Combining an epithelial repair factor and anti-fibrotic with a corticosteroid offers optimal treatment for allergic airways disease

Abstract

Background and purpose: We evaluated the extent to which individual versus combination treatments that specifically target airway epithelial damage [trefoil factor-2 (TFF2)], airway fibrosis [serelaxin (RLX)] or airway inflammation [dexamethasone (DEX)] reversed the pathogenesis of chronic allergic airways disease (AAD).

Experimental approach: Following induction of ovalbumin (OVA)-induced chronic AAD in 6–8 week female Balb/c mice, animals were i.p. administered naphthalene (NA) on day 64 to induce epithelial damage, then received daily intranasal administration of RLX (0.8 mg·mL(−1)), TFF2 (0.5 mg·mL(−1)), DEX (0.5 mg·mL(−1)), RLX + TFF2 or RLX + TFF2 + DEX from days 67–74. On day 75, lung function was assessed by invasive plethysmography, before lung tissue was isolated for analyses of various measures. The control group was treated with saline + corn oil (vehicle for NA).

Key results: OVA + NA-injured mice demonstrated significantly increased airway inflammation, airway remodelling (AWR) (epithelial damage/thickness; subepithelial myofibroblast differentiation, extracellular matrix accumulation and fibronectin deposition; total lung collagen concentration), and significantly reduced airway dynamic compliance (cDyn). RLX + TFF2 markedly reversed several measures of OVA + NA-induced AWR and normalized the reduction in cDyn. The combined effects of RLX + TFF2 + DEX significantly reversed peribronchial inflammation score, airway epithelial damage, subepithelial extracellular matrix accumulation/fibronectin deposition and total lung collagen concentration (by 50–90%) and also normalized the reduction of cDyn.

Conclusions and implications: Combining an epithelial repair factor and anti-fibrotic provides an effective means of treating the AWR and dysfunction associated with AAD/asthma and may act as an effective adjunct therapy to anti-inflammatory corticosteroids

Figure 1

Individual versus combined effects of RLX, TFF2 and DEX on peribronchial inflammation and goblet cell metaplasia. Representative images of H&E‐ (A) and ABPAS‐stained (C) lung sections from each of the groups studied show the extent of bronchial wall inflammatory cell infiltration (A) and goblet cells (indicated by arrows) present within the epithelial layer (C) respectively. Scale bar (A and C) = 100 μm. Also shown is the mean ± SEM inflammation score (B) and goblet cell count (per 100 μm of basement membrane length) (D) from five airways/mouse, where H&E‐stained section were scored for the number and distribution of inflammatory aggregates on a scale of 0 (no apparent inflammation) to 4 (severe inflammation). Numbers in parenthesis represent the number of animals analysed per group. *P < 0.05 versus saline/CO‐treated group; ^# P < 0.05 versus OVA + NA‐treated group; ^† P < 0.05 versus OVA + NA + RLX‐treated group; ⁺ P < 0.05 vs. OVA + NA + TFF2‐treated group; ^§ P < 0.05 versus OVA + NA + RLX + TFF2‐treated group.

Figure 2

Individual versus combined effects of RLX, TFF2 and DEX on TSLP‐associated epithelial damage and extent of airway epithelial thickness. Representative TSLP‐ (A) and Masson's trichrome‐stained (C) lung sections from each of the groups studied show the extent of airway epithelial damage (A) and associated thickness (C) respectively. Scale bar (A and C) = 100 μm. Also shown is the mean ± SEM TSLP‐stained cell counts (per 100 μm of basement membrane length) (B) and epithelial thickness (μm) relative to basement membrane length (D) from five airways/mouse. Numbers in parenthesis represent the number of animals analysed per group. *P < 0.05 versus saline/CO‐treated group; ^# P < 0.05 versus OVA + NA‐treated group; ^† P < 0.05 versus OVA + NA + RLX‐treated group.

Figure 3

Individual versus combined effects of RLX, TFF2 and DEX on subepithelial ECM thickness, lung collagen concentration and subepithelial fibronectin deposition, as measures of fibrosis. Representative fibronectin‐stained (C) lung sections from each of the groups studied show the extent of subepithelial fibronectin deposition within the airways. Scale bar = 100 μm. Also shown is the mean ± SEM subepithelial ECM thickness (μm) relative to basement membrane length (which was morphometrically evaluated from Masson's trichrome‐stained sections) (A), total lung collagen concentration (% collagen content per dry weight lung tissue) (B) and subepithelial fibronectin deposition (D) from five airways/mouse. Numbers in parenthesis represent the number of animals analysed per group. *P < 0.05 versus saline/CO‐treated group; ^# P < 0.05 versus OVA + NA‐treated group; ⁺ P < 0.05 versus OVA + NA + TFF2‐treated group; ^¶ P < 0.05 versus OVA + NA + DEX‐treated group.

Figure 4

Individual versus combined effects of RLX, TFF2 and DEX on epithelial TGF‐β1 expression and subepithelial myofibroblast accumulation. Representative images of immunohistochemically stained lung sections from each of the groups studied show the extent and distribution of epithelial TGF‐β1 expression (A) and subepithelial myofibroblast accumulation (C) respectively. Scale bar (A and C) = 100 μm. Also shown is the mean ± SEM epithelial TGF‐β1 staining (% per field) (B) and α‐SMA‐stained cells (per 100 μm of basement membrane length) within the subepithelial region (D) from 5 airways/mouse. Numbers in parenthesis represent the number of animals analysed per group. *P < 0.05 versus saline/CO‐treated group; ^# P < 0.05 versus OVA + NA‐treated group; ^† P < 0.05 versus OVA + NA + RLX‐treated group; ^¶ P < 0.05 versus OVA + NA + DEX‐treated group.

Figure 5

Individual versus combined effects of RLX, TFF2 and DEX on lung MMP‐9 and MMP‐2 expression and activity. A representative gelatin zymograph (A) shows latent (L) MMP‐9 (gelatinase B; 92 kDa) and MMP‐2 (gelatinase A; 72 kDa) expression levels and their corresponding active (A) forms from each of the groups studied (2 samples per group). Two separate zymographs, each analysing two additional samples per group, produced similar results. Also shown in the mean ± SEM relative OD (of the combined L‐form and A‐forms of MMP‐9 (B) and MMP‐2 (C)), to that in the saline/CO‐treated control group, which is expressed as 1 in each case; from n = 6 mice per group. *P < 0.05 versus saline/CO‐treated group; ^# P < 0.05 versus OVA + NA‐treated group; ^† P < 0.05 versus OVA + NA + RLX‐treated group; ⁺ P < 0.05 versus OVA + NA + TFF2‐treated group; ^¶ P < 0.05 versus OVA + NA + DEX‐treated group.

Figure 6

Individual versus combined effects of RLX, TFF2 and DEX on cDyn. cDyn was measured in response to increasing concentrations of methacholine‐induced airway bronchoconstriction, as an indicator of the lung's ability to stretch and expand. Shown is the mean ± SEM loss of cDyn to each dose of methacholine tested. Numbers in parentheses represent the number of animals analysed per group. *P < 0.05 versus saline/CO‐treated group; ^# P < 0.05 versus OVA + NA‐treated group.