Figure 1.
Treatment with the GSNOR inhibitor caused a reduction in allergic airway inflammation.
DO11.10 CD4+ Th2 cells were adoptively transferred into BALB/c recipient mice that were then exposed to aerosolized OVA for 7 d. Mice were treated intranasally with either the GSNOR inhibitor SPL334 (Th2+ SPL-334, 0.1 or 1 mg/kg daily) or vehicle (Th2). Control mice did not receive Th2 cells but inhaled OVA aerosols. (A) Cell differential counts in the BALF were determined by light microscopic evaluation of stained cytospin preparations. Results are expressed as absolute numbers (per mouse) of lymphocytes (Lym), macrophages (Mac), eosinophils (Eos), and neutrophils (Neu). (B) EPO levels in the BALF from Th2 recipient or control mice were assessed by colorimetric analysis. Results are mean ± SE of four to six individual mice analyzed per group in triplicates and are representative of four independent experiments. *p, 0.05, compared with Th2 group.
Figure 2.
GSNOR inhibitor reduced the number of OVA-specific T cells and eosinophils during allergic airway inflammation.
DO11.10 CD4+ Th2 cells were transferred into BALB/c mice that were then challenged with aerosolized OVA for 7 d. Mice were treated with either SPL-334 (Th2+ SPL-334) or vehicle (Th2). Control mice did not receive Th2 cells but inhaled OVA aerosols. The number of CD4+KJ-126+ T cells (A) and CD11b+Siglec-F+ eosinophils or CD11b+GR-1+ neutrophils (B) in the BALF following OVA inhalation was determined using flow cytometry. Data are representative of two independent experiments.
Figure 3.
GSNOR inhibitor caused a reduction in peribronchial inflammation and mucus secretion during airway inflammation.
DO11.10 CD4+ Th2 cells were transferred into BALB/c mice that were then challenged with aerosolized OVA for 7 d. Mice were treated with either SPL-334 (Th2+ SPL-334) or vehicle (Th2). Control mice did not receive Th2 cells but inhaled OVA aerosols. (A) Peribronchial inflammation and (B) mucus production were determined by histological analysis by staining lung tissue sections with H&E or PAS, respectively (20x). PAS staining was expressed as % PAS+ area per bronchiole, and bronchial wall thickness expressed in µm. Data are representative of two separate experiments. *p, 0.05, compared with Th2 group.
Figure 4.
Treatment with the GSNOR inhibitor caused a reduction in AHR
. DO11.10 CD4+ Th2 cells were transferred into BALB/c mice that were then challenged with aerosolized OVA for 7 d. Mice were treated with either SPL-334 (Th2+ SPL-334) or vehicle (Th2). Control mice did not receive Th2 cells but inhaled OVA aerosols. Lung resistance (RL) and dynamic compliance (CDyn) was assessed in anesthetized and tracheotomized mice that were mechanically ventilated in response to increasing concentrations of methacholine inhalation. Data are mean ±SE of ten individual mice analyzed per group. *p, 0.05, compared with Th2 group.
Figure 5.
Treatment with the GSNOR inhibitor reduced Th2 cytokine production during allergic airway inflammation.
LMCs were obtained from control mice and Th2 recipient mice that have been treated with either SPL-334 or vehicle and inhaled OVA for 7 d. LMCs were stimulated with anti-CD3 (2 µg/ml) or OVA323–334 peptide (1 µg/ml) for 24 h and supernatant analyzed for IL-4, IL-5 or IL-13 production by ELISA. Results are mean ±SE of four mice analyzed per group in triplicates and are representative two independent experiments. *p, 0.05, compared with Th2 group.
Figure 6.
Treatment with the GSNOR inhibitor reduced CCL11 production during allergic lung inflammation.
LMCs were obtained from control mice and Th2 recipient mice that have been treated with either SPL-334 or vehicle and inhaled OVA for 7 d. (A) LMCs were stimulated with anti-CD3 (2 µg/ml) or OVA323–334 peptide (1 µg/ml) for 24 h and supernatant analyzed for eotaxin production by ELISA. (B) CCL11 levels in the BALF were measured by ELISA. Results are mean ±SE of four to six individual mice analyzed per group in triplicates and are representative two independent experiments. *p, 0.05, compared with Th2 group.
Figure 7.
Schematic representation of the anti-inflammatory effects mediated by GSNOR inhibition in asthma.
Nitric Oxide produced by a nitric oxide synthase reacts with glutathione to form S-nitrosoglutathione. This reactive molecule promotes bronchodilation and anti-inflammatory effects, and is ultimately degraded to oxidized glutathione and ammonia by S-nitrosoglutathione reductase (GSNOR). We propose that inhibition of GSNOR (which is over-expressed in inflammatory states and asthma) using the GSNOR inhibitor SPL-334 will increase steady-state levels of S-nitrosoglutathione and thus inhibit inflammation and AHR.