Infection by Toxoplasma gondii, a severe parasite in neonates and AIDS patients, causes impaired anion secretion in airway epithelia

Abstract

The airway epithelia initiate and modulate the inflammatory responses to various pathogens. The cystic fibrosis transmembrane conductance regulator-mediated Cl(-) secretion system plays a key role in mucociliary clearance of inhaled pathogens. We have explored the effects of Toxoplasma gondii, an opportunistic intracellular protozoan parasite, on Cl(-) secretion of the mouse tracheal epithelia. In this study, ATP-induced Cl(-) secretion indicated the presence of a biphasic short-circuit current (Isc) response, which was mediated by a Ca(2+)-activated Cl(-) channel (CaCC) and the cystic fibrosis transmembrane conductance regulator. However, the ATP-evoked Cl(-) secretion in T. gondii-infected mouse tracheal epithelia and the elevation of [Ca(2+)]i in T. gondii-infected human airway epithelial cells were suppressed. Quantitative reverse transcription-PCR revealed that the mRNA expression level of the P2Y2 receptor (P2Y2-R) increased significantly in T. gondii-infected mouse tracheal cells. This revealed the influence that pathological changes in P2Y2-R had on the downstream signal, suggesting that P2Y2-R was involved in the mechanism underlying T. gondii infection in airways. These results link T. gondii infection as well as other pathogen infections to Cl(-) secretion, via P2Y2-R, which may provide new insights for the treatment of pneumonia caused by pathogens including T. gondii.

Keywords: ATP; Cl− secretion; P2Y2 receptor; cystic fibrosis; pneumonia.

Fig. 1.

Effects of T. gondii infection on ATP-induced I_sc currents. Addition of 10 μM ATP to the apical side (ap) of the proximal trachea tissues caused a rise in I_sc response (A). This response was found to be partially inhibited by T. gondii pretreatment (B). (C) A summary of the effects of T. gondii on the dual phases of ATP-induced I_sc response. Data are presented as mean change in I_sc ± SEM (n = 4) and are representative of four independent experiments. *P < 0.05; **P < 0.01.

Fig. 2.

Effect of P2 receptor blocker suramin on intracellular Ca²⁺ induced by ATP in 16HBE cells. Intracellular Ca²⁺ measurement in single 16HBE cells using Fluo 3/AM as a probe. (A) The addition of ATP (10 μM) significantly increased the intracellular Ca²⁺, which was inhibited in the presence of the P2-R antagonist, suramin (100 μM); the addition of 1 μM Thapsigargin (Tg, an inducer of stored Ca²⁺ release) was done to check the activity of cells. (B) A summary of the effects of suramin on ATP-induced calcium elevation. Data are presented as mean of fluorescence intensity ± SEM (n = 4) and are based on four independent experiments. **P < 0.01.

Fig. 3.

Effect of T. gondii on intracellular Ca²⁺ induced by ATP in 16HBE cells. Intracellular Ca²⁺ measurement in single 16HBE cells using Fluo 3/AM as a probe. (A) Addition of ATP (10 μM) significantly increased the intracellular Ca²⁺ inhibited after T. gondii pretreatment for 3 h or 6 h. (B) A summary of the effects of T. gondii on ATP-induced calcium elevation. Data are presented as mean fluorescence intensity ± SEM (n = 4) and are based on four independent experiments. ***P < 0.001; NS, P > 0.05.

Fig. 4.

T. gondii infection results in increased P2Y2-R expression. Quantitative real-time PCR analysis of mRNA expression of P2Y2-R in mice trachea treated without or with T. gondii for 3 d. Absolute values were corrected using GAPDH as a reference gene. Gene expression was reported as the relative variation to unstimulated control tissue mRNA levels. Bars represent means ± SEM (n = 3) and are based on three independent experiments. *P < 0.05.

Fig. 5.

Mechanisms underlying the impairment action of T. gondii on chloride secretion in airway epithelia. The schematic model shows that the impairment action of T. gondii on chloride secretion was related to its ability to inhibit P2Y2-Rs. Black arrows represent the P2Y2-R activation signaling pathways, or cellular events identified in the present and previous studies (42, 43), and a red block symbol denotes the P2Y2-R inhibition signaling pathways and cellular events described in airway epithelia [PIP2, phosphatidylinositol (4,5) bisphosphate].