Intestinal barrier dysfunction orchestrates the onset of inflammatory host-microbiome cross-talk in a human gut inflammation-on-a-chip

Abstract

The initiation of intestinal inflammation involves complex intercellular cross-talk of inflammatory cells, including the epithelial and immune cells, and the gut microbiome. This multicellular complexity has hampered the identification of the trigger that orchestrates the onset of intestinal inflammation. To identify the initiator of inflammatory host-microbiome cross-talk, we leveraged a pathomimetic "gut inflammation-on-a-chip" undergoing physiological flow and motions that recapitulates the pathophysiology of dextran sodium sulfate (DSS)-induced inflammation in murine models. DSS treatment significantly impaired, without cytotoxic damage, epithelial barrier integrity, villous microarchitecture, and mucus production, which were rapidly recovered after cessation of DSS treatment. We found that the direct contact of DSS-sensitized epithelium and immune cells elevates oxidative stress, in which the luminal microbial stimulation elicited the production of inflammatory cytokines and immune cell recruitment. In contrast, an intact intestinal barrier successfully suppressed oxidative stress and inflammatory cytokine production against the physiological level of lipopolysaccharide or nonpathogenic Escherichia coli in the presence of immune elements. Probiotic treatment effectively reduced the oxidative stress, but it failed to ameliorate the epithelial barrier dysfunction and proinflammatory response when the probiotic administration happened after the DSS-induced barrier disruption. Maintenance of epithelial barrier function was necessary and sufficient to control the physiological oxidative stress and proinflammatory cascades, suggesting that "good fences make good neighbors." Thus, the modular gut inflammation-on-a-chip identifies the mechanistic contribution of barrier dysfunction mediated by intercellular host-microbiome cross-talk to the onset of intestinal inflammation.

Keywords: barrier function; disease model; gut inflammation-on-a-chip; inflammation; microbiome.

Fig. 1.

Administration of DSS specifically induces epithelial barrier dysfunction in a gut inflammation-on-a-chip. (A) An experimental design that describes the microenvironment of the human intestine undergoing DSS-mediated epithelial barrier dysfunction and subsequent transmigration of gut bacteria and immune cells (+DSS) compared with the normal healthy condition (−DSS). AP, apical; BL, basolateral. (B) Morphology of the villus epithelium visualized by the differential interference contrast (DIC) (gray, top views) and immunofluorescence (IF) microscopy (colored, side views) at 0 and 48 h after DSS treatment (2%, wt/vol). A white dashed line indicates the location of a porous basement membrane. A white dotted line indicates the contour of villous microarchitecture. The height of villi was measured by analyzing IF micrographs of the vertical cross-cut view (n = 7). (C) Intestinal barrier function of the control intestinal villi (circle; n = 10) compared with the villi challenged to 2% (wt/vol) DSS (square; n = 10) quantitated by TEER. (D) Apparent permeability of a paracellular marker (FITC-dextran; 20 kDa) through the villous epithelial layer in the absence (Control) or the presence of DSS (DSS) (n = 3). Permeability values of the Control was below the detection range. Localization of E-cadherin adherens junction (E) and ZO-1 tight junction proteins (F) (Upper) and the line scan of corresponding IF images (Lower) in the absence (Control) or the presence of DSS treatment (+DSS) for 48 h. (G) Visualization of the mucus layer via IF staining with Alexa Fluor 633-conjugated WGA at 48 h after DSS treatment (Upper) and its quantification (Lower) (n = 8). (H) Epithelial cytotoxicity in response to the DSS treatment quantitated by an lactic acid dehydrogenase (LDH) assay. The culture medium collected from the AP and BL microchannels in the gut inflammation-on-a-chip challenged to DSS for 48 h did not show any LDH release. Cell lysate of Caco-2 cells grown on a chip was used as a positive control (n = 10). (Scale bars, 50 μm.) *P < 0.05, **P < 0.001, ***P < 0.0001.

Fig. 2.

Recovery of barrier dysfunction after the cessation of DSS treatment. (A) Restoration of the barrier function in response to the DSS treatment (+DSS) and its cessation (−DSS) measured by TEER (n = 2). Intestinal villi were challenged to DSS (2%, wt/vol) for 2 d and then further cultured without DSS treatment for an additional 5 d. (B) Phase contrast images showing intestinal villous microstructure before (Control) and after the DSS treatment for 48 h (+DSS). Microengineered villus structure was recovered when DSS was ceased for an additional 48 h (DSS ceased), and the quantification of the height of villi (Right, n = 5). (C) Visualization of the mucus production highlighted by the Alexa Fluor 633-conjugated WGA. A 3D reconstruction of Z-stacked images is shown. Quantification of the averaged intensity of each 3D reconstructed image was performed using ImageJ (Right, n = 2). (D) Localization of tight junction protein ZO-1 (Left) and a line scan snapshot in each experimental group (Right). (Scale bars, 50 μm.) NS, not significant. **P < 0.001, ***P < 0.0001.

Fig. 3.

Direct cross-talk of the barrier-compromised epithelium and immune components induces cytoplasmic oxidative stress. (A) Production of ROS visualized by the fluorescence probe (CellROX) that detects free radicals in the cytoplasm at 24 h after introduction of PBMCs. Images were analyzed to quantify the fluorescence intensity using ImageJ (n = 10). (B) Expression profile of Nrf2 gene in PBMC after the epithelial–immune cross-talk was performed in the presence of DSS alone or DSS and LPS together in the gut inflammation-on-a-chip. Cells were harvested at 24 h after PBMCs were introduced into the basolateral microchannel (n = 2). (C) Direct contact effect between PBMC and DSS-sensitized epithelium was assessed in the Transwell insert with either 0.4- or 8.0-μm pores, respectively. Phase contrast images of a polyester track-etched (PETE) membrane in each Transwell insert were provided below the schematic. White holes show the pores in each membrane. Quantification of the oxidative stress of Caco-2 cells grown on each porous insert (0.4 vs. 8.0 μm) that underwent costimulation with DSS and PBMC (2 × 10⁴ cells per insert) in the AP and BL compartments, respectively (n = 89). (Scale bars, 50 μm.) NS, not significant. **P < 0.001, ***P < 0.0001.

Fig. 4.

Pathophysiological cross-talk between the barrier-impaired villus epithelium, gut microbiome or microbial LPS, and immune components exerts inflammatory responses. (A) Directional secretion of proinflammatory cytokines after costimulation of LPS (10 ng/mL) or E. coli cells (1 × 10⁶ cfu/mL; MOI, 0.25) with PBMC (4 × 10⁶ cells per milliliter) for 24 h in the presence or the absence of DSS treatment (n = 4). Statistical analysis was performed compared with the control group. (B) A cross-sectional view of the villus morphology (Left) and recruited immune cells (Right Inset) in response to apical DSS treatment. The villus epithelium was challenged to LPS (10 ng/mL) in the presence or the absence of DSS for 48 h, and then PBMC (4 × 10⁶ cells per milliliter) was added to the BL side for 24 h. Villi were visualized by staining the plasma membrane of epithelial cells (gray) and PBMC (green). White dotted lines and dashed lines represent the contour of the villus epithelium and the location of porous membranes, respectively. (C) Quantification of the number of recruited PBMCs on the basolateral surface of the villus epithelium. In the LPS panel, “+” indicates the physiological concentration of LPS (10 ng/mL), whereas “++” represents the extremely high concentration of LPS (5 μg/mL) (n = 8). (D) Height of villi in response to the DSS treatment in the presence of LPS at the physiological level (10 ng/mL). (E) Effect of DSS-mediated barrier disruption of villus epithelium in response to LPS at physiological concentration (10 ng/mL). Intestinal barrier function displayed by the normalized TEER was declining in the presence of both DSS and LPS (open square), whereas the presence of LPS alone did not compromise any barrier function (filled square). All of the experimental groups include PBMCs (4 × 10⁶ cells per milliliter). (Scale bars, 50 μm.) NS, not significant. *P < 0.05, **P < 0.001, ***P < 0.0001.

Fig. 5.

Barrier dysfunction alters the probiotic efficacy. (A) Effect of the administration of probiotic VSL#3 and the DSS-mediated barrier dysfunction on the intestinal barrier function. VSL#3 (1 × 10⁷ cfu/mL; MOI, 2.5) was treated at 0 h in +VSL#3 (filled circle). VSL#3 cells were inoculated to intestinal epithelium before (+DSS, Pre-VSL#3, filled square; preculture of VSL#3 for 24 h) or subsequent to (+DSS, Post-VSL#3, open square; postculture of VSL#3 for 24 h) the DSS treatment. Control (open circle); +DSS without VSL#3 (inverted triangle). (B) Visualization of the localized tight junction ZO-1 (ZO-1) and the mucus production (WGA), and the growth profile of VSL#3 cells collected from the effluent in each outlet (upper and lower microchannels) (Effluent culture) in Pre-VSL#3 vs. Post-VSL#3 in the presence of DSS (2%, wt/vol). (C) Assessment of the oxidative stress visualized by the fluorescence probe before (+DSS, Pre-VSL#3) or after the coculture of VSL#3 bacteria (+DSS, Post-VSL#3) in the presence of DSS and PBMC. Quantification of generated ROS using ImageJ (n = 10). (D) Polarized secretion of the proinflammatory cytokines after 24 h since the PBMC coculture (n = 4). Statistical analysis was performed comparing pre- and post-VSL#3 treatment. All experimental groups include PBMC (4 × 10⁶ cells per milliliter). (Scale bar, 50 μm.) NS, not significant. *P < 0.05, **P < 0.001, ***P < 0.0001.