Long-term activation of TLR3 by poly(I:C) induces inflammation and impairs lung function in mice

Abstract

Background: The immune mechanisms associated with infection-induced disease exacerbations in asthma and COPD are not fully understood. Toll-like receptor (TLR) 3 has an important role in recognition of double-stranded viral RNA, which leads to the production of various inflammatory mediators. Thus, an understanding of TLR3 activation should provide insight into the mechanisms underlying virus-induced exacerbations of pulmonary diseases.

Methods: TLR3 knock-out (KO) mice and C57B6 (WT) mice were intranasally administered repeated doses of the synthetic double stranded RNA analog poly(I:C).

Results: There was a significant increase in total cells, especially neutrophils, in BALF samples from poly(I:C)-treated mice. In addition, IL-6, CXCL10, JE, KC, mGCSF, CCL3, CCL5, and TNFalpha were up regulated. Histological analyses of the lungs revealed a cellular infiltrate in the interstitium and epithelial cell hypertrophy in small bronchioles. Associated with the pro-inflammatory effects of poly(I:C), the mice exhibited significant impairment of lung function both at baseline and in response to methacholine challenge as measured by whole body plethysmography and an invasive measure of airway resistance. Importantly, TLR3 KO mice were protected from poly(I:C)-induced changes in lung function at baseline, which correlated with milder inflammation in the lung, and significantly reduced epithelial cell hypertrophy.

Conclusion: These findings demonstrate that TLR3 activation by poly(I:C) modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.

Figure 1

Poly(I:C) induces a dose dependent influx of inflammatory cells into the airways of mice. Mice were administered PBS or, 10, 20, 50 or 100 μg poly(I:C) (I.N.) every 24 h for three days. 24 hours after the last administration, mice were euthanized and BALs were performed. The total number of cells (1A), neutrophils (1B) and mononuclear cells (1C) were measured in the BAL. Data are the mean ± SEM of 6–15 mice from two separate experiments. The Kruskal-Wallace test was used to compare the treatment groups. When this test showed a difference among the treatment groups, selected pairs of treatments were compared using Dunn's multiple comparison test. ** p < 0.001 when compared to PBS-treated mice.

Figure 2

TLR3 KO mice are partially protected from poly(I:C)-induced inflammation in lung interstitium. Representative H&E-stained lung sections from WT- PBS treated (A,E, I)WT poly(I:C)-treated (B, F, J), TLR3 KO PBS treated mice (C ,G, K) and TLR3 KO poly(I:C)-treated (D, H, L). Figures A-L are representative images from each group. Figure A-D are at 10×, Figures E-H are at 40 × and Figures I-L are at 60 ×.

Figure 3

Poly(I:C) induces cytokine secretion from BEAS-2B cells. BEAS-2B cells were incubated for 24 hours at 37°C with serial dilutions of polyI:C. Supernatants were collected after 24 hours and assayed for cytokine levels of IL-6 (A), IL-8 (B), CCL2 (C), CCL5 (D), and CXCL10 (E). Data is representative of 2 different experiments.

Figure 4

Poly(I:C) induces impairment of lung function and AHR. Mice were administered PBS or 10, 20, 50 or 100 μg polyI:C (I.N.) every 24 h for three days. 24 h after the last poly(I:C) administration, baseline lung function and AHR to increasing doses of methacholine was measured by whole body plethysmography (A & B). The 100 ug poly I:C group had higher penh levels than the PBS, 10, and 20 ug groups, p < 0.05 (B). Methacholine challenge resulted in a larger increase from baseline in the poly(I:C)-treated groups than in the PBS group, p < 0.001 for each methacholine dose. Invasive measurements of lung function were performed 24 h following three administrations (24 h apart) of 100 μg poly(I:C) (C). Peak airway resistance after i.v. injection of methacholine at 240 ug/kg are shown. Methacholine challenge resulted in a larger increase from baseline in the poly(I:C)-treated group than in the PBS group, p = 0.015. Repeated measures ANOVA was used to assess the Penh values over increasing methacholine doses as well as to compare increases in resistance in response to methacholine from baseline among the groups. Data are the mean ± SEM of 5–7 mice.

Figure 5

TLR3 KO mice are partially protected from poly(I:C)-induced inflammatory cell influx in the airways. Mice were administered PBS or 100 μg poly(I:C) I.N. every 24 h for three days. 24 hours after the last poly(I:C) administration, mice were euthanized and the lungs were lavaged. The total number of cells (5A), neutrophils (5B) and mononuclear cells(5C) were measured in the BAL. Data are the mean ± SEM of 6 mice. Treatment groups (PBS or 100 μg poly(I:C)) and mouse types were compared using 2-way ANOVA, including an interaction term. *p < 0.05, **p < 0.01 compared to PBS-treated mice. When comparing the impact of poly(I:C) treatment on cell populations in the lavage, there was a significantly larger increase in the response of wild type mice than knockout mice, with respect to total cells and mononuclear cells alone, **p < 0.01 in each case. Similar trends were observed in neutrophils alone but failed to reach statistical significance (p = 0.056).

Figure 6

TLR3 KO mice are partially protected from poly(I:C)-induced impairment of lung function and AHR. Mice were exposed to three doses of 100 mg poly(I:C) (I.N.; 24 h apart). Baseline lung function and AHR to increasing doses of methacholine was measured by whole body plethysmography 24 hours following the last dose of poly(I:C). Data are the mean ± SEM of 6 mice. Prior to challenge, the groups given poly(I:C) had higher Penh values than those given PBS, p < 0.001. This difference was greater in the WT mice than in the KO mice, p = 0.047. Increasing methacholine challenges lead to higher mean penh values for the Poly I:C treated groups than for the PBS groups, p < 0.001, but there was not a statistically significant difference between the poly I:C-treated KO and WT groups p = 0.115. A repeated measure ANOVA was used to assess the change from pre-challenge penh values over increasing methacholine doses. ANOVA was used to compare the peak resistance levels at baseline among the groups.