Development of an Inflammation-Triggered In Vitro "Leaky Gut" Model Using Caco-2/HT29-MTX-E12 Combined with Macrophage-like THP-1 Cells or Primary Human-Derived Macrophages

Abstract

The "leaky gut" syndrome describes a damaged (leaky) intestinal mucosa and is considered a serious contributor to numerous chronic diseases. Chronic inflammatory bowel diseases (IBD) are particularly associated with the "leaky gut" syndrome, but also allergies, autoimmune diseases or neurological disorders. We developed a complex in vitro inflammation-triggered triple-culture model using 21-day-differentiated human intestinal Caco-2 epithelial cells and HT29-MTX-E12 mucus-producing goblet cells (90:10 ratio) in close contact with differentiated human macrophage-like THP-1 cells or primary monocyte-derived macrophages from human peripheral blood. Upon an inflammatory stimulus, the characteristics of a "leaky gut" became evident: a significant loss of intestinal cell integrity in terms of decreased transepithelial/transendothelial electrical resistance (TEER), as well as a loss of tight junction proteins. The cell permeability for FITC-dextran 4 kDa was then increased, and key pro-inflammatory cytokines, including TNF-alpha and IL-6, were substantially released. Whereas in the M1 macrophage-like THP-1 co-culture model, we could not detect the release of IL-23, which plays a crucial regulatory role in IBD, this cytokine was clearly detected when using primary human M1 macrophages instead. In conclusion, we provide an advanced human in vitro model that could be useful for screening and evaluating therapeutic drugs for IBD treatment, including potential IL-23 inhibitors.

Keywords: Caco-2; FITC-dextran 4 kDa (FD4); HT29-MTX-E12; TEER; THP-1; inflammation; inflammatory bowel disease (IBD); leaky gut; monocyte-derived macrophage; pro-inflammatory cytokines; triple-culture.

Figure 1

Schematic diagram of the inflammation-triggered, triple-culture in vitro “leaky gut” model. 19-day-differentiated Caco-2/HT29-MTX-E12 co-cultures were either rested in fresh medium (control), or primed for 24 h with IFN-γ. On the following day, a custom-designed three dimension (3D)-printed cap was carefully placed into the insert of the cultures to confine the medium, before placing the insert upside down in a Petri dish. After that, phorbol 12-myristate 13-acetate (PMA)-differentiated macrophage-like THP-1 cells, or primary monocyte-derived macrophages were transferred on the bottom side of the inverted inserts for 1.5 h, before flipping it back to the regular orientation. Then, for generating the inflammation-mediated “leaky gut” condition, macrophages were activated for 24 h by adding a combination of LPS and IFN-γ. At the same time, for the control model, macrophages were rested in medium for this time.

Figure 2

Custom-designed 3D-printed cap. The design sketches of the cap are: top view (A), front view (B), and 3D view (C). The dimensions of this cap (D) were determined to fit into the insert perfectly to safely confine the medium within the insert during the macrophage adherence procedure (E).

Figure 3

Characterization of the Caco-2/HT29-MTX-E12 (90:10 ratio) co-culture during 21 days of cultivation. (A) Transepithelial-transendothelial electrical resistance (TEER) values of Caco-2, HT29-MTX-E12, and Caco-2/HT29-MTX-E12 (90:10) cell cultures. Values are shown as mean ± standard deviation (SD) (n ≥ 3). (B) Representative microscopy images of cell layers were obtained before and after staining with Alcian blue for mucus production (evident by blue color and black arrows). Scale bar = 100 µm.

Figure 4

IFN-γ priming on the Caco-2/HT29-MTX-E12 co-culture. After 24 h incubation with 50 ng/mL IFN-γ, TEER (A) and permeability value (P_app) (B) of Caco-2/HT29-MTX-E12 co-cultures were measured. TEER values were expressed as a percentage of the initial TEER value (100%). Permeability was expressed as fold change of the untreated control (SC) used as a reference. Bars are the means ± SD (n ≥ 3). * p < 0.05, or *** p < 0.001 were considered significantly different versus SC.

Figure 5

Characterization of the in vitro “leaky gut” model. Intestinal barrier function of the triple cell culture was assessed by (A) TEER and (B) permeability value (P_app) after 6 h and 24 h incubation. The secretion of the key pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the basolateral compartment were quantified after (C) 6 h and (D) 24 h. The release of Occludin, Tight Junction Protein-1 (TJP1), and Zonulin in the apical, and basolateral compartment were measured after (E) 6 h and (F) 24 h. Data are given as mean ± SD (n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 as compared to the control model.

Figure 6

Comparison of different modifications on the “leaky gut” model using macrophage-like THP-1 cells (“−“: non-treatment, “+”: treatment). Intestinal barrier function was assessed by (A) TEER and (B) permeability value (P_app) as compared to the control model. (C) The secretion of key pro-inflammatory cytokines IL-1β, IL-6, IL-23, and TNF-α was quantified in the basolateral compartment of the models after 24 h cultivation. Bars are mean ± SD (n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 as compared to the control model.

Figure 7

Comparison of the modifications on the “leaky gut” model using primary human-derived macrophages (“−”: non-treatment, “+”: treatment). Intestinal barrier function in comparison to the control model was assessed after 24 h by (A) TEER and (B) permeability values. (C) The secretion of key pro-inflammatory cytokines IL-6, IL-23, and TNF-α in the basolateral compartment of control and “leaky gut” models were quantified after 24 h. Bars are mean ± SD (n ≥ 3). * p < 0.05, ** p < 0.01, **** p < 0.0001 as compared to the control model.