The Role of Alveolar Epithelial Type II-Like Cells in Uptake of Structurally Different Antigens and in Polarisation of Local Immune Responses

<div><p>Background</p><p>Our previous studies on intranasal tolerance induction demonstrated reduction of allergic responses with different allergen constructs. The underlying mechanisms varied depending on their conformation or size.</p><p>Objective</p><p>The aim of the present study was to compare the uptake of two structurally different allergen molecules within the respiratory tract following intranasal application.</p><p>Methods</p><p>The three-dimensional Bet v 1 (Bv1-Protein) and the T cell epitope peptide of Bet v 1 (Bv1-Peptide) were labelled with 5,6-Carboxyfluorescein (FAM) and their uptake was investigated in lung cells and cells of the nasal associated lymphoid tissue from naive and sensitised BALB/c mice. Phenotypic characterisation of FAM⁺ lung cells after antigen incubation <i>in vitro</i> and after intranasal application was performed by flow cytometry. Impact of Bv1-Protein and Bv1-Peptide on cytokine profiles and gene expression <i>in vivo</i> or in an alveolar epithelial type II (ATII) cell line were assessed in mono- and co-cultures with monocytes using ELISA and quantitative real-time PCR.</p><p>Results</p><p>Both antigens were taken up preferably by ATII-like cells (ATII-LCs) in naive mice, and by macrophages in sensitised mice. After intranasal application, Bv1-Peptide was taken up faster and more efficiently than Bv1-Protein. <i>In vivo</i> and <i>in vitro</i> experiments revealed that Bv1-Protein induced the transcription of thymic stromal lymphopoietin mRNA while Bv1-Peptide induced the transcription of IL-10 and MCP1 mRNA in ATII-LCs.</p><p>Conclusion and Clinical Relevance</p><p>Both tested antigens were taken up by ATII-LCs under steady state conditions and induced different polarisation of the immune responses. These data may have an important impact for the generation of novel and more effective prophylactic or therapeutic tools targeting the respiratory mucosa.</p></div

Diet Matters: Endotoxin in the Diet Impacts the Level of Allergic Sensitization in Germ-Free Mice

<div><p>Germ-free animals have been used to define the vital role of commensal bacteria on the maturation of the host immune system. However, the role of bacterial residues in diet in this setting is poorly understood. Here we investigated the effect of bacterial contamination in sterile diet on the level of allergic sensitization in germ-free mice. Sterile grain-based diets ST1 and R03 were tested for the level of bacterial contamination. ST1 contained higher amount of bacterial DNA, approximately ten times more endotoxin, and induced higher, TLR4-dependent, cytokine production in dendritic cells compared to R03. In a germ-free mouse model of sensitization to the major birch pollen allergen Bet v 1, feeding on ST1 for at least two generations was associated with decreased production of allergen-specific IgE and IgG1 antibodies in sera in comparison to R03. Furthermore, reduced levels of allergen-specific and ConA-induced cytokines IL-4, IL-5 and IL-13 accompanied by increased levels of IFN-γ were detected in splenocytes cultures of these mice. Our results show that contamination of experimental diet with bacterial residues, such as endotoxin, significantly affects the development of allergic sensitization in germ-free mice. Therefore, careful selection of sterile food is critical for the outcomes of germ-free or gnotobiotic experimental models of immune-deviated diseases.</p></div

Levels of Bet v 1-specific IgG1 and IgA in gut lavage.

<p>Bet v 1-specific IgG1 (A) and IgA (B) in gut lavage were measured by ELISA. Data are plotted as mean values ± SEM. Pooled values of two independent experiments (n = 9–10 sensitized groups, n = 5–6 sham-treated groups) are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.</p

Bv1-Protein markedly increases levels of TSLP mRNA, whereas Bv1-Peptide induces IL-10 and MCP1 mRNA transcription.

<p>(<b>A-C</b>) A549 cells (5x10^<b>5</b> cells/well) were stimulated with 5 μg/ml of Bv1-Protein or Bv1-Peptide for 1 and 24 hours. A549 cells were harvested to evaluate gene expression. Reactions were executed in triplicates and mRNA gene expression of TSLP, IL-10 and MCP1 in A549 cells was normalised to the expression of the housekeeping gene β-actin and relative gene quantification was performed by comparing RNA samples at 1 and 24 hour intervals to control samples at 0 hours. Data are the pool from three independently performed experiments of identical design. (<b>D-F</b>) A549 cells (5x10^<b>5</b> cells/well) were co-cultured in a transwell system with THP1 cells (5x10^<b>5</b> cell/well) and stimulated with 5 μg/ml of Bv1-Protein and Bv1-Peptide for 6 days. A549 and THP1 cells were harvested separately to evaluate gene expression. Reactions were executed in triplicates and mRNA gene expression of TSLP, IL-10 and MCP1 in A549 cells was normalised to the expression of the housekeeping gen β-actin and relative gene quantification was performed by comparing RNA samples of stimulated cells to control samples. Data are the pool from three independently performed experiments of identical design. (<b>G-I</b>) 20 μg of Bv1-Protein or Bv1-Peptide were intranasal administered to naive BALB/c mice (n = 3 per time point). Primary ATII-LCs were isolated from lungs 24 hours after intranasal application to evaluate gene expression. Reactions were executed in triplicates and mRNA gene expression of TSLP, IL-10 and MCP1 in ATII-LCs was normalised to the expression of the housekeeping gene β-actin and relative gene quantification was performed by comparing RNA samples of stimulated cells to control samples. Data are the pool from two independently performed experiments of identical design. (<b>A-I</b>) Values represent means ± SEM. A value P<0.05 was considered to be significant. **P<0.01 and ***P<0.001 indicate levels significantly different between A549 or ATII-LCs stimulated with Bv1-Protein and Bv1-Peptide. ATII-LCs = ATII-like cells; MCP1 = Monocyte chemoattractant protein-1; TSLP = Thymic stromal lymphopoietin.</p

Direct and transwell co-culture of A549 and THP1.

<p>A549 cells (5x10^<b>5</b> cells/well) were co-cultured with THP1 cells (5x10^<b>5</b> cell/well) directly or in a transwell system and stimulated with 5 μg/ml of Bv1-Protein and Bv1-Peptide or with 1 μg of LPS or 1 μg of Pam3-Cys for 6 days. IL-8 (<b>A</b>) and IL-6 (<b>B</b>) levels were measured in cell culture supernatants using ELISA. Values represent means ± SEM. A value P<0.05 was considered to be significant. **P<0.01 and ***P<0.001.</p

Influence of endotoxin-low (R03) and endotoxin-high (ST1) diet on mitogen-induced cytokine production in splenocytes.

<p>Germ-free mice fed endotoxin-low (R03) and endotoxin-high (ST1) diet were sensitized as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167786#pone.0167786.g002" target="_blank">Fig 2</a>. Spleen cell cultures derived from these animals were incubated with 1.5 μg/ml of ConA for 60 h <i>in vitro</i>. Levels of IL-4 (A), IL-5 (B), IL-13 (C), IL-6 (D), IFN-γ (E), TNF-α (F), IL-17 (G) and IL-10 (H) in culture supernatants were measured by MILLIPLEX MAP Mouse Cytokine/Chemokine Panel. Cytokine levels are expressed after subtraction of base line levels of unstimulated splenocytes. Pooled values from two independent experiments (n = 9–10 sensitized groups, n = 5–6 sham-treated groups) are shown as mean values ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.</p

Influence of endotoxin-low (R03) and endotoxin-high (ST1) diet on Bet v 1-specific cytokine production in splenocytes.

<p>Germ-free mice fed endotoxin-low (R03) and endotoxin-high (ST1) diet were sensitized as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167786#pone.0167786.g002" target="_blank">Fig 2</a>. Spleen cell cultures derived from these animals were incubated with 20 μg/ml of Bet v 1 for 60 h <i>in vitro</i>. Levels of IL-4 (A), IL-5 (B), IL-13 (C), IL-6 (D), IFN-γ (E), TNF-α (F), IL-17 (G), and IL-10 (H) in culture supernatants were measured by MILLIPLEX MAP Mouse Cytokine/Chemokine Panel. Cytokine levels are expressed after subtraction of base line levels of unstimulated splenocytes. Pooled values from two independent experiments (n = 9–10 sensitized groups, n = 5–6 sham-treated groups) are shown as mean values ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.</p

Uptake of Bv1-Protein and Bv1-Peptide in A549 cells.

<p>The human ATII cell line A549 was incubated with 5 μg/ml of Bv1-Protein-FAM and Bv1-Peptide-FAM for 0.5, 1, 4, 24, and 48 hours. (<b>A</b>) The uptake was measured via flow cytometry. Dead cells were identified via 7-AAD staining and excluded from analysis. Data are the pool from three independently performed experiments of identical design. Values represent means ± SEM. A value P<0.05 was considered to be significant. *P<0.05, **P<0.01 and ***P<0.001 indicate levels significantly different between A549 cells stimulated with Bv1-Protein or Bv1-Peptide. (<b>B</b>) A549 cells were cultured for 4 hours with Bv1-Protein-FAM and Bv1-Peptide-FAM (green), transferred on glass slides, stained and mounted. Examination was performed with a confocal microscope. Nuclear counterstain was performed with DAPI (blue). ns = not significant.</p

Systemic sensitization to Bet v 1 in mice bred on endotoxin-low (R03) and endotoxin-high (ST1) diet.

<p>(A) Experimental design: Mice were bred on the respective diet for at least two generations. Eight-week-old female germ-free mice fed with R03 or ST1 diet were sensitized by subcutaneous immunization (s.c.) three times with 1 μg of recombinant Bet v 1 in Alum (Bet v 1/Al(OH)₃). Age-matched sham-treated mice were used as controls. At sacrifice, blood, spleens, and small intestines were collected for further analysis. (B) Functional IgE in serum was measured by Bet v 1-mediated β-hexosaminidase release from rat basophil leukemia cells. Bet v 1-specific IgE (C), IgG1 (D), IgA (E), total IgE (F) and total IgA (G) in sera were measured by ELISA. Data are plotted as mean values ± SEM. Pooled values of two independent experiments (n = 9–10 sensitized groups, n = 5–6 sham-treated groups) are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.</p

Time-dependent uptake of Bv1-Protein and Bv1-Peptide in NALT and lungs and phenotypic characterisation of FAM⁺ lung celIs <i>in vivo</i>.

<p>20 μg of Bv1-Protein-FAM or Bv1-Peptide-FAM were intranasal administered to naive BALB/c mice (n = 3 per time point). (<b>A</b> and <b>B</b>) After 1, 6, 24, and 48 hours NALT and lungs were harvested, and analysed by flow cytometry. Dead cells were identified via 7-AAD staining and excluded from analysis. Data are the pool from three independently performed experiments of identical design. (<b>C</b>) FAM^<b>+</b> cells were gated and antigen uptake capacity of macrophages (CD11b^<b>+</b>/CD11c^<b>-</b>), dendritic cells (CD11b^<b>-</b>/CD11c^<b>+</b>), B cells (B220^<b>+</b>/CD19^<b>+</b>), and ATII-LCs (CD11b^<b>-</b>/CD11c^<b>-</b>/CD16/32^<b>-</b>/CD19^<b>-</b>/CD31^<b>-</b>/CD45^<b>-</b>/F4/80^<b>-</b>/MHCII^<b>+</b>) in lungs of naive mice was investigated by flow cytometry. Data are the pool of two independently performed experiments of identical design. Values represent means ± SEM. A value P<0.05 was considered to be significant. *P<0.05 and ***P<0.001 indicate levels significantly different from time point 0 hours. ATII-LCs = ATII-like cells; DCs = dendritic cells; Mϕ = macrophages; NALT = nasal associated lymphoid tissue.</p