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

Innate immune activation by inhaled lipopolysaccharide, independent of oxidative stress, exacerbates silica-induced pulmonary fibrosis in mice

Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress. © 2012 Brass et al

Iron supplementation decreases severity of allergic inflammation in murine lung.

The incidence and severity of allergic asthma have increased over the last century, particularly in the United States and other developed countries. This time frame was characterized by marked environmental changes, including enhanced hygiene, decreased pathogen exposure, increased exposure to inhaled pollutants, and changes in diet. Although iron is well-known to participate in critical biologic processes such as oxygen transport, energy generation, and host defense, iron deficiency remains common in the United States and world-wide. The purpose of these studies was to determine how dietary iron supplementation affected the severity of allergic inflammation in the lungs, using a classic model of IgE-mediated allergy in mice. Results showed that mice fed an iron-supplemented diet had markedly decreased allergen-induced airway hyperreactivity, eosinophil infiltration, and production of pro-inflammatory cytokines, compared with control mice on an unsupplemented diet that generated mild iron deficiency but not anemia. In vitro, iron supplementation decreased mast cell granule content, IgE-triggered degranulation, and production of pro-inflammatory cytokines post-degranulation. Taken together, these studies show that iron supplementation can decrease the severity of allergic inflammation in the lung, potentially via multiple mechanisms that affect mast cell activity. Further studies are indicated to determine the potential of iron supplementation to modulate the clinical severity of allergic diseases in humans

Iron supplementation decreases severity of allergic asthma.

<p>WT and mast cell-deficient <i>Sash</i> mice on the indicated diets were sensitized to OVA then challenged with OVA or saline. <b>A</b>. Airway resistance (measured in units of cm H₂O/ml/sec) in response to methacholine was significantly decreased following allergen challenge for mice that received iron supplementation (*indicates p≤0.0005). <b>B</b>. Iron supplementation decreased infiltration of eosinophils into bronchoalveolar spaces (decrease of 52%; *indicates p = 0.0007).</p

Iron Status of Mice Studied.^**

*<p>indicates p≤0.05 for comparisons of unsupplemented vs. iron-supplemented diets.</p>**<p>Data shown are mean ± SEM for 6 mice/diet group for red blood cells (RBC), hemoglobin (Hgb), hematocrit (Hct), and mean cellular volume (MCV). The numbers of mice studied for serum and tissue iron ranged from 10–25 per group. Hepcidin measurements are given as the geometric mean (upper - lower confidence interval) for 10 allergen-sensitized, saline-challenged mice/group.</p

Iron supplementation does not affect mucus production.

<p>Periodic acid-Schiff (PAS)-stained formalin-fixed, paraffin-embedded lung sections show allergen-induced goblet cell differentiation in the large airways of mice on both 8 ppm (<b>A</b>  =  saline-challenged, <b>B</b>  =  OVA-challenged) and 200 ppm diets (<b>C</b>  =  saline-challenged, <b>D</b>  =  OVA-challenged). Expression of Muc5AC mRNA in the lungs was similar for both diet groups (<b>E</b>).</p

Effects of iron supplementation on allergen-induced cytokine secretion into bronchoalveolar spaces.

<p>Cytokine and chemokines present in bronchoalveolar lavage (BAL) fluid obtained from OVA-sensitized mice 24 hrs following the 3d challenge with aerosolized antigen or saline were markedly increased in unsupplemented compared with iron-supplemented mice (*indicate p<0.01). The percentage increase ranged from 25–35% for IL-1β, IL-5, IL-6, and IL-13 to 170% for IL-17.</p