Hyaluronan fragments contribute to the ozone-primed immune response to lipopolysaccharide

Abstract

Hyaluronan is a high-molecular mass component of pulmonary extracelluar matrix, and lung injury can generate a low-molecular mass hyaluronan (HA) fragment that functions as endogenous ligand to cell surface receptors CD44 and TLR4. This leads to activation of intracellular NF-κB signaling and proinflammatory cytokine production. Based on previous information that ozone exposure causes increased HA in bronchial alveolar lavage fluid and ozone pre-exposure primes immune response to inhaled LPS, we hypothesized that HA production during ozone exposure augments the inflammatory response to LPS. We demonstrate that acute ozone exposure at 1 part per million for 3 h primes the immune response to low-dose aerosolized LPS in C57BL/6J mice, resulting in increased neutrophil recruitment into the airspaces, increased levels of protein and proinflammatory cytokines in the bronchoalveolar lavage fluid, and increased airway hyperresponsiveness. Intratracheal instillation of endotoxin-free HA (25 μg) enhances the biological response to inhaled LPS in a manner similar to ozone pre-exposure. In vitro studies using bone marrow-derived macrophages indicate that HA enhances LPS responses measured by TNF-α production, while immunofluorescence staining of murine alveolar macrophages demonstrates that HA induces TLR4 peripheralization and lipid raft colocalization. Collectively, our observations support that ozone primes macrophage responsiveness to low-dose LPS, in part, due to HA-induced TLR4 peripheralization in lung macrophages.

Figure 1. Comparison of biological effects of ozone exposure at different doses

Ozone-induced inflammatory response in the lung (A) and level of protein in the lung lavage (B) are exposure dose-dependent. C) 1ppm and 2ppm of ozone did not produce different level of airway hyperresponsiveness (* p<0.05, compared with filtered air group; # p<0.05, 2 ppm vs. 1 ppm; N=6).

Figure 2. Ozone exposure primed pulmonary response to inhaled LPS

Animals were exposed to either filtered air (FA) or ozone at 1ppm for 3 hours. After 24 hours, mice were then exposed to aerosolized LPS 2.5 hours. At 4 h post exposure, animals were phenotyped. Ozone pre-exposure significantly increased LPS inhalation-induced lung inflammation (A) and epithelial injury (B). C) Airway hyperresponsiveness was significantly augmented in co-exposed group, when compared with single exposed groups. D) LPS-induced IL-6, MCP-1, and TNFα production were enhanced by ozone pre-exposure. (* p<0.05, **p<0.01, N=6).

Figure 3. Ozone exposure increased HA level in lavage fluid

Lavage fluid from ozone-exposed group contained higher level of HA, when compared with FA group. Although low dose LPS exposure did not increase HA level, HA was significantly enhanced in ozone/LPS co-exposed group. (** p<0.01, N= 8 for FA and O3 groups, N=5 for LPS and co-exposure groups).

Figure 4. zone-enhanced LPS responsiveness was partially blocked by HA binding peptide (HABP)

Oropharygeal aspiration of HABP was performed immediately prior to ozone exposure. 24 h later, animals were then exposed to LPS aerosol. A) HABP partially inhibited inflammatory cell recruitment. B) HABP did not affect the lavage level of total protein. C) Enhanced AHR were completely blocked by HABP pretreatment. D) HABP pretreatment blocked ozone-augmented IL-6, MCP-1 and TNFα production in response to LPS. (* p<0.05, N=6).

Figure 5. Hyaluronan fragments enhanced pulmonary response to inhaled LPS

50μl of HA at 0.5 mg/ml was intratracheally instilled into the lung 2 hours prior to LPS inhalation. A) HA pretreatment significantly enhanced inflammatory cell infiltration in response to LPS exposure. B) HA instillation enhanced LPS-induced lung injury marked by increased lavage protein. C) HA-LPS coexposed group demonstrated increased airway reactivity to 100 mg/ml of methacholine challenge, compared with LPS-exposed group. D) HA instillation increased LPS-induced IL-6 production. (* p<0.05, N=10).

Figure 6. HA primed macrophages for increased response to LPS

BMDM were treated with lavage fluid (BAL) from air/O3-treated mice or HA for 2 hours prior to LPS exposure. Supernatant were collected after 4 h LPS stimulation and evaluated for TNFα production. BAL from O3-treated animals but not HABP-pretreated animals significantly increased TNFα production in BMDM in response to 0.1ng/ml LPS (A) or 1ng/ml LPS (B) stimulation (*p<0.05, N=6). C) Dose and time response of BMDM to HA treatment (* p<0.05, compared with vehicle control; # p<0.05, compared with 4h incubation; N=6); D) HA enhanced LPS-induced TNFα production in BMDM in response to LPS stimulation (*p<0.05, N=6).

Figure 7. Low dose ozone induced TLR4 peripheralization on macrophages

Alveolar macrophages (AM) collected from BAL were analyzed by flow cytometry for TLR4 surface expression by MFI A), geometric mean B), and % high TLR4 positive C). 1ppm of ozone increased surface expression of TLR4 as demonstrated by median fluorescence intensity, geometric mean fluorescence, and percentage of alveolar macrophages with high TLR4 expression (*p<0.05, N=3 per group, every sample was pooled samples from two mice).

Figure 8. HA induced TLR4 peripheralization is associated with increased functional response to LPS

A) Studies with MHS cells demonstrate that HA increased TLR4 peripheralization and colocalization with lipid raft in a NF-κB dependent manner. Cultured MH-S cells were pre-treated with or without PDTC (400 μM) for 1 hour, then cells were incubated with 25 μg/ml HA for 1 hour. TLR4 surface expression (red) and co-localization with lipid raft (green) were showed in overlay images. Confocal images were taken under 100x magnification. B) HA increased MH-S cell susceptibility to LPS. MH-S cells were treated with 25 μg/ml of HA for 30 min, and then exposed to LPS for 2 hours. Multiple TNFα productions in supernatant were enhanced by HA pre-treatment (*p<0.05, N=4).