Intranasal CpG therapy attenuated experimental fungal asthma in a TLR9-dependent and -independent manner

Abstract

Background: CpG administration abolishes airway inflammation and remodeling in acute models of allergic airway disease.

Methods: Herein, we investigated the therapeutic effect of CpG in a chronic fungal model of asthma. TLR9+/+ and TLR9-/- mice were sensitized to soluble Aspergillus fumigatus antigens and challenged with live A. fumigatus conidia. Mice were treated with intraperitoneal (IP) or intranasal (IN) CpG, or left untreated 14-28 days after conidium challenge. All features of allergic airway disease were attenuated in TLR9+/+ mice treated with IN CpG, including airway hyperresponsiveness (AHR), mucus production, and peribronchial fibrosis.

Results: TLR9-/- mice treated with IN CpG exhibited attenuated airway remodeling but not AHR. Whole-lung IL-12 levels were significantly elevated in both TLR9+/+ and TLR9-/- mice receiving IN CpG but not in either group receiving IP CpG. Whole-lung IL-10 levels were significantly elevated in IN CpG-treated TLR9+/+ mice but not in TLR9-/- mice receiving IN CpG. Increased whole-lung transcript and protein levels of the scavenger receptors SR-A and MARCO were observed in TLR9-/- mice compared with TLR9+/+ mice, possibly accounting for the CpG responsiveness in the knockout group.

Conclusions: Together, these data show that IN CpG has a therapeutic effect during established fungal asthma, which is TLR9 dependent and independent.

Fig. 1

IN CpG treatment attenuated AHR in TLR9+/+ but not TLR9–/– mice. Airway resistance analysis in TLR9+/+ and TLR9–/– Aspergillus-sensitized and conidium-challenged mice treated with IN CpG (a) or IP CpG (b) on alternate days from day 14 to day 28 after conidium challenge. Peak increases in airway resistance or hyperresponsiveness were determined at each time point after the intravenous injection of methacholine (MTH). Airway resistance was 1.89 ± 0.11 cm H₂O/ml/s in untreated TLR9+/+ mice and 16.96 ± 3.86 cm H₂O/ml/s after intravenous MTH administration. Means ± SEM; n = 5/group/time point. ∗ p ≤ 0.05; ∗∗∗ p ≤ 0.001, vs. the appropriate baseline measurement or the indicated treatment group following MTH challenge.

Fig. 2

CpG treatment altered systemic immunoglobulin levels in TLR9–/– mice alone. ELISA analysis of serum IgE (a) and IgG2a (b) from Aspergillus-sensitized and conidium-challenged TLR9+/+ and TLR9–/– mice either untreated, or treated with IN or IP CpG. Serum was removed from all groups of mice on day 28 after conidium challenge. Means ± SEM; n = 10/group/time point.

Fig. 3

Peribronchial fibrosis and perifungal fibrotic capsules were markedly decreased or absent following IN but not IP CpG treatment in TLR9+/+ and TLR9–/– mice. Whole-lung tissue sections from Aspergillus-sensitized and challenged TLR9+/+ and TLR9–/– mice were stained with Masson trichrome stain. Representative panels of whole-lung sections from TLR9+/+ (a–c) and TLR9–/– mice (d–f) given no treatment (a, d), IN CpG (b, e), or IP CpG (c, f) are shown. Of note, no fungal masses were detected in whole-lung sections from IN CpG-treated TLR9–/– mice. Collagen deposition is stained blue in these photomicrographs (original magnification: ×200).

Fig. 4

Mucus was markedly reduced following IN but not IP CpG treatment in TLR9+/+ and TLR9–/– mice. Mouse whole-lung tissue sections from Aspergillus-sensitized and challenged TLR9+/+ and TLR9–/–mice were stained with PAS. Representative whole lung sections from TLR9+/+ (a–c) and TLR9–/– mice (d–f) that were given no treatment (a, d), IN CpG (b, e), or IP CpG (c, f) are shown. Mucus stains purple using the PAS staining procedure (original magnification: ×200).

Fig. 5

Fungal growth is markedly reduced following IN but not IP CpG treatment in TLR9+/+ and TLR9–/– mice. Mouse whole-lung tissue sections from Aspergillus-sensitized and challenged TLR9+/+ and TLR9–/–mice were stained with GMS. Aspergillus conidia and hyphae stain black using the GMS staining procedure. Representative whole-lung sections from TLR9+/+ (a–c) and TLR9–/– mice (d–f) that were given no treatment (a, d), IN CpG (b, e), or IP CpG (c, f) are shown. Black arrows highlight conidia engulfed by macrophages. Open circles denote macrophages that have not engulfed conidia. No macrophages containing engulfed conidia were visible in TLR9+/+ mice given IN CpG treatment. Original magnification: ×200 (a, c, d, f) and ×400 (b, e).

Fig. 6

Elevated TLR3 levels were observed in IP CpG-treated TLR9–/– mice. TaqMan analysis of TLR9+/+ and TLR9–/– mouse lung TLR4 (a), TLR6 (b), and TLR3 (c) levels from Aspergillus-sensitized and conidium-challenged TLR9+/+ and TLR9–/– mice that were either untreated, treated with IN CpG, or treated with IP CpG. Means ± SEM; n = 5/group/time point. ∗ p ≤ 0.05 vs. the appropriate untreated control group. WT = Wild type.

Fig. 7

Elevated IFN-α levels were observed in IP CpG treated TLR9–/–mice, and CXCL10 levels were increased following IN CpG treatment in TLR9–/– mice. TaqMan analysis of TLR9+/+ and TLR9–/– mouse lung IFN-α (a) and CXCL10 (b) levels from Aspergillus-sensitized and conidium-challenged TLR9+/+ and TLR9–/– mice that were either untreated, treated with IN CpG, or treated with IP CpG. Means ± SEM; n = 5/group/time point. ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001, vs. the appropriate untreated control group.

Fig. 8

IN CpG inhibited Th2-associated cytokine levels in a TLR9-dependent manner in chronic fungal asthma. ELISA or Bio-Plex of TGF-β (a), TSLP (b), and CCL17 (c) in whole-lung samples from Aspergillus-sensitized and conidium-challenged TLR9+/+ and TLR9–/– mice. Both groups of mice were left untreated or treated with IN CpG, or IP CpG on alternate days from day 14 to day 28 after conidium challenge. Means ± SEM; n = 5/group/time point. ∗ p ≤ 0.05 vs. the appropriate untreated control group.

Fig. 9

TLR9-independent induction of IL-12 and TLR9-dependent induction of IL-10 following IN CpG treatment in chronic fungal asthma. ELISA or Bio-Plex of IL-12 (a) and IL-10 (b) in whole-lung samples from Aspergillus-sensitized and conidium-challenged TLR9+/+ and TLR9–/– mice. Both groups of mice were left untreated or treated with IN or IP CpG on alternate days from day 14 to day 28 after conidium challenge. Note that the levels of IL-12 and IL-10 in untreated TLR9+/+ and TLR9–/– mice were below the limits of detection for these assays. Means ± SEM; n = 10/group/time point. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01, vs. the appropriate IP CpG group.

Fig. 10

Scavenger receptor transcript levels were elevated in TLR9–/– mice with chronic fungal asthma. TaqMan analysis of TLR9+/+ and TLR9–/– mouse lung SR-A (a) and MARCO (b) levels in Aspergillus-sensitized and conidium-challenged TLR9+/+ and TLR9–/– mice. Both groups of mice were left untreated and transcript levels for these scavenger receptors were determined from whole-lung samples taken on day 28 after conidium challenge. Means ± SEM; n = 5/group/time point. ∗ p ≤ 0.05 vs. the TLR9+/+ group.

Fig. 11

Immunohistochemical analysis of SR-A and MARCO expression in whole-lung sections from TLR9+/+ and TLR9–/– mice on day 28 after conidium challenge. Whole-lung tissue sections from Aspergillus-sensitized and challenged TLR9+/+ and TLR9–/– mice were stained using routine immunohistochemical techniques. Representative whole-lung sections from TLR9+/+ (a–c) and TLR9–/– mice (d–f) mice stained with IgG control (a, d), anti-SR-A antibody (b, e), or anti-MARCO antibody (c, f) are shown. Receptor expression stains brown with this immunohistochemical procedure (original magnification: ×200).

Fig. 12

Immunoreactive levels of MARCO and SR-A remain elevated in TLR9–/– mice compared with TLR9+/+ mice after CpG treatment. ELISA analysis of SR-A (a) and MARCO (b) in whole-lung samples from Aspergillus-sensitized and conidium-challenged TLR9+/+ and TLR9–/– mice. Both groups were treated with IN CpG or IP CpG on alternate days from day 14 to day 28 after conidium challenge. Means ± SEM; n = 5/group/time point. ∗ p ≤ 0.05 vs. IN CpG TLR9+/+ group.