TLR4 is necessary for hyaluronan-mediated airway hyperresponsiveness after ozone inhalation

Abstract

Rationale: Ozone is a common environmental air pollutant that contributes to hospitalizations for respiratory illness. The mechanisms, which regulate ozone-induced airway hyperresponsiveness, remain poorly understood. We have previously reported that toll-like receptor 4 (TLR4)-deficient animals are protected against ozone-induced airway hyperresponsiveness (AHR) and that hyaluronan (HA) mediates ozone-induced AHR. However, the relation between TLR4 and hyaluronan in the airway response to ozone remains unexplored.

Objectives: We hypothesized that HA acts as an endogenous TLR4 ligand for the development of AHR after ozone-induced environmental airway injury.

Methods: TLR4-deficient and wild-type C57BL/6 mice were exposed to either inhaled ozone or intratracheal HA and the inflammatory and AHR response was measured.

Measurements and main results: TLR4-deficient mice have similar levels of cellular inflammation, lung injury, and soluble HA levels as those of C57BL/6 mice after inhaled ozone exposure. However, TLR4-deficient mice are partially protected from AHR after ozone exposure as well as after direct intratracheal instillation of endotoxin-free low molecular weight HA. Similar patterns of TLR4-dependent cytokines were observed in the bronchial alveolar lavage fluid after exposure to either ozone or HA. Exposure to ozone increased immunohistological staining of TLR4 on lung macrophages. Furthermore, in vitro HA exposure of bone marrow-derived macrophages induced NF-kappaB and production of a similar pattern of proinflammatory cytokines in a manner dependent on TLR4.

Conclusions: Our observations support the observation that extracellular matrix HA contributes to ozone-induced airways disease. Furthermore, our results support that TLR4 contributes to the biological response to HA by mediating both the production of proinflammatory cytokines and the development of ozone-induced AHR.

Figure 1.

Ozone-induced airway hyperresponsiveness (AHR) and proinflammatory factors in bronchoaveolar lavage fluid. Mice were exposed to either filtered air or ozone (2 ppm × 3 hrs) and invasive measurements of airway response to methacholine were determined by flexivent. (A) Toll-like receptor 4 (TLR4) is essential for the development of ozone-induced AHR (n = 6; *P < 0.05). (B) Proinflammatory cytokines were measured from the cell-free supernatant by luminex bead profiling. The level of keratinocyte-derived chemokine, IL-1β, IL-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α were increased with exposure to ozone and dependent on the presence of TLR4 (n = 6; *P < 0.01).

Figure 2.

Ozone enhanced soluble hyaluronan in the bronchoaveolar lavage fluid. (A) Lung lavage fluid hyaluronan (HA) levels were measured and elevated 24 hours after ozone exposure (n = 10; *P < 0.05). (B) Agar gel electrophoresis of concentrated lung lavage fluid HA, visualized with staining with Stains-All (Sigma, St. Louis, MO). Lane 1: low molecular weight HA ladder. Lane 2: high molecular weight HA ladder. Lane 3: high molecular weight HA (Healon). Lane 4: sonicated Healon (low molecular weight HA). Lane 5: Toll-like receptor-4^−/− (TLR-4^−/−) mouse lavage fluid after ozone exposure. Lane 6: C57BL/6 mouse lavage fluid after ozone exposure. Lane 7: representative C57BL/6 and TLR4^−/− mouse lavage fluid after free air exposure. Lane 8: Ozone-exposed mouse lavage fluid after treatment with hyaluronidase.

Figure 3.

Hyaluronan-induced airway hyperresponsiveness (AHR) and cytokines are dependent on toll-like receptor-4 (TLR4). (A) Direct instillation of hyaluronan (HA) into the lungs of mice resulted in AHR in C57BL6J mice, when compared with vehicle instillation. TLR4-deficient mice were relatively protected from HA-induced AHR, when compared with C57BL/6J (n = 5; *P < 0.05). (B) Levels of proinflammatory cytokines in cell-free supernatant as measured by luminex bead profiling. The levels of keratinocyte-derived chemokine, IL-1β, IL-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α were increased with HA challenge and dependent on the presence of TLR4 (n = 5; *P < 0.01).

Figure 4.

Hyaluronan and toll-like receptor 4 (TLR4) staining in the airways after ozone exposure. Confocal immunohistochemistry was evaluated on lung histology; HA is colored green, TLR4 is colored red, and colocalization is colored yellow (400× magnification). (A) In air-exposed TLR4-deficient mice, there is some visible subepithelial HA. (B) In air-exposed wild-type mice, faint TLR4 staining is observed in bronchial epithelial cells. (C) No significant change in ozone-exposed TLR4-deficient mice. (D) Wild-type mice show up-regulation of TLR4 expression in bronchial epithelia. No significant colocalization of TLR4 with HA is observed.

Figure 5.

Hyaluronan and toll-like receptor 4 (TLR4) costaining in the parenchyma after ozone exposure. Confocal immunohistochemistry was evaluated on lung histology; hyaluronan (HA) is colored green, TLR4 is colored red, and colocalization is colored yellow (1200× magnification). In (A) air-exposed TLR4-deficient (B) and wild-type mice, there is very little HA staining (green) on alveolar macrophages. (B) Faint TLR4 staining (red) is observed on air-exposed alveolar macrophage. No significant colocalization of HA and TLR4 is observed. After ozone exposure, HA staining (green) is observed in the macrophage membrane (arrows) in both (C) TLR4-deficient and (D) wild-type mice. TLR4 staining on alveolar macrophages appears significantly enhanced after ozone exposure in (D) wild-type mice. (E) Close-up of merged image (D) shows colocalization of TLR4 and HA on macrophage membranes (arrows). (F) Semiquantitative analysis of TLR4-HA colocalization in murine airways and alveoli at 600× magnification shows increased colocalization after ozone exposure. (G) Coimmunoprecipitation for CD44-TLR4 (Immunoprecipitation: CD44, blot: TLR4) shows presence of CD44-TLR4 complexes in alveolar macrophage lysates from lung lavage fluid (AM = alveolar macrophages) and whole-lung lysates. Each band represents a sample from a separate mouse.

Figure 6.

Ozone activates nuclear factor-κB (NF-κB) in alveolar macrophages and airway epithelia. (A) Using NF-κB reporter mice, we identify a significant increase in NF-κB activity by luminescence in whole lung homogenates both 3 hours and 24 hours after exposure to ozone when compared with naive (unexposed mice). No increase in luminescence was detected immediately after completion of ozone exposure (time = 0 hr) (n = 5; *P < 0.05). To determine the specific cell types with NF-κB activation, tissue histology was immunostained for green flourescent protein (GFP)-reporter 24 hours after either air (B–C) or ozone (D–E) challenge. Immunostaining is present in many cell types in the lung only after exposure to ozone. RLU = Relative Luminescence Units.

Figure 7.

Hyaluronan activates nuclear factor-κB (NF-κB) in alveolar macrophages and airway epithelia. (A) Using NF-κB reporter mice, we identify a significant increase in NF-κB activity by luminescence in whole lung homogenates both 1 hour and 12 hours after exposure to HA when compared with vehicle (n = 6 per group; *P < 0.05). To determine the specific cell types with NF-κB activation, tissue histology was immunostained for green flourescent protein (GFP)-reporter 2 hours after either vehicle (B–C) or HA challenge (D–E). Dramatically increased immuno-staining is present on alveolar macrophages and airway epithelia after exposure to hyaluronan (arrows).

Figure 8.

Macrophages generate pro-inflammatory cytokines when exposed to hyaluronan. Bone marrow–derived macrophages were collected from C57BL/6J and toll-like receptor 4^−/− (TLR4^−/−) mice. (A) Bone marrow macrophages were directly challenged to increasing doses of hyaluronan (HR). HR can lead to a modest increase of tumor necrosis factor (TNF)-α in the supernatant from C57BL/6J macrophages (n = 5; *P < 0.01). (B) Level of proinflammatory cytokines in supernatant as measured by luminex bead profiling. The level of keratinocyte-derived chemokine (KC), IL-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), and TNF-α were increased with HA (100 μg/ml) challenge and dependent on the presence of TLR4 (n = 5; *P < 0.01).

Figure 9.

Hyaluronan activation of nuclear factor-κB (NF-κB) in macrophages is dependent on toll-like receptor 4 (TLR4). Bone-marrow derived macrophages were collected from NF-κB reporter mice (C57BL/6J and TLR4^−/−). The intensity of NF-κB activation was increased with either hyaluronan (50 μg/ml) or LPS (50 ng/ml) challenge and dependent on the presence of TLR4 (n = 5; *P < 0.01).