Mouse Protocadherin-1 gene expression is regulated by cigarette smoke exposure in vivo

Protocadherin-1 (PCDH1) is a novel susceptibility gene for airway hyperresponsiveness, first identified in families exposed to cigarette smoke and is expressed in bronchial epithelial cells. Here, we asked how mouse Pcdh1 expression is regulated in lung structural cells in vivo under physiological conditions, and in both short-term cigarette smoke exposure models characterized by airway inflammation and hyperresponsiveness and chronic cigarette smoke exposure models. Pcdh1 gene-structure was investigated by Rapid Amplification of cDNA Ends. Pcdh1 mRNA and protein expression was investigated by qRT-PCR, western blotting using isoform-specific antibodies. We observed 87% conservation of the Pcdh1 nucleotide sequence, and 96% conservation of the Pcdh1 protein sequence between men and mice. We identified a novel Pcdh1 isoform encoding only the intracellular signalling motifs. Cigarette smoke exposure for 4 consecutive days markedly reduced Pcdh1 mRNA expression in lung tissue (3 to 4-fold), while neutrophilia and airway hyperresponsiveness was induced. Moreover, Pcdh1 mRNA expression in lung tissue was reduced already 6 hours after an acute cigarette-smoke exposure in mice. Chronic exposure to cigarette smoke induced loss of Pcdh1 protein in lung tissue after 2 months, while Pcdh1 protein levels were no longer reduced after 9 months of cigarette smoke exposure. We conclude that Pcdh1 is highly homologous to human PCDH1, encodes two transmembrane proteins and one intracellular protein, and is regulated by cigarette smoke exposure in vivo

Airway responsiveness, lung cell infiltration and OVA-specific IgE serum levels.

<p>Mice (n = 8) were sensitized with OVA/Alum and received 3 OVA or PBS inhalation challenges. TLR-2 agonist (20 µg per mouse in PBS) or PBS was administered intranasally 1 hour before each challenge. Asthma manifestations were measured one day after the last challenge. A: Airway responsiveness to increasing doses of methacholine was measured by whole-body plethysmography and is expressed as enhanced pause (PenH) (gray symbols: PBS-challenged groups, white symbols: OVA-challenged groups, squares: Pam3Cys-treated groups, triangles: PBS-treated groups). B, C: total (B) and differential (C) cell counts in the BAL of OVA-challenged mice. D: OVA-specific IgE levels in the serum of OVA-challenged mice. (E) Lung tissue cytokines measured in lung homogenates of PBS or Pam3Cys (P3C) treated mice as indicated. All values are displayed as average ± SEM (panels A–D) or individual values (dots)+mean (bar) (panel E). Significance is indicated (*) when p<0.05.</p

Airway responsiveness, lung cells infiltration and OVA-specific IgE serum levels.

<p>Mice (n = 8) were sensitized with OVA/Alum and received a first series of 3 OVA inhalation challenges, during which TLR-2 agonist (20 µg per mouse in PBS) or PBS was administered intranasally 1 hour before challenge. After 3 weeks, mice received a second series of 3 inhalation challenges (OVA or PBS). Asthma manifestations were measured one day after the last challenge. A: Airway responsiveness to increasing doses of methacholine was measured by whole-body plethysmography and is expressed as enhanced pause (PenH) (gray symbols: PBS-challenged groups, white symbols: OVA-challenged groups, squares: Pam3Cys-treated groups, triangles: PBS-treated groups). B: total cell counts in the BAL of PBS and OVA-challenged mice as indicated. C: differential cell counts in the BAL of OVA-challenged mice only. D: OVA-specific IgE levels in pre-challenge and post-challenge sera of OVA-challenged mice. (E) Lung tissue cytokines measured in lung homogenates of PBS or Pam3Cys (P3C) treated mice after OVA challenge at timepoint 1 (left-hand panel) or at timepoint 2 (right-hand panel) as indicated. All values in panels A–D are displayed as average ± SEM, panel E displays individual values (dots)+mean (bar). Significance is indicated (*) when p<0.05.</p

Time schedule of the experimental procedures.

<p>Days of sensitization, OVA/PBS challenges, Pam3Cys/PBS treatments and lung function measurements/section are indicated for both the short protocol (A) and the long-term protocol (B).</p

Airway responsiveness, BAL cell counts and composition, and OVA-specific IgE serum levels.

<p>Mice (n = 8) were sensitized with OVA/Alum and received 4 OVA or PBS inhalation challenges. TLR-2 agonist (20 µg per mouse in PBS) or PBS was administered intranasally 1 hour before the 2 first challenges. Asthma manifestations were measured one day after the last challenge. A: Airway responsiveness to increasing doses of methacholine was measured by whole-body plethysmography and is expressed as enhanced pause (PenH) (gray symbols: PBS-challenged groups, white symbols: OVA-challenged groups, squares: Pam3Cys-treated groups, triangles: PBS-treated groups). B, C: total (B) and differential (C) cell counts in the BAL of PBS and OVA-challenged mice as indicated. D: OVA-specific IgE levels in the serum of PBS and OVA-challenged mice as indicated. All values are displayed as average ± SEM.</p

TLR-2 Activation Induces Regulatory T Cells and Long-Term Suppression of Asthma Manifestations in Mice

<p>Asthma is a chronic inflammatory disease of the airways characterized by variable airway obstruction and airway hyperresponsiveness (AHR). The T regulatory (Treg) cell subset is critically important for the regulation of immune responses. Adoptive transfer of Treg cells has been shown to be sufficient for the suppression of airway inflammation in experimental allergic asthma. Intervention strategies aimed at expanding the Treg cell population locally in the airways of sensitized individuals are therefore of high interest as a potential therapeutic treatment for allergic airway disease. Here, we aim to test whether long-term suppression of asthma manifestations can be achieved by locally expanding the Treg cell subset via intranasal administration of a TLR-2 agonist. To model therapeutic intervention aimed at expanding the endogenous Treg population in a sensitized host, we challenged OVA-sensitized mice by OVA inhalation with concomitant intranasal instillation of the TLR-2 agonist Pam3Cys, followed by an additional series of OVA challenges. Pam3Cys treatment induced an acute but transient aggravation of asthma manifestations, followed by a reduction or loss of AHR to methacholine, depending on the time between Pam3Cys treatment and OVA challenges. In addition, Pam3Cys-treatment induced significant reductions of eosinophils and increased numbers of Treg cells in the lung infiltrates. Our data show that, despite having adverse acute effects, TLR2 agonist treatment as a therapeutic intervention induces an expansion of the Treg cell population in the lungs and results in long-term protection against manifestation of allergic asthma upon subsequent allergen provocation. Our data indicate that local expansion of Tregs in allergic airway disease is an interesting therapeutic approach that warrants further investigation.</p>