Intranasal Administration of poly(I:C) and LPS in BALB/c Mice Induces Airway Hyperresponsiveness and Inflammation via Different Pathways

BACKGROUND: Bacterial and viral infections are known to promote airway hyperresponsiveness (AHR) in asthmatic patients. The mechanism behind this reaction is poorly understood, but pattern recognizing Toll-like receptors (TLRs) have recently been suggested to play a role. MATERIALS AND METHODS: To explore the relation between infection-induced airway inflammation and the development of AHR, poly(I:C) activating TLR3 and LPS triggering TLR4, were chosen to represent viral and bacterial induced interactions, respectively. Female BALB/c or MyD88-deficient C57BL/6 mice were treated intranasally with either poly(I:C), LPS or PBS (vehicle for the control group), once a day, during 4 consecutive days. RESULTS: When methacholine challenge was performed on day 5, BALB/c mice responded with an increase in airway resistance. The maximal resistance was higher in the poly(I:C) and LPS treated groups than among the controls, indicating development of AHR in response to repeated TLR activation. The proportion of lymphocytes in broncheoalveolar lavage fluid (BALF) increased after poly(I:C) treatment whereas LPS enhanced the amount of neutrophils. A similar cellular pattern was seen in lung tissue. Analysis of 21 inflammatory mediators in BALF revealed that the TLR response was receptor-specific. MyD88-deficient C57BL/6 mice responded to poly (I:C) with an influx of lymphocytes, whereas LPS caused no inflammation. CONCLUSION: In vivo activation of TLR3 and TLR4 in BALB/c mice both caused AHR in conjunction with a local inflammatory reaction. The AHR appeared to be identical regardless of which TLR that was activated, whereas the inflammation exhibited a receptor specific profile in terms of both recruited cells and inflammatory mediators. The inflammatory response caused by LPS appeared to be dependent on MyD88 pathway. Altogether the presented data indicate that the development of AHR and the induction of local inflammation might be the result of two parallel events, rather than one leading to another

Poly(I:C) and LPS induced cytokine release in BALF.

<p>Levels of (A) IL-5, (B) IL-12, (C) MCP-1, (D) KC, (E) MIG and (F) VEGF in BALB/c mice treated during 4 days with vehicle (white), poly(I:C) (black) or LPS (gray) i.n. Results are expressed as mean ± SEM. PBS n = 10; poly I:C n = 12; LPS n = 13; ***p<0.001.</p

Poly(I:C) and LPS induced cell recruitment in lung tissue.

<p>Haematoxylin and eosin stained cells in BALB/c mice treated i.n. during 4 days with (A) vehicle, (B) poly(I:C) (mainly neutrophils) and (C) LPS (mainly lymphocytes). Magnification is ×200. (D) Semi quantitative grading score of cell infiltration in the lung.</p

Poly(I:C) and LPS induced cell recruitment in BALF.

<p>(A, C) Absolute and (B, D) relative cell numbers in BALF in BALB/c mice (A, B) respectively MyD88deficient C57BL/6 mice (C, D) treated during 4 days with vehicle (PBS), poly(I:C) or LPS i.n.. Results are expressed as mean ± SEM. *p<0.05.</p

LPS and poly(I:C) induced airway hyperresponsiveness.

<p>(A) Changes in lung resistance (R_L) and (B) compliance (C_L) in response to methacholine in BALB/c mice treated during 4 days with vehicle (PBS), poly(I:C) or LPS i.n.. Results are expressed as mean ± SEM. *p<0.05, ***p<0.001 vs. PBS.</p

Toll-Like Receptor Ligands LPS and Poly (I:C) Exacerbate Airway Hyperresponsiveness in a Model of Airway Allergy in Mice, Independently of Inflammation

<div><p>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 <i>in vivo</i>; 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.</p></div

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

<p>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.</p

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

<p>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.</p