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

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

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

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

Uptake of Bv1-Protein and Bv1-Peptide in lungs of naive and sensitised mice <i>in vivo</i>.

<p>(<b>A</b>) Experimental design: Mice were sensitised 3 times intraperitonealy with 1 μg of Bet v 1 on days 0, 14 and 28. Intranasal challenge with 100 μg of birch pollen extract was performed one week after last sensitisation on days 35, 36 and 37. On day 40, 20 μg of Bv1-Protein-FAM and Bv1-Peptide-FAM were intranasal administered to naive and allergic BALB/c (n = 3) mice and lungs were collected after 1 (for Bv1-Peptide) or 6 hours (for Bv1-Protein). Dead cells were identified via 7-AAD staining and excluded from analysis. (<b>B</b> and <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>/CD45^<b>-</b>/F4/80^<b>-</b>) in lungs of naive and allergic mice was investigated by flow cytometry. 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 naive and allergic mice. ATII-LCs = ATII-like cells; DCs = dendritic cells; Mϕ = macrophages.</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

Cytokine production in splenocytes and BMDC and maturation of BMDC.

<p>Splenocytes (A) and BMDC (B) derived from naive BALB/c mice were stimulated with OLA or cultured with medium alone. IFN-γ, IL-6 and IL-10 levels were determined in supernatants by ELISA. Results show pooled values ± SEM of three to four independent experiments performed with cell suspensions prepared from one individual mouse per experiment. * <i>p</i> < 0.05; ** <i>p</i> < 0.01; *** <i>p</i> < 0.001. BMDC cultured with Pam3Cys, LPS, OLA or medium alone were analysed by flow cytometry for the expression of the maturation markers CD40, CD80, CD86 and MHCII after gating on CD11c⁺ cells (C). Representative histograms from one out of three repeated experiments.</p

Oocyst-Derived Extract of <i>Toxoplasma Gondii</i> Serves as Potent Immunomodulator in a Mouse Model of Birch Pollen Allergy

<div><p>Introduction</p><p>Previously, we have shown that oral infection with <i>Toxoplasma gondii</i> oocysts prevented type I allergy in mice. Here we investigated whether the application of a <i>T</i>. <i>gondii</i> oocyst lysate antigen (OLA) could also reduce allergy development. BALB/c mice were immunised twice with OLA followed by sensitisation with the major birch pollen (BP) allergen Bet v 1 and an aerosol challenge with BP extract.</p><p>Methods</p><p>First, we tested OLA <i>in vitro</i>. Stimulation of splenocytes and bone marrow-derived dendritic cells (BMDC) with OLA led to the production of pro-inflammatory and regulatory cytokines such as IL-6, IFN-γ and IL-10. Moreover, BMDC exposed to OLA upregulated the maturation markers CD40, CD80, CD86, and MHCII. Furthermore, OLA was recognised by TLR2-transfected human embryonic kidney cells.</p><p>Results</p><p>Immunisation of mice with OLA induced high levels of <i>Toxoplasma</i>-specific IgG antibodies in sera along with increased production of IFN-γ and IL-10 in <i>Toxoplasma</i>-antigen restimulated splenocytes. OLA reduced allergic airway inflammation as manifested by significant reduction of eosinophils in bronchoalveolar fluids, decreased cellular infiltrates and mucus production in the lungs. Accordingly, Bet v 1-specific IgE was decreased in OLA-pretreated mice. The reduced allergic immune responses were accompanied by increased numbers of CD4⁺CD25^highFoxp3⁺ regulatory T cells in spleens as well as by increased numbers of granulocytic myeloid-derived suppressor cells in lungs when compared to sensitised controls suggesting that these two cell populations might be involved in the suppression of the allergic immune responses.</p><p>Conclusion</p><p>Our data demonstrate that pretreatment with the oocyst extract can exert anti-allergic effects comparable to <i>T</i>. <i>gondii</i> infection. Thus, the immunomodulatory properties of the parasite extract indicate that this extract and in the future defined molecules thereof might serve as immunomodulatory adjuvants in allergy treatment and prophylaxis.</p></div

TLR activation by OLA.

<p>TLR2- (A), TLR4- (B) and TLR9- (C) transfected human embryonic kidney cells (HEK293) cells were incubated with OLA at three different concentrations and the respective positive controls (Pam3Cys, LPS and CpG) for 24h. Activation was assessed by measuring human (h)IL-8 production by ELISA. Results represent mean values ± SEM from one representative experiment.</p

Regulatory T cells in spleen and myeloid-derived suppressor cells in lung.

<p>Splenocytes, lung and BLN cells were sampled on day 49. Regulatory T cells were identified as CD4⁺CD25^highFoxp3⁺ lymphocytes in spleen cell suspension by flow cytometry analysis (A). Results represent values from one representative experiment with seven to eight mice per group. Expression of IL-10, TGF-β and Foxp3 was measured in pooled BLN cell samples by real-time RT-PCR and is presented as relative ratio to the house keeping gene ALAS (B-D). Results represent data from one representative experiment. G-MDSC (CD11b⁺Ly6G⁺Ly6C^low) were identified by gating on live (7AAD^-) CD11b⁺ lung cells by flow cytometry analysis (E). Results represent pooled values from two experiments with four to eight mice per group. * <i>p</i> < 0.05; ** <i>p</i> < 0.01.</p