Abstract
Agriculture workers have increased rates of airway and skeletal disease. Inhalant exposure to agricultural organic dust extract (ODE) induces bone deterioration in mice; yet, mechanisms underlying lung-bone crosstalk remain unclear. Because Toll-like receptor 2 (TLR2) and TLR4 are important in mediating the airway consequences of ODE, this study investigated their role in regulating bone responses. First, swine facility ODE stimulated wild-type (WT) bone marrow macrophages to form osteoclasts, and this finding was inhibited in TLR4 knock-out (KO), but not TLR2 KO cells. Next, using an established intranasal inhalation exposure model, WT, TLR2 KO and TLR4 KO mice were treated daily with ODE or saline for 3 weeks. ODE-induced airway neutrophil influx and cytokine/chemokine release were similarly reduced in TLR2 and TLR4 KO animals as compared to WT mice. Utilizing micro-computed tomography (CT), analysis of tibia showed loss of bone mineral density, volume and deterioration of bone micro-architecture and mechanical strength induced by ODE in WT mice were significantly reduced in TLR4 but not TLR2 KO animals. Bone marrow osteoclast precursor cell populations were analyzed by flow cytometry from exposed animals. In WT animals, exposure to inhalant ODE increased osteoclast precursor cell populations as compared to saline, an effect that was reduced in TLR4 but not TLR2 KO mice. These results show that TLR2 and TLR4 pathways mediate ODE-induced airway inflammation, but bone deterioration consequences following inhalant ODE treatment is strongly dependent upon TLR4. Thus, the TLR4 signaling pathway appears critical in regulating the lung-bone inflammatory axis to microbial component-enriched organic dust exposures.
Highlights
Bone diseases, osteoporosis and fracture, are recognized as debilitating systemic features of several types of chronic inflammatory lung diseases including chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, and post-lung transplant [1,2,3,4,5]
Osteoclastogenesis was determined by membrane receptor activator of NF-κB ligand (mRANKL) expression from murine bone marrow cells pre-treated with Macrophage colony stimulating factor (M-CSF) and Receptor activator of NF-κB ligand (RANKL) for 3 days and subsequently stimulated with organic dust extract (ODE), LPS (TLR4 agonist), or PGN (TLR2 agonist) for an additional 3 days
These studies demonstrate that TLR4, but not Toll-like receptor 2 (TLR2), is sufficient to promote ODE-induced osteoclastogenesis from M-CSF and RANKL-pretreated bone marrow macrophages in vitro
Summary
Osteoporosis and fracture, are recognized as debilitating systemic features of several types of chronic inflammatory lung diseases including chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, and post-lung transplant [1,2,3,4,5]. Recent studies have described a predominance of gram positive bacteria in agriculture dust samples [18,19,20], and genetic polymorphisms in Toll-like receptor 2 (TLR2) gene have been associated with airway disease in swine confinement workers [21]. In mice deficient in TLR2 or TLR4 and challenged with organic dust environments or extracts, it has been demonstrated there is up to a 50% reduction in various airway inflammatory consequences [22,24]. Their respective role in mediating systemic bone consequences has not been previously described. It is well recognized that osteoclasts express TLRs, the potential impact of inhalant ODE exposures on potentially regulating osteoclast progenitor cell populations is not known
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