and enteric glial homeostasis at an overt neurodegenerative stage. To our knowledge, this is the first study to investigate gut inflammation in patients with iRBD. A few studies have explored neuroinflammation in iRBD patients, showing alterations in the peripheral adaptive immune system, abnormal upregulation of inflammatory cytokine secretion in response to immune stimuli 57 , and increased TNF-α levels in patients with iRBD and multiple phenoconversion risk markers 58 . Nevertheless, none of these studies investigated gut inflammation. Combined with our results, these findings raise the possibility that this immune activation may represent an early, modifiable event in the pathogenesis of synucleinopathies. This hypothesis was recently reinforced by a study showing that patients with inflammatory bowel disease are three times more likely to have probable RBD 59 . Given the increasing recognition of the role of gut-brain axis in PD, the therapeutic targeting of intestinal inflammation has emerged as a promising strategy to delay disease progression or prevent phenoconversion in individuals at risk. One promising anti-inflammatory approach for treating neurodegenerative diseases involves glucagon-like peptide-1 receptor agonists (GLP-1RA). GLP-1RA attenuate T-cell-mediated gut and systemic inflammation, by reducing intestinal cytokine release and enhancing epithelial barrier integrity 60 – 62 . This makes GLP-1RA particularly attractive candidates for modulating early gut-driven inflammation in PD. Dietary modulation, prebiotics, and microbiota-targeted interventions such as fecal microbiota transplantation have been effective in preclinical models. Some early clinical data suggest that these therapeutic strategies may improve PD symptoms 63 . Our findings show that IL-1β and IL-8 are already upregulated in the colon of individuals with iRBD; therefore, it is plausible that immune-based interventions administered at the prodromal stage of PD could attenuate neuroinflammatory cascades and limit propagation of α-synuclein pathology along the gut–brain axis, delaying or preventing conversion to overt PD. Several limitations must be acknowledged. Our patient cohorts were relatively small, although adequate for colonic biopsy studies. The cross-sectional design of the study precludes drawing conclusions about temporal progression. Although iRBD is a validated prodromal marker of PD, the iRBD group may include individuals at different stages of disease progression, or who may eventually develop dementia with Lewy bodies or, less commonly, multisystem atrophy, rather than PD. Detecting alpha-synuclein seeding in colonic biopsies would enable this group to be stratified, although the small sample size may prevent complex association analysis. Sampling was restricted to the colonic mucosa and submucosa, which may prevent the observation of focal changes in other GI segments (e.g., the myenteric plexus or rostral GI organs). Finally, we did not control for dietary and environmental factors, which may contribute to inter-individual variability. More broadly, our study provides insights into the potential role of the digestive tract, and more specifically digestive inflammation, in PD. We showed that, although low-grade digestive inflammation is present in some individuals with iRBD, its extent is similar to that observed in established PD. This evidence challenges the notion that digestive inflammation represents an early pathogenic event in PD. Declarations Ethics Statement The study protocol for the sampling of sigmoid biopsies was approved by the local Committee on Ethics and Human Research (Comité de Protection des Personnes Ouest VI), and registered on ClinicalTrials.gov (identifier NCT04652843) Competing Interests A.C. is an employee of PiLeJe Laboratoire. Other authors declare no financial or non-financial competing interests. Funding Nantes University Hospital was the study promoter. This work was supported by a grant from Nantes University Hospital (Appel d'offre interne 2019, RC19_0401) and France Parkinson (2019). France Parkinson played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. Work at Inserm U1235 on intestinal permeability is supported in part by the ‘Fédération pour la Recherche sur le Cerveau (FRC)’, the ‘Association AMADYS’, ‘Vaincre Parkinson’ and by an EU Joint Programme - Neurodegenerative Disease Research (JPND) supported project ( www.jpnd.eu ). Simon Lassozé, Loïc Sellier-Montaigne and Adrien de Guilhem de Lataillade are recipients of ‘année recherche’ from CHU de Nantes. Author Contribution **LSM** : formal analysis, data curation, investigation, writing-review & editing; **SL** : formal analysis, data curation, investigation; **AdGdL** : data curation, investigation, supervision; **TO** : supervision, validation; data curation, investigation; **MAV** : statistical design and analysis, writing –– original draft,; **KBN** : analysis of calprotectin levels in feces, writing –– original draft; **MRD** : validation, supervision, methodology; **TD:** supervision, validation; data curation, investigation **; SLD** : patient recruitment, clinical data collection and characterization; **IJA** : patient recruitment, clinical data collection and characterization; **EC** : investigation, colonoscopy and biopsy processing; **QM** : investigation, colonoscopy and biopsy processing; **MN:** validation, supervision, writing-review & editing; **GM:** validation, supervision, writing-review & editing; **AC:** methodology, data interpretation; **PD** : writing –– original draft, visualization, validation, supervision, resources, project administration, methodology, investigation, funding acquisition, formal analysis, data curation, conceptualization. **LLV** : writing –– original draft, visualization, validation, supervision, resources, project administration, methodology, investigation, funding acquisition, formal analysis, data curation, conceptualization Acknowledgement The authors would like to thank Patrice Chauveau, Aziz Ouach, Maelle Ningre and Marion Rigot for their administrative and technical support, data management and monitoring, and participants for taking part in the study. 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Endoscopic biopsies in Ussing chambers evaluated for studies of macromolecular permeability in the human colon. Scand J Gastroenterol 40, 586–595 (2005). Wüllner, U. et al. The heterogeneity of Parkinson’s disease. J Neural Transm 130, 827–838 (2023). Tong, J. et al. Low Levels of Astroglial Markers in Parkinson’s Disease: Relationship to α-Synuclein Accumulation. Neurobiol Dis 82, 243–253 (2015). Mark, J. R. et al. Peripheral immune cell response to stimulation stratifies Parkinson’s disease progression from prodromal to clinical stages. Commun Biol 8, 716 (2025). Kim, R. et al. Serum TNF-α and neurodegeneration in isolated REM sleep behavior disorder. Parkinsonism & Related Disorders 81, 1–7 (2020). Reddy, V. L. et al. Assessing prevalence and risk factors for REM sleep behavior disorder among patients with inflammatory bowel disease. npj Parkinsons Dis. 11, 282 (2025). Bang-Berthelsen, C. H. et al. GLP-1 Induces Barrier Protective Expression in Brunner’s Glands and Regulates Colonic Inflammation. Inflammatory Bowel Diseases 22, 2078–2097 (2016). Nozu, T. et al. Glucagon-like peptide-1 analog, liraglutide, improves visceral sensation and gut permeability in rats. J Gastroenterol Hepatol 33, 232–239 (2018). Wong, C. K. et al. Central glucagon-like peptide 1 receptor activation inhibits Toll-like receptor agonist-induced inflammation. Cell Metabolism 36, 130–143.e5 (2024). Cheng, Y. et al. Efficacy of fecal microbiota transplantation in patients with Parkinson’s disease: clinical trial results from a randomized, placebo-controlled design. Gut Microbes 15, 2284247 (2023). Additional Declarations Competing interest reported. A.C. is an employee of PiLeJe Laboratoire. Other authors declare no financial or non-financial competing interests. Supplementary Files SupplementaryTables101025.docx suppfigure1.jpg suppfigure2.jpg suppfigure3.jpg suppfigure4.jpg Cite Share Download PDF Status: Published Journal Publication published 27 Apr, 2026 Read the published version in npj Parkinson's Disease → Version 1 posted Editorial decision: Revision requested 02 Dec, 2025 Reviews received at journal 01 Dec, 2025 Reviewers agreed at journal 17 Nov, 2025 Reviews received at journal 08 Nov, 2025 Reviewers agreed at journal 24 Oct, 2025 Reviewers invited by journal 22 Oct, 2025 Editor assigned by journal 20 Oct, 2025 Submission checks completed at journal 19 Oct, 2025 First submitted to journal 10 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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16:21:47","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52354,"visible":true,"origin":"","legend":"","description":"","filename":"suppfigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/9cdd25807cb4b1cad3893706.jpg"},{"id":95064128,"identity":"207fccd9-2fd6-4d54-b7d0-ddef174dbc36","added_by":"auto","created_at":"2025-11-04 01:20:57","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107174,"visible":true,"origin":"","legend":"","description":"","filename":"suppfigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/6d6cea85c7d7e9c40191cc52.jpg"},{"id":95064136,"identity":"c428c9ad-5901-4b07-90ac-8e7ed9848656","added_by":"auto","created_at":"2025-11-04 01:20:57","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":149984,"visible":true,"origin":"","legend":"","description":"","filename":"12f3f1d3300443a1933f170a8a883ecb1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/20e3d81180ac48475c24dec0.xml"},{"id":95222861,"identity":"55472baa-54ce-49a1-aa31-262c4261086a","added_by":"auto","created_at":"2025-11-05 16:21:16","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15740,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/eb5fc49176c8168be09c9c46.png"},{"id":95222815,"identity":"8cda3fd4-eeb1-4b5f-9b94-1e126bfe98ef","added_by":"auto","created_at":"2025-11-05 16:21:10","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25055,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/ba287a39a2d7827badf2e638.png"},{"id":95223168,"identity":"c03e53a8-1722-4ee0-a55f-ed478c510549","added_by":"auto","created_at":"2025-11-05 16:21:46","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49036,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/1ed842397d1536b388b91268.png"},{"id":95064137,"identity":"a156dd52-42ab-46f4-99f3-df5842d31353","added_by":"auto","created_at":"2025-11-04 01:20:57","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":149112,"visible":true,"origin":"","legend":"","description":"","filename":"12f3f1d3300443a1933f170a8a883ecb1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/743e8bfbffb574e43ec5c85c.xml"},{"id":95064135,"identity":"6400528f-4dc8-48d7-a4e7-0a810f214670","added_by":"auto","created_at":"2025-11-04 01:20:57","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":167846,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/097395e2f7e5f05ded927445.html"},{"id":95064115,"identity":"385328dc-628e-48dc-ac3f-2a0f7f0aade2","added_by":"auto","created_at":"2025-11-04 01:20:56","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProcessing of colonic biopsies. \u003c/strong\u003eFor each subject, 7 biopsies were taken in the sigmoid colon during the course of a rectosigmodoscopy for subjects with iRBD and PD and colonoscopy for controls. Three biopsies were immersed in HBSS and immediately processed in Ussing chambers for the measurement of intestinal permeability. Two biopsies were immersed in RA1 buffer and stored at -80°C until analysis. The two remaining biopsies were dry frozen and stored at -80°C until analysis. Parts of the figure were drawn by using pictures from Biorender and Servier Medical Art (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).\u003c/p\u003e","description":"","filename":"floatimage115.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/95c1693d2251da42a0b5a5b3.jpeg"},{"id":95064116,"identity":"72669816-7974-4147-8e92-da4482143e3f","added_by":"auto","created_at":"2025-11-04 01:20:57","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75875,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of gut inflammation in iRBD and PD. (a)\u003c/strong\u003e Two pooled colonic biopsies per subjects (iRBD, n = 20; PD, n=34 and controls, C, n=20) were analyzed by qPCR to measure the expression levels of TNF-α, IL-1β, and IL-8 mRNA. Data correspond to mean ± SEM; *p \u0026lt; 0.05, **p \u0026lt; 0.05 and ***p \u0026lt; 0.001 as compared to C \u003cstrong\u003e(b)\u003c/strong\u003eCorrelations between IL-8 and IL-1b levels in subjects with iRBD and PD patients. Scatter plot depicting the relationship between IL-8 and IL-1b levels in subjects with iRBD on the left panel and PD on the right panel. The lines represent the linear regression analysis; r = 0.78; p \u0026lt; 0.0001 for iRBD; r = 0.92; p \u0026lt; 0.0001 for PD \u003cstrong\u003e(c)\u003c/strong\u003e Fecal calprotectin levels (μg/g) were measured in stool from iRBD subjects (n = 14), PD patients (n = 25) and controls (n = 19); no differences were observed between groups (158 ± 32 µg/g, 132 ± 37 µg/g and 101 ± 26 µg/g, respectively, p=0.14). Data correspond to mean ± SEM. GraphPad Prism version 10.6.0 was used for designing graphs.\u003c/p\u003e","description":"","filename":"floatimage212.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/65657128cc7adf635afa77da.jpeg"},{"id":95064123,"identity":"1e124005-c934-4897-83bb-90eb5262974f","added_by":"auto","created_at":"2025-11-04 01:20:57","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":134364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of intestinal barrier function and integrity in iRBD and PD.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Illustration depicting the analysis of transcellular intestinal permeability and paracellular permeability by measuring HRP flux and sulfonic acid/dextran flux, respectively. A schematic diagram of the tight junction organization is also provided. Claudins and occludin are transmembrane proteins, which binds to the scaffold peripheral membrane protein ZO-1, which in turn binds to actin cytoskeleton to form a paracellular seal between epithelial intestinal cells \u003cstrong\u003e(b)\u003c/strong\u003eFor the evaluation of transcellular permeability, the flux of horseradish peroxidase (HRP flux, expressed in ng/mL/min) was measured in colonic biopsies mounted in Ussing chambers in subjects with iRBD (n = 18), patients with PD patients (n = 30) and controls (C, n = 18). For the evaluation of paracellular permeability, the flux of dextran (dextran flux, expressed in arbitrary units) and sulfonic acid (SA flux, expressed in arbitrary units) was measured in colonic biopsies mounted in Ussing chambers, in controls (C, n = 14 and 17, respectively), in iRBD subjects (n=16 for both reagents) and in PD patients (n = 28 and 30, respectively). Data correspond to mean ± SEM \u003cstrong\u003e(c)\u003c/strong\u003e Biopsies lysates were subjected to immunoblot analysis using antibodies against occludin, ZO-1, claudin-1 and 2. GAPDH was used as a loading control. The optical densities of occluding, ZO-1, claudin-1 and 2 immunoreactive bands were measured, normalized to the optical densities of GAPDH and expressed as percentages of controls. For ZO-1 and occludin, data correspond to mean ± SEM of 18 samples for controls (C), 20 for iRBD and 34 for PD. For claudin-1 and 2, data correspond to mean ± SEM of 16 samples for controls (C), 16 for iRBD and 26-30 for PD. Parts of the figure were drawn using pictures from Biorender and Servier Medical Art (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). GraphPad Prism version 10.6.0 was used for designing graphs.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/151a5a3567518dce03ea3f10.jpeg"},{"id":108441255,"identity":"00b260a2-aa3b-4dad-8a24-018c252d11ab","added_by":"auto","created_at":"2026-05-04 16:47:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":664343,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/6fb8eac6-86af-49ae-b763-70add83269e5.pdf"},{"id":95223031,"identity":"702c1bdb-9e74-4626-b14a-f080889fb5cd","added_by":"auto","created_at":"2025-11-05 16:21:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22904,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables101025.docx","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/1f7c10defd9177b49c35a6de.docx"},{"id":95064119,"identity":"0bd7999f-8be0-4176-8125-6ca427ac75b9","added_by":"auto","created_at":"2025-11-04 01:20:57","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":110396,"visible":true,"origin":"","legend":"","description":"","filename":"suppfigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/93116416a58f79de9785cd48.jpg"},{"id":95064124,"identity":"71088d10-540e-4462-b032-ed9af3a795b6","added_by":"auto","created_at":"2025-11-04 01:20:57","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":89744,"visible":true,"origin":"","legend":"","description":"","filename":"suppfigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/023f83e9b915ce4ec76024d8.jpg"},{"id":95223045,"identity":"4c3331ee-d600-41e0-95b0-b2776151566f","added_by":"auto","created_at":"2025-11-05 16:21:34","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":52354,"visible":true,"origin":"","legend":"","description":"","filename":"suppfigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/158a05fab7fe8a7e3d03edf5.jpg"},{"id":95064133,"identity":"4fcc5592-0a46-4e2e-9fac-957cb1db5c99","added_by":"auto","created_at":"2025-11-04 01:20:57","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":107174,"visible":true,"origin":"","legend":"","description":"","filename":"suppfigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7829903/v1/7908a16ad1ce0bd00c05b3a3.jpg"}],"financialInterests":"Competing interest reported. A.C. is an employee of PiLeJe Laboratoire. Other authors declare no financial or non-financial competing interests.","formattedTitle":"Elevated levels of colonic interleukin-1beta and interleukin-8 in isolated REM sleep behavior disorder without associated changes in permeability","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOver the last decade it has become increasingly clear that the gastrointestinal (GI) tract plays a central role in Parkinson\u0026rsquo;s disease (PD) (see\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e for reviews). GI symptoms occur in almost every PD patient during the course of the disease\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and autopsy studies have consistently reported the presence of alpha-synuclein deposits, the pathological hallmark of the disease, in the enteric nervous system\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Not only has the functional and pathological involvement of the GI tract been observed in patients with PD but also in subjects with isolated REM sleep behavior disorder (iRBD), which is considered a prodromal stage of the disease \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These and other observations led Braak\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and more recently Borghammer\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e to hypothesize that PD pathology may originate in the GI tract, at least in a patient subpopulation.\u003c/p\u003e\u003cp\u003eIn addition to alpha-synuclein pathology, accumulating evidence indicates the presence of a low-grade inflammatory state of the gut in PD\u003csup\u003e14\u003c/sup\u003e. Increased expression levels of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and interferon-gamma (IFN-γ) have been reported in sigmoid biopsies from PD patients\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Additionally, higher levels of interleukin-1 alpha, IL-8 and calprotectin (a marker of gut inflammation used to monitor inflammatory bowel disease) have been reported in the stool of patients with PD compared with controls\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In addition to immune cells, enteric glial cells (EGC) play crucial roles in regulating GI inflammation\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and this logically prompted the study of the expression of EGC markers, such as glial fibrillary acidic protein (GFAP) and S100-β in the PD gut. Higher mRNA and protein levels of both GFAP\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and S100-β\u003csup\u003e15,24\u003c/sup\u003e have been found in GI biopsies of PD patients. These data suggest that in the PD gut, EGC respond to intestinal inflammation by converting to a \u0026lsquo;reactive phenotype\u0026rsquo;\u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBoth the enteric nervous system and gut inflammation play a regulatory role in maintaining the function and integrity of the intestinal barrier (reviewed in\u003csup\u003e25\u003c/sup\u003e). These findings provided the framework for investigating the intestinal barrier in PD. Intestinal barrier function and integrity are maintained via protein networks called tight junctions (TJs), which primarily consist of transmembrane proteins, including occludin and claudin and scaffold proteins such as zonula-occludens-1 (ZO-1)\u003csup\u003e26\u003c/sup\u003e. Existing studies on intestinal permeability in PD have yielded conflicting results. While some authors have reported evidence of increased colonic permeability in PD\u003csup\u003e16,27\u003c/sup\u003e, others reports have been inconclusive\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e or have shown no difference between PD and control subjects\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. From a structural standpoint, reduced levels of claudin-1\u003csup\u003e24\u003c/sup\u003e, occludin\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e or ZO-1\u003csup\u003e16\u003c/sup\u003e have been observed in colonic biopsies from patients with PD when compared with controls.\u003c/p\u003e\u003cp\u003eTaken together, these findings support the hypothesis that GI inflammation could be an early event in PD, triggering the disease process in the gut. In this scenario, enteric inflammation is thought to increase intestinal permeability, providing access to the nearby enteric neurons and triggering alpha-synuclein pathology. While all studies published to date on GI inflammation in PD have been carried out in patients with overt disease, no data are available for iRBD patients. Here, we examined the expression levels of a panel of proinflammatory cytokines and glial markers in sigmoid colon biopsy specimens from iRBD patients and compared them with samples from PD patients and controls. Intestinal barrier function and integrity were evaluated by measuring gut permeability in sigmoid biopsies mounted in Ussing chambers and by analyzing the expression levels of TJ proteins, respectively. Additional experiments were performed to measure fecal calprotectin in patients with iRBD, PD and controls.\u003c/p\u003e"},{"header":"Patients and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy design and participants\u003c/h2\u003e\u003cp\u003eThis study was a monocentric, prospective interventional study with minimal risks and burdens whose objectives were exploratory. From December 2020 to October 2023, a total of 74 participants in three groups were recruited: 20 participants with iRBD, 34 patients with PD and 20 healthy volunteers. Patients with PD and iRBD, aged over 18 years were recruited from the movement disorder clinic and sleep disorder unit at Nantes University Hospital, France, respectively. The diagnosis of PD was made according to criteria provided by the United Kingdom Parkinson\u0026rsquo;s Disease Survey Brain Bank. The diagnosis of REM sleep behavior disorder (RBD) followed the International Classification of Sleep Disorders \u0026ndash; third edition (ICSD-3) criteria, and neurological examination ruled out secondary causes. The collected demographic data included sex, age at onset and disease duration, as well as age at colonoscopy. Controls were healthy participants who had a clinical evaluation by a gastroenterologist and a routine colonoscopy performed for colorectal cancer screening. All control participants underwent a detailed neurological examination to rule out symptoms of PD, cognitive impairment, and RBD. Participants were not included if they had suffered from irritable bowel syndrome for more than 5 years prior to PD or iRBD, had a history of colorectal cancer, had a history of antibiotic treatment, acute gastrointestinal illness, or hospitalization for an acute medical condition or surgery in the previous month, had a Mini-Mental State Examination (MMSE) score of less than 24. The study protocol was approved in September 2020 by the National Committee on Ethics and Human Research (Comit\u0026eacute; de Protection des Personnes Nord Ouest IV) and registered on ClinicalTrials.gov (identifier NCT04652843). Written informed consent was obtained from each patient and from each control volunteer according to the principles of Helsinki. Healthy participants received a compensation for taking part in the study.\u003c/p\u003e\u003cp\u003eThe clinical trial consisted of an inclusion visit, followed by a rectosigmoidoscopy or a colonoscopy (during which biopsies were taken) within 90 days after inclusion, and ended with a phone call visit for safety. Participants who did not have biopsies taken during either rectosigmoidoscopy or colonoscopy were excluded from the trial.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eClinical assessment\u003c/h3\u003e\n\u003cp\u003eAll participants were screened for specific symptoms of RBD using the RBD screening questionnaire (RBDSQ)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The Unified Parkinson's Disease Rating Scale part III (UPDRS-III) was used to assess motor features of PD; the UPDRS-III axial subscore was calculated as the sum of items 18, 19, 22 and 27\u0026ndash;30, which evaluates symptoms such as dysarthria or postural instability\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Global cognition was assessed with the Montreal Cognitive Assessment (MoCA) and the MMSE. Olfaction was assessed using the Sniffin\u0026rsquo; Sticks Identification 12-Test\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The SCOPA-Aut questionnaire was used to evaluate autonomic symptoms\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Constipation was diagnosed as defined by the Rome IV Diagnostic Criteria for Functional Constipation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eEndoscopic procedure and processing of colonic biopsies\u003c/h3\u003e\n\u003cp\u003eSeven sigmoid colon biopsies were collected from each subject during rectosigmoidoscopy, for patients with PD and iRBD, or colonoscopy, for controls. Three of these biopsies were immediately processed for the assessment of para- and transcellular permeability in Ussing chambers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two other biopsies were pooled, immediately frozen in RA1 buffer (Macherey-Nagel, Hoerdt, France) and stored indefinitely at -80\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For qPCR and western blot analyses, RA1-preserved biopsies were lysed using a \u0026ldquo;Precellys 24\u0026rdquo; tissue homogenizer (Bertin Technologies, St Quentin-en-Yvelines, France) followed by sonication with a \u0026ldquo;Vibracell 75 186\u0026rdquo; device (Sonics, Newton, CT, USA). RNA and protein extraction was then performed with the NucleoSpin RNA/Protein Kit (Macherey-Nagel, Hoerdt, France) according to the manufacturer\u0026rsquo;s instructions. The two remaining biopsies were pooled, snap-frozen on dry-ice, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for further western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Snap-frozen biopsies were lysed and sonicated in radioimmunoprecipitation assay buffer (RIPA, Merck Millipore, Fontenay sous Bois, France, Cat# 20\u0026ndash;188) supplemented with 2 mM orthovanadate (Sigma, Cat# S6505), Phosphatase Inhibitor Cocktail 3 (Sigma, Cat# P5726), and cOmplete\u0026trade; Mini EDTA-free Protease Inhibitor Cocktail (Sigma, Cat# 11697498001). Protein concentration was assessed using a BCA Protein Assay Kit (Thermo Scientific\u0026trade; Pierce\u0026trade;). Samples were diluted with adjusted volumes of Sample Reducing Agent (Life Technologies, Saint-Aubin, France, Cat# NP0009) and NuPAGE Sample Buffer (Life Technologies, Cat# NP0008), incubated at 95\u0026deg;C for 5 min and indefinitely stored at -20\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePara- and transcellular permeability of colonic biopsies in Ussing chambers\u003c/h3\u003e\n\u003cp\u003eThree biopsies per subject were mounted in Ussing chambers (World Precision Instruments; WPI, Hertfordshire, UK) exposing a surface of 0.0314 cm\u0026sup2;. Both the apical and the basolateral sides of each tissue were incubated in 2 ml of Dulbecco\u0026rsquo;s Modified Eagle Medium F12 (DMEM F12; ThermoFisher Scientific, Cat# 31330038) supplemented with 0.1% (v/v) fetal bovine serum (Biowest, Nuaill\u0026eacute;, France, Cat# S1400), 2 mM Glutamine (ThermoFisher Scientific, Cat# 25030024) and 45 g/L of NaHCO\u003csub\u003e3\u003c/sub\u003e (Sigma S5761). The medium was continuously oxygenated with a gas flow (95% O\u003csub\u003e2\u003c/sub\u003e/5% CO\u003csub\u003e2\u003c/sub\u003e) and maintained at 37\u0026deg;C. After equilibration for 30 min, 300 \u0026micro;l of apical medium was replaced with 300 \u0026micro;l of medium containing FITC-5,6-sulfonic acid (0.4 kDa, final concentration 0.1 mg/mL, ThermoFischer Scientific, Cat# F1130), TRITC-Dextran (4 kDa, Sigma, Cat# 60842-46-8) and horse radish peroxidase (HRP, Life Technologies Cat# \u0026hellip;?). Aliquots of 150 \u0026micro;L conditioned media were collected, analyzed and returned to the basolateral compartment every 30 min for a total of 3 h. Paracellular transit from the apical compartment was assessed by measuring FITC and TRITC fluorescence intensities (excitation/emission wavelengths: 487/528 nm and 544/585 nm, respectively) using a BioTek Synergy H1 microplate reader (Agilent). Transcellular transit from the apical compartment was assessed on 30 \u0026micro;l samples of conditioned basolateral media by performing an HRP enzymatic activity assay using the 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethylbenzidine substrate (TMB; BD Bioscience, Le Pont de Claix, France). TMB absorbance was measured at 450 nm on a BioTek Synergy H1 microplate reader. Paracellular and transcellular permeabilities were determined, respectively, by averaging the temporal gradient of fluorescence and absorbance intensities of three technical replicates using a linear regression fit model (GraphPad Prism 5, La Jolla, USA)\u003c/p\u003e\n\u003ch3\u003eqRT-PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA obtained from the two pooled biopsies stored in RA1 buffer was quantified using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific). Purified mRNA was denatured and processed for reverse transcription using Superscript III reverse transcriptase (ThermoFisher Scientific). PCR amplifications were performed using the Fast SYBR\u0026trade; Green kit (ThermoFisher Scientific) and run on a StepOnePlus system (ThermoFisher Scientific). The following primers were used:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eS6 forward\u003c/em\u003e: 5\u0026rsquo;-AAGCACCCAAGATTCAGCGT-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eS6 reverse\u003c/em\u003e: 5\u0026rsquo;-TAGCCTCCTTCATTCTCTTGGC-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eTNF-α forward\u003c/em\u003e: 5\u0026rsquo;-CCCGAGTGACAAGCCTGTAG-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eTNF-α reverse\u003c/em\u003e: 5\u0026rsquo;-TGAGGTACAGGCCCTCTGAT-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eIL-6 forward\u003c/em\u003e: 5\u0026rsquo;-CAATGAGGAGACTTGCCTGGTGAA \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eIL-6 reverse\u003c/em\u003e: 5\u0026rsquo;-TGTGGTTGGGTCAGGGGTGGTT-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eIFN-γ forward\u003c/em\u003e: 5\u0026rsquo;-CCAGAGCATCCAAAAGAGTGTGGAG-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eIFN-γ reverse\u003c/em\u003e: 5\u0026rsquo;-GCTGGCGACAGTTCAGCCATCA-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eIL-1β forward\u003c/em\u003e: 5\u0026rsquo;-GAGCAACAAGTGGTGTTCTCC-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eIL-1β reverse\u003c/em\u003e: 5\u0026rsquo;-TTGGGATCTACACTCTCCAGC-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eIL-8 forward\u003c/em\u003e: 5\u0026rsquo;-CTGGCCGTGGCTCTCTTGG-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eIL-8 reverse\u003c/em\u003e: 5\u0026rsquo;-ATTTCTGTGTTGGCGCAGTGTG-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eIL-10 forward\u003c/em\u003e: 5\u0026rsquo;-TGAAAACAAGAGCAAGGCCG-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eIL-10 reverse\u003c/em\u003e: 5\u0026rsquo;-GCCACCCTGATGTCTCAGTT-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eGFAPk forward\u003c/em\u003e: 5\u0026rsquo;-GGACTGAGGATCAGGGCAAA-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eGFAPk reverse\u003c/em\u003e: 5\u0026rsquo;-CACCCAGTTCTGCTGTCGAA-3\u0026rsquo;\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eWestern Blot\u003c/h2\u003e\u003cp\u003e For the analysis of ZO-1 and occludin expression levels, protein fractions obtained with the NucleoSpin RNA/Protein Kit\u0026trade; were precipitated using Protein Precipitator buffer (PP; NucleoSpin RNA/Protein Kit) and resuspended in Protein Solving Buffer supplemented with tris(2-carboxyethyl)phosphine reducing agent (PSB/TCEP; NucleoSpin RNA/Protein Kit\u0026trade;) according to the manufacturer\u0026rsquo;s instructions. Protein concentration was quantified using a Nanodrop2000 spectrophotometer (ThermoFisher Scientific, Cillebon sur Yvette, France). Equal amounts of purified protein extract were separated using NuPAGE Tris-Acetate 3\u0026ndash;8% gels\u0026trade; (Invitrogen) together with NuPAGE Tris-Acetate SDS running buffer\u0026trade; (Invitrogen) and then transferred onto nitrocellulose membranes with the iBlot\u0026trade; Dry Blotting System (ThermoFisher Scientific). For the analysis of claudin-1 and 2 expression, Equal amounts of RIPA protein lysates were separated using the NuPAGE\u0026trade; Bis-Tris Mini 4\u0026ndash;12%\u0026trade; Protein Gels (Invitrogen), together with NuPAGE MOPS SDS running buffer\u0026trade; (ThermoFisher Scientific) before electrophoretic transfer to nitrocellulose membranes. Membranes were blocked with tris-buffered saline (TBS) with 0.1% (v/v) Tween-20 and 5% (w/v) non-fat dry milk and incubated overnight at 4\u0026deg;C with mouse monoclonal antibody against ZO-1 (1:200, Cat# 33-9100, Thermo Fisher Scientific), rabbit polyclonal antibody against occludin (1:250, Cat# ab31721, Abcam, Paris, France), rabbit polyclonal anti-claudin-1 (1:250, Cat# 51-9000, ThermoFisher Scientific) or rabbit polyclonal anti-claudin-2 (1:250, Cat# 51-6100, ThermoFisher Scientific). To confirm equal protein loading, membranes were probed with mouse monoclonal anti-GAPDH antibody (1:2000, Cat# ab8245, Abcam). Bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit (1:5000; Life technologies, Cat# 31460) or anti-mouse (1:5000; Sigma, Cat# A9044) antibodies and visualized by enhanced chemiluminescent detection using either Clarity\u0026trade; Western ECL (Biorad, Marnes-la-Coquette, France, Cat# 1705060) or Supersignal\u0026trade; West Femto (ThermoFisher Scientific, Cat# 34094) substrates. For quantification, the relevant immunoreactive bands were quantified with laser-scanning densitometry and analyzed with imageJ software (NIH, Bethesda, MD; version 1.51). The values of ZO-1, occludin, claudin-1 and 2 immunoreactivities were normalized to the amount of GAPDH. To allow comparison between different membranes, the density of the bands was expressed as a percentage of the average of controls. Lysates of Caco-2 cells, a human epithelial cell line that expresses the main TJs proteins\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e were used as a positive control to confirm co-migration with ZO-1, occludin and claudins from human colonic biopsies (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCalprotectin measures\u003c/h3\u003e\n\u003cp\u003eFor some iRBD, PD and control participants, a stool sample, ideally the first stool of the morning, was collected and transported to the laboratory at +\u0026thinsp;4\u0026deg;C within 24 hours of collection. Fecal extracts were obtained using the B-CAL-Ex extraction buffer (CALEX\u0026reg; Cap device, B\u0026Uuml;HLMANN Laboratories AG, Sch\u0026ouml;nenbuch, Switzerland). Levels of calprotectin in the patients' fecal extracts were measured using particle-enhanced turbidimetric immunoassay (PETIA; BUHLMANN fCAL\u0026reg; turbo, B\u0026Uuml;HLMANN Laboratories AG, Sch\u0026ouml;nenbuch, Switzerland) on the Optilite\u0026reg; automated analyzer (Binding site, Birmingham, UK). Results were reported in micrograms/gram of stool.\u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eThe exploratory study was designed to include 20 participants in 4 groups: iRBD, PD within less than 5 years from diagnosis, PD with 5 to more years from diagnosis, and healthy (non-PD, non iRBD) volunteers. The total sample size of 80 participants was deemed sufficient to reach exploratory conclusions. Continuous parameters (expression levels of cytokines, inflammation and gut permeability markers) were compared across groups (iRBD, PD and controls) using Kruskall-Wallis nonparametric test. No imputation for missing values was used. Linear correlations between two continuous parameters were estimated using Pearson\u0026rsquo;s correlation and tested towards 0 (two-tailed) for normal values (all participants), and Spearman\u0026rsquo;s correlation in subgroups analysis. Associations between categorical variables (constipation, straining during defecation), inflammation and permeability parameters were tested using Wilcoxon-Mann-Whitney test. All statistical analyses were performed using R Studio software, version 4.4.2 from the R Foundation for Statistical Computing (Vienna, Austria). The level of statistical significance was set at 5% for all analyses. No adjustment for multiplicity was used.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eInclusions\u003c/p\u003e\u003cp\u003eSeventy-five participants were enrolled between December 2020 and October 2023, 20 patients with iRBD, 34 patients with PD and 21 healthy controls. One control participant was subsequently excluded because no biopsies were taken (withdrew consent before colonoscopy).\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePopulation Description\u003c/h2\u003e\u003cp\u003eDemographic and clinical characteristics, including motor and cognitive screening scores are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. There were no differences in demographics between groups. Among patients with PD, 15 individuals (44%) had probable RBD, as indicated by an RBDSQ score\u0026thinsp;\u0026ge;\u0026thinsp;5. MoCA scores were lower in patients with iRBD compared to patients with PD. As expected, both patients with iRBD and PD had higher scores on the UPDRS part III (both total and axial subscore), RBDSQ, and the Rome IV constipation questionnaire as well as lower scores on the Sniffin\u0026rsquo; Sticks Identification Test when compared to controls. Patients with PD had higher scores on the UPDRS part III (both total and axial subscore) in comparison to patients with iRBD. MMSE (on pairwise comparisons) and SCOPA-Aut did not differ between groups.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003ePatient demographics and clinical characteristics.\u003c/b\u003e iRBD: isolated rapid eye movement sleep behavior disorder; MMSE: Mini Mental State Examination; MoCA: Montreal Cognitive Assessment; PD: Parkinson\u0026rsquo;s disease; RBDSQ: RBD screening questionnaire; SCOPA-AUT: Scale for Outcomes in Parkinson's disease for Autonomic symptoms; SSIT: Sniffin\u0026rsquo; Sticks Identification 12-Test; UPDRS part III total: MDS-UPDRS III Movement Disorder Society\u0026rsquo;s Unified Parkinson\u0026rsquo;s Disease Rating Scale Part III, N/A not available.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eiRBD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eControls\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale/female (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16/4 (80/20)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24/10 (71/29)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14/6 (70/30)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge, y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e68.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e63.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e67.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody mass index, kg/m\u0026sup2;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSymptom duration, y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUPDRS part III total (0-132)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19.2\u0026thinsp;\u0026plusmn;\u0026thinsp;11.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001 iRBD vs C, PD vs C, iRBD vs PD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUPDRS part III axial (0\u0026ndash;36)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001 iRBD vs C, PD vs C, iRBD vs PD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRBDSQ (0\u0026ndash;10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001, iRBD vs C, PD vs C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMMSE (0\u0026ndash;30)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e29.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMoCA (0\u0026ndash;30)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.002, iRBD vs PD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSSIT (0\u0026ndash;12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001, iRBD vs C, PD vs C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCOPA-Aut (0\u0026ndash;69)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;10.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConstipation Rome IV (no., %)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11 (55)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13 (38)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001, iRBD vs C, PD vs C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eIL-1β and IL-8 levels in colonic biopsies are higher in iRBD patients compared to controls\u003c/h2\u003e\u003cp\u003eFirst, we used qPCR to analyze the expression levels of TNF-α, IL-1β, IL-8, IL-6, IFN-γ and interleukin-10 (IL-10) in colonic biopsies from patients with iRBD and PD as well as from controls. This panel of cytokines was selected because it has been consistently associated with alteration in gut permeability\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. mRNA expression levels of IL-1β and IL-8 were elevated in both patients with iRBD (p\u0026thinsp;=\u0026thinsp;0.02 and p\u0026thinsp;=\u0026thinsp;0.04, respectively) and patients with PD compared with controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for both cytokines, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). No significant differences in IL-1β or IL-8 expression were observed between the iRBD and PD groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). TNF-α expression was increased in patients with PD compared with controls (p\u0026thinsp;=\u0026thinsp;0.006), but not in the iRBD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The mRNA expression levels of IL-6, IFN-γ and IL-10 did not differ across groups (p\u0026thinsp;=\u0026thinsp;0.93, p\u0026thinsp;=\u0026thinsp;0.4 and p\u0026thinsp;=\u0026thinsp;0.23, respectively) (Supplementary Fig.\u0026nbsp;2a). Except for TNF-α expression, which was higher in men with iRBD (1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 vs 0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 in women, p\u0026thinsp;=\u0026thinsp;0.049), no difference in cytokine levels was observed between men and women (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe mRNA expression levels for IL-1β and IL-8 were strikingly variable among iRBD and PD patients; some showed levels comparable to controls while others showed a 3- to 6-fold increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). We therefore performed a correlation analysis of IL-1β and IL-8 mRNA expression levels to determine whether these cytokines are upregulated in the same individuals. IL-1β and IL-8 expression positively correlated in both iRBD and PD patient groups (r\u0026thinsp;=\u0026thinsp;0.78; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for iRBD; r\u0026thinsp;=\u0026thinsp;0.92; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for PD; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In addition, a correlation between IL-1β and TNF-α (r\u0026thinsp;=\u0026thinsp;0.35, p\u0026thinsp;=\u0026thinsp;0.02) was observed in the PD group (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\u003cp\u003eIL-1β, IL-8, IL-6 and IL-10 mRNA levels were positively associated with UPDRS Part III total and axial scores (see Supplementary Table\u0026nbsp;2 for details), while IL-10 was negatively correlated with olfaction scores in the entire population. However, these correlations were no longer significant when we performed subgroup analyses. In the PD group, IL-8 mRNA levels were positively associated with SCOPA-Aut (p\u0026thinsp;=\u0026thinsp;0.04, r\u0026thinsp;=\u0026thinsp;0.36). No significant correlations were found between cytokine levels and age, symptom duration, UPDRS motor and axial scores, MMSE, MoCA, olfaction score, or SCOPA-Aut in the iRBD group.\u003c/p\u003e\u003cp\u003eConstipation, as evaluated by Rome-IV criteria, was only observed in patients with PD or iRBD (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and was not associated with changes in cytokine expression levels in those patients (Supplementary Table\u0026nbsp;2). Straining during defecation (Rome IV criteria), which occurred in 32 participants (iRBD n\u0026thinsp;=\u0026thinsp;13, PD n\u0026thinsp;=\u0026thinsp;16, controls n\u0026thinsp;=\u0026thinsp;3) was associated with increased expression levels of IL-8 in the entire study population (p\u0026thinsp;=\u0026thinsp;0.04); this relationship was driven more by controls (p\u0026thinsp;=\u0026thinsp;0.004) than by patients with PD (p\u0026thinsp;=\u0026thinsp;0.39) or with iRBD (p\u0026thinsp;=\u0026thinsp;0.44) (Supplementary Fig.\u0026nbsp;4a).\u003c/p\u003e\u003cp\u003eEnteric glial cells, which are involved in the regulation of intestinal permeability\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, respond to gut inflammation by adopting a \u0026lsquo;reactive phenotype\u0026rsquo;\u003csup\u003e20,38\u003c/sup\u003e. This phenotypic switch is associated with the upregulation of several glial markers including GFAP\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e (which includes GFAPκ, the main isoform expressed by enteric glial cells\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e), S100β\u003csup\u003e40\u003c/sup\u003e, and Sox10\u003csup\u003e41\u003c/sup\u003e. qPCR analysis of colonic biopsies revealed no differences in the expression levels of reactive glial markers were between groups (p\u0026thinsp;=\u0026thinsp;0.14 for GFAPκ, p\u0026thinsp;=\u0026thinsp;0.95 for S100β and p\u0026thinsp;=\u0026thinsp;0.98 for Sox10) (Supplementary Fig.\u0026nbsp;2b). No associations were found between glial markers and sex, age, BMI, symptom duration, UPDRS motor and axial scores, MMSE, MoCA, olfaction score, SCOPA-Aut, or constipation (Rome IV criteria, Supplementary Table\u0026nbsp;2). However, straining during defecation (Rome IV criteria) was associated with higher expression levels of Sox-10 in patients with PD (p\u0026thinsp;=\u0026thinsp;0.02, Supplementary Fig.\u0026nbsp;4b).\u003c/p\u003e\u003cp\u003eFecal calprotectin levels, which were measured in a subset of participants (14 iRBD, 25 PD and 19 controls), did not differ between groups (158\u0026thinsp;\u0026plusmn;\u0026thinsp;32 \u0026micro;g/g, 132\u0026thinsp;\u0026plusmn;\u0026thinsp;37 \u0026micro;g/g and 101\u0026thinsp;\u0026plusmn;\u0026thinsp;26 \u0026micro;g/g, respectively, p\u0026thinsp;=\u0026thinsp;0.14, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eColonic barrier function and integrity are comparable between subjects with iRBD and controls\u003c/h2\u003e\u003cp\u003eTo assess intestinal barrier function and integrity, we evaluated colonic permeability and analyzed the expression levels of key TJ proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The para- and transcellular permeability of colonic biopsies from iRBD, PD and control subjects were measured in Ussing chambers using sulfonic acid/dextran and HRP, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). No differences in sulfonic acid, dextran or HRP flux were observed between groups (p\u0026thinsp;=\u0026thinsp;0.33, p\u0026thinsp;=\u0026thinsp;0.16, p\u0026thinsp;=\u0026thinsp;0.05, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). No associations were found between permeability measures and demographic or clinical characteristics, or with mRNA levels of cytokines and glial markers (Supplementary Table\u0026nbsp;3). No differences in the expression levels of the four main TJ proteins- ZO-1, occludin, claudin-1 claudin-2- were observed between groups (p\u0026thinsp;=\u0026thinsp;0.20, p\u0026thinsp;=\u0026thinsp;0.63, p\u0026thinsp;=\u0026thinsp;0.29 and p\u0026thinsp;=\u0026thinsp;0.19, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, c).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSeveral studies have reported abnormalities in gut inflammatory markers and intestinal barrier in patients with overt PD (reviewed in\u003csup\u003e14,42\u003c/sup\u003e). Here, we have used sigmoid colon biopsy specimens from patients with iRBD and compared them to samples from patients with PD and controls to show for the first time that low-grade intestinal inflammation is present in prodromal PD. Among the cytokines and glial markers tested, only IL-8 and IL-1β were augmented in the gut of iRBD patients. In addition to IL-1β and IL-8, TNF-α gene expression was significantly increased in GI biopsies from PD patients. IL-1β is a pro-inflammatory cytokine primarily produced by monocytes, macrophages and dendritic cells\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, whereas, IL-8 is a powerful chemoattractant for neutrophils and is commonly released in the gut by macrophages and epithelial cells\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. However, it is unsurprising, that these two cytokines are concomitantly upregulated, as IL-1β tightly regulates IL-8 expression in various cell types, including intestinal epithelial cells\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The strong correlation between IL-1β and IL-8 expression observed in both iRBD and PD patients supports the hypothesis that a shared underlying inflammatory pathway is activated early in disease progression in certain individuals. These results are consistent with our previous study, in which we found increased expression levels of both IL-1β and TNF-α expression in colon biopsies from subjects with symptomatic PD versus controls (IL-8 expression was not measured)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Furthermore, our data corroborate other publications showing increased TNF-α and IL-1β levels in stool samples from PD patients compared to controls \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. We observed higher TNF-α levels in men with iRBD. Although limited data are available on sex differences in iRBD\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, the higher level of gut inflammation observed in men with prodromal synucleinopathy is consistent with previously reported sex differences in immune reactivity and PD vulnerability\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Fecal calprotectin, an established marker of intestinal inflammation, did not differ between groups. This discordance may indicate that immune activity in iRBD and PD is highly localized or below the detection threshold of this assay.\u003c/p\u003e\u003cp\u003eInterestingly, we observed a significant correlation between IL-8 expression and straining during defecation. Unexpectedly, the strongest association was observed in the control group. While straining is generally regarded as a benign functional symptom, our findings suggest that altered defecatory function may be associated with subclinical gut immune activation, even in individuals without neurological symptoms. One possible interpretation is that straining could be an early marker of gut dysfunction linked to neurodegenerative disease, given some epidemiological evidence linking constipation with an increased risk of PD\u003csup\u003e49\u003c/sup\u003e. An alternative hypothesis is that the upregulation of IL-8 in association with straining in controls may reflect a pathological change in the gut of individuals without neurodegenerative pathological features who are subjected to mechanical stress and remodeling, as observed in patients with spinal cord injury\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. We observed a positive correlation between enteric IL-8 mRNA levels and dysautonomic symptoms in the PD group, indicating a widespread pattern of peripheral alterations\u0026mdash;including in the gut\u0026mdash;in some PD patients. However, in a previous study, no association was found between dysautonomic symptoms and colonic alpha-synucleinopathy\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Unlike other observations based on blood or CSF samples in patients with PD, we did not observe any correlations between inflammatory cytokines and the motor or cognitive phenotype. Previous reports have associated worse motor function with higher blood levels of IL-6, while some studies also demonstrated associations with higher blood levels of IL-1 β or TNFα, or higher CSF levels of IL-6 and IL-8 (reviewed in\u003csup\u003e52\u003c/sup\u003e). Similarly, altered cognitive function or dementia has been correlated with higher blood levels of IL-6, TNF-α, or higher CSF levels of IL-6 or IL-8 (reviewed in\u003csup\u003e52\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eAlthough IL-1β is a known modulator of intestinal barrier function\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, we could not find evidence of intestinal barrier dysfunction and/or alteration in iRBD and PD patients. Beyond the present work, 5 other studies have evaluated the intestinal barrier function in PD and they have provided conflicting results \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. A recent review has discussed this inconsistency in detail, attributing it primarily to small sample sizes and methodological heterogeneity across studies\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In this study, intestinal permeability was assessed using a validated method (Ussing chambers)\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e in a relatively large number of subjects, and no differences were observed between iRBD, PD and controls. In line with this, no changes in the expression levels of TJs proteins were detected. The absence of significant findings underscores the importance of employing more sensitive methods to determine whether subtle intestinal barrier dysfunction occurs in PD and prodromal PD\u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMoreover, and in sharp contrast to previous reports, we found no evidence of increased expression of glial markers, including as GFAP and S-100β in the PD gut. Likewise, these markers were unchanged in iRBD. Although there is no definitive explanation for these diverging results, two points are worth discussing. First, symptomatic PD are clinically and pathologically heterogeneous\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, which can cause variability between studies. Second, prior studies analyzed glial markers expression in the GI tract of patients with advanced PD, whose mean disease duration ranged from 8 to 13 years\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. By contrast, our study included non-demented patients with a mean disease duration of 5.9 years. Third, autopsy studies have shown that levels of GFAP in the substantia nigra, caudate and putamen of subjects with PD were positively correlated with the extent of dopamine loss\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. It is therefore plausible that activation of GFAP-positive cells in both the CNS and peripheral autonomic networks represents a late event in PD. Regarding the correlations analyses between glial markers and clinical characteristics, the only significant relationship was observed is the positive association between straining during defecation and Sox10 expression in PD patients. While this finding may reflect local glial activation induced by mechanical stress, it also raises the possibility of reciprocal influences between specific motor disturbances in the colon and enteric glial homeostasis at an overt neurodegenerative stage.\u003c/p\u003e\u003cp\u003eTo our knowledge, this is the first study to investigate gut inflammation in patients with iRBD. A few studies have explored neuroinflammation in iRBD patients, showing alterations in the peripheral adaptive immune system, abnormal upregulation of inflammatory cytokine secretion in response to immune stimuli\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, and increased TNF-α levels in patients with iRBD and multiple phenoconversion risk markers\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Nevertheless, none of these studies investigated gut inflammation. Combined with our results, these findings raise the possibility that this immune activation may represent an early, modifiable event in the pathogenesis of synucleinopathies. This hypothesis was recently reinforced by a study showing that patients with inflammatory bowel disease are three times more likely to have probable RBD\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Given the increasing recognition of the role of gut-brain axis in PD, the therapeutic targeting of intestinal inflammation has emerged as a promising strategy to delay disease progression or prevent phenoconversion in individuals at risk. One promising anti-inflammatory approach for treating neurodegenerative diseases involves glucagon-like peptide-1 receptor agonists (GLP-1RA). GLP-1RA attenuate T-cell-mediated gut and systemic inflammation, by reducing intestinal cytokine release and enhancing epithelial barrier integrity\u003csup\u003e\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. This makes GLP-1RA particularly attractive candidates for modulating early gut-driven inflammation in PD. Dietary modulation, prebiotics, and microbiota-targeted interventions such as fecal microbiota transplantation have been effective in preclinical models. Some early clinical data suggest that these therapeutic strategies may improve PD symptoms\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Our findings show that IL-1β and IL-8 are already upregulated in the colon of individuals with iRBD; therefore, it is plausible that immune-based interventions administered at the prodromal stage of PD could attenuate neuroinflammatory cascades and limit propagation of α-synuclein pathology along the gut\u0026ndash;brain axis, delaying or preventing conversion to overt PD.\u003c/p\u003e\u003cp\u003eSeveral limitations must be acknowledged. Our patient cohorts were relatively small, although adequate for colonic biopsy studies. The cross-sectional design of the study precludes drawing conclusions about temporal progression. Although iRBD is a validated prodromal marker of PD, the iRBD group may include individuals at different stages of disease progression, or who may eventually develop dementia with Lewy bodies or, less commonly, multisystem atrophy, rather than PD. Detecting alpha-synuclein seeding in colonic biopsies would enable this group to be stratified, although the small sample size may prevent complex association analysis. Sampling was restricted to the colonic mucosa and submucosa, which may prevent the observation of focal changes in other GI segments (e.g., the myenteric plexus or rostral GI organs). Finally, we did not control for dietary and environmental factors, which may contribute to inter-individual variability.\u003c/p\u003e\u003cp\u003eMore broadly, our study provides insights into the potential role of the digestive tract, and more specifically digestive inflammation, in PD. We showed that, although low-grade digestive inflammation is present in some individuals with iRBD, its extent is similar to that observed in established PD. This evidence challenges the notion that digestive inflammation represents an early pathogenic event in PD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics Statement\u003c/h2\u003e\u003cp\u003e The study protocol for the sampling of sigmoid biopsies was approved by the local Committee on Ethics and Human Research (Comit\u0026eacute; de Protection des Personnes Ouest VI), and registered on ClinicalTrials.gov (identifier NCT04652843)\u003c/p\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eA.C. is an employee of PiLeJe Laboratoire. Other authors declare no financial or non-financial competing interests.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eNantes University Hospital was the study promoter. This work was supported by a grant from Nantes University Hospital (Appel d'offre interne 2019, RC19_0401) and France Parkinson (2019). France Parkinson played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. Work at Inserm U1235 on intestinal permeability is supported in part by the \u0026lsquo;F\u0026eacute;d\u0026eacute;ration pour la Recherche sur le Cerveau (FRC)\u0026rsquo;, the \u0026lsquo;Association AMADYS\u0026rsquo;, \u0026lsquo;Vaincre Parkinson\u0026rsquo; and by an EU Joint Programme - Neurodegenerative Disease Research (JPND) supported project (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.jpnd.eu\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.jpnd.eu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Simon Lassoz\u0026eacute;, Lo\u0026iuml;c Sellier-Montaigne and Adrien de Guilhem de Lataillade are recipients of \u0026lsquo;ann\u0026eacute;e recherche\u0026rsquo; from CHU de Nantes.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e**LSM** : formal analysis, data curation, investigation, writing-review \u0026amp;amp; editing; **SL** : formal analysis, data curation, investigation; **AdGdL** : data curation, investigation, supervision; **TO** : supervision, validation; data curation, investigation; **MAV** : statistical design and analysis, writing \u0026ndash;\u0026ndash; original draft,; **KBN** : analysis of calprotectin levels in feces, writing \u0026ndash;\u0026ndash; original draft; **MRD** : validation, supervision, methodology; **TD:** supervision, validation; data curation, investigation **; SLD** : patient recruitment, clinical data collection and characterization; **IJA** : patient recruitment, clinical data collection and characterization; **EC** : investigation, colonoscopy and biopsy processing; **QM** : investigation, colonoscopy and biopsy processing; **MN:** validation, supervision, writing-review \u0026amp;amp; editing; **GM:** validation, supervision, writing-review \u0026amp;amp; editing; **AC:** methodology, data interpretation; **PD** : writing \u0026ndash;\u0026ndash; original draft, visualization, validation, supervision, resources, project administration, methodology, investigation, funding acquisition, formal analysis, data curation, conceptualization. **LLV** : writing \u0026ndash;\u0026ndash; original draft, visualization, validation, supervision, resources, project administration, methodology, investigation, funding acquisition, formal analysis, data curation, conceptualization\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank Patrice Chauveau, Aziz Ouach, Maelle Ningre and Marion Rigot for their administrative and technical support, data management and monitoring, and participants for taking part in the study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLionnet, A. \u003cem\u003eet al.\u003c/em\u003e Does Parkinson\u0026rsquo;s disease start in the gut? \u003cem\u003eActa Neuropathol\u003c/em\u003e 135, 1\u0026ndash;12 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChapelet, G., Leclair-Visonneau, L., Clairembault, T., Neunlist, M. \u0026amp; Derkinderen, P. 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K. \u003cem\u003eet al.\u003c/em\u003e Central glucagon-like peptide 1 receptor activation inhibits Toll-like receptor agonist-induced inflammation. \u003cem\u003eCell Metabolism\u003c/em\u003e 36, 130\u0026ndash;143.e5 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheng, Y. \u003cem\u003eet al.\u003c/em\u003e Efficacy of fecal microbiota transplantation in patients with Parkinson\u0026rsquo;s disease: clinical trial results from a randomized, placebo-controlled design. \u003cem\u003eGut Microbes\u003c/em\u003e 15, 2284247 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-parkinsons-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjparkd","sideBox":"Learn more about [npj Parkinson's Disease](http://www.nature.com/npjparkd/)","snPcode":"41531","submissionUrl":"https://submission.springernature.com/new-submission/41531/3","title":"npj Parkinson's Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"idiopathic REM sleep behavior disorder, Parkinson’s disease, GI inflammation, intestinal permeability, enteric glial cells, tight junctions","lastPublishedDoi":"10.21203/rs.3.rs-7829903/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7829903/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSome recent studies suggest that gastrointestinal inflammation could play a role in the development of Parkinson\u0026rsquo;s disease (PD) by increasing intestinal permeability and triggering early neuropathological changes in the gut. Therefore, we set out to analyze gut inflammation as well as intestinal barrier function and integrity in patients with isolated REM sleep behavior disorder (iRBD), a prodromal stage of PD. Sigmoid colon biopsy specimens from patients with iRBD (n\u0026thinsp;=\u0026thinsp;20), patients with PD (n\u0026thinsp;=\u0026thinsp;34) and controls (n\u0026thinsp;=\u0026thinsp;20) were analyzed by qPCR to measure the expression levels of proinflammatory cytokines and enteric glial markers. Fecal calprotectin levels were also measured to further assess gastrointestinal inflammation. Gut permeability was evaluated in biopsies mounted in Ussing chambers, and the integrity of the intestinal epithelial barrier was assessed by analyzing the expression of tight junction proteins by western blot. We found that the mRNA expression levels of interleukin-1β and interleukin-8 were increased in both patients with iRBD and PD relative to controls; the expression of TNF-α was also higher in PD but not in iRBD patients. We did not observe any differences in colonic permeability, tight junction proteins expression and calprotectin levels between iRBD, PD and control participants. Our study is the first to characterize the inflammatory profile in the gut in iRBD. As a whole, our findings provide evidence that enteric inflammation is present at a moderate level in prodromal PD, not higher than in individuals with established PD, and without concurrent changes in intestinal permeability.\u003c/p\u003e","manuscriptTitle":"Elevated levels of colonic interleukin-1beta and interleukin-8 in isolated REM sleep behavior disorder without associated changes in permeability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 01:20:52","doi":"10.21203/rs.3.rs-7829903/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-02T20:16:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-01T23:09:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246263085674325765024727230868955446166","date":"2025-11-17T19:20:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-08T15:22:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114094710221958280419563039373995726237","date":"2025-10-24T14:09:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-22T14:05:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-20T04:15:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-19T11:00:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Parkinson's Disease","date":"2025-10-10T19:48:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-parkinsons-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjparkd","sideBox":"Learn more about [npj Parkinson's Disease](http://www.nature.com/npjparkd/)","snPcode":"41531","submissionUrl":"https://submission.springernature.com/new-submission/41531/3","title":"npj Parkinson's Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ce90e686-2e1b-488d-b311-4aa891de9c24","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57273961,"name":"Health sciences/Diseases"},{"id":57273962,"name":"Health sciences/Gastroenterology"},{"id":57273963,"name":"Biological sciences/Immunology"},{"id":57273964,"name":"Health sciences/Neurology"},{"id":57273965,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-05-04T16:47:41+00:00","versionOfRecord":{"articleIdentity":"rs-7829903","link":"https://doi.org/10.1038/s41531-026-01340-9","journal":{"identity":"npj-parkinsons-disease","isVorOnly":false,"title":"npj Parkinson's Disease"},"publishedOn":"2026-04-27 15:56:54","publishedOnDateReadable":"April 27th, 2026"},"versionCreatedAt":"2025-11-04 01:20:52","video":"","vorDoi":"10.1038/s41531-026-01340-9","vorDoiUrl":"https://doi.org/10.1038/s41531-026-01340-9","workflowStages":[]},"version":"v1","identity":"rs-7829903","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7829903","identity":"rs-7829903","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}