Regenerative Effects of Live Probiotics on Mucosal Barrier and Inflammation in a Microengineered Pathomimetic Leaky Gut Chip

Introduction:: “Leaky gut” refers to a clinical condition where the epithelial permeability is abnormally elevated¹. Although probiotics have been shown to improve intestinal inflammation and remedy epithelial barrier dysfunction, their effectiveness and reliability have been a matter of controversy². The lack of appropriate surrogate models has hindered the experimental investigation and validation of probiotic therapeutic intervention, which should demonstrate in vivo-relevant cytodifferentiation, morphogenesis, and long-term host-probiotics interaction. To address this issue, we utilized a biomimetic gut-on-a-chip (Gut Chip) microphysiological system^{3, 4}. This system consists of two microfluidic channels separated by a porous membrane, which mimic the lumen and vasculature of the intestinal tissue microenvironment under fluid flow. Additionally, it undergoes mechanical deformation to mimic the peristalsis-like motion of the intestine. The Gut Chip effectively reproduces physiodynamic intestinal barrier function, mucosal microenvironment, three-dimensional (3D) tissue structure, and long-term host-microbe co-cultures in a living human gut. Using a disease modeling approach^{5, 6}, we established a pathomimetic Leaky Gut Chip to simulate increased epithelial permeability and intestinal inflammation and assess probiotic interventions as live biotherapeutics. Our research also emphasizes the significance of utilizing a stretchable, flow cell system like our Gut Chip for conducting steady-state co-culture experiments over extended periods. This study, therefore, could unveil the underexplored therapeutic potential of probiotics in treating abnormal gastrointestinal conditions such as “Leaky gut.”


Materials and Methods:: We fabricated gut-on-a-chip microfluidic devices from polydimethylsiloxane (PDMS) using a photolithography and soft-lithography protocol³. We then created a gut chip model by introducing Caco-2 cells (final density, 5 × 10⁶ cells/mL) and culturing them under a fluidic flow of 50 μL/h (corresponding shear stress, ~ 0.003 dyne/cm²) and stretching motions (10% in cell strain, 0.15 Hz in frequency) in both the apical and basolateral channels at 37^oC and 5% CO₂. Upon forming the 3D microstructure of the intestinal cells^{4, 7}, we induced intestinal barrier impairment and mucosal inflammation by introducing a cocktail of pro-inflammatory cytokines, TNF-α and IL-1β (100 ng/mL), to both channels. After 3 days, we ceased the cytokine challenge and administered either Lactobacillus rhamnosus GG (LGG, ATCC 53103) or VSL#3, a mixture of 8 probiotic species, for 3 additional days under an anoxic-oxic interface (AOI) to support anaerobic respiration of the bacteria. We have previously shown that LGG³ or VSL#3⁶ administration could enhance the barrier function of Caco-2 epithelium without a cytokine challenge. We evaluated the intestinal permeability by measuring the apparent permeability coefficient (P_app) using fluorescein (376.27 Da) or transepithelial electrical resistance (TEER) using Ag/AgCl^- electrodes. We also performed immunofluorescence (IF) imaging by confocal microscopy to evaluate the expression of tight junction proteins ZO-1, mucin protein MUC-2, and inflammatory proteins, NFκB p65, pSTAT3, and MYD88, in response to the cytokine treatment and the probiotic intervention under a defined experimental timeline (Fig. 1).


Results, Conclusions, and Discussions:: Results and Discussions: The Caco-2 epithelium successfully underwent 3D morphogenesis in the Gut Chip, which was significantly abrogated following the introduction of cytokines (Fig. 2a). The cytokine insult resulted in a disrupted junctional protein ZO-1 (Fig. 2b) and increased permeability of the intestinal barrier quantified as TEER and P_app (Fig. 2c-d). Notably, discontinuing the cytokine treatment alone was insufficient to improve the transepithelial permeability or the expression of tight junction proteins (Fig. 2c). Conversely, the administration of either LGG or VSL#3 for 3 days (Fig 3a) was effective in restoring the permeability (Fig. 3b). Additionally, confocal IF imaging revealed an increase in the organized expression of tight junction ZO-1 and occludin as well as the generation of gel-forming mucin proteins MUC2 (Fig. 3d), indicating the ability to enhance the gut barrier of probiotics. Furthermore, the co-culture reduced the inflammation caused by the cytokine challenge by significantly suppressing the expression of inflammatory markers NFκB-p65, pSTAT3, and MYD88 (Fig. 3d), demonstrating the anti-inflammatory benefits of probiotics. These findings underscore the potential of using probiotics as live biotherapeutics to alleviate mucosal barrier disruption and exert an anti-inflammatory effect in the leaky human gut.

Conclusions: We demonstrated the regenerative ability of probiotics LGG and VSL#3 in treating the leaky gut condition. Using a gut disease-on-a-chip approach, we highlighted an unprecedented proof-of-principle model that can be offered for future translational research, specifically in the testing of first- and next-generation probiotics, synbiotic intervention, and the human gut microbiome. To address the issues of interpersonal variability and reproducibility in the future, we can employ advanced culture techniques like patient-derived organoids that capture the individual's genetic background and reflect their distinct disease status. This approach enables us to model various intestinal diseases, including ulcerative colitis, Crohn’s Disease, or colorectal cancer, using the patient's tissues that carry their genetic makeup and pathological signature in a microphysiological environment. Ultimately, we aim to leverage patient-specific facilitate clinical trials and bridge the bench to the bedside by contributing to the field of personalized medicine.


Acknowledgements (Optional): : This work was supported in part by the National Institutes of Health (K00CA245801, K01OD030515, and R21CA236690), the CJ Cheiljedang, the Leona M. & Harry B. Helmsley Charitable Trust, and Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (No. 2021R1A6A3A14045370).


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