Toll-like receptor ligands LPS and poly (I:C) exacerbate airway hyperresponsiveness in a model of airway allergy in mice, independently of inflammation

Abstract

It is well-established that bacterial and viral infections have an exacerbating effect on allergic asthma, particularly aggravating respiratory symptoms, such as airway hyperresponsiveness (AHR). The mechanism by which these infections alter AHR is unclear, but some studies suggest that Toll-like receptors (TLRs) play a role. In this study, we investigated the impact of TLR3 and TLR4 ligands on AHR and airway inflammation in a model of pre-established allergic inflammation. Female BALB/c mice were sensitised and challenged intranasally (i.n.) with either PBS or ovalbumin (OVA) and subsequently i.n. challenged with poly (I:C) (TLR3) or LPS (TLR4) for four consecutive days. The response to methacholine was measured in vivo; cellular and inflammatory mediators were measured in blood, lung tissue and broncheoalveolar lavage fluid (BALF). OVA challenge resulted in an increase in AHR to methacholine, as well as increased airway eosinophilia and TH2 cytokine production. Subsequent challenge with TLR agonists resulted in a significant increase in AHR, but decreased TLR-specific cellular inflammation and production of immune mediators. Particularly evident was a decline in LPS-induced neutrophilia and neutrophil-associated cytokines following LPS and poly (I:C) treatment. The present data indicates that TLRs may play a pivotal role in AHR in response to microbial infection in allergic lung inflammation. These data also demonstrate that aggravated AHR occurs in the absence of an exacerbation in airway inflammation and that allergic inflammation impedes a subsequent inflammatory response to TLRs. These results may parallel clinical signs of microbial asthma exacerbation, including an extended duration of illness and increased respiratory symptoms.

Figure 1. Airway hyperresponsiveness in OVA-sensitised and challenged mice treated with LPS or poly (I:C).

All animals were sensitised i.p. with OVA/Al (OH)₃ and subsequently challenged i.n. with PBS or OVA (3 days) and PBS, LPS or Poly (I:C) (4 days). 24 hrs after the final challenge, changes in lung resistance (R_L) in response to increasing doses of methacholine (MCh) was measured using the flexiVent animal respirator. Data is represented as mean resistance (R_L) ± SEM. (A) Animals were treated with PBS or LPS for 4 days following OVA or PBS treatment. (B) Animals were treated with PBS or Poly (I:C) for 4 days following OVA or PBS treatment. ¤ p<0.05 comparing EC₅₀ values between OVA-PBS and OVA-LPS or OVA-PBS and OVA-Poly (I:C) using one-way ANOVA followed by a Tukey multiple-comparison post-test. ****p<0.0001 comparing R_Lmax of OVA and PBS challenged groups (OVA-PBS vs. PBS-PBS; OVA-LPS vs. PBS-LPS; OVA-Poly (I:C) vs. PBS-Poly (I:C)) using one-way ANOVA followed by a Tukey multiple-comparison post-test. n = 13–16 animals per group.

Figure 2. Inflammatory cells in blood (A–C), tissue (D–F) and BALF (G–I) following TLR ligand challenge in OVA-sensitised mice.

All animals were sensitised i.p. with OVA/Al (OH)₃ and subsequently challenged i.n. with PBS (grey bars) or OVA (black bars) (3 days) and PBS, LPS or Poly (I:C) (4 days). 24 hrs after the final challenge, blood, tissue and BALF were collected. Neutrophils (A, D), eosinophils (B, E) and CD3⁺ cells (C, F) in blood and tissue were determined using flow cytometry; a differential cell count was used to determine neutrophil (G), eosinophil (H) and lymphocyte (I) number in BALF. Data is represented as mean ± SEM. Data was analysed using a two-way ANOVA followed by a Bonferroni multiple comparison post-test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. n = 6 (blood, tissue) or 14 (BALF) animals per group.

Figure 3. Inflammatory mediators in BALF following TLR ligand challenge in OVA-sensitised mice.

All animals were sensitised i.p. with OVA/Al (OH)₃ and subsequently challenged i.n. with PBS or OVA (3 days) and PBS, LPS or Poly (I:C) (4 days). 24 hrs after the final challenge, BALF was extracted and cytokine levels were measured using the Cytokine Mouse 20-Plex Panel and the RANTES Mouse Singleplex Bead Kit. Data is represented as mean ± SEM. Data was analysed using a two-way ANOVA followed by a Bonferroni multiple comparison post-test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. n = 6–14 animals per group.

Figure 4. Neutrophil and T-lymphocyte populations in blood and tissue following TLR ligand challenge in OVA-sensitised mice.

All animals were sensitised i.p. with OVA/Al (OH)₃ and subsequently challenged i.n. with PBS (grey bars) or OVA (black bars) (3 days) and PBS, LPS or Poly (I:C) (4 days). 24 hrs after the final challenge, blood (C, D) and lung tissue (A, B) were collected and percent of Ly6C^lo neutrophils (A, C) and CD8⁺ CD3⁺ lymphocytes (B, D) were measured by flow cytometry. Data is represented as mean percent ± SEM. Data was analysed using a two-way ANOVA followed by a Bonferroni multiple comparison post-test *p<0.05, **p<0.01, ***p<0.001; n = 6 per animals per group.