Effector and regulatory dendritic cells display distinct patterns of miRNA expression.

INTRODUCTION: MicroRNAs (miRNAs) contribute to the regulation of dendritic cell (DC) polarization, thereby influencing the balance of adaptive immune responses. Herein, we studied the expression of miRNAs in polarized DCs and analyzed whether expression of these miRNAs could be associated with allergic rhinitis and allergen immunotherapy (AIT) outcome. METHOD: Using specific culture conditions, we differentiated immature human monocyte-derived DCs into DC1, DC2, and DCreg subsets (supporting the differentiation of TH 1, TH 2 or regulatory T cells, respectively). Profiling of miRNA expression was performed in these DC subpopulations using microarrays. Levels of miRNAs specific for polarized DCs were then evaluated in a cohort of 58 patients with allergic rhinitis and 25 non-allergic controls, as well as in samples from 30 subjects treated with sublingual grass pollen tablets or placebo for four months. RESULTS: We successfully identified 16 miRNAs differentially regulated between immature DCs, DC1, DC2, and DCreg cells. In allergic rhinoconjunctivitis patients, the expression of two of those miRNAs (miR-132 and miR-155), was down-regulated compared to non-allergic individuals. However, the levels of these miRNAs were not significantly modified following four months of grass pollen immunotherapy. CONCLUSIONS: Studying polarized DCs and clinical samples from subjects with or without allergic rhinoconjunctivitis, we demonstrated that the expression of two miRNAs linked to effector DCs (i.e., DC1 and/or DC2 cells), was reduced in the blood of patients with allergic rhinoconjunctivitis. Nevertheless, these miRNAs did not represent relevant biomarkers to predict or follow-up AIT efficacy.

Changes in markers associated with dendritic cells driving the differentiation of either TH2 cells or regulatory T cells correlate with clinical benefit during allergen immunotherapy.

BACKGROUND: Regulatory dendritic cell (DC) markers, such as C1Q, are upregulated in PBMCs of patients with grass pollen allergy exhibiting clinical benefit during allergen immunotherapy (AIT). OBJECTIVES: We sought to define markers differentially expressed in human monocyte-derived DCs differentiated toward a proallergic (DCs driving the differentiation of TH2 cells [DC2s]) phenotype and investigate whether changes in such markers in the blood correlate with AIT efficacy. METHODS: Transcriptomes and proteomes of monocyte-derived DCs polarized toward DCs driving the differentiation of TH1 cells (DC1s), DC2s, or DCs driving the differentiation of regulatory T cells (DCreg cells) profiles were compared by using genome-wide cDNA microarrays and label-free quantitative proteomics, respectively. Markers differentially regulated in DC2s and DCreg cells were assessed by means of quantitative PCR in PBMCs from 80 patients with grass pollen allergy before and after 2 or 4 months of sublingual AIT in parallel with rhinoconjunctivitis symptom scores. RESULTS: We identified 20 and 26 new genes/proteins overexpressed in DC2s and DCreg cells, respectively. At an individual patient level, DC2-associated markers, such as CD141, GATA3, OX40 ligand, and receptor-interacting serine/threonine-protein kinase 4 (RIPK4), were downregulated after a 4-month sublingual AIT course concomitantly with an upregulation of DCreg cell-associated markers, including complement C1q subcomponent subunit A (C1QA), FcγRIIIA, ferritin light chain (FTL), and solute carrier organic anion transporter family member 2B1 (SLCO2B1), in the blood of clinical responders as opposed to nonresponders. Changes in such markers were better correlated with clinical benefit than alterations of allergen-specific CD4(+) T-cell or IgG responses. CONCLUSIONS: A combination of 5 markers predominantly expressed by blood DCs (ie, C1Q and CD141) or shared with lymphoid cells (ie, FcγRIIIA, GATA3, and RIPK4) reflecting changes in the balance of regulatory/proallergic responses in peripheral blood can be used as early as after 2 months to monitor the early onset of AIT efficacy.

Efficacy of sublingual vectorized recombinant Bet v 1a in a mouse model of birch pollen allergic asthma.

BACKGROUND: Second generation sublingual allergy vaccines based upon recombinant allergens combined with vector systems are being developed as an alternative to conventional allergen extracts. Herein, we evaluated the efficacy of a recombinant form of the major allergen Bet v 1a (rBet v 1a) formulated as a mucoadhesive particle in a preclinical model of birch pollen (BP) respiratory allergy. MATERIALS AND METHODS: BALB/c mice were sensitized to BP extracts by intraperitoneal injections followed by aerosol exposures. Sensitized mice underwent sublingual immunotherapy (SLIT) twice a week for eight weeks with either a BP extract or rBet v 1a formulated in amylopectin-based microparticles (MPA). SLIT efficacy was assessed using whole body plethysmography, lung histology and cell counts in broncho-alveolar lavages (BAL) as read outs. BP and/or rBet v 1a-specific T cell and antibody responses were monitored in lung and serum, respectively. IgA levels were measured in saliva. RESULTS: Mice sensitized to BP exhibit chronic airway hyperresponsiveness (AHR), lung inflammation (documented by compliance and resistance measurements), eosinophil infiltrates in BAL, as well as Bet v 1-specific Th2 biased responses. Both SLIT with soluble rBet v 1a (50µg/dose) and BP extract (equivalent to 50µg rBet v 1 per dose) lead to a significant reduction in AHR, lung eosinophilia and Th2 responses. A sub-optimal dose of 5µg of rBet v 1a displays a similar level of efficacy with a significant decrease of Th2 responses when formulated with MPA microparticles. In addition, allergen vectorization with mucoadhesive particles allows a faster reduction in AHR in sensitized animals. CONCLUSION: We demonstrate in a murine model of chronic BP respiratory allergy the efficacy of SLIT with vectorized rBet v 1a. Thus, combining recombinant allergens with mucoadhesive vector systems paves the ground for improved second generation sublingual allergy vaccines.

A regulatory dendritic cell signature correlates with the clinical efficacy of allergen-specific sublingual immunotherapy.

BACKGROUND: Given their pivotal role in the polarization of T-cell responses, molecular changes at the level of dendritic cells (DCs) could represent an early signature indicative of the subsequent orientation of adaptive immune responses during immunotherapy. OBJECTIVE: We sought to investigate whether markers of effector and regulatory DCs are affected during allergen immunotherapy in relationship with clinical benefit. METHODS: Differential gel electrophoresis and label-free mass spectrometry approaches were used to compare whole proteomes from human monocyte-derived DCs differentiated toward either regulatory or effector functions. The expression of those markers was assessed by using quantitative PCR in PBMCs from 79 patients with grass pollen allergy enrolled in a double-blind, placebo-controlled clinical study evaluating the efficacy of sublingual tablets in an allergen exposure chamber over a 4-month period. RESULTS: We identified several markers associated with DC1 and/or DC17 effector DCs, including CD71, FSCN1, IRF4, NMES1, MX1, TRAF1. A substantial phenotypic heterogeneity was observed among various types of tolerogenic DCs, with ANXA1, Complement component 1 (C1Q), CATC, GILZ, F13A, FKBP5, Stabilin-1 (STAB1), and TPP1 molecules established as shared or restricted regulatory DC markers. The expression of 2 of those DCs markers, C1Q and STAB1, was increased in PBMCs from clinical responders in contrast to that seen in nonresponders or placebo-treated patients. CONCLUSION: C1Q and STAB1 represent candidate biomarkers of early efficacy of allergen immunotherapy as the hallmark of a regulatory innate immune response predictive of clinical tolerance.

Lactococcus lactis NCC 2287 alleviates food allergic manifestations in sensitized mice by reducing IL-13 expression specifically in the ileum.

OBJECTIVE: Utilizing a food allergy murine model, we have investigated the intrinsic antiallergic potential of the Lactococcus lactis NCC 2287 strain. METHODS: BALB/c mice were sensitized at weekly intervals with ovalbumin (OVA) plus cholera toxin (CT) by the oral route for 7 weeks. In this model, an oral challenge with a high dose of OVA at the end of the sensitization period leads to clinical symptoms. Lactococcus lactis NCC 2287 was given to mice via the drinking water during sensitization (prevention phase) or after sensitization (management phase). RESULTS: Lactococcus lactis NCC 2287 administration to sensitized mice strikingly reduced allergic manifestations in the management phase upon challenge, when compared to control mice. No preventive effect was observed with the strain. Lactococcus lactis NCC 2287 significantly decreased relative expression levels of the Th-2 cytokine, IL-13, and associated chemokines CCL11 (eotaxin-1) and CCL17 (TARC) in the ileum. No effect was observed in the jejunum. CONCLUSION/SIGNIFICANCE: These results taken together designate Lactococcus lactis NCC 2287 as a candidate probiotic strain appropriate in the management of allergic symptoms.

Bifidobacterium bifidum NCC 453 promotes tolerance induction in murine models of sublingual immunotherapy.

BACKGROUND: Enhancing clinical efficacy remains a major goal in allergen-specific immunotherapy. In this study, we tested three strains of bifidobacteria as candidate adjuvants for sublingual allergy vaccines. METHODS: Probiotic candidates were evaluated in human monocyte-derived dendritic cell (h-DC) maturation and CD4(+) T-cell polarization in vitro models and further tested in murine models of sublingual immunotherapy in BALB/c mice sensitized to either ovalbumin or birch pollen. RESULTS: Bifidobacterium adolescentis, B. bifidum and B. longum induced h-DC maturation and polarized naïve CD4(+) T cells toward interferon-γ and interleukin-10 production. B. bifidum increased CD25(high), Foxp3(+) cells within CD4(+) T lymphocytes and was the most potent inducer of interferon-γ in Th2-skewed peripheral blood mononuclear cells and h-DC T-cell cocultures. It also induced a significant decrease in airway hyperresponsiveness in BALB/c mice sensitized to ovalbumin. Sublingual administration of B. bifidum together with recombinant Bet v 1 enhanced tolerance induction in BALB/c mice sensitized to birch pollen, with a downregulation of both airway hyperresponsiveness, lung inflammation and Bet v 1-specific Th2 responses. CONCLUSIONS: Due to its capacity to reorient established Th2 responses toward Th1/regulatory T-cell profiles, B. bifidum represents a valid candidate adjuvant for specific immunotherapy of type I allergies.

Lactic acid bacteria as adjuvants for sublingual allergy vaccines.

We compared immunomodulatory properties of 11 strains of lactic acid bacteria as well as their capacity to enhance sublingual immunotherapy efficacy in a murine asthma model. Two types of bacterial strains were identified, including: (i) potent inducers of IL-12p70 and IL-10 in dendritic cells, supporting IFN-gamma and IL-10 production in CD4+ T cells such as Lactobacillus helveticus; (ii) pure Th1 inducers such as L. casei. Sublingual administration in ovalbumin-sensitized mice of L. helveticus, but not L. casei, reduced airways hyperresponsiveness, bronchial inflammation and proliferation of specific T cells in cervical lymph nodes. Thus, probiotics acting as a Th1/possibly Treg, but not Th1 adjuvant, potentiate tolerance induction via the sublingual route.

Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells.

BACKGROUND: A detailed characterization of oral antigen-presenting cells is critical to improve second-generation sublingual allergy vaccines. OBJECTIVE: To characterize oral dendritic cells (DCs) within lingual and buccal tissues from BALB/c mice with respect to their surface phenotype, distribution, and capacity to polarize CD4(+) T-cell responses. METHODS: In situ analysis of oral DCs was performed by immunohistology. Purified DCs were tested in vitro for their capacity to capture, process, and present the ovalbumin antigen to naive CD4(+) T cells. In vivo priming of ovalbumin-specific T cells adoptively transferred to BALB/c mice was analyzed by cytofluorometry in cervical lymph nodes after sublingual administration of mucoadhesive ovalbumin. RESULTS: Three subsets of oral DCs with a distinct tissue distribution were identified: (1) a minor subset of CD207(+) Langerhans cells located in the mucosa itself, (2) a major subpopulation of CD11b(+)CD11c(-) and CD11b(+)CD11c(+) myeloid DCs at the mucosal/submucosal interface, and (3) B220(+)120G8(+) plasmacytoid DCs found in submucosal tissues. Purified myeloid and plasmacytoid oral DCs capture and process the antigen efficiently and are programmed to elicit IFN-gamma and/or IL-10 production together with a suppressive function in naive CD4(+) T cells. Targeting the ovalbumin antigen to oral DCs in vivo by using mucoadhesive particles establishes tolerance in the absence of cell depletion through the stimulation of IFN-gamma and IL-10-producing CD4(+) regulatory T cells in cervical lymph nodes. CONCLUSION: The oral immune system is composed of various subsets of tolerogenic DCs organized in a compartmentalized manner and programmed to induce T(H)1/regulatory T-cell responses.

IL-10-inducing adjuvants enhance sublingual immunotherapy efficacy in a murine asthma model.

BACKGROUND: IL-10-inducing adjuvants could enhance the efficacy of allergy vaccines in establishing allergen-specific tolerance. The aim of this study was to identify such adjuvants using in vitro cultures of human and murine cells and to evaluate them in a therapeutic murine model of sublingual immunotherapy (SLIT). METHODS: Adjuvants stimulating IL-10 gene expression by human or murine immune cells were tested sublingually in BALB/c mice sensitized to ovalbumin (OVA), assessing the reduction in airway hyperresponsiveness (AHR) by whole-body plethysmography. The induction of regulatory T cells (T(reg)) was evaluated using phenotypic and functional assays. T-cell proliferation in cervical lymph nodes (LNs) was assessed following intravenous transfer of CFSE-labelled OVA-specific T cells and FACS analysis. RESULTS: A combination of 1,25-dihydroxyvitamin D3 plus dexamethasone (VitD3/Dex) as well as Lactobacillus plantarum were found to induce IL-10 production by human and murine dendritic cells (DCs). The former inhibits LPS-induced DC maturation, whereas L. plantarum induces DC maturation. Following stimulation with VitD3/Dex-pretreated DCs, CD4+ naïve T cells exhibit a T(reg) profile. In contrast, a Th1/T(reg) pattern of differentiation is observed in the presence of DCs treated with L. plantarum. Both adjuvants significantly enhance SLIT efficacy in mice, in association with either induction of Foxp3+ T(reg) cells (for VitD3/Dex) or proliferation of OVA-specific T cells in cervical LNs (for L. plantarum). CONCLUSIONS: Both VitD3/Dex and L. plantarum polarize naïve T cells towards IL-10-expressing T cells, through distinct mechanisms. As adjuvants, they both enhance SLIT efficacy in a murine asthma model.

A synthetic triacylated pseudo-dipeptide molecule promotes Th1/TReg immune responses and enhances tolerance induction via the sublingual route.

In this study, we tested two triacylated pseudo-dipeptidic molecules, OM-197-MP-AC and OM-294-BA-MP as candidate adjuvants for allergy vaccines. Both molecules induce human dendritic cell (h-DC) maturation and polarize naïve T cells toward the Th1 type with IFNgamma production. Only OM-294-BA-MP induces IL10 gene expression both in monocyte-derived DCs and CD4+ naïve T cells. Sublingual administration of OM-294-BA-MP plus the antigen enhances tolerance induction in BALB/c mice with established asthma to ovalbumin with an impact on both airways hyperresponsiveness and lung inflammation. Given its Th1/Treg polarizing properties, OM-294-BA-MP is a valid candidate for sublingual allergy vaccines.

Phospholipid scramblase, a new effector of FcepsilonRI signaling in mast cells.

The signaling network of the high-affinity receptor for IgE in mast cells involves tyrosine phosphorylation of a number of proteins. Here we report evidence that phospholipid scramblase, a protein involved in the distribution of phospholipids between the two leaflets of the plasma membrane, is a target for tyrosine kinases in this signal. The implication of this observation is briefly discussed.