# Advanced integrated human in vitro approach to explore gut-brain barrier interactions with gut microbiota metabolites and pesticides

Maria Abou Diwan, Juliette Poiriez, Avénie Huet, Claire Joly Condette, Emmanuel Sevin, Stéphane Delanaud, Halima Ouadid-Ahidouch, Véronique Bach, Fabien Gosselet, Hafida Khorsi-Cauet, et al.

► To cite this version:

Maria Abou Diwan, Juliette Poiriez, Avénie Huet, Claire Joly Condette, Emmanuel Sevin, et al. Advanced integrated human in vitro approach to explore gut-brain barrier interactions with gut microbiota metabolites and pesticides. Ecotoxicology and Environmental Safety, 2026, 309, 10.1016/j.ecoenv.2025.119539. hal-05421671

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# Advanced integrated human in vitro approach to explore gut-brain barrier interactions with gut microbiota metabolites and pesticides

Maria Abou Diwan<sup>a,b,1</sup>, Juliette Poiriez<sup>a,1</sup>①, Avénie Huet<sup>b,c</sup>, Claire Joly Condette<sup>b</sup>, Emmanuel Sevin<sup>a</sup>, Stéphane Delanaud<sup>b</sup>, Halima Ouadid-Ahidouch<sup>c</sup>, Véronique Bach<sup>b</sup>, Fabien Gosselet<sup>a</sup>, Hafida Khorsi-Cauet<sup>b,2</sup>, Pietra Candela<sup>a,*2</sup>

## ARTICLE INFO

### Keywords:

Human in vitro models
SHIME® model
Gut microbiota
Intestinal barrier
Blood-brain barrier
Organophosphates

## ABSTRACT

The gut microbiota (GM) plays a central role in host barrier homeostasis. Alterations in GM due to chronic dietary pesticide residues can have far-reaching consequences on human health. Although in vitro models are increasingly used to study organophosphate effects, especially Chlorpyrifos (CPF), accurately modeling complex GM-host interactions remains challenging. Here we investigated how CPF-altered GM metabolites influence epithelial and brain barrier integrity. An integrated human-relevant in vitro approach combining three complementary models: the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) under control and CPF-exposed conditions to evaluate both short-term and long-term effects (15 and 30 days), a Caco-2-based intestinal barrier (IB) and a blood-brain barrier (BBB) co-culture. Two parallel experimental setups (SHIME-IB and SHIME-BBB) were developed: SHIME®-derived microbial metabolites and pesticide residues supernatants were directly applied to each barrier model for 24 h. Confocal imaging revealed discontinuous localization of tight junction proteins (occludin, claudin-5 and ZO-1) without concomitant increase in FITC-dextran apparent permeability or overt cytotoxicity. This structural disorganization occurred despite unchanged transcriptional expression of tight junctions, except for a significant reduction of CLDN5 mRNA in the BBB at CPF15, suggesting early molecular signals. In contrast, we showed a selective decrease in P-GP (P-glycoprotein) expression in both barriers, confirmed at the protein level. Concurrently, IL-8 (Interleukin-8) secretion increased markedly in IB, particularly at CPF15, highlighting its potential as an early biomarker chemokine-driven inflammatory activation. This integrated approach provides an ethical model to study environmental contaminants' effects on human barriers, revealing microbiota-mediated disruption by CPF.

## 1. Introduction

The intricate relationship between the gut and the brain, often referred to as the gut-brain axis, has garnered significant attention in recent years. This bidirectional communication system involves the gut microbiota (GM), a diverse community of microorganisms residing in the gut, and the barriers that separate it from the rest of the body (Morais et al., 2021). These barriers include the intestinal barrier (IB), which limits the absorption of food contaminants, and the blood-brain barrier

(BBB), which restricts the entry of harmful molecules and cells into the brain (Abou Diwan et al., 2023; Daneman and Rescigno, 2009). This axis of communication was long overlooked in medical and scientific research but has received increasing recognition in recent decades, as extensive studies have uncovered its critical role in both mental and physical health (Dinan and Cryan, 2017; Long-Smith et al., 2020; Cryan et al., 2019).

Recent animal studies indicate that disruptions in the GM due to contaminants, such as pesticides, can impact human health by altering

<footnote><sup>a</sup> Laboratoire de la Barrière Hémato-Encéphalique (LBHE), UR 2465, Université d'Artois, Lens 62300, France</footnote>

<footnote><sup>b</sup> PERITOX—Pépinatalité et Risques Toxiques—UMR I 01 UPJV/INERIS, Centre Universitaire de Recherche en Santé, CURS-UPJV, Université Picardie Jules Verne, Amiens 80054 CEDEX 1, France</footnote>

<footnote><sup>c</sup> Laboratoire de physiologie cellulaire et moléculaire (LPCM), UR UPJV 4667, Université Picardie Jules Verne, UPJV, Amiens 80039, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: pietra.candela@univ-artois.fr (P. Candela).</footnote>

<footnote n="1">¹ First co-authors contributed equally to this work</footnote>

<footnote n="2">² These authors contributed equally to this work.</footnote>
the IB and potentially causing long-term effects on the BBB (Djekkoun et al., 2022; Guibourdenche et al., 2021a; Jin et al., 2016; Zhao et al., 2016). Among these pesticides, the organophosphate insecticide Chlorpyrifos (CPF) is widely used and known for its environmental persistence, which has garnered significant attention (EFSA, 2019, 2021; Supreeth and Raju, 2017). While its well-known neurotoxic effects, primarily through acetylcholinesterase inhibition, are well-documented (Cresto et al., 2023; Miller et al., 2021), recent studies indicate that CPF may also impair GM diversity and function (Reygner et al., 2016a; Joly Condette et al., 2022; Onchoi et al., 2020; Gama et al., 2022; Perez-Fernandez et al., 2020). These microbial alterations can impair the intestinal immune response, compromise barrier integrity, and potentially exacerbate neuroinflammation by destabilizing the BBB (Parran et al., 2005; Matsuzaki et al., 2023). Despite these findings, it is still unclear how GM dysregulation by CPF leads to downstream effects on both the IB and BBB.

In light of this, there is a pressing need to explore the mechanisms underlying these interactions in order to better understand how CPF may impact both gut and brain barriers.

In this context, there is a growing need for ecotoxicological approaches that integrate mechanistic human-relevant in vitro models to assess the systemic effects of environmental contaminants on human health. While advances have been made in modeling the GM-brain axis, progress in developing in vitro models that closely replicate in vivo conditions remains limited (Moysidou and Owens, 2021). Consequently, creating a protocol to simulate and analyze the gut-brain axis in vitro for human applications remains a significant challenge (Loh et al., 2024).

This study addresses this gap by employing a multi-model in vitro strategy to investigate the effects of CPF-induced GM modifications on biological barriers. We used three well-established human in vitro models to investigate the impact of GM on barriers: the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®), a Caco-2 based IB model, and a human BBB co-culture model. This multi-model approach aims to better replicate the complexity of the GM-brain axis and provide more reliable insights into the effects of GM changes on biological barriers in human health. By integrating ecotoxicological exposure scenarios with quantitative cellular endpoints such as barrier permeability, cytokine secretion, and tight junction integrity, this study contributes to the development of mechanistic tools for environmental safety assessment.

The SHIME® is a promising and well-established model that replicates the human GM (Van de Wiele et al., 2015; Molly et al., 1993) and allows for the study of microbial composition (aerobes and anaerobes), metabolic activities (Short-chain fatty acids SCFAs), and gut interactions. This model provides valuable insights into how the GM influences health and disease, including its role in immune modulation, IB integrity, and metabolic processes (Joly Condette et al., 2022; Šuligoj et al., 2020; Di Vincenzo et al., 2023). Moreover, it has been successfully employed in studies investigating the impact of environmental contaminants, such as pesticides, on gut health and GM dynamics by quantifying SCFAs and pesticide metabolites (Joly Condette et al., 2022; Reygner et al., 2016b; Abou Diwan et al., 2025). Importantly, the SHIME® enables chronic exposure studies using pesticide concentrations that are environmentally relevant and reflective of potential human exposure levels, thereby enhancing the ecological and toxicological relevance of the findings. This makes it a powerful tool for exploring how pesticides like CPF may impact gut health and, in turn, influence the gut-brain axis.

For investigating IB function, we used an optimized version of the Caco-2-based IB model, which accelerates cell culture processes, providing a more efficient way to study IB integrity (Sevin et al., 2019).

The BBB model, developed in our laboratory, consists of co-culturing human brain-like endothelial cells (hBLEC) derived from CD34 + stem cells from human umbilical cord blood with human brain pericytes. This system displaying BBB properties close to those observed in vivo (Cecchelli et al., 2014), forms two distinct compartments: the luminal

compartment, containing endothelial cells, and the abluminal compartment, containing human pericytes. It allows for the assessment of toxicity and the passage of compounds and cells across the BBB, as demonstrated in our numerous publications (Versele et al., 2022a; Lucana et al., 2024; Porkoláb et al., 2024).

These models are typically used individually, each providing insights into different aspects of barrier function. The SHIME® model focuses on studying GM and its impact on gut health, while Caco-2 cells are primarily used to investigate IB integrity, and the BBB model is essential for exploring neurological and systemic effects.

In our previous studies, a SHIME-IB-BBB configuration was published. It involves a stepwise inoculation process: SHIME® samples are first applied to the luminal side of the IB model for 4 h, followed by the inoculation of the abluminal supernatants onto the BBB model for an additional 4 h. This setup allows for the sequential exposure of microbial metabolites together with bacterial cells from the gut to the brain to study the bacterial translocation across barriers (Abou Diwan et al., 2025). This setup is essential for understanding how gut-derived metabolites and bacteria, including those influenced by pesticide exposure, affect both intestinal and BBB functions in a more integrated, whole-body context.

In contrast, in this current study, we combined these models into a configuration adapted to address different research questions. We employed a direct exposure configuration SHIME-IB and SHIME-BBB to isolate and evaluate the local effects of gut-derived metabolites on each barrier independently. In this setup, filtered SHIME® supernatants (devoid of bacteria) are applied directly to either the IB or BBB models for 24 h to examine the specific, localized interactions at each barrier, providing more detailed insights into how these gut metabolites and pesticide residues influence barrier integrity in a more targeted manner.

Our primary objective is to validate this integrated approach by assessing the stability of the SHIME® model and evaluating the permeability, viability and IL-8 (Interleukin-8) secretion of Caco-2 cells and hBLEC after exposure to SHIME®-derived samples. Additionally, we aim to examine subsequent impact of CPF-induced GM dysbiosis on the barriers using this model setup.

A key novelty of this research is the integration of distinct human in vitro models, the SHIME® model, the IB model, and the BBB model, to create a comprehensive system for studying the gut-brain axis. By simulating the complex interactions between GM, IB, and BBB, this integrated approach enhances our understanding of gut-brain axis dynamics and the health risks posed by environmental contaminants and paves the way for developing new therapeutic strategies to protect barrier integrity.

## 2. Materials and methods

### 2.1. *In vitro* models

#### 2.1.1. SHIME® model

The Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) (Van de Wiele et al., 2015) was developed in 1993 (Molly et al., 1993). This dynamic simulator was designed to replicate the human gastrointestinal tract (GIT), including the stomach (reactor 1), duodenum/jejunum (R2), ileum/caecum (R3), proximal (R4), transverse (R5) and distal colon (R6). The SHIME® model evolved from earlier single stage chemostats and semi-continuous reactors, which were limited in their ability to maintain stable microbial communities over time. By incorporating multiple compartments, the SHIME® model developed at the PeriTox Laboratory currently includes six double-wall reactors and better simulates the complex and heterogeneous environment of the human GM, allowing for more accurate studies of microbial composition and metabolic activities (Joly Condette et al., 2022; Réquilé et al., 2018). Peristaltic pumps connect these fermenters that are maintained at a constant temperature of 37°C, with continuous monitoring of pH and redox potential. To simulate the GIT ecosystem and
provide nutrients for the intestinal flora, each reactor is filled with a nutritional medium (primary supply) before fecal inoculum, with a volume that corresponds to the real volume of each intestinal segment. This supply mainly contains a mineral mix, carbohydrates, protein sources, yeast, tween and mucins in water, adjusted to a pH between 2 and 2.5 (with 1 M HCl) and then sterilized at 121 °C for 20 min (Molly et al., 1993). Once the fecal inoculum is added, the system is activated to initiate pump operation. This nutritional medium is added to the stomach along with the pancreatic juice to the duodenum through a fill-and-draw system. The reactor's content is continuously stirred (Fig. 1).

The model initiation requires a human feces inoculum. This research targeted women individuals because the focus of this study is the unique GM composition of women. Our criteria of inclusion selected five healthy adults' volunteers: free of gastrointestinal symptoms and those who had not taken antibiotics in the six months preceding fecal collection. The collection of women feces from the Amiens University Hospital, with the help of Dr. Arthur Foulon, and their inoculation in the SHIME® were approved by the Ethical committee for non-interventional research (CERNI) of Picardy Jules Verne University (Evaluation number 2023-07-5/2023-27). For one SHIME® experiment, 50 g of feces are needed. For a TWIN-SHIME, a configuration that provides analysis of two different conditions or treatments of GM, 100 g of feces are needed if both treatments are tested on the same population (e.g. women of reproductive age).

### 2.1.2. System and GM monitoring

To assess the stability of the SHIME® model, we monitored key physiological parameters including temperature, pH, and redox voltage.

The system was maintained at a constant temperature of 37°C to simulate human body conditions. pH levels were measured at regular intervals (twice a day) using pH meters. Electrodes constantly detect pH and redox potential (,< -200 mV) guaranteeing anaerobic conditions in colon reactors (pH=5.5-6 in proximal, 6-6.4 in transverse and 6.4-6.8 in distal colon), ensuring they remain within the physiological range of human GM. Continuous monitoring and control of these environmental conditions is crucial for maintaining a stable and representative microbial community.

The stabilization phase of the GM of the SHIME® model has been extensively researched and is generally set between 10 and 20 days. During this phase, the GM adjusts to the conditions within the SHIME® compartments, forming a stable and representative microbial ecosystem. Studies have confirmed the SHIME® model's reliability in maintaining GM stability, showing that essential microbial populations and their metabolic functions reach a steady state within this period (Van de Wiele et al., 2015). In our previous published studies, we set and validated the stabilization phase of GM at 15 days (Joly Condette et al., 2022; Abou Diwan et al., 2025). This validation supports the SHIME® model as a dependable and reproducible method for long-term studies, effectively enabling the examination of GM interactions and reactions to various treatments: chronic exposure to pesticides.

### 2.1.3. Exposure of GM to pesticides

In this study, we employed a chronic exposure protocol to investigate the effects of CPF on GM and its downstream impact on biological barriers. Samples from the in vitro digestion were collected on the 15th day of stabilization, representing the control condition (CPF0). After a 15-day stabilization period of GM, daily exposure to the pesticide began.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Experimental design and in vitro methodology of the SHIME® model (Réquilé et al. 2018; Joly Condette et al. 2022).</figcaption></figure>
Extracts are then collected 15 days and 30 days post-exposure to study respectively short-term and long-term effects of CPF on GM and barriers. The sampling process was repeated three times in the final week of each period to allow statistical analysis. A daily aliquot of CPF stock solution (0.1 mg/mL) was added to the stomach reactor to achieve a nominal dose of 1 mg CPF per day. Practically, 10 mL of the 0.1 mg/mL stock solution (10 mL × 0.1 mg/mL = 1 mg) was added each day to the 200 mL stomach reactor feed (final nominal concentration in the stomach feed immediately after addition = 1 mg/200 mL = 0.0048 mg/mL = 5 µg/mL) (Joly Condette et al., 2022; Abou Diwan et al., 2025). For human-exposure context, 1 mg/day corresponds to ≈ 0.017 mg/kg/day for a 60-kg adult (1 mg ÷ 60 kg). This selected CPF dose (1 mg/day) was determined based on estimated dietary exposure levels, using data from ANSES (Joly et al., 2013) on pesticide residues commonly detected in food. While this value exceeds the former ADI (Acceptable Daily Intake) of 0.001 mg/kg/day previously proposed by EFSA, it remains within the range of potential real-world exposure scenarios reported in food residue monitoring studies (EFSA, 2019, 2021). Additionally, this concentration allows for the investigation of both immediate and cumulative effects of CPF on the GM and barrier function. The rationale for this exposure design, including dose selection and sampling strategy, is grounded in our previously published work (Abou Diwan et al., 2025), where we comprehensively characterized the CPF-induced perturbations in GM composition and metabolic activity and where CPF and its principal metabolites were quantified in the actual SHIME® samples (Abou Diwan et al., 2025). In that study, we employed both culture-based methods under aerobic and anaerobic conditions to measure the abundance of key microbial species and explore bacterial translocation across barriers, and gas chromatography to quantify microbial metabolites (for the same SHIME® samples of this present study). These findings confirmed the responsiveness and functionality of the SHIME® model under CPF exposure and provide the foundational context for the current work.

In the present methodological study, we focus on the application of CPF-exposed GM supernatants to advance in vitro models of the intestinal and blood-brain barriers. This approach allows us to isolate and examine the functional consequences of CPF-induced microbial variations on barrier integrity, building directly upon our previously validated GM exposure framework.

### 2.1.4. Intestinal barrier model

The protocol for culturing Caco-2 cells has been extensively described in previous studies by our colleagues (Sevin et al., 2019, 2013).

For this study, Caco-2 cells at passage 026 were initially seeded at a density of $0.4 \times 10^5$ cells in 25 cm² plastic flasks. These cells were maintained in a complete medium, which was replenished every two days. The medium used was Dulbecco's Modified Eagle's Medium (DMEM).

Caco-2 cells were incubated for three days until 80-90 % confluence, then trypsinized and reseeded in flasks. Upon reaching high density ($approx. 0.5 \times 10^5$ cells/cm²) after 5-6 days, they were transferred to 12-well plates with 0.4 µm Transwell filters. Cells were seeded at 100,000 cells/cm² insert filters. The cells were then cultured for 21 days in complete medium, with the medium being changed every two days.

### 2.1.5. Human in vitro blood-brain barrier model

The in vitro human BBB model we used provides a reliable framework for studying interactions at the BBB and CNS pharmacology. It also allows for a better understanding of transport mechanisms of molecules and cells and the effects of pharmacological or toxic agents on the human BBB. The BBB model was established using a co-culture system comprising human brain pericytes (hBPs) in the lower compartment and hBLEC on the 0.4 µm Transwell insert. The hBPs were provided by Professor Takashi Kanda's team at the Department of Neurology and Clinical Neuroscience, Yamaguchi University, Japan (Shimizu et al.,

2011). These cells were isolated following the Declaration of Helsinki guidelines, with the protocol approved by the ethics committee of the Medical Faculty at Yamaguchi University (IRB#: H18-033-6) (Menaceur et al., 2023).

The co-culture of endothelial cells (hBLEC) with pericytes permits replicating the specific cellular interactions of the BBB (Daneman et al., 2010; Sweeney et al., 2018). After thawing, hBPs (passages 15-25) were cultured in gelatin-coated Petri dishes using DMEM high glucose medium. In accordance with the consent and approval from the French Ministry of Higher Education and Research (CODECOH Number DC2011-1321), human hematopoietic stem cells were isolated from umbilical cord blood (Cecchelli et al., 2014; Heymans et al., 2020). Cells were cultured, sorted and differentiated into endothelial cells in EGM-2 medium containing VEGF (PeproTech Inc, Cranbury, NJ, USA). After six days, the co-culture was ready for incubation with SHIME® supernatants.

### 2.1.6. Experimental design of the human in vitro models

In the indirect configuration SHIME-IB-BBB (previously published), unfiltered SHIME® samples (1:5 dilution) were added on the luminal side of the IB model for 4 h after which the abluminal side was transferred to the BBB model to study the translocation of bacteria from the luminal to the abluminal side of the IB model and then to the BBB model (Abou Diwan et al., 2025). In contrast, in this current research, the direct configuration SHIME-IB and SHIME-BBB consists of adding centrifuged and filtered (using Startorius filters, Minisart, 0.2 µM, Germany) SHIME® supernatants (1:5 dilution) to the luminal side of the IB model and the luminal side of BBB model during 24 h (Fig. 2). Filtered samples do not contain bacteria but only bacterial metabolites to assess the effects of microbial metabolic activity on IB and BBB cells in different conditions.

In the direct combination, the supernatants were collected from reactors 4, 5, and 6 (R4, R5, R6), centrifuged, diluted at a 1:5 ratio, and then pooled in equal volumes to create a representative colon-derived mixture (MIX) for downstream application. The MIX was subsequently used to expose the IB epithelium cells (Caco-2 cells) and BBB hBLEC for 24 h. Here the study focuses on assessing bacterial metabolites effects on cells viability, permeability and tight junction integrity, including ZO-1 (Zonula Occludens) and occludin (ocln).

## 2.2. Permeability and viability of IB and BBB models

### 2.2.1. Cell viability

Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay as previously described (Sevin et al., 2019; Versele et al., 2022a), which measures mitochondrial metabolic activity as an indicator of cell health. Following 24 h treatment with SHIME® samples (1/5 dilution), the viability of hBLEC and Caco-2 cells was assessed using the MTT assay (Sigma-Aldrich, USA). For this assay, 0.5 mL of a 1 mg/mL MTT solution was incubated with the cells (apical compartment), along with 1.5 mL of Ringer-Hepes (RH) buffer in the basolateral compartment, until the formation of purple formazan crystals at 37°C and 5 % CO₂. These crystals form as a result of the reduction of MTT by mitochondrial succinate dehydrogenase in metabolically active cells.

After crystal formation, the solution was removed, and 250 µL of dimethyl sulfoxide (DMSO) was added per filter to solubilize the crystals. Absorbance was then measured at 560 nm using the spectophotometer (Synergy H1, Biotek) to calculate the optical density as a percentage relative to the control. Caco-2 cells and the hBLEC of the BBB co-culture model were incubated with filtered and centrifuged SHIME® supernatants for 24 h.

### 2.2.2. Permeability analysis

After 24 h of exposure to SHIME®, the luminal compartment was incubated with 0.5 mL of a 25 µM FD4 (4 kDa FITC-dextran) solution
<figure><img src="image_4.png" /><figcaption>Fig. 2. Experimental design of gut and brain human in vitro models.</figcaption></figure>

prepared in Ringer-Hepes buffer (RH; NaCl, KCl, CaCl2, MgCl2·6H2O, NaHCO3, Hepes, Glucose) for BBB but 100 µM FD4 solution for IB model, while the basolateral wells contained 1.5 mL of RH buffer. After one hour of incubation with the integrity marker, the contents of each filter were collected. The amount of FD4 was then determined by measuring fluorescence using a spectrophotometer (Synergy H1, BioTek). The excitation/emission wavelength used was 432 nm/538 nm. Based on a previously established calibration curve, the quantities of FD4 were determined from the Relative Fluorescence Units (RFU). The fluorescence values were then used to calculate the apparent permeability (P app) in cm/s of the hBLEC or Caco-2 cells. P app to FD4 was determined as previously published (Sevin et al., 2019). Triton X-100 (10 %) was used as a positive control for barrier disruption and cytotoxicity.

## 2.3. Immunofluorescence Analysis

After exposure of Caco-2 and hBLEC to SHIME® supernatants during 24 h, we evaluated cell layer integrity by immunostaining ZO-1 and occludin (in Caco-2 cells) and claudin-5 (specific to hBLEC cells), key proteins involved in maintaining barrier function. Cells were fixed with paraformaldehyde 4 %, permeabilized with Triton X-100 (0.1 %), and incubated with primary antibodies against ZO-1 (Invitrogen) and occlin (Invitrogen). After washing, cells were incubated for 1 h with fluorescently labeled secondary antibodies (Alexa Fluor™ 488 or 568 anti-rabbit, Invitrogen) ( versele et al., 2020). Biotek Cytation C10

Confocal Imaging Reader (Agilent, Santa Clara, California) microscope was used to analyze the expression and localization of these proteins in hBLEC and Caco-2 cells. Tight junction immunofluorescence was analyzed using the Cytation C10 Confocal Imaging Reader and ImageJ Java 1.8.0_345 (5 randomly selected fields per Transwell membrane and triplicates/condition were used for quantification).

## 2.4. RT-qPCR Analysis

### 2.4.1. Extraction and quantification of mRNA

After their respective treatments, endothelial cells and Caco-2 cells were rinsed with cold Ringer's H and lysed using RP1 lysis buffer (Macherey-Nagel GmbH & Co., Germany). Cellular lysates were processed according to the NucleoSpin RNA/Protein kit protocol (Macherey-Nagel GmbH & Co., Germany). Extracted RNA was quantified by spectrophotometry (Synergy H1, BioTek) by measuring absorbance at 260, 280, and 320 nm, corresponding to nucleic acids, proteins, and solvent contamination, respectively. RNA concentration (ng·µL⁻¹) was calculated using the Beer–Lambert law, and sample purity was assessed by the A260/A280 ratio; values ≈ 2.1 were considered indicative of pure RNA.

### 2.4.2. Reverse transcription

RNA samples meeting quality criteria were reverse-transcribed to complementary DNA (cDNA) using iScript™ 5 × SuperMix (Bio-Rad). Each reaction contained 250 ng RNA and reagents supplied in the
SuperMix, including reverse transcriptase, RNase inhibitors, random primers, dNTPs, and MgCl2. Reaction volumes were brought to 20 µL with nuclease-free water. Two controls were prepared for each run: a no-template control (NTC) where RNA was replaced by water to detect possible contamination, and a no-RT control to verify absence of genomic DNA in RNA samples. Reverse transcription was performed in an MJ Research thermocycler using the manufacturer's recommended program.

### 2.4.3. Quantitative PCR

Quantitative real-time PCR (qPCR) was used to measure expression of target genes (ZO-1, OCLN, CLDN5, BCRP Breast Cancer Resistance Protein, P-gp P-glycoprotein, ICAM-1) using gene-specific primer pairs (table 1 in *supplementary material 3*) designed with BLAST and ordered from Invitrogen (Massachusetts, USA). Primer efficiency was validated by standard curves prior to experimental runs. qPCR reactions were assembled using SsoFast™ EvaGreen® Supermix (Bio-Rad), which contains DNA polymerase, cofactors, dNTPs, and the EvaGreen fluorescent dye. Amplifications were performed on a CFX96 Real-Time System (Bio-Rad) for 40 cycles with an annealing temperature of 60 °C. Gene expression levels were calculated using the ΔΔCt method and normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

### 2.5. Western Blot assay

Protein extracts were thawed on ice before centrifugation at 10,000 rpm for 10 min at 4 °C, and protein concentration in the supernatants was measured by the Bradford assay at 595 nm. Aliquots containing 20 µg total protein were mixed with Laemmli sample buffer containing β-mercaptoethanol, denatured at 95 °C for 5 min and at RT during 15 min for P-gp, and electrophoresed using resolution gel 10 % (Biorad). Proteins were then transferred to nitrocellulose membranes, which were blocked in TBS-Tween (0.1 %) with 5 % skimmed milk and probed overnight at 4 °C with primary antibodies (table 2 in *supplementary material 4*). After TBST washes, membranes were incubated with HRP-conjugated secondary antibodies (table 3 in *supplementary material 4*), developed with enhanced chemiluminescence, and imaged. Band intensities (imaged with the Azure c600 Western blot Imaging system) were quantified using AzureSpot 2.0 Gel Analysis software (Azure Biosystems, Dublin, Ireland), and normalized to β-actin.

### 2.6. IL-8 assay in IB model and BBB

In our study, we assessed the levels of IL-8 in the IB and BBB model using the Human IL-8 ELISA kit (cat. No KHC0081, Invitrogen, Thermo Fischer Scientific) to evaluate the potential impact of SHIME® supernatants on barriers and the effects CPF exposure on IL-8 secretion. After 24 h exposure of IB and BBB models to 1:5 diluted SHIME® supernatants, IL-8 levels were measured in apical and basolateral compartments.

The Human IL-8 ELISA assay was performed following the manufacturer standardized protocol to quantify IL-8 levels in samples. The concentration of IL-8 in each sample was determined by comparing the absorbance (detected at 450 nm using Biotek Cytation C10 Confocal Imaging Reader (Agilent, Santa Clara, California) microplate reader) to a standard curve generated with known IL-8 concentrations. The data were expressed as IL-8 concentrations (pg/mL).

### 2.7. Statistical analysis

Data were expressed as mean ± standard error to the mean (SEM). Statistical analysis was conducted using GraphPad Prism 8.0.1 statistical software (GraphPad Software, Inc., San Diego, CA, USA). T-test, one-way and two-way analyses of variance (ANOVA) were employed, with $p < 0.05$ being the statistical significance threshold for all analyses. When statistical analysis showed non-parametric values, data were

expressed as mean ± SEM using the non-parametric Mann Whitney test or Kruskal-Wallis test.

## 3. Results

### 3.1. Assessment of SHIME® model stability

In our previously published work, we thoroughly validated the GM of the SHIME® model in terms of composition and metabolic activity, specifically SCFAs. Our results demonstrated that the SHIME® model successfully replicates the human gut environment, exhibiting consistent microbial composition and SCFA production over extended periods (Joly Condette et al., 2022; Abou Diwan et al., 2025; Réquilé et al., 2018).

Here, we focused on the stability evaluation of the SHIME® system during the GM stabilization and exposure periods with an emphasis on the methodological process. This stability was ensured through routine monitoring of pH and redox potential, as well as maintaining a constant temperature (37 °C) using a controlled water bath that circulates water through the double walls of the reactors.

Our daily measurements in the proximal (ascending), transverse, and distal (descending) colon compartments demonstrated stable temperature evolution, reflecting system stability (supplementary material 1 A). Additionally, we observed stable pH (supplementary material 1 B) and redox potential (supplementary material 1 C), indicating the stability of microbial flora. The initial pH value corresponded to the acidic pH of the artificial supply (sterile feed medium). After the addition of fecal samples, we began daily pH adjustments, resulting in consistent pH levels in the colon reactors: 5.5–6 in Asc colon, 6–6.4 in the Trans colon, and 6.4–6.8 in the Desc colon. Similarly, the redox potential was initially greater than 0 at the start (inoculum) but rapidly decreased due to the addition of H₂, N₂ and CO₂ to ensure anaerobic conditions in colon, reaching levels below -200 mV. The redox potential remained constant, staying below -300 mV in Trans and Desc colon and between –300 mV and –200 mV in Asc colon. This stability confirms the efficiency of our system assembly, ensuring leak-proof reactors, as we only needed to add gas once and did not require additional gas during the 45 days of SHIME® experiments. These conditions allowed the growth of strict anaerobes and facultative aerobes.

In conclusion, the stability of the SHIME® system was successfully maintained throughout the GM stabilization and exposure periods, with consistent temperature, pH, and redox potential values across all colon compartments. This ensured the proper growth of microbial flora under anaerobic conditions, validating the reliability and efficiency of the SHIME® model for further experimentation.

### 3.2. IB and BBB integrity analysis: cell viability, apparent permeability and immunofluorescence

To ensure that the addition of SHIME® supernatants (a mix of colon reactor samples) is not toxic to Caco-2 and hBLEC, we first centrifuged the SHIME® samples to remove bacterial cells and then evaluated cell viability and apparent permeability (P app) to FITC-dextran after 24 h of exposure to the filtered and centrifuged supernatants.

The suitable dilution of SHIME® supernatants was determined based on comparative permeability testing in the IB (Fig. 3.A) and BBB (Fig. 3.B) models. Non-diluted samples (+ SHIME ND) exhibited cytotoxic effects, as evidenced by elevated permeability levels (P app = 13.27 ×10⁻⁶ cm/s in IB and 21.79 ×10⁻⁶ cm/s in BBB compared to control: - SHIME). We tested dilutions of 1:2, 1:5, and 1:10. While all three dilutions were non-toxic in the IB model, the 1:2 dilution resulted in high permeability in the BBB model (P app = 16.97 ×10⁻⁶ cm/s). Both the 1:5 (P app = 0.03 ×10⁻⁶ cm/s in IB and 1.88 ×10⁻⁶ cm/s in BBB) and 1:10 (P app = 0.02 ×10⁻⁶ cm/s in IB and 1.55 ×10⁻⁶ cm/s in BBB) dilutions produced acceptable permeability results in both models. Ultimately, the 1:5 dilution of filtered supernatants was selected for cell
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Effect of SHIME® supernatants (a mix of colon reactor samples) on barriers integrity with the study of (A) apparent permeability (P app 10⁻⁶ cm/s FD4), (B) relative cell viability to FITC-Dextran and (E,F,G,H) confocal microscopy of ZO-1 immunofluorescence in IB and BBB models under control conditions (- SHIME) and with 1:5 diluted SHIME supernatants. Data is illustrated as mean ± SEM using analysis of variance (ANOVA), unpaired *t*-test and Mann Whitney test. Significance *p* < 0.05; **p** < 0.01, ***p** < 0.001, ****p** < 0.0001. - SHIME: without SHIME addition = control, + SHIME ND: non-diluted.</figcaption></figure>
viability, permeability, and immunostaining assays. This selection was based on its effectiveness in previous bacterial translocation experiments where a 1:5 dilution of non-filtered samples also yielded reliable results (Abou Diwan et al., 2025).

We next assessed its impact on cell metabolic activity using the MTT assay. The results indicated a slight 37 % increase in Caco-2 cell viability compared to the control (p < 0.05) after 24 h of exposure to the 1:5 diluted SHIME. Conversely, a 24-hour exposure to the same diluted sample significantly decreased the viability of hBLEC.

We included Triton X-100 (10 %) as a positive control for P app and viability to validate assay sensitivity to overt barrier disruption. As expected, triton exposure induced a marked increase in permeability in both models (Fig. 3A–B), exceeding values obtained with non-diluted and 1:5 or 1:10 diluted SHIME® samples, and caused a pronounced loss of cell viability (Fig. 3C–D) compared with all SHIME conditions, confirming assay sensitivity to major barrier disruption.

Following the metabolic activity assays, we next examined the effect of the 1:5 dilution on tight junction integrity, a key determinant of barrier permeability. We performed immunofluorescence staining to analyze the localization of tight junction proteins, such as ZO-1, in Caco-2 and hBLEC.

Immunofluorescence analysis revealed that in the control condition (- SHIME), the staining of ZO-1 was well-defined and continuous along the cell borders in both the IB (Fig. 3.E) and BBB (Fig. 3.F) models, indicating intact tight junctions.

Similarly, both the IB (Fig. 3 G) and BBB (Fig. 3 H) models exhibit preserved tight junction cohesion under the 1:5 dilution of SHIME supernatants, as indicated by the consistent and continuous ZO-1 staining. These findings suggest that the 1:5 dilution of SHIME supernatants does not induce significant impairment of tight junctions, indicating that it is likely non-toxic and does not compromise barrier function in either

model.

Overall, the immunofluorescence results indicate that the 1:5 dilution of SHIME supernatants is suitable for maintaining barrier integrity in both IB and BBB models, while non-diluted samples are detrimental to tight junctions.

### 3.3. Effects of CPF-treated SHIME® on barriers integrity

In this section, we examined the impact of CPF-treated SHIME® supernatants, diluted to a 1:5 ratio, on the integrity of the IB and BBB models (Fig. 4).

Under exposure to this dilution during 24 h, CPF exposure did not significantly affect Caco-2 (Fig. 4 A) and hBLEC (Fig. 4 B) viability after 15 and 30 days of exposure in both models.

Furthermore, exposure of SHIME® supernatants to CPF did not significantly impact permeability in either model (Fig. 4 C-D), confirming the suitability of this dilution for future GM and environmental contaminant studies.

### 3.4. Effects of CPF-treated SHIME® on immunofluorescence of tight junctions (Occludin) and associated proteins (ZO-1)

To further investigate the impact of SHIME® supernatants on barrier integrity, we performed confocal microscopy analysis of key tight junction proteins, occludin (ocln) and ZO-1, in Caco-2 cells exposed to both untreated and CPF-treated SHIME® supernatants. Confocal microscopy analysis of ocln (tight junction protein) and ZO-1 (associated with tight junctions) in Caco-2 cells exposed to SHIME® supernatants (1:5 dilution without CPF) revealed that ZO-1 was consistently localized along the cell membrane (Fig. 5A), indicating intact tight junctions. Similarly, ocln staining showed continuous membrane localization

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Effect of CPF-treated SHIME® (1:5 dilution) on relative cell viability and permeability of Caco-2 and hBLEC. Data is illustrated as mean ± SEM using ANOVA. Significance * p < 0.05; ** p < 0.01, *** p < 0.001. – SHIME: without SHIME addition = control, + 1/5 SHIME exposure: dilution of SHIME® to 1:5 deprived of CPF = CPF0; CPF15: 15 days of exposure; CPF30: 30 days of exposure.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Effect of CPF-treated SHIME (1:5 dilution) on ZO-1 and Occludin (Ocln) localization (A to F) (confocal microscopy, scale bar = 100 µM), immunofluorescence quantification (G and H), transcriptional expression of tight junctions and efflux pumps (I to L) and protein expression of P-gp efflux pump (M) in Caco-2 cells. White arrows indicate discontinuous staining. Data is illustrated as mean ± SEM using ANOVA. Significance * p < 0.05. + SHIME 1:5: dilution of SHIME® to 1:5 = CPF0, CPF15: 15 days of exposure; CPF30: 30 days of exposure.</figcaption></figure>

(Fig. 5D), further confirming intact intercellular barrier function under control conditions. This suggests that the addition of SHIME® supernatants, devoid of pesticide residues, does not impact membrane integrity in Caco-2 cells membrane.

In contrast, exposure to CPF-treated SHIME® supernatants (CPF15 and CPF30) resulted in discontinuous staining pattern of ZO-1 (indicated by white arrows) (Fig. 5B and C) and ocln (Fig. 5E and F) along the cell membrane, indicating localized discontinuity of tight junction protein localization within specific regions of the monolayer. This reduced tight junction cohesion was observed even though the P app of Caco-2 cells was not significantly increased compared to CPF0 SHIME® samples (Fig. 4C).

For hBLECs, in addition to ZO-1 analyzed in the IB model, claudin-5 (cldn5) was evaluated as it is a key tight junction protein critical for maintaining BBB integrity, as supported by multiple studies (Greene et al., 2019; Versele et al., 2022b). Confocal microscopy of hBLECs exposed to SHIME® supernatants (1:5 dilution without CPF), showed that ZO-1 was consistently localized along the cell membrane (Fig. 6) similarly to the negative control (supplementary material), indicating intact tight junctions. This suggests that SHIME® supernatants, free from pesticide residues, do not alter ZO-1 localization in hBLEC membrane.

However, exposure to CPF-treated SHIME® supernatants (CPF15 and CPF30) induced discontinuous ZO-1 and cldn5 staining (Fig. 6). Cldn5 staining appeared less uniform, with more frequent discontinuities (white arrows) compared to both the negative control (supplementary material 2) and CPF0 SHIME® supernatants (Fig. 6 D), with the highest occurrence of discontinuity in cells exposed to CPF15 SHIME® samples (Fig. 6 E).

Overall, compared to the negative controls, both ZO-1 and Cldn5 in CPF-treated hBLECs exhibited reduced junctional continuity, even

though the P app of hBLEC was not significantly increased compared to CPF0 SHIME® samples.

In conclusion, CPF-treated SHIME® supernatants induced a discontinuous tight junction pattern. Despite these localized alterations, P app values remained unchanged, suggesting that CPF exposure can compromise tight junctions locally without disrupting overall barrier permeability. In contrast, SHIME® supernatants without pesticide residues did not affect cell-cell junction stability in Caco-2 and hBLEC. These results confirmed that the selected SHIME® sample dilution maintains barrier function without inducing cytotoxicity.

To enhance the understanding of this observation, we quantified tight junction immunofluorescence (Figs. 5 and 6 G-H) and performed RT-qPCR analysis (after 24 h of exposure) and Western blot (after 48 h of exposure) of tight junctions and efflux pumps (P-gp and BCRP). While immunofluorescence quantification did not reveal statistically significant differences, mRNA expression analysis showed altered transcriptional profiles, indicating molecular responses that may precede or accompany protein discontinuous localization.

### 3.5. Effects of CPF-treated SHIME® on expression of barrier key proteins

We assessed the transcriptional expression of tight junction and related gene expression (relative to GAPDH) in Caco-2 cells (Fig. 5) and hBLECs (Fig. 6) after 24 h exposure to 1:5 diluted SHIME® mix treated with CPF.

In Caco-2 cells, mRNA levels of ZO-1 and OCLN showed no significant change (Fig. 5 I and J). Among efflux transporters, BCRP mRNA did not vary significantly (Fig. 5K) whereas P-GP was significantly down-regulated after exposure to CPF15 1:5 (≈29% decrease; 1.00 vs 0.71; p = 0.0231 vs CPF0), with CPF30 showing no effect at the mRNA level (Fig. 5L) but a significant downregulation at the protein level (Fig. 5M).
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Effect of CPF-treated SHIME (1:5 dilution) on ZO-1 and claudin-5 (Cldn5) localization (A to F) (confocal microscopy, scale bar = 100 µM), immunofluorescence quantification (G and J), transcriptional expression of tight junctions and efflux pumps (H, I, K, L and M) and protein expression (N) in hBLEC cells. White arrows indicate discontinuous staining. Data is illustrated as mean ± SEM using ANOVA. Significance * p < 0.05; ** p < 0.01. + 1/5 SHIME exposure: dilution of SHIME® to 1:5 deprived of CPF = CPF0, CPF15: 15 days of exposure; CPF30: 30 days of exposure.</figcaption></figure>

In hBLECs (Fig. 6), ZO-1, OCLN, VE-cadherin and BCRP transcripts were unaltered (data not shown in Fig. 6 for VE-cadherin). By contrast, CLDN5 mRNA levels were significantly decreased after 24 h exposure to CPF15 1:5 (37% reduction versus CPF0; 1.00 vs 0.63; p = 0.0017),

whereas CPF30 did not differ from CPF0 (p = 0.5701) (Fig. 6 K). P-GP mRNA was significantly reduced after 24 h exposure to both CPF15 and CPF30 1:5 samples (≈40% decrease for CPF15: 0.60 vs 1.00, p = 0.0025; ≈37% decrease for CPF30: 0.63 vs 1.00, p = 0.0037)

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Effect of CPF-treated SHIME (1:5 dilution) on IL-8 secretion in the luminal compartment (A) by Caco-2 cells in the IB model and (B) hBLEC in the BBB model. Data is illustrated as mean ± SEM using ANOVA. Significance * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001. – SHIME: without SHIME addition = control; 1/5: dilution of SHIME to 1:5; CPF0: SHIME Control without CPF; CPF15: 15 days of exposure; CPF30: 30 days of exposure.</figcaption></figure>
(Fig. 6 M). P-GP downregulation was not observed at the protein level by Western Blot.

Overall, these results indicate that, under the tested conditions, CPF-treated SHIME® selectively downregulated CLDN5 and P-GP without affecting the expression of other tight junctions and efflux pumps analyzed.

### 3.6. IL-8 assay in IB and BBB models

Given that IL-8 is a key mediator of inflammatory responses and a sensitive indicator of barrier cell activation, especially under environmental stress, we focused on quantifying IL-8 levels to capture early signs of inflammatory changes. Our ELISA assay aimed to evaluate IL-8 levels (in luminal and abluminal compartments of both models) under different experimental conditions to assess the impact of SHIME® supernatants exposure with or without CPF.

In the IB model, the control condition (-SHIME), representing Caco-2 cells without SHIME® supernatants, exhibited very low IL-8 levels (relatively to other conditions), around 40 pg/mL (Fig. 7 A). However, the 1:5 dilution of CPF0 SHIME® supernatant (without CPF), showed a significant increase in IL-8 levels to approximately 380 pg/mL (p < 0.05) compared to control condition (- SHIME). The +SHIME CPF15 1:5 and +SHIME CPF30 1:5 conditions, where SHIME® supernatants were exposed to CPF demonstrated even higher IL-8 levels when exposed to 15 days of CPF (p < 0.01) compared to control. Specifically, IL-8 concentrations were approximately 580 pg/mL for +SHIME CPF15 1/5 (p < 0.01) and 250 pg/mL for +SHIME CPF30 1/5 (p < 0.05). In contrast, IL-8 was not detected in the abluminal compartment which indicates that secretion was only in the luminal compartment and there was no passage of cytokines across the IB.

Similarly, in the BBB model, control conditions without SHIME® supernatants (-SHIME) showed very low IL-8 levels (Fig. 7 B). Exposure to the 1:5 dilution of CPF0 SHIME® supernatant resulted in increased IL-8 levels (p < 0.0001), indicating an inflammatory response. Further exposure to CPF-treated SHIME® supernatants (+SHIME CPF15 1:5) led to slightly higher IL-8 levels, with concentrations significantly higher than the control (p < 0.0001).

Interestingly, unlike the IB model, where IL-8 was not detected in the abluminal compartment, IL-8 was detected in the abluminal compartment of the BBB model reflecting IL-8 secretion by pericytes and/or cytokine translocation between both compartments. Additionally, while the IL-8 increase range in the IB model was between 200 and 600 pg/mL, in the BBB model, the increase range was between 200 and 4000 pg/mL indicating differences in cytokine handling and barrier properties between the two systems under exposure to gut metabolites and pesticides residues.

In summary, the results indicate that SHIME® supernatants, both with and without CPF, induce an inflammatory response in the IB and BBB models, as evidenced by increased IL-8 levels. CPF exposure further amplifies this response, particularly in the IB model after 15 days of exposure, suggesting that prolonged exposure to CPF exacerbates inflammation.

## 4. Discussion

### 4.1. Key findings interpretation: confirmation and comparison with literature

The present study aimed to explore the effects of GM-derived metabolites and the environmental contaminant CPF on the IB and the BBB, using distinct human in vitro models. Our findings highlight how chronic exposure to CPF, even at low doses, can subtly disrupt barrier structure and trigger inflammatory signaling, emphasizing the crucial role of GM in modulating these effects. These results provide new perspectives on the interplay between microbial metabolites and environmental contaminants in maintaining barrier homeostasis, confirming

some aspects of previous reports while revealing novel, localized impacts on barrier integrity. Overall, this work underscores the potential risks associated with dietary pesticide residues and their relevance to human health and environmental safety.

We first verified model stability and non-cytotoxic exposure conditions to ensure observed effects reflect biologically relevant responses rather than experimental artifacts. Notably, 1:5 dilution of SHIME® supernatants was identified as an optimal concentration for subsequent experiments, based on permeability data and its suitability for bacterial translocation studies (Abou Diwan et al., 2025). This dilution exhibited non-toxic effects on cell viability and barrier permeability, making it appropriate for assessing the effects of GM metabolites and contaminants like CPF on barrier function. These findings align with previous studies that demonstrated that microbial metabolites, under normal conditions, do not harm epithelial or endothelial cell integrity, and may even contribute to the maintenance of IB homeostasis (Ghosh et al., 2021).

One of the key findings of this study is that exposure to SHIME® supernatants without pesticide residues did not significantly affect tight junction protein localization or cell viability. In contrast, exposure to SHIME® supernatants treated with CPF led to early signs of tight junction protein localization impairment evidenced by discontinuous staining of occl and ZO-1 in IB and cldn5 and ZO-1 in BBB models. These results corroborate prior research on the impact of the environmental contaminant CPF on tight junction proteins (Tirelli et al., 2007; Guibourdenche et al., 2021b, Abou Diwan et al., 2024). Interestingly, despite these changes in membrane organization and the significant decrease in expression of cldn5 in BBB and P-gp in both IB and BBB, overall barrier permeability did not increase significantly, even though our previous GM metabolites analysis on the same samples showed significant shifts in SCFAs ratios and the detection of CPF in the SHIME® reactors (Abou Diwan et al., 2025).

This pattern is consistent with literature indicating that subtle changes in tight junction protein localization can lead to barrier dysfunction that may not be immediately apparent through conventional permeability assays (Versele et al., 2022a).

To understand how this microbial and pesticides concentration changes in GM led to localized tight junction discontinuity without impacting permeability, we analyzed early molecular and inflammatory signals that may precede measurable increases in paracellular permeability. We measured the transcriptional expression of these junctions as well as efflux pumps and we quantified IL-8 secretion because this cytokine serves as an early indicator of epithelial and endothelial barrier cell activation (Bishara, 2012; Vitucci et al., 2024). Its quantification allows detection of subtle inflammatory responses preceding broad cytokine induction.

The concurrent reduction of CLDN5 mRNA specifically in hBLECs at CPF15, and the selective decrease of P-gp mRNA at CPF15 in the intestinal model (and at CPF15/CPF30 in the BBB model), provides a molecular sign compatible with this early perturbation: decreased synthesis of key junctional and detoxification proteins.

The inflammatory readout further supports an acute, dysbiosis-linked stress at CPF15. IL-8 levels peaked at CPF15, concordant with our earlier SHIME® study (Abou Diwan et al., 2025) showing maximal CPF accumulation and loss of beneficial *Lactobacillus* and *Bifidobacterium* spp. within 15 days, concomitant with shifts in SCFA ratios. By day 30, the microbial community composition had shifted toward increased abundance of potentially pathogenic bacteria *Clostridium* and *Enterococcus* spp., which possess xenobiotic-transforming capacities. This suggests that the early phase (15 days) corresponds to a state of acute dysbiosis, limited CPF detoxification capacity by GM, and accumulation of pro-inflammatory metabolites, while later exposure (30 days) reflects a stage characterized by enhanced CPF biotransformation and altered metabolic outputs (we observed a progressive decrease in CPF concentrations between day 15 and 30). Consequently, the apparent "recovery" of barrier integrity at CPF30 should not be interpreted as a full
restoration of epithelial or endothelial health and does not indicate
restoration of a healthy GM but rather a shift toward an adapted,
CPF-tolerant community producing distinct metabolite profiles with
reduced direct toxicity to epithelial and endothelial cells that could
explain higher IL-8 levels at CPF15 (Chen et al., 2025).

Increased IL-8 secretion in both IB and BBB models following expo-
sure to SHIME® supernatants, particularly after 15 days of CPF expo-
sure, underscores an early inflammatory response triggered by pesticide
contamination as previously evidenced (Réquilé et al., 2018). These data
suggest that GM metabolites, especially in the presence of environ-
mental contaminants like CPF, can induce inflammatory cascades that
may compromise barrier function over time. This is particularly con-
cerning given the critical role of inflammation in the pathogenesis of
various diseases, including inflammatory bowel disease (IBD), neuro-
logical disorders, and other conditions related to gut-brain communi-
cation (Bercik et al., 2011).

Furthermore, the results of this study suggest that prolonged expo-
sure to pesticides like CPF may exacerbate inflammatory responses. This
prolonged exposure may mirror real-life scenarios, where continuous
pesticide exposure could lead to sustained inflammation and barrier
dysfunction (Lopes-Ferreira et al., 2023). Given the pivotal role of the
GM in modulating immune signaling (Huang et al., 2021), these findings
emphasize the importance of considering both direct and GM-mediated
effects of environmental contaminants on human health, especially in
relation to the gut-brain axis.

Moreover, the difference we observed in IL-8 compartmentalization
between IB and BBB likely reflects distinct secretion polarities or cyto-
kine transport properties between intestinal epithelial cells and brain
endothelial cells (Verma et al., 2006; Sonnier et al., 2010; Rossi et al.,
2013). Nevertheless, while the observed increase in IL-8 suggests that
CPF exposure could be triggering an inflammatory process, it does not
capture the full complexity of the inflammatory state. To robustly
confirm and characterize the complex inflammatory cascade, future
analysis should consider quantifying additional pro-inflammatory cy-
tokines such as IL-1β, IL-6, and TNF-α and assess markers of inflamma-
some activation like NLRP3.

## 4.2. Innovative and robust in vitro approach

The configuration combining SHIME® with IB and BBB models (SHIME-IB and SHIME-BBB) is an innovative approach. The SHIME® model is a validated in vitro model for simulating the human GM metabolic processes. The resulting SHIME-IB-BBB system provided a more comprehensive understanding of how GM metabolites and environmental contaminants like pesticides affect the integrity of multiple biological barriers in sequence, mimicking the potential translocation of microbial metabolites from the gut to the brain. This setup offers a more holistic model for studying the gut-brain axis, which is crucial for understanding how disturbances in gut health may impact brain function and disease.

Previous studies have typically employed isolated barrier models or
simplified microbial exposure systems (Réquilé et al., 2018; Tirelli et al.,
2007; Li and Ehrich, 2013). By integrating SHIME® with IB and BBB
models, our study overcomes these constraints, establishing a novel
approach within environmental contaminant research. This configura-
tion allows for a more precise simulation of the complex interactions
between the GM, contaminants, and the biological barriers, improving
our understanding of the potential systemic effects of microbial me-
tabolites and environmental toxins. Moreover, the SHIME-IB-BBB
configuration (Abou Diwan et al., 2025) provides an opportunity to
study how sequential exposure to microbiota-derived metabolites and
environmental contaminants might impact not just gut function, but also
brain health, thus offering a more integrated perspective on the
gut-brain axis.

4.3. Limits and perspectives

While our study provides valuable insights into the effects of GM
metabolites and environmental contaminants as CPF on the integrity of
biological barriers, several limitations should be acknowledged.

The CPF concentration used in this study (1 mg/day) was selected to reflect realistic human dietary exposure scenarios, as previously estimated by ANSES and applied in earlier SHIME® studies (Joly et al., 2013). This dose has been shown to induce measurable effects on GM composition and epithelial barrier in vitro without causing acute toxicity, supporting its relevance for modeling environmentally realistic exposures. Importantly, this concentration is substantially lower than doses associated with acute toxicity in humans (typically above 2–5 mg/kg/day) (Eaton et al., 2008; Algharably et al., 2023), yet higher than average background exposures, allowing us to capture potential subclinical effects. While our current study focuses on short-term exposure, we acknowledge that chronic and cumulative effects warrant further investigation. Future studies will consider testing lower, sub-ADI (Acceptable Daily intake) doses in both IB and BBB models to explore dose-dependent effects.

In addition, the present work specifically focused on GM derived
from women of reproductive age, a population particularly vulnerable to
dietary pesticide exposure and hormonal modulation of gut-brain in-
teractions. This choice aligns with the broader scope of our research
project, which compares pregnant and non-pregnant women. Sex-
specific differences in GM composition are increasingly recognized as
critical factors influencing host physiology, immune responses, and
xenobiotic metabolism. Estrogen and other sex hormones can shape
microbial communities, which in turn modulate neuroinflammatory
pathways and BBB integrity. For instance, female GM has been shown to
promote distinct barrier responses compared to male GM. In our pre-
vious work (Abou Diwan et al., 2024), we demonstrated that offspring of
chlorpyrifos-exposed dams exhibited sex-dependent alterations in BBB
protein expression, further supporting the relevance of sex as a biolog-
ical variable in GM-brain interactions. We also note that previous
studies using the SHIME® model, such as those by Réquilé et al. (2018)
and Joly et al., were based on mixed-derived inocula (men and women)
and reported gut dysbiosis and bacterial translocation across IB
following chlorpyrifos exposure. Building on this foundation, future
studies will compare male and female-derived GM to assess differential
barrier responses to CPF exposure.

Despite combining advanced models of the intestinal (Caco-2) and
brain (hBLEC) barriers with the SHIME® system, the experimental
design cannot fully reproduce the complex physiological interactions
along the gut-brain axis, including neural and immune signaling.

In particular, immune-related processes are not represented, as the
BBB model does not include immune cells. Moreover, while our in vitro
models focus on barrier-specific mechanisms using epithelial cells
(Caco-2) and brain endothelial cells, they do not incorporate additional
cell types such as mucus-secreting cells or astrocytes. These models
nonetheless provide an ethical approach to dissect barrier-specific mo-
lecular mechanisms. Although our 2D models effectively characterized
CPF-induced effects on the IB and BBB, future studies using 3D co-
cultures or organ-on-chip platforms could provide greater physiolog-
ical relevance (Lozoya-Agullo et al., 2017; Rizzi et al., 2021; Izadifar
et al., 2022) by integrating astrocytes, immune cells, and
mucus-producing goblet cells (Haddad et al., 2023), thereby better
reflecting in vivo gut-brain interactions and enabling analysis of
multicellular signaling dynamics.

5. Conclusion

This study successfully established a novel, human-relevant in vitro
approach by integrating the SHIME® system with IB and BBB models to
analyze microbiota-mediated effects on epithelial and endothelial bar-
rier integrity. Chronic low-dose exposure to CPF, delivered via
microbiota-derived metabolites, subtly altered tight junction localization, reduced CLDN5 and P-GP expression, and increased IL-8 secretion, without inducing overt cytotoxicity or major permeability changes. These findings provide mechanistic insight into how environmental contaminants may prime the gut-brain axis through early molecular and inflammatory signals, underscoring the value of this integrated, non-animal approach for mechanistic and screening studies in environmental toxicology.

## Environmental Implication

This study introduces a novel in vitro approach that integrates human-relevant models of the gut microbiota, intestinal barrier, and blood-brain barrier to assess the systemic effects of environmental contaminants. By simulating realistic exposure to Chlorpyrifos (CPF)-altered microbial metabolites, our experimental setup revealed how chronic pesticide exposure may compromise epithelial and endothelial barrier integrity through indirect, microbiota-mediated mechanisms. This mechanistic insight into barrier disruption and inflammation contributes to the understanding of sub-lethal, long-term effects of environmental pollutants on human health. Given the environmental persistence of CPF, this approach offers a versatile and ethical tool for ecotoxicological risk assessment, supporting more comprehensive evaluations of emerging contaminants under environmentally relevant conditions.

## Ethical approval

The study protocol received approval from the Ethical Committee for Non-Interventional Research (CERNI) at Picardy Jules Verne University (Evaluation number 2023-07-5/2023-27). The ethics committee of the Medical Faculty at the University of Yamaguchi Graduate School (IRB#: H18-033-6) approved the protocol for isolating pericytes from human tissue, ensuring adherence to the Declaration of Helsinki as revised in Somerset West in 1996. Additionally, the French Ministry of Higher Education and Research (CODECOH Number DC2011-1321) approved the protocol for isolating endothelial cells, and all related experiments were conducted in accordance with this approved protocol.

## Funding

This research received funding from the French State and the Hauts-de-France Region as part of CPER MOSOPS 2021-2027 project and through the Ecophyto II + action plan, led by the ministries of agriculture, ecology, health, and research, with financial support from the French Office for Biodiversity (OFB-24-1845 PESTIPREDIS). Maria Abou Diwan received a doctoral fellowship (2021-2024) from the ‘Conseil régional du Nord-Pas-de-Calais’ (50 %), Picardy University (25 %), and Artois University (25 %). Juliette Poiriez received a doctoral fellowship (2024-2027) from the ‘Conseil régional du Nord-Pas-de-Calais’ (50 %) and Artois University (50 %).

## CRediT authorship contribution statement

**Hafida Khorsi-Cauet:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.
**Fabien Gosselet:** Writing – review & editing, Writing – original draft, Visualization, Validation, Resources.
**Véronique Bach:** Writing – review & editing, Writing – original draft, Validation, Resources.
**Halima Ouadid-Ahidouch:** Visualization.
**Stéphane Delanaud:** Software, Methodology, Formal analysis, Data curation.
**Emmanuel Sevin:** Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation.
**Claire Joly Condette:** Visualization.
**Avénie Huet:** Writing – original draft, Methodology.
**Juliette Poiriez:**

Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation.
**Maria Abou Diwan:** Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation.
**Pietra Candela:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

## Consent to participate

Participation in this study was entirely voluntary, and participants could withdraw at any time without any penalty. Participants were given a detailed explanation of the study's purpose, procedures, potential risks, and benefits. Confidentiality of the participants' data was maintained throughout the study, since the protocol ensures that the processed data is anonymous. It does not allow for the identification of individuals, even indirectly, in any way; therefore, it is not considered personal data, and there is no correspondence table linking each person's identity to a random number referring to a series of individual data.

## Consent to publish

Not applicable.

## Declaration of Competing Interest

There are no competing interest to declare.

## Acknowledgements

The authors would like to express their sincere gratitude to Dr. Arthur Foulon, gynecologist at the University Hospital of Amiens, for his invaluable assistance in identifying suitable participants and providing fecal samples. We are deeply appreciative of the women volunteers who participated in this study and donated their fecal samples for this research. Our thanks also go to the research management team at the University of Picardy Jules Verne for their support with the ethics approval application. Lastly, we extend our thanks to Professor Fumitaka Shimizu and Professor Takashi Kanda from the Department of Neurology and Clinical Neuroscience at Yamaguchi University in Japan for providing the human brain pericytes.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2025.119539.

## Data availability

Data will be made available on request.

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