Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

Abstract

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers. We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Figure 1:. The ileal loop model.

(A) Schematic overview of the ileal loop model. Median laparotomy is performed on mice under anesthesia and placed on a temperature-controlled surgery board. (B) Exteriorization of the caecum (*), ileum and mesentery. Two adequate sites for ligation are identified (1,2). (C) Isolate a segment of 4 cm length: the first ligature (1) is placed close to the ileo-caecal junction and a second ligature (2) is placed 4 cm away from the first ligature. (D) Two small incisions are made in the mesentery (1, 2) to create a 4 cm length ileal loop. After removal of luminal content and ligation of cut-ends, reagents such as fluorescent markers and chemoattractants can be injected into the lumen. The ileal loop is well vascularized (black arrowheads).

Figure 2:. The proximal colon loop model.

(A) Schematic overview of the pcLoop model. Median laparotomy is performed on mice under anesthesia placed on a temperature-controlled surgery board. (B) Exteriorization of the caecum (*), proximal colon, mesocolon and ileum. Two adequate sites for ligation are identified (1,2). (C) The first ligature (1) is placed close to the caecum and a second ligature (2) is placed 2 cm more distal from the first ligature. (D) The pcLoop is exteriorized, cleaned of luminal content and inflated with reagents such as fluorescent markers and chemoattractants. The pcLoop is a well-vascularized 2 cm segment of proximal colon (black arrowheads indicate blood supply).

Figure 3:. JAM-A regulates intestinal permeability in vivo.

(A) JAM-A deficiency (Jam-a^−/−) led to increased colonic permeability to 4 kDa FITC-dextran. Jam-a^−/− (13x animals; black dots) were compared with Jam-a^+/+ controls (12x animals; white dots). 4 kDa FITC-dextran (1 mg/mL) in HBSS was injected into the pcLoop lumen. Fluorescence was measured in blood serum after a 120 min incubation period. Data are expressed as means ± SEM; n = 3 independent experiments. ****P < 0.0001; Mann-Whitney U test. (B) Increased colonic permeability to 4 kDa FITC-dextran in Villin-cre; Jam-a^fl/fl (18x animals, black dots) compared to controls (Jam-a^fl/fl, 12x animals, white dots). Data are means ± SEM; n = 4 independent experiments. ****P < 0.0001; Mann-Whitney U test. This figure has been modified from Flemming S, Luissint AC et al..

Figure 4:. JAM-A promotes LTB₄-dependent recruitment of PMN into the lumen of the pcLoop.

(A) Gating strategy to quantify PMN (CD45⁺, CD11b⁺, and Ly-6G/Gr1⁺ cells) in luminal content by flow cytometry with fluorescent counting beads. Leukocytes from blood samples were used as a positive control for the gating strategy. (B) Number of PMN recruited into the pcLoop lumen after cytokine (TNFα+IFNγ, 100ng each) treatment (10x animals; white dots) or after a combination of cytokines and 1 nM LTB₄ (10x animals; black dots). Black squares represent the number of PMN at baseline as assessed in an intact colonic segment identical in length to the pcLoop that was not subjected to any surgery or treatment with proinflammatory cytokines and LTB₄ (9x animals). Data are the mean ± SEM (n = 3 independent experiments), Kruskal-Wallis test with Dunn's multiple comparison test. *P < 0.05, ****P < 0.0001. (C) Immunohistochemical staining of PMN (anti-Ly6G/Gr1 antibody) in the epithelium of the pcLoop after treatment with cytokines alone (left panel, TNFα+IFNγ) or a combination of cytokines and LTB₄ (right panel). The number of PMN recruited in the pcLoop is increased in the presence of LTB₄ (black arrowheads). Scale bar: 100 μm. (D) Number of PMN recruited in the pcLoop lumen in Villin-cre; Jam-a^fl/fl mice (11x animals; black dots) compared to Jam-a^fl/fl mice (10x animals; white dots) in response to 1 nM LTB₄. Data are means ± SEM; n = 3 independent experiments. *P < 0.05; 2-tailed Student's t test. This figure has been modified from Flemming S, Luissint AC et al..