Tissue-specific antigen-presenting cells contribute to distinct phenotypes of allergy.

Antigen-presenting cells (APCs) are critical cells bridging innate and adaptive immune responses by taking up, processing, and presenting antigens to naïve T cells. At steady state, APCs thus control both tissue homeostasis and the induction of tolerance. In allergies however, APCs drive a Th2-biased immune response that is directed against otherwise harmless antigens from the environment. The main types of APCs involved in the induction of allergy are dendritic cells, monocytes, and macrophages. However, these cell types can be further divided into local, tissue-specific populations that differ in their phenotype, migratory capacity, T-cell activating potential, and production of effector molecules. Understanding if distinct populations of APCs contribute to either tissue-specific immune tolerance, allergen sensitization, or allergic inflammation will allow us to better understand disease pathology and develop targeted treatment options for different stages of allergic disease. Therefore, this review describes the main characteristics, phenotypes, and effector molecules of the APCs involved in the induction of allergen-specific Th2 responses in affected barrier sites, such as the skin, nose, lung, and gastrointestinal tract. Furthermore, we highlight open questions that remain to be addressed to fully understand the contribution of different APCs to allergic disease.

Rescue from Pseudomonas aeruginosa Airway Infection via Stem Cell Transplantation.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to impaired ion transport in epithelial cells. Although lung failure due to chronic infection is the major comorbidity in individuals with cystic fibrosis, the role of CFTR in non-epithelial cells has not been definitively resolved. Given the important role of host defense cells, we evaluated the Cftr deficiency in pulmonary immune cells by hematopoietic stem cell transplantation in cystic fibrosis mice. We transplanted healthy bone marrow stem cells and could reveal a stable chimerism of wild-type cells in peripheral blood. The outcome of stem cell transplantation and the impact of healthy immune cells were evaluated in acute Pseudomonas aeruginosa airway infection. In this study, mice transplanted with wild-type cells displayed better survival, lower lung bacterial numbers, and a milder disease course. This improved physiology of infected mice correlated with successful intrapulmonary engraftment of graft-derived alveolar macrophages, as seen by immunofluorescence microscopy and flow cytometry of graft-specific leucocyte surface marker CD45 and macrophage marker CD68. Given the beneficial effect of hematopoietic stem cell transplantation and stable engraftment of monocyte-derived CD68-positive macrophages, we conclude that replacement of mutant Cftr macrophages attenuates airway infection in cystic fibrosis mice.

IL-17 regulates DC migration to the peribronchial LNs and allergen presentation in experimental allergic asthma.

IL-17 is associated with different phenotypes of asthma, however, it is not fully elucidated how it influences induction and maintenance of asthma and allergy. In order to determine the role of IL-17 in development of allergic asthma, we used IL-17A/F double KO (IL-17A/F KO) and WT mice with or without neutralization of IL-17 in an experimental allergic asthma model and analyzed airway hyperresponsiveness, lung inflammation, T helper cell polarization, and DCs influx and activation. We report that the absence of IL-17 reduced influx of DCs into lungs and lung draining LNs. Compared to WT mice, IL-17A/F KO mice or WT mice after neutralization of IL-17A showed reduced airway hyperresponsiveness, eosinophilia, mucus hypersecretion, and IgE levels. DCs from draining LNs of allergen-challenged IL-17A/F KO mice showed a reduction in expression of migratory and costimulatory molecules CCR7, CCR2, MHC-II, and CD40 compared to WT DCs. Moreover, in vivo stimulation of adoptively transferred antigen-specific cells was attenuated in lung-draining LNs in the absence of IL-17. Thus, we report that IL-17 enhances airway DC activation, migration, and function. Consequently, lack of IL-17 leads to reduced antigen-specific T cell priming and impaired development of experimental allergic asthma.

A comparison of curated gene sets versus transcriptomics-derived gene signatures for detecting pathway activation in immune cells.

BACKGROUND: Despite the significant contribution of transcriptomics to the fields of biological and biomedical research, interpreting long lists of significantly differentially expressed genes remains a challenging step in the analysis process. Gene set enrichment analysis is a standard approach for summarizing differentially expressed genes into pathways or other gene groupings. Here, we explore an alternative approach to utilizing gene sets from curated databases. We examine the method of deriving custom gene sets which may be relevant to a given experiment using reference data sets from previous transcriptomics studies. We call these data-derived gene sets, "gene signatures" for the biological process tested in the previous study. We focus on the feasibility of this approach in analyzing immune-related processes, which are complicated in their nature but play an important role in the medical research. RESULTS: We evaluate several statistical approaches to detecting the activity of a gene signature in a target data set. We compare the performance of the data-derived gene signature approach with comparable GO term gene sets across all of the statistical tests. A total of 61 differential expression comparisons generated from 26 transcriptome experiments were included in the analysis. These experiments covered eight immunological processes in eight types of leukocytes. The data-derived signatures were used to detect the presence of immunological processes in the test data with modest accuracy (AUC = 0.67). The performance for GO and literature based gene sets was worse (AUC = 0.59). Both approaches were plagued by poor specificity. CONCLUSIONS: When investigators seek to test specific hypotheses, the data-derived signature approach can perform as well, if not better than standard gene-set based approaches for immunological signatures. Furthermore, the data-derived signatures can be generated in the cases that well-defined gene sets are lacking from pathway databases and also offer the opportunity for defining signatures in a cell-type specific manner. However, neither the data-derived signatures nor standard gene-sets can be demonstrated to reliably provide negative predictions for negative cases. We conclude that the data-derived signature approach is a useful and sometimes necessary tool, but analysts should be weary of false positives.

B cells control maternofetal priming of allergy and tolerance in a murine model of allergic airway inflammation.

BACKGROUND: Allergic asthma is a chronic lung disease resulting from inappropriate immune responses to environmental antigens. Early tolerance induction is an attractive approach for primary prevention of asthma. OBJECTIVE: We analyzed the mechanisms of perinatal tolerance induction to allergens, with particular focus on the role of B cells in preconception and early intrauterine immune priming. METHODS: Wild-type (WT) and B cell-deficient mice received ovalbumin (OVA) intranasally before mating. Their offspring were analyzed in a murine model of allergic airway inflammation. RESULTS: Although antigen application before conception protected WT progeny from allergy, it aggravated allergic airway inflammation in B cell-deficient offspring. B-cell transfer restored protection, demonstrating the crucial role of B cells in perinatal tolerance induction. Effective diaplacentar allergen transfer was detectable in pregnant WT mice but not in pregnant B-cell knockout dams, and antigen concentrations in WT amniotic fluid (AF) were higher than in IgG-free AF of B cell-deficient dams. Application of OVA/IgG immune complexes during pregnancy boosted OVA uptake by fetal dendritic cells (DCs). Fetal DCs in human subjects and mice expressed strikingly higher levels of Fcγ receptors compared with DCs from adults and were highly efficient in taking up OVA/IgG immune complexes. Moreover, murine fetal DCs effectively primed antigen-specific forkhead box P3+ regulatory T cells after in vitro coincubation with OVA/IgG-containing AF. CONCLUSION: Our data support a decisive role for B cells and immunoglobulins during in utero tolerance priming. These findings improve the understanding of perinatal immunity and might support the development of effective primary prevention strategies for allergy and asthma in the future.

B cell subsets are modulated during allergic airway inflammation but are not required for the development of respiratory tolerance in a murine model.

Allergic asthma is a widespread chronic inflammatory disease of the airways. The role of different B cell subsets in developing asthma and respiratory tolerance is not well known. Especially regulatory B (Breg) cells are proposed to be important in asthma regulation. Using wild-type (WT) and B cell-deficient (µMT) mice we investigated how B cells are affected by induction of allergic airway inflammation and respiratory tolerance and whether they are necessary to develop these conditions. WT mice with an asthma-like phenotype, characterized by increased airway hyper reactivity, eosinophilic airway inflammation, mucus hypersecretion and elevated Th2 cytokines, exhibited increased MHCII and CD23 expression on follicular mature B cells in lung, bronchial lymph nodes (bLN) and spleen, which contributed to allergen-specific T cell proliferation in vitro. Germinal center B cell numbers were elevated and associated with increased production of allergen-specific immunoglobulins especially in bLN. In contrast, respiratory tolerance clearly attenuated these B cell alterations and directly enhanced marginal zone precursor B cells, which induced regulatory T cells in vitro. However, µMT mice developed asthma-like and tolerized phenotypes like WT mice. Our data indicate that although B cell subsets are affected by asthma-like and respiratory tolerant phenotypes, B cells are not required for tolerance induction.

IL-27 Is Essential for Suppression of Experimental Allergic Asthma by the TLR7/8 Agonist R848 (Resiquimod).

Different models of experimental allergic asthma have shown that the TLR7/8 agonist resiquimod (R848) is a potential inhibitor of type 2 helper cell-driven inflammatory responses. However, the mechanisms mediating its therapeutic effects are not fully understood. Using a model of experimental allergic asthma, we show that induction of IL-27 by R848 is critical for the observed ameliorative effects. R848 significantly inhibited all hallmarks of experimental allergic asthma, including airway hyperreactivity, eosinophilic airway inflammation, mucus hypersecretion, and Ag-specific Ig production. Whereas R848 significantly reduced IL-5, IL-13, and IL-17, it induced IFN-γ and IL-27. Neutralization of IL-27 completely reversed the therapeutic effect of R848 in the experimental asthma model, demonstrating dependence of R848-mediated suppression on IL-27. In vitro, R848 induced production of IL-27 by murine alveolar macrophages and dendritic cells and enhanced expression of programmed death-ligand 1, whose expression on monocytes and dendritic cells has been shown to regulate peripheral tolerance in both murine and human studies. Moreover, in vitro IL-27 enhanced secretion of IFN-γ whereas it inhibited IL-5 and IL-13, demonstrating its direct effect on attenuating Th2 responses. Taken together, our study proves that R848-mediated suppression of experimental asthma is dependent on IL-27. These data provide evidence of a central role of IL-27 for the control of Th2-mediated allergic diseases.

Improved protocol for simultaneous analysis of leukocyte subsets and epithelial cells from murine and human lung.

PURPOSE: To study and isolate lung cells by flow cytometry, enzymatic digestion and generation of single cell suspensions is required. This significantly influences expression of cellular epitopes and protocols need to be adapted for the best isolation and subsequent analysis of specific cellular subsets. MATERIALS AND METHODS: We optimized protocols for the simultaneous isolation and characterization of specific human and murine lung cell types. For alveolar epithelial cells (AEC), a primarily dispase based digestion method and for leukocytes, a primarily collagenase based technique was adapted. Protocols were applied in parallel in either single experimental mice or human lung specimens. RESULTS: Optimized dispase/DNase digestion yielded a high percentage of Epcam+CD45-CD31- AEC as assessed by flow cytometry. Epcam+CD45-CD3-CD11b-CD11c-CD16/32-CD19-CD31-F4/80- AEC were readily sortable with high purity and typical morphology and function upon in vitro stimulation with lipopolysaccharide or respiratory-syncytial-virus (RSV) infection. To analyze lung leukocytes, specimens were digested with an adapted collagenase/DNase protocol yielding high percentages of viable leukocytes with typical morphology, function, and preserved subset specific leukocyte markers. Both protocols could be applied simultaneously in a single experimental mouse post mortem. Application of both digestion methods in primary human lung specimens yielded similar results with high proportions of Epcam+CD45- human AEC after dispase/DNase digestion and preservation of human T cell epitopes after collagenase/DNase digestion. CONCLUSION: The here described protocols were optimized for the simple and efficient isolation of murine and human lung cells. In contrast to previously described techniques, they permit simultaneous in-depth characterization of pulmonary epithelial cells and leukocyte subsets such as T helper, cytotoxic T, and B cells from one sample. As such, they may help to comprehensively and sustainably characterize murine and human lung specimens and facilitate studies on the role of lung immune cells in different respiratory pathologies.

Gain-of-function STAT1 mutations are associated with intracranial aneurysms.

Chronic mucocutaneous candidiasis, characterized by persistent or recurrent fungal infections, represents the clinical hallmark in gain-of-function (GOF) signal transducer and activator of transcription 1 (STAT1) mutation carriers. Several cases of intracranial aneurysms have been reported in patients with GOF STAT1 mutation but the paucity of reported cases likely suggested this association still as serendipity. In order to endorse this association, we link the development of intracranial aneurysms with STAT1 GOF mutation by presenting the two different cases of a patient and her mother, and demonstrate upregulated phosphorylated STAT4 and IL-12 receptor ß1 upon stimulation in patient's blood cells. We also detected increased transforming growth factor (TGF)-ß type 2 receptor expression, particularly in CD14+ cells, and a slightly higher phosphorylation rate of SMAD3. In addition, the mother of the patient developed disseminated bacille Calmette-Guérin disease after vaccination, speculating that GOF STAT1 mutations may confer a predisposition to weakly virulent mycobacteria.

Monocyte/macrophage lineage commitment and distribution are affected by the lack of regulatory T cells in scurfy mice.

Foxp3(+) regulatory T (Treg) cells play a pivotal role in maintaining immunological tolerance. Loss-of-function mutations in the Foxp3 gene result in multiorgan inflammation known as immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in humans and scurfy (Sf) disease in mice. While the impact of missing Treg cells on adaptive immune cells is well documented, their role in regulation of myeloid cells remains unclear. Here we report that Sf mice exhibit an altered composition of stem and progenitor cells, characterized by increased numbers of myeloid precursors and higher efficiency of macrophage generation ex vivo. The proportion of monocytes/macrophages in the bone marrow, blood, and spleen was significantly elevated in Sf mice, which was accompanied with tissue-specific monocyte expression of homing receptor and phagocytic activity. Sf mice displayed high levels of M-CSF and other inflammatory cytokines, including monocyte-recruiting chemokines. Adoptive transfer of WT CD4(+) cells and in vivo neutralization of M-CSF normalized frequencies of monocyte subsets and their progenitors and reduced high levels of monocyte-related cytokines in Sf mice, while Treg cell transfer to RAG2(-/-) mice had no effect on myelopoiesis and monocyte/macrophage counts. Our findings illustrate that deregulated myelopoiesis in Sf mice is mainly caused by the inflammatory reaction resulting from the lack of Treg cells.

TLR4 preconditioning is associated with low success of OK-432 treatment for lymphatic malformations in children.

PURPOSE: We have recently shown that the relative TLR4 expression on monocytes of low responding pediatric patients after OK-432 treatment is significantly reduced after stimulation with lipopolysaccharide (LPS) compared with high responding children. The aim of this study was to perform further analysis to explain this observation. METHODS: Monocytes from children with high (HR, n = 5) and low response (LR, n = 6) after previous OK-432 treatment were stimulated with LPS for 20 h and analyzed with fluorescence-activated cell sorting (mean fluorescence intensity, MFI; level of significance P ≤ 0.05). RESULTS: Mean MFI after LPS stimulation was comparable in both groups (HR 1142 ± 652 units, LR 839 ± 427 units, P = 0.85). Significant changes after LPS stimulation are explained by higher pre-stimulation values in the LR group compared with the HR group (950 ± 718 vs. 477 ± 341, P = 0.25) with considerable differences of the mean expression changes after LPS stimulation (HR 665 ± 683 vs. LR -111 ± 605, P = 0.08). CONCLUSION: The previously shown reduced TLR4 upregulation on monocytes after LPS stimulation in the LR group compared with the HR group can be primarily explained by TLR preconditioning. This observation implies the use of absolute values with definite thresholds.

Priming of CD8+ T cells against cytomegalovirus-encoded antigens is dominated by cross-presentation.

CMV can infect dendritic cells (DCs), and direct Ag presentation could, therefore, lead to the priming of CMV-specific CD8(+) T cells. However, CMV-encoded immune evasins severely impair Ag presentation in the MHC class I pathway; thus, it is widely assumed that cross-presentation drives the priming of antiviral T cells. We assessed the contribution of direct versus cross priming in mouse CMV (MCMV) infection using recombinant viruses. DCs infected with an MCMV strain encoding the gB498 epitope from HSV-1 were unable to stimulate in vitro naive gB498-specific CD8(+) T cells from TCR transgenic mice. Infection of C57BL/6 mice with this recombinant virus led, however, to the generation of abundant numbers of gB498-specific T cells in vivo. Of the DC subsets isolated from infected mice, only CD8α(+) DCs were able to stimulate naive T cells, suggesting that this DC subset cross-presents MCMV-encoded Ag in vivo. Upon infection of mice with MCMV mutants encoding Ag that can either be well or hardly cross-presented, mainly CD8(+) T cells specific for cross-presented epitopes were generated. Moreover, even in the absence of immune evasion genes interfering with MHC class I-mediated Ag presentation, priming of T cells to Ag that can only be presented directly was not observed. We conclude that the host uses mainly DCs capable of cross-presentation to induce the CMV-specific CD8(+) T cell response during primary, acute infection and discuss the implications for the development of a CMV vaccine.

Polysialic acid on neuropilin-2 is exclusively synthesized by the polysialyltransferase ST8SiaIV and attached to mucin-type o-glycans located between the b2 and c domain.

Neuropilin-2 (NRP2) is well known as a co-receptor for class 3 semaphorins and vascular endothelial growth factors, involved in axon guidance and angiogenesis. Moreover, NRP2 was shown to promote chemotactic migration of human monocyte-derived dendritic cells (DCs) toward the chemokine CCL21, a function that relies on the presence of polysialic acid (polySia). In vertebrates, this posttranslational modification is predominantly found on the neural cell adhesion molecule (NCAM), where it is synthesized on N-glycans by either of the two polysialyltransferases, ST8SiaII or ST8SiaIV. In contrast to NCAM, little is known on the biosynthesis of polySia on NRP2. Here we identified the polySia attachment sites and demonstrate that NRP2 is recognized only by ST8SiaIV. Although polySia-NRP2 was found on bone marrow-derived DCs from wild-type and St8sia2(-/-) mice, polySia was completely lost in DCs from St8sia4(-/-) mice despite normal NRP2 expression. In COS-7 cells, co-expression of NRP2 with ST8SiaIV but not ST8SiaII resulted in the formation of polySia-NRP2, highlighting distinct acceptor specificities of the two polysialyltransferases. Notably, ST8SiaIV synthesized polySia selectively on a NRP2 glycoform that was characterized by the presence of sialylated core 1 and core 2 O-glycans. Based on a comprehensive site-directed mutagenesis study, we localized the polySia attachment sites to an O-glycan cluster located in the linker region between b2 and c domain. Combined alanine exchange of Thr-607, -613, -614, -615, -619, and -624 efficiently blocked polysialylation. Restoration of single sites only partially rescued polysialylation, suggesting that within this cluster, polySia is attached to more than one site.

Dynamic Toll-like receptor expression predicts outcome of sclerotherapy for lymphatic malformations with OK-432 in children.

BACKGROUND: Sclerotherapy with OK-432 is recommended as a first-line treatment for lymphatic malformations. However, 40% of patients show poor response, defined by involution to <50% of the original size. It has been suggested that the OK-432 effect is highly dependent on the Toll-like receptor (TLR) 4-dependent expression of TLR7 in antigen-presenting cells. We hypothesized that the ability for TLR expression in monocytes after treatment with the TLR4-ligand lipopolysaccharide (LPS) can be used to predict successful OK-432 treatment. METHODS: Blood was taken from children with low responder (LR, n = 6) and high responder (HR, n = 5) of previous OK-432 treatment. Monocytes were stimulated with LPS for 20 h. TLR expression was analyzed with fluorescence-activated cell sorting (mean fluorescence intensity). The level of significance was P ≤ 0.05. RESULTS: The mean age of patients in the HR group was 1.4 ± 0.9 y and in the LR group 2.8 ± 2.9 y (P = 0.31). The mean TLR4 upregulation after LPS stimulation in the HR group was significantly higher than in the LR group (factor 3.6 versus factor 1 compared with nonstimulated controls; P = 0.037). The mean TLR7 expression did not show significant differences between the groups. CONCLUSIONS: Dynamic TLR4 expression represents most probably a predictive parameter for the treatment of lymphatic malformations with OK-432 and should be further investigated.

Standardized generation of human iPSC-derived hematopoietic organoids and macrophages utilizing a benchtop bioreactor platform under fully defined conditions.

BACKGROUND: There is a significant demand for intermediate-scale bioreactors in academic and industrial institutions to produce cells for various applications in drug screening and/or cell therapy. However, the application of these bioreactors in cultivating hiPSC-derived immune cells and other blood cells is noticeably lacking. To address this gap, we have developed a xeno-free and chemically defined intermediate-scale bioreactor platform, which allows for the generation of standardized human iPSC-derived hematopoietic organoids and subsequent continuous production of macrophages (iPSC-Mac). METHODS: We describe a novel method for intermediate-scale immune cell manufacturing, specifically the continuous production of functionally and phenotypically relevant macrophages that are harvested on weekly basis for multiple weeks. RESULTS: The continuous production of standardized human iPSC-derived macrophages (iPSC-Mac) from 3D hematopoietic organoids also termed hemanoids, is demonstrated. The hemanoids exhibit successive stage-specific embryonic development, recapitulating embryonic hematopoiesis. iPSC-Mac were efficiently and continuously produced from three different iPSC lines and exhibited a consistent and reproducible phenotype, as well as classical functionality and the ability to adapt towards pro- and anti-inflammatory activation stages. Single-cell transcriptomic analysis revealed high macrophage purity. Additionally, we show the ability to use the produced iPSC-Mac as a model for testing immunomodulatory drugs, exemplified by dexamethasone. CONCLUSIONS: The novel method demonstrates an easy-to-use intermediate-scale bioreactor platform that produces prime macrophages from human iPSCs. These macrophages are functionally active and require no downstream maturation steps, rendering them highly desirable for both therapeutic and non-therapeutic applications.

Competitive survival of clonal serial Pseudomonas aeruginosa isolates from cystic fibrosis airways in human neutrophils.

Chronic airway infections with Pseudomonas aeruginosa are the major co-morbidity in most people with cystic fibrosis (CF) sustained by neutrophils as the major drivers of lung inflammation, damage, and remodeling. Phagocytosis assays were performed with clonal consortia of longitudinal P. aeruginosa airway isolates collected from people with CF since the onset of lung colonization until patient's death or replacement by another clone. The extra- and intracellular abundance of individual strains was assessed by deep amplicon sequencing of strain-specific single nucleotide variants in the bacterial genome. The varied microevolution of the accessory genome of the P. aeruginosa clones during mild and severe courses of infection corresponded with a differential persistence of clonal progeny in the neutrophil phagosome. By simultaneously exposing the ancestor and its progeny to the same habitat, the study recapitulated the time lapse of the temporal change of the fitness of the clone to survive in neutrophils.

Single cell RNA sequencing reveals distinct clusters of Irf8-expressing pulmonary conventional dendritic cells.

A single population of interferon-regulatory factor 8 (Irf8)-dependent conventional dendritic cell (cDC type1) is considered to be responsible for both immunogenic and tolerogenic responses depending on the surrounding cytokine milieu. Here, we challenge this concept of an omnipotent single Irf8-dependent cDC1 cluster through analysis of pulmonary cDCs at single cell resolution. We report existence of a pulmonary cDC1 cluster lacking Xcr1 with an immunogenic signature that clearly differs from the Xcr1 positive cDC1 cluster. The Irf8+Batf3+Xcr1- cluster expresses high levels of pro-inflammatory genes associated with antigen presentation, migration and co-stimulation such as Ccr7, Cd74, MHC-II, Ccl5, Il12b and Relb while, the Xcr1+ cDC1 cluster expresses genes corresponding to immune tolerance mechanisms like Clec9a, Pbx1, Cadm1, Btla and Clec12a. In concordance with their pro-inflammatory gene expression profile, the ratio of Xcr1- cDC1s but not Xcr1+cDC1 is increased in the lungs of allergen-treated mice compared to the control group, in which both cDC1 clusters are present in comparable ratios. The existence of two distinct Xcr1+ and Xcr1- cDC1 clusters is furthermore supported by velocity analysis showing markedly different temporal patterns of Xcr1- and Xcr1+cDC1s. In summary, we present evidence for the existence of two different cDC1 clusters with distinct immunogenic profiles in vivo. Our findings have important implications for DC-targeting immunomodulatory therapies.

Toll-like receptor-2 agonist-allergen coupling efficiently redirects Th2 cell responses and inhibits allergic airway eosinophilia.

Toll-like receptor (TLR) agonists beneficially modulate allergic airway inflammation. However, the efficiency of TLR agonists varies considerably, and their exact cellular mechanisms (especially of TLR 2/6 agonists) are incompletely understood. We investigated at a cellular level whether the administration of the pharmacologically improved TLR2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPP) conjugated to antigenic peptide (BPP-OVA) could divert an existing Th2 response and influence airway eosinophilia. The effects of BPP-OVA on airway inflammation were assessed in a classic murine sensitization/challenge model and an adoptive transfer model, which involved the adoptive transfer of in vitro differentiated ovalbumin (OVA)-specific Th2 cells. Functional T-cell stimulation by lung dendritic cells (DCs) was determined both in vitro and in vivo, combined with a cytokine secretion analysis. A single mucosal application of BPP-OVA efficiently delivered antigen, led to TLR2-mediated DC activation, and resulted in OVA-specific T-cell proliferation via lung DCs in vivo. In alternative models of allergic airway disease, a single administration of BPP-OVA before OVA challenge (but not BPP alone) significantly reduced airway eosinophilia, most likely through altered antigen-specific T-cell stimulation via DCs. Analyses of adoptively transferred Th2-biased cells after BPP-OVA administration in vivo suggested that BPP-OVA guides antigen-specific Th2 cells to produce significantly higher amounts of IFN-γ upon allergen challenge. In conclusion, our data show for the first time that a single mucosal administration of a TLR 2/6 agonist-allergen conjugate can provoke IFN-γ responses in Th2-biased cells and alleviate allergic airway inflammation.

Timing of Blood Sample Processing Affects the Transcriptomic and Epigenomic Profiles in CD4+ T-cells of Atopic Subjects.

Optimal pre-analytical conditions for blood sample processing and isolation of selected cell populations for subsequent transcriptomic and epigenomic studies are required to obtain robust and reproducible results. This pilot study was conducted to investigate the potential effects of timing of CD4+ T-cell processing from peripheral blood of atopic and non-atopic adults on their transcriptomic and epigenetic profiles. Two heparinized blood samples were drawn from each of three atopic and three healthy individuals. For each individual, CD4+ T-cells were isolated from the first blood sample within 2 h (immediate) or from the second blood sample after 24 h storage (delayed). RNA sequencing (RNA-Seq) and histone H3K27 acetylation chromatin immunoprecipitation sequencing (ChIP-Seq) analyses were performed. A multiplicity of genes was shown to be differentially expressed in immediately processed CD4+ T-cells from atopic versus healthy subjects. These differences disappeared when comparing delayed processed cells due to a drastic change in expression levels of atopy-related genes in delayed processed CD4+ T-cells from atopic donors. This finding was further validated on the epigenomic level by examining H3K27 acetylation profiles. In contrast, transcriptomic and epigenomic profiles of blood CD4+ T-cells of healthy donors remained rather unaffected. Taken together, for successful transcriptomics and epigenomics studies, detailed standard operation procedures developed on the basis of samples from both healthy and disease conditions are implicitly recommended.

Interleukin-17A and interleukin-22 production by conventional and non-conventional lymphocytes in three different end-stage lung diseases.

Objectives: The contribution of adaptive vs. innate lymphocytes to IL-17A and IL-22 secretion at the end stage of chronic lung diseases remains largely unexplored. In order to uncover tissue- and disease-specific secretion patterns, we compared production patterns of IL-17A and IL-22 in three different human end-stage lung disease entities. Methods: Production of IL-17A, IL-22 and associated cytokines was assessed in supernatants of re-stimulated lymphocytes by multiplex assays and multicolour flow cytometry of conventional T cells, iNKT cells, γδ T cells and innate lymphoid cells in bronchial lymph node and lung tissue from patients with emphysema (n = 19), idiopathic pulmonary fibrosis (n = 14) and cystic fibrosis (n = 23), as well as lung donors (n = 17). Results: We detected secretion of IL-17A and IL-22 by CD4+ T cells, CD8+ T cells, innate lymphoid cells, γδ T cells and iNKT cells in all end-stage lung disease entities. Our analyses revealed disease-specific contributions of individual lymphocyte subpopulations to cytokine secretion patterns. We furthermore found the high levels of microbial detection in CF samples to associate with a more pronounced IL-17A signature upon antigen-specific and unspecific re-stimulation compared to other disease entities and lung donors. Conclusion: Our results show that both adaptive and innate lymphocyte populations contribute to IL-17A-dependent pathologies in different end-stage lung disease entities, where they establish an IL-17A-rich microenvironment. Microbial colonisation patterns and cytokine secretion upon microbial re-stimulation suggest that pathogens drive IL-17A secretion patterns in end-stage lung disease.