Facilitation of Allergic Sensitization and Allergic Airway Inflammation by Pollen-Induced Innate Neutrophil Recruitment

Abstract

Neutrophil recruitment is a hallmark of rapid innate immune responses. Exposure of airways of naive mice to pollens rapidly induces neutrophil recruitment. The innate mechanisms that regulate pollen-induced neutrophil recruitment and the contribution of this neutrophilic response to subsequent induction of allergic sensitization and inflammation need to be elucidated. Here we show that ragweed pollen extract (RWPE) challenge in naive mice induces C-X-C motif ligand (CXCL) chemokine synthesis, which stimulates chemokine (C-X-C motif) receptor 2 (CXCR2)-dependent recruitment of neutrophils into the airways. Deletion of Toll-like receptor 4 (TLR4) abolishes CXCL chemokine secretion and neutrophil recruitment induced by a single RWPE challenge and inhibits induction of allergic sensitization and airway inflammation after repeated exposures to RWPE. Forced induction of CXCL chemokine secretion and neutrophil recruitment in mice lacking TLR4 also reconstitutes the ability of multiple challenges of RWPE to induce allergic airway inflammation. Blocking RWPE-induced neutrophil recruitment in wild-type mice by administration of a CXCR2 inhibitor inhibits the ability of repeated exposures to RWPE to stimulate allergic sensitization and airway inflammation. Administration of neutrophils derived from naive donor mice into the airways of Tlr4 knockout recipient mice after each repeated RWPE challenge reconstitutes allergic sensitization and inflammation in these mice. Together these observations indicate that pollen-induced recruitment of neutrophils is TLR4 and CXCR2 dependent and that recruitment of neutrophils is a critical rate-limiting event that stimulates induction of allergic sensitization and airway inflammation. Inhibiting pollen-induced recruitment of neutrophils, such as by administration of CXCR2 antagonists, may be a novel strategy to prevent initiation of pollen-induced allergic airway inflammation.

Keywords: CXCR2; Toll-like receptor 4; allergic inflammation; neutrophil; reactive oxygen species.

Figure 1.

Ragweed pollen extract (RWPE) challenge induces an innate immune response in the airways. (A) Single-challenge model protocol. (B) Bronchoalveolar lavage fluid (BALF) neutrophil number. RWPE challenge in naive wild-type (WT) mice recruited neutrophils into airways 16 and 72 hours after challenge (n = 5–7 per group). (C) BALF levels of chemokine (C-X-C motif) ligand (CXCL) 1 and CXCL2 4 hours after challenge. RWPE challenge in naive WT mice increases CXCL1 and CXCL2 in airways 4 hours after challenge. (D) Ex vivo superoxide generation from BALF cells of WT mice. WT mice were killed at 30 minutes and 16 hours after RWPE challenge (n = 3–5 per group), and ex vivo superoxide generation from BALF cells was quantified. There was no difference in superoxide generation in any treatment group 30 minutes after challenge. However, at 16 hours after challenge, the RWPE challenge group produced more superoxides. Data are expressed as means ± SEM. *P < 0.05. **P < 0.01. ****P < 0.0001. NS, not significant.

Figure 2.

Deletion of Toll-like receptor 4 inhibits RWPE challenge–induced innate immune response. (A) CXCL1 and CXCL2 mRNA expression in lungs. RWPE challenge increased the expression of CXCL1 and CXCL2 mRNA 1 hour after challenge in naive WT mice but not in Tlr4 knockout (KO) mice (n = 3 per group). (B) BALF levels of CXCL1 and CXCL2. RWPE challenge increased CXCL1 and CXCL2 levels 4 hours after RWPE challenge in naive WT mice but not in Tlr4 KO mice (n = 3–5 per group). (C) BALF neutrophil numbers. RWPE challenge increased the number of neutrophils in BALF at 16 hours after challenge in naive WT mice but not in Tlr4 KO mice (n = 5–7 per group). (D) Ex vivo superoxide generation from BALF cells. After PBS or RWPE challenge, ex vivo superoxide generation from BALF was quantified. Ex vivo superoxide generation from BALF cells increased 16 hours after RWPE challenge in naive WT mice but not in Tlr4 KO mice (n = 3–5 per group). Data are expressed as means ± SEM. *P < 0.05. **P < 0.01. ****P < 0.0001.

Figure 3.

Effect of Toll-like receptor 4 on RWPE-induced allergic airway inflammation. (A) Repeated-challenge model protocol. (B–F) RWPE repeated-challenge model in WT mice and Tlr4 KO mice. (B) BALF eosinophil and total inflammatory cell numbers. Multiple challenges with RWPE induced a greater increase in the number of eosinophils and total inflammatory cells in WT mice compared with Tlr4 KO mice. (C and D) Mucin secretion in airway epithelial cells. Multiple challenges with RWPE induced a greater increase in mucin secretion in WT mice compared with Tlr4 KO mice. (C) Mucin secretion in airway epithelial cells. Original magnification: ×400. (D) Epithelial mucin score. (E) Serum ragweed-specific IgE. Multiple challenge with RWPE induced an increase in serum ragweed-specific IgE in WT mice but not in Tlr4 KO mice. (F) T helper type 2 (Th2) cytokines in BALF. Multiple challenges with RWPE induced an increase in BALF levels of IL-5, IL-13, thymic stromal lymphopoietin (TSLP), and IL-33 in WT mice but not in Tlr4 KO mice. For all groups, 5 to 21 mice per group were used. Data are expressed as means ± SEM. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 4.

Effect of forced neutrophil recruitment in Tlr4 KO mice on RWPE-induced allergic airway inflammation. (A) BALF CXCL1 levels in Tlr4 KO mice. Intranasal challenge with RWPE alone or xanthine with xanthine oxidase (X+XO) alone failed to induce secretion of CXCL1. Administration of a cocktail of RWPE and X+XO induced secretion of CXCL1. (B) BALF neutrophil numbers in Tlr4 KO mice. Intranasal challenge with RWPE alone or X+XO alone failed to increase neutrophil recruitment in BALF 16 hours after challenge. Administration of a cocktail of RWPE and X+XO increased recruitment of neutrophils. (C–E) Effect of repeated RWPE challenge in the presence or absence of X+XO in Tlr4 KO mice. (C) BALF eosinophil and total inflammatory cell numbers in Tlr4 KO mice. RWPE+X+XO challenge increased the number of eosinophils and total inflammatory cells in BALF. (D and E) Mucin secretion in airway epithelial cells of Tlr4 KO mice. RWPE multiple challenges induced a greater increase in mucin secretion in mice challenged with RWPE+X+XO compared with RWPE. (D) Mucin secretion in airway epithelial cells. Original magnification: ×400. (E) Epithelial mucin score. For all groups, five to eight mice per group were used. Data are expressed as means ± SEM. **P < 0.01. ***P < 0.001. ****P < 0.0001.

Figure 5.

Effect of chemokine (C-X-C motif) receptor 2 (CXCR2) inhibitor (INH) on RWPE challenge–induced innate and allergic inflammation. (A) Protocol for single-challenge model after intranasal administration of CXCR2 inhibitor. (B) BALF neutrophil numbers in WT mice. Administration of CXCR2 inhibitor before RWPE challenge inhibited neutrophil recruitment 16 hours after challenge (n = 5–9 per group). (C) Ex vivo superoxide generation from BALF cells. Intranasal administration of CXCR2 inhibitor before RWPE challenge inhibited ex vivo superoxide generation. *P < 0.05 compared with all other groups (n = 3–4 per group). (D) Protocol for repeated-challenge model in naive WT mice with or without CXCR2 inhibitor. (E–I) Effect of repeated RWPE challenge in the presence or absence of CXCR2 inhibitor in WT mice (n = 5–9 per group). (E) BALF eosinophil and total inflammatory cell numbers. Administration of CXCR2 inhibitor before RWPE challenge inhibited the number of eosinophils and total inflammatory cells. (F and G) Mucin secretion in airway epithelial cells. Administration of CXCR2 inhibitor before RWPE challenge inhibited the increase in mucin secretion. (F) Mucin secretion in airway epithelial cells. Original magnification: ×400. (G) Epithelial mucin score. (H) Serum ragweed-specific IgE. Administration of CXCR2 inhibitor before RWPE challenge inhibited serum ragweed-specific IgE levels. (I) Th2 cytokines in BALF. Administration of CXCR2 inhibitor before RWPE challenge inhibited secretion of IL-5, IL-13, TSLP, and IL-33 in BALF. Data are expressed as means ± SEM. *P < 0.05. **P < 0.01.

Figure 6.

Effect of repeated intranasal administration of neutrophils from donor mice into Tlr4 KO recipient mice after RWPE challenge. (A) Protocol for the repeated-challenge model in Tlr4 KO mice with or without neutrophil replacement 8 hours prior to each instillation of RWPE. (B–F) Effect of repeated RWPE challenge with or without replacement of activated neutrophils in Tlr4 KO mice, assessed on Day 14, 72 hours after the final RWPE challenge on Day 11. (B) BALF eosinophil and total inflammatory cell numbers in Tlr4 KO mice. Intranasal administration of neutrophils after RWPE challenge increased the number of late-phase (72 h) BALF eosinophils and total inflammatory cells. (C and D) Mucin secretion in airway epithelial cells of Tlr4 KO mice. Intranasal administration of neutrophils after RWPE challenge stimulated mucin secretion in Tlr4 KO mice. (C) Mucin secretion in airway epithelial cells. Original magnification: ×400. (D) Epithelial mucin score. (E) Serum ragweed-specific IgE. Intranasal administration of neutrophils after RWPE challenge increased levels of serum ragweed-specific IgE. (F) Th2 cytokines in BALF. Intranasal administration of neutrophils after RWPE challenge increased secretion of IL-5, IL-13, TSLP, and IL-33 in BALF. For all groups, 6 to 18 mice per group were used. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.