Cyclophilin A Plays Potential Roles in a Rat Model of Asthma and Suppression of Immune Response

Abstract

Purpose: Cyclophilin A (CypA) inhibits CD4⁺ T cell signal transduction via interleukin-2-inducible T-cell kinase (Itk), a tyrosine kinase required for T helper (Th) 2 cells function. Furthermore, mice with CypA silencing developed allergic diseases associated with increased Th2 cytokines production. CD4⁺ T cells with a Th2-cytokine pattern have been demonstrated to have a pivotal role in the pathogenesis of asthma. However, the effects of CypA in regulating immunity in asthma and in relieving asthmatic symptoms in vivo are entirely unknown.

Methods: Recombinant CypA protein (rCypA) was generated and purified. Ovalbumin (OVA)-challenged asthmatic rats model and acetylcholine chloride (ACh)-induced contraction of tracheal spirals were established. The pulmonary resistance (RL) value of asthmatic rats in vivo and the isometric tension of tracheal spirals ex vivo were recorded by MFLab 3.01 software. The levels of Th1 and Th2 cytokines and the quantities of immunoglobulin (IgA, IgG, IgM and IgE) in the supernatants of rat spleen lymphocytes were detected and analysed by bio-plex Suspension Array System and ELISA, respectively. CD4⁺ T cells were separated by MicroBeads, and the levels of interleukin (IL)-4 and interferon-γ (IFN-γ) were detected by ELISA.

Results: rCypA (10 ng/kg) significantly reduced RL within 2-7 min in OVA-challenged asthmatic rats in vivo, and there were no significant differences compared with terbutaline (TB) and hydrocortisone (HC). Furthermore, rCypA (10 ng/mL) significantly reduced the isometric tension in the ACh-induced contraction of the tracheal spiral ex vivo, and the effect of rCypA was better than that of TB. Additionally, rCypA suppressed the secretion of both Th1 and Th2 cytokines, and the suppressive effects of rCypA were stronger than those of HC, especially on Th2 cytokines.

Conclusion: These findings indicate that CypA may serve as a potential novel therapeutic strategy for asthma.

Keywords: Th2 cytokines; asthma; cyclophilin A; immunoregulation.

Figure 1

Effects of rCypA on pulmonary resistance (RL) in asthmatic rats. (A) Time–response curves of blank, asthmatic rats (AS), terbutaline (TB), hydrocortisone (HC) and recombinant CypA (rCypA) in asthmatic rats. The data are presented as the mean ± SEM (n = 8). (B) Boxplot analysis of the sum value in RL during the 2–4 min of the early airway response (RL_2–4min). *P < 0.05 for TB, HC and rCypA groups vs AS group.

Figure 2

Effects of rCypA on isometric tension of tracheal spirals. (A) Time–response curves of the blank, ACh group (ACh), terbutaline (TB), hydrocortisone (HC) and recombination CypA (rCypA) groups in isolated tracheal spirals. The data are presented as the mean ± SEM (n = 4). (B) Boxplot analysis of the sum of isometric tension among all the groups. *P < 0.05 for TB, HC and rCypA groups vs ACh group.

Figure 3

Suppression effects of recombinant CypA on cytokines and immunoglobulins. (A–J) The concentrations of cytokines and immunoglobulins. The unit of each cytokine is described as pg/mL. The unit of each immunoglobulin is described as ng/mL. Data are presented as the mean ± SEM (n = 8). *P < 0.05 vs baseline in the Non-stim group; ^#P < 0.05 vs baseline in the PWM-stim group. (K) Heatmap of the cytokines profile for Th1, Th2, TNF-α and immunoglobulin in supernatants of lymphocytes.

Figure 4

Effects of recombinant CypA on IL-4 and IFN-γ levels in separated CD4⁺ T cells. (A) The concentrations of IL-4 in different groups. (B) The concentrations of IFN-γ in different groups. The unit of each cytokine is described as pg/mL. Data are presented as the mean ± SEM (n = 4). *P < 0.05, when compared with the Con A group.

Figure 5

Schematic illustration of the suppressive effect of CypA on the levels of cytokines in the Non-stim and PWM-stim groups. The Y-axis represents an arbitrary measure of cytokines.