Treating obesity decreases lung dysfunction in the obesity-related asthma model.

<p>DIO mice performed voluntary exercise or consumed a normal chow diet to treat obesity. (a) Airway hyperresponsiveness and (b) inflammatory cell infiltration in the bronchoalveolar lavage fluid were measured in the weight-reduced, obesity-related asthma mice. *, Statistical significance to lean mice (p<0.05); ^#, Statistical significance to DIO mice. <i>DIO-N-OVA</i>, DIO-OVA mice with diet-restriction; <i>DIO-Ex-OVA</i>, DIO-OVA mice with voluntary exercise. Error bars indicated mean±SEM of five mice per group. All data are representative of three independent experiments.</p

Mouse models in this study.

<p>(a) Scheme of this study. C57BL/6 mice fed HFD for 16 weeks and some of the DIO mice underwent OVA sensitization and challenge (DIO-OVA). Some of the DIO-OVA mice were treated with TNF-α neutralizing antibody for TNF-α blockade or a Cl₂MDP-containing liposome for alveolar macrophage depletion. For the treatment of obesity, the DIO-OVA mice performed voluntary exercise (DIO-OVA-Ex) or underwent dietary restriction (DIO-OVA-N) after 12 weeks of HFD feeding. (b) Body weight and (c) blood glucose tolerance was measured at the end of 16 weeks after HFD feeding. *, Statistical significance to lean mice (p<0.05); ^#, Statistical significance to DIO mice. <i>i.p.</i>, intraperitoneal injection; <i>i.n.</i>, intranasal injection; <i>TNF</i>, TNF-α neutralizing antibody. Error bars indicated mean±SEM of five mice per group. All data are representative of three independent experiments.</p

Baseline lung function in obese mice.

<p>Baseline lung function in obese mice.</p

Obesity exacerbates asthmatic symptoms in the asthma model.

<p>(a) AHR, (b) inflammatory cell infiltrations in the BAL fluids, (c) total IgE and (d) OVA-IgE levels in the sera were measured in the asthma model (lean-OVA) and obesity-related asthma model (DIO-OVA). *, Statistical significance to their control group (lean or DIO; p<0.05); ^#, Statistical significance between lean-OVA and DIO-OVA (p<0.05). Error bars indicated mean±SEM of five mice per group. All data are representative of three independent experiments.</p

Treating obesity decreases TNF-α levels in the obesity-related asthma model.

<p>DIO mice performed voluntary exercise or consumed a normal chow diet to treat obesity. TNF-α levels in the lung homogenates were measured in the weight-reduced, obesity-related asthma mice. The solid lines indicate statistical significance between each group (p<0.05). Error bars indicated mean±SEM of five mice per group. All data are representative of three independent experiments.</p

Alveolar Macrophages Play a Key Role in Cockroach-Induced Allergic Inflammation via TNF-α Pathway

<div><p>The activity of the serine protease in the German cockroach allergen is important to the development of allergic disease. The protease-activated receptor (PAR)-2, which is expressed in numerous cell types in lung tissue, is known to mediate the cellular events caused by inhaled serine protease. Alveolar macrophages express PAR-2 and produce considerable amounts of tumor necrosis factor (TNF)-α. We determined whether the serine protease in German cockroach extract (GCE) enhances TNF-α production by alveolar macrophages through the PAR-2 pathway and whether the TNF-α production affects GCE-induced pulmonary inflammation. Effects of GCE on alveolar macrophages and TNF-α production were evaluated using in vitro MH-S and RAW264.6 cells and in vivo GCE-induced asthma models of BALB/c mice. GCE contained a large amount of serine protease. In the MH-S and RAW264.7 cells, GCE activated PAR-2 and thereby produced TNF-α. In the GCE-induced asthma model, intranasal administration of GCE increased airway hyperresponsiveness (AHR), inflammatory cell infiltration, productions of serum immunoglobulin E, interleukin (IL)-5, IL-13 and TNF-α production in alveolar macrophages. Blockade of serine proteases prevented the development of GCE induced allergic pathologies. TNF-α blockade also prevented the development of such asthma-like lesions. Depletion of alveolar macrophages reduced AHR and intracellular TNF-α level in pulmonary cell populations in the GCE-induced asthma model. These results suggest that serine protease from GCE affects asthma through an alveolar macrophage and TNF-α dependent manner, reflecting the close relation of innate and adaptive immune response in allergic asthma model.</p> </div

GCE promotes allergic phenotype through the TNF-α pathway.

<p>Lung homogenates were harvested and used for measuring (a) IL-5, (b) IL-13 and (c) IFN-γ. (d) The level of serum IgE in the blood. Lung tissues were stained with (e) PAS and (f) Masson's Trichrome. (g) PAS-positive cells in peri-bronchial regions and (h) total collagen deposition in the lung tissue were quantitatively calculated. * indicates statistical significance between “Sham” and “GCE” (n = 5, <i>p</i><0.05). ^# indicates statistical significance between “GCE” and “a-TNF” (n = 5, <i>p</i><0.05). All data are representative of three independent experiments.</p

GCE induces TNF-α production in macrophages.

<p>(a) TNF-α production in MH-S cells incubated with GCE, GCE+PMB, or GCE+aprotinin. (b) Relative MFI ratio in “a” panel. (c) TNF-α secretion in the culture supernatant of “a” panel. * indicates statistical significance between “Control” and “GCE” (n = 3, <i>p</i><0.05). ^# indicates statistical significance between “GCE” and “GCE+Aprotinin” (n = 3, <i>p</i><0.05). (d) Scheme for short-term GCE exposure model. Kinetics of intracellular (e) PAR-2 and (g) TNF-α expression from alveolar macrophages in the BAL fluid of the short-term GCE exposure model. (f) Relative MFI ratio in “e” panel. (h) Relative MFI ratio in “g” panel. * indicates statistical significance to “D0” (n = 5, <i>p</i><0.05). ^# indicates statistical significance between “D12” and “a-TNF” (n = 5, <i>p</i><0.05). All data are representative of three independent experiments.</p

Serine protease in GCE develops pulmonary inflammation, alveolar macrophage infiltration and TNF-α expression.

<p>(a) Scheme for the inhibition of GCE protease activity of GCE-induced asthma model. (b) AHR and (c) BAL cell count. (d) Alveolar macrophage, interstitial macrophage, and dendritic cell population in the lung tissue. (e) Quantitative analysis of alveolar macrophage from “d” panel. (f) Intracellular expressions of TNF-α in the alveolar macrophages of the GCE-induced asthma experiment. (g) Relative MFI ratio in “f” panel. * indicates statistical significance between “Sham” and “GCE” (n = 5, p<0.05). ^# indicates statistical significance between “GCE” and “GCE+Aprotinin” (n = 5, <i>p</i><0.05). All data are representative of three independent experiments. <i>AMs</i>, alveolar macrophages; <i>IMs</i>, interstitial macrophages; <i>DCs</i>, dendritic cells.</p

GCE promotes pulmonary inflammation through the TNF-α pathway.

<p>(a) Scheme for TNF-α neutralization of GCE-induced asthma model. <i>a-TNF</i>, anti-TNF-α mAb; <i>goat</i>, goat IgG. (b) AHR and (c) BAL cell count. (d) TNF-α-producing macrophage population in the lung tissue. (e) Quantitative analysis of CD11b⁺TNF-α⁺ cell population from “d” panel. (f) TNF-α levels in lung homogenates. * indicates statistical significance between “Sham” and “GCE” (n = 5, p<0.05). ^# indicates statistical significance between “GCE” and “a-TNF” (n = 5, <i>p</i><0.05). All data are representative of three independent experiments. <i>R_L</i>, pulmonary resistance; <i>Mac</i>, macrophage; <i>Lym</i>, lymphocyte; <i>Eos</i>, eosinophil; <i>Neu</i>, neutrophil.</p