Hyaluronan signaling during ozone-induced lung injury requires TLR4, MyD88, and TIRAP

Abstract

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.

Figure 1. Ozone inhalation increased airway sensitivity to methacholine challenge and was dependent on the TLR4-MyD88-TIRAP signaling pathway.

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. A) Ozone-induced AHR was increased in WT mice but not in TLR4−/− mice, B) MyD88−/−mice, or C) TIRAP−/− mice (*p<0.05 vs. FA-WT; #p<0.05 vs. O3-WT, N = 5–6 per group).

Figure 2. Ozone exposure increased neutrophilic lung inflammation in a manner partially dependent on MyD88, but not TLR4 and TIRAP.

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 (A) and TIRAP (C), but was partially dependent of MyD88 (B) (* p<0.05, vs. FA exposed group; # p<0.05 vs. O₃-WT, N = 4–6 per group).

Figure 3. Ozone inhalation increased the level of pro-inflammatory factors in alveolar lavage fluid in a manner partially dependent on TLR4, MyD88, and TIRAP.

The level of (A) KC, (B) IL-1β, (C) IL-6, (D) MCP-1 and (E) 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).

Figure 4. 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).

Figure 5. Level of HA in the BALF was increased after exposure to ozone.

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).

Figure 6. Airway reactivity to HA challenge was dependant on TLR4, MyD88, and TIRAP.

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

Figure 7. HA challenge was not sufficient for either neutrophilic inflammation or epithelial injury.

(A) There were no observed differences in cellular inflammation in the airspace 2 hours after direct challenge to HA, (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).

Figure 8. 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.

The level of (A) KC, (B) IL-1β, (C) IL-6, (D) MCP-1 and (E) 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).