Dietary fat enhanced chylomicron-mediated LPS translocation in a Caco-2 transwell model

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Tomassen, Coen Govers, A. Paul Vos, Nicole J.W. Wit This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1575818/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jan, 2023 Read the published version in Lipids in Health and Disease → Version 2 posted 10 You are reading this latest preprint version Show more versions Abstract Background There is increasing evidence that dietary fat, and especially saturated fat, promotes the absorption of lipopolysaccharide (LPS) via enhanced chylomicron production. The latter can subsequently transport LPS to other parts of the body, where it can induce low-grade chronic inflammation that links to various metabolic and gut-related diseases. To identify promising (food) compounds that can prevent or ameliorate LPS-related low grade inflammation, we developed and optimized an in vitro model for dietary fat-induced LPS translocation that closely mimics the in vivo situation and facilitates high-throughput screening. Methods For this, epithelial Caco-2 cells were differentiated in 21 days to a small intestinal phenotype. Thereafter, the cells were exposed to a dilution range of in vitro digested palm oil and sunflower oil, with or without starvation, to determine optimal conditions for fat-induced chylomicron production as measured by basolateral levels of the chylomicron-related marker apolipoprotein B. Next, LPS was co-incubated in various concentrations with the digested oils and fat-induced LPS translocation to the basolateral side was assessed. Results We found that dietary fat-induced LPS translocation in Caco-2 cells was optimal after apical exposure to digested oils in a 1:50 dilution, in combination with 750ng/ml LPS, preceded by one-week starvation (cultured without fetal bovine serum). Co-incubation with chylomicron blocker Pluronic L81 confirmed that fat-induced LPS translocation is mediated via chylomicron production in this Caco-2 model. Conclusion We developed a robust Caco-2 cell model for dietary fat-induced LPS translocation that can be used for high-throughput screening of (food) compounds that can reduce LPS-related low grade inflammation. Lipopolysaccharide dietary fat chylomicrons intestine translocation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background There is growing evidence for a role of the intestinal barrier in low-grade inflammation. Impaired intestinal barrier function could facilitate the passage of luminal antigens or other molecules that lead to a mucosal immune response. Such a luminal antigen that is commonly linked to chronic inflammation is lipopolysaccharide (LPS) which consists of a lipid, an inner- plus outer core and a polysaccharide composed of 0-antigens joined by a covalent binding. LPS is a cell wall component of Gram-negative bacteria that is naturally present in the gut in high quantities. LPS can cross the intestinal barrier from the intestinal lumen via paracellular and/or transcellular routes. Paracellular translocation can occur when tight junction function is impeded, resulting in a reduced intestinal integrity. For example, ethanol induces disruption of the tight junctions and can therefore enforce paracellular translocation of LPS [ 1 , 2 ], which can subsequently contribute to alcoholic liver disease (ALD)[ 3 ]. Transcellular translocation of LPS, on the other hand, is especially linked to a high load of dietary fat [ 4 ]. In vitro , animal and human studies have demonstrated that the ingestion of a high fat meal, especially high in saturated fat, facilitates the translocation of luminal LPS across the intestinal barrier. Chylomicrons, transporter molecules of dietary triglycerides, seem to play an important role in this process, as after a high fat challenge the postprandial LPS levels peak at the same time as chylomicrons and LPS is highest in the chylomicron-rich fraction [ 5 – 10 ]. Via chylomicrons, LPS can subsequently be transported to other organs in the body, where its endotoxin activity can induce low-grade chronic inflammation linked to various metabolic diseases. In people with obesity, insulin resistance and/or type 2 diabetes, increased plasma levels of LPS were found compared to a group of healthy subjects after ingestion of a high fat diet [ 11 ]. Moreover, LPS activity was reported to be highly correlated with metabolic and cardiovascular risk factors and the number of components of the metabolic syndrome [ 12 , 13 ]. This phenomena is referred to as metabolic endotoxemia [ 14 ]. Next to metabolic endotoxemia, there is growing evidence that (micro)-inflammation of the intestinal mucosa plays a role in the pathogenesis of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) [ 15 , 16 ]. IBS symptoms have been reported to correlate with increased permeability and subclinical inflammation [ 17 ]. Furthermore, Dlugosz and colleagues [ 18 ] have shown that especially patients with IBS-D (diarrhea predominant IBS subtype) have higher serum levels of LPS, indicating a subclinical inflammation [ 19 , 20 ]. In IBD patients elevated serum LPS were found to be associated with disease activity [ 15 , 21 ]. Furthermore, an increased risk for developing IBD was linked to the intake of dietary fat [ 22 ], supporting a role for LPS in IBD pathology. To identify promising interventions that can prevent or ameliorate LPS-related low grade inflammation, in vitro models are valuable for high-throughput screening. Several studies have suggested that intestinal epithelial Caco-2 cells can serve as an in vitro model to study fat-induced LPS translocation via chylomicron secretion. However most of these studies used free fatty acids to induce chylomicron secretion [ 7 , 23 ] and not the unrefined dietary oils that are typically part of the western diet. The purpose of our study was to develop an in vitro model of fat-induced transcellular translocation of LPS that more accurately mimics the in vivo situation. We investigated the effects of digested palm- and sunflower oil on LPS translocation in Caco-2 cells. Methods Chemicals All chemicals were purchased from Sigma Aldrich (St Louis, Missouri, USA) unless otherwise stated. In vitro gastrointestinal digestion To mimic oral digestion, palm oil (mainly C16:0) (Research Diet Services BV) and sunflower oil (mainly C18:2) (Research Diet Services BV) were both diluted to 0.2 g/ml with 140 mM NaCl + 5 mM KCl to a total volume of 15 ml. This mixture was gently vortexed, and heated for 15 min at 56°C. To mimic gastric digestion, the pH was set to 2 with 1 M HCl and 40 mg/ml of pepsin solution (1092 U/ml dissolved in 0.1 M HCl) was added. The samples were incubated for 1 h at 37°C while gently shaking. To mimic intestinal digestion, the pH was set to 5.8 with 1 M NaHCO 3 and 4 mg/ml pancreatin (6.84 U/mg trypsin activity), 5.9 units/ml α-chromotrypsin (65.62 U/mg), 1 mg/ml lipase (all from porcine) and bile salts (94.6 mg/ml sodiumtaurocholate and 83 mg/ml sodium glycodeoxycholate) dissolved in 0.1 M NaHCO 3 were added. Next, the pH was adjusted to 6.5 using 1 M NaHCO 3 , after which the samples were incubated at 37°C for 2 h while gently shaking. After incubation, the pH was adjusted to 7.5 with 1 M NaHCO 3 and the volume of the digest was filled up to 40 ml with 140 mM NaCl + 5 mM KCl. As control a digest was prepared which contained all buffers and enzymes, but without oils. Caco-2 culture Caco-2 cells (ATCC-HTB-37) were cultured in Dulbecco’s Modified Eagles Medium (DMEM) with high glucose (4.5 g/l) and 25 mM HEPES (Life technologies, 42430), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone) at 37°C and 5% CO 2 . Translucent, 0.4 µm transwell inserts (Greiner Bio-one) were seeded on the apical side with 500 µl (12 wells) or 150 µl (24 wells) of 0.225x10 6 cell/ml Caco- 2, with 1500 µl (12 wells) or 750 µl (24 wells) of basolateral medium, respectively. The cells were incubated for 21 days at 37°C with 5% CO 2 to differentiate in small-intestinal like epithelial cells. Apical and basolateral medium was replaced three times a week and one day prior to the investigational exposure. To monitor integrity of the Caco-2 monolayer, transepithelial electrical Resistance (TEER) was measured by using a MilliCell ERS (Millipore Amsterdam, The Netherlands). Differentiated Caco-2 monolayers were considered of acceptable quality if TEER values were higher than 700 Ω/cm 2 (24 wells) and 450 Ω/cm 2 (12 wells) before exposure to the investigational treatment. Fat-induced LPS translocation experiments Prior to exposure to fats and LPS (Sigma L4391), Caco-2 cells were generally starved for one week, meaning that culture medium without added FBS was used in the apical compartment for the last 3 medium replacements. Subsequently, Caco-2 cells were apically exposed to digested oil samples (1:50 diluted in DMEM, unless stated otherwise) for 24 hours. DMEM and control digest samples (also 1:50 diluted in DMEM, unless stated otherwise) were included as negative controls. To determine fat-induced translocation of LPS, additionally 100, 250, 500, 750 or 1000 ng/ml LPS was apically added for 24 hours. The TEER was measured during the exposure experiments at 0, 1, 3, 6 and/or 24 hours. Chylomicron blockage experiments To investigate whether LPS is translocated via chylomicrons in our fat-induced LPS translocation model, we also performed Caco-2 experiments with and without the chylomicron blocker Pluronic L81 (PL81, BASF Corporation, Germany), as described by Ghoshal et al. (J Lipid Res, 2009, 50:90–7). All these experiments were performed in DMEM without phenol-red (Life Technologies) and FBS, as this seemed to interfere with the chylomicron detection. Caco-2 cells were first pre-incubated overnight with LPS before exposure to PL81 and dietary fats, to prevent paracellular LPS leakage by PL81 exposure. Therefore, 20-day differentiated starved Caco-2 cells were apically challenged overnight with 1 mg/ml LPS (diluted in DMEM without FBS and phenol red). Next, Caco-2 were washed 3 times with DMEM without FBS and phenol red, after which digested oil samples were apically added, mixed with 500 ug/ml PL81. After 24-hour incubation, chylomicron production and LPS translocation were assessed in the basolateral compartment. Detection of chylomicron-marker ApoB Chylomicron production by Caco-2 cells was assessed by measuring ApoB levels in the basolateral medium using an ApoB ELISA (ABIN612664, Antibodies-online), which detects both ApoB-48 and ApoB-100, according to the manufacturer’s protocol. In in vivo settings, adult human intestines secrete chylomicrons which only contain ApoB-48, not ApoB-100, which is predominantly associated with VLDL derived from liver [ 24 ]. However, Caco-2 cells are known to produce and secrete chylomicrons with both ApoB-48 and ApoB-100 [ 25 , 26 ]. Detection of LPS LPS was measured in the apical as well in basolateral medium with a chromogenic assay from Nodia (Assiociates Cap. Cod Inc). The assay was performed in an microtiterplate reader (Tecan) as well in a Pyros Kinetix Flex tube reader (pKFlex) (Nodia). Pyrochrome lysate was reconstituted with 3.2 ml pyrochrome reconstitution buffer (C1500-5). Samples were diluted with LPS free MQ and incubated for 15 min in a water bath at 70°C. After 1 h at 4°C the samples were placed for 10 min at room temperature and directly measured with an endpoint measurement on the microtiter plate reader or on the pKFlex. For both methods a calibration from Limulus Amebocyte lysate control standard endotoxin 0.5 µg/vial (CSE E0005-1) was used. For the endpoint method 125 µl pyrochrome was added to 125 µl treated sample or standard and the mixture was incubated at 37°C. After 35, 45 and 60 min, 80 µl sample was taken and added to 20 µl Acetic acid. Finally, the absorbance was measured at 405 nm on the plate reader. For the pyros kinetix flex method 200 µl treated sample or standard was added in a pKflex glass tube. 50 µl pyrochrome was added and the mixture was briefly stirred and placed in the pKFlex. Using the Pyros express 21 CFR Part 11 compliant software the endotoxin concentration was calculated. Another method used for functional LPS quantification is the HEK-Blue hTLR4 assay [ 27 ]. HEK-Blue hTLR4 cells are transgenic for the cell surface expressed hTLR4 MD-2 and CD14 receptors and contain a downstream reporter system resulting in secretion of secreted embryonic alkaline phosphtase (SEAP) under the control of NFkB and AP1 promotors. HEK-Blue hTLR4 cells (InvivoGen, Toulouse, France) were sub-cultured in DMEM with 10% FBS once per week and medium was refreshed twice per week. For experiments, HEK-Blue hTLR4 cells were detached using a cell scraper when 90–95% confluence was reached and 0.5×10 6 cells were transferred to each well of a 96-well Poly-D-Lysine coated plate. After overnight incubation at 37°C in an atmosphere containing 5% CO 2, we added 0.0001-100 EU/ml LPS (E. coli O11: B4, 1 EU ≡ 0.15 ng/ml, Sigma, St. Louis, MO, USA) or 100 ul basolateral medium from the Fat-induced LPS translocation experiments to the cells and the plate was incubated for 8 h (37°C; 5% CO 2 ). TLR4 stimulation resulted in SEAP secretion, which was quantified by mixing 20 µl of supernatant (depleted from cells by centrifugation at 450 g for 5 min) with 180 µl of Quanti-Blue™ in a new flat bottom 96-well plate. The plate was incubated for 3 h at 37°C and absorption at 655 nm was determined every hour using a spectrophotometer (TECAN, Giessen, The Netherlands). Detection of paracellular translocation by FD4 To determine paracellular translocation of LPS in the Caco-2 exposure experiments, 250 µg/ml 4 kDa FITC-dextran (FD4) was added to the apical side at the start of the investigational exposures. After 24 hours, fluorescence (Excitation 485 nm / Emission 528 nm) in the basolateral compartment was determined. The fluorescent values were compared to a calibrations curve for FD4 and the percentage of paracellular translocation was calculated. Data analysis Exposures performed with a concentration range to determine optimal conditions for the model were conducted as a single cell experiment. Verification exposures were performed with a biological triplicate. These data are represented as average ± STD. Statistical significance of difference was analysed by ANOVA followed by Student’s t -test for pair wise comparison. Differences between groups are considered statistically significant when P < 0.05. Results Dietary fat-induced chylomicron secretion in Caco-2 cells To study the potential of saturated and unsaturated dietary fats to induce chylomicron production in Caco-2 cells, in vitro digested palm oil and sunflower oil were used, respectively. A first step before applying these dietary fats to Caco-2 cells consisted of in vitro digestion to better mimic intestine luminal conditions. The digested oils were tested in different dilutions to determine the optimal condition for chylomicron production. The digested oils did not appear to negatively impacted the barrier integrity of small-intestinal-like Caco-2 cells at 1:10, 1:50 and 1:100 dilutions. In contrast, 1:1 dilutions reduced the TEER to some extend when compared to the control digest (Fig. 1 A and B), indicative for a compromised intestinal integrity. This seems to fit the detected basolateral ApoB levels, as only 1:10, 1:50 and 1:100 dilutions of digested palm oil and sunflower oil induced an increase in basolateral ApoB levels (Fig. 2 ). At these concentrations palm oil showed the most pronounced induction of about a 1.2 times increase (20%) compared to digest control. The (middle) 1:50 dilution was selected to continue optimisation of the model, as Luchoomun et al previously showed that too high or low levels of fatty acids (in combination with bile acids) can result in reduced ApoB secretion [ 23 ]. To mimic the in vivo situation more closely [ 28 ], Caco-2 cells were starved (meaning deprived from FBS on the apical side) in the last week of differentiation and thus one week before dietary fat exposure. After 24 hours of exposure to the 50 times diluted digested oils, intestinal integrity of the starved 21-day old Caco-2 was not affected when compared to the control digest (Fig. 3 A). In contrast, chylomicron secretion was significantly increased by palm oil compared to the digest control (Fig. 3 B). This indicates that starvation of Caco-2 cells prior to fat exposure increases chylomicron production, thereby enlarging the window of opportunity for modulation. Based on these data we determined that a 1:50 dilution of in vitro digested oils and 1-week starving of the Caco-2 cells prior to fat exposure were the most optimal conditions for our Caco-2 model on fat-induced chylomicron secretion. Dietary fat-induced LPS translocation in Caco-2 cells Having established the optimal culture and fat-exposure conditions to enhance fat-induced chylomicron secretion in Caco-2 cells, we investigated whether this model could be extended to study dietary fat-induced LPS translocation. To this end, Caco-2 cells were co-incubated with the digested oils and different concentrations of LPS (0, 50, 100, 250, 500, 750 or 1000 ng/ml). A 24-hour exposure with these samples did not affect intestinal integrity, indicating that apical LPS has no detrimental effect on the Caco-2 cells (Fig. 4 ). Furthermore, LPS does not seem to drastically affect fat-induced ApoB secretion, although, there seems to be a reduced ApoB secretion upon high apical LPS exposure (Fig. 5 A). However, only at a high level of apical LPS exposure (≥ 750ng/ml), a clear distinction in basolateral LPS can be found between the digested oil samples and control digest (Fig. 5 B). This indicates that at least 750 ng/ml LPS is needed in conjunction with digested oils to detect dietary fat-induced LPS translocation in our Caco-2 model. Thus, we continued the experiments using 750 ng/mL LPS. To exclude paracellular LPS translocation, FD4 was apically added to the digested oils (1:50 dilution) and LPS (750 ng/ml) in the Caco-2 model. No effect was seen on TEER, and thus intestinal integrity (Fig. 6 ). After 24h exposure, no FD4 translocation to the basolateral compartment was detected, whereas basolateral LPS levels were still increased by palm oil and sunflower oil (Fig. 7 A and B). This indicates that the paracellular route is not involved in fat-induced LPS translocation in this Caco-2 model. Dietary fat-induced LPS translocation is mediated by chylomicron production in Caco-2 cells. To verify that the dietary fat-induced LPS translocation is mediated by chylomicron production, the chylomicron blocker PL81 was added to the Caco-2 model. As PL81 is known to affect the intestinal barrier function [ 29 ], the timing of exposures in the model was adapted to avoid paracellular leakage of LPS due to PL81-induced barrier disruption. Cario et al described that LPS can be internalized and stored in intestinal epithelial cells [ 30 ], therefore Caco-2 cells were preincubated overnight with LPS, instead of directly co-incubated with the digested dietary fats. Prior to co-exposure with PL81, we first confirmed that the overnight stored LPS could still be released upon fat exposure to the Caco-2 cells. Co-exposure of the Caco-2 cells with PL81, showed an expected strong reduction in TEER (Fig. 8 ), especially in control digest conditions. Surprisingly, a less dramatic drop in TEER was seen in cells co-incubated with the oils. Particularly palm oil seemed to protect against the PL81-induced deterioration of the intestinal layer. Next, we measured basolateral ApoB secretion (Fig. 9 A) and LPS translocation (Fig. 9 B) and found that PL81 significantly reduced basolateral ApoB levels independent of the applied digests. This indicates that PL81 accurately blocked chylomicron production in our Caco-2 cell model. When measuring LPS translocation with PL81 co-exposure, we observed a strong increase in basolateral LPS when Caco-2 were exposed to the digest control, (Fig. 9 B). This is probably linked to the strong disruption of the barrier integrity (Fig. 8 ), and thus mainly represents paracellular leakage. Interestingly, when Caco-2 cells were co-exposed to either palm or sunflower oil digests, the PL81 treatment (Fig. 9 B; grey bars) even reduced the LPS translocation in Caco-2 compared to without PL81 treatment (Fig. 9 B; black bars). Even though this reduction in basolateral LPS did not reach significance (p = 0.1), the data strongly suggest that dietary fat-induced LPS translocation in our model is (at least partly) mediated by chylomicron production. Discussion We developed an in vitro Caco-2 model to study digested dietary fat-induced LPS translocation, which can serve as a screening tool to identify (food) compounds with potential beneficial and preventive effects on LPS-related low grade inflammation. In this study we determined the optimal concentrations of digested oils and apical LPS to stimulate chylomicron production by small-intestinal-like Caco-2 cells, and subsequent LPS translocation, without inducing cytotoxic effects and disrupting the intestinal barrier. As both TEER and FD4 were not affected after the challenge with digested oils and LPS, the transport of LPS is unlikely to be facilitated through paracellular transport. Similarly, Mani et al. [ 31 ] observed no difference in intestinal integrity in pigs after ex vivo treatments with endotoxins in relation to dietary oil compositions. Taken together, these data suggest that fat-induced LPS translocation is routed via transcellular transport and not paracellular transport. The use of PL81, a known inhibitor of chylomicron production [ 7 ], suggested that fat-induced chylomicron production is involved in the LPS translocation, as the dietary fat-induced secretion of ApoB was diminished with a consequent created reduction of LPS translocation. PL81 uptake by differentiated Caco-2 cells is known to be a protein-facilitated active process and PL81 inhibits fat absorption by decreasing the triacylglycerols transport from the cytosol to the endoplasmic reticulum. This in turn inhibits assembly and secretion of chylomicrons [ 29 ]. In case of adding PL81 without a dietary fat (digest control), we found that the integrity of the layer was severely disturbed and the TEER was considerably decreased. In other studies that use PL81 as a chylomicron inhibitor the barrier disruption was not mentioned. However, our study shows that this is important to take into account when using this chylomicron blocking-compound, especially when not used in combination with lipid-like molecules in the apical compartment. Upon co-incubation with digested oils, we found that the disruption of the intestinal barrier by PL81 was much less pronounced, indicating a ‘protective effect’ of the digested fats against PL81-induced barrier disruption. The mechanism behind this is unclear, but it might be related to the cellular distribution of PL81 that can be affected by coincubation with dietary fat(ty acids) [ 29 ]. Fatty acid-induced chylomicron production and LPS translocation were previously studied in Caco-2 cells using free fatty acids by Luchoomun [ 23 ] and Ghoshal et al [ 7 ], respectively. The incubation with digested oils is more closely mimicking the in vivo situation and therefore should be preferred over the fatty acid-induced LPS model. Furthermore, it was previously reported that exposure of Caco-2 cells to palmitic acids alone can disrupt the intestinal barrier and thereby reduce ApoB secretion [ 32 ] and induce paracellular transport [ 33 ], which is not desirable for a fat-induced LPS model. However, in that study only fatty acids were used, not digested oils. Luchoomun et al. [ 23 ] previously showed that bile acids are important for efficient chylomicron production in Caco-2 cells. The in vitro digestion integrated in our model, also includes the addition of bile acids which are therefore also included in our studies as the total digest is applied to Caco-2 cells. Ghoshal et al. [ 34 ] showed that LPS translocation is dependent on long-chain fatty acid exposure in Caco-2 cells, whereas short chain fatty acids, which are not transported via chylomicrons [ 35 ], did not induce LPS transport. This indicates that chylomicron production is involved in LPS translocation, which was supported by their PL81 experiments abolishing the long chain fatty acid-induced effect. Similar effects of PL81 were also found in our Caco-2 model, indicating that also with digested oils the fat-induced LPS translocation is linked to chylomicron production. However, our data suggest that this LPS translocation induced by digested oils is only partly dependent on chylomicron production, as ApoB secretion was almost completely abolished by PL81, whereas LPS is only reduced by approximately 50%. This would however fit with recent findings of Akiba et al, that showed a second lipid-dependent route for LPS transport in the intestine, which is independent of chylomicron secretion [ 36 ]. In their rat model they also found that PL81 could not completely block fat-induced LPS translocation, and they additionally found (labelled) LPS back in the portal vein. They found no evidence for paracellular leakage of LPS. Alternatively, it has previously been shown that high fat consumption can induce internalization of tight junction proteins which increases paracellular transport, including LPS transport [ 31 ]. This potential process for fat-induced LPS translocation was not reflected in TEER levels and FD4 translocation in our model. Based on our findings, we can however not exclude that paracellular leakage of LPS might still play a role in fat-induced LPS translocation. Several studies have shown that composition of dietary oil has a substantial effect on postprandial endotoxemia. Especially saturated fats are linked to higher postprandial LPS levels in blood [ 31 , 37 ]. In our model we recapitulated this by showing that palm oil more strongly induced chylomicron secretion and LPS translocation when compared to sunflower oil. In contrast, Laugerette et al. [ 8 ] showed that palm oil had no effect on plasma endotoxin concentrations while sunflower oil augmented plasma endotoxemia by 59–70%. In western diets, fats and oils are common components of the diet [ 38 ]. In recent years, the development of obesity, inflammation and other metabolic diseases has been linked to low grade endotoxemia associated with high dietary fat and energy intake [ 12 ]. These studies have raised questions whether diet-induced endotoxemia is caused by changes in permeability of the intestine, variations in the gut microbiota or simply by a change in intake of the dietary fat and energy content of the food. There is increasing evidence that the low grade endotoxemia is induced by a combination of all the above factors. For instance, next to the effect of dietary fat on LPS translocation, a high saturated fat diet, like palm oil, is reported to result in higher gram negative bacterial populations such as E. coli which produce gut LPS [ 39 ]. Life style changes, and especially also dietary interventions that reduce chylomicron production and lower LPS production by the gut microbiome, are thought to be effective in reducing low grade inflammation and thereby reducing the risk for CVD and other co-morbidities linked to metabolic syndrome. Next to this impact on metabolic health, also gastrointestinal health has a link with dietary fat and LPS-related inflammation. Associations between fatty meal consumption and IBS symptom induction have also been identified in a number of studies. In fact, in many IBS patients the symptoms were triggered after the consumption of fatty or fried food [ 40 , 41 ]. Dietary fat is also linked to an increased risk for developing IBD [ 22 ]. Interestingly, also higher serum and/or fecal LPS levels are found in IBD and IBS patients [ 42 ], especially in patients with predominant diarrhoea-related symptoms [ 18 ]. This link with high fat intake and the previously found association with high LPS levels, might indicate a role for fat-induced LPS translocation in IBS and IBD pathology. If confirmed in future studies, dietary interventions that influence fat-induced LPS translocation might also be beneficial for this patient population. Conclusion We developed an in vitro model that closely mimics the in vivo situation to study dietary fat-induced LPS translocation, linked to LPS-related low grade inflammation. This Caco-2 model can be used to screen dietary fats and other food components that can prevent or reduce inflammatory conditions. In this way, this model contributes to research identifying beneficial food (compounds) for human metabolic and gastrointestinal health. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article. Competing interests The authors declare that they have no competing interests. Funding This research was supported by the Dutch ministry of Economic affairs via Public Private Partnership grants from the Top consortium for Knowledge and Innovation Agri & Food, project AF12203 “CCC CarboHealth” and project AF16012 “Nutrition to improve quality of life of IBS patients”. The funding agencies had no role in the collection, analysis, and interpretation of data or in the preparation, review, or approval of the manuscript. Authors' contributions M.T. was in the lead for conceptualization, methodology, performance and analysis of the experiments, and writing of the manuscript. C.G. contributed to acquisition, conceptualization and manuscript reviewing and revising. P.V. contributed to acquisition and manuscript reviewing and revising. N.W. contributed to acquisition, conceptualization, analysis of the experiments and writing, reviewing and revising of the manuscript. Acknowledgements Not applicable. References Bala S, Marcos M, Gattu A, Catalano D, Szabo G: Acute Binge Drinking Increases Serum Endotoxin and Bacterial DNA Levels in Healthy Individuals. PLOS ONE 2014, 9:e96864. Elamin E, Jonkers D, Juuti-Uusitalo K, van Ijzendoorn S, Troost F, Duimel H, Broers J, Verheyen F, Dekker J, Masclee A: Effects of Ethanol and Acetaldehyde on Tight Junction Integrity: In Vitro Study in a Three Dimensional Intestinal Epithelial Cell Culture Model. PLOS ONE 2012, 7:e35008. 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Porras M, Martín MT, Yang PC, Jury J, Perdue MH, Vergara P: Correlation between cyclical epithelial barrier dysfunction and bacterial translocation in the relapses of intestinal inflammation. Inflamm Bowel Dis 2006, 12:843–852. Dlugosz A, Nowak P, D'Amato M, Mohammadian Kermani G, Nyström J, Abdurahman S, Lindberg G: Increased serum levels of lipopolysaccharide and antiflagellin antibodies in patients with diarrhea-predominant irritable bowel syndrome. Neurogastroenterol Motil 2015, 27:1747–1754. Vivinus-Nébot M, Frin-Mathy G, Bzioueche H, Dainese R, Bernard G, Anty R, Filippi J, Saint-Paul MC, Tulic MK, Verhasselt V, et al: Functional bowel symptoms in quiescent inflammatory bowel diseases: role of epithelial barrier disruption and low-grade inflammation. Gut 2014, 63:744–752. Halpin SJ, Ford AC: Prevalence of symptoms meeting criteria for irritable bowel syndrome in inflammatory bowel disease: systematic review and meta-analysis. Am J Gastroenterol 2012, 107:1474–1482. Magro DO, Kotze PG, Martinez CAR, Camargo MG, Guadagnini D, Calixto AR, Vasques ACJ, Ayrizono MdLS, Geloneze B, Pareja JC, et al: Changes in serum levels of lipopolysaccharides and CD26 in patients with Crohn's disease. Intestinal research 2017, 15:352–357. Ananthakrishnan AN, Khalili H, Konijeti GG, Higuchi LM, de Silva P, Fuchs CS, Willett WC, Richter JM, Chan AT: Long-term intake of dietary fat and risk of ulcerative colitis and Crohn's disease. Gut 2014, 63:776–784. Luchoomun J, Hussain MM: Assembly and secretion of chylomicrons by differentiated Caco-2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly. J Biol Chem 1999, 274:19565–19572. Traber M, Kayden H, Rindler M: Polarized secretion of newly synthesized lipoproteins by the Caco-2 human intestinal cell line. Journal of lipid research 1987, 28:1350–1363. Hughes TE, Ordovas JM, Schaefer EJ: Regulation of intestinal apolipoprotein B synthesis and secretion by Caco-2 cells. Lack of fatty acid effects and control by intracellular calcium ion. Journal of Biological Chemistry 1988, 263:3425–3431. Wang Y, Lin Q, Zheng P, Li L, Bao Z, Huang F: Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on Chylomicron and VLDL Synthesis and Secretion in Caco-2 Cells. BioMed Research International 2014, 2014:684325. Govers C, Tomassen MMM, Rieder A, Ballance S, Knutsen SH, Mes JJ: Lipopolysaccharide quantification and alkali-based inactivation in polysaccharide preparations to enable in vitro immune modulatory studies. Bioactive Carbohydrates and Dietary Fibre 2016, 8:15–25. Hiebl V, Schachner D, Ladurner A, Heiss EH, Stangl H, Dirsch VM: Caco-2 Cells for Measuring Intestinal Cholesterol Transport - Possibilities and Limitations. Biological Procedures Online 2020, 22:7. Fatma S, Yakubov R, Anwar K, Hussain MM: Pluronic L81 enhances triacylglycerol accumulation in the cytosol and inhibits chylomicron secretion. J Lipid Res 2006, 47:2422–2432. Cario E, Rosenberg IM, Brandwein SL, Beck PL, Reinecker HC, Podolsky DK: Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors. J Immunol 2000, 164:966–972. Mani V, Hollis JH, Gabler NK: Dietary oil composition differentially modulates intestinal endotoxin transport and postprandial endotoxemia. Nutrition & metabolism 2013, 10:6–6. Bateman PA, Jackson KG, Maitin V, Yaqoob P, Williams CM: Differences in cell morphology, lipid and apo B secretory capacity in caco-2 cells following long term treatment with saturated and monounsaturated fatty acids. Biochim Biophys Acta 2007, 1771:475–485. Gori M, Altomare A, Cocca S, Solida E, Ribolsi M, Carotti S, Rainer A, Francesconi M, Morini S, Cicala M, Guarino MPL: Palmitic Acid Affects Intestinal Epithelial Barrier Integrity and Permeability In Vitro. Antioxidants (Basel, Switzerland) 2020, 9:417. Ghoshal S, Witta J, Zhong J, de Villiers W, Eckhardt E: Chylomicrons promote intestinal absorption of lipopolysaccharides. Journal of Lipid Research 2009, 50:90–97. Schönfeld P, Wojtczak L: Short- and medium-chain fatty acids in energy metabolism: the cellular perspective. J Lipid Res 2016, 57:943–954. Akiba Y, Maruta K, Takajo T, Narimatsu K, Said H, Kato I, Kuwahara A, Kaunitz JD: Lipopolysaccharides transport during fat absorption in rodent small intestine. Am J Physiol Gastrointest Liver Physiol 2020, 318:G1070-g1087. Lyte JM, Gabler NK, Hollis JH: Postprandial serum endotoxin in healthy humans is modulated by dietary fat in a randomized, controlled, cross-over study. Lipids in health and disease 2016, 15:186–186. Cordain L, Eaton SB, Sebastian A, Mann N, Lindeberg S, Watkins BA, O'Keefe JH, Brand-Miller J: Origins and evolution of the Western diet: health implications for the 21st century. Am J Clin Nutr 2005, 81:341–354. Fuke N, Nagata N, Suganuma H, Ota T: Regulation of Gut Microbiota and Metabolic Endotoxemia with Dietary Factors. Nutrients 2019, 11:2277. Böhn L, Störsrud S, Törnblom H, Bengtsson U, Simrén M: Self-reported food-related gastrointestinal symptoms in IBS are common and associated with more severe symptoms and reduced quality of life. Am J Gastroenterol 2013, 108:634–641. Rijnaarts I, Witteman BJM, Zoetendal EG, Govers C, de Wit NJW, de Roos NM: Subtypes and Severity of Irritable Bowel Syndrome Are Not Related to Patients’ Self-Reported Dietary Triggers: Results From an Online Survey in Dutch Adults. Journal of the Academy of Nutrition and Dietetics 2021, 121:1750–1762.e1758. Zhou SY, Gillilland M, 3rd, Wu X, Leelasinjaroen P, Zhang G, Zhou H, Ye B, Lu Y, Owyang C: FODMAP diet modulates visceral nociception by lipopolysaccharide-mediated intestinal inflammation and barrier dysfunction. J Clin Invest 2018, 128:267–280. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 12 Jan, 2023 Read the published version in Lipids in Health and Disease → Version 2 posted Editorial decision: Major revision 31 Jul, 2022 Reviews received at journal 28 Jul, 2022 Reviews received at journal 21 Jun, 2022 Reviewers agreed at journal 09 Jun, 2022 Reviews received at journal 02 Jun, 2022 Reviewers agreed at journal 23 May, 2022 Reviewers invited by journal 22 May, 2022 Editor assigned by journal 17 May, 2022 Submission checks completed at journal 17 May, 2022 First submitted to journal 17 May, 2022 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1575818","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2022-04-22 14:29:57","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":109031473,"identity":"05148eab-cc9a-4d9b-9600-5185271f772d","order_by":0,"name":"Monic M.M. Tomassen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYJCCA0hsGwj1gLAWAxg7DUIlELYIruUwYS267b0HD/xg+COn23788YePO84nbrh9gO0BPi1mZ84lHOxhMDA2O5NjJjnzzO3EDecS2A3warmRYwB0jEHitgM5bMy8bbdzN5xhYJMgTsv5548//207R4qWGwkG0oxtB4jQcuaMwcEeA2NjsxtvzCR725LrZ55hbMOv5XiP8YcfFXJyZufTH3/42WZnzHeG+ZjEBzxaIMAAhcfYQFDDKBgFo2AUjAL8AAAhHlMmx191rwAAAABJRU5ErkJggg==","orcid":"","institution":"Wageningen Food \u0026 Biobased Research – Food Health \u0026 Consumer Research group","correspondingAuthor":true,"prefix":"","firstName":"Monic","middleName":"M.M.","lastName":"Tomassen","suffix":""},{"id":109031474,"identity":"f72962ae-dec6-446a-b076-44b7095a44a3","order_by":1,"name":"Coen Govers","email":"","orcid":"","institution":"Wageningen Food \u0026 Biobased Research – Food Health \u0026 Consumer Research group","correspondingAuthor":false,"prefix":"","firstName":"Coen","middleName":"","lastName":"Govers","suffix":""},{"id":109031475,"identity":"1029c732-3628-4ed4-aea4-758fc218ee08","order_by":2,"name":"A. Paul Vos","email":"","orcid":"","institution":"Wageningen Food \u0026 Biobased Research – Food Health \u0026 Consumer Research group","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"Paul","lastName":"Vos","suffix":""},{"id":109031476,"identity":"b7e89b74-4da5-4a95-b4b2-88a9caa87389","order_by":3,"name":"Nicole J.W. Wit","email":"","orcid":"","institution":"Wageningen Food \u0026 Biobased Research – Food Health \u0026 Consumer Research group","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"J.W.","lastName":"Wit","suffix":""}],"badges":[],"createdAt":"2022-04-20 09:29:16","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-1575818/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1575818/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12944-022-01754-3","type":"published","date":"2023-01-12T18:17:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":22068921,"identity":"ff9c8286-8d91-4238-8615-3e92cb540160","added_by":"auto","created_at":"2022-05-31 14:07:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":197398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of digested palm oil and sunflower oil dilutions on intestinal integrity of Caco-2 cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eEffect of different dilutions of digested palm oil (Left) and sunflower oil (right) on TEER in 21 days old differentiated Caco-2 cells. TEER: % compared to digest control (1:1), which was set to 100%. Oils and control digest were diluted in DMEM w/o phenol red and w/o FBS. These exposures were conducted as a single cell experiment. \u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1575818/v2/04fb90f021fd7cd4708da51a.jpg"},{"id":22067737,"identity":"3087da64-c679-4ee4-ac13-e0cef02aeab9","added_by":"auto","created_at":"2022-05-31 13:57:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":213497,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of digested palm oil and sunflower oil dilutions on basolateral ApoB secretion by Caco-2 cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eEffect of different dilutions of control digest, digested palm oil and sunflower oil on basolateral ApoB secretion from 21 days differentiated Caco-2 cells without starving. Reflected ApoB levels are relative levels correlated to control digest. Exposures were conducted as a single cell experiment.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1575818/v2/a9bcaaff4dea97d47760a17b.jpg"},{"id":22066587,"identity":"b8b4e062-619d-450b-a8cc-5ab8a6f93b71","added_by":"auto","created_at":"2022-05-31 13:52:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":234467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of 1-week starvation prior to digested palm oil and sunflower oil exposure, on intestinal integrity and basolateral ApoB secretion of Caco-2 cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003ea) Effect of palm oil and sunflower oil (both 1:50 diluted) on TEER of 21 days differentiated Caco-2 cells after 1 week starvation. TEER % compared to control digest (also 1:50 diluted) which was set to 100%. Oils and control digest were diluted with DMEM w/o FBS and w/o phenol. Exposures were conducted in technical duplicate and a biological triplicate (N=3). (no signification was observed between the samples). b) Effect of digested palm oil and sunflower oil (both 1:50 diluted) on basolateral ApoB secretion from 21 days differentiated Caco-2 cells after 1 week starvation. Depicted ApoB levels are relative levels correlated to control digest (also 1:50 diluted). Exposur\u003c/p\u003e\u003cp\u003ees were conducted in technical duplicate and a biological triplicate (N=3) (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1575818/v2/606bd5ab1f677e67a2546a20.jpg"},{"id":22068333,"identity":"b0880dec-ad0a-48e1-b199-ddf75a30714e","added_by":"auto","created_at":"2022-05-31 14:02:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":197590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of LPS concentrations on intestinal integrity of Caco-2 cells, co-exposed to digested palm oil and sunflower oil\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eEffect of different concentrations LPS (ng/ml) in time on TEER in fat-exposed, 21 days differentiated and 1 week starved Caco-2 cells. TEER % compared to digest control (100%).\u0026nbsp;Digest control (a), digested palm oil (b) and digested Sunflower (c) were diluted 1:50 in DMEM w/o phenol-red and w/o FBS. Caco-2 cells were co-incubated after starvation with 0, 100, 250, 500, 750 and 1000 ng/ml LPS. These exposures were conducted as a single cell experiment.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1575818/v2/5502d2e95121a30e1c30a0cf.jpg"},{"id":22066590,"identity":"40914ac0-e0d9-443d-bc7a-8d4b321d8f55","added_by":"auto","created_at":"2022-05-31 13:52:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":273398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of LPS concentrations on basolateral ApoB secretion and LPS translocation by Caco-2 cells after co-exposure to digested palm oil and sunflower oil\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eEffect of different concentrations LPS (ng/ml) on ApoB (a) and LPS (b) secretion by fat-exposed Caco-2 cells Digest control, digested palm oil and digested sunflower oil were diluted 1:50 in DMEM w/o phenol-red and w/o FBS. Caco-2 cells were co-incubated with LPS concentrations 0,100, 250, 500, 750 and 1000ng/ml for 24 hours. APO B was measured with an Elisa assay and the LPS was measured with the HEK-TLR4 assay. The OD represent the LPS concentration of the samples. These exposures were conducted as a single cell experiment.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1575818/v2/50158d8cf273d05e1361002f.jpg"},{"id":22066585,"identity":"66a4dbfb-9087-4742-bfd7-a0d92d721c67","added_by":"auto","created_at":"2022-05-31 13:52:50","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":226167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of digested palm oil, digested sunflower oil, FD4 and LPS on intestinal integrity of Caco-2 cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eEffect of digested palm oil (a) and sunflower oil (b), FD4 and LPS on TEER in Caco-2 cells. TEER (%) was compared to control digest without FD4 and LPS, which was set to 100%. TEER t=0 is measured immediately after adding oils, FD4 and LPS. The absolute TEER values of control digest were highly stable in time. Control digest is sample with digestion enzymes, but without oil. These exposures were conducted as a single cell experiment.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1575818/v2/3135115c7d7aff3ab97b63e9.jpg"},{"id":22068336,"identity":"61f7fe64-ad4f-43e1-b4b6-7ffad8f1a000","added_by":"auto","created_at":"2022-05-31 14:02:50","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":236873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of digested palm oil and sunflower oil on paracellular and transcellular translocation by Caco-2 cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eEffect of palm oil and sun flower oil on relative basolateral levels of FD4 (a) and LPS (b) secretion by fat-exposed Caco-2 cells Digest control, digested palm oil and digested sunflower oil were diluted 1:50 in DMEM w/o phenol-red and w/o FBS. Caco-2 cells were co-incubated with 0 and 750 ng/ml LPS for 24 hours. FD4 was calculated using a calibration curve and compared to the control digest. LPS was measured with the microtiter pyrochrome assay. These exposures were conducted as a single cell experiment.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1575818/v2/c25e539b35c96d2b68f86d1a.jpg"},{"id":22067740,"identity":"c3cd4d2b-b454-48b4-85b0-7569442926b3","added_by":"auto","created_at":"2022-05-31 13:57:50","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":173800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChylomicron blockage by PL81 and its effect on intestinal integrity of Caco-2 cells \u003c/strong\u003e\u003c/p\u003e\u003cp\u003eEffect of PL-81 with or without palm oil and sunflower oil on TEER in fat and LPS exposed Caco-2 cells. Caco-2 cells were overnight incubated with LPS (1mg/ml). TEER t=0 is measured immediately after adding oils and PL-81. TEER (%) was compared to control digest with LPS without PL-81, which was set to 100%. Control digest is sample with digestion enzymes, but without oil. These exposures were conducted as technical duplicate and a biological triplicate (N=3) experiment.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1575818/v2/aa207c0f9f889c71d1a778ce.jpg"},{"id":22068922,"identity":"a39ac3b5-aed8-4de6-8888-8fc2a8ce70bf","added_by":"auto","created_at":"2022-05-31 14:07:50","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":251412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChylomicron blockage by PL81 and its effect on basolateral ApoB secretion and LPS translocation\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eEffect of PL-81 with or without palm oil and sunflower oil on relative levels of Apo-B (a) and LPS (b) secretion by fat exposed, starved Caco-2 cells. Caco-2 cells were incubated with 0 and 1000 ng/ml LPS overnight. Digest control, digested palm oil and digested sunflower oil were diluted 1:50 in DMEM w/o phenol-red and w/o FBS with 500 µg/ml PL-81 blocker. Apo-B was measured with an elisa assay and LPS was measured with the pKFlex assay. These exposures were conducted as a technical duplicate and a biological triplicate (N=3) (p\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1575818/v2/73eb8862bcb2ae772c96c3e4.jpg"},{"id":44716436,"identity":"f59a860a-dbec-47ff-9fda-dfaf5f634be3","added_by":"auto","created_at":"2023-10-16 18:25:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":850162,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1575818/v2/c2782c1f-4768-4a98-aecd-d4dc61ae98aa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dietary fat enhanced chylomicron-mediated LPS translocation in a Caco-2 transwell model","fulltext":[{"header":"Background","content":"\u003cp\u003eThere is growing evidence for a role of the intestinal barrier in low-grade inflammation. Impaired intestinal barrier function could facilitate the passage of luminal antigens or other molecules that lead to a mucosal immune response. Such a luminal antigen that is commonly linked to chronic inflammation is lipopolysaccharide (LPS) which consists of a lipid, an inner- plus outer core and a polysaccharide composed of 0-antigens joined by a covalent binding. LPS is a cell wall component of Gram-negative bacteria that is naturally present in the gut in high quantities. LPS can cross the intestinal barrier from the intestinal lumen via paracellular and/or transcellular routes. Paracellular translocation can occur when tight junction function is impeded, resulting in a reduced intestinal integrity. For example, ethanol induces disruption of the tight junctions and can therefore enforce paracellular translocation of LPS [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], which can subsequently contribute to alcoholic liver disease (ALD)[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Transcellular translocation of LPS, on the other hand, is especially linked to a high load of dietary fat [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eIn vitro\u003c/em\u003e, animal and human studies have demonstrated that the ingestion of a high fat meal, especially high in saturated fat, facilitates the translocation of luminal LPS across the intestinal barrier. Chylomicrons, transporter molecules of dietary triglycerides, seem to play an important role in this process, as after a high fat challenge the postprandial LPS levels peak at the same time as chylomicrons and LPS is highest in the chylomicron-rich fraction [\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Via chylomicrons, LPS can subsequently be transported to other organs in the body, where its endotoxin activity can induce low-grade chronic inflammation linked to various metabolic diseases. In people with obesity, insulin resistance and/or type 2 diabetes, increased plasma levels of LPS were found compared to a group of healthy subjects after ingestion of a high fat diet [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, LPS activity was reported to be highly correlated with metabolic and cardiovascular risk factors and the number of components of the metabolic syndrome [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This phenomena is referred to as metabolic endotoxemia [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNext to metabolic endotoxemia, there is growing evidence that (micro)-inflammation of the intestinal mucosa plays a role in the pathogenesis of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. IBS symptoms have been reported to correlate with increased permeability and subclinical inflammation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, Dlugosz and colleagues [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] have shown that especially patients with IBS-D (diarrhea predominant IBS subtype) have higher serum levels of LPS, indicating a subclinical inflammation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In IBD patients elevated serum LPS were found to be associated with disease activity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, an increased risk for developing IBD was linked to the intake of dietary fat [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], supporting a role for LPS in IBD pathology.\u003c/p\u003e \u003cp\u003eTo identify promising interventions that can prevent or ameliorate LPS-related low grade inflammation, \u003cem\u003ein vitro\u003c/em\u003e models are valuable for high-throughput screening. Several studies have suggested that intestinal epithelial Caco-2 cells can serve as an \u003cem\u003ein vitro\u003c/em\u003e model to study fat-induced LPS translocation via chylomicron secretion. However most of these studies used free fatty acids to induce chylomicron secretion [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and not the unrefined dietary oils that are typically part of the western diet. The purpose of our study was to develop an \u003cem\u003ein vitro\u003c/em\u003e model of fat-induced transcellular translocation of LPS that more accurately mimics the \u003cem\u003ein vivo\u003c/em\u003e situation. We investigated the effects of digested palm- and sunflower oil on LPS translocation in Caco-2 cells.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eAll chemicals were purchased from Sigma Aldrich (St Louis, Missouri, USA) unless otherwise stated.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vitro\u003c/span\u003e \u003cb\u003egastrointestinal digestion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo mimic oral digestion, palm oil (mainly C16:0) (Research Diet Services BV) and sunflower oil (mainly C18:2) (Research Diet Services BV) were both diluted to 0.2 g/ml with 140 mM NaCl\u0026thinsp;+\u0026thinsp;5 mM KCl to a total volume of 15 ml. This mixture was gently vortexed, and heated for 15 min at 56\u0026deg;C. To mimic gastric digestion, the pH was set to 2 with 1 M HCl and 40 mg/ml of pepsin solution (1092 U/ml dissolved in 0.1 M HCl) was added. The samples were incubated for 1 h at 37\u0026deg;C while gently shaking. To mimic intestinal digestion, the pH was set to 5.8 with 1 M NaHCO\u003csub\u003e3\u003c/sub\u003e and 4 mg/ml pancreatin (6.84 U/mg trypsin activity), 5.9 units/ml α-chromotrypsin (65.62 U/mg), 1 mg/ml lipase (all from porcine) and bile salts (94.6 mg/ml sodiumtaurocholate and 83 mg/ml sodium glycodeoxycholate) dissolved in 0.1 M NaHCO\u003csub\u003e3\u003c/sub\u003e were added. Next, the pH was adjusted to 6.5 using 1 M NaHCO\u003csub\u003e3\u003c/sub\u003e, after which the samples were incubated at 37\u0026deg;C for 2 h while gently shaking. After incubation, the pH was adjusted to 7.5 with 1 M NaHCO\u003csub\u003e3\u003c/sub\u003e and the volume of the digest was filled up to 40 ml with 140 mM NaCl\u0026thinsp;+\u0026thinsp;5 mM KCl. As control a digest was prepared which contained all buffers and enzymes, but without oils.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCaco-2 culture\u003c/h2\u003e \u003cp\u003eCaco-2 cells (ATCC-HTB-37) were cultured in Dulbecco\u0026rsquo;s Modified Eagles Medium (DMEM) with high glucose (4.5 g/l) and 25 mM HEPES (Life technologies, 42430), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Translucent, 0.4 \u0026micro;m transwell inserts (Greiner Bio-one) were seeded on the apical side with 500 \u0026micro;l (12 wells) or 150 \u0026micro;l (24 wells) of 0.225x10\u003csup\u003e6\u003c/sup\u003e cell/ml Caco- 2, with 1500 \u0026micro;l (12 wells) or 750 \u0026micro;l (24 wells) of basolateral medium, respectively. The cells were incubated for 21 days at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e to differentiate in small-intestinal like epithelial cells. Apical and basolateral medium was replaced three times a week and one day prior to the investigational exposure. To monitor integrity of the Caco-2 monolayer, transepithelial electrical Resistance (TEER) was measured by using a MilliCell ERS (Millipore Amsterdam, The Netherlands). Differentiated Caco-2 monolayers were considered of acceptable quality if TEER values were higher than 700 Ω/cm\u003csup\u003e2\u003c/sup\u003e (24 wells) and 450 Ω/cm\u003csup\u003e2\u003c/sup\u003e (12 wells) before exposure to the investigational treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFat-induced LPS translocation experiments\u003c/h2\u003e \u003cp\u003ePrior to exposure to fats and LPS (Sigma L4391), Caco-2 cells were generally starved for one week, meaning that culture medium without added FBS was used in the apical compartment for the last 3 medium replacements. Subsequently, Caco-2 cells were apically exposed to digested oil samples (1:50 diluted in DMEM, unless stated otherwise) for 24 hours. DMEM and control digest samples (also 1:50 diluted in DMEM, unless stated otherwise) were included as negative controls. To determine fat-induced translocation of LPS, additionally 100, 250, 500, 750 or 1000 ng/ml LPS was apically added for 24 hours. The TEER was measured during the exposure experiments at 0, 1, 3, 6 and/or 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eChylomicron blockage experiments\u003c/h2\u003e \u003cp\u003eTo investigate whether LPS is translocated via chylomicrons in our fat-induced LPS translocation model, we also performed Caco-2 experiments with and without the chylomicron blocker Pluronic L81 (PL81, BASF Corporation, Germany), as described by Ghoshal et al. (J Lipid Res, 2009, 50:90\u0026ndash;7). All these experiments were performed in DMEM without phenol-red (Life Technologies) and FBS, as this seemed to interfere with the chylomicron detection.\u003c/p\u003e \u003cp\u003eCaco-2 cells were first pre-incubated overnight with LPS before exposure to PL81 and dietary fats, to prevent paracellular LPS leakage by PL81 exposure. Therefore, 20-day differentiated starved Caco-2 cells were apically challenged overnight with 1 mg/ml LPS (diluted in DMEM without FBS and phenol red). Next, Caco-2 were washed 3 times with DMEM without FBS and phenol red, after which digested oil samples were apically added, mixed with 500 ug/ml PL81. After 24-hour incubation, chylomicron production and LPS translocation were assessed in the basolateral compartment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDetection of chylomicron-marker ApoB\u003c/h2\u003e \u003cp\u003eChylomicron production by Caco-2 cells was assessed by measuring ApoB levels in the basolateral medium using an ApoB ELISA (ABIN612664, Antibodies-online), which detects both ApoB-48 and ApoB-100, according to the manufacturer\u0026rsquo;s protocol. In \u003cem\u003ein vivo\u003c/em\u003e settings, adult human intestines secrete chylomicrons which only contain ApoB-48, not ApoB-100, which is predominantly associated with VLDL derived from liver [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, Caco-2 cells are known to produce and secrete chylomicrons with both ApoB-48 and ApoB-100 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetection of LPS\u003c/h2\u003e \u003cp\u003eLPS was measured in the apical as well in basolateral medium with a chromogenic assay from Nodia (Assiociates Cap. Cod Inc). The assay was performed in an microtiterplate reader (Tecan) as well in a Pyros Kinetix Flex tube reader (pKFlex) (Nodia). Pyrochrome lysate was reconstituted with 3.2 ml pyrochrome reconstitution buffer (C1500-5). Samples were diluted with LPS free MQ and incubated for 15 min in a water bath at 70\u0026deg;C. After 1 h at 4\u0026deg;C the samples were placed for 10 min at room temperature and directly measured with an endpoint measurement on the microtiter plate reader or on the pKFlex. For both methods a calibration from Limulus Amebocyte lysate control standard endotoxin 0.5 \u0026micro;g/vial (CSE E0005-1) was used. For the endpoint method 125 \u0026micro;l pyrochrome was added to 125 \u0026micro;l treated sample or standard and the mixture was incubated at 37\u0026deg;C. After 35, 45 and 60 min, 80 \u0026micro;l sample was taken and added to 20 \u0026micro;l Acetic acid. Finally, the absorbance was measured at 405 nm on the plate reader. For the pyros kinetix flex method 200 \u0026micro;l treated sample or standard was added in a pKflex glass tube. 50 \u0026micro;l pyrochrome was added and the mixture was briefly stirred and placed in the pKFlex. Using the Pyros express 21 CFR Part 11 compliant software the endotoxin concentration was calculated.\u003c/p\u003e \u003cp\u003eAnother method used for functional LPS quantification is the HEK-Blue hTLR4 assay [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHEK-Blue hTLR4 cells are transgenic for the cell surface expressed hTLR4 MD-2 and CD14 receptors and contain a downstream reporter system resulting in secretion of secreted embryonic alkaline phosphtase (SEAP) under the control of NFkB and AP1 promotors. HEK-Blue hTLR4 cells (InvivoGen, Toulouse, France) were sub-cultured in DMEM with 10% FBS once per week and medium was refreshed twice per week.\u003c/p\u003e \u003cp\u003eFor experiments, HEK-Blue hTLR4 cells were detached using a cell scraper when 90\u0026ndash;95% confluence was reached and 0.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells were transferred to each well of a 96-well Poly-D-Lysine coated plate. After overnight incubation at 37\u0026deg;C in an atmosphere containing 5% CO\u003csub\u003e2,\u003c/sub\u003e we added 0.0001-100 EU/ml LPS (E. coli O11: B4, 1 EU\u0026thinsp;\u0026equiv;\u0026thinsp;0.15 ng/ml, Sigma, St. Louis, MO, USA) or 100 ul basolateral medium from the Fat-induced LPS translocation experiments to the cells and the plate was incubated for 8 h (37\u0026deg;C; 5% CO\u003csub\u003e2\u003c/sub\u003e). TLR4 stimulation resulted in SEAP secretion, which was quantified by mixing 20 \u0026micro;l of supernatant (depleted from cells by centrifugation at 450 \u003cem\u003eg\u003c/em\u003e for 5 min) with 180 \u0026micro;l of Quanti-Blue\u0026trade; in a new flat bottom 96-well plate. The plate was incubated for 3 h at 37\u0026deg;C and absorption at 655 nm was determined every hour using a spectrophotometer (TECAN, Giessen, The Netherlands).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetection of paracellular translocation by FD4\u003c/h2\u003e \u003cp\u003eTo determine paracellular translocation of LPS in the Caco-2 exposure experiments, 250 \u0026micro;g/ml 4 kDa FITC-dextran (FD4) was added to the apical side at the start of the investigational exposures. After 24 hours, fluorescence (Excitation 485 nm / Emission 528 nm) in the basolateral compartment was determined. The fluorescent values were compared to a calibrations curve for FD4 and the percentage of paracellular translocation was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eExposures performed with a concentration range to determine optimal conditions for the model were conducted as a single cell experiment. Verification exposures were performed with a biological triplicate. These data are represented as average\u0026thinsp;\u0026plusmn;\u0026thinsp;STD. Statistical significance of difference was analysed by ANOVA followed by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test for pair wise comparison. Differences between groups are considered statistically significant when P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDietary fat-induced chylomicron secretion in Caco-2 cells\u003c/h2\u003e \u003cp\u003eTo study the potential of saturated and unsaturated dietary fats to induce chylomicron production in Caco-2 cells, \u003cem\u003ein vitro\u003c/em\u003e digested palm oil and sunflower oil were used, respectively. A first step before applying these dietary fats to Caco-2 cells consisted of \u003cem\u003ein vitro\u003c/em\u003e digestion to better mimic intestine luminal conditions. The digested oils were tested in different dilutions to determine the optimal condition for chylomicron production. The digested oils did not appear to negatively impacted the barrier integrity of small-intestinal-like Caco-2 cells at 1:10, 1:50 and 1:100 dilutions. In contrast, 1:1 dilutions reduced the TEER to some extend when compared to the control digest (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B), indicative for a compromised intestinal integrity. This seems to fit the detected basolateral ApoB levels, as only 1:10, 1:50 and 1:100 dilutions of digested palm oil and sunflower oil induced an increase in basolateral ApoB levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At these concentrations palm oil showed the most pronounced induction of about a 1.2 times increase (20%) compared to digest control. The (middle) 1:50 dilution was selected to continue optimisation of the model, as Luchoomun et al previously showed that too high or low levels of fatty acids (in combination with bile acids) can result in reduced ApoB secretion [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo mimic the \u003cem\u003ein vivo\u003c/em\u003e situation more closely [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], Caco-2 cells were starved (meaning deprived from FBS on the apical side) in the last week of differentiation and thus one week before dietary fat exposure. After 24 hours of exposure to the 50 times diluted digested oils, intestinal integrity of the starved 21-day old Caco-2 was not affected when compared to the control digest (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In contrast, chylomicron secretion was significantly increased by palm oil compared to the digest control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This indicates that starvation of Caco-2 cells prior to fat exposure increases chylomicron production, thereby enlarging the window of opportunity for modulation. Based on these data we determined that a 1:50 dilution of \u003cem\u003ein vitro\u003c/em\u003e digested oils and 1-week starving of the Caco-2 cells prior to fat exposure were the most optimal conditions for our Caco-2 model on fat-induced chylomicron secretion.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDietary fat-induced LPS translocation in Caco-2 cells\u003c/h2\u003e \u003cp\u003eHaving established the optimal culture and fat-exposure conditions to enhance fat-induced chylomicron secretion in Caco-2 cells, we investigated whether this model could be extended to study dietary fat-induced LPS translocation. To this end, Caco-2 cells were co-incubated with the digested oils and different concentrations of LPS (0, 50, 100, 250, 500, 750 or 1000 ng/ml). A 24-hour exposure with these samples did not affect intestinal integrity, indicating that apical LPS has no detrimental effect on the Caco-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, LPS does not seem to drastically affect fat-induced ApoB secretion, although, there seems to be a reduced ApoB secretion upon high apical LPS exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, only at a high level of apical LPS exposure (\u0026ge;\u0026thinsp;750ng/ml), a clear distinction in basolateral LPS can be found between the digested oil samples and control digest (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This indicates that at least 750 ng/ml LPS is needed in conjunction with digested oils to detect dietary fat-induced LPS translocation in our Caco-2 model. Thus, we continued the experiments using 750 ng/mL LPS.\u003c/p\u003e\u003cp\u003eTo exclude paracellular LPS translocation, FD4 was apically added to the digested oils (1:50 dilution) and LPS (750 ng/ml) in the Caco-2 model. No effect was seen on TEER, and thus intestinal integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). After 24h exposure, no FD4 translocation to the basolateral compartment was detected, whereas basolateral LPS levels were still increased by palm oil and sunflower oil (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B). This indicates that the paracellular route is not involved in fat-induced LPS translocation in this Caco-2 model.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDietary fat-induced LPS translocation is mediated by chylomicron production in Caco-2 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo verify that the dietary fat-induced LPS translocation is mediated by chylomicron production, the chylomicron blocker PL81 was added to the Caco-2 model. As PL81 is known to affect the intestinal barrier function [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], the timing of exposures in the model was adapted to avoid paracellular leakage of LPS due to PL81-induced barrier disruption. Cario et al described that LPS can be internalized and stored in intestinal epithelial cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], therefore Caco-2 cells were preincubated overnight with LPS, instead of directly co-incubated with the digested dietary fats. Prior to co-exposure with PL81, we first confirmed that the overnight stored LPS could still be released upon fat exposure to the Caco-2 cells. Co-exposure of the Caco-2 cells with PL81, showed an expected strong reduction in TEER (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), especially in control digest conditions. Surprisingly, a less dramatic drop in TEER was seen in cells co-incubated with the oils. Particularly palm oil seemed to protect against the PL81-induced deterioration of the intestinal layer.\u003c/p\u003e \u003cp\u003eNext, we measured basolateral ApoB secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA) and LPS translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB) and found that PL81 significantly reduced basolateral ApoB levels independent of the applied digests. This indicates that PL81 accurately blocked chylomicron production in our Caco-2 cell model. When measuring LPS translocation with PL81 co-exposure, we observed a strong increase in basolateral LPS when Caco-2 were exposed to the digest control, (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). This is probably linked to the strong disruption of the barrier integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), and thus mainly represents paracellular leakage. Interestingly, when Caco-2 cells were co-exposed to either palm or sunflower oil digests, the PL81 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB; grey bars) even reduced the LPS translocation in Caco-2 compared to without PL81 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB; black bars). Even though this reduction in basolateral LPS did not reach significance (p\u0026thinsp;=\u0026thinsp;0.1), the data strongly suggest that dietary fat-induced LPS translocation in our model is (at least partly) mediated by chylomicron production.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe developed an \u003cem\u003ein vitro\u003c/em\u003e Caco-2 model to study digested dietary fat-induced LPS translocation, which can serve as a screening tool to identify (food) compounds with potential beneficial and preventive effects on LPS-related low grade inflammation.\u003c/p\u003e \u003cp\u003eIn this study we determined the optimal concentrations of digested oils and apical LPS to stimulate chylomicron production by small-intestinal-like Caco-2 cells, and subsequent LPS translocation, without inducing cytotoxic effects and disrupting the intestinal barrier. As both TEER and FD4 were not affected after the challenge with digested oils and LPS, the transport of LPS is unlikely to be facilitated through paracellular transport. Similarly, Mani et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] observed no difference in intestinal integrity in pigs after \u003cem\u003eex vivo\u003c/em\u003e treatments with endotoxins in relation to dietary oil compositions. Taken together, these data suggest that fat-induced LPS translocation is routed via transcellular transport and not paracellular transport.\u003c/p\u003e \u003cp\u003eThe use of PL81, a known inhibitor of chylomicron production [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], suggested that fat-induced chylomicron production is involved in the LPS translocation, as the dietary fat-induced secretion of ApoB was diminished with a consequent created reduction of LPS translocation. PL81 uptake by differentiated Caco-2 cells is known to be a protein-facilitated active process and PL81 inhibits fat absorption by decreasing the triacylglycerols transport from the cytosol to the endoplasmic reticulum. This in turn inhibits assembly and secretion of chylomicrons [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In case of adding PL81 without a dietary fat (digest control), we found that the integrity of the layer was severely disturbed and the TEER was considerably decreased. In other studies that use PL81 as a chylomicron inhibitor the barrier disruption was not mentioned. However, our study shows that this is important to take into account when using this chylomicron blocking-compound, especially when not used in combination with lipid-like molecules in the apical compartment. Upon co-incubation with digested oils, we found that the disruption of the intestinal barrier by PL81 was much less pronounced, indicating a \u0026lsquo;protective effect\u0026rsquo; of the digested fats against PL81-induced barrier disruption. The mechanism behind this is unclear, but it might be related to the cellular distribution of PL81 that can be affected by coincubation with dietary fat(ty acids) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFatty acid-induced chylomicron production and LPS translocation were previously studied in Caco-2 cells using free fatty acids by Luchoomun [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and Ghoshal et al [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], respectively. The incubation with digested oils is more closely mimicking the \u003cem\u003ein vivo\u003c/em\u003e situation and therefore should be preferred over the fatty acid-induced LPS model. Furthermore, it was previously reported that exposure of Caco-2 cells to palmitic acids alone can disrupt the intestinal barrier and thereby reduce ApoB secretion [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and induce paracellular transport [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which is not desirable for a fat-induced LPS model. However, in that study only fatty acids were used, not digested oils. Luchoomun et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] previously showed that bile acids are important for efficient chylomicron production in Caco-2 cells. The \u003cem\u003ein vitro\u003c/em\u003e digestion integrated in our model, also includes the addition of bile acids which are therefore also included in our studies as the total digest is applied to Caco-2 cells. Ghoshal \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] showed that LPS translocation is dependent on long-chain fatty acid exposure in Caco-2 cells, whereas short chain fatty acids, which are not transported via chylomicrons [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], did not induce LPS transport. This indicates that chylomicron production is involved in LPS translocation, which was supported by their PL81 experiments abolishing the long chain fatty acid-induced effect. Similar effects of PL81 were also found in our Caco-2 model, indicating that also with digested oils the fat-induced LPS translocation is linked to chylomicron production. However, our data suggest that this LPS translocation induced by digested oils is only partly dependent on chylomicron production, as ApoB secretion was almost completely abolished by PL81, whereas LPS is only reduced by approximately 50%. This would however fit with recent findings of Akiba et al, that showed a second lipid-dependent route for LPS transport in the intestine, which is independent of chylomicron secretion [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In their rat model they also found that PL81 could not completely block fat-induced LPS translocation, and they additionally found (labelled) LPS back in the portal vein. They found no evidence for paracellular leakage of LPS. Alternatively, it has previously been shown that high fat consumption can induce internalization of tight junction proteins which increases paracellular transport, including LPS transport [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This potential process for fat-induced LPS translocation was not reflected in TEER levels and FD4 translocation in our model. Based on our findings, we can however not exclude that paracellular leakage of LPS might still play a role in fat-induced LPS translocation.\u003c/p\u003e \u003cp\u003eSeveral studies have shown that composition of dietary oil has a substantial effect on postprandial endotoxemia. Especially saturated fats are linked to higher postprandial LPS levels in blood [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In our model we recapitulated this by showing that palm oil more strongly induced chylomicron secretion and LPS translocation when compared to sunflower oil. In contrast, Laugerette et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] showed that palm oil had no effect on plasma endotoxin concentrations while sunflower oil augmented plasma endotoxemia by 59\u0026ndash;70%.\u003c/p\u003e \u003cp\u003eIn western diets, fats and oils are common components of the diet [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In recent years, the development of obesity, inflammation and other metabolic diseases has been linked to low grade endotoxemia associated with high dietary fat and energy intake [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These studies have raised questions whether diet-induced endotoxemia is caused by changes in permeability of the intestine, variations in the gut microbiota or simply by a change in intake of the dietary fat and energy content of the food. There is increasing evidence that the low grade endotoxemia is induced by a combination of all the above factors. For instance, next to the effect of dietary fat on LPS translocation, a high saturated fat diet, like palm oil, is reported to result in higher gram negative bacterial populations such as \u003cem\u003eE. coli\u003c/em\u003e which produce gut LPS [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Life style changes, and especially also dietary interventions that reduce chylomicron production and lower LPS production by the gut microbiome, are thought to be effective in reducing low grade inflammation and thereby reducing the risk for CVD and other co-morbidities linked to metabolic syndrome.\u003c/p\u003e \u003cp\u003eNext to this impact on metabolic health, also gastrointestinal health has a link with dietary fat and LPS-related inflammation. Associations between fatty meal consumption and IBS symptom induction have also been identified in a number of studies. In fact, in many IBS patients the symptoms were triggered after the consumption of fatty or fried food [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Dietary fat is also linked to an increased risk for developing IBD [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Interestingly, also higher serum and/or fecal LPS levels are found in IBD and IBS patients [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], especially in patients with predominant diarrhoea-related symptoms [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This link with high fat intake and the previously found association with high LPS levels, might indicate a role for fat-induced LPS translocation in IBS and IBD pathology. If confirmed in future studies, dietary interventions that influence fat-induced LPS translocation might also be beneficial for this patient population.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe developed an \u003cem\u003ein vitro\u003c/em\u003e model that closely mimics the \u003cem\u003ein vivo\u003c/em\u003e situation to study dietary fat-induced LPS translocation, linked to LPS-related low grade inflammation. This Caco-2 model can be used to screen dietary fats and other food components that can prevent or reduce inflammatory conditions. In this way, this model contributes to research identifying beneficial food (compounds) for human metabolic and gastrointestinal health.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Dutch ministry of Economic affairs via Public Private Partnership grants from the Top consortium for Knowledge and Innovation Agri \u0026amp; Food, project AF12203 “CCC CarboHealth” and project AF16012 “Nutrition to improve quality of life of IBS patients”.\u0026nbsp;The funding agencies had no role in the collection, analysis, and interpretation of data or in the preparation, review, or approval of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors' contributions\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.T. was in the lead for conceptualization, methodology, performance and analysis of the experiments, and writing of the manuscript. C.G. contributed to acquisition, conceptualization and manuscript reviewing and revising. P.V. contributed to acquisition and manuscript reviewing and revising. N.W. contributed to acquisition, conceptualization, analysis of the experiments and writing, reviewing and revising of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBala S, Marcos M, Gattu A, Catalano D, Szabo G: Acute Binge Drinking Increases Serum Endotoxin and Bacterial DNA Levels in Healthy Individuals. 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Nutrients 2019, 11:2277.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB\u0026ouml;hn L, St\u0026ouml;rsrud S, T\u0026ouml;rnblom H, Bengtsson U, Simr\u0026eacute;n M: Self-reported food-related gastrointestinal symptoms in IBS are common and associated with more severe symptoms and reduced quality of life. Am J Gastroenterol 2013, 108:634\u0026ndash;641.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRijnaarts I, Witteman BJM, Zoetendal EG, Govers C, de Wit NJW, de Roos NM: Subtypes and Severity of Irritable Bowel Syndrome Are Not Related to Patients\u0026rsquo; Self-Reported Dietary Triggers: Results From an Online Survey in Dutch Adults. Journal of the Academy of Nutrition and Dietetics 2021, 121:1750\u0026ndash;1762.e1758.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou SY, Gillilland M, 3rd, Wu X, Leelasinjaroen P, Zhang G, Zhou H, Ye B, Lu Y, Owyang C: FODMAP diet modulates visceral nociception by lipopolysaccharide-mediated intestinal inflammation and barrier dysfunction. J Clin Invest 2018, 128:267\u0026ndash;280.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"lipids-in-health-and-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lhad","sideBox":"Learn more about [Lipids in Health and Disease](http://lipidworld.biomedcentral.com/)","snPcode":"12944","submissionUrl":"https://submission.nature.com/new-submission/12944/3","title":"Lipids in Health and Disease","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lipopolysaccharide, dietary fat, chylomicrons, intestine, translocation","lastPublishedDoi":"10.21203/rs.3.rs-1575818/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1575818/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThere is increasing evidence that dietary fat, and especially saturated fat, promotes the absorption of lipopolysaccharide (LPS) via enhanced chylomicron production. The latter can subsequently transport LPS to other parts of the body, where it can induce low-grade chronic inflammation that links to various metabolic and gut-related diseases. To identify promising (food) compounds that can prevent or ameliorate LPS-related low grade inflammation, we developed and optimized an \u003cem\u003ein vitro\u003c/em\u003e model for dietary fat-induced LPS translocation that closely mimics the \u003cem\u003ein vivo\u003c/em\u003e situation and facilitates high-throughput screening.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFor this, epithelial Caco-2 cells were differentiated in 21 days to a small intestinal phenotype. Thereafter, the cells were exposed to a dilution range of \u003cem\u003ein vitro\u003c/em\u003e digested palm oil and sunflower oil, with or without starvation, to determine optimal conditions for fat-induced chylomicron production as measured by basolateral levels of the chylomicron-related marker apolipoprotein B. Next, LPS was co-incubated in various concentrations with the digested oils and fat-induced LPS translocation to the basolateral side was assessed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that dietary fat-induced LPS translocation in Caco-2 cells was optimal after apical exposure to digested oils in a 1:50 dilution, in combination with 750ng/ml LPS, preceded by one-week starvation (cultured without fetal bovine serum). Co-incubation with chylomicron blocker Pluronic L81 confirmed that fat-induced LPS translocation is mediated via chylomicron production in this Caco-2 model.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eWe developed a robust Caco-2 cell model for dietary fat-induced LPS translocation that can be used for high-throughput screening of (food) compounds that can reduce LPS-related low grade inflammation.\u003c/p\u003e","manuscriptTitle":"Dietary fat enhanced chylomicron-mediated LPS translocation in a Caco-2 transwell model","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-05-31 13:52:48","doi":"10.21203/rs.3.rs-1575818/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-07-31T07:46:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-07-28T19:11:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-06-21T12:50:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4b86e2c8-e54c-4b20-9e2b-5ccd2b1f8d17","date":"2022-06-09T15:10:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-06-02T20:35:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"768f190e-5d68-40f3-8969-5dd87edac26b","date":"2022-05-23T12:48:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-05-22T14:14:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-05-18T02:35:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-05-18T02:35:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Lipids in Health and Disease","date":"2022-05-17T13:44:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"lipids-in-health-and-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lhad","sideBox":"Learn more about [Lipids in Health and Disease](http://lipidworld.biomedcentral.com/)","snPcode":"12944","submissionUrl":"https://submission.nature.com/new-submission/12944/3","title":"Lipids in Health and Disease","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"27c23ce9-aed1-4aa6-acc5-5445e098412a","owner":[],"postedDate":"May 31st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T18:22:04+00:00","versionOfRecord":{"articleIdentity":"rs-1575818","link":"https://doi.org/10.1186/s12944-022-01754-3","journal":{"identity":"lipids-in-health-and-disease","isVorOnly":false,"title":"Lipids in Health and Disease"},"publishedOn":"2023-01-12 18:17:58","publishedOnDateReadable":"January 12th, 2023"},"versionCreatedAt":"2022-05-31 13:52:48","video":"","vorDoi":"10.1186/s12944-022-01754-3","vorDoiUrl":"https://doi.org/10.1186/s12944-022-01754-3","workflowStages":[]},"version":"v2","identity":"rs-1575818","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1575818","identity":"rs-1575818","version":["v2"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}