Blocking pannexin1 reduces airway inflammation in a murine model of asthma

Abstract

Stressed or injured cells release ATP into the extracellular milieu via the pannexin1 (Panx1) channels, which is the basis of inflammation in a variety of conditions, including allergic lung inflammation. Although the role of Panx1 in mediating inflammation has been well established, the role of its mimetic peptide, ¹⁰Panx1, which inhibits ATP release from Panx1 channels, in allergic asthma remains understudied. The aim of this study was to evaluate the effects of using ¹⁰Panx1 to inhibit Panx1 channel in a murine model of ovalbumin (OVA)-induced asthma. We demonstrate that blockade of Panx1 significantly attenuated goblet cell hyperplasia and inflammatory cell infiltration into the lungs of OVA-sensitized mice. Inhibition of Panx1 also reduced the total and eosinophil cell numbers in the bronchoalveolar lavage fluid (BALF) and reduced expression of CCL11 and CCL2 in lung tissues from mice. Moreover, we detected lower levels of IL-5 and IL-13 in the culture supernatant of OVA-restimulated splenocytes from ¹⁰Panx1-treated mice. Collectively, our findings suggest that Panx1 inhibition of allergen-mediated lung inflammation has the potential to suppress allergic responses in asthma.

Keywords: 10Panx1; EXtracellular ATP; asthma; chemokine; lung inflammation; pannexin1.

Figure 1

Sensitization model of ovalbumin (OVA)-induced allergic airway inflammation. This diagram depicts the OVA-induced airway inflammation model used in the study. On days 0 and 7, mice were sensitized by intraperitoneal injection (I.P.) with 100 µg OVA with 2 mg Alum in 200 µl normal saline. On days 19, 22, 26, 29, and 33, mice were challenged with 10 µg of OVA intratracheally (I.T.) together with 1.5 or 3 mg/kg ¹⁰Panx1 for the treatment groups and 3 mg/kg of scrambled Panx1 peptide for the control group. Twenty four hours after the last challenge, mice were sacrificed and analyzed.

Figure 2

Blockade of Panx1 suppresses airway inflammation and goblet cell hyperplasia in the lungs of OVA-sensitized mice. A. Representative micrographs of hematoxylin and eosin (H&E) stained lung sections to determine eosinophil infiltration (top panel) and periodic acid-Schiff (PAS) stained lung sections to determine goblet cell hyperplasia (bottom panel) (original magnification; 200 ×). Digital images obtained from histological sections were quantified using MetaMorph software. The integrated intensity of H&E and PAS-positive cells was obtained. B. Quantification of H&E-stained cells per bronchia showing airway inflammation. C. Quantification of PAS-stained cells per bronchia showing mucus production. Intensity fold changes were calculated from the average values of the OVA group. PBS, normal control mice; OVA, OVA-sensitized and challenged mice; SCR, 3 mg/kg (scrambled Panx1 peptide) treatment + OVA-sensitized and challenged mice; P1.5 and P3 (1.5 or 3 mg/kg ¹⁰Panx1, respectively) treatment + OVA-sensitized and challenged mice. All data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Intratracheal administration of ¹⁰Panx1 reduced cell numbers and ATP levels in bronchoalveolar lavage fluid (BALF) of mice. BALF from each mouse was collected to assess cell infiltration in the airways and the accumulation of ATP. A. Total cell counts in BALF were determined using a hemocytometer. B. Differential cell counts were performed according to the morphological characteristics of the cells. C. The percentage of eosinophils in each mouse was determined. D. The level of ATP in each BALF sample was measured by a luminometer. PBS, normal control mice; OVA, OVA-sensitized and challenged mice; SCR, 3 mg/kg (scrambled Panx1 peptide) treatment + OVA-sensitized and challenged mice; P1.5 and P3 (1.5 or 3 mg/kg ¹⁰Panx1, respectively) treatment + OVA-sensitized and challenged mice. All data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01.

Figure 4

Inhibition of Panx1 suppresses chemokine expression. The mRNA expression of (A) CCL11 and (C) CCL2 was analyzed by real-time PCR. Both genes were normalized to β-actin, and their fold changes were normalized to the OVA-sensitized group. (B) Serum levels of CCL11 and (D) CCL2 were also determined by ELISA (9-12 mice per group). PBS, normal control mice; OVA, OVA-sensitized and challenged mice; SCR, 3 mg/kg (scrambled panx1 peptide) treatment + OVA-sensitized and challenged mice; P1.5 and P3 (1.5 or 3 mg/kg ¹⁰Panx1, respectively) treatment + OVA-sensitized and challenged mice. All data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01.

Figure 5

¹⁰Panx1 administration influences Th2 responses in mice. At sacrifice, splenic cells were isolated and restimulated with 100 µg/ml OVA for six days without ¹⁰Panx1 or scrambled peptide to detect the level of Th2 cytokines. (A) There was no change in IL-4 levels. (B) IL-5 and (C) IL-13 levels were lower in mice after ¹⁰Panx1 administration. The level of OVA-specific antibodies was also determined by ELISA. (D) OVA-specific IgG1 levels were lower in mice treated with ¹⁰Panx1. (E) No change in OVA-specific IgE. PBS, normal control mice; OVA, OVA-sensitized and challenged mice; SCR, 3 mg/kg (scrambled Panx1 peptide) treatment + OVA-sensitized and challenged mice; P1.5 and P3 (1.5 or 3 mg/kg ¹⁰Panx1, respectively) treatment + OVA-sensitized and challenged mice. All data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.