ISO-1, a macrophage migration inhibitory factor antagonist, inhibits airway remodeling in a murine model of chronic asthma

Abstract

Airway remodeling is the process of airway structural change that occurs in patients with asthma in response to persistent inflammation and leads to increasing disease severity. Drugs that decrease this persistent inflammation play a crucial role in managing asthma episodes. Mice sensitized (by intraperitoneal administration) and then challenged (by inhalation) with ovalbumin (OVA) develop an extensive eosinophilic inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening, and airway wall area increase, similar to pathologies observed in human asthma. We used OVA-sensitized/challenged mice as a murine model of chronic allergic airway inflammation with subepithelial fibrosis (i.e., asthma). In this OVA mouse model, mRNA and protein of macrophage migration inhibitory factor (MIF) are upregulated, a response similar to what has been observed in the pathogenesis of acute inflammation in human asthma. We hypothesized that MIF induces transforming growth factor-β1 (TGF-β1) synthesis, which has been shown to play an important role in asthma and airway remodeling. To explore the role of MIF in the development of airway remodeling, we evaluated the effects of an MIF small-molecule antagonist, (S,R)3-(4-hy-droxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), on pathologies associated with the airway-remodeling process in the OVA mouse model. We found that administration of ISO-1 significantly mitigated all symptoms caused by OVA treatment. In addition, the treatment of OVA-sensitized mice with the MIF antagonist ISO-1 significantly reduced TGF-β1 mRNA levels in pulmonary tissue and its protein level in bronchial alveolar lavage fluid supernatants. We believe the repression of MIF in the ISO-1 treatment group led to the significant suppression observed in the inflammatory responses associated with the allergen-induced lung inflammation and fibrosis in our murine asthma (OVA) model. Our results implicate a possible function of MIF in the pathogenesis of chronic asthma and suggest that MIF might be an important therapeutic target for airway remodeling.

Figure 1

Pulmonary expression of MIF mRNA. Reverse transcription–polymerase chain reaction was performed to examine mRNA expression of MIF in mice lung tissue. Expression of housekeeping gene β-actin 1 mRNA was detected as a control. Lane 1: marker; lane 2: control; lane 3: OVA; lane 4: ISO-1; lane 5: DEX.

Figure 2

Immunohistochemistry of macrophage MIF in the lung was assessed quantitatively by using IPP 6.0 and expressed as integrated absorbency (IA). (A,B) MIF was weakly detected in airway in the control group (Control; n = 8). (C,D) MIF was prominent in airway in the ovalbumin group(OVA; n = 8) after the OVA challenge. (E,F) MIF was prominent in the airway in the ISO-1 group (ISO-1; n = 8). (G,H) MIF was weakly detected in the airway in the DEX group (DEX; n = 8) (original magnification: ×200) ^#P < 0.01 versus OVA group; ^ΔP < 0.01 versus control group.

Figure 3

Total and differential cell counts in BALF. The numbers of total cells, lymphocytes and eosinophils were significantly elevated in the OVA group compared with the control group (×10⁵). Treatment with ISO-1 and DEX significantly decreased the numbers of total cells, lymphocytes and eosinophils. ^ΔP < 0.01 versus control group; ^#P < 0.01 versus OVA group.

Figure 4

ISO-1 and DEX reduced OVA-induced increased airway wall area. Lung tissue was stained with H&E. (A) Control; (B) OVA; (C) ISO-1; (D) DEX. ^ΔP < 0.01 versus control group; ^⋆P < 0.05 versus control group; ^#P < 0.01 versus OVA group. (Magnification: 200×, μm²/μm [B,D]; 100×, μm²/μm [A,C]).

Figure 5

ISO-1 and DEX reduced OVA-induced increased airway smooth muscle thickness. Lung tissue was stained with H&E (original magnification: 200×, μm²/μm). ^ΔP < 0.01 versus control group; ^⋆P < 0.05 versus control group; ^#P < 0.01 versus OVA group.

Figure 6

ISO-1 and DEX reduced OVA-induced increased subepithelial collagen deposition. Lung tissue was stained with Masson′s trichrome stain (original magnification: 200×, μm²/μm). (A) Control; (B) OVA; (C) ISO-1; (D) DEX. ^ΔP < 0.01 versus control group; ^#P < 0.01 versus OVA group.

Figure 7

ISO-1 and DEX decreased OVA-induced GC metaplasia. Lung tissue was stained with Alcian blue and periodic acid-Schiff (original magnification: ×200, cells per 100 μm of basement membrane). (A) Control; (B) OVA; (C) ISO-1; (D) DEX. ^ΔP < 0.01 versus control group; ^⋆P < 0.05 versus control group; ^#P < 0.01 versus OVA group; ^⋄P < 0.05 versus OVA group; ^★P < 0.01 versus DEX group.

Figure 8

ISO-1 and DEX reduced OVA-induced pulmonary expression of TGF-β1 mRNA. Lane 1: marker; lane 2: control; lane 3: OVA; lane 4: ISO-1; lane 5: DEX.

Figure 9

ISO-1 and DEX reduced OVA-induced increased TGF-β1 levels in BALF. ^ΔP < 0.01 versus control group; ^#P < 0.01 versus OVA group.