KA/E2 induced allergic airway inflammation via TLR2.

Lung inflammation was induced via intranasal inoculation of 4x105 KA/E2 trophozoites (A). Airway resistance values in response to methacholine (0 to 50 mg/ml) were compared between WT and TLR2 KO mice treated with KA/E2 (B). Differential inflammatory cells were counted in BAL from WT or TLR2 KO mice using a microscope (C). (n = 5 mice/group, 3 independent experiments, *; p < 0.05).</p

Comparison of the histopathological changes after KA/E2 treatment in WT and TLR2 KO mice.

Tissue inflammation was observed in the images of lung sections after H&E or PAS staining (A). Representative inflammation scoring and PAS-positive cells (mean ± SEM). A grade of 0 indicates no detectable inflammation and a grade 4 indicates high percentages of airways and blood vessels in section cuffing by inflammatory cells (0 = normal tissue; 1 = 75%). Severity scoring was based on the thickness of the bronchi or vessels surrounding inflammatory cells (0 = no cells; 1 = 1–3; 2 = 4–6; 3 = 7–9 cells thick; 4 = 10 or more cells thick). Means of the p-value were calculated for comparison to the control (n = 5 mice/group, 3 independent experiments, *; p < 0.05).</p

Comparison of the Th2 cytokine expression level after treatment with KA/E2 in WT and TLR2 KO mice.

Th2 related cytokine (IL-4, IL-5, and IL-13) concentrations in the BALF (A) and culture supernatants of CD3-stimulated lymphocytes from LLN (B) were determined using ELISA. The plates were read at 450 nm on a standard ELISA reader, VICTOR 3 (Each p-value was determined using t-test methods compared to the control) (n = 5 mice/group, 3 independent experiments, *p p < 0.01).</p

KA/E2 ES-P induced inflammatory-related gene expression by TLR2 in MLE12 cells.

Comparison of the expression of inflammatory response-related genes in MLE 12 cells from the KA/E2 ES-P group or TLRs antagonist pre-treated group. (n = 3/group, 3 independent experiments, *; p p p < 0.001).</p

Comparison of DC surface marker activation and the production of Th2-related cytokine expressing cells.

Activated BMDCs surface marker (A) and IL-4 expressing T cells (B) were compared between the WT and TLR2 KO groups. (n = 3/group, 3 independent experiments, *; p p p < 0.001).</p

Evaluation of the inflammatory-related gene expression owing to KA/E2 ES-P in primary lung cells from WT and TLR2 KO mice.

Assessment of the expression of inflammatory response related genes in primary lung cells from WT and TLR2 KO mice. (n = 3/group, 3 independent experiments, *; p p p < 0.001).</p

Effect of adipose-derived stem cells (ASCs) on cytokine levels in the bronchoalveolar lavage fluid.

<p>IL-4, IL-5, and IL-13 were significantly higher in the OVA group than PBS group. ASC treatment significantly decreased IL-4, IL-5, and IL-13 but increased IL-10 and TGF—β in asthmatic mice. However, the PGE2 inhibitor (A) or TGF-β neutralizing Ab (B) eliminated these immunomodulatory effects of ASCs. Data are expressed as the mean ± SEM of four independent experiments each performed in triplicate. *,§,ǁ,¶,_**, §§§,§§§§, ¶¶¶¶¶ <i>p</i><0.001, †,_******<i>p</i> = 0.007, ‡ <i>p</i> = 0.027, ††,¶¶¶ <i>p</i> = 0.028, ‡‡ <i>p =</i> 0.030, §§ <i>p =</i> 0.010, ǁǁ <i>p</i> = 0.022, ¶¶ <i>p</i> = 0.032, ***,‡‡‡ <i>p</i> = 0.038, ††† <i>p =</i> 0.049, ǁǁǁ,†††† <i>p</i> = 0.003, ****,‡‡‡‡ <i>p =</i> 0.004, ǁǁǁǁǁ <i>p =</i> 0.026, ¶¶¶¶ <i>p</i> = 0.029, ***** <i>p</i> = 0.006, ††††† <i>p</i> = 0.012, ‡‡‡‡‡ <i>p</i> = 0.036, §§§§§ <i>p =</i> 0.042, ǁǁǁǁǁ <i>p =</i> 0.046.</p

Activation of BMDCs by ES proteins.

<p>Expression of cell surface markers (MHCII, CD40, CD80, and CD86) on mouse BMDCs pulsed with ES proteins or LPS for 48 hr, compared with expression in untreated cells. (three independent experiments).</p

<i>Acanthamoeba</i> Protease Activity Promotes Allergic Airway Inflammation via Protease-Activated Receptor 2

<div><p><i>Acanthamoeba</i> is a free-living amoeba commonly present in the environment and often found in human airway cavities. <i>Acanthamoeba</i> possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of <i>Acanthamoeba</i> has not been reported. We repeatedly inoculated mice with <i>Acanthamoeba</i> trophozoites or excretory-secretory (ES) proteins intra-nasally and evaluated symptoms and airway immune responses. <i>Acanthamoeba</i> trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (<i>CCL11</i>, <i>CCL17</i>, <i>CCL22</i>, <i>TSLP</i>, and <i>IL-25</i>) in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF) inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher <i>Acanthamoeba</i>-specific IgE titers than healthy controls and we found <i>Acanthamoeba</i> specific antigen from house dust in typical living room. Our findings suggest that <i>Acanthamoeba</i> elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that <i>Acanthamoeba</i> may be one of the triggers human airway allergic disease.</p></div

The experimental protocol.

<p>(A) Mice were sensitized on days 0, 1, 7, and 8 by intraperitoneal injection of ovalbumin (OVA) and challenged intranasally on days 14, 15, 21, and 22. Purified adipose-derived stem cells (ASCs; 1 × 10⁶) were injected via the tail vein on days 12, 13, 19, and 20. PGE2 and TGF-β were blocked by intraperitoneal injection of a PGE2 inhibitor or anti-TGF-β-Ab on days 13, 14, 15, 16, 17, 20, 21, 22, and 23. (B) The mice were divided into five treatment groups.</p