Causative Role of Anoxic Environment in Bacterial Regulation of Human Intestinal Function

Abstract

Introduction

Life on Earth depends on oxygen; human tissues require oxygen signaling, whereas many microorganisms, including bacteria, thrive in anoxic environments. Despite these differences, human tissues and bacteria coexist in close proximity to each other such as in the intestine. How oxygen governs intestinal-bacterial interactions remains poorly understood.

Methods

To address to this gap, we created a dual-oxygen environment in a microfluidic device to study the role of oxygen in regulating the regulation of intestinal enzymes and proteins by gut bacteria. Two-layer microfluidic devices were designed using a fluid transport model and fabricated using soft lithography. An oxygen-sensitive material was integrated to determine the oxygen levels. The intestinal cells were cultured in the upper chamber of the device. The cells were differentiated, upon which bacterial strains, a facultative anaerobe, Escherichia coli Nissle 1917, and an obligate anaerobe, Bifidobacterium Adolescentis, were cultured with the intestinal cells.

Results

The microfluidic device successfully established a dual-oxygen environment. Of particular importance in our findings was that both strains significantly upregulated mucin proteins and modulated several intestinal transporters and transcription factors but only under the anoxic–oxic oxygen gradient, thus providing evidence of the role of oxygen on bacterial-epithelial signaling.

Conclusions

Our work that integrates cell and molecular biology with bioengineering presents a novel strategy to engineer an accessible experimental system to provide tailored oxygenated environments. The work could provide new avenues to study intestine-microbiome signaling and intestinal tissue engineering, as well as a novel perspective on the indirect effects of gut bacteria on tissues including tumors.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12195-022-00735-x.

Introduction

The survival and development of organisms on Earth depends on oxygen levels. For example, in the human body, tissues require oxygen for survival and maintaining functional properties, whereas many microorganisms, including bacteria, thrive in the absence of oxygen. Despite these differences, human tissues and bacteria coexist in the intestine, often in close proximity to each other. One such example is the dual-oxygen environment in the intestine. The human intestine is normally partitioned oxygenated,²⁵ whereas the different gut bacterial species, which comprise aerobes, facultative anaerobes and obligate anaerobes, are distributed radially from the epithelium to the mucosal layer into the lumen.³ The oxygen gradient is not only vital for regulating transcription factors¹⁵ and synthesis of proteins,²⁹ which help control numerous metabolic and physiologic pathways, but also critical for maintaining intestinal functions.⁴⁵ The dual-oxygen environment and local oxygen gradients from the epithelium to the lumen influence the development, colonization, and function of the mammalian and bacterial cells. For aerobes and facultative anaerobes, which prefer oxygen for respiration, the lack of oxygen influences the metabolic pathways.³⁸ For obligate anaerobes, the existing of oxygen not only suppresses their growth but also upregulates the synthesis of superoxide that damages enzymes.^{11, 12} It is thought that the dysregulation of oxygen gradients could play a critical role in intestinal inflammation and resolution of inflammatory bowel disease (IBD). In fact, it has been observed in IBD patients that there is an increase in the facultative anaerobes and a corresponding decrease in obligate anaerobes,^{6, 26} guiding the hypothesis that the disrupted barrier function in these patients results in a change in the oxygen levels which in turn redistributes the gut microbiota.³²

The intestine is the primary site for the absorption of small molecule drugs. Absorption of the drugs in the intestinal epithelium is facilitated by efflux (MDR1, BCRP) and absorptive (OAT1, and OATPB) transporters.^{35, 37} The regulation of transporter function is regulated by the corresponding transcription factors.⁸ Transcription factors regulate membrane transporters.⁸ For example, MDR1 is related to a number of the same transcription factors, such as the pregnane x receptor (PXR) and the constitutive androstane receptor (CAR).^{17, 18} Interestingly, Shah and colleagues implicated the role of CAR in downregulation of drug-metabolizing enzymes and drug transporters.³³ Oxygen levels influence transcription factors,¹⁵ however, whether oxygenation would regulate microbial signaling to regulate intestinal transporters has not been shown.

Although the discovery that gut bacteria can affect drug metabolism dates back nearly a century, the link between the microbiota and pharmacology remains critically underexplored. Gut bacteria can modify drugs through a direct transformation or an indirect modulation of intestinal absorption. While direct transformation has been studied such as for the metabolism of levodopa³⁹ and digoxin,^{10, 23, 31} indirect effects, which are more likely to affect drug absorption, although more complex to study, remain unexplored.

Interestingly, the intestine expresses many of the same drug metabolizing enzymes that the liver does, and therefore, can have drug metabolism capability similar to that of the liver.⁴³ Of the phase I metabolizing enzymes, the cytochrome P450 family (CYPs) dominates the metabolism of drugs.³⁰ Fifty-seven human CYPs have been identified to be responsible for the biotransformation of most foreign substances, including 70–80% of all drugs in clinical use.¹⁹ Remarkably, almost 70% of human drug metabolism and pharmacokinetics can be attributed to seven major CYPs, which include CYP1A2, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5.^{27, 44} The pharmacokinetics of drugs generally varies significantly among people⁷; it is, therefore, plausible that some of the variability stems from the contributions of the gut bacteria in absorption and metabolism of the drug in the intestine.

There has been a substantial interest in creating experimental models of the intestinal microenvironment.^{22, 42} Studying the direct effects of bacteria, where bacterial strains affect compounds and vice versa, can utilize anaerobic chambers as these studies require a single concentration of oxygen. In contrast, the principles pertaining to the indirect effects of the gut bacteria on intestinal function remain unexplored. A part of the reason for this is the lack of easy-to-use experimental systems that can provide a dual-oxygen environment. For example, while the breakdown of xenobiotics by gut bacterial strains has been reported,⁴⁶ we do not know how bacteria would signal to the intestinal epithelium to affect absorption of these compounds. The current experimental systems, including the microfluidic devices, are complex,⁴ lack perfusion,¹⁶ culture anaerobic bacteria under aerobic conditions,³⁴ or use bulky anaerobic chambers.¹³ Given the importance of oxygen on bacterial function, it is important to study how oxygen mediated changes to bacterial function would affect intestinal function. The major bottleneck to understanding the impact of bacteria on drug absorption and metabolism is the lack of fundamental analytical approaches to quantify the transporters, transcription factors, and enzymes that are part of the important functions of the intestinal epithelium.

In this paper, we report the development of a dual-oxygen environment to study the role of oxygen in regulating the regulation of intestinal enzymes and proteins by gut bacteria. We modeled the transport of oxygen through the different sources to design the microfluidic device. An oxygen-sensitive film was integrated within the device that provided a measure of the oxygen levels. Using two different bacterial strains, E. coli Nissle 1917 (ECN) and Bifidobacterium adolescentis (B. adolescentis, Bifido), we show for the first time how oxygen levels influence the signaling from the bacteria to the intestinal cells. We relate these findings to absorptive and efflux transporters, transcription factors, and enzymes. Our results illustrate the complex role the local environment can play in regulating bacterial signaling of the host function.

Materials and Methods

Device Fabrication

We designed a microfluidic device to create a dual-oxygen tension environment to support the culture of normoxic intestinal cells in conjunction with the anoxic gut bacteria.⁴⁰ The two layers of the device were created using soft lithography on a lithographically patterned SU-8 photoresist mold. Each layer contained a fluidic channel approximately 1 mm wide, 1 cm long, and 100 μm tall. The channel in the top chamber was for culturing the cells and the bacteria whereas that in the bottom chamber was for providing the perfused culture media. SYLGARD 184 Silicone Kit containing a silicone elastomer base and curing agent (Dow Chemical) was used to fabricate the polydimethylsiloxane (PDMS) devices. Approximately 20 g of 9:1 of the base and curing agent was added to the wafer mold to form the top chamber of the device. To determine the oxygen levels in the device, an oxygen sensor was developed and imbedded in device according to our previous work.⁴⁰ The fluorescence signal from the oxygen film indicated lack of oxygen or a hypoxic environment. The thickness of the top layer was designed to limit the diffusive transport of oxygen from the atmosphere to the cells. A 10 µm thick porous polyester membrane (AR Brown-US, Pittsburgh, PA) were cut using a laser cutter (Epilog Laser, Golden, CO). The porous membrane was attached to the PDMS top chamber, the one with oxygen sensors, using SYLGARD 184 Silicone Elastomer as glue, cured overnight in 65 °C oven. Finally, the top chamber with porous membrane attached and the bottom chamber on the microscope slide were bonded together through plasma cleaner (Harrick Plasma).

Cell Culture

Human intestinal epithelial cells Caco-2 (HTB-37) were obtained from ATCC (Manassas, VA) and grown in 1X Eagle’s Modified Eagle Medium (EMEM) (VWR) containing 4.5 g/L glucose and 25 mM HEPES supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% pen-strep (Gibco). The cells were cultured at 37 °C in a humidified incubator with 5% CO₂. Cells were cultured in a 75 cm² cell culture flask until the cells were 80% confluent. For standard tissue culture experiments, the cells were seeded in 48 well Transwell inserts (Corning, NY) and grown to confluence (~ 1 week) at which point there were approximately 0.04 × 10⁶ cells in each well. Before seeding the cells in the devices, the devices were coated with 50 µg/mL Bovine Plasma Fibronectin (Gibco) and then with 100 µg/mL Type I collagen (BD); each for 30 min at 37 °C. The cells were then seeded at a density of 2 × 10⁴ cells/cm². After allowing for the cells to attach, fresh cell culture media was perfused at 5 µL/h on top of the cells for a week at which point there were approximately 0.0215 (± 0.003) $\times$ 10⁶ cells per device. Prior to initiating the coculture with bacteria, the intestinal cells were cultured in antibiotic free medium for 24-h.

Bacteria Culture

In this work, two strains of gut bacteria, facultative aerobic E. coli Nissle 1917 (Ardeypharm GmbH, Germany) and obligate anaerobic B. adolescentis (Manassas, VA) were used. ECN was cultured in LB containing 1% (W/V) Tryptone, 0.5% (W/V) yeast extract, and 1% (W/V) sodium chloride and B. adolescentis was cultured in Reinforced Clostridial Medium.⁴⁰ The standard curve between number of bacteria cells and optical density (OD) was then determined used serious dilution, as described in supplementary document.

Experimental Design

O2C− refers to the condition when the oxygen levels were not controlled. In other words, the mammalian and bacterial strains were cultured under aerobic conditions. O2C+ refers to the condition that epithelial cells were in aerobic conditions whereas anaerobic conditions were created in the bacterial layer. To create this condition, we used the process presented in our recent work.⁴⁰ The O2C+ condition represents the device with oxygen control where the top chamber is hypoxic but intestinal cells are oxygenated. In contrast, the O2C− condition represents the device without oxygen control where the top chamber and cells are both oxygenated. In general, the microfluidic device was developed with a thick PDMS top layer which restricted transport of the atmospheric oxygen into the device. We found that at a certain thickness, oxygen transport into the device could be completely blocked.⁴⁰ For the cultures in the microfluidic device, the different bacterial strains were introduced in the top chamber over the epithelial cells (MOI of 10:1) while antibiotic free media was perfused in the bottom chamber (Fig. 1a). The colon cells were cultured in the top chamber along with the bacteria, ECN and B. adolescentis. As a facultative bacterium, although ECN can survive in both anoxic and normoxic environments, it exhibits a slower growth rate in anoxic conditions. For ECN, the flow rate of the perfused media was 30 µL/h.⁴⁰ Unlike ECN, B. adolescentis is an obligate anaerobe, which grows rapidly in anoxic conditions. To create the anoxic environment for this bacteria, a perfusion rate of 7.2 µL/h was used.⁴⁰ The viability of the intestinal cells when cocultured with bacteria was determined using a live/dead stain. The results are presented in the supplementary material.

Figure 1.

(a) Schematic of the two-chamber microfluidic device with oxygen sensors at three locations in the top chamber. (b) Isometric view of the coculture in the microfluidic device. (c) Schematic of the O2C− and O2C+ experimental conditions: oxygen transport from the atmosphere and bottom chamber media into the top chamber through the top cover and porous membrane. (d) ZO-1 immunostaining in O2C+ and O2C− culture conditions. The insets show the signal from the oxygen sensor. Green: ZO-1; Blue: nuclei; Red: oxygen sensor. (e) Hypoxia in the two different conditions. Green: hypoxia; Blue: nuclei. (f, g). The growth of GFP labeled E.coli Nissle 1917 and (f) anaerobic bacteria B. adolescentis (g) in different culture conditions. Scale bar is 20 µm for all the images.

Characterization of Transporters and Cytochrome P450s

As experimental controls, the different transporters and CYP450s were induced or inhibited (Tables S1, S2) and the relative gene expression was measured and plotted. Detailed procedure is provided in the supplementary file.

Quantitative Polymerase Chain Reaction (qPCR)

To obtain enough RNA sample for the qPCR test, we pooled cells from five devices. The cells were washed with sterile PBS (Sigma) and trypsinized (Gibco). mRNA was isolated using the RNeasy Mini Kit (Qiagen). The RNA concentration of each sample was quantified using NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA). cDNA samples were prepared with the qScript™ cDNA SuperMix (QuantaBio, Beverly, MA) and (QuantaBio, Beverly, MA) using Thermo Cycler C1000 Touch (BIO-RAD, Hercules, CA) following the manufacture’s protocol. qPCR was performed using the Thermo Cycler (BIO-RAD, Hercules, CA). The primers are listed in Table S3. Expression of mucin was determined using qPCR at 0, 4 and 18 h of coculture. Controls were cells without bacteria and bacteria without cells; in each case, the mRNA was extracted and stored at -80C until further use.

Relative Gene Expression Analysis

The relative gene expression was calculated using 2^−∆∆Ct Method.²⁰ In brief, we first calculated the ΔCt of each target genes to the housekeeping gene—the ΔCt value is the difference between Ct of target genes and of housekeeping gene (HKG) of each experimental and control group. By subtracting the ΔCt values of experimental group from the control, ΔΔCt was calculated for each gene which was used to calculate the relative gene expression = 2^{(−∆∆Ct)}. In the bar plot, “log2 relative gene expression” was used to represent gene expression, in which the positive number indicates upregulation and negative number indicates downregulation. The fold change between the relative gene express of O2C+ and O2C− was calculated and plotted as well.

CYP 450 Activity

P450-Glow CYP3A4 Assay with Luciferin-IPA and P450-Glo CYP1A2 were purchased from Promega (Madison, WI) and used according to the manufacturer’s instructions. For the microfluidic devices, the assay reagents were perfused through the devices at 30 µL/h for 1 h in dark. The eluted samples were collected, and luminescence of samples was measured immediately using a plate reader (Spectra Max M2). In all culture conditions, to compare the degree that each bacterium affected CYP activity, each group were normalized to its respective control of only the bacteria condition. As experimental controls, the inducer and inhibitors of the different cytochrome P450 enzymes were used (Table S2).

Immunofluorescence Imaging

The cells were fixed with 3.7% paraformaldehyde (PFA) for 10 min after which they were permeabilized with 0.1% (v/v) Triton X100 (Sigma Aldrich) in phosphate-buffered saline (PBS) (Gibco) for 10 min at room temperature. The cells were washed with PBS three times after which non-specific binding were blocked with 3% (v/v) BSA-PBS at room temperature for 30 min. Then we followed the manufacturer’s protocol to stain each protein. To stain, the mucin proteins and tight junction (ZO-1), 1/200 dilution of the primary antibody [anti-MUC2 (Bioss) and anti-ZO-1 (Bioss)] was added to the cells and incubated at 4 °C overnight. The cells were washed with PBS and the secondary antibody was added and incubated at room temperature for 1 h. A 1/1000 (v/v) dilution of Hoechst (Thermo Fisher) was added for 10 min to stain the nuclei. The cells were washed with PBS and imaged on a Nikon fluorescent microscope (Nikon Ti, Tokyo, Japan). The results were quantified by measuring the fluorescence intensity and normalizing it by the number of cells in each image, using ImageJ. The fluorescence intensity of MUC2 (green) and the number of nuclei (blue) in each image were used to determine the fluorescence intensity per nuclei; data at 4 and 18 h was normalized against the control (0 h). Cells cultured in device with the bacteria co-cultured at 0 h timepoint were used as the control. To image hypoxic cells, we followed the protocol we developed previously.⁴⁰ Briefly, the cells were treated with 200 µM pimonidazole HCl (Hypoxyprobe, Burlington, MA) for 2 h, and then were fixed and stained with FITC-Mab (Hypoxyprobe, Burlington, MA), FITC HRP MAb (Hypoxyprobe, Burlington, MA), and Hoechst 33,342 (Thermo Fisher Scientific, Waltham, MA). The principle is that pimonidazole HCl reduces in hypoxic cells to form stable covalent adducts with thiol groups that can be measured under a fluorescent microscope.

Data Analysis

For each group, the cells from five microfluidic devices were pooled. A minimum of three independent pooled samples (in other words, 15 microfluidic devices) were used in each group. Unless otherwise indicated, data is expressed as means ± SEM. Differences between experimental groups were compared using one-way ANOVA and t test. Statistical analyses were done with Excel. Differences with a p < 0.05 were considered statistically significant.

Results

Microfluidic Device with Dual-Oxygen Environment

We created a two-layer microfluidic device using soft lithography of polydimethylsiloxane (PDMS) (Figs. 1a and 1b). The dual-oxygen environment representing the intestinal-bacterial interface was created by first mathematically modeling the oxygen dynamics in the device. The two sources of oxygen were: the diffusion of environmental (atmospheric) oxygen into the device through the permeable PDMS; and the transport of oxygen dissolved in the media perfusing in the bottom chamber (Fig. 1c). We first passively restricted the diffusive transport of environmental (atmospheric) oxygen into the device by increasing the thickness of the top PDMS layer. Modeling the atmospheric oxygen transport in PDMS using Fick’s first law (Eq. 1), we found that a ~ 5 mm thick PDMS layer would be effective.⁴⁰

$$\frac{\partial c}{\partial t}=D\frac{{\partial }^{2}c}{\partial y^2}$$ 1

Here, $c$ is the oxygen concentration and $D$ is the oxygen diffusion coefficient in PDMS. The concentration of oxygen in the atmosphere was taken to be 21%. Comparisons of this condition (O2C+) (Fig. 1c right) were made with the normal device without the thick top (O2C−) (Fig. 1c left). The second source of oxygen was the culture media. This oxygen would be consumed by the intestinal cells. We seeded intestinal cells on the membrane and cultured them under different media perfusion rates in the bottom chamber. According to our previous work, at very high flow rates (30 μL/h), there was excess oxygen which was measured by the oxygen sensor; while at low flow rates (5 μL/h), when the oxygen levels were not sufficient, the cells consumed all the oxygen and turned hypoxic, and the oxygen sensor registered no oxygen.⁴⁰ Through experimentation, we found an optimum flow rate at which the cells were no hypoxic and the oxygen sensor did not register any oxygen, meaning the layer above the cells was anoxic. We selected this value of media perfusion (7.2 μL/h) as our working flow rate.⁴⁰ Interestingly, we found the O2C+ condition led to a higher ZO-1 expression (Fig. 1d), suggestting the formation of a tighter barrier function. To confirm the hypoxic status of cells in the two different oxygen conditions, we checked the hypoxia by using pimonidazole staining (Fig. 1e). The growth profiles of the two bacterial strains confirmed the appropriate oxygen levels (Figs. 1f and 1g): the growth of ECN normal in the O2C− condition and, as expected, a bit lower in O2C+; in contrast, the growth of B. adolescentis was negligible in the O2C− condition while robust growth was observed in the O2C+ condition.

Regulation of Drug Transporters

We first determined the viability of the cells when co-cultured with bacteria (Fig. S2). For both O2C− and O2C+, the cell viability remained high over 8-h. We then looked at intestinal membrane transporters, which included efflux (MDR1, MRP2 and BCRP) and absorptive (OAT1 and OATPB) transporters. In the O2C− condition, ECN upregulated MDR1and OAT1 while significantly lowered the expression of MRP2 and BCRP, whereas no change was observed for OATPB (Fig. 2a). B. adolescentis upregulated the expression of MDR1 while downregulated the other transporters. For the O2C+ culture condition, ECN increased MRP2 and OATPB expression and significantly decreased the expression of BCRP and OAT1. B. adolescentis only lowered BCRP, OAT1, and OATPB, while has no effects on MDR1 and MRP2 (Fig. 2b). Generally, the efflux transporters were affected more by ECN in O2C− condition, while the absorptive transporters were influenced in the O2C+ condition. The expression of two efflux transporters, MDR1 and MRP2 were significantly influenced by both bacteria in the O2C− condition, whole no significant change was found in O2C+ condition. These results suggest that there is variability in the regulation of the transporters in the intestinal cells by the different bacteria in both culture conditions. Interestingly, oxygen played a significant role in regulating the bacterial influence on the drug transporters. Since B. adolescentis is an obligate anaerobe, as expected, carrying out the coculture in the O2C+ condition (when the bacteria were anoxic) caused significant changes to the intestinal efflux transporters (Fig. 2c). The behavior of ECN was, however, unexpected. Since ECN can survive in both anoxic and normoxic environments, we did not expect many significant uniform changes on switching the culture conditions. To our surprise, we found that changing from normoxic to anoxic conditions led to significant functional differences in the transporters (Fig. 2c).

Figure 2.

(a, b) Log2 Relative expression of transporters in the intestinal cells under O2C− (a) and O2C+ (b) conditions. Cells were cultured for 1 week and before coculturing with different bacterium strains. Expression data of each experimental condition was normalized by its control. The control is cells without bacteria cocultured in each condition, referring as 0. The positive value means upregulation and negative value means downregulation. (c) Relative fold change of the transporter function between the two conditions: O2C+ vs. O2C−. The value larger than 1 means upregulation, otherwise indicates downregulation. *p ≤ 0.05; **p ≤ 0.01. Error bars indicate ± SEM. Sample replicates n ≥ 3.

Regulation of Transcription Factors

Oxygen levels influence transcription factors¹⁵ that in turn regulate the membrane transporters.⁸ To further our understanding of the mechanism for oxygen regulating bacterial signaling to the intestinal cells, we looked at the expression of several relevant transcription factors. Our results show that in the O2C− condition (Fig. 3a), most of the transcription factors were inhibited by ECN and B. adolescentis. Upon changing to the oxygen-controlled condition, there were substantial changes to most of the transcription factors, again indicating the strong influence of oxygen in regulating bacterial effects on the intestinal cells (Fig. 3b). The most dramatic change was observed in the case of VDR (Vitamin D Receptor) and PXR (Pregnane X Receptor) (Fig. 3c), which were upregulated by ECN while switched the culture conditions from O2C− to O2C+, both master regulators of key genes related to transporters and metabolic enzymes.

Figure 3.

(a, b) Log2 Relative expression of transcription factors in the intestinal cells under O2C− (a) and O2C+ (b). Cells were cultured for 1 week and before coculturing with different bacterium strains. Expression data of each experimental condition was normalized by its control. The control is cells without bacteria cocultured in each condition, referring as 0. The positive value means upregulation and negative value means downregulation. (c) Relative change in transcription factors between the two conditions. The value larger than 1 means the expression is upregulation in O2C+ compared to O2C−, otherwise indicates downregulation. *p ≤ 0.05; **p ≤ 0.01. Error bars indicate ± SEM. Sample replicates n ≥ 3.

Regulation of Intestinal Enzymes

Since the human intestinal epithelium expresses cytochrome P450 enzymes, we measured the expression of the important human CYPs under the two conditions. Remarkably, the two bacterial strains caused significant changes to the intestinal cytochromes. CYP3A4, the most abundant cytochrome, was affected the most (Fig. 4). Except for CYP2E1 and CYP2D6, the two bacterial strains appeared to have quite similar effects on the other CYPs. Except for CYP1A2, CYP3A4 and CYP2E1, oxygen conditions did not appear to have a significant effect on the expression of the CYPs (Fig. 4c).

Figure 4.

(a, b) Log2 Relative expression of six important cytochrome P450s in the intestinal cells under O2C− (a) and O2C+ (b). Cells were cultured for 1 week and before coculturing with different bacterium strains. Expression data of each experimental condition was normalized by its control. The control is cells without bacteria cocultured in each condition, referring as 0. The positive value means upregulation and negative value means downregulation. (c) Relative change in important cytochrome P450 enzymes between the two conditions. The value larger than 1 means the expression is upregulation in O2C+ compared to O2C−, otherwise indicates downregulation. *p ≤ 0.05; **p ≤ 0.01. Error bars indicate ± SEM. Sample replicates n ≥ 3.

Influence of Oxygen on Enzymatic Activity

We determined how oxygen and bacteria change the activity of two of the important CYPs, CYP1A2 and CYP3A4. The control group we used here was the cells-only condition in the microfluidic device. All the results were compared against the control group to obtain the fold change. We found that the intestinal cells alone showed relatively modest activities that were slightly upregulated in the O2C+ condition (Fig. 5). We found that both bacterial strains significantly upregulated the activities of the two intestinal CYPs; not surprisingly, bacteria alone had negligible activity towards the CYPs. Again, oxygen control has a significant effect on signaling by ECN. In each case, providing oxygen control further upregulated the activities, reinforcing that bacterial signaling is dependent on oxygen.

Figure 5.

Influence of oxygen levels and bacterial strains on activity of CYP450 3A4 and 1A2. Cells were (a) monocultured or cocultured with (b) E.coli Nissle 1917 or (c) B. adolescentis. Values represent mean ± SEM; (*p < 0.05).

Bacteria Upregulated Mucins

Mucins are important for the intestinal cells as they help in the formation of mucus layers, which for one, provide a physical barrier between the bacteria and the cells as well as provide several other functions.^{14, 28} Previous studies have shown that microfluidic system can help cells to upregulate synthesis of mucins, however, it is not known whether bacteria play a role as well. We first compared the expression of MUC2 across the different culture conditions with or without oxygen control. We found that the O2C+ condition significantly upregulated the expression of MUC2 (Fig. 6a). Interestingly, coculture with bacterial strains in the O2C− condition downregulated MUC2 (Fig. 6b). However, in the O2C+ condition, there was a dramatic increase in MUC2 (Fig. 6c). These results were supported by the immunofluorescence and quantification of MUC2 protein in the cells (Figs. 6d and 6e).

Figure 6.

(a–c) Influence of oxygen levels and bacterial strains on MUC2. The control is monocultured cells. The O2C+ condition represents the device with oxygen control where the top chamber is hypoxic but intestinal cells are oxygenated. In this case, the obligate Bifido showed rapid growth. In contrast, the O2C− condition represents the device without oxygen control where the top chamber and cells are both oxygenated. In the O2C+ means the cells received proper amount of oxygen like in vivo, that is the cells were normoxic but the chamber was anoxic. In this case, while the facultative ECN showed robust growth, Bifido’s growth was inhibited. (d) MUC2 protein at different time points. The cells were cocultured with ECN under O2C+ condition. (e) Quantification of MUC2 from the immunofluorescent image.

Discussion

Oxygen has shaped the life on Earth. Organisms have adapted to survive in different oxygenated conditions. As is often the case, nature finds unique scenarios where organisms with varying requirements coexist, supported by subtle properties of the local environment. One such place is the human intestine where while the epithelium requires oxygen, most of the bacterial constituents thrive in anoxic environments. We created a microfluidic device that recapitulated this dual-oxygen environment of the intestine. The standalone device accounted for balanced oxygen from the different sources to provide the intestinal cells with oxygen, while the anaerobes without oxygen. Simultaneously, by utilizing the in-situ oxygen sensors, the oxygen consumption by the epithelial layer was balanced by the perfused media, which resulted in the surrounding microenvironment for the bacterial cells becoming anoxic. This was verified by measuring cellular hypoxia (or the lack thereof) and bacterial cell proliferation under different oxygen conditions.

We selected to investigate two species of gut bacteria, E. coli Nissle 1917 and B. adolescentis. E. coli Nissle 1918 (ECN), a facultative anaerobe, is a Gram-negative probiotic bacteria.^{24, 36} B. adolescentis (Bifido), an obligate anaerobe and Gram-positive, has been shown to positively influence the treatment of gastrointestinal infections and disorders.^{1, 2} In the oxygen-controlled condition (O2C+), the epithelial cells were in the normoxic environment and the bacteria in anoxic condition whereas for the case without oxygen control (O2C−), both strains were in the normoxic condition. We determined how oxygen regulated bacterial signaling to the epithelium. In particular, the absorptive and efflux transporters and transcription factors showed significant variation between the O2C+ and O2C− cases. Even the specific activity of important intestinal enzymes was significantly different between the two cases. Remarkably, these effects were often comparable to the results obtained from control experiments using standard inhibitors and inducers (Figs. S3, S4). We further found the dual-oxygen environment upregulated the expression of markers related to mucin as well as the production of mucins. Intriguingly, even for the facultative bacteria, ECN, the change from aerobic to anaerobic environment led to significant changes in the signaling to the intestinal cells, possibly due to the change from aerobic to anaerobic respiration.

The results prompted development of a proposed mechanism in which bacterial metabolites would likely utilize the absorptive transporters and signal through the nuclear pore complex to regulate the transcription factors which in turn would regulate the activity of the cytochrome P450 enzymes (Fig. S5). This hypothesis is supported by our observation that bacterial metabolites regulate P450 enzymes (Fig. S6). In addition, previous reports have indicated that metabolites produced by bacteria can regulate transcription factors (TFs)^{5, 21}; our work provides insights into oxygen’s regulation of the transporters through bacterial signaling and the downstream effects on transcription factors and enzymes. Future studies could investigate the role of oxygen in influencing pathogenic bacteria and their effect on barrier function.

Our work integrates cell and molecular biology with bioengineering and presents a novel strategy to engineer an accessible experimental system to provide tailored oxygenated environments. The work could provide new avenues for intestine-microbiome signaling and intestinal tissue engineering, as well as a novel perspective on the indirect effects of gut bacteria on tissues including tumors. Lastly, these new insights into transporters and enzymes could be combined with direct metabolism of drugs by gut bacteria⁴⁶ to develop improved pharmacokinetic models.^{9, 41}

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

O2C−

Culture condition with no control of oxygen

O2C+

Culture condition with oxygen control

ECN

E. coli Nissle 1917

Bifido

Bifidobacterium adolescentis (B. adolescentis)

MOI

Multiplicity of Infection of bacteria to cells

CYP

Cytochrome P450

MUC2

Mucin 2

Author Contributions

Participated in research design: CW, AB. Conducted experiments: CW, AC, JB, DM, AAJ, AN. Contributed new reagents or analytic tools: CW, AB. Performed data analysis: CW, JB, AAJ, AB. Performed data visualization: CW, JB, AC, DM. Wrote or contributed to the writing of the manuscript: CW, AC, JB, DM, AB.

Funding

This work was partly funded by the Nayar Prize II, Alternatives Research and Development Foundation (ARDF) and student scholarships from the Armor College of Engineering.

Data Availability

The data and material are listed in supplementary documents.

Code Availability

Not applicable.

Conflict of interest

The authors Chengyao Wang, Andrea Cancino, Jasmine Baste, Daniel Marten, Advait Anil Joshi, Amreen Nasreen, Abhinav Bhushan have declared that no conflict of interest exists.

Human Studies

No human studies were carried out by the authors for this article.

Animal Studies

No animal studies were carried out by the authors for this article.