Langerhans cell precursors acquire RANK/CD265 in prenatal human skin

AbstractThe skin is the first barrier against foreign pathogens and the prenatal formation of a strong network of various innate and adaptive cells is required to protect the newborn from perinatal infections. While many studies about the immune system in healthy and diseased adult human skin exist, our knowledge about the cutaneous prenatal/developing immune system and especially about the phenotype and function of antigen-presenting cells such as epidermal Langerhans cells (LCs) in human skin is still scarce. It has been shown previously that LCs in healthy adult human skin express receptor activator of NF-κB (RANK), an important molecule prolonging their survival. In this study, we investigated at which developmental stage LCs acquire this important molecule. Immunofluorescence double-labeling of cryostat sections revealed that LC precursors in prenatal human skin either do not yet [10–11 weeks of estimated gestational age (EGA)] or only faintly (13–15 weeks EGA) express RANK. LCs express RANK at levels comparable to adult LCs by the end of the second trimester. Comparable with adult skin, dermal antigen-presenting cells at no gestational age express this marker. These findings indicate that epidermal leukocytes gradually acquire RANK during gestation – a phenomenon previously observed also for other markers on LCs in prenatal human skin

Germ-Free Mice Exhibit Mast Cells With Impaired Functionality and Gut Homing and Do Not Develop Food Allergy

Background: Mucosal mast cells (MC) are key players in IgE-mediated food allergy (FA). The evidence on the interaction between gut microbiota, MC and susceptibility to FA is contradictory.Objective: We tested the hypothesis that commensal bacteria are essential for MC migration to the gut and their maturation impacting the susceptibility to FA.Methods: The development and severity of FA symptoms was studied in sensitized germ-free (GF), conventional (CV), and mice mono-colonized with L. plantarum WCFS1 or co-housed with CV mice. MC were phenotypically and functionally characterized.Results: Systemic sensitization and oral challenge of GF mice with ovalbumin led to increased levels of specific IgE in serum compared to CV mice. Remarkably, despite the high levels of sensitization, GF mice did not develop diarrhea or anaphylactic hypothermia, common symptoms of FA. In the gut, GF mice expressed low levels of the MC tissue-homing markers CXCL1 and CXCL2, and harbored fewer MC which exhibited lower levels of MC protease-1 after challenge. Additionally, MC in GF mice were less mature as confirmed by flow-cytometry and their functionality was impaired as shown by reduced edema formation after injection of degranulation-provoking compound 48/80. Co-housing of GF mice with CV mice fully restored their susceptibility to develop FA. However, this did not occur when mice were mono-colonized with L. plantarum.Conclusion: Our results demonstrate that microbiota-induced maturation and gut-homing of MC is a critical step for the development of symptoms of experimental FA. This new mechanistic insight into microbiota-MC-FA axis can be exploited in the prevention and treatment of FA in humans

Antigen-specific and antigen-non-specific intervention strategies against allergy in mice

Frühere Untersuchungen bezüglich intranasaler Toleranzinduktion zeigten die erfolgreiche Reduktion von allergischen Reaktionen mit Hilfe verschiedener Allergenkonstrukte (z.B. rekombinanten Allergenproteine, Polypeptide, Chimären und Peptiden). Es wurde jedoch nicht gezeigt wie Antigene mit verschiedenen Konformationen in den Schleimhäuten der Atemwege aufgenommen werden. In dieser Studie haben wir versucht, die Aufnahme in verschiedenen Zellpopulationen der Atemwege nach der intranasalen Verabreichung von drei strukturell unterschiedlichen Allergenen zu vergleichen. Hierzu wurden folgende drei Konstrukte verwendet: das Hauptbirkenpollenallergen Bet v 1-Protein, ein lineares synthetisch hergestelltes Hybrid Polypeptid (Hybrid-Peptide) und das Bet v 1 Monopeptid (Bet v 1-Peptide). Das Markieren der drei verwendeten Konstrukte mittels 5,6-Carboxyfluorescein ermöglicht es deren Aufnahme in Zellen des nasal assoziierten lymphatischen Gewebes und in Lungenzellen von naiven konventionellen, Bet v 1 sensibilisierten oder naiven keimfreien BALB/c Mäusen zu untersuchen. Die phänotypische Zellcharakterisierung in Lungenzellen sowohl nach einer in vitro Inkubation als auch nach einer intranasalen in vivo Verabreichung der drei Antigene ergab für naiven konventionellen Mäuse, dass alveolar epitheliale Typ 2 Zellen (ATII), jedoch nicht professionelle Antigenpräsentierende Zellen (wie Dendritische Zellen, Makrophagen oder B Zellen) für die Antigenaufnahme in der Lunge verantwortlich sind. Ähnliche Beobachtungen wurden in in vivo Studien an naiven keimfreien Mäusen gemacht. Im Falle von sensibilisierten Mäusen wurde festgestellt, dass Makrophagen hauptsächlich an der Antigenaufnahme in der Lunge verantwortlich sind. Um den Aufnahmemodus in die Zelle zu untersuchen, wurden rezeptorvermittelte Endozytose und Makropinozytose in der ATII-Zelllinie (A549) und aus Knochenmark generierten Dendritischen Zellen (BM-DCs) blockiert. Diese Inhibitionsversuche zeigen, dass sowohl in A549 Zellen als auch in BM-DCs rezeptorvermittelte Endozytose in der Aufnahme von Bet v 1-Protein und Hybrid-Peptide involviert ist. Die Blockade von Makropinozytose zeigt einen geringen Effekt in der Antigenaufnahme von Bet v 1-Protein und Hybrid-Peptide durch A549 Zellen, jedoch keinen Effekt in BM-DCs. Im Falle von Bet v 1-Peptide ist anzunehmen, dass diese durch direkte Zellpenetration in die Zelle aufgenommen wird. Desweiteren wurden ATII Zellen aus naiven konventionellen Mäusen nach der intranasalen Verabreichung von Bet v 1-Protein oder Bet v 1-Peptide, zur Untersuchung der mRNA isoliert. Unsere Untersuchungen ergaben, dass Bet v 1-Protein einen Anstieg von TSLP mRNA verursacht, jedoch Bet v 1-Peptide die Transkription von IL-10 und MCP1 steigert. Zusätzlich wurden A549 Zellen zusammen (in direktem Kontakt oder durch eine Transmembran getrennt) mit einer Monozytenzelllinie in der Gegenwart von Bet v 1-Protein, Hybrid-Peptide oder Bet v 1-Peptide stimuliert. Im Anschluss wurden diverse Zytokine (IL-6, IL-8 und TGF[beta]) als auch die mRNA Transkription (IL-6, IL-8, IL-10, MCP1 und TSLP) gemessen. Die Stimulation von A549 Zellen welche in direktem Kontakt mit der Monozytenzellline standen führte zu einer vermehrten Ausschüttung von IL-6 und IL-8. Werden die Zellen durch eine Transmembran voneinander getrennt und zusammen mit Bet v 1-Peptide oder Hybrid-Peptide inkubiert, konnten eine erhöhte Transkription von IL-10 mRNA in A549 Zellen beobachtet werden. Im Falle von einer Inkubation mit Bet v 1-Protein wurde vermehrt TSLP mRNA in A549 Zellen transkripiert. Diese Daten sind in Einklang mit unseren in vivo Beobachtungen. Unsere Daten zeigen, dass ATII Zellen nicht nur Zellen der Atemweg mit Barrierefunktion sind, sondern dass sie auch als wichtige Schlüsselfiguren in der Polarisation des Immunsystems fungieren können. Diese Funktion scheint aber vorsätzlich von der Größe und der Konformation des aufgenommenen Allergens beeinflusst zu werden.Our previous studies on intranasal tolerance induction demonstrated successfully the reduction of allergic responses with different allergen constructs (i.e. recombinant allergen proteins, polypeptides, chimers, and peptides). However, it has not been shown yet how these antigens with different conformations were taken up in the respiratory mucosa. In this study we therefore sought to compare the uptake of three structurally different allergens, i.e. the major birch pollen allergen Bet v 1-Protein, a linear synthetic hybrid poly-peptide and a Bet v 1 mono-peptide, by different cell populations of the respiratory tract following intranasal application. The three-dimensional recombinant Bet v 1-Protein, the poly-peptide (Hybrid-Peptide) and the mono-peptide of Bet v 1 (Bet v 1-Peptide) were labelled with 5,6-Carboxyfluorescein and their uptake was investigated in cells of the nasal associated lymphoid tissue (NALT) and lung cells from conventional naive and Bet v 1-sensitised or naive germ-free BALB/c mice. Phenotypic cell-characterisation of lung cells after antigen incubation in vitro, and after intranasal application in vivo was performed by flow cytometry. In vivo and in vitro studies in naive conventional mice indicated that alveolar epithelia type 2 (ATII) cells, but not the professional antigen presenting cells - dendritic cells, macrophages or B cells - were the major cell population in lungs taking up all three investigated antigens. Similar observations were made in in vivo studies with naive germ-free mice, whereas macrophages represent the dominant antigen-internalising cell population in sensitised mice. In order to investigate the pathway of antigen uptake within the cell, receptor mediated endocytosis or macropinocytosis were blocked in cells of an ATII cell line (A549) and bone marrow-derived dendritic cells (BM-DCs). From these blocking experiments it is possible to suggest that receptor mediated endocytosis is partly involved in the internalisation of Bet v 1-Protein and Hybrid-Peptide in A549 cells and BM-DCs. On the other hand, blockade of macropinocytosis had only a minor effect on the antigen uptake of Bet v 1-Protein and Hybrid-Peptide in A549 cells but not in BM-DCs. In contrast, the used blocking strategies had no effect on the internalisation of Bet v 1-Peptide in A549 cells or in BM-DCs, suggesting that Bet v 1-Peptide is internalised via direct cell penetration. Furthermore, ATII cells were isolated from lungs of naive conventional mice after intranasal application of Bet v 1-Protein or Bet v 1-Peptide in order to investigate the mRNA profile in vivo. Our analyses show an increase of TSLP mRNA levels after Bet v 1-Protein application, whereas Bet v 1-Peptide induced transcription of IL-10 and MCP1 in ATII cells. Additionally, A549 cells were co-cultured (directly or in transwells) with monocytes in the presence of Bet v 1-Protein, Hybrid-Peptide and Bet v 1-Peptide, followed by investigations on cytokine release (IL-6, IL-8 and TGF[beta]) and mRNA transcription (IL-6, IL-8, IL-10, MCP1, and TSLP). Stimulation of direct co-culture with Bet v 1-Peptide and Bet v 1-Protein resulted in enhanced secretion of IL-6 and IL-8, whereas incubation with Hybrid-Peptide induced only IL-6 release. Stimulation with Bet v 1-Peptide and Hybrid-Peptide gave rise to enhanced levels of IL-10 mRNA, while stimulation with Bet v 1-Protein led to increased transcription of TSLP mRNA in A549 cells of transwell co-cultures. While freshly isolated ATII cells are described to transdifferentiate towards an ATI phenotype in vitro, our data generated in A549 cells are in line with our in vivo findings, demonstrating that A549 cells can be used for further investigations, Our data indicate that ATII cells are not only respiratory cells with barrier functions but may also function as important key players in polarisation of the immune system in dependence of the size of the internalised allergen under steady state conditions.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2015OeBB(VLID)171645

Oocyst-Derived Extract of Toxoplasma Gondii Serves as Potent Immunomodulator in a Mouse Model of Birch Pollen Allergy.

INTRODUCTION:Previously, we have shown that oral infection with Toxoplasma gondii oocysts prevented type I allergy in mice. Here we investigated whether the application of a T. gondii 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. METHODS:First, we tested OLA in vitro. 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. RESULTS:Immunisation of mice with OLA induced high levels of Toxoplasma-specific IgG antibodies in sera along with increased production of IFN-γ and IL-10 in Toxoplasma-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+CD25highFoxp3+ 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. CONCLUSION:Our data demonstrate that pretreatment with the oocyst extract can exert anti-allergic effects comparable to T. gondii 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

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

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

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

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