Repetitive inhalant organic dust extract (ODE)-induced airway inflammatory responses were reduced in TLR2 and TLR4 KO mice.

<p>WT, TLR2 KO, and TLR4 KO mice were <i>i</i>.<i>n</i>. treated with ODE daily or saline for 3 weeks whereupon animals were euthanized 5 hrs following final exposure. Neutrophil recruitment (<b>A</b>), IL-6 (<b>B</b>), TNF-α (<b>C</b>), IL-1β (<b>D</b>), CXCL1 (<b>E</b>), and CXCL2 (<b>F</b>) were determined in bronchoalveolar lavage fluid. Bar graphs represent the mean with standard error bars shown (N = minimum of 6 mice/group from 3 independent studies). Statistical significance denoted by asterisks (*p<0.01, **p<0.01, ***p<0.001) as compared to respective saline treatment group. Line denotes statistical significance (#p<0.05, ##p<0.01, ###p<0.001) of WT vs. TLR2 and TLR4 KO mice.</p

Organic dust extract (ODE) promotes osteoclastogenesis <i>in vitro</i> with WT and TLR2 KO, but not TLR4 KO, bone marrow macrophages.

<p>Bone marrow derived cells from C57BL/6 WT mice (<b>A, top panel</b>), TLR2 KO mice (<b>B, middle panel</b>), and TLR4 KO (<b>C, bottom panel</b>) were cultured with M-CSF and RANKL and ± ODE (0.5%). In side-by-side experiments, cells were also cultured with a TLR4 agonist (lipopolysaccharide; LPS; 10 ng) or TLR2 agonist (peptidoglycan; PGN; 1 μg) as additional controls. Membrane receptor activator of NF-κB ligand (mRANKL) expression was determined by flow cytometry analysis. A representative contour plot from each experimental group is shown with a rightward shift demonstrating gating of positive mRANKL expression after exclusion of debris. Bar graphs show the mean with standard error bars of percentage of mRANKL expression (P2 gate). N = 6/group from three separate studies ran in duplicate. Statistical significance denoted by asterisks (***p<0.001) vs. unstimulated control.</p

Inhalant exposure with organic dust extract (ODE) increases osteoclast precursor populations (OCP) in murine bone marrow cells.

<p>WT mice were <i>i</i>.<i>n</i>. treated with saline or ODE daily for three weeks whereupon mice were euthanized and bone marrow cells were collected and analyzed by flow cytometry. After exclusion of debris and dead cells, triple negative (TN) cells were gated based upon CD45R^-CD3^-CD11b^lo phenotype with <b>panel A</b> depicting the frequency of TN cells as a percentage of live cells in a bar graph. <b>Panel B</b> depicts a representative dot plot of TN cells expressing CD115 with associated frequency distribution shown in bar graph. Next, representative dot plots with frequency distribution of osteoclast precursor populations: TN CD115+CD117+ (<b>panel C</b>), and TN CD115+CD117+CD27+ (<b>panel D)</b> are shown. All bar graphs represent mean percentage with SEM bars. N = minimum of 6 mice/group from three independent experiments. Statistical significance denoted as asterisks (*p<0.05) vs. saline.</p

Loss of trabecular bone demonstrated in WT and TLR2 KO, but not TLR4 KO, mice treated repetitively with inhalant ODE.

<p>WT, TLR2 KO, and TLR4 KO animals were <i>i</i>.<i>n</i>. treated daily with saline or ODE for 3 weeks. A representative three-dimensional (3D) reconstructed image from region of interest of proximal tibia from one mouse per treatment group (minimum of 6 mice/group from 3 independent studies).</p

Repetitive inhalant ODE exposure increased serum TRACP 5b levels in WT and TLR2 KO but not TLR4 KO animals.

<p>WT, TLR2 KO, and TLR4 KO mice were <i>i</i>.<i>n</i>. treated with ODE daily or saline for 3 weeks whereupon animals were euthanized 5 hrs following final exposure. Levels of tartrate-resistant acid phosphatase 5b (TRACP 5b) were determined in serum. Bar graphs represent the mean with standard error bars shown (N = minimum of 6 mice/group from 3 independent studies). Statistical significance denoted by asterisks (*p<0.05) as compared to respective saline treatment group.</p

Increased osteoclast precursor populations (OCP) in murine bone marrow following inhalant ODE treatment is dependent upon TLR4, but not TLR2, signaling pathway.

<p>TLR2 KO (<b>A, top panel</b>) and TLR4 KO (<b>B, bottom panel</b>) mice were <i>i</i>.<i>n</i>. treated with saline or ODE daily for three weeks whereupon mice were euthanized and bone marrow cells were collected and analyzed by flow cytometry. After exclusion of debris and dead cells, triple negative (TN) cells were gated based upon CD45R^-CD3^-CD11b^lo phenotype. Distribution of TN cells are shown as mean percentage with SEM bars of live cells. Distribution of TN cells expressing CD115, CD115CD117, and CD115CD117CD27 are shown as mean percentage with SEM bars of TN cells. N = 4 mice/group. Statistical significance denoted by asterisks (**p<0.01) vs. saline.</p

WT and TLR2 KO, but not TLR4 KO, mice were susceptible to the systemic bone deterioration response following repetitive ODE inhalation exposure by micro-CT analysis.

<p>WT, TLR4 KO, and TLR2 KO mice were <i>i</i>.<i>n</i>. treated daily with saline or ODE for 3 weeks whereupon trabecular bone of proximal tibia was analyzed micro-CT analysis. Changes in bone quality and bone quantity were observed in ODE-treated WT mice as compared to saline. TLR4 KO, but not TLR2 KO, animals were generally less responsiveness to bone changes induced by ODE. To compare findings across animal strains, bar graph depicts the mean with SEM bars of the percent change induced by ODE treatments (difference between ODE and saline treatment divided by saline groups multiplied by 100) in bone parameters compiled from three independent studies of 2–3 mice per study (N = 6–9 mice). Asterisks denote statistical significance (*p<0.05) vs. WT.</p