# Development of an in vitro triculture model of intestine inflammation to assess the immunomodulatory properties of cassava and roselle, two African green leafy vegetables

Nelly Fioroni, Maria del Carmen Ponce de León Rodríguez, Nicolas Leconte, Claire Mouquet-Rivier, Caroline Guzman, Frédéric Boudard, Claudie Dhuique-Mayer, Myriam Collin, Anaïs Deglos, Emmanuelle Reboul, et al.

► To cite this version:

Nelly Fioroni, Maria del Carmen Ponce de León Rodríguez, Nicolas Leconte, Claire Mouquet-Rivier, Caroline Guzman, et al. Development of an in vitro triculture model of intestine inflammation to assess the immunomodulatory properties of cassava and roselle, two African green leafy vegetables. NFS Journal, 2025, 38, pp.100213. 10.1016/j.nfs.2025.100213. hal-04922852

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Submitted on 8 Dec 2025

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Original article

# Development of an *in vitro* triculture model of intestine inflammation to assess the immunomodulatory properties of cassava and roselle, two African green leafy vegetables

Nelly Fioroni<sup>a,1,*</sup>, Maria Del Carmen Ponce de León Rodríguez<sup>a,1</sup>, Nicolas Leconte<sup>b</sup>, Claire Mouquet-Rivier<sup>a</sup>, Caroline Guzman<sup>a</sup>, Frédéric Boudard<sup>a</sup>, Claudie Dhuique-Mayer<sup>a,c</sup>, Myriam Collin<sup>d</sup>, Anaïs Deglos<sup>a</sup>, Emmanuelle Reboul<sup>e</sup>, Ángela Bravo-Núñez<sup>e</sup>, Caroline Laurent-Babot<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Caco-2/HT29 biculture

THP-1 macrophages

Leaf extract

*In vitro* digestion

Intestinal permeability

Cytokines

## ABSTRACT

The intestine integrity and function are crucial for nutrient absorption and to prevent the entry of harmful antigens. In sub-Saharan Africa, frequent infections and enteric dysfunction can cause intestinal inflammation that is associated with stunting. Therefore, to study the anti-inflammatory effects of cassava and roselle leaves, commonly consumed as sauces, we developed an *in vitro* triculture model of intestine inflammation using intestinal Caco-2 (colorectal adenocarcinoma) and HT29-MTX (goblet cell-like) together with the macrophage-like THP-1 cells. Stimulation of this model with lipopolysaccharide/interferon-γ resulted in mucus over-production, higher pro-inflammatory cytokine release and loss of intestinal barrier integrity due to increased permeability. Polar/non-polar extracts and digested sauces from cassava and roselle leaves reduced cytokine production in both intestinal and THP-1 cells to different extents and restored barrier integrity and permeability. The developed and validated triculture model of inflamed intestine thus demonstrated the anti-inflammatory properties of cassava and roselle leaves, despite moderate responses.

## 1. Introduction

The primary function of the intestine is to absorb (micro)-nutrients, but it also regulates molecule entry and protects the body against antigens. Intestinal epithelial cells (IEC) and their apical junction complex form a semi-permeable barrier that limits translocation of luminal material to the lamina propria, where immune cells (e.g. macrophages, dendritic cells, lymphocytes) reside. The microbial community of the gastrointestinal tract is vital for maintaining intestinal homeostasis. In physiological conditions, the intestinal mucosa tolerates commensal microorganisms and food antigens through a finely regulated immunity mechanism. Imbalances can lead to inflammation, barrier defects, immune dysfunction, and nutrient malabsorption [1,2]. The aetiology of

intestinal inflammation, the incidence of which is rising worldwide, remains unclear and involves multiple factors ([3]; [4]). Gut inflammation is linked to pathological conditions, such as enteric infections, diarrhoea, inflammatory bowel disease, metabolic syndrome, and non-communicable diseases (e.g. obesity and undernutrition) [5,6]. Stunting affects 22 % of children under five years old globally and 32 % in sub-Saharan Africa [7]. The environmental enteric dysfunction hypothesis suggests that chronic exposure to enteropathogens causes gut inflammation, contributing to stunted growth in these children [8,9]. Environmental enteric dysfunction is characterized by intestinal inflammation, villus atrophy, and crypt hyperplasia, leading to altered epithelial permeability and reduced intestinal absorptive capacity and ultimately in poor digestion and nutrient malabsorption [10,11].

<footnote><sup>a</sup> UMR QualiSud, Université de Montpellier, Avignon Université, CIRAD, Institut Agro, IRD, Université de la Réunion, Montpellier, France</footnote>

<footnote><sup>b</sup> UMR HSM, Université de Montpellier, CNRS, IRD, Montpellier, France</footnote>

<footnote><sup>c</sup> CIRAD, UMR QualiSud, F-34398 Montpellier, France</footnote>

<footnote><sup>d</sup> UMR DIADE, IRD/CIRAD F2F-Palmiers, Université de Montpellier, IRD, Montpellier, France</footnote>

<footnote><sup>e</sup> Aix-Marseille Université, INRAE, INSERM, C2VN, Marseille, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: nelly.fioroni@umontpellier.fr (N. Fioroni), caroline.babot@umontpellier.fr (C. Laurent-Babot).</footnote>

<footnote n="1">¹ Nelly Fioroni and Maria del Carmen Ponce de León Rodríguez are co-first authors, they contributed equally to this work.</footnote>
Research on gut inflammation is crucial to understand the underlying mechanisms and find effective nutritional solutions, especially for low and middle-income countries. Improving the anti-inflammatory and antioxidant status by increasing the intake of fruits and vegetables rich in bioactive compounds can prevent malnutrition and the associated inflammation [12,13]. In sub-Saharan Africa, green leafy vegetables (GLVs) are widely consumed by the population and are key foods for their nutrition and dietary diversity [14]. Traditionally, GLVs are cooked as sauces to serve with cereal-based dishes. GLVs are excellent sources of iron, zinc, magnesium, potassium, and vitamin C, thus contributing to the daily requirements of these micronutrients, especially in children [15,16]. They are also rich in polyphenols (e.g. phenolic acids or flavonoids) and carotenoids (e.g. lutein, β-carotene), giving them strong antioxidant capacity [17]. Moreover, GLVs have demonstrated anti-inflammatory effects in vitro (murine and human immune cell models) [18,19] and in vivo using animal models (rats, mice, pigs) [20,21]. However, their impact on intestinal inflammation remains underexplored, particularly for cassava and African roselle leaves, which are widely consumed in Africa yet notably understudied.

In the past decades, *in vitro*, *ex vivo* and *in vivo* models have been
developed to study intestinal epithelium functions, metabolism and
inflammation. Animal models replicate organism-level physiology, but
they raise ethical concerns and show species-specific responses,
complicating their extrapolation to humans (Ponce de León Rodríguez,
[22]). *In vitro* models reduce animal use and allow simpler and more
reproducible studies of molecular mechanisms. The Caco-2 cell line
(immortalized human colorectal adenocarcinoma cells) mimics enter-
ocytes [23], but has limitations, including the absence of mucus layer
[24]. To better replicate the human intestinal barrier, Caco-2 cells can
be co-cultured with HT29-MTX cells, a stable clone derived from HT29
colorectal adenocarcinoma cells [25]. While not all HT29 cells produce
mucus, those that differentiate into goblet cells like the HT29-MTX clone
can produce a dense mucus layer due to the secretion of gel-forming
mucins (MUC2, MUC5AC) [26,27]. Regarding inflammation, the Caco-
2 cell line can produce some inflammatory molecules in response to
cytokines or stimuli (e.g. lipopolysaccharides, LPS, from microorgan-
isms), such as interleukin (IL)-1β, tumour necrosis factor (TNF)-α,
interferon (IFN)-γ [28,29]. Several stimuli, such as IFN-γ and TNF-α, are
known to act synergically on intestinal epithelial cells (F. [30,31]).
However, for modelling intestinal inflammation, Caco-2 and HT29-MTX
cells lack immune cells that are crucial for pro-inflammatory cytokine
production [32]. Therefore, some groups tried to integrate immune
cells, such as peripheral blood mononuclear cells or murine
macrophage-like cells, in co-culture models [33,34]. As mixing human
and murine cells in culture models is debated, the human THP-1 cell
line, which can differentiate into macrophages, provides a solution
[35,36] to better mimic physiological conditions. However, although
this approach could be particularly useful in gut inflammation research,
studies using co-culture of Caco-2, HT29-MTX, and THP-1 cells remain
rare.

Based on literature, and particularly the works of [37–39,40] we
developed an *in vitro* triculture model to evaluate the anti-inflammatory
effects of food-derived bioactive compounds in the intestine. To mimic
the intestinal barrier, we co-cultured Caco-2 cells with mucus-producing
HT29-MTX cells grown on a filter and macrophage-like THP-1 cells at
the basolateral side. The model was fully characterized (morphology,
pro-inflammatory cytokine release, and barrier permeability changes)
before using it to assess the anti-inflammatory effects of polar/non-polar
extracts and of *in vitro* digested sauces of cassava and roselle leaves.

**2. Materials and methods**

**2.1. Chemical and reagents**

Dulbecco's Modified Eagle Medium GlutaMax™ (DMEM), RPMI 1640-GlutaMAX™ medium, nonessential amino acids, foetal bovine

serum (FBS), penicillin-streptomycin (10,000 U-10 mg), trypsin-
ethylenediamine tetraacetate (EDTA), Dulbecco's phosphate-buffered
saline with or without calcium and magnesium (DPBS), IFN-γ, were
purchased from Thermo Fisher Scientific (Saint-Quentin Fallavier,
France). Phorbol 12-myristate 13-acetate (PMA), dexamethasone, LPS
from Escherichia coli O55 B5, acid acetic, 4-(2-hydroxyethyl)-1-piper-
azineethanesulfonic acid (HEPES), Triton X-100, Bacillus sp. α-amylase
(≥40.0 U/mg protein), porcine pepsin (≥2500 U/mg protein), porcine
pancreatin (activity, 8 × USP specifications) and porcine bile extract
were from Sigma Aldrich (Saint-Quentin Fallavier, France). Alcian blue
and fast red were from Sigma-Aldrich (St. Louis, USA). Ethanol, hexane
and sodium hydroxide (NaOH) were from HoneyWell Riedel-de Haën
(Seelze, Germany).

**2.2. Plant materials**

**2.2.1. GLV supply**

The two African GLVs (one batch per species), cassava (*Manihot esculenta*) and roselle (*Hibiscus sabdariffa*), were purchased frozen from African grocery stores in Montpellier (France) in 2021. Leaves were then freeze dried, powdered, and stored at –20 °C.

**2.2.2. Preparation of GLV polar and non-polar extracts**

Solid-phase extraction, using the method reported by Fioroni et al. [17], was used to obtain polar (ethanol/water, 80/20) and non-polar (ethanol/hexane, 60/40) extracts from the freeze-dried GLVs. Extracts were stored at –20 °C until use.

**2.2.3. Preparation of GLV-based model sauces**

Freeze-dried GLVs were used to prepare simplified model sauces with dry matter content (12 %) and oil content (4 %) similar to those of field recipes based on Burkinabe and Beninese recipes [41]. Briefly, 5 g of powdered freeze-dried GLVs were mixed with 2.4 g peanut oil and 52 g water, then boiled for 5 min. After cooling, sauces were weighed, adjusted to 60 g with water, and stored at –20 °C until use.

**2.3. Simulated GLV sauce digestion and micelle formation**

A three-step *in vitro* digestion protocol, validated in human studies [42], was used after adaptations [43]. GLV model sauces (6 g) or peanut oil (control) were mixed with 16 mL 0.9 % NaCl and 1.25 mL salivary solution (pH 7) to mimic the oral phase. After 10 min at 37 °C, pH was adjusted to 4.00 ± 0.02 with 1 M HCl, and 1 mL porcine pepsin (P7012) (40 g/L in 0.1 M HCl) was added to mimic the gastric phase at 37 °C for 30 min. Then, the pH was adjusted to 6.00 ± 0.02 with 0.9 M sodium bicarbonate, followed by addition of 4.5 mL porcine pancreatin (P7545) solution (3 g/L in 0.1 M sodium citrate, pH 6) and 2 mL porcine bile (B8631) extract (127 g/L in 0.1 M sodium citrate, pH 6) to simulate the intestinal phase at 37 °C for 30 min.

Micellar fraction was separated from oil droplets and food particles by ultracentrifugation at 3000 rpm (Thermo Scientific SL 40 FR centrifuge in a TX-750 rotor), at 10 °C for 72 min, then filtered through 0.80 and 0.22 µm filters (Millipore, Burlington, MA, USA). The micellar fraction consists of micellar fractions containing non-polar compounds (including carotenoids) and a continuous polar phase that contains polyphenols. Aliquots of micellar fraction were diluted 1:5 with DMEM before assessing their anti-inflammatory effect.

**2.4. Cell lines and culture conditions**

The TC-7 subclone (ATCC: HTB-37) of the Caco-2 cell line [44] was
generously provided by Monique Rousset (Nancy University, France).
The HT29-MTX cell line (HT29-MTX 10–6) was obtained from the Cel-
luloNet biobank BB-0033-00072 (UMS3444/US8/SFR Biosciences,
Paris). Cells were maintained in 75 cm² flasks (Falcon™) at 37 °C and 5
% CO₂ in DMEM GlutaMax™ with 1 % nonessential amino acids, 10 %
heat-inactivated FBS, and 1 % penicillin-streptomycin (10 000 U–10 mg). At 80 % of confluence, cells were trypsinized with 0.05 % trypsin/ 1 mM EDTA. For experiments, Caco-2-TC7 cells were used between passages 56 and 65 and HT29-MTX cells between passages 22 and 67.

The human THP-1 cell line (monocytes; ATCC-TIB-202TM) was maintained in suspension in RPMI 1640-GlutaMax™ medium with 10 % FBS and 1 % penicillin-streptomycin at 37 °C and 5 % CO2. THP-1 cells were differentiated into macrophage-like cells by adding 20 ng/mL PMA for 48 h. After 24 h of rest in complete RPMI medium, cells were washed with DPBS (Ca²⁺[+] Mg²⁺[+]) containing 1 % FBS before use. THP-1 cells were used between passages 9 and 29.

## 2.5. Triculțure model

Our first aim was to establish a stable Caco-2-TC7/HT29-MTX biculture system to mimic the human intestine with barrier integrity and mucus secretion. Caco-2-TC7 and HT29-MTX cells were plated alone or together (Caco-2-TC7/HT29-MTX biculture; 9:1 ratio) on 6-well ThinCert™ inserts (0.4 µm pore size, Greiner bio-one) at a density of 10⁵ cells/cm² (day 1) and cultured in complete culture medium (changed every 2 days) for 15 days to allow their spontaneous differentiation into IEC. For the biculture, a 9:1 ratio was used to simulate the proportion of enterocytes and goblet cells in the intestine [45].

We then established a stable triculture of Caco-2-TC7/HT29-MTX cells with PMA-differentiated THP-1 cells to mimic the human intestine in a homeostatic state adapted from published protocols [37–39,40]. At day 12 of Caco-2-TC7/HT29-MTX biculture, THP-1 cells (2.6 × 10⁴ cells/cm²) were seeded in new 6-well plates (Greiner bio-one) and differentiated with 20 ng/mL PMA for 48 h, followed by washes with DPBS (Ca²⁺[+] Mg²⁺[+])/1 % FBS. Then, the Caco-2-TC7/HT29-MTX cell inserts were added to the wells containing the differentiated THP-1 cells (day 14) and grown in DMEM for 24 h (Fig. 1). Barrier integrity was assessed by measuring the transepithelial electrical resistance (TEER) and phenol red apparent permeability. Mucus secretion was assessed by histological staining and glycoprotein quantification.

The third objective was to induce an inflammation-like response in the stable triculture model. To this aim, at day 15, a pro-inflammatory

cocktail of LPS-IFN-γ (1 µg/mL-50 ng/mL) was added to both compartments. These concentrations were chosen based on the literature and after preliminary adjustments. After 4 or 18 h of stimulation, the inflammatory markers including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), cytokines (TNF-α, IL-6, IL-8), and epithelial barrier integrity (TEER, apparent permeability) were measured. TNF-α release was measured after 4 h of stimulation, reflecting its early secretion during inflammation, while IL-6 and IL-8, secreted later, were measured after 18 h [35]. To confirm the protocol effectiveness, triculture were pre-incubated with 10 µM dexamethasone, a known anti-inflammatory compound, for 2 h before adding the pro-inflammatory cocktail.

## 2.6. GLV exposure effects

### 2.6.1. Effect of GLV extracts on cytokine production by LPS-stimulated THP-1 cells

THP-1 cells (5 × 10⁴ cells/well) were seeded in 24-well plates in complete RPMI 1640-GlutaMAX™ medium with 20 ng/mL PMA and differentiated for 48 h, as described in Section 2.5. After 24 h, cells were incubated with GLV extracts (6.25–50 µg/mL) at 37 °C for 2 h. Then, cells were stimulated with LPS (100 ng/mL) and supernatants were collected at 4 and 18 h for cytokine (TNF-α, IL-6, and IL-8) measurements.

### 2.6.2. Effect of GLV extracts and digested sauces on the triculture model of intestine inflammation

The apical compartment of stable triculture was pre-incubated with GLV extracts (50 µg/mL) or micellar fractions (diluted 1:5 with DMEM) for 2 h before the addition of LPS-INF-γ (1 µg/mL-50 ng/mL) to induce inflammation. After 4 h or 18 h of incubation, cytokine production was measured in the apical and basolateral medium as well as epithelial barrier integrity and cytotoxicity.

## 2.7. Lactate dehydrogenase (LDH) assay to measure cytotoxicity

To test cell viability in the different experimental conditions, LDH

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Schematic representation of the *in vitro* triculture model protocol.</figcaption></figure>
release into the apical medium was quantified by spectrophotometry using the LDH Activity Assay Kit (Sigma-Aldrich, Saint-Quentin Fallaviers, France) following the supplier's instructions.

## 2.8. Barrier integrity assessment

### 2.8.1. TEER measurement

The integrity of intestinal monolayers (single and biculture) was monitored daily by TEER measurement with the Millicell®-ERS Voltmeter until confluence and during medium changes (three times per week). In the triculture system, TEER was assessed before co-culture with THP-1 cells, after 24 h of rest, and after incubation with LPS/IFN-γ for 18 h. TEER values were expressed as Ohms (Ω).

### 2.8.2. Phenol red apparent permeability (Papp)

Phenol red apparent permeability (Papp) was measured as previously described [46]. After two washes with DPBS (Ca⁺²[+] Mg⁺²[+]), 1 mL of 1 mM phenol red in DPBS (Ca⁺²[+] Mg⁺²[+]) with 20 mM HEPES at pH 7.4 was added to the apical side and 1 mL of DPBS with 20 mM HEPES in the basolateral side. Following incubation at 37 °C for 1 h, phenol red concentration in the basolateral compartment was measured by reading the absorbance at 560 nm after the addition of 10 % NaOH (0.1 N). The trans-epithelial flux was expressed as Papp coefficient according to the following equation:

$$Papp = \frac{C}{C_0} \cdot \frac{V}{t} \cdot \frac{1}{A}$$

where C is the phenol red concentration after 60 min (mM), C₀ is the initial phenol red concentration (mM), t is the diffusion time (s), V is the basolateral compartment volume (mL), and A is the filter membrane area (cm²).

## 2.9. Mucus staining with alcian blue in Caco-2-TC7/HT29-MTX cell biculture

In Caco-2-TC7 and HT29-MTX cell single cultures and in Caco-2-TC7/HT29-MTX cell biculture (9:1), mucus from HT29-MTX cells was stained with alcian blue and alkaline phosphatase from differentiated Caco-2 cells with fast red (SIGMAFAST™ Fast Red TR/Naphthol AS-MX Tablets, St. Louis, USA). Alcian blue was dissolved in 3 % acetic acid and added to the apical side at room temperature for 30 min. After washing with DPBS (Ca⁺²[−] Mg⁺²[−]), cells were stained with fast red at 37 °C. The reaction was stopped with water and samples were observed under an inverted optical microscope (Nikon Eclipse TE200, Minato, Tokyo, Japan).

## 2.10. Histological staining of mucus in Caco-2-TC7/HT29-MTX cell biculture

To observe mucus production by histochemistry in Caco-2-TC7/HT29-MTX cell biculture (9:1), at day 16, cells were gently rinsed with cold DPBS (Ca + 2[−] Mg + 2[−]) / 4 % sucrose/3 % acetic to avoid mucus elimination. Cells were fixed with ice-cold methanol-Carnoy's fixative solution (60 % anhydrous methanol, 30 % chloroform, 10 % acetic acid) for 15 min. After fixation, cells were gently rinsed 3 times and rehydrated with ice-cold DPBS/4 % sucrose/3 % acetic. Culture inserts were removed and membrane were cut in small piece and embedded in DPBS/2.5 % degassed agarose, dehydrated through a graded series of ethanol, and embedded in cold-polymerizing resin Technovit 7100 methacrylate (Heraeus Kulzer GmbH, Wehrheim, Germany). Specimens were mounted on bloc/histobloc with Technovit resin 3040 (Heraeus Kulzer GmbH, Wehrheim, Germany). Samples were cut (4 µm of thickness) with a Microtome Microm HM355S (Thermo Scientific, Waltham, USA), placed on clean slides and dried. Tissue sections were stained with Mayer's Mucihematein Modified reagent, ready to use

(Electron Microscopy Sciences, Hatfield, England) for 20 min at room temperature and briefly washed with tap water to remove excess dye. The slides were then washed three times in milliQ water bath, each for 5 min. After drying, staining intensity was evaluated microscopically and slides were mounted with Q Path® Isomount 2000 mounting medium (VWR international, Radnor, USA) and 0.15 mm coverslips.

## 2.11. Mucus quantification by glycoprotein measurement

The periodic acid/Schiff (PAS) stain was used to quantify mucin, the main mucus glycoprotein in cultures [47]. Cells were lysed with 200 µL 1 % Triton X-100 in DPBS and incubated with 0.5 % periodic acid at 37 °C for 2 h. Then, the Schiff reagent was added at room temperature for 30 min. Absorption was measured at 555 nm using a Spectro UV-Vis Infinite 200 Pro plate reader (Tecan Group Ltd., Grödig, Austria), with a calibration curve from 10 to 600 µg/mL using porcine mucin.

## 2.12. Quantification of cytokines and inflammation-related enzymes

### 2.12.1. Enzyme-Linked Immuno-Sorbent Assay (ELISA)

TNF-α, IL-6 and IL-8 were quantified using ELISA kits (Human TNF alpha Uncoated ELISA, Human IL-6 Uncoated ELISA, Human IL-8 Uncoated ELISA) according to the manufacturer's instructions (Invitrogen™ by Thermo Fisher Scientific, Vienna, Austria).

### 2.12.2. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis

For gene expression analysis by RT-qPCR, total RNA was extracted from harvested cells using the NucleoSpin® RNA/Protein kit (Macherey-Nagel, Hoerdt, France) and concentrated, if needed, using the Nucleo-Spin® RNA Clean-up Kit XS (Macherey-Nagel, Hoerdt, France). RNA purity was assessed by spectrometry (Thermo Scientific™ NanoDrop™ One/OneC, Waltham, USA) and for finer quantification, with a Qubit™ 4 fluorometer (Invitrogen™, Carlsbas, USA) and the Qubit™ RNA BR Assay kit (Invitrogen™, Carlsbas, USA). Then, cDNA was synthesized by RT with 1 µg of total RNA and the LunaScript® RT SuperMix kit (New England Biolabs Inc., Ipswich, USA) following the manufacturer's instructions. The RT-qPCR analyzes were carried out at the qPCR platform of the University of Montpellier, France, using the LightCycler® 480 System thermocycler in 384-well block (Roche, Penzberg, Germany) with 0.5 µL diluted cDNA (1/20) and 1 µL of master mix prepared with the SensiFAST™ SYBR® No-ROX kit (Bioline Meridian Bioscience, Cincinnati, USA). The programme used was as follows: 5 min at 95 °C for enzyme activation, 45 cycles with denaturation at 95 °C for 10 s, 10 s pairing step at 64 °C, and 10 s elongation step at 72 °C. The primer sequences (Table 1) were selected with the NCBI Primer Blast application. The relative expression level for each gene was normalized to that of the housekeeping genes RPLP0 and RPL37A for Caco-2 and HT29-MTX cells, and RPLP0 and ACTB for THP-1 cells [48,49,50].

## 2.13. Immunofluorescence staining

Immunofluorescence staining was used to assess the expression of ZO-1, occludin, claudin (tight junction proteins) and of MUC5AC (mucin) in the biculture and triculture models. Cells were rinsed with ice-cold DPBS/4 % sucrose/3 % acetic acid, fixed with 4 % paraformaldehyde/4 % sucrose in DPBS for 20 min, and incubated with 0.1 % Triton X-100 for 3 min. Then, cells were incubated in Image-IT® Fx Signal Enhancer (Cell Signaling Technology, Danvers, USA) for 30 min and blocked with DPBS/Fish Gelatin Blocking Agent 1× solution (Biotium, Fremont, USA) for 60 min. Cells were incubated (4 °C overnight) in BlockAid™ Blocking Solution (Thermo Fisher Scientific, Waltham, USA) with the following primary antibodies against occludin (2 µg/mL) from Atlas antibodies, and ZO-1 (2.5 µg/mL), claudin (5 µg/mL) and MUC5AC (3 µg/mL) from Abcam. After washing with PBS-Fish Gelatin Blocking Agent 1×, cells were incubated with Alexa-conjugated
<table><caption>**Table 1**

Primer pairs used to quantify cytokine gene expression in intestinal and immune cells.</caption><thead><tr><th>Genes</th><th>Primers sequences (5'→3')</th><th>Concentration</th><th>PCR efficiency</th></tr></thead><tbody><tr><td>NOS2</td><td>F-CCGAGGCAAACAGCACATTC<br>R-CTGAGGGCTTTGCTGAGGTC</td><td>0.6 µM</td><td>1.95</td></tr><tr><td>COX2</td><td>F-GCTGTTCCACCCATGTCAA<br>R-TTCCGGTGTTGAGCAGTTTTCT</td><td>0.4 µM</td><td>2</td></tr><tr><td>TNF</td><td>F-TTCCCCAGGGACCTCTCTCT<br>R-AGGGTTTGCTACAACATGGGC</td><td>0.4 µM</td><td>1.97</td></tr><tr><td>IL6</td><td>F-CCAGAGCTGTGCAGATGAGTACA<br>R-GGCATTTGTGGTTGGTCAGG</td><td>0.6 µM</td><td>1.97</td></tr><tr><td>IL8</td><td>F-AGACAGCAGAGCACACAAGC<br>R-CACAGTGAGATGGTTCCTTCC</td><td>0.6 µM</td><td>2</td></tr><tr><td>IL10</td><td>F-GGCGCTGTCATCGATTTCTTC<br>R-CACTCATGGCTTTGTAGATGCC</td><td>0.4 µM</td><td>1.96</td></tr><tr><td>RPLP0</td><td>F-TCTACAACCCCTGAAGTGCTTGAT<br>R-CAATCTGCAGACAGACACTGG</td><td>0.4 µM</td><td>2</td></tr><tr><td>RPL37A</td><td>F-<br>TCTGTGGCAAAACCAAGATGAAGA<br>R-TTACCGTGACAGCGGAAGTG</td><td>0.4 µM</td><td>1.93</td></tr><tr><td>ACTB</td><td>F-GATCATTGCTCCTCCTGAGC<br>R-CCGGACTCGTCATACTCCTG</td><td>0.4 µM</td><td>1.87</td></tr></tbody></table>

secondary antibodies at 37 °C for 60 min; anti-rabbit Alexa Fluor 488 (4 µg/mL); and anti-mouse Alexa Fluor 633 (4 µg/mL) from Thermo Fisher Scientific (Saint-Quentin Fallavier, France). Cell nuclei were counterstained with DAPI (3 µg/mL in DPBS-sucrose) in the dark for 15 min (Euromedex, Souffelweyersheim, France). Samples were mounted on clean slides with Prologold™ Gold antifade reagent (Thermo Fisher Scientific, Waltham, USA) and 0.17 mm coverslips. Fluorescent images were captured with a 880 Multiphoton confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) using Zeiss ZEN software at the Confocal Microscopy Platform - MRI in Montpellier, France. The Imaris imaging software (Andor Technology, Belfast, Northern Ireland) was used to reconstruct 3D images from z-stack data.

## 2.14. Statistical analysis

All data were expressed as the mean ± standard deviation (SD). Data were analysed with Statgraphics Centurion 19.0 using one-way ANOVA followed by the Fisher's least significant difference (LSD) test. A p-value <0.05 was considered significant. When homogeneity of variance was not respected (Levene's test), a logarithmic nonparametric test (Games-Howell test) was used.

# 3. Results

## 3.1. Characterization of Caco-2-TC7/HT29-MTX cell biculture

### 3.1.1. Barrier integrity assessment

TEER values increased progressively in both Caco-2-TC7 cell single-culture and Caco-2-TC7/HT29-MTX cell biculture monolayers for 3 days after seeding (Fig. 2A). The highest TEER values (at confluence; day 3) were significantly lower in biculture (580 Ω.cm²) than in Caco-2-TC7 cells alone (775 Ω.cm²). Conversely, membrane permeability was similar in monocultures and biculture (data not shown).

3.1.1.1. Mucus secretion. After 16 days of culture, the apical side of Caco-2-TC7/HT29-MTX cell biculture was stained with alcian blue (acidic mucins secreted by HT29-MTX cells) and fast red (alkaline phosphatase, a marker of Caco-2 cell differentiation) (Fig. 2B). The staining patterns showed well-differentiated Caco-2-TC7 cells and a mucus layer produced by HT29-MTX cells. The presence of both red and blue staining in Caco-2-TC7/HT29-MTX cell biculture confirmed the growth and distribution of both cell types. Moreover, red multicellular "bump-like" areas, characteristics of Caco-2-TC7 cells, were interspersed

with blue HT29-MTX cells. Quantification using PAS staining confirmed the enhanced mucus secretion in Caco-2-TC7/HT29-MTX cell biculture compared with Caco-2-TC7 cell monocultures (+49 %, p < 0.05) (Fig. 2C).

Lastly, staining with mucicarmine (mucins) (Fig. 2D) of Caco-2-TC7/HT29-MTX cell biculture at day 16 showed the presence of both cell types, of an apical portion distended by abundant mucus-laden granules in HT29-MTX cells, and of a thin mucus layer.

## 3.2. Optimization of the triculture model conditions

### 3.2.1. Characterization of stable triculture of Caco-2-TC7/HT29-MTX cells with PMA-differentiated THP-1 cells

To assess the impact of THP-1 cells on the Caco-2-TC7/HT29-MTX cell barrier integrity, TEER and Papp were monitored over 48 h. In triculture, TEER values slightly but significantly decreased after 24 h (–15 % compared with control biculture) (Fig. 3A). This indicated that differentiated THP-1 cells induced a weak barrier disruption that did not lead to Papp increase (Fig. 3B). LDH release quantification showed no significant increase in stable triculture, in both apical and basolateral compartments, compared with biculture (data not shown).

### 3.2.2. Characterization of triculture after incubation with the LPS/IFN-γ cocktail to induce inflammation

3.2.2.1. Barrier integrity assessment. Incubation with LPS/IFN-γ strongly and progressively decreased the TEER values in stable triculture compared with control biculture (–25 %) (Fig. 3A). This indicates that the barrier-disrupting effects of THP-1 cells was exacerbated by inflammation induction. As expected, inflammation induction increased Papp, indicating higher barrier permeability (Fig. 3B). Pretreatment with dexamethasone prevented LPS/IFN-γ effects on TEER and permeability. The similar LDH concentrations in both apical and basolateral compartments of triculture incubated or not with LPS/IFN-γ (with/without dexamethasone) indicated that LPS/IFN-γ and dexamethasone did not have any cytotoxic effect (data not shown).

3.2.2.2. Immunofluorescence staining of tight junctions. To determine whether the permeability increase induced by incubation with LPS/IFN-γ was linked to tight junction disruption, the expression of tight junction proteins (ZO-1, occludin, claudin) was assessed by immunofluorescence (Fig. 4A). In biculture, ZO-1, occluding and claudin were homogeneously and clearly expressed, defining the cell perimeter and enabling the apical membrane identification. In triculture incubated with LPS/IFN-γ, the distribution of tight junction proteins was altered: staining decreased in intensity and became irregular. The intracytoplasmic fluorescence signal increased, indicating a delocalization of tight junction proteins from the apical membranes and a rearrangement of their cellular organization. The specific analysis of ZO-1, an essential component of the tight junctional complex that is normally located toward the apical side of epithelial cells, showed that upon incubation with LPS/IFN-γ, in triculture, ZO-1 signal intensity decreased at the apical side and increased at the basolateral side with intracytoplasmic accumulation (Fig. 4B). This indicated ZO-1 expression delocalization and reorganization.

3.2.2.3. Mucus production. MUC5AC staining was increased in triculture incubated with LPS/IFN-γ compared with biculture (Fig. 4C), indicating higher mucus production by HT29-MTX cells in inflammatory conditions.

3.2.2.4. TNF-α, IL-6, and IL-8 production. Cytokine release (expressed as the percentage of the concentration in stable triculture) was measured in the apical and basolateral compartments of biculture, stable triculture, and triculture incubated with LPS/IFN-γ for 18 h (with/without
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Characterization of Caco-2-TC7/HT29-MTX cell biculture A. TEER values of Caco2-TC7 cells alone and bicultured with HT29-MTX cells at different time points expressed as ohms.cm². B. Caco2-TC7 cells (single culture) stained with fast red (left), HT29-MTX cells (single culture) stained with alcian blue (right), and Caco-2-TC7/HT29-MTX cell biculture (9:1 ratio) stained with fast red and alcian blue (bottom). Arrows show “bump-like” areas characteristic of Caco-2-TC7 cells. C. Mucin quantification by PAS stain in Caco2-TC7 and HT29-MTX single cultures and Caco-2-TC7/HT29-MTX cell biculture, expressed as percentage of positive-Caco-2-TC7 cells. \*p < 0.05 versus Caco2-TC7 cells. D. Histological staining of biculture Caco-2-TC7/HT29-MTX with Mayer's Mucihematein. Arrows show mucus layer. TEER: transepithelial electrical resistance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

dexamethasone pretreatment) (Fig. 3C). In the apical medium, IL-6 and TNFα levels were very low (<15 pg/mL) in all culture models with no significant difference. Only IL-8 concentration significantly increased upon inflammation induction (1200 pg/mL) compared with stable triculture (280 pg/mL). This increase was prevented by pre-incubation with dexamethasone (300 pg/mL). In the basolateral compartment, the concentration of all three cytokines increased upon incubation with LPS/IFN-γ (values up to 15,000 pg/mL for TNFα, 300 pg/mL for IL-6, and 180,000 pg/mL for IL-8) compared with stable triculture. Dexamethasone prevented this inflammatory response.

### 3.2.2.5. Gene expression by RT-qPCR.
The expression of genes encoding inflammation-related enzymes (COX-2 and NOS2) and cytokines (TNF-α, IL-6, and IL-8) was evaluated in both Caco-2-TC7/HT29-MTX biculture and in triculture (after incubation or not with LPS/IFN-γ for 18 h) (Fig. 5). In IEC (apical compartment), NOS2 (iNOS) and IL8 (IL-8) expression was upregulated after exposure to LPS/IFN-γ for 18 h, while COX2 and TNF gene expression did not change. IL6 expression was not detectable (data not shown). In THP-1 cells (basolateral compartment), gene expression of all cytokines as well as COX2 was enhanced by LPS/

IFN-γ exposure, but not NOS2.

### 3.3. Application: assessment of inflammation modulation by cassava and roselle leaves

#### 3.3.1. Effect of the leaf extract concentration on cytokine production by THP-1 macrophages stimulated with LPS

The effects of four concentrations of GLV polar and non-polar extracts on cytokine production by LPS-stimulated THP-1 cells were tested to select the most appropriate concentration for further studies. First, 0.1 % ethanol (the vehicle used, corresponding to the maximum ethanol concentration in the GLV extract at 50 µg/mL) had no effect on cytokine production (data not shown). Incubation of THP-1 cells with polar and non-polar cassava extracts (6.25–50 µg/mL) induced a concentration-dependent inhibition of TNF-α compared with the positive control (LPS-stimulated THP-1 cells): 34 % and 21 % of decrease at 50 µg/mL, respectively (Fig. 6A). Roselle extracts also attenuated TNF-α production, particularly the non-polar extract (−35 % at 50 µg/mL). Both cassava and roselle extracts induced a concentration-dependent inhibition of IL-6 production compared with the positive control: 50 % and 60
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Characterization of triculture of Caco-2-TC7/HT29-MTX cells with PMA-induced THP-1 cells. To induce inflammation, 24 h after the triculture set-up (stable triculture), cells were incubated with LPS-INF-γ (1 µg/mL-50 ng/mL) for 18 h (inflamed triculture). Dexamethasone (10 µM) was added as positive control (+dex) 2 h before the pro-inflammatory stimulation. (A) TEER (barrier integrity) measurement over 48 h after the triculture set-up, expressed as percentage of the biculture values. (B) Phenol red apparent permeability (Papp) at 48 h in triculture in which inflammation was induced or not. (C) TNF-α, IL-8 and IL-6 release in the apical and basolateral compartments in biculture, stable triculture and triculture in which inflammation was induced or not, expressed as percentage of the values in the stable triculture. Data are the mean ± SD (bars) (n = 12) from 4 experiments performed in triplicate. \*p < 0.05, stable triculture versus biculture. \*\*p < 0.05, inflamed triculture versus stable triculture. TEER: transepithelial electrical resistance; TNF: tumour necrosis factor; IL: interleukin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

% decrease at 50 µg/mL for polar and non-polar extracts, respectively (Fig. 6B). Polar and non-polar cassava extracts also inhibited IL-8 pro-duction at 50 µg/mL by 40 % and 27 %. Conversely, only the non-polar roselle extract had an effect (33 % decrease) (Fig. 6C).

These experiments identified 50 µg/mL as the most effective con-centration for inhibiting cytokine production by LPS-stimulated THP-1 cells. Moreover, LDH release was minimal at all tested extract concen-trations and also with 0.1 % ethanol vehicle, confirming the absence of cytotoxic effects (data not shown).

### 3.3.2. Modulation of inflammation markers by GLV extracts and micellar fractions in the triculture model after inflammation induction by LPS/INF-γ

#### 3.3.2.1. Barrier integrity assessment.
In triculture stimulated with LPS/ INF-γ, polar and non-polar cassava and roselle extracts maintained TEER values to levels comparable to those of stable triculture and even biculture (100–120 Ohm) (Fig. 7A and D). Polar cassava extracts (Fig. 7B) and both polar and non-polar roselle extracts (Fig. 7E) signif-icantly limited the increase of barrier permeability observed upon

incubation with LPS/INF-γ and also with ethanol. Similarly, cassava and roselle micellar fractions prevented the TEER decrease induced by LPS/ INF-γ stimulation and maintained its values close to those observed in biculture (Fig. 7G). This was more pronounced with roselle micellar fractions, as confirmed by the permeability decrease (Fig. 7H). Thus, both polar and non-polar extracts and the digested sauces of cassava and roselle leaves protected the intestinal barrier integrity in inflammation conditions. Neither extracts nor micellar fractions caused cytotoxicity (evaluated by measuring LDH release) (data not shown).

#### 3.3.2.2. TNF-α, IL-6, and IL-8 cytokine production.
In the inflamed tr-culture, TNF-α, IL-6 and IL-8 production increased, compared with sta-ble triculture. Analysis of the effects of vehicles (i.e. oil for micellar fractions and 0.1 % ethanol for extracts) on cytokine production showed that oil did not have any inhibitor effect, while 0.1 % ethanol exhibited some effects (described later). Neither cassava nor roselle extracts and micellar fractions decreased IL-6 production in triculture stimulated with LPS/INF-γ (data not shown).

In apical medium, only IL-8 could be quantified. Surprisingly,
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Immunostaining of tight junction proteins (A,B) and MUC5AC (C) in Caco-2-TC7/HT29-MTX cell biculture and inflamed triculture of Caco-2-TC7/HT29-MTX cells with PMA-differentiated THP-1 cells (incubation with LPS-INF-γ, 1 µg/mL-50 ng/mL, for 18 h). (A) Images show occludin (yellow), claudin (orange) and ZO-1 (green) expression; cell nuclei are in blue. Arrows indicate the increased intracellular pool of tight junction proteins in inflamed triculture versus biculture. (B) 3D confocal laser scanning microscopy image of ZO-1 expression in a Caco-2-TC7/HT29-MTX cell biculture and in inflamed triculture. Images were reconstructed from z-stack files. Arrows show ZO-1 translocation from the apical to basolateral side. (C) MUC5AC staining in a Caco-2-TC7/HT29-MTX cell biculture and in a LPS-INF-γ-stimulated triculture. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

ethanol decreased IL-8 secretion in triculture incubated with LPS/INF-γ (Fig. 7C). Compared with the 0.1 % ethanol control, non-polar cassava extracts showed a significant inhibition (Fig. 7C). Roselle polar and particularly non-polar extracts decreased IL-8 secretion by intestinal cells in LPS/INF-γ-stimulated triculture (Fig. 7F). Roselle micellar fractions also decreased IL-8 secretion by intestinal cells (–27 %), but not cassava micellar fractions (Fig. 7I).

In the basolateral medium, cassava extracts decreased the secretion of TNF-α and IL-8 by THP-1 cells compared with the 0.1 % ethanol control (Fig. 7C). Roselle extracts had no significant effect on TNF-α secretion, but the polar extract had a slight inhibitory effect on IL-8 (Fig. 7F). Cassava and roselle micellar fractions significantly and similarly decreased the secretion of TNF-α but not of IL-8 (Fig. 7I).

## 4. Discussion

In this study, we developed an *in vitro* intestinal model to assess the potential anti-inflammatory effects of bioactive compounds from foods. Given the context of high prevalence of malnutrition and environmental stress in sub-Saharan Africa, we focused on two African GLVs, cassava and roselle which are rich in micronutrients and bioactive compounds with antioxidant and anti-inflammatory properties [17].

Based on a literature review of *in vitro* models of intestine inflammation and their use (Ponce de León Rodríguez, [22]), we selected the

most commonly used intestinal epithelial cell lines: Caco-2-TC7 cells with absorption capacity and mucus-secreting HT29-MTX cells (goblet cells) [51,25,24]. The Caco-2-TC7 clone could differentiate faster (15 days) than the parental line (30 days), while retaining the same characteristics and forming a more homogeneous and stable monolayer [52,44,53]. Mucus from HT29-MTX cells is crucial for estimating the intestinal permeability and absorption [27]. These cell lines are used in co-culture to closely mimic the *in vivo* intestinal physiology [54,26,55]. In our model, mucus production by HT29-MTX cells was demonstrated by alcian blue/fast red staining. Histological sections showed HT29-MTX cells among enterocytes, identifiable by their mucus pockets, and clusters of mucus on the apical membrane. Fluorescent labelling of MUC5AC, mucins produced by HT29-MTX cells [56], further confirmed the presence of the mucus layer.

Caco-2 cells can produce inflammatory mediators (e.g. cytokines or enzymes) in response to stimuli (LPS, IL-1β, TNF-α, IFN-γ) [28,29], but this response is often low. The presence of both epithelial and immune cells would better represent the physiological conditions than epithelial cells alone in an inflammatory context. The aim of our study was to establish a triculture model of Caco-2-TC7/HT29-MTX cells with human immune cells to mimic the human intestine in homeostatic conditions and upon inflammation. We selected the human monocyte-like THP-1 cell line because it can differentiate into macrophages and is widely used in inflammation models [35,36].
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Analysis by RT-PCR of the following genes: (A) *NOS2*, (B) *COX2*, (C) *TNF*, (D) *IL8*, and (E) *IL6* in cells from the apical and basolateral compartments in biculture, stable triculture and LPS-INF-γ-stimulated triculture (LPS-INF-γ, 1 µg/mL-50 ng/mL for 18 h). The relative expression level of each target gene was normalized to that of two housekeeping genes previously validated (*RPLP0* and *RPL37A* for biculture, *RPLP0* and *β*-actin (ACTB) for THP-1) and compared to stable triculture. Data represent means ± SD (bars) (n = 3, triplicate measurements in one experiment). \*p < 0.05, LPS-INF-γ-stimulated *versus* stable triculture. *TNF*, tumour necrosis factor; *IL*, interleukin.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Effect of different concentrations of polar and non-polar extracts of cassava and roselle leaves on TNF-α (A), IL-6 (B) and IL-8 (C) production by LPS-stimulated THP-1 macrophages. Cells were pre-incubated with GLV extracts (6.25, 12.5, 25, or 50 µg/mL) for 2 h before stimulation with LPS (100 ng/mL) for 4 h (TNF-α) or 18 h (IL-6 and IL-8). Data are the mean ± SD (n = 6) of two independent experiments performed in triplicate and expressed as the percentage of the positive control value (LPS). Different letters indicate significant differences (p < 0.05).</figcaption></figure>

To validate the model, we tested various parameters in normal and inflammatory conditions. TEER measurements indicated the establishment of barrier integrity (Caco-2-TC7/HT29-MTX biculture) through tight junction formation during the 14 days of differentiation, at a lower level than in monoculture, consistent with the literature [26], and probably due to differences in the rearrangement of the tight junction complex between cell types [57]. Addition of THP-1 cells slightly decreased TEER (–15 %), suggesting disruption of the cell monolayer integrity, likely via soluble factors, such as TNF-α [58,59]. The LPS-INF-γ cocktail strongly reduced TEER (–30 %), indicating increased membrane permeability during inflammation, confirmed by the higher phenol red transport. As shown in several studies, production of pro-inflammatory cytokines alters the tight junction complex, resulting in increased permeability [60,61]. In our model, TNF-α, IL-6, and IL-8 production and gene expression were strongly increased after pro-inflammatory stimulation, particularly in THP-1 cells, as expected and according to the literature [39,40]. Caco-2-TC7/HT29-MTX cell biculture showed limited TNF-α and IL-6 production, compared with other studies [62,63,64], highlighting the importance of including immune cells. The gene expression analysis showed NOS2 upregulation in both Caco-2-TC7/HT29-MTX and THP-1 cells, and COX2 upregulation only in THP-1 cells, likely due to the lower Caco-2 cell response to LPS [65]. This aligns with studies on intestinal inflammation, indicating that nitric oxide production is linked to colon lesions in inflammatory conditions

[66–69]. COX-2, an oxidoreductase that produces prostanoids (prostaglandins and thromboxanes), is overexpressed during inflammation and is an inflammation marker.

Immunofluorescence microscopy revealed disruptions in the apical junction complex, with delocalization of claudin, occludin, and particularly ZO-1, which was translocated from the apical to the basolateral side, forming an undulating network, as previously reported [70,71]. This tight junction complex reorganization under inflammation conditions is consistent with previous studies [37,70,72]. Similarly, confocal fluorescence microscopy showed increased MUC5AC production upon stimulation, indicating heightened inflammation. This response supports mucus overproduction to trap bacteria, sustaining inflammation and to repair the damaged mucus layer [73,2]. Previous studies have also demonstrated a link between pro-inflammatory cytokines, such as TNF-α, IL-6 or LPS, and an increased production of MUC2 [74] and MUC5AC [75] both in HT-29 cells. Thus, upon incubation with LPS/IFN-γ, our triculture model exhibited an inflammation-like response, characterized by increased permeability, altered apical junction complex, mucus overproduction, and elevated pro-inflammatory cytokine and enzyme release [76,77,61]. Pre-incubation with dexamethasone confirmed the model responsiveness to anti-inflammatory agents, by partially restoring barrier integrity, permeability and reducing cytokine production.

Then, we investigated the anti-inflammatory effects of cassava and
<figure><img src="image_8.png" /><figcaption>**Fig. 7.** Effect of polar and non-polar extracts of cassava (A-C) and roselle (D-F) on the production of various inflammation markers in the inflamed triculture model. Cells were pre-incubated with 50 µg/mL extracts for 2 h and stimulated with LPS-INF-γ (1 µg/mL-50 ng/mL) for 4 or 18 h. (A,D,G) TEER over 48 h after triculture set-up and expressed as percentage of the biculture values. (B,E,H) Phenol red apparent permeability (Papp). (C,F,J) Cytokine release in the apical or basolateral compartment after 4 h (TNF-α) or 18 h (IL-8) of stimulation, expressed as percentage of the values in the inflamed triculture. Data are the mean ± SD (n = 3) of triplicate measurements from one experiment. Different letters indicate significant differences (p < 0.05). \#p < 0.05, stable triculture versus biculture. \*p < 0.05, inflamed triculture (all conditions) versus stable triculture. TEER: transepithelial electrical resistance; TNF: tumour necrosis factor; IL: interleukin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

roselle leaves. Previous studies highlighted the *in vitro* antioxidant capacity of polar/non-polar extracts of these leaves due to the presence of bioactive compounds, such as carotenoids and flavonoids [17]. In LPS-stimulated THP-1 cells, polar/non-polar extracts of these GLVs inhibited TNF-α, IL-6 and IL-8 production by macrophages in a concentration-dependent manner, with the highest effect observed at 50 µg/mL, a concentration within plasma physiological levels. Cassava extracts showed stronger anti-inflammatory activity than roselle extracts, likely due to their twice higher flavonoid content (quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside; 80 % of phenolic compounds). Previous studies showed flavonoid anti-inflammatory effects in LPS-induced murine macrophages (RAW 264.7) *via* the NF-κB and MAPK pathways

[78,79]. Roselle effects may be partly explained by the presence of chlorogenic acid [17] that inhibits pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) in RAW 264.7 macrophages [80]. The both non-polar extract effects were likely due to lutein and β-carotene [17] that also inhibit the production of pro-inflammatory cytokines and enzymes (iNOS, COX-2) in murine macrophages following inflammation induction [81,82]. Our findings confirmed the anti-inflammatory effects of cassava and roselle leaves observed in previous studies in immune cells where they inhibit the production of pro-inflammatory cytokines and enzymes [83,20,84].

Overall, addition of polar/non-polar GLV extracts (50 µg/mL) to the apical side of the triculture model after incubation with LPS/IFN-γ
partially restored the cell monolayer integrity. This effect is likely due to their flavonoid and carotenoid contents that influence TEER and tight junction protein expression. For instance, quercetin and kaempferol (present in the polar extracts of cassava leaves) [17] enhance tight junction protein expression (claudin-4, ZO-2, occludin, claudin-1) and restore intestinal barrier function in Caco-2 cells [85,86]. Lutein (present in non-polar roselle extracts) reduces tissue damage and inflammatory factors and enhances tight junction protein expression (ZO-1, claudin-1, occludin) in dextran sulphate sodium-induced colitis in mice [87]. In an *ex vivo* mouse jejunum model, β-carotene, also present in non-polar roselle extracts, enhanced intestinal barrier function, as indicated by the increased TEER values [88].

Cytokine production modulation varied according to the GLV and cell type. In IEC (Caco-2-TC7/HT29-MTX biculture), only IL-8 was detectable and was decreased by polar/non-polar roselle extracts and non-polar cassava extracts. This suggests that roselle phenolic compounds and carotenoids could reduce IL-8 production in IEC, while only cassava carotenoids had an impact. Cytokine inhibition was less pronounced in THP-1 cells, with a slight inhibition of IL-8 and TNF-α by cassava extracts. This could be explained by the fact that the bioactive compounds must cross the IEC barrier to reach THP-1 cells. The effects on THP-1 cells may be mediated by polyphenols or their metabolites after absorption by enterocytes. Indeed, carotenoids require specific vehicles to be efficiently absorbed [89], which was not done in this study.

Isolated molecules provide initial insights, but they do not fully replicate the nutritional and physiological conditions due to the absence of the food matrix that determines the bioaccessibility and bioavailability after digestion. Therefore, we also evaluated the effects of cassava and roselle micellar fractions obtained after *in vitro* digestion of GLV sauces. Globally, micellar fractions showed weaker effects than extracts. Both micellar fractions restored TEER and barrier integrity after their alteration upon incubation with LPS/IFN-γ, but only roselle micellar fractions significantly reduced permeability. Roselle micellar fractions decreased IL-8 production by intestinal cells (apical side), and both micellar fractions modestly reduced TNF-α production by THP-1 cells with no effect on IL-8 (basolateral side). This suggests that the roselle bioactive compounds from digested sauce were more bioaccessible than those of cassava. Previous work in our laboratory showed that the bioaccessibility of roselle compounds [total polyphenols (58 %), β-carotene (2 %) and lutein (10 %)] was about two times higher than that of cassava compounds [total polyphenols (33 %), β-carotene (1 %) and lutein (4 %)]. Moreover, the modest inhibition of cytokine production by THP-1 cells could potentially be attributed to limited transport of micellar fractions, and thus bioactive compounds, across the IEC barrier. This could be explained by poor absorption of carotenoids due to low bioaccessibility, although other studies reported higher bioaccessibility rates for GLV carotenoids [90,42]. Alternatively, the low bioaccessibility of carotenoids observed in our study may be due to the low oil proportion in our sauces, chosen to increase dry matter and bioactive compound concentration. Nevertheless, according to the literature, carotenoid absorption by Caco-2 cells is generally low (~10 %) [91]. Besides, the low absorption of polyphenols can be explained by the need for hydrolysis by colonic microbiota before absorption. For instance, chlorogenic acid from roselle is absorbed as caffeic acid. Furthermore, rutin and the 3-O-rutinoside kaempferol present in cassava and roselle leaves are phenolic acids in esterified form and are less absorbed than in free form [92].

Overall, this study suggests a potential protective effect of cassava and particularly roselle sauces, after *in vitro* digestion, on intestinal barrier integrity and cytokine production related to intestinal inflammation. Although no study specifically examined these GLV effects on intestinal inflammation, many *in vitro* and *in vivo* works explored the role of polyphenols and carotenoids (also found in roselle and cassava leaves) on intestinal inflammation. For example, rutin has positive effects on intestinal inflammation in mice models, by reducing TNF-α, IL-1β, IL-6 or IFN-γ levels and increasing epithelial integrity [93,94].

β-carotene also improved the intestinal barrier function in a murine jejunum *ex vivo* model, by reversing villi shortening and lymphocyte infiltration and reducing IL-6 and TNF-α levels [88].

## 5. Conclusion

The *in vitro* cell culture model developed in this study replicated the intestinal responses to inflammatory stimulation. As this model provides a straightforward and reproducible method for screening the preventive effects on gut inflammation of plant extracts and foods after *in vitro* digestion, it is a valuable tool for preliminary screening before *in vivo* validation. Both cassava and roselle extracts differently reduced inflammation markers (TNF-α and IL-8), likely due to differences in polyphenol and carotenoid contents and their mechanisms of action. Results obtained with digested GLV sauces also supported the anti-inflammatory effect of GLV consumption, likely due to their high bioactive compound contents. However, these findings require confirmation through further *in vivo* experiments using animal models to consider the whole digestive system including intestinal microbiota.

## Funding

This research received no external funding.

## Ethical statement

This work does not include experiments with humans or animals, and therefore does not require approval by an ethics committee.

## CRediT authorship contribution statement

**Nelly Fioroni:** Writing – review & editing, Writing – original draft, Methodology, Formal analysis.
**María Del Carmen Ponce de León**
**Rodríguez:** Writing – original draft, Methodology, Formal analysis.
**Nicolas Leconte:** Writing – original draft, Methodology, Formal analysis.
**Claire Mouquet-Rivier:** Writing – review & editing, Supervision, Conceptualization.
**Caroline Guzman:** Methodology.
**Frédéric Bou-dard:** Methodology, Formal analysis.
**Claudie Dhuique-Mayer:** Methodology, Formal analysis.
**Myriam Collin:** Methodology.
**Anaïs Deglos:** Methodology.
**Emmanuelle Reboul:** Methodology, Formal analysis.
**Ángela Bravo-Núñez:** Methodology.
**Caroline Laurent-Babot:** Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We thank Philippe Clair from the qPCR platform/Montpellier GenomiX for useful advice.

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