Modeling CDI using a GOC-based system.

(A) Schematic of the GOC system design. (B) Schematic of the GOC-based in vitro CDI model. (C) Confocal micrographs of the intestinal epithelium cultured for 24 hours in the GOC. The upper microchannel was perfused with normoxic (+ Oxygen) or anaerobic (-Oxygen) culture medium. Under anaerobic condition, the intestinal epithelium was either infected (+ CDI) or not infected (– CDI) by C. difficile. Tight junctions were immunostained for ZO-1 (red), the mucus layer was labeled with wheat germ agglutinin (WGA, green), and nuclei were stained with DAPI (blue). Scale bar: 50 µm. (D-F) Quantitative analysis of (D) C. difficile cell counts in the GOCs at 0, 3 and 24 hours post-infection; (E) the average ZO-1 fluorescence intensity of intestinal epithelium in GOCs; and (F) secretion of pro-inflammatory cytokines IL-8 and IFN-γ in GOCs with or without C. difficile infection. Data are presented as mean ± SD (n = 4 independent replicates). Significance was determined by unpaired t-test (* P < 0.05, ** P < 0.01, *** P < 0.001). Figure 1—figure supplement 1. Characterization of the GOC device and on-chip cell culture.

Prevention effect of LAX4 against CDI in the GOC-based in vitro system.

(A) Schematic of experimental timeline for LXA4 administration in the control (CDI), prevention and therapy groups. (B) Confocal micrographs of the intestinal epithelium in GOCs subjected to prevention and therapy groups. Tight junctions were immunostained for ZO-1 (red) and nuclei were stained with DAPI (blue). Scale bar, 25 µm. (C-F) Quantitative analysis of (C) the average ZO-1 fluorescence intensity in the intestinal epithelium in CDI control, LXA4 prevention and therapy groups (n = 4 independent replicates); (D) epithelial cell viability with or without LXA4 prevention (n = 3 independent replicates); (E) secretion of the pro-inflammatory cytokines IL-8 and IFN-γ with or without LXA4 prevention (n = 3 independent replicates); and (F) C. difficile cell counts in GOCs with or without LXA4 prevention (n = 9 independent replicates). (G) Differentially expressed genes of intestinal epithelium cells from GOCs with or without LAX4 prevention. Genes with |log2(fold change)| >= 1 and adjusted P value < 0.05 were considered significantly differentially expressed. (H) GO analysis of up- and down-regulated genes in the LXA4 prevention group. The top five enriched terms are displayed. Data in (C–F) are presented as mean ± SD; significance was determined by unpaired t-test (* P < 0.05, ** P < 0.01, *** P < 0.001). Figure 2—figure supplement 1. Cytotoxicity assessment of LXA4 and its impact on the transcriptomic landscape.

Protective effect of LXA4 against CDI in a murine model.

(A) Experimental scheme for establishing the CDI model in mice, including the timeline of LXA4 administration. (B) Body weight changes in the CDI control and LXA4 prevention groups. (C) C. difficile bacterial loads in feces at day 3 post-infection with or without LXA4 prevention. (D) Histopathological scores of ileum, cecum, and colon from H&E-stained sections with or without LXA4 prevention. (E) Representative H&E-stained images of ileum, cecum, and colon tissues with or without LXA4 prevention. Arrows indicate key pathological features: immune cell infiltration (blue), villous epithelium loss (yellow), edema (purple), and crypt loss (green). Scale bar: 50 µm. Data in (B–D) are presented as mean ± SD (n = 5 mice per group). Statistical significance was determined by unpaired t-test (* P < 0.05, ** P < 0.01).

Adjunctive effect of LXA4 with vancomycin for the treatment of CDI.

(A) Experimental scheme for the combined administration of LXA4 and vancomycin. (B) Confocal micrographs of the intestinal epithelium in GOCs under different conditions: CDI control, vancomycin monotherapy (VAN), LXA4 combined with full-dose vancomycin (LXA4 + 1× VAN), and LXA4 combined with half-dose vancomycin (LXA4 + 0.5× VAN). Tight junctions are immunostained for ZO-1 (red) and nuclei are stained with DAPI (blue). Scale bar: 50 µm. (C-D) Quantitative analysis of (C) the average ZO-1 fluorescence intensity and (D) the secretion of pro-inflammatory cytokines (IL-8 and IFN-γ) in GOCs from the CDI control, VAN, and LXA4 + 1× VAN groups. (E-F) Quantitative analysis of (E) the average ZO-1 fluorescence intensity and (F) the secretion of IL-8 and IFN-γ in GOCs from the CDI control, VAN, LXA4 + 1× VAN, and LXA4 + 0.5× VAN groups. Data in (C-F) are presented as mean ± SD (n = 4 independent replicates). Statistical significance was determined by unpaired t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Characterization of the GOC device and on-chip cell culture.

(A) Photograph of the fabricated GOC device (left) and a micrograph of its porous membrane (right). Scale bar: 20 µm. (B) Brightfield images depicted the endothelial cell layer formation procedure in the GOC. Scale bar: 100 µm. (C) Fluorescence images from a Live/Dead assay under normoxic and anaerobic conditions. Live and dead cells were stained green and red, respectively. Scale bar: 100 µm.

Cytotoxicity assessment of LXA4 and its impact on the transcriptomic landscape.

(A) Confocal immunofluorescence images of ZO-1 (red) in Caco-2 cells cultured in the presence or absence of 200 nM LXA4. Scale bar: 50 µm. The quantitative analysis (right) shows no significant change in ZO-1 expression, indicating that LXA4 didn’t cause observable toxicity to epithelial cells in the GOC. Data are mean ± SD (n = 6 independent replicates). (B) Heatmap of RNA-sequencing data illustrating distinct gene expression profiles between the CDI and LXA4 prevention groups. Color scale corresponds to row-scaled Z-score (red: upregulated; blue: downregulated).