Hyaluronan Signaling during Ozone-Induced Lung Injury Requires TLR4, MyD88, and TIRAP

Ozone exposure is associated with exacerbation of reactive airways disease. We have previously reported that the damage-associated molecular pattern, hyaluronan, is required for the complete biological response to ambient ozone and that hyaluronan fragments signal through toll-like receptor 4 (TLR4). In this study, we further investigated the role of TLR4 adaptors in ozone–induced airway hyperresponsiveness (AHR) and the direct response to hyaluronan fragments (HA). Using a murine model of AHR, C57BL/6J, TLR4−/−, MyD88−/−, and TIRAP−/− mice were characterized for AHR after exposure to either ozone (1 ppm×3 h) or HA fragments. Animals were characterized for AHR with methacholine challenge, cellular inflammation, lung injury, and production of pro-inflammatory cytokines. Ozone-exposed C57BL/6J mice developed cellular inflammation, lung injury, pro-inflammatory cytokines, and AHR, while mice deficient in TLR4, MyD88 or TIRAP demonstrated both reduced AHR and reduced levels of pro-inflammatory cytokines including TNFα, IL-1β, MCP-1, IL-6 and KC. The level of hyaluronan was increased after inhalation of ozone in each strain of mice. Direct challenge of mice to hyaluronan resulted in AHR in C57BL/6J mice, but not in TLR4−/−, MyD88−/−, or TIRAP−/− mice. HA-induced cytokine production in wild-type mice was significantly reduced in TLR4−/−, MyD88−/−, or TIRAP−/− mice. In conclusion, our findings support that ozone-induced airway hyperresponsiveness is dependent on the HA-TLR4-MyD88-TIRAP signaling pathway

Ozone inhalation increased airway sensitivity to methacholine challenge and was dependent on the TLR4-MyD88-TIRAP signaling pathway.

<p>Animals were exposed to filtered air (FA) or 1 ppm of ozone for 3 hours. Airway responsiveness to methacholine challenge was measured 24 h later. <b>A</b>) Ozone-induced AHR was increased in WT mice but not in TLR4−/− mice, <b>B</b>) MyD88−/−mice, or <b>C</b>) TIRAP−/− mice (*p<0.05 vs. FA-WT; #p<0.05 vs. O3-WT, N = 5–6 per group).</p

HA challenge was not sufficient for either neutrophilic inflammation or epithelial injury.

<p>(<b>A</b>) There were no observed differences in cellular inflammation in the airspace 2 hours after direct challenge to HA, (<b>B</b>) When compared to ozone challenge, HA challenge had no observed effect on the level of BALF total protein (*p<0.05, vs. FA exposed group, N = 5).</p

Direct challenge to hyaluronan fragments increased the level of pro-inflammatory factors in alveolar lavage fluid in a manner partially dependent on TLR4, MyD88, and TIRAP.

<p>The level of (<b>A</b>) KC, (<b>B</b>) IL-1β, (<b>C</b>) IL-6, (<b>D</b>) MCP-1 and (<b>E</b>) TNFα in BALF from vehicle or HA-challenged WT, TLR4−/−, MyD88−/− and TIRAP−/− mice were measured by luminex beads (*p<0.05, vs. FA exposed group; #: p<0.05, vs. O₃-exposed group comparisons between strains; N = 4 per group).</p

Airway reactivity to HA challenge was dependant on TLR4, MyD88, and TIRAP.

<p>HA increased AHR in WT mice, but not in (<b>A</b>) TLR4−/−mice, (<b>B</b>) MyD88−/−mice, or (<b>C</b>) TIRAP−/− mice (*p<0.05 vs. vehicle-WT; #p<0.05 vs. HA-WT, N = 5 per group).</p

Level of HA in the BALF was increased after exposure to ozone.

<p>HA levels were significantly increased in all ozone-exposed groups (*p<0.01, vs. FA exposed group; #p<0.05, vs. O3-exposed WT, group, N = 4–5 per group).</p

Increased lavage total protein level after ozone exposure was independent of TLR4, MyD88, and TIRAP (*p<0.01, vs. FA exposed group; N = 5–6 per group).

<p>Increased lavage total protein level after ozone exposure was independent of TLR4, MyD88, and TIRAP (*p<0.01, vs. FA exposed group; N = 5–6 per group).</p

Ozone inhalation increased the level of pro-inflammatory factors in alveolar lavage fluid in a manner partially dependent on TLR4, MyD88, and TIRAP.

<p>The level of (<b>A</b>) KC, (<b>B</b>) IL-1β, (<b>C</b>) IL-6, (<b>D</b>) MCP-1 and (<b>E</b>) TNFα in BALF from air and ozone-exposed WT, TLR4−/−, MyD88−/− and TIRAP−/− mice were measured by luminex beads (*p<0.05, vs. FA exposed group; #p<0.05, vs. O₃-exposed group comparisons between strains; N = 4–5 per group).</p

Ozone exposure increased neutrophilic lung inflammation in a manner partially dependent on MyD88, but not TLR4 and TIRAP.

<p>Ozone-exposed mice demonstrated increased neutrophil cell counts in BALF when compared to air-exposed animals. Neutrophil recruitment to the airspace was independent of TLR4 (<b>A</b>) and TIRAP (<b>C</b>), but was partially dependent of MyD88 (<b>B</b>) (* p<0.05, vs. FA exposed group; # p<0.05 vs. O₃-WT, N = 4–6 per group).</p