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. 2016 Aug;64(8):459-69.
doi: 10.1369/0022155416656349. Epub 2016 Jul 1.

Toxoplasma gondii Infection Promotes Epithelial Barrier Dysfunction of Caco-2 Cells

Affiliations

Toxoplasma gondii Infection Promotes Epithelial Barrier Dysfunction of Caco-2 Cells

Marisol Pallete Briceño et al. J Histochem Cytochem. 2016 Aug.

Abstract

After oral infection, Toxoplasma gondii invades intestinal cells, induces breakdown of intestinal physiology and barrier functions, and causes intestinal pathology in some animal species. Although parasites' invasion into host cells is a known phenomenon, the effects of T. gondii infection in the intestinal barrier are still not well established. To evaluate morphological and physiological modifications on the colorectal adenocarcinoma-derived Caco-2 cell line during T. gondii infection, microvilli, tight junction integrity, and transepithelial electrical resistance (TEER) were investigated under infection. It was observed that the dextran uptake (endocytosis) and distribution were smaller in infected than in noninfected Caco-2 cells. The infection leads to the partial loss of microvilli at the cell surface. Claudin-1, zonula occludens-1 (ZO-1), and occludin expressions were colocalized by immunofluorescence and presented discontinuous net patterns in infected cells. Immunoblotting analysis at 24 hr postinfection revealed decreasing expression of occludin and ZO-1 proteins, whereas claudin-1 presented similar expression level compared with noninfected cells. T. gondii decreased TEER in Caco-2 cells 24 hr after infection. Our results suggest that T. gondii infection may lead to the loss of integrity of intestinal mucosa, resulting in impaired barrier function.

Keywords: Caco-2 cells; Toxoplasma gondii; epithelial barrier dysfunction; tight junctions.

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Conflict of interest statement

Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Toxoplasma gondii infection affects endocytosis and paracellular permeability of FITC-Dextran in Caco-2 cells. (A) Caco-2 cells were incubated with Dulbecco’s modified Eagle’s medium and 20% fetal bovine serum (FBS) for 20 days. Fluorescence assay was performed incubating Caco-2 cells with 1.0 mg/ml of 4-kDa FITC-Dextran for 1 hr, after 24 hr of T. gondii infection. Cells were stained for nuclei with TO-PRO-3 (blue; Life Technologies) and FITC-Dextran (green). The histograms on the right panels represent the FITC-Dextran fluorescence intensity. (B) The T. gondii infection decreases paracellular permeability of FITC-Dextran in Caco-2 cell monolayers. The apical-to-basolateral flux of FITC-Dextran through Caco-2 monolayers cultured in transwell system was monitored for 1 hr after dextran incubation in the presence or absence of tachyzoites. Result represents the mean ± standard error of the mean of three independent experiments. ***p<0.0001. Scale bar is 10 μm.
Figure 2.
Figure 2.
Toxoplasma gondii tachyzoites decrease transepithelial electrical resistance (TEER) of Caco-2 monolayers. Caco-2 cells were cultured in the transwell system for 20 days in Dulbecco’s modified Eagle’s medium with 20% fetal bovine serum (FBS) and then were infected with T. gondii. The TEER was measured after 20 and 24 hr of infection. White column represents TEER of the insert with medium. Gray columns represent TEER of Caco-2 cells without tachyzoites. Black columns represent TEER of Caco-2 cells after 20 and 24 hr of infection. Data are expressed as the mean ± standard error of the mean for triplicate samples. ***p<0.0001.
Figure 3.
Figure 3.
Loss of actin filaments in the presence of Toxoplasma gondii infection. Caco-2 cells were cultured in coverslips for 20 days in Dulbecco’s modified Eagle’s medium supplemented with 20% of fetal bovine serum (FBS). The cells were infected with T. gondii, and 24 hr later, the actin cytoskeleton was analyzed using Alexa Fluor 594–conjugated phalloidin (which has a high affinity for actin filaments). The coverslips were observed in a confocal microscope. (A) Confocal microscopy images of microvilli and actin cytoskeleton of Caco-2 cells after 24 hr of T. gondii infection. At the apical surface, on the left and upper panel, phalloidin was detected in developed microvilli structures on noninfected Caco-2 monolayer (arrows). At right side, poorly developed microvilli on the apical surface of infected Caco-2 cells can be observed by phalloidin detection (arrows). At the basolateral surface, the actin filament net was detected by phalloidin. On left panel (below), a thick net of actin filament is revealed, whereas a weak net is detected in the infected cells (right panel, below). (B) Optical x–y sections from full projection of the epithelium are presented by captured images of phalloidin and T. gondii detection using SAG-1 antibody (top side). The histograms show the relative quantification analysis of phalloidin–actin detection (bottom side). The relative quantification of the full projection images confirms the decrease of actin filament on infected cells. Scale bar is 10 μm.
Figure 4.
Figure 4.
Expressions of claudin-1, occludin, and zonula occludens-1 (ZO-1) are altered in Caco-2 cells 24 hr after Toxoplasma gondii infection. Caco-2 cells were cultured in coverslips for 20 days in Dulbecco’s modified Eagle’s medium supplemented with 20% of fetal bovine serum (FBS). The cells were infected with T. gondii for 24 hr and immunostained with specific antibodies to detect claudin-1, ZO-1, and occludin. Continuous net pattern of claudin-1, ZO-1, and occludin in Caco-2 cells (panels A, C, D, F, G, I) is observed after 20 days of culture. Discontinuous and disorganized net patterns of claudin-1, ZO-1, and occludin (panels J, L, M, O, P, R) in infected cells are indicated with asterisks. Immunofluorescence assays for claudin-1, ZO-1, and occludin were performed using rabbit polyclonal anti-claudin-1 (green), mouse monoclonal anti-ZO-1 (green), and rabbit anti-occludin (green). Panels A to C, D to F, and G to I represent claudin-1, ZO-1, and occludin immunostaining, respectively. B, E, and H images represent negative SAG-1 immunolocalization in noninfected cells. C, F, and I images are a merge of images from noninfected cells. Panels J, L; M, O; and P, R represent claudin-1, ZO-1, and occludin immunostaining, respectively, in infected cells. K, N, and Q images show T. gondii parasites (red) that were labeled with mouse monoclonal anti-SAG-1 antibody, and L, O, and R merged pictures show SAG-1 immunolocalization within Caco-2 cells. The immunoblot analysis was done to measure the claudin-1, ZO-1, and occludin proteins expression in infected and noninfected cells (S). Whole cell lyses were separated on SDS-PAGE gel and immunoblotted with antibodies against the corresponding tight junction proteins, ZO-1, occludin, and claudin-1, followed by chemiluminescence detection. Protein bands were visualized using a ChemiDoc MP system. Quantification of immunoblotting by using β-actin as an internal control in ImageJ software (T). Scale bar is 10 μm.

References

    1. Dubey JP. The history and life cycle of Toxoplasma gondii. In: Weiss LM, Kim K. editors. Toxoplasma gondii. The model Apicomplexan: perspective and methods. San Diego: Academic Press; 2007, p. 1–17.
    1. Dimier IH, Bout DT. Inhibition of Toxoplasma gondii replication in IFN-gamma-activated human intestinal epithelial cells. Immunol Cell Biol. 1997;75:511–4. - PubMed
    1. Dubey JP, Ferreira LR, Martins J, McLeod R. Oral oocyst-induced mouse model of toxoplasmosis: effect of infection with Toxoplasma gondii strains of different genotypes, dose, and mouse strains (transgenic, out-bred, in-bred) on pathogenesis and mortality. Parasitology. 2012;139:1–13. - PMC - PubMed
    1. Gregg B, Taylor BC, John B, Tait-Wojno ED, Girgis NM, Miller N, Wagage S, Roos DS, Hunter CA. Replication and distribution of Toxoplasma gondii in the small intestine after oral infection with tissue cysts. Infect Immun. 2013;81:1635–43. - PMC - PubMed
    1. Coombes JL, Charsar BA, Han SJ, Halkias J, Chan SW, Koshy AA, Striepen B, Robey EA. Motile invaded neutrophils in the small intestine of Toxoplasma gondii-infected mice reveal a potential mechanism for parasite spread. Proc Natl Acad Sci U S A. 2013;110:E1913–22. - PMC - PubMed

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